Decellularized tissue engineered hyaline cartilage graft for articular cartilage repair

Journal Pre-proof Decellularized tissue engineered hyaline cartilage graft for articular cartilage repair Xiaolei Nie, Yon Jin Chuah, Wenzhen Zhu, Pengfei He, Yvonne Peck, Dong-An Wang PII:

S0142-9612(20)30067-3

DOI:

https://doi.org/10.1016/j.biomaterials.2020.119821

Reference:

JBMT 119821

To appear in:

Biomaterials

Received Date: 20 October 2019 Revised Date:

3 January 2020

Accepted Date: 23 January 2020

Please cite this article as: Nie X, Chuah YJ, Zhu W, He P, Peck Y, Wang D-A, Decellularized tissue engineered hyaline cartilage graft for articular cartilage repair, Biomaterials (2020), doi: https:// doi.org/10.1016/j.biomaterials.2020.119821. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier Ltd.

Decellularized Tissue Engineered Hyaline Cartilage Graft for Articular Cartilage Repair

Xiaolei Nie1, Yon Jin Chuah1, Wenzhen Zhu1, Pengfei He1, Yvonne Peck1 and Dong-An Wang*1,2

1

School of Chemical and Biomedical Engineering, Nanyang Technological University,

Singapore 2

Department of Biomedical Engineering, City University of Hong Kong, Kowloon, Hong

Kong SAR

*Corresponding author:

Dong-An WANG, Ph.D. Department of Biomedical Engineering City University of Hong Kong 83 Tat Chee Avenue, Kowloon, Hong Kong SAR Email: [email protected]

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Abstract Articular cartilage repair has been a long-standing challenge in orthopaedic medicine due to the limited self-regenerative capability of cartilage tissue. Currently, cartilage lesions are often treated by microfracture or autologous chondrocyte implantation (ACI). However, these treatments are frequently reported to result in a mixture of the desired hyaline cartilage and mechanically inferior fibrocartilage. In this study, by combining the advantages of cartilage tissue engineering and decellularization technology, we developed a decellularized allogeneic hyaline cartilage graft, named dLhCG, which achieved superior efficacy in articular cartilage repair and surpassed living autologous chondrocyte-based cartilaginous engraftment and ACI. By the 6-month time point after implantation in porcine knee joints, the fine morphology, composition, phenotype, microstructure and mechanical properties of the regenerated hyaline-like cartilaginous neo-tissue have been demonstrated via histology, biochemical assays, DNA microarrays and mechanical tests. The articular cartilaginous engraftment with allogeneic dLhCG was indicated to be well consistent, compatible and integrated with the native cartilage of the host. The successful repair of articular chondral defects in large animal models suggests the readiness of allogeneic dLhCG for clinical trials.

Keywords: Tissue engineering, cartilage, scaffold-free, decellularization, large animal model, pre-clinical

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1. Introduction Articular cartilage, as a connective tissue of the diarthrodial joint that is devoid of blood vessels, lymphatic tissues and nerves, is highly specialized for adaptation to harsh biomechanical environments.[1] The demanded superior biomechanical property of articular cartilage is critically endowed as a result of its unique, highly pure, hyaline-type cartilaginous composition and structural organization of the extracellular matrices (ECMs), specifically, a tight network of type II collagen (Col II) fibrils and abundant negatively charged proteoglycan chains. Consequently, once cartilage is damaged by trauma, disease or degeneration, the avascular nature of cartilage tissue and the low metabolic rate of the chondrocytes severely limits the capability of intrinsic healing and repair.[2] Hence, effective therapies for the treatment of significant articular cartilage lesions ultimately rely on surgical intervention and engraftments that resemble the high purity of hyaline cartilage tissue in situ. Current articular cartilage repairing strategies mainly include bone marrow stimulation (“microfracture”), osteochondral autograft transfer system (OATS, also known as “mosaicplasty”) and autologous chondrocyte implantation (ACI).[3] Small, symptomatic articular cartilage lesions are commonly treated via microfracture, which results in the formation of mechanically inferior fibrocartilage.[4,5] OATS can temporarily restore the smooth surface of the defect; however, graft subsidence due to postoperative weight bearing and the absence of repair within the dead space between the cylindrical grafts remains as unsolved flaws that may eventually result in the failure of the repair.[6] The inefficacy of OATS indicates that, in addition to the limited donor source of autologous native cartilage grafts, another major hurdle in cartilage transplantation lies in the host-graft integration, because both the diffusion between hard tissues and in situ re-cellularization from the host are hampered. ACI is a cellular engineering-based surgical procedure that is currently practised as a popular, second-line treatment for relatively larger-sized articular cartilage lesions,

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especially in young patients.[7] However, due to the liquid nature of the implant, the loss of therapeutic chondrocytes severely affects the quality of the final repair.[8,9] In addition, the uncontrollable overgrowth of neo-tissue and the secondary damage that is caused by suturing during the operation are frequently encountered as negative complications.[8–12] In recent decades, the decellularization of natural tissue has been developed and applied as a new technology to provide grafting materials for the use of regenerative medicine; in this method, immunogenic cellular components are removed and active biological factors are preserved together with the genuine 3D microstructures and architecture that made up the original tissue ECM. [13],[14] These decellularized ECMs have been reported to enhance tissue regeneration and function recovery, because they resemble native matrix environments and provide critical biological cues for cell adhesion, growth and development. Moreover, the application of the decellularization approach broadens the source of tissue graft from autologous to allogeneic and xenogeneic since the cellular components, as the major sources of the immunogens, are substantially eliminated.[15] However, the decellularization of native cartilage, as a hard tissue composed of condensed ECM, suffers from either over-residue of cellular immunogens after inadequate treatment or overdestruction of the ECM by harsh procedures, either of which defeats the purpose of the decellularization technology.[16] In this study, we developed a new strategy that combines the advantages of cartilage tissue engineering and decellularization technology, specifically meaning to decellularize a tissue-engineered cartilage graft instead of native cartilage explants. Through these methods, we designed and developed a novel, decellularized, tissue-engineered cartilage graft that is composed of pure hyaline-like cartilaginous ECM and is endowed with a macro-porous (hundreds of microns in diameter) structure. As a porous, sponge-like graft, it provides a softer and more accessible platform that can be much more easily and thoroughly

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decellularized via milder processes, thereby allowing for the original composition and microstructure of the graft’s ECM to be maximally preserved. Simultaneously, the cellular components can be maximally cleared. For the same reason, this graft (being that it is softer and more porous than decellularized native cartilage) also allows host chondrocytes to easily migrate across the host-graft interface and to re-cellularize the acellular graft, thus enabling the development of neo-tissue in situ and simultaneously facilitating the host-graft connection and integration.[17] This decellularized, tissue-engineered hyaline cartilage graft is derived via decellularization process from an autologous chondrocyte-based tissue engineering product that we had previously developed, which is known as “living hyaline cartilage graft” (LhCG).[18,19] Following the naming of LhCG, here, the decellularized product is named dLhCG. As previously reported, LhCG is produced by a 3D culture of autologous chondrocytes in an alginate-based macro-porous hydrogel. For the creation of this product, an inter-penetrating network (IPN) combining chondrocytes-derived cartilaginous neo-tissue and alginate scaffolding material was first formed, after which the alginate component was instantly removed from the IPN via rinsing with citrate saline. Thus, LhCG, as a tissue engineering product that is only composed of living cartilaginous neo-tissue, consists of a form of a macroscopic, sponge-like construct (Figure 1.).[19] The setting-up process like this determines that LhCG is free of any non-cartilaginous impurities. Moreover, as the 3D culture of chondrocytes has always been mediated by non-cell-adhesive hydrogel substrates, focal adhesions and the consequent fibrogenic de-differentiation of chondrocytes are substantially prevented or minimized. Hence, LhCG possesses a relatively pure hyaline-type cartilaginous composition and phenotype, which is characterized by the predominant content of Col II over type I collagen (Col I).[18] As a direct product of LhCG via decellularization, dLhCG is designed to be exclusively made of the ECM of LhCG (Figure 1.); therefore,

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dLhCG naturally inherits the hyaline-type cartilaginous composition and property, as well as the structural traits, of LhCG. Further taking the advantages of decellularization, namely being an acellular product, dLhCG is designed to possess immuno-compatibility and recellularization accessibility for transplantation practice while also providing a logistic convenience for transportation and storage, as well as a much more flexible time-window for surgical operation. Collectively, dLhCG is expected to become a competent and practical offthe-shelf product for articular cartilage engraftment in the clinical setting, and the production of dLhCG is no longer limited by a dependence on an autologous source of cells or materials because it is open to much more abundant allogeneic and xenogeneic sources. In this study, via an optimized decellularizing approach, dLhCG samples were produced with autologous and allogeneic porcine chondrocytes, respectively, and implanted into the knee articular chondral defects of domestic pig models for 6 months. Living autologous chondrocyte-based engraftments of LhCG and ACI were also conducted in parallel as controls to highlight the impact of decellularization. Besides, the selection of ACI as a control was also based on the fact that in comparison with another clinically practiced treatment - microfracture, ACI may result in better phenotype in the regenerated neotissue[20–22] despite the higher complication in operation and modest superiority in terms of cartilage repair in general[23–25]. The evaluation of cartilage repair was performed via histology, biochemical assays, mechanical tests and medical imaging; in addition, gene profiling via DNA microarrays was also performed to reveal the results of re-cellularization and cell repopulation at the operation sites.

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Figure 1.

Preparation and implantation of living hyaline cartilage graft (LhCG) and decellularized

LhCG (dLhCG) in comparison with autologous chondrocyte implantation (ACI) for articular cartilage repair: a schematic illustration with a timeline. Porcine chondrocytes are harvested from domestic pig models on -15 Day (15 days before designated Day 0). Primary and Passage 1 (P1) chondrocytes are derived by -14 Day and Day 0, respectively. P1 porcine chondrocytes and gelatin microsphere porogens are co-suspended and then co-encapsulated in alginate hydrogel under 4
on Day 0. Cavities are automatically created in the cell-laden hydrogel by the dissolution of microsphere porogens under elevated temperature at 37
by 1 Day. The chondrocytes outgrow the gel phase into the cavities and fill them up with cartilage neo-tissue to form an interpenetrating network (IPN) of neo-tissue and alginate hydrogel by 35 Day. The alginate hydrogel component is instantly removed from the IPN by rinsing with citrate saline to form LhCG on Day 35.[19] Some of maturated LhCG are decellularized to form dLhCG on Day42. dLhCG and the remaining LhCG are respectively implanted into knee articular chondral defects of domestic pig models on Day60 (2-Mon). ACI is conducted according to standard protocols provided by Carticel® that suspension of P2 autologous chondrocytes are injected into the articular chondral defect and sealed with a membrane made of decellularized porcine skin by sutures.[63] The samples of repaired cartilage are harvested for evaluation after 6 months since implantation (by the end of 8-Mon since gel encapsulation of chondrocytes).

2. Results and Discussion 2.1. Decellularization of tissue-engineered cartilage graft: from LhCG to dLhCG The dLhCG samples were prepared via decellularizing LhCG. Decellularization technology aims and works to substantially remove the cellular components whereas maximally preserve 7

the ECM of a biological tissue or organ of interest. Specifically, for the decellularization of LhCG, which is a tissue-engineered hyaline cartilage graft, the clearance of the chondrocyte components was demonstrated by elimination of DNA content, and the maintenance of the cartilaginous ECM was measured by retention of Col II and proteoglycan. The retention of cartilaginous proteoglycan, namely aggrecan, was demonstrated by the measurement of chondroitin sulfate and keratin sulfate glycosaminoglycan (GAG) residues that are the subbranch components in the “brush-like” supramolecular structure of aggrecan. In this study, we attempted to compare four different decellularization approaches (Methods G1, G2, G3 and G4, as seen in Supplemental Figure 1) to identify an optimal method for the generation of a high quality dLhCG from LhCG; specifically, we aimed to generate a derived dLhCG product that simultaneously maintained the lowest DNA content and the highest contents of Col II and GAG. For this purpose, the dLhCG products that were derived via the four decellularization methods were examined via histology and biochemical assays. (Figure 2) According to haematoxylin and eosin (H&E) and 4',6-diamidino-2-phenylindole (DAPI) staining, the absence of nucleic acid was observed in both dLhCG G2 and G4 groups (Figure 2a); meanwhile, the lowest DNA content was detected from dLhCG G2 (Figure 2b). This outcome highlighted the efficacy of the repeated freeze-thawing processes between ultralow (-80°C) and room temperatures, which were only implemented in the G2 method. In terms of ECM preservation, as revealed by GAG and collagen assays, associated with Safranin O (Saf O) staining for GAG and immunohistochemistry (IHC) staining for Col II, the greatest preservation of both GAG and Col II were also observed from the dLhCG G2 method (Figure 2a and 2b). Collectively, the G2 method appears to be an optimal approach for the decellularization of LhCG, and dLhCG G2 is the best among all of the decellularized LhCG products.

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Figure 2.

Decellularization of tissue engineering hyaline cartilage graft: from LhCG to dLhCG. A).

Histology of LhCG and various groups of dLhCG samples achieved from different decellularizing methods, namely dLhCG G1, G2, G3 and G4 (sample diameter ~8mm, thickness 1~2mm), by Haematoxylin and eosin (H&E) staining, Safranin-O (Saf O) staining, and immuno-histochemical (IHC) staining with primary antibody of type II collagen (Col II), respectively. DAPI staining has been overlaid with the IHC staining of Col II. Positive Haematoxylin stain of nuclei appears in purple; positive Eosin stain of ECM appears in pink; positive Saf O stain of GAG appears in bright red; positive IHC stain of Col II emits red florescence; and positive 4',6-diamidino-2-phenylindole (DAPI) stain of cell nuclei emits blue florescence. Scale bar: 200µm. B). Quantification of DNA residue, glycosaminoglycan (GAG) and total collagen contents, respectively, in LhCG and various groups of dLhCG samples: dLhCG G1, G2, G3 and G4, in comparison with native articular cartilage (NAC) and decellularized native articular cartilage using the Method G2 (dNAC G2). The measurements of the bar graphs are overlaid in Supplemental Table 3, 4 and 5. Statistical analysis has been conducted. The exact p values of the one-way ANOVA tests are presented in Supplemental Table 6, 7 and 8. For each group, n=3. Error bar represents standard deviation. Confidence interval is 95%.

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Notably, the DNA content remaining in dLhCG G2 was averagely about 1.76 µg per mg dry weight of the total ECM in contrast to the 85.73 µg/mg, 204.3 µg/mg and 94.57 µg/mg DNA that remained in the samples of the same sourced LhCG (before decellularization), native articular cartilage explants (NAC) and decellularized NAC via the G2 method (dNAC G2), respectively (Figure 2b). This level of DNA residue in dLhCG G2 demonstrates a substantial clearance of the immunogenic cellular components. Also compared with LhCG, NAC and dNAC G2, the retention of Col II in dLhCG G2 appears to be nearly 90%; whilst, the retention of GAG in dLhCG G2 appears nearly 50% of that in LhCG or NAC, but still slightly greater than that in dNAC G2 (Figure 2b). The results of the ECM preservation indicated that, given the same decellularizing treatment, the retention rates of Col II are generally very high due to the great molecular size, intermolecular tangling and relatively lower solubility of Col II. Contrastively, the generally much lower retention rates of GAGs in all samples are caused by the significantly smaller molecular sizes, linear molecular structure and higher solubility levels. In summary, as a decellularized cartilage product, the quality of dLhCG G2 not only excelled among all of the parallel groups but also significantly surpassed the decellularized native cartilage (dNAC G2). Therefore, in all of the following procedures, only dLhCG G2 was adopted as the dLhCG sample for this study and, in all of the following contexts, the denotation of “dLhCG” exclusively refers to the dLhCG G2 product by default. 2.2. In vivo biocompatibility of dLhCG Despite the successful decellularization, the non-autologous ECM and the trace amounts of non-autologous cellular residues that remain in dLhCG may still pose potential risks of provoking transplant rejections in the recipients of allogeneic or xenogeneic engraftments. Accordingly, the test of in vivo biocompatibility of dLhCG was designed and performed by embedding the porcine-sourced dLhCG into the greater omentums of rats and checking host

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responses at the end of the 1st and 2nd weeks, respectively. The rationale of this design is based on the idea that if dLhCG, as a xenograft, does not provoke transplant rejections in the highly vascularized greater omentum, then it should be able to be safely used as an allograft in articular chondral defects, which is believed to be an “immune-privileged” location due to the avascular and nerveless nature of the cartilaginous tissues.[26] Table 1. Scoring on host reaction to porcine sourced dLhCG in rat greater omentum using five-point

1 week

2 weeks

Lymphocytes

3

2

Multinucleated giant cells

1

0

Neutrophils

2

2

Macrophages

3

2

Mast cells

2

1

Eosinophils

2

1

ordinal severity scale* * 5-point ordinal severity-scale: no inflammation–0, minimal–1; mild–2; moderate–3; and severe–4.

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Figure 3.

Test of in vivo biocompatibility of porcine sourced dLhCG in rat greater omentum.

Haematoxylin and eosin (H&E), Safranin-O (Saf O) Masson’s trichrome (MT), Alizarin red staining (AR) and immunohistochemical (IHC) staining with primary antibody of CD3 and CD68 for dLhCG after being implanted in rat greater omentum for one and two weeks, respectively. Scale bar: 500µm. The rectangles in the left panel encloses the region of the entire graft. The arrows on the image of “Saf O, 2-Week” indicate the capillaries. Positive Haematoxylin stain of nuclei appears in purple; positive Eosin stain of ECM appears in pink; positive Saf O stain of GAG appears in bright red; positive MT stain of collagens appears bluish; positive AR stain of calcification appears in dark red; and positive IHC staining by polymerization of 3',3'-diaminobenzidine (DAB) catalyzed by horse radish peroxidase (HRP) conjugated on secondary antibody appears in golden brown.

Host reactions to a biocompatible graft proceed similarly to a wound healing process, of which inflammation, angiogenesis and tissue remodelling are the necessary sequential procedures, in contrast to rejection reactions.[27,28] By the end of the 1st week after implantation, a moderate amount of macrophages, neutrophils and lymphocytes that is accompanied by a minimum amount of multinucleated giant cells was observed within and around the embedded dLhCG; these cell types decreased in number by the end of the 2nd week, which indicated that a normal inflammatory reaction had occurred rather than a phagocytic transplant rejection[29,30] (Table 1). The decreases in macrophages and Tlymphocytes were also evidenced by the weakened immunostaining of CD68 and CD3 cells, respectively (Figure 3). Moreover, in both CD3 and CD68 staining, at Week 1, the graft was prominent with a weaker staining than the surrounding tissue. It indicated that neither T-cells nor macrophages was attracted by the graft. The strong staining at the surrounding tissue is due to the natively in situ T-cells and macrophages in the greater omentum. Furthermore, at Week 2, the graft gradually became unrecognizable from the surrounding tissue with similar intensity of staining in both CD3 and CD68 to the surrounding tissue. However, this was not due to increase of T-cells and macrophages in the graft region as confirmed by the scoring in Table 1. Instead, it was due to the weakened staining in the surrounding tissue. It suggested that the surrounding omentum tissue was rearranging and reorganizing itself to sustain the 12

growth of new tissues, which is an indicator of the tissue remodelling process. In combination with other staining, most obviously in Saf O staining, small capillaries were observed around the grafting area, which indicated the occurrence of angiogenesis to supply nutrients and oxygen and remove wastes to support neo-tissue growth. In addition, the higher magnification image exhibited a more thorough infiltration of cells into the graft from Week 1 to Week 2, which indicated the avoidance of fibrous encapsulation of the graft by the surrounding tissue. At the same time, morphological changes occurred from the avascular cartilage-like tissue (as dLhCG) towards the vascularized omentum-like tissue as revealed by the weakened staining of the cartilaginous total collagen (Masson’s Trichrome, MT) and GAG (Saf O) that was accompanied by the strengthened staining of calcification (Alizarin red), which indicated the beginning of tissue remodelling[31] (Figure 3). Collectively, the host reactions to the implanted dLhCG appeared to be indicative of a normal healing process; therefore, the in vivo biocompatibility of dLhCG, as a xenograft in an immune-sensitive part of the recipient body, was verified so that it could be safely used as a cartilage allograft in “immune-privileged” articular chondral defects in the following study. 2.3.Articular cartilage regeneration via implantation of dLhCG For articular cartilage repair, there is a significant challenge in how to endow the regenerated neo-tissue with a pure hyaline cartilaginous phenotype. Another challenge exists in how to manage optimal host-graft integration. In this study, the dLhCG series of products were designed and developed to address these key challenges. The donor sites of the cartilage explants for cell sourcing were adopted on the non-weight bearing femoral trochlear grooves in the stifle joint of the pigs. As the operation site for implantation and repair, articular chondral defects beyond critical size (8 mm in diameter) were created on the femoral condyles that represented the most weight bearing positions. The experimental defects were made to penetrate the full thickness of the cartilage layer, but these defects were carefully

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controlled by keeping the subchondral bones untouched. The grafts were excised and examined at 6 months to observe the potential fibrosis and hypertrophy in the regenerated cartilaginous neo-tissue, if they occurred, would become detectable after such a period,[32– 34] which are demonstrated as observable upregulations of Col I and type X collagen (Col X), respectively, over the existed abundant Col II deposited earlier. The parallel samples of the implants included autologous chondrocytes (ACI), autologous chondrocyte-laden and derived LhCG (Auto LhCG), autologous chondrocyte-derived but decellularized dLhCG (Auto dLhCG) and allogeneic chondrocyte-derived but decellularized dLhCG (Allo dLhCG). Furthermore, untreated defects were also created and employed as controls.

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Figure 4.

Histology for in situ biocompatibility of Allo dLhCG, Auto dLhCG and Auto LhCG in

comparison with ACI at time point of 6 months after implantation in porcine knee chondral defects. The negative control, namely the untreated defects that are left void of grafts, is marked as “Untreated defect”. A) Immuno-histochemical (IHC) staining with primary antibody of CD3 and B) CD68. Two arrows are used to indicate the location and width of the experimental defects originally made. Scale bar: 2 mm. Positive IHC staining by polymerization of 3',3'-diaminobenzidine (DAB) catalyzed by horse radish peroxidase (HRP) conjugated on secondary antibody appears in golden brown.

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Figure 5. Histology of cartilage repair by engraftment of Allo dLhCG, Auto dLhCG and Auto LhCG in comparison with ACI at time point of 6 months after implantation in porcine knee chondral defects. The negative control, namely the untreated defects that are left void of grafts, is marked as “Untreated defect”. A) Hematoxylin and eosin (H&E) staining, B) Masson’s trichrome (MT) staining, C) Safranin-O (Saf O) staining, and immuno-histochemistry (IHC) staining with D) primary antibody of Type II collagen (Col II), E) Type I collagen (Col I), and F) Type X collagen (Col X). Two arrows are used to indicate the location and width of the experimental defects originally made. Scale bar: 2 mm. Positive Haematoxylin stain of nuclei appears in purple; positive Eosin stain of ECM appears in pink; positive Saf O stain of GAG appears in bright red; positive MT staining of collagens appears blue; positive IHC staining by polymerization of 3',3'-diaminobenzidine (DAB) catalyzed by horse radish peroxidase (HRP) conjugated on secondary antibody appears in golden brown. G) The histological scores based on both Histological Histochemical Grading System (HHGS) and Wakitani scoring system. A low score (min. 0) defines a native-like cartilage tissue with full thickness defect filling and good integration with adjacent host cartilage. * indicates statistical significance (p < 0.05); ** indicates p ≤ 0.01, *** indicates p ≤ 0.001. For each group, n=3. Error bar represents standard deviation. Confidence interval is 95%. The measurements of the bar graphs are overlaid in Supplemental Table 9 and 10. The exact p values of the one-way ANOVA tests are presented in Supplemental Table 11 and 12.

After 6 months in vivo, biocompatibility was first specifically assessed in the sole sample that had the allogeneic sourced implant, Allo dLhCG, compared with all of the other autologous cell-laden or derived samples. The immunostaining of CD3 and CD68 indicated that no significant difference could be identified among all of these samples but equally negative indications. This result suggested that, as a decellularized, allogeneic sourced

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implant, Allo dLhCG did not provoke detectable levels of immune reactions from the host (Figure 4 and Supplemental Figure 2). On the basis of this survey of histological biocompatibility, the outcome of articular cartilage regeneration was revealed and examined by further histological staining. All cartilaginous engraftments by dLhCGs, LhCG and ACI were able to restore the defective cartilage with full thicknesses of regenerated cartilaginous neo-tissue in contrast to the untreated defects in which the experimental chondral lesions remained unrepaired (Figure 5 and Supplemental Figure 3). Biochemical assays were conducted on the parallel samples to measure the contents of DNA, GAG and total collagen in regenerated cartilaginous neotissues, respectively, representing the cell number (via DNA: 7.7 pg per chondrocyte)[35] and ECM contents (via GAG and total collagen). These quantitative outcomes were normalized by the total wet weight in order to demonstrate the value of density per construct of the neotissue and was also normalized by the total dry weight to demonstrate the relative net magnitudes of the solid contents excluding water. (Figure 6; comparative data normalized against cell number/DNA are presented in Supplemental Figure 4) Compared with the intact adjacent cartilage (NAC) and as revealed via biochemical assays, the cell densities in the untreated defects did not appear lower, albeit the ECM contents were much smaller. These quantitative data appeared to be in accordance with the histological results. Collectively, these results verified that the autogenous compensation occurred in the untreated defects via cell migration and ECM deposition; however, this self-reparation was not able to repair the defect beyond critical size. In contrast, in the samples treated by implantations of Auto LhCG and ACI, both of which delivered autologous chondrocytes via ex vivo processing, the cell densities appeared to be lower than that in NAC, especially in the ACI product; the ECM contents appeared to be similar to that in NAC in the ACI product but were slightly lower in the Auto LhCG, particularly in GAG content (Figure 6). These results were also in line with

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the histological staining for total collagen and GAG (Figure 5b-c). The histological staining for both Auto and Allo dLhCG implanted samples indicated the abundant presence of total collagen and GAG, which appeared to be comparably positive with that of NAC (Figure 5bc). The quantitative results from the biochemical assays also indicated similar contents of ECM between NAC and dLhCG samples except for a slightly lower cell density in the Auto dLhCG sample. This result confirmed the successful migration of chondrocytes from the adjacent host cartilage into the originally acellular dLhCG implants and the fine development of cartilaginous neo-tissue upon these decellularized implants, especially in Allo dLhCG.

Figure 6. Biochemical analyses of cartilage repair by engraftment of Allo dLhCG, Auto dLhCG and Auto LhCG in comparison with ACI at time point of 6 months after implantation in porcine knee chondral defects. The negative control, namely the untreated defects that are left void of grafts, is marked as “Untreated defect”; the positive control, namely the intact surrounding native articular cartilage of the host, is marked as “NAC”. The analyses include DNA assay, glycosaminoglycan (GAG) assay, and total soluble collagen assay. The quantity of each composition are normalized respectively by wet weight and dry weight of the total extracellular matrices (ECM). * indicates statistical significance (p ≤0.05); ** indicates p ≤ 0.01, *** indicates p ≤ 0.001, **** indicates p ≤ 0.0001. For each group, n=5. Error bar represents standard deviation. Confidence interval is 95%. The measurements of the bar graphs are overlaid in Supplemental Table 13 to 18. The exact p values of the one-way ANOVA tests are presented in Supplemental Table 19 to 24.

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Histological analyses were also performed on the morphology and integration of the regenerated cartilaginous neo-tissues within the surrounding host NAC. It was observed that the ACI sample exhibited an irregular surface with pannus-like formations outgrowing from the top edge of the regenerated cartilage (Figure 5). This morbidity has also been mentioned in clinical case reports.[36] Here, the overgrowth of cartilaginous neo-tissue is induced by the covering treatment with the porcine skin derived membrane, which is performed to seal the opening of the defect in order to prevent the leakage of the liquid-formed ACI cell suspension from the operation site. After 6 months in vivo, the covering membrane was mostly metabolized except for minor residues that remained at the top, as indicated by faint Saf O staining for the lower quantity of GAG (Figure 5c). This is because the porcine skin-derived membrane is composed of rich Col I and type III collagen (Col III) that are not of hyaline cartilaginous collagen fibrils, but that are fine substrates for chondrocytes to attach to and commit focal adhesion. Being a specialized member of fibroblast lineages, the adhered chondrocytes will commit dedifferentiation and result in the formation of fibrocartilage, instead of pure hyaline cartilage. Due to the same reason, overgrowth of neo-fibrocartilage on the surface was also enabled and mediated by contact with the cell-affinitive membrane.[37] This overgrowth morbidity compromises the smoothness and regularity of the cartilage surface and therefore poses a significant drawback of ACI. In contrast, the implantations of solid-phased dLhCGs or LhCG resulted in the seamless integration between the regenerated cartilaginous neo-tissues and the adjacent host NAC, which shared a common, continuous and smooth chondral surface (Figure 5). It benefited from the porous, sponge-like formation of the dLhCG and LhCG implants. Besides the convenience of the operation as being in solid form versus the liquid implant of ACI, this type of physical formation enables better physical fit of the implants within the chondral defects via a vaster contact area, and also facilitates

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better access for cell migration and ECM connection with the ambient host tissues, which is particularly more effectual in the more porous acellular dLhCGs rather than in the cell-laden LhCG counterpart. Moreover, the composition of the pure, hyaline-like cartilaginous ECM in dLhCG and LhCG makes them specifically favourable for the invasion, expansion and development of chondrocytes due to the preserved chondro-conductive biological cues and the specific microstructures in the ECM framework of the implants.[15,38] Whilst, on the other hand, the same ECM framework also functions to physically confine the development of the regenerated cartilaginous neo-tissue from outgrowing the general boundaries of the chondral surface like that occurred in the ACI treatment. The avoidance of overgrowth was also attributed to the nature of the hyaline cartilaginous ECM in dLhCG and LhCG implants, which does not favour cell focal adhesion, therefore prevents the over proliferation like the dedifferentiated chondrocytes’ behaviour. Based on the general outcomes of histology, peer scoring and grading evaluations following two sets of scoring systems, the Wakitani Scoring System and the Histology Histopathology Grading System, were carried out in the form of a blind test (Supplemental Table 1-2, a zero score indicating the same as the native counterpart - NAC). The results from the two evaluations appeared to be consistent with each other, and both evaluation results further confirmed that both Allo and Auto dLhCG samples possess high histological similarity to NAC, which appeared better than the autologous cell-laden samples of Auto LhCG and ACI, particularly much superior to the ACI sample (Figure 5g). As designed and practised, the subchondral bones were deliberately protected from experimental trauma in all of the samples when the experimental chondral defects were created. After 6 months in vivo, and according to the histology (Figure 5), as well as the micro-CT (Supplemental Figure 5), the integrity of the subchondral bones appeared to be finely maintained in both Allo and Auto dLhCG samples. However, in the (Auto) LhCG samples, an observable plateau of bony

22

overgrowth distorted the line of tidemark upward into the cartilage layer; whilst in the ACI sample, an observable bony collapse led to a downward subsidence of the cartilaginous neotissue into the subchondral bones, which also broke the integrity of the tidemark; and, in the sample with the untreated defects, the subchondral bones appeared to be fragmented. These results again indicated that the articular cartilage plays a critical role in shielding the integrity of the subchondral bones, while the subchondral bones also provide critical support to the articular cartilage. Untreated penetrating lesions in the articular cartilage, once expand into the subchondral bones, will initiate a destructive cycle that destroys both of these tissues and worsens the osteochondral lesions. In this case, the key therapy relies on the repair of the non-self-regenerative articular cartilage via cartilaginous engraftments, herein - the implantations of Allo and Auto dLhCG. 2.4.Phenotypic analysis of regenerated cartilage neo-tissue by histology and DNA microarray Further histological analyses were performed particularly focusing on the cell phenotype in the regenerated cartilaginous neo-tissues. Col II dominates the collagen content in healthy adult articular cartilage, which marks the hyaline cartilaginous phenotype.[39] Significant upregulations of Col I and Col X in articular cartilage respectively marks the fibrotic dedifferentiation and hypertrophic degeneration of chondrocytes. From the histology results, the regenerated cartilaginous neo-tissue via implantation of dLhCGs and LhCG exhibited a high purity of Col II occupancy with free or minimum of Col I or Col X (Figure 5d-f). Conversely, ACI-derived neo-tissue exhibited observable upregulations of Col I and Col X particularly in the central area, suggesting phenotypic changes of the implanted chondrocytes in the ACI products leading to both fibrosis and hypertrophy (Figure 5d-f). These results indicate that the chondrocytes in suspending culture as conducted in the ACI approach are prone to precipitation and adhesion upon the surfaces of the chondral defects and also at the

23

interface with the covering membrane, both of which can lead to the loss of hyaline cartilaginous phenotype.[40–43] In contrast, the hyaline-like cartilaginous phenotype of the chondrocytes in LhCG was well preserved because the cells had always been cultivated via a 3D culture without focal adhesive substrates.[44] The final cell population and phenotype in the initially acellular dLhCG implants were determined by the general outcomes from competitions of cell migration and proliferation that had originated from different ambient host tissues, including the adjacent articular cartilage, the subchondral bones and the synovial fluid. According to histological staining, after 6 months in vivo, hyaline-like cartilaginous ECM apparently dominated the matrix content of the regenerated neo-tissue in both Auto and Allo dLhCG derived products, which suggests that the chondrocytes from the adjacent articular cartilage surpassed all of the other sourced cells, dominated the occupancy in the grafting area and developed abundant hyaline-like cartilaginous neo-tissue in situ.

Figure 7.

DNA microarray profiles indicating gene expression of the cells harvested from the

grafting areas by Allo dLhCG, Auto dLhCG, Auto LhCG and ACI, as well as the ambient host tissues of native articular cartilage (NAC), subchondral bones and synovium at time point of 6 months after implantation in porcine knee chondral defects. The sample from untreated defect was also employed as a

24

control. Expression level are sorted to observe patterns on a list of 13 marker genes, including cartilaginous and chondrogenic markers of Type II collagen (Col2a1), aggrecan (ACAN), cartilage oligomeric matrix protein (COMP), Type IX and XI collagen (Col 9a1 and Col 11a1), transcription factor Sox9 and transforming growth factor-β1 (TGFβ1); and some other osteochondral related markers of Type I collagen (Col1a1), Type X collagen (Col10a1), testican-1 (SPOCK1), Type III collagen (Col3a1), integrin binding sialoprotein (IBSP) and alkaline phosphate (ALPL). Bars represent the fold change in gene expression i.e. up-regulation (upwards) or down-regulation (downwards).

These observational conclusions were examined and confirmed by transcriptome profiling of the resident cells in the grafting and ambient areas via DNA microarrays. Comparative analyses on the profiles of the most relevant gene expressions were performed (Figure 7). The parallel samples were harvested from the regenerated neo-tissues in the grafting areas of the dLhCGs, LhCG and ACI as well as the ambient host tissues including the NAC, subchondral bones and synovium. The sample from the untreated defect was also employed as a control. The relevant genes included cartilaginous and chondrogenic markers of Col II (Col2a1), aggrecan (ACAN), cartilage oligomeric matrix protein (COMP), Type IX and XI collagen (Col 9a1 and Col 11a1, respectively), transcription factor Sox9 and transforming growth factor-β1 (TGFβ1); additionally, several other osteochondral-related markers of Col I (Col1a1), Col X (Col10a1), testican-1 (SPOCK1), Col III (Col3a1), integrin binding sialoprotein (IBSP) and alkaline phosphate (ALPL) were also identified. As a benchmark, in the profile of NAC, the expressions of key chondrogenic markers were mostly and exclusively upregulated, thus clearly indicating a mature and healthy phenotype of the hyaline cartilage. Conversely, the profiles of samples from the untreated defects, subchondral bones and synovium clearly demonstrated a non-cartilaginous phenotype. From the selfregenerated tissues in the untreated defects, almost all of the related marker gene expression levels were downregulated, except for testican-1 and Col III, which are generally indicative of non-hyaline cartilaginous proteoglycan and collagen, respectively. The profile of the subchondral bones indicated mixed expressions of bony and calcified (degenerated) 25

cartilaginous genes, such as alkaline phosphate, IBSP and Col X. In the profile of the synovium, the expressions of almost all of the cartilaginous and chondrogenic markers were also downregulated. In contrast with these control samples, the general tendency of gene expression levels in Allo- and Auto dLhCG-derived samples appeared to be in line with that of NAC, although the extents of upregulation were observably weaker. The major difference between the NAC and dLhCG-derived samples lies in opposed regulating tendencies of Col IX, COMP and Sox9. The significant upregulation of Col IX in NAC indicated the stabilization of the fibrillary collagen network in the cartilaginous ECM[45] and prevention of fibril aggregation[46], whereas a flat expression of Col IX in dLhCG-derived samples indicated that the remodelling of the collagen network did not appear to be complete yet in these regenerated cartilaginous neo-tissues. The relatively greater upregulation of COMP in dLhCG-derived samples indicated more active cartilage turnover,[47,48] and the upregulation of Sox9, which is a chondrogenic transcription factor, indicated that the process of chondrogenesis was still active in these regenerated neo-tissues. Collectively, the cells residing in dLhCG-derived cartilaginous neo-tissues demonstrated a clear feature of having freshly committed chondrocytes that possess a hyaline cartilaginous phenotype and continue to experience a remodelling process towards maturation. The DNA microarray outcomes from Auto and Allo dLhCG appeared to be very similar to each other. These results reinforced and explained the conclusions drawn from the histological analyses that, after 6 months in vivo, chondrocytes migrated from the adjacent host articular cartilage, dominated the occupancy in the originally acellular dLhCG implants and maintained their hyaline cartilaginous phenotype; therefore, these chondrocytes exhibited the capability to develop hyaline-like cartilaginous neo-tissue in situ. This was attributed to the superior physical features and fine chondro-conductivity of the dLhCG implants. Comparatively, the DNA microarray profiles of the cells that were extracted from the Auto LhCG-derived cartilaginous

26

neo-tissues provided mixed indications. On the one hand, after 6 months in vivo, this autologous chondrocyte-derived, living cell-laden tissue-engineered implant had regenerated hyaline cartilaginous neo-tissues that possessed the highest similarity to NAC in terms of chondrocytic and chondrogenic gene expressions; but on the other hand, the upregulations of hypertrophic cartilaginous and bony markers, which were respectively represented by Col X and alkaline phosphatase, suggested the beginning of a phenotypic transition from “overmature” cartilage to the formation of bones via a pathway like endochondral ossification. These hyaline cartilaginous tissue formations again reflect the unique advantages of LhCG for articular cartilaginous engraftment. However, the upregulation of hypertrophic and bony markers may demonstrate a flaw of LhCG. It may be induced by prolonged ex vivo processes to which the employed and delivered autologous chondrocytes were exposed, specifically, the initial enzymatic harvest and the subsequent 2D subculture, followed by prolonged 3D culture in vitro. Additionally, after being implanted into the chondral defect, the vast population of these “over-processed” and pre-occupied chondrocytes could hardly be repopulated by fresh chondrocytes migrating from the adjacent host cartilage. This flaw of the cell-laden LhCG was completely avoided in the dLhCGs via decellularization to remove the “old” cells followed by in situ recellularization via the migration of fresh cells from the host. Due to a similar reason that affected the autologous cell-laden LhCG sample, the tendencies of chondrocytic hypertrophy and cartilaginous calcification in the ACI-derived neo-tissues appeared to be even more significant as revealed by the intensive upregulations of Col X and IBSP in the DNA microarray profiles. Besides the influence of prolonged ex vivo processing as well, another notable cause was that, according to the standard protocols, P2 cells were employed for ACI versus P1 cells that were used for LhCG. An additional passage of subculture in monolayer before implantation caused more severe problem of dedifferentiation of chondrocytes; additionally, after implantation, further dedifferentiation was

27

induced in the operation site due to cell adhesion upon the inner surfaces of the chondral defects, as well as the cover membrane. The gradual loss of the hyaline cartilaginous phenotype was revealed via the downregulation of Col II that was accompanied with an observable upregulation of Col I and a significant upregulation of Col X. Collectively, the outcomes from the DNA microarrays confirmed and explained the phenotypic findings from histological analyses in the regenerated cartilaginous neo-tissues. Phenotypic variation was also reflected in the superfine microstructure of the regenerated cartilaginous neo-tissues, as revealed in picrosirius staining by polarization and bright field microscopy (Figure 8). Also as a benchmark, in NAC, a typical zonal structure was illustrated: in the superficial zone, collagen fibrils and chondrocytes were packed and aligned in parallel with the general surface of the articular cartilage; in the middle zone, chondrocytes were dispersed within the oblique collagen fibrillary network; and, in the deep zone, chondrocytes were aligned in a columnar orientation, along with the collagen fibrils perpendicular to the general surface (also perpendicular to the subchondral tidemark) of the articular cartilage. Among all of the experimental samples, this type of tri-zonal microstructure with the neat alignment of cells and ECM was clearly formed in the Allo dLhCG-derived cartilaginous neo-tissue, which indicates a nearly accomplished reparatory remodelling of the articular cartilage. Comparatively, the zonal structure hadn’t been fully established in the Auto dLhCG- or LhCG-derived cartilaginous neo-tissue, thus indicating that the remodelling had not yet implemented, and that the maturation was still pending. In contrast, the microstructures of the ACI-derived cartilaginous neo-tissue and the autogenously regenerated tissue in the untreated chondral defects appeared to be rather random and lacked any alignment or orientation. These results further confirmed the superior chondro-conductivity of dLhCG implants, especially in regard to Allo dLhCG.

28

Figure 8.

Microstructural histology of articular cartilage by Picrosirius staining under polarized

light and bright. The staining reveals the grafting areas by Allo dLhCG, Auto dLhCG and Auto LhCG in comparison with ACI at time point of 6 months after implantation in porcine knee chondral defects. The negative control, namely the untreated defects that are left void of grafts, is marked as “Untreated defect”; and the positive control – intact, surrounding native articular cartilage of the host – is marked as “NAC”. Scale bar: 200 µm. Picrosirius stains relatively raw matrix fibers in brown or dark yellow and relatively delicate fibrils in green or light yellow under polarized light.

Table 2. Mechanical Test of regenerated cartilaginous neo-tissue

Sample *

Compressive modulus (MPa) **

NAC

0.46±0.02

Untreated defect

0.29±0.33

ACI

0.29±0.27

Allo dLhCG

0.39±0.16

Auto dLhCG

0.30±0.27

Auto LhCG

0.32±0.17

29

*

“NAC”: the intact, adjacent native articular cartilage of the host. “Untreated defect”: the created

defect that were left empty without any engraftment or treatment. “ACI”: the regenerated tissue after being treated by clinical available autologous chondrocyte implantation. **

Testing time point is 6 months after implantation. The Young’s modulus is presented as mean ±

standard deviation.

2.5. Mechanical property of repaired articular cartilage The major functions of the articular cartilage are to bear compressive stresses at the joint and to articulate movements of the connected bones. Hence, the ultimate quality evaluation of cartilage repair lies in the mechanical properties of the regenerated cartilaginous neo-tissues. The overall mechanical properties of a hard tissue, specifically articular cartilage, is determined by the abundance, composition, phenotype and microstructure of the cartilaginous ECM, as well as the integration with the surrounding host tissues and the integrity of the subchondral bones. The abundance and composition of the ECM were measured and tested via biochemical assays and histology, the phenotype was characterized via histology and DNA microarrays, and the microstructure was demonstrated via picrosirius staining. Collectively, all of the experimental implants were pure in composition - free of noncartilaginous constituents. The tissue-engineered implants, LhCG and dLhCGs, possessed relatively pure hyaline-like cartilaginous phenotypes; in particular, the implantation of Allo dLhCG had even successfully regenerated the typical zonal cartilaginous microstructures in the neo-tissues, thus indicating the best chondro-conductivity. Herein, to focus on the mechanical properties of the regenerated cartilaginous neo-tissue, a confined mechanical test was conducted on cartilage explants that were harvested from the operation sites without the attachments of the subchondral bones, from which the compressive modulus of each sample was measured (Table 2 and Supplemental Figure 6). The results indicated that the best mechanical property was also detected from the Allo dLhCG-derived cartilaginous neo-tissue, the compressive modulus of which was measured to be 0.39±0.16 MPa, which was 30

approximately 85% of NAC (0.46±0.02 MPa) and approximately 120% of the average of the Auto LhCG group (the second highest group on the average of the Young’s modulus) and approximately 135% of the average of the untreated defect (the lowest group on the average of the Young’s modulus). As a measurement of key functional restoration, this result met and reflected the general outcomes from all aspects of the features and factors of articular cartilage repair: after 6 months in vivo, the implantation of Allo dLhCG had derived the finest neo-tissue of articular cartilage in situ, albeit the final maturation was yet to be completed.

3. Conclusions In this study, by combining the advantages of cartilage tissue engineering and decellularization technology, we developed a decellularized allogeneic hyaline cartilage graft, Allo dLhCG, which achieved a superior efficacy of articular cartilage repair. By the 6-month time point of implantation in porcine knee joints, the fine morphology, composition, phenotype, microstructure and mechanical properties of the regenerated hyaline-like cartilaginous neo-tissue were demonstrated via histology, biochemical assays, DNA microarrays and mechanical tests. The results indicated that cartilaginous engraftment via Allo dLhCG surpassed living autologous chondrocytes-laden and derived cellular or engineered tissue engraftments by ACI or Auto LhCG in almost all aspects of its features and functions. Allo dLhCG also exhibited better microstructural reconstruction and higher average Young’s modulus, which may suggest its better chondro-conductivity than autologous chondrocyte derived, decellularized hyaline cartilage graft, Auto dLhCG. Particularly, Allo dLhCG was proven to be well compatible with the host because it was free of immune rejection regardless of whether it was used as a xenograft in the greater omentum of the rat or as an allograft for articular cartilaginous engraftment in chondral defects of the porcine knee joint. The success of this allogeneic decellularized graft broadens the source of

31

grafting materials, thereby avoiding the risk of donor site morbidity for the purpose of collecting autologous materials. Additionally, due to the advantages of being an acellular product, Allo dLhCG may act as a competent and practical off-the-shelf product, thus allowing patients to experience a flexible time window for clinical operations and providing a logistic convenience for transportation and storage. Collectively, the successful repair of articular chondral defects in large animal models suggests the readiness of Allo dLhCG for clinical applications.

4. Experimental Section Chondrocytes harvest and preparation: All animal experimentation in this study was performed in line with the protocols approved by Institutional Animal Care and Use Committee (IACUC). Unless stated otherwise, all chemicals used in this study were purchased from Sigma Aldrich, Singapore; all cell culture reagents were purchased from Invitrogen and Life Technologies. Twelve (12) domestic pigs (sus scrofa domesticus, body weight range: 47 ~ 53 kg) were employed as the sources of chondrocytes as well as the hosts of articular cartilage repair. The donor sites of cartilage explants, as the source of chondrocytes, were adopted on the femoral trochlear groove in the stifle joints. Porcine chondrocytes were harvested from the cartilage explants. Briefly, eight (8) pieces of cylindershaped (3 mm in diameter) cartilage explants were harvested from the donor sites of each knee joint using biopsy punch. (Supplemental Figure 7) The explants were mechanically homogenized into smaller fragments and then digested overnight with collagenase II (1mg/ml, by Gibco) in chondrocyte culture primary (CCP) medium that is composed of 12% fetal bovine serum (FBS), 0.1% L- proline, 0.1% ascorbic acid, 0.2% 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES) and 0.2% non-essential amino acids (NEAA) in Dulbecco’s modified Eagle medium (DMEM). The obtained primary porcine chondrocytes 32

were sub-cultured from Passage 0 (P0) to P1 in CCP medium and the P1 chondrocytes were employed for all following experimentation. Fabrication of LhCG: The fabrication of LhCG was performed according to the previously established protocol.[18] Briefly, porcine chondrocytes (P1, 1 × 107 cells/mL) and gelatin microspheres (made via double-emulsion method, 150~180 µm diameter, 0.3g/mL) were coencapsulated in alginate hydrogel at 4°C. These cell-laden hydrogel constructs were then translocated onto non-cell-adhesive tissue culture plates and cultivated in chondrocyte culture (CC) medium that is composed of 20% (v/v) FBS, 0.01 M HEPES, NEAA, 0.1 mM, Lproline (0.4 mM), ascorbic acid (0.05 mg/mL), penicillin (100 units/mL) and streptomycin (100 mg/mL) in DMEM. The cultivation was conducted on an orbital shaker (50 rpm) in a humidified incubator at 37 °C with 5% CO2. After 35 days, these hydrogel constructs were subjected to a rinse with solution of sodium citrate (55 mM in 0.15 M NaCl solution) for 10 mins at room temperature, by which the alginate components in the constructs are removed. Thereafter, the obtained scaffold-free constructs, that is LhCG, were further cultivated in CC medium for another 7 days’ maturation and became ready for decellularization and further use. (Figure 1.) Decellularization of LhCG for fabrication of dLhCG: To fabricate dLhCG samples, four (4) sets of decellularization methods, respectively denoted as Method G1, G2, G3 and G4, were attempted on LhCG samples (Supplemental Figure 1.). Method G1 was conducted by cycling the freezing (-20°C, 3 hours) and thawing (room temperature, 4 hours) processes on LhCG for three times, followed by soaking in 1% sodium dodecyl sulfate (SDS) based hypertonic solution (TRIS buffer, pH 8.0, by 1st Base) for 72 hours.[49,50] Method G2 was conducted by cycling the freezing (-80°C, 3 hours) and thawing (room temperature, 4 hours) processes on LhCG for three times and followed by soaking in TRIS hypertonic solution for 24 hours and then in 1% Triton X-100 (1st Base) based TRIS hypotonic solution for 48 hrs.[51–53] 33

Method G3 was conducted by soaking LhCG in TRIS hypotonic solution for 48 hours and then in 1% Triton X-100 based TRIS hypotonic solution for 72 hours.[54] Method G4 was conducted by soaking LhCG in 1% SDS solution based TRIS hypotonic solution for 72 hours and then in 1% Triton X-100 based TRIS hypotonic solution for another 72 hours.[55] After the above treatments, all treated samples were further soaked, in turn, in DNase (0.5 mg/mL DNase I) and RNase (50 µg/mL RNase A) solutions for 3 hours, followed by rinsing in PBS for 24 hours. All soaking procedures were accompanied with agitation at 150 rpm under 37°C. The final products were respectively denoted as dLhCG G1, G2, G3 and G4. For comparison, decellularization following Method G2 was also conducted on porcine NAC (native articular cartilage explant with the same thickness as that of LhCG: 1.5mm) resulting in a decellularized NAC control sample denoted as dNAC G2. (Figure 2) Embedment of dLhCG in rat greater omentum: Under general anaesthesia, each piece of porcine sourced dLhCG was embedded into the greater omentum of each rat (SpragueDawley all female, 8 weeks old) via open surgery and closure by sutures. After surgery, all animals were allowed for unrestricted activity in cages. Two time points were applied for sample harvest, respectively, at the end of the 1st and 2nd week since the surgery. Surgery of dLhCG implantation and ACI in porcine articular chondral defects: Under general anaesthesia, a medial parapatellar approach was performed to expose the stifle joint of the experimental pigs and the patella was laterally dislocated. One critical-sized (8 mm diameter) full-thickness cartilage defect was created using surgical puncher with null or minimal damage to the subchondral bone on each femoral condyle. Given 12 animals, totally 48 experimental defects were created, among which 10 defects were kept untreated as negative controls; 10 defects were filled with allogeneic sourced Allo dLhCGs; 10 defects were filled with autologous sourced Auto dLhCGs; 9 defects were filled with autologous sourced Auto LhCGs; and 9 defects were treated by ACI. Autologous or allogeneic engraftments were 34

respectively performed in the same or different individuals as the cell donors among the same group of animals. Mixing of auto- and allo-grafting was avoided being conducted in the same individual host. Both LhCG and dLhCG implants were press-fit into the defects without deliberate fixation. ACI was performed according to standard protocol provided by Carticel®. Briefly, since 2 weeks before implantation, the cryopreserved P1 autologous chondrocytes were thawed and further expanded to meet the required cell density (12 million cells/ml) as ACI cell suspension (P2 chondrocytes in serum free DMEM) ready for implantation. On surgery, firstly a patch of membrane made of decellularized and devitalized porcine skin (Fortiva® Porcine Dermis) was placed to cover the opening of cartilage defect and fixed by sewing and gluing with commercial fibrin glue (TISSEEL kit, ThermoFisher Scientific Inc.) over the edges to prevent displacement and leakage, subsequently 80 µL of ACI cell suspension was injected into the cartilage defect covered underneath the membrane. After implantation, the patella was restored to the original position and all surgical wounds were closed by sutures. After surgery, all animals were allowed for unrestricted activity all the time till being sacrificed for sample harvest 6 months later when the femur distal ends including the whole osteochondral parts were collected with complete integrity. The samples for mechanical tests, biochemical assays and DNA microarray were washed in PBS and processed within 2 hours; the samples for histology were immersed in 10% neutralized formalin prior to further process. Histology and immunohistochemistry: All samples were paraffin-embedded and then sectioned with a thickness of 10 µm through the centre of the sample with a microtome. H&E, MT, Saf O and Picrosirius red staining were respectively conducted according to standard protocols.[56–59] The picrosirius staining was imaged and observed under polarization and bright-field microscopy. Immunohistochemical staining was conducted with primary antibodies of Col I, Col II, Col X, CD3 and CD68, respectively. For fluorescent imaging, the 35

slides of samples were first blocked in horse serum and then incubated with corresponding primary antibody, followed by incubation with goat anti-rabbit IgG-FITC (Santa Cruz, USA) at 37°C for 1 hour; lastly, the cell nuclei were counterstained with DAPI before imaging and observing under a fluorescence microscope. For non-fluorescent imaging, the slides of samples were stained according to the standard protocol provided by Ultra Vision Quanto Detection System HRP DAB kit (Thermo Scientific).[60] Briefly, the sections were deparaffined and rehydrated, which was soaked in PBS for at least 5 mins before proceeding on. Pepsin was added to the sections for 20 mins under 37 °C to extract the targeted antigen and then washed by PBS. Subsequently, the sections were blocked by UltraVision hydrogen peroxide block under room temperature for 10 mins and washed by PBS. Next, the sections were blocked by protein block for 5 mins at room temperature. The blocking solution was shaken away without PBS wash. The primary antibody was added to the sections which were incubated for 1 hr at room temperature. From now on after each step, the slides were washed by PBS. The primary antibody amplifier quanto and horseradish peroxidase (HRP) polymer quanto were added in sequence each for 10 mins at room temperature. When the HRP polymer quanto was added, light was avoided. Next, the mixture of the 3,3'Diaminobenzidine (DAB) substrate and DAB chromogen at a ratio of 1 ml to 30 µL was added to the sections for at most 3 mins for all types of antigen examined in IHC to avoid overstaining, after which the slides were washed with distilled water. Finally, the slides were dehydrated and mounted before observation under bright field microscope. For the scoring of the histology slides, three experts have marked on the same set of histology slides blindly and independently. The scoring from all three experts have been averaged and statistics has been conducted based on the three experts scoring. Biochemical Assays: The samples were first frozen at −20 °C and then lyophilized for 24 h. The completely freeze-dried samples were digested overnight in papain solution consisting of 36

0.3 mg/mL

papain

dissolved

in

0.2 mM

dithiothreitol

mixed

with

0.1 mM

ethylenediaminetetraacetic acid disodium salt. DNA content was measured by the fluorometric Hoechst 33258 assay. GAG content was measured by 1,9-dimethylmethylene blue (DMMB) dye binding assay.[61] Total collagen content was quantified using Lhydroxyproline assay after hydrolysis in HCl (6M) overnight.[62] DNA microarray: The extraction of total RNA was conducted following TRIzol® protocol. Briefly, 50 mg of tissue sample was homogenized in TRIzol and incubated for 5 mins at room temperature in TRIzol® reagent before adding 0.2 mL of chloroform. The capped tube was shaken vigorously by hand for 15 secs and incubated for 2-3 mins at room temperature. Subsequently, the sample was centrifuged at 12,000 x g for 15 mins at 4 °C. After centrifugation, the aqueous phase of the sample was transferred to a new tube. Next, 0.5 mL of isopropanol was added to the aqueous phase and incubated at room temperature for 10 mins before centrifuging at 12,000 x g for 10 mins at 4 °C. Later, the supernatant was removed from the tube, leaving only the RNA pellet which is washed by 1 mL of 75% ethanol. The tube was vortexed briefly and then centrifuged at 7500 x g for 5 mins at 4 °C. The wash was discarded, and the RNA pellet was air dried for 5 mins. A microarray platform of Agilent SurePrint G3 Custom GE 8x60K 1 colour (Agilent, USA) was used for profiling the global gene expression. Total RNA (100ng) was labelled with Low Input Quick Amp Labelling Kit and purified by Qiagen RNeasy Kit (Qiagen, Singapore). Cyanine 3-CTP labelled cRNA (600ng) was hybridized onto the microarray platform for 17 h under 65°C at 10 rpm in Agilent hybridization oven. After hybridization, the microarray slide was washed in gene expression wash buffer. The hybridized microarray slide was scanned on Agilent High Resolution Microarray Scanner. The data were log-transformed and normalised via (the 75th) Percentile Shift Method using Agilent Feature Extraction software. All normalised data were then baseline-transformed to the median of all samples. 37

Biomechanical Test: A confined mechanical test was conducted on cartilage explants (cylinder shape, 8 mm in diameter and 1.5 mm thick) harvested from the operation sites without attachment of subchondral bones. Young’s modulus of the harvested samples was measured using rheometer (Instron, USA). A constant, compressive crosshead moving was applied at the speed of 1mm/min on samples until the end-of-test criterion was reached at 80% of the specimen height. A stress-strain curve was plotted, from which the Young’s modulus was calculated through linearization of the elastic region of the curve. Statistical analysis: All data in this study were shown as mean ± standard deviation. One-way ANOVA statistical analysis with post hoc comparisons was used to analyse the data. p < 0 .05 was deemed to indicate statistical significance. The measurements were taken from distinct samples. Acknowledgement The authors would like to thank the immunologist from Singapore General Hospital, Dr. Jabed Iqubal, for (independent) inflammatory severity scoring. The authors would like to thank Drs. Zheng Yang, Yingnan Wu and Vinitha Denslin for (independently scoring) histological images. Funding: this work is financially supported by Start-up Grant for Professor (SGP 9380099 to Dong-An Wang) and SRG 7005212 (to Dong-An Wang), City University of Hong Kong, and Tier 2 Academic Research Fund (MOE2016T2-1-138(S) to Wang Dong-An), Singapore. Author Contributions X.L.N. was responsible for the conduct of all experiments, data collection and analyses, and manuscript preparation; P.F.H. and W.Z.Z contributed to in vitro experiment; Y.J.C. and Y.P. contributed to in vivo experiment and D.A.W. was responsible for the design of

38

experimentation, project supervision and coordination, data analyses, and manuscript preparation. Data Availability The raw/processed data required to reproduce these findings cannot be shared at this time due to technical or time limitations. (The authors guarantee that they will be available for downloading by the time of publication.)

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Elsevier Editorial System(tm) for Biomaterials Manuscript Draft Manuscript Number: jbmt50639R1 Title: Decellularized Tissue Engineered Hyaline Cartilage Graft for Articular Cartilage Repair Article Type: FLA Original Research Section/Category: Biomaterials and Regenerative Medicine (BRM) Keywords: Tissue engineering, cartilage, scaffold-free, decellularization, large animal model, pre-clinical Corresponding Author: Dr. Dong-an Wang, Corresponding Author's Institution: City University of Hong Kong First Author: Xiaolei Nie Order of Authors: Xiaolei Nie; Yon Jin Chuah; Wenzhen Zhu; Pengfei He; yvonne peck; Dong-an Wang Abstract: Articular cartilage repair has been a long-standing challenge in orthopaedic medicine due to the limited self-regenerative capability of cartilage tissue. Currently, cartilage lesions are often treated by microfracture or autologous chondrocyte implantation (ACI). However, these treatments are frequently reported to result in a mixture of the desired hyaline cartilage and mechanically inferior fibrocartilage. In this study, by combining the advantages of cartilage tissue engineering and decellularization technology, we developed a decellularized allogeneic hyaline cartilage graft, named dLhCG, which achieved superior efficacy in articular cartilage repair and surpassed living autologous chondrocyte-based cartilaginous engraftment and ACI. By the 6-month time point after implantation in porcine knee joints, the fine morphology, composition, phenotype, microstructure and mechanical properties of the regenerated hyaline-like cartilaginous neo-tissue have been demonstrated via histology, biochemical assays, DNA microarrays and mechanical tests. The articular cartilaginous engraftment with allogeneic dLhCG was indicated to be well consistent, compatible and integrated with the native cartilage of the host. The successful repair of articular chondral defects in large animal models suggests the readiness of allogeneic dLhCG for clinical trials.

Response to reviewers Click here to download Response to reviewers: Responce to Reviewer.docx

Dear Professor Mao, The Editor

Thank you very much for the kind and helpful feedbacks indeed! Enclosed please find our point-by-point responses to your and the reviewer’s comments and suggestions. We have also uploaded the revised Manuscript and the Supplementary File. Please kindly receive and check them out. We hope that this version of our submission could be acceptable for publication in your journal, Biomaterials.

Best regards and many thanks

Dongan Wang, Ph.D. City University of Hong Kong

Responses to the Editor’s Comments and Suggestions 1. Address the comments from the attached reviewer's report, but mostly focus on #3; Please refer to the Responses to the Reviewer’s Comments and Suggestions as stated below.

2. Add the new data you have included in the responses to the Supplementary Information file, mostly the following: Reviewer #1 (Question #1-Defect images); Added as Supplemental Figure 2

Reviewer #2 (Question #1-SEM images; Added as Supplemental Figure 1C

#8-large animal data; Added as Supplemental Figure 3

#10-mechanical testing (clarify the Y-axis label;

Added as Supplemental Figure 6

Minor Question #3-Collagen and GAG deposition; Added as Supplemental Figure 4

Minor Questions #3-surgery images). Added as Supplemental Figure 7

Responses to the Reviewer’s Comments and Suggestions Reviewer #1: This study introduced a novel concept for the design of cartilage regeneration scaffold. However, there seemed to be a lot of lack of exploration about cartilage regeneration mechanism. 1.There should be more supporting data or explanation regarding the improvement of cartilage regeneration after implantation of Allo dLhCG in joint defect. The purpose of this manuscript is to report a novel biological material that was essentially ECM secreted by living cells fabricated using an approach combining 3D cell culture and decellularization technique. The design of the experiment was to characterize the material and verify the tissue repair efficacy. Hence the manuscript was organized in a manner of reporting the fabrication process, the characterization of the biomaterial in vitro and the tissue repair efficacy, especially in vivo, sequentially. Particularly, since we observed the superior performance of Allo dLhCG graft in the classical histology test of the regenerated tissue, we conducted DNA microarray to test the phenotype of the cells and thus to reveal the mechanism of the superior performance in tissue repair in vivo. We have presented and discussed the data in the manuscript. More thorough and specific studies on the mechanisms, such as gene sequencing on top of the conducted microarray, are definitely worth conducting in our immediate future work. 2.It was hard to find a proper evidence for the conclusion, "Allo dLhCG also exhibited better chondro-conductivity than the autologous chondrocyte derived, decellularized hyaline cartilage graft, Auto dLhCG, in terms of the microstructural reconstruction and mechanical properties", in page 30. As indicated in Table 2. Allo dLhCG exhibited higher average Young’s modulus. And, as indicated Figure 8 and stated in Page 28, “among all of the experimental samples, tri-zonal microstructure with the neat alignment of cells and ECM was clearly formed in the Allo dLhCG-derived cartilaginous neo-tissue, which indicates a nearly accomplished reparatory remodelling of the articular cartilage. Comparatively, the zonal structure hadn’t been fully established in the Auto dLhCG- or LhCG-derived cartilaginous neo-tissue, thus indicating that the remodelling had not yet implemented, and that the maturation was still pending.”

Consulting from the reviewer’s comment, we would like to revise this conclusion sentence in Page 31 as follows, “Allo dLhCG also exhibited better microstructural reconstruction and higher average Young’s modulus, which may suggest its better chondro-conductivity than autologous chondrocyte derived, decellularized hyaline cartilage graft, Auto dLhCG.” 3.For the newly added image in Figure 3, it would be much better to add some explanations to support the conclusion.

Thanks for the comment indeed. We would like to add in more explanations as the following into Page 12-13, “Moreover, in both CD3 and CD68 staining, at Week 1, the graft was prominent with a weaker staining than the surrounding tissue. It indicated that neither T-cells nor macrophages was attracted by the graft. The strong staining at the surrounding tissue is due to the natively in situ T-cells and macrophages in the greater omentum. Furthermore, at Week 2, the graft gradually become unrecognizable from the surrounding tissue with similar intensity of staining in both CD3 and CD68 to the surrounding tissue. However, this was not due to increase of T-cells and macrophages in the graft region as confirmed by the scoring in Table 1. Instead, it was due to the weakened staining in the surrounding tissue. It suggested that the surrounding omentum tissue was rearranging and reorganizing itself to sustain the growth of new tissues, which is an indicator of the tissue remodelling process. In combination with other staining, most obviously in Safranin O, small capillaries were observed around the grafting area, which indicated the occurrence of angiogenesis. In addition, the higher magnification image exhibited a more thorough infiltration of cells into the graft from Week 1 to Week 2, which indicated the avoidance of fibrous encapsulation of the graft by the surrounding tissue.”

Manuscript Click here to download Manuscript: Manuscript (3).docx

Click here to view linked References

Decellularized Tissue Engineered Hyaline Cartilage Graft for Articular Cartilage 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

Repair

Xiaolei Nie1, Yon Jin Chuah1, Wenzhen Zhu1, Pengfei He1, Yvonne Peck1 and Dong-An Wang*1,2

1

School of Chemical and Biomedical Engineering, Nanyang Technological University,

Singapore 2

Department of Biomedical Engineering, City University of Hong Kong, Kowloon, Hong

Kong SAR

*Corresponding author:

Dong-An WANG, Ph.D. Department of Biomedical Engineering City University of Hong Kong 83 Tat Chee Avenue, Kowloon, Hong Kong SAR Email: [email protected]

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Abstract 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

Articular cartilage repair has been a long-standing challenge in orthopaedic medicine due to the limited self-regenerative capability of cartilage tissue. Currently, cartilage lesions are often treated by microfracture or autologous chondrocyte implantation (ACI). However, these treatments are frequently reported to result in a mixture of the desired hyaline cartilage and mechanically inferior fibrocartilage. In this study, by combining the advantages of cartilage tissue engineering and decellularization technology, we developed a decellularized allogeneic hyaline cartilage graft, named dLhCG, which achieved superior efficacy in articular cartilage repair and surpassed living autologous chondrocyte-based cartilaginous engraftment and ACI. By the 6-month time point after implantation in porcine knee joints, the fine morphology, composition, phenotype, microstructure and mechanical properties of the regenerated hyaline-like cartilaginous neo-tissue have been demonstrated via histology, biochemical assays, DNA microarrays and mechanical tests. The articular cartilaginous engraftment with allogeneic dLhCG was indicated to be well consistent, compatible and integrated with the native cartilage of the host. The successful repair of articular chondral defects in large animal models suggests the readiness of allogeneic dLhCG for clinical trials.

Keywords: Tissue engineering, cartilage, scaffold-free, decellularization, large animal model, pre-clinical

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1. Introduction 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

Articular cartilage, as a connective tissue of the diarthrodial joint that is devoid of blood vessels, lymphatic tissues and nerves, is highly specialized for adaptation to harsh biomechanical environments.[1] The demanded superior biomechanical property of articular cartilage is critically endowed as a result of its unique, highly pure, hyaline-type cartilaginous composition and structural organization of the extracellular matrices (ECMs), specifically, a tight network of type II collagen (Col II) fibrils and abundant negatively charged proteoglycan chains. Consequently, once cartilage is damaged by trauma, disease or degeneration, the avascular nature of cartilage tissue and the low metabolic rate of the chondrocytes severely limits the capability of intrinsic healing and repair.[2] Hence, effective therapies for the treatment of significant articular cartilage lesions ultimately rely on surgical intervention and engraftments that resemble the high purity of hyaline cartilage tissue in situ. Current articular cartilage repairing strategies mainly include bone marrow stimulation (“microfracture”), osteochondral autograft transfer system (OATS, also known as “mosaicplasty”) and autologous chondrocyte implantation (ACI).[3] Small, symptomatic articular cartilage lesions are commonly treated via microfracture, which results in the formation of mechanically inferior fibrocartilage.[4,5] OATS can temporarily restore the smooth surface of the defect; however, graft subsidence due to postoperative weight bearing and the absence of repair within the dead space between the cylindrical grafts remains as unsolved flaws that may eventually result in the failure of the repair.[6] The inefficacy of OATS indicates that, in addition to the limited donor source of autologous native cartilage grafts, another major hurdle in cartilage transplantation lies in the host-graft integration, because both the diffusion between hard tissues and in situ re-cellularization from the host are hampered. ACI is a cellular engineering-based surgical procedure that is currently practised as a popular, second-line treatment for relatively larger-sized articular cartilage lesions,

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especially in young patients.[7] However, due to the liquid nature of the implant, the loss of 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

therapeutic chondrocytes severely affects the quality of the final repair.[8,9] In addition, the uncontrollable overgrowth of neo-tissue and the secondary damage that is caused by suturing during the operation are frequently encountered as negative complications.[8–12] In recent decades, the decellularization of natural tissue has been developed and applied as a new technology to provide grafting materials for the use of regenerative medicine; in this method, immunogenic cellular components are removed and active biological factors are preserved together with the genuine 3D microstructures and architecture that made up the original tissue ECM. [13],[14] These decellularized ECMs have been reported to enhance tissue regeneration and function recovery, because they resemble native matrix environments and provide critical biological cues for cell adhesion, growth and development. Moreover, the application of the decellularization approach broadens the source of tissue graft from autologous to allogeneic and xenogeneic since the cellular components, as the major sources of the immunogens, are substantially eliminated.[15] However, the decellularization of native cartilage, as a hard tissue composed of condensed ECM, suffers from either over-residue of cellular immunogens after inadequate treatment or overdestruction of the ECM by harsh procedures, either of which defeats the purpose of the decellularization technology.[16] In this study, we developed a new strategy that combines the advantages of cartilage tissue engineering and decellularization technology, specifically meaning to decellularize a tissue-engineered cartilage graft instead of native cartilage explants. Through these methods, we designed and developed a novel, decellularized, tissue-engineered cartilage graft that is composed of pure hyaline-like cartilaginous ECM and is endowed with a macro-porous (hundreds of microns in diameter) structure. As a porous, sponge-like graft, it provides a softer and more accessible platform that can be much more easily and thoroughly

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decellularized via milder processes, thereby allowing for the original composition and 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

microstructure of the graft’s ECM to be maximally preserved. Simultaneously, the cellular components can be maximally cleared. For the same reason, this graft (being that it is softer and more porous than decellularized native cartilage) also allows host chondrocytes to easily migrate across the host-graft interface and to re-cellularize the acellular graft, thus enabling the development of neo-tissue in situ and simultaneously facilitating the host-graft connection and integration.[17] This decellularized, tissue-engineered hyaline cartilage graft is derived via decellularization process from an autologous chondrocyte-based tissue engineering product that we had previously developed, which is known as “living hyaline cartilage graft” (LhCG).[18,19] Following the naming of LhCG, here, the decellularized product is named dLhCG. As previously reported, LhCG is produced by a 3D culture of autologous chondrocytes in an alginate-based macro-porous hydrogel. For the creation of this product, an inter-penetrating network (IPN) combining chondrocytes-derived cartilaginous neo-tissue and alginate scaffolding material was first formed, after which the alginate component was instantly removed from the IPN via rinsing with citrate saline. Thus, LhCG, as a tissue engineering product that is only composed of living cartilaginous neo-tissue, consists of a form of a macroscopic, sponge-like construct (Figure 1.).[19] The setting-up process like this determines that LhCG is free of any non-cartilaginous impurities. Moreover, as the 3D culture of chondrocytes has always been mediated by non-cell-adhesive hydrogel substrates, focal adhesions and the consequent fibrogenic de-differentiation of chondrocytes are substantially prevented or minimized. Hence, LhCG possesses a relatively pure hyaline-type cartilaginous composition and phenotype, which is characterized by the predominant content of Col II over type I collagen (Col I).[18] As a direct product of LhCG via decellularization, dLhCG is designed to be exclusively made of the ECM of LhCG (Figure 1.); therefore,

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dLhCG naturally inherits the hyaline-type cartilaginous composition and property, as well as 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

the structural traits, of LhCG. Further taking the advantages of decellularization, namely being an acellular product, dLhCG is designed to possess immuno-compatibility and recellularization accessibility for transplantation practice while also providing a logistic convenience for transportation and storage, as well as a much more flexible time-window for surgical operation. Collectively, dLhCG is expected to become a competent and practical offthe-shelf product for articular cartilage engraftment in the clinical setting, and the production of dLhCG is no longer limited by a dependence on an autologous source of cells or materials because it is open to much more abundant allogeneic and xenogeneic sources. In this study, via an optimized decellularizing approach, dLhCG samples were produced with autologous and allogeneic porcine chondrocytes, respectively, and implanted into the knee articular chondral defects of domestic pig models for 6 months. Living autologous chondrocyte-based engraftments of LhCG and ACI were also conducted in parallel as controls to highlight the impact of decellularization. Besides, the selection of ACI as a control was also based on the fact that in comparison with another clinically practiced treatment - microfracture, ACI may result in better phenotype in the regenerated neotissue[20–22] despite the higher complication in operation and modest superiority in terms of cartilage repair in general[23–25]. The evaluation of cartilage repair was performed via histology, biochemical assays, mechanical tests and medical imaging; in addition, gene profiling via DNA microarrays was also performed to reveal the results of re-cellularization and cell repopulation at the operation sites.

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

Figure 1.

Preparation and implantation of living hyaline cartilage graft (LhCG) and decellularized

LhCG (dLhCG) in comparison with autologous chondrocyte implantation (ACI) for articular cartilage repair: a schematic illustration with a timeline. Porcine chondrocytes are harvested from domestic pig models on -15 Day (15 days before designated Day 0). Primary and Passage 1 (P1) chondrocytes are derived by -14 Day and Day 0, respectively. P1 porcine chondrocytes and gelatin microsphere porogens are co-suspended and then co-encapsulated in alginate hydrogel under 4℃ on Day 0. Cavities are automatically created in the cell-laden hydrogel by the dissolution of microsphere porogens under elevated temperature at 37℃ by 1 Day. The chondrocytes outgrow the gel phase into the cavities and fill them up with cartilage neo-tissue to form an interpenetrating network (IPN) of neo-tissue and alginate hydrogel by 35 Day. The alginate hydrogel component is instantly removed from the IPN by rinsing with citrate saline to form LhCG on Day 35.[19] Some of maturated LhCG are decellularized to form dLhCG on Day42. dLhCG and the remaining LhCG are respectively implanted into knee articular chondral defects of domestic pig models on Day60 (2-Mon). ACI is conducted according to standard protocols provided by Carticel® that suspension of P2 autologous chondrocytes are injected into the articular chondral defect and sealed with a membrane made of decellularized porcine skin by sutures.[63] The samples of repaired cartilage are harvested for evaluation after 6 months since implantation (by the end of 8-Mon since gel encapsulation of chondrocytes).

2. Results and Discussion 2.1. Decellularization of tissue-engineered cartilage graft: from LhCG to dLhCG The dLhCG samples were prepared via decellularizing LhCG. Decellularization technology aims and works to substantially remove the cellular components whereas maximally preserve 7

the ECM of a biological tissue or organ of interest. Specifically, for the decellularization of 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

LhCG, which is a tissue-engineered hyaline cartilage graft, the clearance of the chondrocyte components was demonstrated by elimination of DNA content, and the maintenance of the cartilaginous ECM was measured by retention of Col II and proteoglycan. The retention of cartilaginous proteoglycan, namely aggrecan, was demonstrated by the measurement of chondroitin sulfate and keratin sulfate glycosaminoglycan (GAG) residues that are the subbranch components in the “brush-like” supramolecular structure of aggrecan. In this study, we attempted to compare four different decellularization approaches (Methods G1, G2, G3 and G4, as seen in Supplemental Figure 1) to identify an optimal method for the generation of a high quality dLhCG from LhCG; specifically, we aimed to generate a derived dLhCG product that simultaneously maintained the lowest DNA content and the highest contents of Col II and GAG. For this purpose, the dLhCG products that were derived via the four decellularization methods were examined via histology and biochemical assays. (Figure 2) According to haematoxylin and eosin (H&E) and 4',6-diamidino-2-phenylindole (DAPI) staining, the absence of nucleic acid was observed in both dLhCG G2 and G4 groups (Figure 2a); meanwhile, the lowest DNA content was detected from dLhCG G2 (Figure 2b). This outcome highlighted the efficacy of the repeated freeze-thawing processes between ultralow (-80°C) and room temperatures, which were only implemented in the G2 method. In terms of ECM preservation, as revealed by GAG and collagen assays, associated with Safranin O (Saf O) staining for GAG and immunohistochemistry (IHC) staining for Col II, the greatest preservation of both GAG and Col II were also observed from the dLhCG G2 method (Figure 2a and 2b). Collectively, the G2 method appears to be an optimal approach for the decellularization of LhCG, and dLhCG G2 is the best among all of the decellularized LhCG products.

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

Figure 2.

Decellularization of tissue engineering hyaline cartilage graft: from LhCG to dLhCG. A).

Histology of LhCG and various groups of dLhCG samples achieved from different decellularizing methods, namely dLhCG G1, G2, G3 and G4 (sample diameter ~8mm, thickness 1~2mm), by Haematoxylin and eosin (H&E) staining, Safranin-O (Saf O) staining, and immuno-histochemical (IHC) staining with primary antibody of type II collagen (Col II), respectively. DAPI staining has been overlaid with the IHC staining of Col II. Positive Haematoxylin stain of nuclei appears in purple; positive Eosin stain of ECM appears in pink; positive Saf O stain of GAG appears in bright red; positive IHC stain of Col II emits red florescence; and positive 4',6-diamidino-2-phenylindole (DAPI) stain of cell nuclei emits blue florescence. Scale bar: 200µm. B). Quantification of DNA residue, glycosaminoglycan (GAG) and total collagen contents, respectively, in LhCG and various groups of dLhCG samples: dLhCG G1, G2, G3 and G4, in comparison with native articular cartilage (NAC) and decellularized native articular cartilage using the Method G2 (dNAC G2). The measurements of the bar graphs are overlaid in Supplemental Table 3, 4 and 5. Statistical analysis has been conducted. The exact p values of the one-way ANOVA tests are presented in Supplemental Table 6, 7 and 8. For each group, n=3. Error bar represents standard deviation. Confidence interval is 95%.

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Notably, the DNA content remaining in dLhCG G2 was averagely about 1.76 µg per 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

mg dry weight of the total ECM in contrast to the 85.73 µg/mg, 204.3 µg/mg and 94.57 µg/mg DNA that remained in the samples of the same sourced LhCG (before decellularization), native articular cartilage explants (NAC) and decellularized NAC via the G2 method (dNAC G2), respectively (Figure 2b). This level of DNA residue in dLhCG G2 demonstrates a substantial clearance of the immunogenic cellular components. Also compared with LhCG, NAC and dNAC G2, the retention of Col II in dLhCG G2 appears to be nearly 90%; whilst, the retention of GAG in dLhCG G2 appears nearly 50% of that in LhCG or NAC, but still slightly greater than that in dNAC G2 (Figure 2b). The results of the ECM preservation indicated that, given the same decellularizing treatment, the retention rates of Col II are generally very high due to the great molecular size, intermolecular tangling and relatively lower solubility of Col II. Contrastively, the generally much lower retention rates of GAGs in all samples are caused by the significantly smaller molecular sizes, linear molecular structure and higher solubility levels. In summary, as a decellularized cartilage product, the quality of dLhCG G2 not only excelled among all of the parallel groups but also significantly surpassed the decellularized native cartilage (dNAC G2). Therefore, in all of the following procedures, only dLhCG G2 was adopted as the dLhCG sample for this study and, in all of the following contexts, the denotation of “dLhCG” exclusively refers to the dLhCG G2 product by default. 2.2. In vivo biocompatibility of dLhCG Despite the successful decellularization, the non-autologous ECM and the trace amounts of non-autologous cellular residues that remain in dLhCG may still pose potential risks of provoking transplant rejections in the recipients of allogeneic or xenogeneic engraftments. Accordingly, the test of in vivo biocompatibility of dLhCG was designed and performed by embedding the porcine-sourced dLhCG into the greater omentums of rats and checking host

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responses at the end of the 1st and 2nd weeks, respectively. The rationale of this design is 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

based on the idea that if dLhCG, as a xenograft, does not provoke transplant rejections in the highly vascularized greater omentum, then it should be able to be safely used as an allograft in articular chondral defects, which is believed to be an “immune-privileged” location due to the avascular and nerveless nature of the cartilaginous tissues.[26] Table 1. Scoring on host reaction to porcine sourced dLhCG in rat greater omentum using five-point

1 week

2 weeks

Lymphocytes

3

2

Multinucleated giant cells

1

0

Neutrophils

2

2

Macrophages

3

2

Mast cells

2

1

Eosinophils

2

1

ordinal severity scale* * 5-point ordinal severity-scale: no inflammation–0, minimal–1; mild–2; moderate–3; and severe–4.

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Figure 3. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

Test of in vivo biocompatibility of porcine sourced dLhCG in rat greater omentum.

Haematoxylin and eosin (H&E), Safranin-O (Saf O) Masson’s trichrome (MT), Alizarin red staining (AR) and immunohistochemical (IHC) staining with primary antibody of CD3 and CD68 for dLhCG after being implanted in rat greater omentum for one and two weeks, respectively. Scale bar: 500µm. The rectangles in the left panel encloses the region of the entire graft. The arrows on the image of “Saf O, 2-Week” indicate the capillaries. Positive Haematoxylin stain of nuclei appears in purple; positive Eosin stain of ECM appears in pink; positive Saf O stain of GAG appears in bright red; positive MT stain of collagens appears bluish; positive AR stain of calcification appears in dark red; and positive IHC staining by polymerization of 3',3'-diaminobenzidine (DAB) catalyzed by horse radish peroxidase (HRP) conjugated on secondary antibody appears in golden brown.

Host reactions to a biocompatible graft proceed similarly to a wound healing process, of which inflammation, angiogenesis and tissue remodelling are the necessary sequential procedures, in contrast to rejection reactions.[27,28] By the end of the 1st week after implantation, a moderate amount of macrophages, neutrophils and lymphocytes that is accompanied by a minimum amount of multinucleated giant cells was observed within and around the embedded dLhCG; these cell types decreased in number by the end of the 2nd week, which indicated that a normal inflammatory reaction had occurred rather than a phagocytic transplant rejection[29,30] (Table 1). The decreases in macrophages and Tlymphocytes were also evidenced by the weakened immunostaining of CD68 and CD3 cells, respectively (Figure 3). Moreover, in both CD3 and CD68 staining, at Week 1, the graft was prominent with a weaker staining than the surrounding tissue. It indicated that neither T-cells nor macrophages was attracted by the graft. The strong staining at the surrounding tissue is due to the natively in situ T-cells and macrophages in the greater omentum. Furthermore, at Week 2, the graft gradually became unrecognizable from the surrounding tissue with similar intensity of staining in both CD3 and CD68 to the surrounding tissue. However, this was not due to increase of T-cells and macrophages in the graft region as confirmed by the scoring in Table 1. Instead, it was due to the weakened staining in the surrounding tissue. It suggested that the surrounding omentum tissue was rearranging and reorganizing itself to sustain the 12

growth of new tissues, which is an indicator of the tissue remodelling process. In combination 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

with other staining, most obviously in Saf O staining, small capillaries were observed around the grafting area, which indicated the occurrence of angiogenesis to supply nutrients and oxygen and remove wastes to support neo-tissue growth. In addition, the higher magnification image exhibited a more thorough infiltration of cells into the graft from Week 1 to Week 2, which indicated the avoidance of fibrous encapsulation of the graft by the surrounding tissue. At the same time, morphological changes occurred from the avascular cartilage-like tissue (as dLhCG) towards the vascularized omentum-like tissue as revealed by the weakened staining of the cartilaginous total collagen (Masson’s Trichrome, MT) and GAG (Saf O) that was accompanied by the strengthened staining of calcification (Alizarin red), which indicated the beginning of tissue remodelling[31] (Figure 3). Collectively, the host reactions to the implanted dLhCG appeared to be indicative of a normal healing process; therefore, the in vivo biocompatibility of dLhCG, as a xenograft in an immune-sensitive part of the recipient body, was verified so that it could be safely used as a cartilage allograft in “immune-privileged” articular chondral defects in the following study. 2.3.Articular cartilage regeneration via implantation of dLhCG For articular cartilage repair, there is a significant challenge in how to endow the regenerated neo-tissue with a pure hyaline cartilaginous phenotype. Another challenge exists in how to manage optimal host-graft integration. In this study, the dLhCG series of products were designed and developed to address these key challenges. The donor sites of the cartilage explants for cell sourcing were adopted on the non-weight bearing femoral trochlear grooves in the stifle joint of the pigs. As the operation site for implantation and repair, articular chondral defects beyond critical size (8 mm in diameter) were created on the femoral condyles that represented the most weight bearing positions. The experimental defects were made to penetrate the full thickness of the cartilage layer, but these defects were carefully

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controlled by keeping the subchondral bones untouched. The grafts were excised and 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

examined at 6 months to observe the potential fibrosis and hypertrophy in the regenerated cartilaginous neo-tissue, if they occurred, would become detectable after such a period,[32– 34] which are demonstrated as observable upregulations of Col I and type X collagen (Col X), respectively, over the existed abundant Col II deposited earlier. The parallel samples of the implants included autologous chondrocytes (ACI), autologous chondrocyte-laden and derived LhCG (Auto LhCG), autologous chondrocyte-derived but decellularized dLhCG (Auto dLhCG) and allogeneic chondrocyte-derived but decellularized dLhCG (Allo dLhCG). Furthermore, untreated defects were also created and employed as controls.

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

Figure 4.

Histology for in situ biocompatibility of Allo dLhCG, Auto dLhCG and Auto LhCG in

comparison with ACI at time point of 6 months after implantation in porcine knee chondral defects. The negative control, namely the untreated defects that are left void of grafts, is marked as “Untreated defect”. A) Immuno-histochemical (IHC) staining with primary antibody of CD3 and B) CD68. Two arrows are used to indicate the location and width of the experimental defects originally made. Scale bar: 2 mm. Positive IHC staining by polymerization of 3',3'-diaminobenzidine (DAB) catalyzed by horse radish peroxidase (HRP) conjugated on secondary antibody appears in golden brown.

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

Figure 5. Histology of cartilage repair by engraftment of Allo dLhCG, Auto dLhCG and Auto LhCG in comparison with ACI at time point of 6 months after implantation in porcine knee chondral defects. The negative control, namely the untreated defects that are left void of grafts, is marked as “Untreated defect”. A) Hematoxylin and eosin (H&E) staining, B) Masson’s trichrome (MT) staining, C) Safranin-O (Saf O) staining, and immuno-histochemistry (IHC) staining with D) primary antibody of Type II collagen (Col II), E) Type I collagen (Col I), and F) Type X collagen (Col X). Two arrows are used to indicate the location and width of the experimental defects originally made. Scale bar: 2 mm. Positive Haematoxylin stain of nuclei appears in purple; positive Eosin stain of ECM appears in pink; positive Saf O stain of GAG appears in bright red; positive MT staining of collagens appears blue; positive IHC staining by polymerization of 3',3'-diaminobenzidine (DAB) catalyzed by horse radish peroxidase (HRP) conjugated on secondary antibody appears in golden brown. G) The histological scores based on both Histological Histochemical Grading System (HHGS) and Wakitani scoring system. A low score (min. 0) defines a native-like cartilage tissue with full thickness defect filling and good integration with adjacent host cartilage. * indicates statistical significance (p < 0.05); ** indicates p ≤ 0.01, *** indicates p ≤ 0.001. For each group, n=3. Error bar represents standard deviation. Confidence interval is 95%. The measurements of the bar graphs are overlaid in Supplemental Table 9 and 10. The exact p values of the one-way ANOVA tests are presented in Supplemental Table 11 and 12.

After 6 months in vivo, biocompatibility was first specifically assessed in the sole sample that had the allogeneic sourced implant, Allo dLhCG, compared with all of the other autologous cell-laden or derived samples. The immunostaining of CD3 and CD68 indicated that no significant difference could be identified among all of these samples but equally negative indications. This result suggested that, as a decellularized, allogeneic sourced

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implant, Allo dLhCG did not provoke detectable levels of immune reactions from the host 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

(Figure 4 and Supplemental Figure 2). On the basis of this survey of histological biocompatibility, the outcome of articular cartilage regeneration was revealed and examined by further histological staining. All cartilaginous engraftments by dLhCGs, LhCG and ACI were able to restore the defective cartilage with full thicknesses of regenerated cartilaginous neo-tissue in contrast to the untreated defects in which the experimental chondral lesions remained unrepaired (Figure 5 and Supplemental Figure 3). Biochemical assays were conducted on the parallel samples to measure the contents of DNA, GAG and total collagen in regenerated cartilaginous neotissues, respectively, representing the cell number (via DNA: 7.7 pg per chondrocyte)[35] and ECM contents (via GAG and total collagen). These quantitative outcomes were normalized by the total wet weight in order to demonstrate the value of density per construct of the neotissue and was also normalized by the total dry weight to demonstrate the relative net magnitudes of the solid contents excluding water. (Figure 6; comparative data normalized against cell number/DNA are presented in Supplemental Figure 4) Compared with the intact adjacent cartilage (NAC) and as revealed via biochemical assays, the cell densities in the untreated defects did not appear lower, albeit the ECM contents were much smaller. These quantitative data appeared to be in accordance with the histological results. Collectively, these results verified that the autogenous compensation occurred in the untreated defects via cell migration and ECM deposition; however, this self-reparation was not able to repair the defect beyond critical size. In contrast, in the samples treated by implantations of Auto LhCG and ACI, both of which delivered autologous chondrocytes via ex vivo processing, the cell densities appeared to be lower than that in NAC, especially in the ACI product; the ECM contents appeared to be similar to that in NAC in the ACI product but were slightly lower in the Auto LhCG, particularly in GAG content (Figure 6). These results were also in line with

19

the histological staining for total collagen and GAG (Figure 5b-c). The histological staining 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

for both Auto and Allo dLhCG implanted samples indicated the abundant presence of total collagen and GAG, which appeared to be comparably positive with that of NAC (Figure 5bc). The quantitative results from the biochemical assays also indicated similar contents of ECM between NAC and dLhCG samples except for a slightly lower cell density in the Auto dLhCG sample. This result confirmed the successful migration of chondrocytes from the adjacent host cartilage into the originally acellular dLhCG implants and the fine development of cartilaginous neo-tissue upon these decellularized implants, especially in Allo dLhCG.

Figure 6. Biochemical analyses of cartilage repair by engraftment of Allo dLhCG, Auto dLhCG and Auto LhCG in comparison with ACI at time point of 6 months after implantation in porcine knee chondral defects. The negative control, namely the untreated defects that are left void of grafts, is marked as “Untreated defect”; the positive control, namely the intact surrounding native articular cartilage of the host, is marked as “NAC”. The analyses include DNA assay, glycosaminoglycan (GAG) assay, and total soluble collagen assay. The quantity of each composition are normalized respectively by wet weight and dry weight of the total extracellular matrices (ECM). * indicates statistical significance (p ≤0.05); ** indicates p ≤ 0.01, *** indicates p ≤ 0.001, **** indicates p ≤ 0.0001. For each group, n=5. Error bar represents standard deviation. Confidence interval is 95%. The measurements of the bar graphs are overlaid in Supplemental Table 13 to 18. The exact p values of the one-way ANOVA tests are presented in Supplemental Table 19 to 24.

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

Histological analyses were also performed on the morphology and integration of the regenerated cartilaginous neo-tissues within the surrounding host NAC. It was observed that the ACI sample exhibited an irregular surface with pannus-like formations outgrowing from the top edge of the regenerated cartilage (Figure 5). This morbidity has also been mentioned in clinical case reports.[36] Here, the overgrowth of cartilaginous neo-tissue is induced by the covering treatment with the porcine skin derived membrane, which is performed to seal the opening of the defect in order to prevent the leakage of the liquid-formed ACI cell suspension from the operation site. After 6 months in vivo, the covering membrane was mostly metabolized except for minor residues that remained at the top, as indicated by faint Saf O staining for the lower quantity of GAG (Figure 5c). This is because the porcine skin-derived membrane is composed of rich Col I and type III collagen (Col III) that are not of hyaline cartilaginous collagen fibrils, but that are fine substrates for chondrocytes to attach to and commit focal adhesion. Being a specialized member of fibroblast lineages, the adhered chondrocytes will commit dedifferentiation and result in the formation of fibrocartilage, instead of pure hyaline cartilage. Due to the same reason, overgrowth of neo-fibrocartilage on the surface was also enabled and mediated by contact with the cell-affinitive membrane.[37] This overgrowth morbidity compromises the smoothness and regularity of the cartilage surface and therefore poses a significant drawback of ACI. In contrast, the implantations of solid-phased dLhCGs or LhCG resulted in the seamless integration between the regenerated cartilaginous neo-tissues and the adjacent host NAC, which shared a common, continuous and smooth chondral surface (Figure 5). It benefited from the porous, sponge-like formation of the dLhCG and LhCG implants. Besides the convenience of the operation as being in solid form versus the liquid implant of ACI, this type of physical formation enables better physical fit of the implants within the chondral defects via a vaster contact area, and also facilitates

21

better access for cell migration and ECM connection with the ambient host tissues, which is 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

particularly more effectual in the more porous acellular dLhCGs rather than in the cell-laden LhCG counterpart. Moreover, the composition of the pure, hyaline-like cartilaginous ECM in dLhCG and LhCG makes them specifically favourable for the invasion, expansion and development of chondrocytes due to the preserved chondro-conductive biological cues and the specific microstructures in the ECM framework of the implants.[15,38] Whilst, on the other hand, the same ECM framework also functions to physically confine the development of the regenerated cartilaginous neo-tissue from outgrowing the general boundaries of the chondral surface like that occurred in the ACI treatment. The avoidance of overgrowth was also attributed to the nature of the hyaline cartilaginous ECM in dLhCG and LhCG implants, which does not favour cell focal adhesion, therefore prevents the over proliferation like the dedifferentiated chondrocytes’ behaviour. Based on the general outcomes of histology, peer scoring and grading evaluations following two sets of scoring systems, the Wakitani Scoring System and the Histology Histopathology Grading System, were carried out in the form of a blind test (Supplemental Table 1-2, a zero score indicating the same as the native counterpart - NAC). The results from the two evaluations appeared to be consistent with each other, and both evaluation results further confirmed that both Allo and Auto dLhCG samples possess high histological similarity to NAC, which appeared better than the autologous cell-laden samples of Auto LhCG and ACI, particularly much superior to the ACI sample (Figure 5g). As designed and practised, the subchondral bones were deliberately protected from experimental trauma in all of the samples when the experimental chondral defects were created. After 6 months in vivo, and according to the histology (Figure 5), as well as the micro-CT (Supplemental Figure 5), the integrity of the subchondral bones appeared to be finely maintained in both Allo and Auto dLhCG samples. However, in the (Auto) LhCG samples, an observable plateau of bony

22

overgrowth distorted the line of tidemark upward into the cartilage layer; whilst in the ACI 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

sample, an observable bony collapse led to a downward subsidence of the cartilaginous neotissue into the subchondral bones, which also broke the integrity of the tidemark; and, in the sample with the untreated defects, the subchondral bones appeared to be fragmented. These results again indicated that the articular cartilage plays a critical role in shielding the integrity of the subchondral bones, while the subchondral bones also provide critical support to the articular cartilage. Untreated penetrating lesions in the articular cartilage, once expand into the subchondral bones, will initiate a destructive cycle that destroys both of these tissues and worsens the osteochondral lesions. In this case, the key therapy relies on the repair of the non-self-regenerative articular cartilage via cartilaginous engraftments, herein - the implantations of Allo and Auto dLhCG. 2.4.Phenotypic analysis of regenerated cartilage neo-tissue by histology and DNA microarray Further histological analyses were performed particularly focusing on the cell phenotype in the regenerated cartilaginous neo-tissues. Col II dominates the collagen content in healthy adult articular cartilage, which marks the hyaline cartilaginous phenotype.[39] Significant upregulations of Col I and Col X in articular cartilage respectively marks the fibrotic dedifferentiation and hypertrophic degeneration of chondrocytes. From the histology results, the regenerated cartilaginous neo-tissue via implantation of dLhCGs and LhCG exhibited a high purity of Col II occupancy with free or minimum of Col I or Col X (Figure 5d-f). Conversely, ACI-derived neo-tissue exhibited observable upregulations of Col I and Col X particularly in the central area, suggesting phenotypic changes of the implanted chondrocytes in the ACI products leading to both fibrosis and hypertrophy (Figure 5d-f). These results indicate that the chondrocytes in suspending culture as conducted in the ACI approach are prone to precipitation and adhesion upon the surfaces of the chondral defects and also at the

23

interface with the covering membrane, both of which can lead to the loss of hyaline 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

cartilaginous phenotype.[40–43] In contrast, the hyaline-like cartilaginous phenotype of the chondrocytes in LhCG was well preserved because the cells had always been cultivated via a 3D culture without focal adhesive substrates.[44] The final cell population and phenotype in the initially acellular dLhCG implants were determined by the general outcomes from competitions of cell migration and proliferation that had originated from different ambient host tissues, including the adjacent articular cartilage, the subchondral bones and the synovial fluid. According to histological staining, after 6 months in vivo, hyaline-like cartilaginous ECM apparently dominated the matrix content of the regenerated neo-tissue in both Auto and Allo dLhCG derived products, which suggests that the chondrocytes from the adjacent articular cartilage surpassed all of the other sourced cells, dominated the occupancy in the grafting area and developed abundant hyaline-like cartilaginous neo-tissue in situ.

Figure 7.

DNA microarray profiles indicating gene expression of the cells harvested from the

grafting areas by Allo dLhCG, Auto dLhCG, Auto LhCG and ACI, as well as the ambient host tissues of native articular cartilage (NAC), subchondral bones and synovium at time point of 6 months after implantation in porcine knee chondral defects. The sample from untreated defect was also employed as a

24

control. Expression level are sorted to observe patterns on a list of 13 marker genes, including 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

cartilaginous and chondrogenic markers of Type II collagen (Col2a1), aggrecan (ACAN), cartilage oligomeric matrix protein (COMP), Type IX and XI collagen (Col 9a1 and Col 11a1), transcription factor Sox9 and transforming growth factor-β1 (TGFβ1); and some other osteochondral related markers of Type I collagen (Col1a1), Type X collagen (Col10a1), testican-1 (SPOCK1), Type III collagen (Col3a1), integrin binding sialoprotein (IBSP) and alkaline phosphate (ALPL). Bars represent the fold change in gene expression i.e. up-regulation (upwards) or down-regulation (downwards).

These observational conclusions were examined and confirmed by transcriptome profiling of the resident cells in the grafting and ambient areas via DNA microarrays. Comparative analyses on the profiles of the most relevant gene expressions were performed (Figure 7). The parallel samples were harvested from the regenerated neo-tissues in the grafting areas of the dLhCGs, LhCG and ACI as well as the ambient host tissues including the NAC, subchondral bones and synovium. The sample from the untreated defect was also employed as a control. The relevant genes included cartilaginous and chondrogenic markers of Col II (Col2a1), aggrecan (ACAN), cartilage oligomeric matrix protein (COMP), Type IX and XI collagen (Col 9a1 and Col 11a1, respectively), transcription factor Sox9 and transforming growth factor-β1 (TGFβ1); additionally, several other osteochondral-related markers of Col I (Col1a1), Col X (Col10a1), testican-1 (SPOCK1), Col III (Col3a1), integrin binding sialoprotein (IBSP) and alkaline phosphate (ALPL) were also identified. As a benchmark, in the profile of NAC, the expressions of key chondrogenic markers were mostly and exclusively upregulated, thus clearly indicating a mature and healthy phenotype of the hyaline cartilage. Conversely, the profiles of samples from the untreated defects, subchondral bones and synovium clearly demonstrated a non-cartilaginous phenotype. From the selfregenerated tissues in the untreated defects, almost all of the related marker gene expression levels were downregulated, except for testican-1 and Col III, which are generally indicative of non-hyaline cartilaginous proteoglycan and collagen, respectively. The profile of the subchondral bones indicated mixed expressions of bony and calcified (degenerated) 25

cartilaginous genes, such as alkaline phosphate, IBSP and Col X. In the profile of the 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

synovium, the expressions of almost all of the cartilaginous and chondrogenic markers were also downregulated. In contrast with these control samples, the general tendency of gene expression levels in Allo- and Auto dLhCG-derived samples appeared to be in line with that of NAC, although the extents of upregulation were observably weaker. The major difference between the NAC and dLhCG-derived samples lies in opposed regulating tendencies of Col IX, COMP and Sox9. The significant upregulation of Col IX in NAC indicated the stabilization of the fibrillary collagen network in the cartilaginous ECM[45] and prevention of fibril aggregation[46], whereas a flat expression of Col IX in dLhCG-derived samples indicated that the remodelling of the collagen network did not appear to be complete yet in these regenerated cartilaginous neo-tissues. The relatively greater upregulation of COMP in dLhCG-derived samples indicated more active cartilage turnover,[47,48] and the upregulation of Sox9, which is a chondrogenic transcription factor, indicated that the process of chondrogenesis was still active in these regenerated neo-tissues. Collectively, the cells residing in dLhCG-derived cartilaginous neo-tissues demonstrated a clear feature of having freshly committed chondrocytes that possess a hyaline cartilaginous phenotype and continue to experience a remodelling process towards maturation. The DNA microarray outcomes from Auto and Allo dLhCG appeared to be very similar to each other. These results reinforced and explained the conclusions drawn from the histological analyses that, after 6 months in vivo, chondrocytes migrated from the adjacent host articular cartilage, dominated the occupancy in the originally acellular dLhCG implants and maintained their hyaline cartilaginous phenotype; therefore, these chondrocytes exhibited the capability to develop hyaline-like cartilaginous neo-tissue in situ. This was attributed to the superior physical features and fine chondro-conductivity of the dLhCG implants. Comparatively, the DNA microarray profiles of the cells that were extracted from the Auto LhCG-derived cartilaginous

26

neo-tissues provided mixed indications. On the one hand, after 6 months in vivo, this 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

autologous chondrocyte-derived, living cell-laden tissue-engineered implant had regenerated hyaline cartilaginous neo-tissues that possessed the highest similarity to NAC in terms of chondrocytic and chondrogenic gene expressions; but on the other hand, the upregulations of hypertrophic cartilaginous and bony markers, which were respectively represented by Col X and alkaline phosphatase, suggested the beginning of a phenotypic transition from “overmature” cartilage to the formation of bones via a pathway like endochondral ossification. These hyaline cartilaginous tissue formations again reflect the unique advantages of LhCG for articular cartilaginous engraftment. However, the upregulation of hypertrophic and bony markers may demonstrate a flaw of LhCG. It may be induced by prolonged ex vivo processes to which the employed and delivered autologous chondrocytes were exposed, specifically, the initial enzymatic harvest and the subsequent 2D subculture, followed by prolonged 3D culture in vitro. Additionally, after being implanted into the chondral defect, the vast population of these “over-processed” and pre-occupied chondrocytes could hardly be repopulated by fresh chondrocytes migrating from the adjacent host cartilage. This flaw of the cell-laden LhCG was completely avoided in the dLhCGs via decellularization to remove the “old” cells followed by in situ recellularization via the migration of fresh cells from the host. Due to a similar reason that affected the autologous cell-laden LhCG sample, the tendencies of chondrocytic hypertrophy and cartilaginous calcification in the ACI-derived neo-tissues appeared to be even more significant as revealed by the intensive upregulations of Col X and IBSP in the DNA microarray profiles. Besides the influence of prolonged ex vivo processing as well, another notable cause was that, according to the standard protocols, P2 cells were employed for ACI versus P1 cells that were used for LhCG. An additional passage of subculture in monolayer before implantation caused more severe problem of dedifferentiation of chondrocytes; additionally, after implantation, further dedifferentiation was

27

induced in the operation site due to cell adhesion upon the inner surfaces of the chondral 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

defects, as well as the cover membrane. The gradual loss of the hyaline cartilaginous phenotype was revealed via the downregulation of Col II that was accompanied with an observable upregulation of Col I and a significant upregulation of Col X. Collectively, the outcomes from the DNA microarrays confirmed and explained the phenotypic findings from histological analyses in the regenerated cartilaginous neo-tissues. Phenotypic variation was also reflected in the superfine microstructure of the regenerated cartilaginous neo-tissues, as revealed in picrosirius staining by polarization and bright field microscopy (Figure 8). Also as a benchmark, in NAC, a typical zonal structure was illustrated: in the superficial zone, collagen fibrils and chondrocytes were packed and aligned in parallel with the general surface of the articular cartilage; in the middle zone, chondrocytes were dispersed within the oblique collagen fibrillary network; and, in the deep zone, chondrocytes were aligned in a columnar orientation, along with the collagen fibrils perpendicular to the general surface (also perpendicular to the subchondral tidemark) of the articular cartilage. Among all of the experimental samples, this type of tri-zonal microstructure with the neat alignment of cells and ECM was clearly formed in the Allo dLhCG-derived cartilaginous neo-tissue, which indicates a nearly accomplished reparatory remodelling of the articular cartilage. Comparatively, the zonal structure hadn’t been fully established in the Auto dLhCG- or LhCG-derived cartilaginous neo-tissue, thus indicating that the remodelling had not yet implemented, and that the maturation was still pending. In contrast, the microstructures of the ACI-derived cartilaginous neo-tissue and the autogenously regenerated tissue in the untreated chondral defects appeared to be rather random and lacked any alignment or orientation. These results further confirmed the superior chondro-conductivity of dLhCG implants, especially in regard to Allo dLhCG.

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

Figure 8.

Microstructural histology of articular cartilage by Picrosirius staining under polarized

light and bright. The staining reveals the grafting areas by Allo dLhCG, Auto dLhCG and Auto LhCG in comparison with ACI at time point of 6 months after implantation in porcine knee chondral defects. The negative control, namely the untreated defects that are left void of grafts, is marked as “Untreated defect”; and the positive control – intact, surrounding native articular cartilage of the host – is marked as “NAC”. Scale bar: 200 µm. Picrosirius stains relatively raw matrix fibers in brown or dark yellow and relatively delicate fibrils in green or light yellow under polarized light.

Table 2. Mechanical Test of regenerated cartilaginous neo-tissue

Sample *

Compressive modulus (MPa) **

NAC

0.46±0.02

Untreated defect

0.29±0.33

ACI

0.29±0.27

Allo dLhCG

0.39±0.16

Auto dLhCG

0.30±0.27

Auto LhCG

0.32±0.17

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* 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

“NAC”: the intact, adjacent native articular cartilage of the host. “Untreated defect”: the created

defect that were left empty without any engraftment or treatment. “ACI”: the regenerated tissue after being treated by clinical available autologous chondrocyte implantation. **

Testing time point is 6 months after implantation. The Young’s modulus is presented as mean ±

standard deviation.

2.5. Mechanical property of repaired articular cartilage The major functions of the articular cartilage are to bear compressive stresses at the joint and to articulate movements of the connected bones. Hence, the ultimate quality evaluation of cartilage repair lies in the mechanical properties of the regenerated cartilaginous neo-tissues. The overall mechanical properties of a hard tissue, specifically articular cartilage, is determined by the abundance, composition, phenotype and microstructure of the cartilaginous ECM, as well as the integration with the surrounding host tissues and the integrity of the subchondral bones. The abundance and composition of the ECM were measured and tested via biochemical assays and histology, the phenotype was characterized via histology and DNA microarrays, and the microstructure was demonstrated via picrosirius staining. Collectively, all of the experimental implants were pure in composition - free of noncartilaginous constituents. The tissue-engineered implants, LhCG and dLhCGs, possessed relatively pure hyaline-like cartilaginous phenotypes; in particular, the implantation of Allo dLhCG had even successfully regenerated the typical zonal cartilaginous microstructures in the neo-tissues, thus indicating the best chondro-conductivity. Herein, to focus on the mechanical properties of the regenerated cartilaginous neo-tissue, a confined mechanical test was conducted on cartilage explants that were harvested from the operation sites without the attachments of the subchondral bones, from which the compressive modulus of each sample was measured (Table 2 and Supplemental Figure 6). The results indicated that the best mechanical property was also detected from the Allo dLhCG-derived cartilaginous neo-tissue, the compressive modulus of which was measured to be 0.39±0.16 MPa, which was 30

approximately 85% of NAC (0.46±0.02 MPa) and approximately 120% of the average of the 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

Auto LhCG group (the second highest group on the average of the Young’s modulus) and approximately 135% of the average of the untreated defect (the lowest group on the average of the Young’s modulus). As a measurement of key functional restoration, this result met and reflected the general outcomes from all aspects of the features and factors of articular cartilage repair: after 6 months in vivo, the implantation of Allo dLhCG had derived the finest neo-tissue of articular cartilage in situ, albeit the final maturation was yet to be completed.

3. Conclusions In this study, by combining the advantages of cartilage tissue engineering and decellularization technology, we developed a decellularized allogeneic hyaline cartilage graft, Allo dLhCG, which achieved a superior efficacy of articular cartilage repair. By the 6-month time point of implantation in porcine knee joints, the fine morphology, composition, phenotype, microstructure and mechanical properties of the regenerated hyaline-like cartilaginous neo-tissue were demonstrated via histology, biochemical assays, DNA microarrays and mechanical tests. The results indicated that cartilaginous engraftment via Allo dLhCG surpassed living autologous chondrocytes-laden and derived cellular or engineered tissue engraftments by ACI or Auto LhCG in almost all aspects of its features and functions. Allo dLhCG also exhibited better microstructural reconstruction and higher average Young’s modulus, which may suggest its better chondro-conductivity than autologous chondrocyte derived, decellularized hyaline cartilage graft, Auto dLhCG. Particularly, Allo dLhCG was proven to be well compatible with the host because it was free of immune rejection regardless of whether it was used as a xenograft in the greater omentum of the rat or as an allograft for articular cartilaginous engraftment in chondral defects of the porcine knee joint. The success of this allogeneic decellularized graft broadens the source of

31

grafting materials, thereby avoiding the risk of donor site morbidity for the purpose of 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

collecting autologous materials. Additionally, due to the advantages of being an acellular product, Allo dLhCG may act as a competent and practical off-the-shelf product, thus allowing patients to experience a flexible time window for clinical operations and providing a logistic convenience for transportation and storage. Collectively, the successful repair of articular chondral defects in large animal models suggests the readiness of Allo dLhCG for clinical applications.

4. Experimental Section Chondrocytes harvest and preparation: All animal experimentation in this study was performed in line with the protocols approved by Institutional Animal Care and Use Committee (IACUC). Unless stated otherwise, all chemicals used in this study were purchased from Sigma Aldrich, Singapore; all cell culture reagents were purchased from Invitrogen and Life Technologies. Twelve (12) domestic pigs (sus scrofa domesticus, body weight range: 47 ~ 53 kg) were employed as the sources of chondrocytes as well as the hosts of articular cartilage repair. The donor sites of cartilage explants, as the source of chondrocytes, were adopted on the femoral trochlear groove in the stifle joints. Porcine chondrocytes were harvested from the cartilage explants. Briefly, eight (8) pieces of cylindershaped (3 mm in diameter) cartilage explants were harvested from the donor sites of each knee joint using biopsy punch. (Supplemental Figure 7) The explants were mechanically homogenized into smaller fragments and then digested overnight with collagenase II (1mg/ml, by Gibco) in chondrocyte culture primary (CCP) medium that is composed of 12% fetal bovine serum (FBS), 0.1% L- proline, 0.1% ascorbic acid, 0.2% 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES) and 0.2% non-essential amino acids (NEAA) in Dulbecco’s modified Eagle medium (DMEM). The obtained primary porcine chondrocytes 32

were sub-cultured from Passage 0 (P0) to P1 in CCP medium and the P1 chondrocytes were 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

employed for all following experimentation. Fabrication of LhCG: The fabrication of LhCG was performed according to the previously established protocol.[18] Briefly, porcine chondrocytes (P1, 1 × 107 cells/mL) and gelatin microspheres (made via double-emulsion method, 150~180 μm diameter, 0.3g/mL) were coencapsulated in alginate hydrogel at 4°C. These cell-laden hydrogel constructs were then translocated onto non-cell-adhesive tissue culture plates and cultivated in chondrocyte culture (CC) medium that is composed of 20% (v/v) FBS, 0.01 M HEPES, NEAA, 0.1 mM, Lproline (0.4 mM), ascorbic acid (0.05 mg/mL), penicillin (100 units/mL) and streptomycin (100 mg/mL) in DMEM. The cultivation was conducted on an orbital shaker (50 rpm) in a humidified incubator at 37 °C with 5% CO2. After 35 days, these hydrogel constructs were subjected to a rinse with solution of sodium citrate (55 mM in 0.15 M NaCl solution) for 10 mins at room temperature, by which the alginate components in the constructs are removed. Thereafter, the obtained scaffold-free constructs, that is LhCG, were further cultivated in CC medium for another 7 days’ maturation and became ready for decellularization and further use. (Figure 1.) Decellularization of LhCG for fabrication of dLhCG: To fabricate dLhCG samples, four (4) sets of decellularization methods, respectively denoted as Method G1, G2, G3 and G4, were attempted on LhCG samples (Supplemental Figure 1.). Method G1 was conducted by cycling the freezing (-20°C, 3 hours) and thawing (room temperature, 4 hours) processes on LhCG for three times, followed by soaking in 1% sodium dodecyl sulfate (SDS) based hypertonic solution (TRIS buffer, pH 8.0, by 1st Base) for 72 hours.[49,50] Method G2 was conducted by cycling the freezing (-80°C, 3 hours) and thawing (room temperature, 4 hours) processes on LhCG for three times and followed by soaking in TRIS hypertonic solution for 24 hours and then in 1% Triton X-100 (1st Base) based TRIS hypotonic solution for 48 hrs.[51–53] 33

Method G3 was conducted by soaking LhCG in TRIS hypotonic solution for 48 hours and 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

then in 1% Triton X-100 based TRIS hypotonic solution for 72 hours.[54] Method G4 was conducted by soaking LhCG in 1% SDS solution based TRIS hypotonic solution for 72 hours and then in 1% Triton X-100 based TRIS hypotonic solution for another 72 hours.[55] After the above treatments, all treated samples were further soaked, in turn, in DNase (0.5 mg/mL DNase I) and RNase (50 µg/mL RNase A) solutions for 3 hours, followed by rinsing in PBS for 24 hours. All soaking procedures were accompanied with agitation at 150 rpm under 37°C. The final products were respectively denoted as dLhCG G1, G2, G3 and G4. For comparison, decellularization following Method G2 was also conducted on porcine NAC (native articular cartilage explant with the same thickness as that of LhCG: 1.5mm) resulting in a decellularized NAC control sample denoted as dNAC G2. (Figure 2) Embedment of dLhCG in rat greater omentum: Under general anaesthesia, each piece of porcine sourced dLhCG was embedded into the greater omentum of each rat (SpragueDawley all female, 8 weeks old) via open surgery and closure by sutures. After surgery, all animals were allowed for unrestricted activity in cages. Two time points were applied for sample harvest, respectively, at the end of the 1st and 2nd week since the surgery. Surgery of dLhCG implantation and ACI in porcine articular chondral defects: Under general anaesthesia, a medial parapatellar approach was performed to expose the stifle joint of the experimental pigs and the patella was laterally dislocated. One critical-sized (8 mm diameter) full-thickness cartilage defect was created using surgical puncher with null or minimal damage to the subchondral bone on each femoral condyle. Given 12 animals, totally 48 experimental defects were created, among which 10 defects were kept untreated as negative controls; 10 defects were filled with allogeneic sourced Allo dLhCGs; 10 defects were filled with autologous sourced Auto dLhCGs; 9 defects were filled with autologous sourced Auto LhCGs; and 9 defects were treated by ACI. Autologous or allogeneic engraftments were 34

respectively performed in the same or different individuals as the cell donors among the same 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

group of animals. Mixing of auto- and allo-grafting was avoided being conducted in the same individual host. Both LhCG and dLhCG implants were press-fit into the defects without deliberate fixation. ACI was performed according to standard protocol provided by Carticel®. Briefly, since 2 weeks before implantation, the cryopreserved P1 autologous chondrocytes were thawed and further expanded to meet the required cell density (12 million cells/ml) as ACI cell suspension (P2 chondrocytes in serum free DMEM) ready for implantation. On surgery, firstly a patch of membrane made of decellularized and devitalized porcine skin (Fortiva® Porcine Dermis) was placed to cover the opening of cartilage defect and fixed by sewing and gluing with commercial fibrin glue (TISSEEL kit, ThermoFisher Scientific Inc.) over the edges to prevent displacement and leakage, subsequently 80 µL of ACI cell suspension was injected into the cartilage defect covered underneath the membrane. After implantation, the patella was restored to the original position and all surgical wounds were closed by sutures. After surgery, all animals were allowed for unrestricted activity all the time till being sacrificed for sample harvest 6 months later when the femur distal ends including the whole osteochondral parts were collected with complete integrity. The samples for mechanical tests, biochemical assays and DNA microarray were washed in PBS and processed within 2 hours; the samples for histology were immersed in 10% neutralized formalin prior to further process. Histology and immunohistochemistry: All samples were paraffin-embedded and then sectioned with a thickness of 10 m through the centre of the sample with a microtome. H&E, MT, Saf O and Picrosirius red staining were respectively conducted according to standard protocols.[56–59] The picrosirius staining was imaged and observed under polarization and bright-field microscopy. Immunohistochemical staining was conducted with primary antibodies of Col I, Col II, Col X, CD3 and CD68, respectively. For fluorescent imaging, the 35

slides of samples were first blocked in horse serum and then incubated with corresponding 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

primary antibody, followed by incubation with goat anti-rabbit IgG-FITC (Santa Cruz, USA) at 37°C for 1 hour; lastly, the cell nuclei were counterstained with DAPI before imaging and observing under a fluorescence microscope. For non-fluorescent imaging, the slides of samples were stained according to the standard protocol provided by Ultra Vision Quanto Detection System HRP DAB kit (Thermo Scientific).[60] Briefly, the sections were deparaffined and rehydrated, which was soaked in PBS for at least 5 mins before proceeding on. Pepsin was added to the sections for 20 mins under 37 °C to extract the targeted antigen and then washed by PBS. Subsequently, the sections were blocked by UltraVision hydrogen peroxide block under room temperature for 10 mins and washed by PBS. Next, the sections were blocked by protein block for 5 mins at room temperature. The blocking solution was shaken away without PBS wash. The primary antibody was added to the sections which were incubated for 1 hr at room temperature. From now on after each step, the slides were washed by PBS. The primary antibody amplifier quanto and horseradish peroxidase (HRP) polymer quanto were added in sequence each for 10 mins at room temperature. When the HRP polymer quanto was added, light was avoided. Next, the mixture of the 3,3'Diaminobenzidine (DAB) substrate and DAB chromogen at a ratio of 1 ml to 30 µL was added to the sections for at most 3 mins for all types of antigen examined in IHC to avoid overstaining, after which the slides were washed with distilled water. Finally, the slides were dehydrated and mounted before observation under bright field microscope. For the scoring of the histology slides, three experts have marked on the same set of histology slides blindly and independently. The scoring from all three experts have been averaged and statistics has been conducted based on the three experts scoring. Biochemical Assays: The samples were first frozen at −20 °C and then lyophilized for 24 h. The completely freeze-dried samples were digested overnight in papain solution consisting of 36

0.3 mg/mL 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

papain

dissolved

in

0.2 mM

dithiothreitol

mixed

with

0.1 mM

ethylenediaminetetraacetic acid disodium salt. DNA content was measured by the fluorometric Hoechst 33258 assay. GAG content was measured by 1,9-dimethylmethylene blue (DMMB) dye binding assay.[61] Total collagen content was quantified using Lhydroxyproline assay after hydrolysis in HCl (6M) overnight.[62] DNA microarray: The extraction of total RNA was conducted following TRIzol® protocol. Briefly, 50 mg of tissue sample was homogenized in TRIzol and incubated for 5 mins at room temperature in TRIzol® reagent before adding 0.2 mL of chloroform. The capped tube was shaken vigorously by hand for 15 secs and incubated for 2-3 mins at room temperature. Subsequently, the sample was centrifuged at 12,000 x g for 15 mins at 4 °C. After centrifugation, the aqueous phase of the sample was transferred to a new tube. Next, 0.5 mL of isopropanol was added to the aqueous phase and incubated at room temperature for 10 mins before centrifuging at 12,000 x g for 10 mins at 4 °C. Later, the supernatant was removed from the tube, leaving only the RNA pellet which is washed by 1 mL of 75% ethanol. The tube was vortexed briefly and then centrifuged at 7500 x g for 5 mins at 4 °C. The wash was discarded, and the RNA pellet was air dried for 5 mins. A microarray platform of Agilent SurePrint G3 Custom GE 8x60K 1 colour (Agilent, USA) was used for profiling the global gene expression. Total RNA (100ng) was labelled with Low Input Quick Amp Labelling Kit and purified by Qiagen RNeasy Kit (Qiagen, Singapore). Cyanine 3-CTP labelled cRNA (600ng) was hybridized onto the microarray platform for 17 h under 65°C at 10 rpm in Agilent hybridization oven. After hybridization, the microarray slide was washed in gene expression wash buffer. The hybridized microarray slide was scanned on Agilent High Resolution Microarray Scanner. The data were log-transformed and normalised via (the 75th) Percentile Shift Method using Agilent Feature Extraction software. All normalised data were then baseline-transformed to the median of all samples. 37

Biomechanical Test: A confined mechanical test was conducted on cartilage explants 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

(cylinder shape, 8 mm in diameter and 1.5 mm thick) harvested from the operation sites without attachment of subchondral bones. Young’s modulus of the harvested samples was measured using rheometer (Instron, USA). A constant, compressive crosshead moving was applied at the speed of 1mm/min on samples until the end-of-test criterion was reached at 80% of the specimen height. A stress-strain curve was plotted, from which the Young’s modulus was calculated through linearization of the elastic region of the curve. Statistical analysis: All data in this study were shown as mean ± standard deviation. One-way ANOVA statistical analysis with post hoc comparisons was used to analyse the data. p < 0 .05 was deemed to indicate statistical significance. The measurements were taken from distinct samples. Acknowledgement The authors would like to thank the immunologist from Singapore General Hospital, Dr. Jabed Iqubal, for (independent) inflammatory severity scoring. The authors would like to thank Drs. Zheng Yang, Yingnan Wu and Vinitha Denslin for (independently scoring) histological images. Funding: this work is financially supported by Start-up Grant for Professor (SGP 9380099 to Dong-An Wang) and SRG 7005212 (to Dong-An Wang), City University of Hong Kong, and Tier 2 Academic Research Fund (MOE2016T2-1-138(S) to Wang Dong-An), Singapore. Author Contributions X.L.N. was responsible for the conduct of all experiments, data collection and analyses, and manuscript preparation; P.F.H. and W.Z.Z contributed to in vitro experiment; Y.J.C. and Y.P. contributed to in vivo experiment and D.A.W. was responsible for the design of

38

experimentation, project supervision and coordination, data analyses, and manuscript 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

preparation. Data Availability The raw/processed data required to reproduce these findings cannot be shared at this time due to technical or time limitations. (The authors guarantee that they will be available for downloading by the time of publication.)

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*Declaration of Interest Statement

Declaration of interests ☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. ☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: