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3D printing electrospinning fiber-reinforced decellularized extracellular matrix for cartilage regeneration
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3D printing electrospinning fiber-reinforced decellularized extracellular matrix for cartilage regeneration Weiming Chena,c,1, Yong Xub,1, Yaqiang Lid,1, Litao Jiae, Xiumei Mof, , Gening Jiangb, , ⁎ Guangdong Zhoua,c,e, ⁎
⁎
a
Department of Plastic and Reconstructive Surgery, Shanghai Key Lab of Tissue Engineering, Shanghai 9th People’s Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China b Department of Thoracic Surgery, Shanghai Pulmonary Hospital, Tongji University School of Medicine, Shanghai, China c National Tissue Engineering Center of China, Shanghai, China d Department of Orthopaedic Surgery, Shanghai 9th People’s Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China e Research Institute of Plastic Surgery, Wei Fang Medical College, Shandong, China f College of Chemistry, Chemical Engineering and Biotechnology, Donghua University, Shanghai, China
HIGHLIGHTS
GRAPHICAL ABSTRACT
decellularized matrix (CDM) • Cartilage is processed into inks for 3D printing. scaffolds with controlled • CDM-based 3D shape and pore structure are obtained.
can improve the mechanical • Fibers property of 3D-printed CDM scaffolds. fiber-reinforced CDM scaf• 3D-printed folds exhibit elasticity in wet condition.
CDM scaffolds can • Fiber-reinforced repair articular cartilage defects in rabbits.
ARTICLE INFO
ABSTRACT
Keywords: 3D printing Cartilage decellularized matrix Electrospinning fiber 3D scaffold Tissue engineering
Cartilage decellularized matrix (CDM) is considered a promising biomaterial for fabricating cartilage tissue engineering scaffolds. An ideal CDM-based scaffold should possess customizable 3D shape for complex tissue regeneration and proper pore size for cell infiltration, as well as provide mechanical support for cell growth. 3D printing is an efficiently technique for preparing customizable 3D scaffolds, however, fabricating CDM-based 3D-printed scaffolds with customizable shapes, proper pore structure and satisfactory mechanical properties remains a challenge. In the current study, to achieve customizable CDM-based 3D scaffolds, CDM was successfully processed into inks suitable for 3D printing. Further, the poor mechanics of CDM-based scaffolds were significantly improved by adding electrospinning fiber into the CDM-based inks for 3D printing. Importantly, the 3D-printed electrospinning fiber-reinforced CDM-based scaffold presented good biocompatibility and can enhance repair articular cartilage defects in rabbits. The current study provides a novel strategy for printing electrospinning fiber-reinforced CDM-based scaffolds for tissue regeneration.
Corresponding authors at: Department of Plastic and Reconstructive Surgery, Shanghai Key Lab of Tissue Engineering, Shanghai 9th People’s Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China (G. Zhou). E-mail addresses: [email protected] (X. Mo), [email protected] (G. Jiang), [email protected] (G. Zhou). 1 These authors contributed equally to this work. ⁎
https://doi.org/10.1016/j.cej.2019.122986 Received 28 March 2019; Received in revised form 14 September 2019; Accepted 27 September 2019 1385-8947/ © 2019 Elsevier B.V. All rights reserved.
Please cite this article as: Weiming Chen, et al., Chemical Engineering Journal, https://doi.org/10.1016/j.cej.2019.122986
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1. Introduction
powder while maintaining the suitability for 3D printing and improving the mechanical properties of CDM-based scaffolds. To confirm this concept, in this study, dispersed electrospinning gelatin/poly (lactic-coglycolic acid) (PLGA) fibers were successfully prepared via electrospinning, dehydration, homogenizing, and evaporation drying. Adding dispersed electrospinning fiber to CDM-based inks did not influence the printability. Further, as expected, the incorporation of fibers not only enhanced the stiffness of the 3D-printed CDM-based scaffolds, but also improve their toughness. Moreover, 3D-printed electrospinning fiberreinforced CDM scaffolds (CDM-Fiber) presented good biocompatibility both in vitro and in vivo, as well as could enhance repair articular cartilage defects in rabbits.
Decellularized extracellular matrix (DECM) provides a physiological microenvironment to support cell survival and differentiation, as well as biochemical signals to modulate cell behavior; hence, it has aroused extensive interest in the field of tissue engineering [1]. CDM, as one kind of DECM, has been suggested as a popular candidate for preparing cartilage tissue engineering scaffolds owing to its constructive remodeling properties [2–4]. In previous research [3], total decellularized cartilage was successfully used for preparing cartilage scaffolds with tissue-specific 3D shapes and microarchitecture. However, the native cartilage extracellular matrix (ECM) is a dense connective tissue with a pore size of only a few nanometers, which evidently prevents cell infiltration [5,6]. Therefore, using advanced processing techniques, fabricating CDM-based scaffold suitable for cell seeding and infiltration is a popular approach for cartilage regeneration. To meet the requirement of tissue engineering scaffolds with appropriate porosity for cell seeding, CDMs have been successfully fabricated into many forms including sheets [7], hydrogels [8], and powder [9]. For example, CDM powder can be packed into molds and freeze-dried for preparing 3D porous scaffolds beneficial to cell ingrowth [9]. Although CDM-based scaffolds have led to the tremendous progress for first-generation engineered cartilage, fabricating CDMbased scaffolds with complex architecture and appropriate mechanical properties remains a challenge [10]. 3D printing has emerged as a versatile and efficiently technique to build ECM-mimicking 3D scaffolds with designed architecture and controlled pore structure. Currently, limited biomaterials can be used for preparing cartilage tissue engineering scaffolds by 3D printing, these can be basically divided into two groups: natural materials (such as collagen [11], gelatin [12], and alginate [13]) and synthetic materials (such as polycaprolactone (PCL) [14] and polylactic acid (PLA) [15]), However, in view of the complexity composition and structure of natural cartilage ECM, fabricating scaffolds that fully mimic the features of ECM is currently not possible using the above materials [1,16]. To address this unmet challenge, designing CDM-based scaffolds via 3D printing is an alternative strategy. Efforts have been made towards creating CDM (or DECM)-based inks for 3D printing. Notably, for the first time, Cho and co-worker used tissue-specific DECM bio-inks for 3D cell printing [10,16]. Recently, DECMs derived from cartilage [16], heart [16,17] adipose [16], liver [17,18], and skeletal muscle [17,19], tissues have been successfully processed into inks for preparing 3D-printed scaffolds, which present tremendous advantages in terms of tissue regeneration. However, owing to the soft nature and mechanical instability of CDM, current 3Dprinted CDM-based scaffolds present weak mechanical properties, which clearly limited the tissue regeneration application of 3D-printed scaffolds [10,20]. To improve the mechanical performance and maintain the stability of CDM-based scaffolds, a supportive PCL framework is always required [10,16]. But the disadvantages of this requirement are clear. 1) Preparing multi-material scaffold requires multiple nozzles (one for the CDM, the other for the PCL) with accurate alignment of each nozzle during the 3D printing; 2) The long-term degradation of PCL might be problematic. Therefore, developing a simple pathway to prepare 3D-printed CDM-based scaffolds and improving the mechanical properties has been a challenging and attractive direction. To fabricate 3D-printed CDM-based scaffolds for cartilage tissue engineering, in the current study, for the first time, CDM was processed into powder and blended with hyaluronic acid (HA, which is one of the main components of the cartilage matrix) solution as an ink, that is suitable for 3D printing and preparing 3D scaffolds (CDM-3DP). However, CDM-3DP suffers from weak mechanical properties. In natural articular cartilage tissue, collagen fibers have an important role in the mechanical properties [21,22]. Inspired by this, adding fibers into CDM-3DP could enhance the mechanical properties. To date, no studies have demonstrated that electrospinning fibers can be added to CDM
2. Materials and method 2.1. Preparation of CDM CDM was obtained from the cow scapular cartilage, which was purchased from a local slaughterhouse, the acellularization procedure was performed according to the previously literatures [23,24]. After complete cleaning and excision of fibrous connective tissue, the resulting cartilage was sliced. First, the sliced cartilage was incubated in a 1 M sodium hydroxide solution for 4 h at room temperature. Secondly, the cartilage matrix was freeze-dried and crushed into particle with a grinder. Thirdly, the cartilage particles were rinsed in distilled water with continuous shaking until the pH value of the phosphate buffer solution (PBS) became neutral. Subsequently, the cartilage particles were homogenized by high speed homogenizer. Finally, the CDM was freeze dried. 2.2. Preparation of short electrospinning gelatin/PLGA fibers Gelatin (Gel strength approximately 240 g Bloom, Aladdin) and PLGA (Mw: 8 × 104, Daigang Biomaterials) were separately dissolved in 1,1,1,3,3,3-hexafluoro-2-propanol; the concentration of gelatin and PLGA were 16% and 8%, respectively. The gelatin solution and PLGA solutions were mixed in a ratio of 10:3 (V/V). Then, gelatin/PLGA fiber membranes were fabricated by an electrospinning technique. The voltage was 16 KV, the flow rate was controlled at 3 mL/h, and the spinneret-collector distance was 15 cm. The gelatin/PLGA fiber membranes were then dehydrated at 180 °C for 60 min in a drying oven. The dehydrated fiber membranes were cut into small pieces and placed in tertbutanol. Using a homogenizer, fiber dispersions were obtained by homogenizing the fiber pieces for 20 min at 5000 rpm. Finally, the dispersed short electrospinning gelatin/PLGA fibers were dried by evaporation. 2.3. 3D printing CDM-based scaffold and fiber-based scaffold Four kinds of inks were prepared for printing different scaffolds: these were CDM-3DP, CDM-Fiber 25%, CDM-Fiber 50%, and Fiber-3DP, respectively (see Table 1). CDM-3DP: 0.7 g of HA was dissolved in 10 mL of deionized water for preparing a 7% HA solution. 3 g of CDM powder were placed into the 7% HA solution, the mixture was stirred well and formed a stable semifluid. CDM-Fiber 25% and CDM-Fiber 50%: 2.25 (or 1.5) g of CDM powder and 0.75 (or 1.5) g of fibers were placed into the 7% HA solution, the mixture was stirred as inks for CDM-Fiber 25% (or CDMFiber 50%). Fiber-3DP: 3 g of fibers were placed into the 7% HA solution, the mixture was stirred well and formed a stable semifluid. Scaffolds fabrication: Four kinds of rectangular scaffolds were fabricated by extruding different inks through nozzles with an inner diameter of 500 µm via a 3D plotting system. The moving speed of the plotting head was 0.5 mm s−1, the dosing speed was 0.002 mm s−1, and 2
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2.4.4. Cell viability on the 3D-printed scaffold The isolation and culture of chondrocytes from rabbit articular cartilage was performed according to previously described study [25]. In general, articular cartilage tissues were harvested from New Zealand white rabbits and digested with 0.15% type II collagenase (Gibco), the isolated chondrocytes were cultured in DMEM containing 10% FBS, 100 µg/mL streptomycin, and100 U/mL penicillin. Chondrocytes of the second generation were harvested for further experiments. Before cell seeding, all scaffolds were disinfected with ethylene oxide. 5 × 105 Chondrocytes were seeded into Fiber-3DP and CDM-Fiber 50% for in vitro culture. Cell seeding efficiency was calculated after 24 h of cell culture according to the formula [26]: cell seeding efficiency (%) = (total cell number − remaining cell number)/total cell number × 100%. The cell viability on the scaffolds was measured using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (Sigma-Aldrich, USA) as previous reported methods [27] after cell culture at 1, 5, and 9 days. The cell proliferation on the scaffolds was determined using a total DNA quantification assay (PicoGreen assay, Invitrogen, USA) after cell culture at 1, 5, and 9 days. The viability of the chondrocyte was also determined using Live & Dead cell viability assays (Invitrogen, USA) after 5 days of cell culture.
Table 1 The list of abbreviations. Number
Abbreviations
Full Names
1 2 3 4 5 6 7 8 9 10 11
CDM ECM DECM 3D PCL PLA PLGA HA PBS CDM-3DP CDM-Fiber 25%
12
CDM-Fiber 50%
13 14 15 16 17 18 19 20 21 22
Fiber-3DP EDC NHS SEM HE GAG Coll Ⅱ G′ G″ UV
Cartilage decellularized matrix Extracellular matrix Decellularized extracellular matrix Three-dimensional Polycaprolactone Polylactic acid Poly (lactic-co-glycolic acid) Hyaluronic acid Phosphate buffer solution 3D-printed CDM scaffold 3D-printed CDM and fiber scaffold (Fiber/CDM = 1/3, w/w) 3D-printed CDM and fiber scaffold (Fiber/CDM = 1/1, w/w) 3D-printed fiber scaffold 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide N-hydroxysuccinimide Scanning electron microscopy Hematoxylin and eosin Glycosaminoglycans Collagen II Storage modulus Loss modulus Ultraviolet
2.4.5. Cartilage regeneration in nude mice All experimental animals (nude mice and rabbit) were treated according to the standard guidelines approved by the Shanghai Jiao Tong University Ethics Committee. 200 µL chondrocytes with a concentration of 5.0 × 108 cells/mL were seeded evenly into Fiber-3DP and CDMFiber 50%, respectively. These cell-scaffold constructs were incubated for 4 h at 37 °C in a humidified atmosphere containing 5% CO2, and then further cultured for one week in vitro. Finally, the cell-scaffold constructs were implanted into the subcutaneous tissue of nude mice (n = 6 per group) for four weeks and eight weeks. Histological evaluation: using a paraffin sections method, cell-scaffold constructs from nude mice were fixed in 4% paraformaldehyde, embedded, and cut into tissues; then, the paraffin sections were stained with hematoxylin and eosin (HE) and Safranin-O. To evaluate the cartilage-specific phenotypes, type collagen II was detected by immunohistochemical staining of the tissue sections.
the strand spacing (the distance between the middle lines of the adjacent strands) was adjusted at 900 µm. Layer slicing was held constant at 380 µm. The strand angle of the orientation between subsequent layers was set at 90°. The 3D-printed scaffolds were freeze dried and crosslinked by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC)/N-hydroxysuccinimide (NHS) solution (5% EDC and 3% NHS in 95% ethanol) for 24 h. Finally, the 3D-printed scaffolds were freeze dried again. 2.4. Characterization 2.4.1. Morphology observation The morphologies of the CDM powder, electrospinning gelatin/ PLGA fiber membrane, short electrospinning gelatin/PLGA fibers, and four kinds of 3D-printed scaffolds (CDM-3DP, CDM-Fiber 25%, CDMFiber 50%, and Fiber-3DP) were observed by scanning electron microscopy (SEM, Hitachi TM-1000) at an accelerating voltage of 15 kV.
2.4.6. Articular cartilage regeneration in rabbits Healthy male New Zealand white rabbits weighing approximately 2.5 kg were randomly selected for in vivo study. Using trephine bur, an osteochondral defect (diameter: 3.5 mm, depth: 4 mm) was created in the trochlear groove of the leg. Then, the rabbits were randomly divided into three groups (n = 5): for the Fiber-3DP and CDM-Fiber 50% groups, defects were implanted; for the control group, the defects were untreated. At 12 weeks after surgery, the rabbits were sacrificed and histological specimens were harvested. Using a paraffin sections method, the histological specimens were fixed, decalcified, embedded in paraffin, cut into sections, then stained with HE for morphological evaluation and stained with Safranin O and fast green for Glycosaminoglycans (GAG) distribution evaluation. Immunohistochemical staining of the repaired cartilage tissue was performed to evaluate cartilage-specific phenotypes. The histological and immunohistochemical staining tissue sections were examined using a light microscope.
2.4.2. Rheological analysis The viscosity and rheological properties of the CDM ink (ink for preparing CDM-3DP) and CDM/Fiber ink (ink for preparing CDM-Fiber 50%) were tested to evaluate their printability. These experiments were measured on an Anton Paar MCR302 Rheometer with parallel-plate (25 mm diameter) geometry at 25 °C. Dynamic viscosity was performed at a 0.5 mm gap from 0.1 to 100 s−1. Dynamic rheology tests were performed to measure the frequency-dependent storage modulus (G′) and loss modulus (G″) of the inks at 1% strain, 1 Hz frequency, and a 0.5 mm gap for 300 s. 2.4.3. Mechanical properties Rectangular scaffolds (10 mm × 10 mm × 2.5 mm) were prepared for compressive strength analysis using a mechanical test machine (HY940FS, China). The compression strain-stress curves of CDM-3DP, CDMFiber 25%, CDM-Fiber 50%, and Fiber-3DP in a wet state were measured with 40% and 60% compression strain under a constant displacement rate of 5 mm/min. One hundred cycles loading-unloading tests were performed with 50% compression strain at a strain rate of 10 mm/min.
2.4.7. Statistical Analysis. All data were collected from at least three testes. Statistical analysis was performed by one-way ANOVA using Origin 8.0. Significant data were indicated with a “*”, the criteria for statistical significance was *p < 0.05. 3
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3. Results
however, all the strands of the CDM-Fiber 25% (Fig. 4b and f), CDMFiber 50% (Fig. 4c and g), and Fiber-3DP (Fig. 4d and h) present a fibrous and porous structure. The CDM powder and fibers seemed to have bonded and formed a uniform structure (Fig. 4f and g), which is different from the conventional structure of 3D-printed scaffolds [12,29] and probably suitable for enhancing nutrient metabolism during tissue regeneration.
3.1. Preparation of fiber-reinforced CDM scaffolds Fig. 1 displays the preparation process of electrospinning fiber-reinforced CDM-based 3D-printed scaffolds for cartilage regeneration and articular cartilage repair. To allow CDM to be squeezed out from needle and meet the requirements for inks, a dense cartilage matrix was processed into CDM powder (Fig. 2a) through a series of processes, including cutting, decellularization, crushing, homogenizing, and freeze drying. Typically, CDM powders present a sheet structure (Fig. 2b) at the micron scale. For obtaining one-dimensional fibers, electrospinning gelatin/PLGA fiber membrane was innovatively processed into dry dispersed fibers by homogenizing and rapid evaporation (Fig. 2c). SEM images conform that the fibers were evenly dispersed (Fig. 2d). Then, the CDM powder was mixed with the gelatin/PLGA fibers, and HA solution to form a stable colloid (Fig. 2e) as a 3D printing ink. After 3D printing, freeze drying, and chemical crosslinking, stable and elastic fiber-reinforced CDM-based scaffold (Fig. 2f) was successfully prepared.
3.4. Mechanical properties of scaffolds Owning the appropriate mechanical properties is one of essential requirements for tissue engineering scaffolds. As indicated in Fig. 5a, the CDM-3DP and CDM-Fiber 25% were easily broken and deformed by compressing, however, the CDM-Fiber 50% presented fine elasticity in the wet state and could retain its initial shape under a compressing and releasing cycle. Another compressing and releasing cycle test results further proved that CDM-3DP presented weak mechanical performance (Fig. 5b), and that the CDM-Fiber 25% exhibited poor elasticity (Fig. 5c). However, CDM-Fiber 50% (Fig. 5d) and Fiber-3DP (Fig. 3e) presented highly nonlinear and closed compressive stress-strain curves, indicating acceptable compressive elasticity and the ability to bear 60% compressive strain. Furthermore, CDM-Fiber 50% presented higher compressive strength than CDM-3DP and CDM-Fiber 25%, as indicated by the compressive stress-strain curves (Fig. 5b-e) and Young’s modulus analysis (Fig. 5f). After 100 loading-unloading cycle compressions (60% strain), CDMFiber 50% still retain the ability to recover its original shape (Fig. 5g). Further, compared with CDM-3DP and CDM-Fiber 25%, CDM-Fiber 50% better preserved the maximum stress value (12.11 Kpa, Fig. 5h) with minimum stress loss (15.08%, Fig. 5i) after 100 cycles of compression at 60% strain. However, both CDM-3DP and CDM-Fiber 25% demonstrated significantly decreased maximum stress values (0.92 Kpa and 7.71 Kpa, respectively) with clear stress loss (82.70% and 38.62%, respectively). These results indicated that the electrospinning fibers not only significantly increased the mechanical strength of the CDM-based 3D-printed scaffold, but also provides a CDM-based scaffold that was elastic. CDM-Fiber 50% possessed the appropriate mechanical properties and was therefore chosen as the optimal scaffold for subsequent study.
3.2. Rheological analysis To enable 3D printing, CDM-based inks must exhibit a relatively low elastic shear modulus under high shear stress to allow them to flow stably passing through the deposition nozzle. As indicated in Fig. 3a, both CDM and CDM/Fiber ink exhibited the shear-thinning behavior throughout the entire shear rate range analyzed. The shear viscosity decreased with increasing shear rates, which is required for a continuous flow of printable ink [28]. As indicated in Fig. 3b, both CDM ink and CDM/Fiber ink exhibited elastic behavior, the storage modulus (G′) of both CDM ink and CDM/Fiber ink were greater than the loss modulus (G″), which enables the stable flow of ink in the extrusion process [28]. 3.3. SEM of scaffolds The microstructure of the four kinds of 3D printed scaffolds was observed via SEM (Fig. 4). As indicated in Fig. 4a, the surface strands of the CDM-3DP present a sheet and rough structure (Fig. 4a and e);
Fig. 1. Schematic illustration of electrospinning fiber-reinforced CDM-based 3D-printed scaffold for cartilage regeneration. 4
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Fig. 2. Materials and preparation of fiber-reinforced CDM scaffold. (a) Optical image of CDM powder. (b) SEM image of CDM powder. (c) Dispersed gelatin/PLGA fibers before (in tert-butanol) and after evaporation drying. (d) SEM image of dispersed gelatin/PLGA fibers. I Colloid consisting of CDM powder, gelatin/PLGA fibers and HA solution as inks. (f) 3D-printed fiber-reinforced CDM scaffold (CDM-Fiber 50%).
3.5. In vitro study
adhesion and proliferation.
The biocompatibility of CDM-based 3D-printed scaffolds was explored in vitro. At 24 h after cell seeding, the CDM-Fiber 50% groups indicated significantly greater cell-seeding efficiency than the Fiber3DP groups (Fig. 6a). MTT assay after 1, 5, and 9 days was shown in Fig. 6b. The cell viability absorbance values increased from day 1 to day 9 for two scaffold groups. It was found that cell viability on CDM-Fiber 50% was significantly higher than that on Fiber-3DP. The DNA content of the CDM-Fiber 50% groups also greater than the Fiber-3DP groups after 9 days of cell culture (Fig. 6c), indicating that the CDM-based scaffold provided a more favorable platform for cell proliferation. From the fluorescence micrograph (Fig. 6d-g), chondrocytes were better distributed on the strands of the CDM-Fiber 50% than Fiber-3DP; a large quantity of living cells (green) and virtually no dead cells (red) could be observed in the CDM-Fiber 50% group. In vitro experiment proved that CDM-Fiber 50% had a good biocompatibility for cell
3.6. Cartilage regeneration in nude mice Despite the fact that CDM-based 3D-printed scaffolds demonstrated the potential for cell growth in vitro, the question of whether the scaffolds were suitable for cartilage regeneration in vivo remained. The feasibility of cartilage regeneration in vivo was explored by implanting the cell-scaffold construct into subcutaneous tissue of nude mice (Fig. 7a). After eight weeks of in vivo implantation, both the Fiber-3DP and CDM-Fiber 50% group formed white cartilage-like tissues (Fig. 7b and c); however, the cell-scaffold constructs from the CDM-Fiber 50% group retained their original square shape (Fig. 7c), which was more regular than the regenerated tissue from the Fiber-3DP group (Fig. 7b). Moreover, the regenerated tissue from the Fiber-3DP group still existed channels and pores, but the cell-scaffold construct from the CDM-Fiber 50% group formed a dense and uniform tissue. Histological
Fig. 3. Rheological behavior of the inks (CDM and CDM/Fiber). (a) The viscosity of inks at 25 °C. (b) Dynamic modulus at varying frequency at 25 °C.
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Fig. 4. SEM images of 3D printed scaffolds. SEM images of (a, e) CDM, (b, f) CDM-Fiber 25%, (c, g) CDM-Fiber 50%, and (d, h) Fiber-3DP.
Fig. 5. Compressive mechanical properties of 3D-printed scaffolds (in wet state). (a) Photographs of the CDM-3DP, CDM-Fiber 25%, and CDM-Fiber 50% under a compressing and releasing cycle (compressive strain = 60%). Compressive stress-strain curves under compressing and releasing cycles (compressive strain = 40% and 60%) of (b) CDM-3DP, (c) CDM-Fiber 25%, (d) CDM-Fiber 50%, and I Fiber-3DP. (f) Compressive Young’s modulus of four kinds of scaffolds. The compressive Young’s modulus is from the slope of the stress-strain curve at a strain range of 0%–40% (*p < 0.05, n = 4). (g) Compressive stress-strain curves of CDM-Fiber 50% under 100 cycles in the compressive test at 60% strain. The history of (h) maximum stress and (i) stress loss as a function of compressive test cycles (100 cycles).
examination further revealed that the specimens in both the Fiber-3DP (Fig. 7d-i) and CDM-Fiber 50% (Fig. 7j-o) groups presented cartilagelike tissue, a cartilage lacuna-typical cartilage structure gradually
formed with increased in vivo culture time. The inner regenerated tissue in the Fiber-3DP specimen was loose; however, a connect and dense cartilage lacuna could be observed in the CDM-Fiber 50% specimen. 6
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Fig. 6. Cell-seeding efficiency, cell viability, and cell proliferation. (a) Cell-seeding efficiency of Fiber-3DP and CDM-Fiber 50% after 24 h cell seeding. (b) Chondrocyte viability of Fiber-3DP and CDM-Fiber 50% with the MTT assay and (c) cell proliferation with the PicoGreen assay after 1, 5, and 9 days. Fluorescence micrographs of chondrocytes cultured on (d, f) Fiber-3DP and (e, g) CDM-Fiber 50% at 5 days (live and dead cells are dyed green and red, respectively). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
3.7. Articular cartilage regeneration in rabbits
the good production of collagen II and the formation of regenerated cartilage tissue. These results demonstrated that CDM-fiber 50% could promote the regeneration and remodeling of a cartilage defect in rabbits.
Cartilage regeneration in situ, especially in an animal model, is the most direct evidence for predicting the potential of future biomedical application. In the current study, pure scaffolds were implanted into rabbit articular cartilage defects to evaluate the feasibility of cartilage repair in situ. After 12 weeks of implantation, from the gross appearance of the cartilage tissues, a clear unrepaired defect could be observed in the Non-treated group (Fig. 8a), and a partially filled defect in the Fiber-3DP group (Fig. 8b). However, completely filling of the defect was observed in the CDM-Fiber 50% group (Fig. 8c). In the Non-treated group, there remained a clear boundary between the defect and normal tissue (Fig. 8a and Fig. S1), the defect was filled with thin fibrous tissue as indicated in the HE staining (Fig. 8d) and virtually no staining (red) with Safranin O-fast green (Fig. 8g) in the joint surface, indicating no GAG deposition. In the Fiber-3DP group, the cartilage defect was not filled overall (Fig. 8b and Fig. S2); histological staining revealed that a thin regenerated cartilage tissue formed on the surface of defect (Fig. 8e and h). However, in the CDM-Fiber 50% group, white and uniform cartilage-like tissue with a flat surface could be observed in the defect area (Fig. 8c and Fig. S3). Numerous new chondrocytes emerged on the surface of the defect, histological staining revealed that dense regenerated cartilage tissue formed, which was well integrated with the original cartilage tissue (Fig. 8f, i, and Fig. S3). Immunohistochemical analysis revealed limited deposition of type collagen II in the Non-treated (Fig. 8j and Fig. S1) and Fiber-3DP groups (Fig. 8k and Fig. S2). Significant deposition of type collagen II was observed in the CDM-fiber 50% group (Fig. 8l and Fig. S3), indicating
4. Discussion CDM has become a favorable biomimetic scaffold sources, and has an important role for tissue regeneration [1]. However, to meet the requirements of complex tissue regeneration, fabricating CDM-based scaffolds with a controllable 3D architecture and the appropriate mechanical properties still remains a challenge. The successfully 3D printing of CDM is a significant milestone for the controllable fabrication of CDM-based scaffolds [16]. However, the soft nature of CDM and the weak mechanical properties of the current 3D-printed CDM scaffolds clearly limit their future clinical application. In this study, CDM scaffolds with controllable 3D architecture and pore size were successfully fabricated by using a 3D printing technique. The mechanical properties of the 3D-printed CDM scaffolds were clearly enhanced with the introduction of electrospinning fibers. The fiber-reinforced CDM scaffolds possessed advantages for tissue regeneration, such as fibrous surface structure and good elasticity in a wet state. Most importantly, fiber-reinforced CDM scaffolds could significantly promote cartilage regeneration in vivo. The first challenge is preparing the CDM-based inks suitable for 3D printing. In the present study, CDM powder and HA solution were successfully processed into inks for fabricating CDM-3DP. However, the poor mechanical strength and brittleness of CDM-3DP severely limited 7
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Fig. 7. Cartilage regeneration in nude mice. (a) The cell-scaffold construct was implanted into subcutaneous tissue of nude mice. Gross view of regenerated cartilage at 8 weeks in nude mice in (b) Fiber-3DP group and (c) CDM-Fiber 50% group. Histological analysis of regenerated cartilage in (d-i) Fiber-3DP group and (j-o) CDMFiber 50% group at four weeks (d-f, j-l) and eight weeks (g-i, m-o) in vivo. Sections are stained with Safranin-O (d, g, j, and m) and HE (e, h, k, and n) to evaluate ECM deposition and structure of regenerated cartilage. F, i, l, and o were taken from e, h, k, and n at higher magnification, respectively.
its application for tissue engineering. To overcome the weak mechanical properties of CDM-3DP, for the first time, dispersed electrospinning gelatin/PLGA fibers were added into CDM-based inks for the preparation of CDM and fiber-composited scaffolds. Interestingly, the
incorporation of electrospinning gelatin/PLGA fibers not only enhanced the stiffness of the CDM-based scaffolds, but also improved their toughness. Actually, in natural articular cartilage tissue, collagen fibers have the major role in mechanical performance [21]. The provided 3DP 8
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Fig. 8. Articular cartilage repair in rabbits. Macroscopic images of the cartilage joints from Non-treated, Fiber-3DP, and CDM-Fiber 50% groups at 12 weeks after surgery. Histological and immunohistochemical analysis of cartilage defect area from (d, g, and j) Non-treated group, (e, h, and k) Fiber-3DP group, and (f, i, and l) CDM-Fiber 50% group at 12 weeks after surgery, stained with HE (d-f) and Safranin O-fast green (j-i), and type collagen II (j-l).
exhibited good mechanical properties, which could be related to the following reasons. Firstly, amino and carboxyl groups formed a chemical bond on the CDM, HA, and gelatin fiber after crosslinking [30,31]; the formed stable network significantly enhanced the mechanical strength. Secondly, for electrospinning fibers, as with reinforced steel bars in armored concrete, increasing the fiber content could improve the mechanical properties of the scaffold. Moreover, electrospinning fibers not only improve mechanical properties of CDMbased scaffold, but also cause the strands of the 3D-printed scaffold to present a fibrous and porous structure. It has been proven that a fibrous structured scaffold possesses advantages for tissue regeneration, such as enhancing cell adhesion and nutrient metabolism [32,33]. 3D printing electrospinning fiber-reinforced CDM-based inks is a significant development for fabricating customizable CDM scaffolds with specific outer shape, controlled inner structure, and proper mechanical properties; however, the question of whether the present scaffold could achieve satisfactory tissue regeneration was the main concern. Today, because of its limited capacity for self-repair, the process of cartilage repair remains a clinical problem [34,35]. CDM is a promising biomaterial for preparing scaffolds to repair injured cartilage. In the current study, the results both in vitro and in vivo indicated that CDM-based 3D-printed scaffolds were favorable for chondrocyte growth and cartilage regeneration. Because CDM provides a satisfactory microenvironment for chondrocyte adhesive and proliferation, CDMbased 3D-printed scaffold (CDM-Fiber 50%) presented considerably more cell seeding efficiency and cell proliferation rate than non-CDM scaffolds (Fiber-3DP), indicating the excellent biocompatibility of CDM-
based scaffold. The controlled pore size and excellent biocompatibility of CDMbased scaffolds were beneficial for cell infiltration and proliferation, allowing cell-scaffold constructs to form uniform and dense cartilage tissue in nude mice subcutaneously. Further, by 3D printing, scaffold of complex shape can be fabricated to be used for specific tissue regeneration; the satisfactory mechanical properties of the scaffolds avoid shape changes and collapses of the cell-scaffold during cartilage regeneration. Most importantly, cell-free CDM-based 3D-printed scaffold could also repair a damaged articular cartilage in rabbits. The CDMFiber 50% group demonstrated that thicker cartilage and significant deposition of type collagen II formed, indicating that CDM-Fiber 50% is a promising candidate for regeneration and remodeling of the cartilage defects. Containing the native component of the cartilage matrix-CDM could be the main reason why CDM-Fiber 50% scaffolds present a superior cartilage repairing effect than non-CDM scaffold (Fiber-3DP). Moreover, owing to the fibrous and interconnected internal structures, growth factor and mesenchymal stem cells from the bone marrow could infiltrate into the scaffolds, which is beneficial to cartilage repair. Recently, 3D bioprinting has aroused more and more attention in tissue engineering. In order to meet the requirements of bioprinting, CDM need to be fabricated into ultraviolet (UV) or chemical crosslinking gelation. UV is known to have a damaging impact on a cell’s nuclear DNA [36]. Photoinitiators or chemical crosslinkers direct contacted with cells in the 3D bio-printed tissues may be cytotoxic. CDM has also been processed into temperature-induced gelation, but the gelatin formed slowly and was unstable [16]. However, in this study, 9
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3D printing scaffolds have more advantages than 3D bio-printed tissues. For example, chemical crosslinkers can be easily removed from 3D printed scaffolds before cell seeding, so that the cell activity is not influenced by chemical agents; Electrospinning fibers can significantly improve the mechanical properties of CDM-based 3D printed scaffold; Compared with 3D bio-printed tissues, 3D printed scaffolds are more convenient for sterilization, storage, and transportation with greater potential for productization. In addition, the stability of product should be concerned to make CDM-based product available for future clinical application. For example, the main compositions of CDM are collagen II and aggrecan, but the product processing may influence collagen II or aggrecan content of CDM. For standardized CDM production, some factors should be well controlled. First, the species, origin and age of animals should be kept as consistent as possible. Second, the production personnel and equipment need to be well controlled. Standards should also be set to ensure the consistency of the composition or structure of different batches of production.
[7] [8] [9] [10] [11] [12] [13]
[14]
5. Conclusions In the current study, by combining a series of processing techniques and chemical treatment, 3D-printed CDM-based scaffolds with customizable shape and controlled inner structure were successfully fabricated. For the first time, electrospinning gelatin/PLGA fibers were dispersed and processed into dry short fibers, serving as a reinforcement to overcome the soft nature and mechanical instability of 3D-printed CDM scaffolds. CDM-Fiber 50% presented several advantages for tissue engineering, such as fibrous structure, good elasticity (in a wet state), and excellent biocompatibility. Moreover, CDM-Fiber 50% could significantly repair cartilage defects in rabbits. The current study provides a promising cartilage regeneration scaffold and develops a novel strategy for printing electrospinning fiber-reinforced CDM-based scaffolds.
[15]
Acknowledgements
[21]
This work was supported by the National Key Research and Development Program of China (2017YFC1103900), the China Postdoctoral Science Foundation funded project (2017M621495), the National Natural Science Foundation of China (81571823, 81570089), the Key Research and Development Program of Shandong Province (2016GGB14002), the Shanghai Committee of Science and Technology (15DZ1941600), and the Program for Shanghai Outstanding Medical Academic Leader.
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Appendix A. Supplementary data
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Supplementary data to this article can be found online at https:// doi.org/10.1016/j.cej.2019.122986.
[27]
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Contents lists available at ScienceDirect
Chemical Engineering Journal journal homepage: www.elsevier.com/locate/cej
3D printing electrospinning fiber-reinforced decellularized extracellular matrix for cartilage regeneration Weiming Chena,c,1, Yong Xub,1, Yaqiang Lid,1, Litao Jiae, Xiumei Mof, , Gening Jiangb, , ⁎ Guangdong Zhoua,c,e, ⁎
⁎
a
Department of Plastic and Reconstructive Surgery, Shanghai Key Lab of Tissue Engineering, Shanghai 9th People’s Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China b Department of Thoracic Surgery, Shanghai Pulmonary Hospital, Tongji University School of Medicine, Shanghai, China c National Tissue Engineering Center of China, Shanghai, China d Department of Orthopaedic Surgery, Shanghai 9th People’s Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China e Research Institute of Plastic Surgery, Wei Fang Medical College, Shandong, China f College of Chemistry, Chemical Engineering and Biotechnology, Donghua University, Shanghai, China
HIGHLIGHTS
GRAPHICAL ABSTRACT
decellularized matrix (CDM) • Cartilage is processed into inks for 3D printing. scaffolds with controlled • CDM-based 3D shape and pore structure are obtained.
can improve the mechanical • Fibers property of 3D-printed CDM scaffolds. fiber-reinforced CDM scaf• 3D-printed folds exhibit elasticity in wet condition.
CDM scaffolds can • Fiber-reinforced repair articular cartilage defects in rabbits.
ARTICLE INFO
ABSTRACT
Keywords: 3D printing Cartilage decellularized matrix Electrospinning fiber 3D scaffold Tissue engineering
Cartilage decellularized matrix (CDM) is considered a promising biomaterial for fabricating cartilage tissue engineering scaffolds. An ideal CDM-based scaffold should possess customizable 3D shape for complex tissue regeneration and proper pore size for cell infiltration, as well as provide mechanical support for cell growth. 3D printing is an efficiently technique for preparing customizable 3D scaffolds, however, fabricating CDM-based 3D-printed scaffolds with customizable shapes, proper pore structure and satisfactory mechanical properties remains a challenge. In the current study, to achieve customizable CDM-based 3D scaffolds, CDM was successfully processed into inks suitable for 3D printing. Further, the poor mechanics of CDM-based scaffolds were significantly improved by adding electrospinning fiber into the CDM-based inks for 3D printing. Importantly, the 3D-printed electrospinning fiber-reinforced CDM-based scaffold presented good biocompatibility and can enhance repair articular cartilage defects in rabbits. The current study provides a novel strategy for printing electrospinning fiber-reinforced CDM-based scaffolds for tissue regeneration.
Corresponding authors at: Department of Plastic and Reconstructive Surgery, Shanghai Key Lab of Tissue Engineering, Shanghai 9th People’s Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China (G. Zhou). E-mail addresses: [email protected] (X. Mo), [email protected] (G. Jiang), [email protected] (G. Zhou). 1 These authors contributed equally to this work. ⁎
https://doi.org/10.1016/j.cej.2019.122986 Received 28 March 2019; Received in revised form 14 September 2019; Accepted 27 September 2019 1385-8947/ © 2019 Elsevier B.V. All rights reserved.
Please cite this article as: Weiming Chen, et al., Chemical Engineering Journal, https://doi.org/10.1016/j.cej.2019.122986
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1. Introduction
powder while maintaining the suitability for 3D printing and improving the mechanical properties of CDM-based scaffolds. To confirm this concept, in this study, dispersed electrospinning gelatin/poly (lactic-coglycolic acid) (PLGA) fibers were successfully prepared via electrospinning, dehydration, homogenizing, and evaporation drying. Adding dispersed electrospinning fiber to CDM-based inks did not influence the printability. Further, as expected, the incorporation of fibers not only enhanced the stiffness of the 3D-printed CDM-based scaffolds, but also improve their toughness. Moreover, 3D-printed electrospinning fiberreinforced CDM scaffolds (CDM-Fiber) presented good biocompatibility both in vitro and in vivo, as well as could enhance repair articular cartilage defects in rabbits.
Decellularized extracellular matrix (DECM) provides a physiological microenvironment to support cell survival and differentiation, as well as biochemical signals to modulate cell behavior; hence, it has aroused extensive interest in the field of tissue engineering [1]. CDM, as one kind of DECM, has been suggested as a popular candidate for preparing cartilage tissue engineering scaffolds owing to its constructive remodeling properties [2–4]. In previous research [3], total decellularized cartilage was successfully used for preparing cartilage scaffolds with tissue-specific 3D shapes and microarchitecture. However, the native cartilage extracellular matrix (ECM) is a dense connective tissue with a pore size of only a few nanometers, which evidently prevents cell infiltration [5,6]. Therefore, using advanced processing techniques, fabricating CDM-based scaffold suitable for cell seeding and infiltration is a popular approach for cartilage regeneration. To meet the requirement of tissue engineering scaffolds with appropriate porosity for cell seeding, CDMs have been successfully fabricated into many forms including sheets [7], hydrogels [8], and powder [9]. For example, CDM powder can be packed into molds and freeze-dried for preparing 3D porous scaffolds beneficial to cell ingrowth [9]. Although CDM-based scaffolds have led to the tremendous progress for first-generation engineered cartilage, fabricating CDMbased scaffolds with complex architecture and appropriate mechanical properties remains a challenge [10]. 3D printing has emerged as a versatile and efficiently technique to build ECM-mimicking 3D scaffolds with designed architecture and controlled pore structure. Currently, limited biomaterials can be used for preparing cartilage tissue engineering scaffolds by 3D printing, these can be basically divided into two groups: natural materials (such as collagen [11], gelatin [12], and alginate [13]) and synthetic materials (such as polycaprolactone (PCL) [14] and polylactic acid (PLA) [15]), However, in view of the complexity composition and structure of natural cartilage ECM, fabricating scaffolds that fully mimic the features of ECM is currently not possible using the above materials [1,16]. To address this unmet challenge, designing CDM-based scaffolds via 3D printing is an alternative strategy. Efforts have been made towards creating CDM (or DECM)-based inks for 3D printing. Notably, for the first time, Cho and co-worker used tissue-specific DECM bio-inks for 3D cell printing [10,16]. Recently, DECMs derived from cartilage [16], heart [16,17] adipose [16], liver [17,18], and skeletal muscle [17,19], tissues have been successfully processed into inks for preparing 3D-printed scaffolds, which present tremendous advantages in terms of tissue regeneration. However, owing to the soft nature and mechanical instability of CDM, current 3Dprinted CDM-based scaffolds present weak mechanical properties, which clearly limited the tissue regeneration application of 3D-printed scaffolds [10,20]. To improve the mechanical performance and maintain the stability of CDM-based scaffolds, a supportive PCL framework is always required [10,16]. But the disadvantages of this requirement are clear. 1) Preparing multi-material scaffold requires multiple nozzles (one for the CDM, the other for the PCL) with accurate alignment of each nozzle during the 3D printing; 2) The long-term degradation of PCL might be problematic. Therefore, developing a simple pathway to prepare 3D-printed CDM-based scaffolds and improving the mechanical properties has been a challenging and attractive direction. To fabricate 3D-printed CDM-based scaffolds for cartilage tissue engineering, in the current study, for the first time, CDM was processed into powder and blended with hyaluronic acid (HA, which is one of the main components of the cartilage matrix) solution as an ink, that is suitable for 3D printing and preparing 3D scaffolds (CDM-3DP). However, CDM-3DP suffers from weak mechanical properties. In natural articular cartilage tissue, collagen fibers have an important role in the mechanical properties [21,22]. Inspired by this, adding fibers into CDM-3DP could enhance the mechanical properties. To date, no studies have demonstrated that electrospinning fibers can be added to CDM
2. Materials and method 2.1. Preparation of CDM CDM was obtained from the cow scapular cartilage, which was purchased from a local slaughterhouse, the acellularization procedure was performed according to the previously literatures [23,24]. After complete cleaning and excision of fibrous connective tissue, the resulting cartilage was sliced. First, the sliced cartilage was incubated in a 1 M sodium hydroxide solution for 4 h at room temperature. Secondly, the cartilage matrix was freeze-dried and crushed into particle with a grinder. Thirdly, the cartilage particles were rinsed in distilled water with continuous shaking until the pH value of the phosphate buffer solution (PBS) became neutral. Subsequently, the cartilage particles were homogenized by high speed homogenizer. Finally, the CDM was freeze dried. 2.2. Preparation of short electrospinning gelatin/PLGA fibers Gelatin (Gel strength approximately 240 g Bloom, Aladdin) and PLGA (Mw: 8 × 104, Daigang Biomaterials) were separately dissolved in 1,1,1,3,3,3-hexafluoro-2-propanol; the concentration of gelatin and PLGA were 16% and 8%, respectively. The gelatin solution and PLGA solutions were mixed in a ratio of 10:3 (V/V). Then, gelatin/PLGA fiber membranes were fabricated by an electrospinning technique. The voltage was 16 KV, the flow rate was controlled at 3 mL/h, and the spinneret-collector distance was 15 cm. The gelatin/PLGA fiber membranes were then dehydrated at 180 °C for 60 min in a drying oven. The dehydrated fiber membranes were cut into small pieces and placed in tertbutanol. Using a homogenizer, fiber dispersions were obtained by homogenizing the fiber pieces for 20 min at 5000 rpm. Finally, the dispersed short electrospinning gelatin/PLGA fibers were dried by evaporation. 2.3. 3D printing CDM-based scaffold and fiber-based scaffold Four kinds of inks were prepared for printing different scaffolds: these were CDM-3DP, CDM-Fiber 25%, CDM-Fiber 50%, and Fiber-3DP, respectively (see Table 1). CDM-3DP: 0.7 g of HA was dissolved in 10 mL of deionized water for preparing a 7% HA solution. 3 g of CDM powder were placed into the 7% HA solution, the mixture was stirred well and formed a stable semifluid. CDM-Fiber 25% and CDM-Fiber 50%: 2.25 (or 1.5) g of CDM powder and 0.75 (or 1.5) g of fibers were placed into the 7% HA solution, the mixture was stirred as inks for CDM-Fiber 25% (or CDMFiber 50%). Fiber-3DP: 3 g of fibers were placed into the 7% HA solution, the mixture was stirred well and formed a stable semifluid. Scaffolds fabrication: Four kinds of rectangular scaffolds were fabricated by extruding different inks through nozzles with an inner diameter of 500 µm via a 3D plotting system. The moving speed of the plotting head was 0.5 mm s−1, the dosing speed was 0.002 mm s−1, and 2
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2.4.4. Cell viability on the 3D-printed scaffold The isolation and culture of chondrocytes from rabbit articular cartilage was performed according to previously described study [25]. In general, articular cartilage tissues were harvested from New Zealand white rabbits and digested with 0.15% type II collagenase (Gibco), the isolated chondrocytes were cultured in DMEM containing 10% FBS, 100 µg/mL streptomycin, and100 U/mL penicillin. Chondrocytes of the second generation were harvested for further experiments. Before cell seeding, all scaffolds were disinfected with ethylene oxide. 5 × 105 Chondrocytes were seeded into Fiber-3DP and CDM-Fiber 50% for in vitro culture. Cell seeding efficiency was calculated after 24 h of cell culture according to the formula [26]: cell seeding efficiency (%) = (total cell number − remaining cell number)/total cell number × 100%. The cell viability on the scaffolds was measured using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (Sigma-Aldrich, USA) as previous reported methods [27] after cell culture at 1, 5, and 9 days. The cell proliferation on the scaffolds was determined using a total DNA quantification assay (PicoGreen assay, Invitrogen, USA) after cell culture at 1, 5, and 9 days. The viability of the chondrocyte was also determined using Live & Dead cell viability assays (Invitrogen, USA) after 5 days of cell culture.
Table 1 The list of abbreviations. Number
Abbreviations
Full Names
1 2 3 4 5 6 7 8 9 10 11
CDM ECM DECM 3D PCL PLA PLGA HA PBS CDM-3DP CDM-Fiber 25%
12
CDM-Fiber 50%
13 14 15 16 17 18 19 20 21 22
Fiber-3DP EDC NHS SEM HE GAG Coll Ⅱ G′ G″ UV
Cartilage decellularized matrix Extracellular matrix Decellularized extracellular matrix Three-dimensional Polycaprolactone Polylactic acid Poly (lactic-co-glycolic acid) Hyaluronic acid Phosphate buffer solution 3D-printed CDM scaffold 3D-printed CDM and fiber scaffold (Fiber/CDM = 1/3, w/w) 3D-printed CDM and fiber scaffold (Fiber/CDM = 1/1, w/w) 3D-printed fiber scaffold 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide N-hydroxysuccinimide Scanning electron microscopy Hematoxylin and eosin Glycosaminoglycans Collagen II Storage modulus Loss modulus Ultraviolet
2.4.5. Cartilage regeneration in nude mice All experimental animals (nude mice and rabbit) were treated according to the standard guidelines approved by the Shanghai Jiao Tong University Ethics Committee. 200 µL chondrocytes with a concentration of 5.0 × 108 cells/mL were seeded evenly into Fiber-3DP and CDMFiber 50%, respectively. These cell-scaffold constructs were incubated for 4 h at 37 °C in a humidified atmosphere containing 5% CO2, and then further cultured for one week in vitro. Finally, the cell-scaffold constructs were implanted into the subcutaneous tissue of nude mice (n = 6 per group) for four weeks and eight weeks. Histological evaluation: using a paraffin sections method, cell-scaffold constructs from nude mice were fixed in 4% paraformaldehyde, embedded, and cut into tissues; then, the paraffin sections were stained with hematoxylin and eosin (HE) and Safranin-O. To evaluate the cartilage-specific phenotypes, type collagen II was detected by immunohistochemical staining of the tissue sections.
the strand spacing (the distance between the middle lines of the adjacent strands) was adjusted at 900 µm. Layer slicing was held constant at 380 µm. The strand angle of the orientation between subsequent layers was set at 90°. The 3D-printed scaffolds were freeze dried and crosslinked by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC)/N-hydroxysuccinimide (NHS) solution (5% EDC and 3% NHS in 95% ethanol) for 24 h. Finally, the 3D-printed scaffolds were freeze dried again. 2.4. Characterization 2.4.1. Morphology observation The morphologies of the CDM powder, electrospinning gelatin/ PLGA fiber membrane, short electrospinning gelatin/PLGA fibers, and four kinds of 3D-printed scaffolds (CDM-3DP, CDM-Fiber 25%, CDMFiber 50%, and Fiber-3DP) were observed by scanning electron microscopy (SEM, Hitachi TM-1000) at an accelerating voltage of 15 kV.
2.4.6. Articular cartilage regeneration in rabbits Healthy male New Zealand white rabbits weighing approximately 2.5 kg were randomly selected for in vivo study. Using trephine bur, an osteochondral defect (diameter: 3.5 mm, depth: 4 mm) was created in the trochlear groove of the leg. Then, the rabbits were randomly divided into three groups (n = 5): for the Fiber-3DP and CDM-Fiber 50% groups, defects were implanted; for the control group, the defects were untreated. At 12 weeks after surgery, the rabbits were sacrificed and histological specimens were harvested. Using a paraffin sections method, the histological specimens were fixed, decalcified, embedded in paraffin, cut into sections, then stained with HE for morphological evaluation and stained with Safranin O and fast green for Glycosaminoglycans (GAG) distribution evaluation. Immunohistochemical staining of the repaired cartilage tissue was performed to evaluate cartilage-specific phenotypes. The histological and immunohistochemical staining tissue sections were examined using a light microscope.
2.4.2. Rheological analysis The viscosity and rheological properties of the CDM ink (ink for preparing CDM-3DP) and CDM/Fiber ink (ink for preparing CDM-Fiber 50%) were tested to evaluate their printability. These experiments were measured on an Anton Paar MCR302 Rheometer with parallel-plate (25 mm diameter) geometry at 25 °C. Dynamic viscosity was performed at a 0.5 mm gap from 0.1 to 100 s−1. Dynamic rheology tests were performed to measure the frequency-dependent storage modulus (G′) and loss modulus (G″) of the inks at 1% strain, 1 Hz frequency, and a 0.5 mm gap for 300 s. 2.4.3. Mechanical properties Rectangular scaffolds (10 mm × 10 mm × 2.5 mm) were prepared for compressive strength analysis using a mechanical test machine (HY940FS, China). The compression strain-stress curves of CDM-3DP, CDMFiber 25%, CDM-Fiber 50%, and Fiber-3DP in a wet state were measured with 40% and 60% compression strain under a constant displacement rate of 5 mm/min. One hundred cycles loading-unloading tests were performed with 50% compression strain at a strain rate of 10 mm/min.
2.4.7. Statistical Analysis. All data were collected from at least three testes. Statistical analysis was performed by one-way ANOVA using Origin 8.0. Significant data were indicated with a “*”, the criteria for statistical significance was *p < 0.05. 3
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3. Results
however, all the strands of the CDM-Fiber 25% (Fig. 4b and f), CDMFiber 50% (Fig. 4c and g), and Fiber-3DP (Fig. 4d and h) present a fibrous and porous structure. The CDM powder and fibers seemed to have bonded and formed a uniform structure (Fig. 4f and g), which is different from the conventional structure of 3D-printed scaffolds [12,29] and probably suitable for enhancing nutrient metabolism during tissue regeneration.
3.1. Preparation of fiber-reinforced CDM scaffolds Fig. 1 displays the preparation process of electrospinning fiber-reinforced CDM-based 3D-printed scaffolds for cartilage regeneration and articular cartilage repair. To allow CDM to be squeezed out from needle and meet the requirements for inks, a dense cartilage matrix was processed into CDM powder (Fig. 2a) through a series of processes, including cutting, decellularization, crushing, homogenizing, and freeze drying. Typically, CDM powders present a sheet structure (Fig. 2b) at the micron scale. For obtaining one-dimensional fibers, electrospinning gelatin/PLGA fiber membrane was innovatively processed into dry dispersed fibers by homogenizing and rapid evaporation (Fig. 2c). SEM images conform that the fibers were evenly dispersed (Fig. 2d). Then, the CDM powder was mixed with the gelatin/PLGA fibers, and HA solution to form a stable colloid (Fig. 2e) as a 3D printing ink. After 3D printing, freeze drying, and chemical crosslinking, stable and elastic fiber-reinforced CDM-based scaffold (Fig. 2f) was successfully prepared.
3.4. Mechanical properties of scaffolds Owning the appropriate mechanical properties is one of essential requirements for tissue engineering scaffolds. As indicated in Fig. 5a, the CDM-3DP and CDM-Fiber 25% were easily broken and deformed by compressing, however, the CDM-Fiber 50% presented fine elasticity in the wet state and could retain its initial shape under a compressing and releasing cycle. Another compressing and releasing cycle test results further proved that CDM-3DP presented weak mechanical performance (Fig. 5b), and that the CDM-Fiber 25% exhibited poor elasticity (Fig. 5c). However, CDM-Fiber 50% (Fig. 5d) and Fiber-3DP (Fig. 3e) presented highly nonlinear and closed compressive stress-strain curves, indicating acceptable compressive elasticity and the ability to bear 60% compressive strain. Furthermore, CDM-Fiber 50% presented higher compressive strength than CDM-3DP and CDM-Fiber 25%, as indicated by the compressive stress-strain curves (Fig. 5b-e) and Young’s modulus analysis (Fig. 5f). After 100 loading-unloading cycle compressions (60% strain), CDMFiber 50% still retain the ability to recover its original shape (Fig. 5g). Further, compared with CDM-3DP and CDM-Fiber 25%, CDM-Fiber 50% better preserved the maximum stress value (12.11 Kpa, Fig. 5h) with minimum stress loss (15.08%, Fig. 5i) after 100 cycles of compression at 60% strain. However, both CDM-3DP and CDM-Fiber 25% demonstrated significantly decreased maximum stress values (0.92 Kpa and 7.71 Kpa, respectively) with clear stress loss (82.70% and 38.62%, respectively). These results indicated that the electrospinning fibers not only significantly increased the mechanical strength of the CDM-based 3D-printed scaffold, but also provides a CDM-based scaffold that was elastic. CDM-Fiber 50% possessed the appropriate mechanical properties and was therefore chosen as the optimal scaffold for subsequent study.
3.2. Rheological analysis To enable 3D printing, CDM-based inks must exhibit a relatively low elastic shear modulus under high shear stress to allow them to flow stably passing through the deposition nozzle. As indicated in Fig. 3a, both CDM and CDM/Fiber ink exhibited the shear-thinning behavior throughout the entire shear rate range analyzed. The shear viscosity decreased with increasing shear rates, which is required for a continuous flow of printable ink [28]. As indicated in Fig. 3b, both CDM ink and CDM/Fiber ink exhibited elastic behavior, the storage modulus (G′) of both CDM ink and CDM/Fiber ink were greater than the loss modulus (G″), which enables the stable flow of ink in the extrusion process [28]. 3.3. SEM of scaffolds The microstructure of the four kinds of 3D printed scaffolds was observed via SEM (Fig. 4). As indicated in Fig. 4a, the surface strands of the CDM-3DP present a sheet and rough structure (Fig. 4a and e);
Fig. 1. Schematic illustration of electrospinning fiber-reinforced CDM-based 3D-printed scaffold for cartilage regeneration. 4
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Fig. 2. Materials and preparation of fiber-reinforced CDM scaffold. (a) Optical image of CDM powder. (b) SEM image of CDM powder. (c) Dispersed gelatin/PLGA fibers before (in tert-butanol) and after evaporation drying. (d) SEM image of dispersed gelatin/PLGA fibers. I Colloid consisting of CDM powder, gelatin/PLGA fibers and HA solution as inks. (f) 3D-printed fiber-reinforced CDM scaffold (CDM-Fiber 50%).
3.5. In vitro study
adhesion and proliferation.
The biocompatibility of CDM-based 3D-printed scaffolds was explored in vitro. At 24 h after cell seeding, the CDM-Fiber 50% groups indicated significantly greater cell-seeding efficiency than the Fiber3DP groups (Fig. 6a). MTT assay after 1, 5, and 9 days was shown in Fig. 6b. The cell viability absorbance values increased from day 1 to day 9 for two scaffold groups. It was found that cell viability on CDM-Fiber 50% was significantly higher than that on Fiber-3DP. The DNA content of the CDM-Fiber 50% groups also greater than the Fiber-3DP groups after 9 days of cell culture (Fig. 6c), indicating that the CDM-based scaffold provided a more favorable platform for cell proliferation. From the fluorescence micrograph (Fig. 6d-g), chondrocytes were better distributed on the strands of the CDM-Fiber 50% than Fiber-3DP; a large quantity of living cells (green) and virtually no dead cells (red) could be observed in the CDM-Fiber 50% group. In vitro experiment proved that CDM-Fiber 50% had a good biocompatibility for cell
3.6. Cartilage regeneration in nude mice Despite the fact that CDM-based 3D-printed scaffolds demonstrated the potential for cell growth in vitro, the question of whether the scaffolds were suitable for cartilage regeneration in vivo remained. The feasibility of cartilage regeneration in vivo was explored by implanting the cell-scaffold construct into subcutaneous tissue of nude mice (Fig. 7a). After eight weeks of in vivo implantation, both the Fiber-3DP and CDM-Fiber 50% group formed white cartilage-like tissues (Fig. 7b and c); however, the cell-scaffold constructs from the CDM-Fiber 50% group retained their original square shape (Fig. 7c), which was more regular than the regenerated tissue from the Fiber-3DP group (Fig. 7b). Moreover, the regenerated tissue from the Fiber-3DP group still existed channels and pores, but the cell-scaffold construct from the CDM-Fiber 50% group formed a dense and uniform tissue. Histological
Fig. 3. Rheological behavior of the inks (CDM and CDM/Fiber). (a) The viscosity of inks at 25 °C. (b) Dynamic modulus at varying frequency at 25 °C.
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Fig. 4. SEM images of 3D printed scaffolds. SEM images of (a, e) CDM, (b, f) CDM-Fiber 25%, (c, g) CDM-Fiber 50%, and (d, h) Fiber-3DP.
Fig. 5. Compressive mechanical properties of 3D-printed scaffolds (in wet state). (a) Photographs of the CDM-3DP, CDM-Fiber 25%, and CDM-Fiber 50% under a compressing and releasing cycle (compressive strain = 60%). Compressive stress-strain curves under compressing and releasing cycles (compressive strain = 40% and 60%) of (b) CDM-3DP, (c) CDM-Fiber 25%, (d) CDM-Fiber 50%, and I Fiber-3DP. (f) Compressive Young’s modulus of four kinds of scaffolds. The compressive Young’s modulus is from the slope of the stress-strain curve at a strain range of 0%–40% (*p < 0.05, n = 4). (g) Compressive stress-strain curves of CDM-Fiber 50% under 100 cycles in the compressive test at 60% strain. The history of (h) maximum stress and (i) stress loss as a function of compressive test cycles (100 cycles).
examination further revealed that the specimens in both the Fiber-3DP (Fig. 7d-i) and CDM-Fiber 50% (Fig. 7j-o) groups presented cartilagelike tissue, a cartilage lacuna-typical cartilage structure gradually
formed with increased in vivo culture time. The inner regenerated tissue in the Fiber-3DP specimen was loose; however, a connect and dense cartilage lacuna could be observed in the CDM-Fiber 50% specimen. 6
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Fig. 6. Cell-seeding efficiency, cell viability, and cell proliferation. (a) Cell-seeding efficiency of Fiber-3DP and CDM-Fiber 50% after 24 h cell seeding. (b) Chondrocyte viability of Fiber-3DP and CDM-Fiber 50% with the MTT assay and (c) cell proliferation with the PicoGreen assay after 1, 5, and 9 days. Fluorescence micrographs of chondrocytes cultured on (d, f) Fiber-3DP and (e, g) CDM-Fiber 50% at 5 days (live and dead cells are dyed green and red, respectively). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
3.7. Articular cartilage regeneration in rabbits
the good production of collagen II and the formation of regenerated cartilage tissue. These results demonstrated that CDM-fiber 50% could promote the regeneration and remodeling of a cartilage defect in rabbits.
Cartilage regeneration in situ, especially in an animal model, is the most direct evidence for predicting the potential of future biomedical application. In the current study, pure scaffolds were implanted into rabbit articular cartilage defects to evaluate the feasibility of cartilage repair in situ. After 12 weeks of implantation, from the gross appearance of the cartilage tissues, a clear unrepaired defect could be observed in the Non-treated group (Fig. 8a), and a partially filled defect in the Fiber-3DP group (Fig. 8b). However, completely filling of the defect was observed in the CDM-Fiber 50% group (Fig. 8c). In the Non-treated group, there remained a clear boundary between the defect and normal tissue (Fig. 8a and Fig. S1), the defect was filled with thin fibrous tissue as indicated in the HE staining (Fig. 8d) and virtually no staining (red) with Safranin O-fast green (Fig. 8g) in the joint surface, indicating no GAG deposition. In the Fiber-3DP group, the cartilage defect was not filled overall (Fig. 8b and Fig. S2); histological staining revealed that a thin regenerated cartilage tissue formed on the surface of defect (Fig. 8e and h). However, in the CDM-Fiber 50% group, white and uniform cartilage-like tissue with a flat surface could be observed in the defect area (Fig. 8c and Fig. S3). Numerous new chondrocytes emerged on the surface of the defect, histological staining revealed that dense regenerated cartilage tissue formed, which was well integrated with the original cartilage tissue (Fig. 8f, i, and Fig. S3). Immunohistochemical analysis revealed limited deposition of type collagen II in the Non-treated (Fig. 8j and Fig. S1) and Fiber-3DP groups (Fig. 8k and Fig. S2). Significant deposition of type collagen II was observed in the CDM-fiber 50% group (Fig. 8l and Fig. S3), indicating
4. Discussion CDM has become a favorable biomimetic scaffold sources, and has an important role for tissue regeneration [1]. However, to meet the requirements of complex tissue regeneration, fabricating CDM-based scaffolds with a controllable 3D architecture and the appropriate mechanical properties still remains a challenge. The successfully 3D printing of CDM is a significant milestone for the controllable fabrication of CDM-based scaffolds [16]. However, the soft nature of CDM and the weak mechanical properties of the current 3D-printed CDM scaffolds clearly limit their future clinical application. In this study, CDM scaffolds with controllable 3D architecture and pore size were successfully fabricated by using a 3D printing technique. The mechanical properties of the 3D-printed CDM scaffolds were clearly enhanced with the introduction of electrospinning fibers. The fiber-reinforced CDM scaffolds possessed advantages for tissue regeneration, such as fibrous surface structure and good elasticity in a wet state. Most importantly, fiber-reinforced CDM scaffolds could significantly promote cartilage regeneration in vivo. The first challenge is preparing the CDM-based inks suitable for 3D printing. In the present study, CDM powder and HA solution were successfully processed into inks for fabricating CDM-3DP. However, the poor mechanical strength and brittleness of CDM-3DP severely limited 7
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Fig. 7. Cartilage regeneration in nude mice. (a) The cell-scaffold construct was implanted into subcutaneous tissue of nude mice. Gross view of regenerated cartilage at 8 weeks in nude mice in (b) Fiber-3DP group and (c) CDM-Fiber 50% group. Histological analysis of regenerated cartilage in (d-i) Fiber-3DP group and (j-o) CDMFiber 50% group at four weeks (d-f, j-l) and eight weeks (g-i, m-o) in vivo. Sections are stained with Safranin-O (d, g, j, and m) and HE (e, h, k, and n) to evaluate ECM deposition and structure of regenerated cartilage. F, i, l, and o were taken from e, h, k, and n at higher magnification, respectively.
its application for tissue engineering. To overcome the weak mechanical properties of CDM-3DP, for the first time, dispersed electrospinning gelatin/PLGA fibers were added into CDM-based inks for the preparation of CDM and fiber-composited scaffolds. Interestingly, the
incorporation of electrospinning gelatin/PLGA fibers not only enhanced the stiffness of the CDM-based scaffolds, but also improved their toughness. Actually, in natural articular cartilage tissue, collagen fibers have the major role in mechanical performance [21]. The provided 3DP 8
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Fig. 8. Articular cartilage repair in rabbits. Macroscopic images of the cartilage joints from Non-treated, Fiber-3DP, and CDM-Fiber 50% groups at 12 weeks after surgery. Histological and immunohistochemical analysis of cartilage defect area from (d, g, and j) Non-treated group, (e, h, and k) Fiber-3DP group, and (f, i, and l) CDM-Fiber 50% group at 12 weeks after surgery, stained with HE (d-f) and Safranin O-fast green (j-i), and type collagen II (j-l).
exhibited good mechanical properties, which could be related to the following reasons. Firstly, amino and carboxyl groups formed a chemical bond on the CDM, HA, and gelatin fiber after crosslinking [30,31]; the formed stable network significantly enhanced the mechanical strength. Secondly, for electrospinning fibers, as with reinforced steel bars in armored concrete, increasing the fiber content could improve the mechanical properties of the scaffold. Moreover, electrospinning fibers not only improve mechanical properties of CDMbased scaffold, but also cause the strands of the 3D-printed scaffold to present a fibrous and porous structure. It has been proven that a fibrous structured scaffold possesses advantages for tissue regeneration, such as enhancing cell adhesion and nutrient metabolism [32,33]. 3D printing electrospinning fiber-reinforced CDM-based inks is a significant development for fabricating customizable CDM scaffolds with specific outer shape, controlled inner structure, and proper mechanical properties; however, the question of whether the present scaffold could achieve satisfactory tissue regeneration was the main concern. Today, because of its limited capacity for self-repair, the process of cartilage repair remains a clinical problem [34,35]. CDM is a promising biomaterial for preparing scaffolds to repair injured cartilage. In the current study, the results both in vitro and in vivo indicated that CDM-based 3D-printed scaffolds were favorable for chondrocyte growth and cartilage regeneration. Because CDM provides a satisfactory microenvironment for chondrocyte adhesive and proliferation, CDMbased 3D-printed scaffold (CDM-Fiber 50%) presented considerably more cell seeding efficiency and cell proliferation rate than non-CDM scaffolds (Fiber-3DP), indicating the excellent biocompatibility of CDM-
based scaffold. The controlled pore size and excellent biocompatibility of CDMbased scaffolds were beneficial for cell infiltration and proliferation, allowing cell-scaffold constructs to form uniform and dense cartilage tissue in nude mice subcutaneously. Further, by 3D printing, scaffold of complex shape can be fabricated to be used for specific tissue regeneration; the satisfactory mechanical properties of the scaffolds avoid shape changes and collapses of the cell-scaffold during cartilage regeneration. Most importantly, cell-free CDM-based 3D-printed scaffold could also repair a damaged articular cartilage in rabbits. The CDMFiber 50% group demonstrated that thicker cartilage and significant deposition of type collagen II formed, indicating that CDM-Fiber 50% is a promising candidate for regeneration and remodeling of the cartilage defects. Containing the native component of the cartilage matrix-CDM could be the main reason why CDM-Fiber 50% scaffolds present a superior cartilage repairing effect than non-CDM scaffold (Fiber-3DP). Moreover, owing to the fibrous and interconnected internal structures, growth factor and mesenchymal stem cells from the bone marrow could infiltrate into the scaffolds, which is beneficial to cartilage repair. Recently, 3D bioprinting has aroused more and more attention in tissue engineering. In order to meet the requirements of bioprinting, CDM need to be fabricated into ultraviolet (UV) or chemical crosslinking gelation. UV is known to have a damaging impact on a cell’s nuclear DNA [36]. Photoinitiators or chemical crosslinkers direct contacted with cells in the 3D bio-printed tissues may be cytotoxic. CDM has also been processed into temperature-induced gelation, but the gelatin formed slowly and was unstable [16]. However, in this study, 9
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3D printing scaffolds have more advantages than 3D bio-printed tissues. For example, chemical crosslinkers can be easily removed from 3D printed scaffolds before cell seeding, so that the cell activity is not influenced by chemical agents; Electrospinning fibers can significantly improve the mechanical properties of CDM-based 3D printed scaffold; Compared with 3D bio-printed tissues, 3D printed scaffolds are more convenient for sterilization, storage, and transportation with greater potential for productization. In addition, the stability of product should be concerned to make CDM-based product available for future clinical application. For example, the main compositions of CDM are collagen II and aggrecan, but the product processing may influence collagen II or aggrecan content of CDM. For standardized CDM production, some factors should be well controlled. First, the species, origin and age of animals should be kept as consistent as possible. Second, the production personnel and equipment need to be well controlled. Standards should also be set to ensure the consistency of the composition or structure of different batches of production.
[7] [8] [9] [10] [11] [12] [13]
[14]
5. Conclusions In the current study, by combining a series of processing techniques and chemical treatment, 3D-printed CDM-based scaffolds with customizable shape and controlled inner structure were successfully fabricated. For the first time, electrospinning gelatin/PLGA fibers were dispersed and processed into dry short fibers, serving as a reinforcement to overcome the soft nature and mechanical instability of 3D-printed CDM scaffolds. CDM-Fiber 50% presented several advantages for tissue engineering, such as fibrous structure, good elasticity (in a wet state), and excellent biocompatibility. Moreover, CDM-Fiber 50% could significantly repair cartilage defects in rabbits. The current study provides a promising cartilage regeneration scaffold and develops a novel strategy for printing electrospinning fiber-reinforced CDM-based scaffolds.
[15]
Acknowledgements
[21]
This work was supported by the National Key Research and Development Program of China (2017YFC1103900), the China Postdoctoral Science Foundation funded project (2017M621495), the National Natural Science Foundation of China (81571823, 81570089), the Key Research and Development Program of Shandong Province (2016GGB14002), the Shanghai Committee of Science and Technology (15DZ1941600), and the Program for Shanghai Outstanding Medical Academic Leader.
[22]
Appendix A. Supplementary data
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Supplementary data to this article can be found online at https:// doi.org/10.1016/j.cej.2019.122986.
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[16] [17]
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[23] [24]
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