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Oriented cartilage extracellular matrix-derived scaffold for cartilage tissue engineering
Journal of Bioscience and Bioengineering VOL. 113 No. 5, 647 – 653, 2012 www.elsevier.com/locate/jbiosc
Oriented cartilage extracellular matrix-derived scaffold for cartilage tissue engineering Shuaijun Jia, 1, † Lie Liu, 2 Weimin Pan, 3 Guolin Meng, 1, † Chunguang Duan, 1, † Laquan Zhang, 4 Zhuo Xiong, 4 and Jian Liu 1,⁎ Institute of Orthopaedics, Xijing Hospital, The Fourth Military Medical University, Xi'an 710032, China, 1 Department of Orthopaedics, Baoji Central Hospital, Baoji 721008, China, 2 Department of Human Movement Studies, Xi'an Physical Education University, Xi'an 710032, China, 3 and Department of Mechanical Engineering, Tsinghua University, Beijing 100084, China 4 Received 14 November 2011; accepted 15 December 2011 Available online 21 January 2012
The structure of a cartilage scaffold is required to mimic native articular cartilage, which has an oriented structure associated with its mechanical function. In this study, an oriented cartilage extracellular matrix (ECM)-derived scaffold was fabricated composed of microtubules arranged in parallel in vertical section. The mechanical property was higher than that of a typical non-oriented scaffold (p b 0.05). Oriented and non-oriented scaffolds were seeded with chondrogenic-induced bone mesenchymal stem cells and cell-scaffold constructs were implanted subcutaneously in the dorsa of nude mice. At 4 weeks, all samples stained positive for safranin O, toluidine blue, and collagen type II, but negative for collagen type I. Oriented-structure constructs contained numerous parallel giant bundles of densely packed collagen fibers with chondrocyte-like cells aligned along the fibers. Total DNA, glycosaminoglycans and collagen contents increased with time and these values were similar in the two groups. Compared with the native articular cartilage, the Young's modulus of the tissue-engineered (TE) cartilage reached 42.9%, 23.0% in oriented and non-oriented scaffolds respectively, at 4 weeks. These results indicate that oriented ECMderived scaffolds enhance the biomechanical property of TE cartilage and thus represent a promising approach to cartilage tissue engineering. © 2011, The Society for Biotechnology, Japan. All rights reserved. [Key words: Oriented scaffold; Extracellular matrix; Bone mesenchymal stem cells; Cartilage tissue engineering; Biomechanical property]
Articular cartilage provides near frictionless motion between articulating surfaces and protects the bones of synovial joints from being damaged when subjected to impact and load bearing (1). Anatomically and functionally, articular cartilage consists of four zones: the superficial, transitional, deep and calcified zones and generally exhibits columnar orientation of cells and anisotropic direction of collagen fibers that run vertically from the tidemark towards the joint surface (2,3). This aligned collagen fiber network is believed to be a critical factor required for the biomechanical properties of articular cartilage (4). Although articular cartilage is highly susceptible to damage, it has limited intrinsic regeneration and self-repair capacity due to its innate avascular nature and low cell-to-matrix ratio (5). Tissue engineering has proved to be the most promising alternative therapy that combines cells, scaffolds and environmental factors for repair of articular cartilage defects (6). The scaffold serves as an extracellular matrix (ECM) providing three-dimensional conformation and orientation to cells and has a vital role in cartilage tissue engineering (7). ⁎ Corresponding author. Tel.: + 86 29 8477 1013; fax: + 86 29 8477 1012. E-mail address: [email protected] (J. Liu). † These authors contributed equally to this work.
The availability of suitable scaffold material is a major component of tissue engineering strategies. Therefore, identification of an optimal matrix for cartilage tissue engineering has led to the development and application of numerous scaffolds derived from both natural and synthetic materials (6). Previous studies have demonstrated that scaffolds derived from naturally decellularized cartilage ECM are devoid of allogeneic or xenogeneic cellular antigens while preserving the majority of ECM structural and functional proteins (8,9). Furthermore, it has been shown that cartilage ECM-derived scaffolds preserve the main constituents of native cartilage including glycosaminoglycan (GAG) and collagen type II, which possess good biocompatibility and provide a natural microenvironment for the support of bone mesenchymal stem cell (BMSC) attachment, proliferation and differentiation into chondrocytes (10). Therefore it is hypothesized that cartilage ECM is a suitable material for use as a cartilage scaffold. More importantly, the designed structure of cartilage scaffolds should mimic native articular cartilage, which has an oriented structure associated with its mechanical and physiological functions (11). Furthermore, the oriented architecture of this scaffold should possess superior biomechanical properties and protect cells from early critical compression prior to secretion of abundant ECM. Therefore, scaffold with a biomimetic-oriented architecture is an important requirement for tissue-engineered (TE) cartilage.
1389-1723/$ - see front matter © 2011, The Society for Biotechnology, Japan. All rights reserved. doi:10.1016/j.jbiosc.2011.12.009
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The source of cells is an additional factor critical to the development of tissue engineering strategies. Although targeted regenerative tissue is formed from autologous chondrocytes, the supply of these cells is very limited. BMSCs, which can be safely harvested by bone marrow biopsy, have been reported as an alternative cell source due to their characteristic capacity for rapid proliferation and multiple differentiation potential (12). Extensive research has demonstrated BMSCs differentiation into chondrocytes by application of transforming growth factor-β (TGF-β) family members in various 3-D scaffolds (13). Therefore, BMSCs represent promising candidates for use in cartilage tissue engineering. The purpose of this study was to fabricate an oriented cartilage ECM-derived scaffold combined with chondrogenic-induced BMSCs for enhancement of the biomechanical property of TE cartilage in vivo. The resultant constructs were cultured for 2 weeks and 4 weeks at subcutaneous sites in nude mice, after which biochemical, histological and biomechanical properties were evaluated. MATERIALS AND METHODS Preparation of the scaffolds Bovine articular cartilage ECM was obtained by decellularization using previously described methods (8). Oriented scaffolds were prepared using a modified temperature gradient-guided thermal-induced phase separation (TIPS) technique followed by freeze-drying (Fig. 1). Briefly, a suspension of cartilage ECM (3%, w/v) was prepared and infused into a cylindrical mould with an inner diameter of 8 mm and a height of 10 mm. The top of the mould was open so that the upper surface of the slurry was maintained at room temperature. The mould was then placed vertically onto a metal plate equilibrated to − 196°C and frozen in liquid nitrogen. This technique allowed orientation of the structure of the scaffold by solvent crystallization under a unidirectional temperature gradient. Frozen samples were lyophilized in a freeze-dryer (Alpha 2–4, Chaist, Germany) for 48 h to form an oriented microtubule structure. Scaffolds were then removed from the mould and cut into cylinders (diameter, 5 mm; thickness, 2 mm) using biopsy punch and scalpel. Typical non-oriented scaffolds were fabricated by simple freeze-drying. Briefly, the suspension was frozen for consecutive periods of 1 h at − 20°C and − 80°C and lyophilized for 48 h. All scaffolds were cross-linked by treatment with genipin solution (0.5%, w/v) (Wako Pure Chemical Industries, Osaka, Japan) for 48 h at room temperature (14) and sterilized by exposure to 20 kGy 60Co radiation prior to cell culture and experimentation. Scaffolds characterization The oriented and non-oriented scaffold samples were sputter-coated with gold at 40 mA for scanning electron microscope analysis (SEM; S-3400N, Hitachi, Tokyo, Japan) at an accelerating voltage of 5.0 kV. The porosity of the two types of scaffold was estimated according to previously reported liquid displacement methods (11). Mechanical property of scaffolds Mechanical property of oriented and non-oriented scaffold was determined by measurement of Young's modulus according to previously reported methods (15). The compressive strength of scaffolds along the longitudinal direction was measured by using a universal material testing machine (Shimadzu, Kyoto, Japan) at a crosshead speed of 1.0 mm/min. Young's modulus was obtained from the initial slopes of the stress–strain curves. Results are expressed as the mean of six independent measurements. Isolation and chondrogenic induction of BMSCs In this study, all experimental procedures involving animals were approved by the Institutional Animal Review Committee of Fourth Military Medical University. BMSCs were obtained from the tibia of New Zealand White rabbits as described previously (16) and cultured to 80% confluence. Adherent cells were released with trypsin (Sigma-Aldrich, St. Louis, MO,
USA) and subcultured in chondrogenic induction medium to induce differentiation of BMSCs. The induction medium contained high glucose Dulbecco's modified Eagle's medium (DMEM; Invitrogen, Camarillo, CA, USA), 10% fetal bovine serum (FBS; HyClone, Utah, USA), 10 ng/ml TGF-β3 (PeproTech, Rocky Hill, NJ, USA), 1% ITS+ premix (BD, Franklin Lakes, NJ, USA), 10− 7 M dexamethasone (Sigma), 50 μg/ml ascorbic acid (Sigma), 1 mM sodium pyruvate (Sigma), 4 mM proline (Sigma), and 1% antibiotics (100 U/ml penicillin, 100 mg/ml streptomycin, Sigma) (8,17). The medium was replaced twice per week and the cells were harvested at day 21 for seeding. Proliferation and morphology of chondrogenic BMSCs on scaffolds Chondrogenic-induced BMSCs were resuspended in culture medium (cell density, 1 × 107 cells/ml) and seeded onto scaffolds (5 × 105 cells/scaffold). Cells were allowed to attach to the scaffolds for 2 h at 37°C prior to the addition of fresh induction medium (1 ml). Cell-scaffold constructs were further incubated at 37°C in an atmosphere of 5% CO2 and 95% humidity and the medium changed twice a week. Cell viability and proliferation within the scaffolds were analyzed by 3-(4,5-Dimethyl-2-thiazolyl)-2, 5diphenyl-2H-tetrazolium bromide (MTT; Sigma) assay (14). Adhesion properties, morphological characteristics and the distribution of the cells within scaffolds were analyzed by SEM (S-3400N, Hitachi, Tokyo, Japan). In vivo implantation Following in vitro culture (1 week), cell-scaffold constructs were implanted subcutaneously in the dorsa of nude mice (aged 4 weeks) (8). Mice were sacrificed at 2 weeks and 4 weeks, and samples of TE cartilage were harvested. Histological and immunohistochemical analysis of TE cartilage Implants were fixed overnight with 4% paraformaldehyde and then embedded in paraffin at 4 weeks after implantation. Sections (thickness, 5 μm) were stained for proteoglycans with safranin O and toluidine blue according to standard protocols (8). For immunohistochemical analysis, sections (thickness, 20 μm) were deparaffinized with xylene, rehydrated in graded ethanol series and washed with PBS. Collagen was detected immunohistochemically using monoclonal antibodies against collagen types I (ab90395; Abcam, Cambridge, MA, USA) and II (433120; Invitrogen, Camarillo, CA, USA) as previously described (18). Rabbit articular cartilage was prepared by the same method described for TE cartilage for use as positive controls. Biochemical analysis of TE cartilage Samples of TE cartilage were harvested at 2 weeks and 4 weeks after implantation for biochemical evaluation and digested by lysis buffer containing 125 μg/ml papain (Sigma), 10 mM cysteine hydrochloride (Sigma), 100 mM sodium phosphate and 10 mM EDTA, for 24 h at 60°C (19). The DNA content was evaluated using the Quant-iT™ PicoGreen® dsDNA Assay Kit (Molecular Probes, Eugene, OR, USA) (20). Fluorescence intensity was measured using a Vicam Series-4 Fluorimeter (Vicam, Milford, MA, USA). A standard curve for the analysis was generated using bacteriophage lambda DNA supplied with the kit. Total GAG content was quantified using a Blyscan™ Sulfated Glycosaminoglycan Assay kit (Biocolor, Carrickfergus, Northern Ireland, UK) and a spectrophotometer (Bio-Tek Instruments, Winooski, VT, USA) at 656 nm, based on dimethylmethylene blue binding (18). A standard curve was established using bovine cartilage chondroitin sulfate (Sigma) (21). Aliquots of the papain-digested sample solution were incubated at 48°C for 48 h in 0.05 M acetic acid containing 0.5 M NaCl (pH 3) and 10 mg/ml pepsin (Sigma). Total collagen was determined by the Sircol™ Soluble Collagen Assay kit (Biocolor) with absorbance at 555 nm measured using a spectrophotometer (BioTek). A standard curve was generated using L-hydroxyproline (Sigma) (22). Six samples from each TE cartilage group were measured. Native rabbit articular cartilage was used as a control. Biomechanical assay of TE cartilage Biomechanical property of the two types of TE cartilage developed during the 4 weeks of implantation was determined by measurement of Young's modulus (E), which was measured by using a universal material testing machine (Shimadzu, Kyoto, Japan). For unconfined compression tests (UCC) (20,23), samples were transferred to a stainless steel dish containing PBS equilibrated to room temperature. The height of each specimen was determined at a compressive force threshold of 0.05 N. Five progressive strain loadings at 4% of the original cartilage height were performed with a test velocity of 0.01 mm/s. Each loading
FIG. 1. Schematic diagram showing the fabrication of oriented scaffolds by thermal-induced phase separation technique: (A) scheme of the cylindrical mould (with an inner diameter of 8 mm and a height of 10 mm) filled cartilage ECM, (B) scheme of oriented scaffold (diameter of the microtubule: 105.2 ± 20.3 μm). TH represents high temperature and TL represents low temperature.
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cycle was followed by a relaxation phase to reach equilibrium for 2000 s. The load and displacement data at the end of each relaxation phase were used to determine Young's modulus, including the information of diameter and height of each cartilage specimen. Six samples from each group were measured. Native rabbit articular cartilage was used as a control. Statistical analysis Statistical analysis was performed using the SPSS 13.0 software package. All values were expressed as mean ± standard deviation. Statistical comparisons of the MTT assay results obtained for oriented and non-oriented scaffolds were performed using independent t-tests. Differences in biochemical content and Young's modulus among groups were analyzed by one-way ANOVA. Values of p b 0.05 were considered statistically significant.
RESULTS Scaffold characterization Following genipin-mediated crosslinking, a scaffold color change was observed (white to dark blue). The microstructure of oriented and non-oriented scaffolds was observed by SEM as shown in Fig. 2. It was noted that the pore structure of the oriented and non-oriented scaffolds was noticeably distinct. Pores within the oriented scaffold were microtubule-like and arranged in parallel in vertical section (Fig. 2A). The microtubules (diameter, 105.2 ± 20.3 μm) were interconnected. In contrast, pores within the non-oriented scaffold were distributed randomly and uniformly in vertical and cross sections (Figs. 2C, D). The diameter of random macropores was 114.3 ± 32.2 μm. There was no significant difference in the porosity between oriented and non-oriented scaffolds (91.6% ± 3.1% and 92.2% ± 3.0% respectively). Mechanical property of scaffolds Young's modulus of oriented and non-oriented scaffolds was 89.35 ± 7.96 kPa and 36.20 ± 4.67 kPa, respectively. The compressive modulus of oriented scaffolds was 2.5-fold higher than that of non-oriented scaffolds (p b 0.05). Proliferation of chondrogenic BMSCs on scaffolds A significant increase in the proliferation of chondrogenic-induced BMSCs seeded on oriented and non-oriented scaffolds was measured by MTT assay (Fig. 3), although proliferation in the oriented scaffold group was higher than in the non-oriented scaffold group from day 3 to day
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9 (p b 0.05). However, there was no significant difference in proliferation between the two groups at day 11 and day 13. Adhesion and morphology of chondrogenic BMSCs on scaffolds SEM micrographs showing adhesion and morphology of cells on scaffolds are presented in Fig. 4. It was observed that chondrogenic-induced BMSCs adhered to the scaffolds and displayed a spherical morphology at day 1 (Figs. 4A, D). Moreover, cells were aligned along the oriented microtubules in the oriented scaffold (Fig. 4A). An improved distribution and level of contact between cells to form larger aggregates on the scaffolds were observed after 3 days in culture (Figs. 4B, E). Most cells exhibited a round or elliptic morphology similar to that of chondrocyte-like cells and were well distributed in the core of pores (Figs. 4B, E). Higher magnification revealed moderate cell adhesion and chondrocyte-like morphology in oriented scaffolds (Fig. 4C, white arrows) and non-oriented scaffolds (Fig. 4F, white arrows). There was no significant difference in the cell morphology between the two types of constructs throughout the period of experiment. Histological and immunohistochemical staining of TE cartilage All samples stained positive for safranin O (Figs. 5A–C), toluidine blue (Figs. 5D–F) and collagen type II (Figs. 5G–I), but negative for collagen type I (Figs. 5J–L) at 4 weeks after implantation. These observations indicated that chondrogenic-induced BMSCs in all implants continued to proliferate and secrete cartilage-specific ECM components to form homogeneous cartilage-like tissue. The staining for sections was uniform in matrices and there were no significant differences in the staining intensity between the two types of TE cartilages. Moreover, the round and elongated chondrocyte-like cells were surrounded by abundant ECM and typical cartilage lacunae were observed (Fig. 5 and Fig. S1, black arrows). In oriented-structure constructs, numerous parallel giant bundles of densely-packed collagen fibers were visible and chondrocyte-like cells were aligned along the oriented collagen fibers (Fig. 5 and Fig. S1, black arrows).
FIG. 2. SEM micrographs of the oriented scaffold in (A) vertical section and (B) cross section, and of the non-oriented scaffold in (C) vertical section and (D) cross section.
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J. BIOSCI. BIOENG., Mechanical property for both experimental groups also increased with time. Compared to native articular cartilage (E of 0.664 ± 0.031 MPa), the Young's modulus of the two types of TE cartilages reached 42.9% and 23.0% in oriented and non-oriented scaffolds respectively, at 4 weeks. DISCUSSION
FIG. 3. Proliferation of chondrogenic-induced BMSCs seeded on oriented scaffold (filled squares) and non-oriented scaffolds (circles). *p b 0.05, significance between the two groups.
Biochemical analysis of TE cartilage Biochemical characterization of the two types of TE cartilages at 2 weeks and 4 weeks after implantation and native cartilage is shown in Figs. 6A–C. The amount of DNA, GAG and collagen in both TE cartilage groups increased in a time-dependent manner (Figs. 6A–C). Total DNA, GAG and collagen contents were similar in the two experimental groups at 2 weeks and 4 weeks (Figs. 6A–C). Total DNA, GAG and collagen in the two experimental groups at both time points were lower compared with native cartilage (p b 0.05) (Figs. 6A–C). Biomechanical assay of TE cartilage The biomechanical properties of the two types of TE cartilages and native rabbit cartilage are shown in Fig. 6D. Constructs formed on oriented scaffolds exhibited superior mechanical property (E of 0.196 ± 0.019 MPa and 0.285 ± 0.021 MPa) than those formed on non-oriented scaffolds (E of 0.091 ± 0.007 MPa and 0.153 ± 0.013 MPa) at 2 weeks and 4 weeks (p b 0.05). It was observed that scaffold structure significantly impacted the Young's modulus of the two experimental groups.
In this study, in vitro and in vivo experiments showed that the oriented cartilage ECM-derived scaffold possessed sufficient mechanical strength to meet the requirements of cartilage regeneration. The value of Young's modulus of the oriented scaffold was 2.5 times higher than that of non-oriented scaffold in vitro (p b 0.05). Seeded chondrogenic-induced BMSCs successfully adhered and distributed onto oriented and non-oriented scaffolds in vitro. Furthermore, implantation of oriented scaffolds combined with differentiated BMSCs in the dorsa of nude mice formed superior biomechanically functional TE cartilage constructs after 4 weeks, and efficient cartilaginous matrix secretion was observed. Oriented scaffolds fabricated from various materials through diverse techniques have been shown to possess superior strength and compression modulus than that of typical non-oriented scaffolds in the dry state in vitro (11,15,24). In this study, a microtubularoriented cartilage scaffold was fabricated using an improved temperature gradient-guided TIPS technique. Due to the existence of a temperature gradient during phase separation, fibrous crystals are formed along the orientation of the temperature gradient with generation of the oriented scaffold resulting in vertical microtubules following solvent removal by freeze-drying (25,26). In accordance with previous studies, the characteristics of this fabricated oriented scaffold included parallel arrangement of microtubules, appropriate pore size and high porosity making these scaffolds suitable for chondrocyte proliferation and differentiation (11,15,25). Furthermore, the compressive modulus of oriented scaffolds was higher than that of non-oriented scaffolds. This may be attributed to an increased
FIG. 4. Adhesion and morphology of chondrogenic-induced BMSCs cultured on (A–C) oriented scaffolds and (D–F) non-oriented scaffold. Cultured cells showed a spherical morphology at day 1 (A, D; magnification is 500×) and a round or elliptic morphology at day 3 (B, E; magnification is 1000 ×). (C, F) Higher magnification of cells adhesion and chondrocyte-like morphology at day 3 (magnification is 3000×). White arrows indicate the locations of the cells.
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FIG. 5. Histological and immunohistochemical staining of oriented and non-oriented TE cartilage at 4 weeks after implantation and native articular cartilage. Black arrows indicate the locations of the chondrocyte-like cells and cartilage lacunae. Scale bars: 100 μm.
capacity to support more compressive stress due to the greater thickness of the microtubule walls (11). Chondrogenic BMSCs successfully adhered and distributed in both oriented and non-oriented cartilage ECM-derived scaffolds in vitro and phenotypic stability of differentiated BMSCs was supported. These observations indicate that chondrogenic-induced BMSCs do not undergo a change in phenotype towards a fibroblast-like shape as observed during monolayer expansion. Moreover, it was shown that the oriented scaffold served as a guide for chondrogenic-induced BMSC adherence and alignment along the orientation of vertical microtubules, thus mimicking the physiological structure of the native cartilage. Although both types of scaffolds supported the proliferation of seeded BMSCs, cell proliferation on oriented scaffold was higher than that on non-oriented scaffold from day 3 to day 9. This result may be attributed to the oriented microtubules structure (congruent alignment and interconnected), which facilitates the transport of
culture medium and the exchange of metabolite during the initial stages of culture. However, this advantage was lost at day 11 and day 13 due to increased cell number and ECM accumulation (27). Following implantation for 4 weeks, the total DNA content of both types of TE cartilages increased with time in vivo, although these values were lower than those of native articular cartilage (p b 0.05). However, the total DNA content was similar in the two experimental groups at 2 weeks and 4 weeks, indicating that the oriented scaffold structure did not significantly affect cell proliferation in vivo after maximum capacity of the scaffold was reached. This demonstrated that the superior mechanical property of oriented TE cartilage may be related to the aligned structure rather than the cell number. Efficient cartilaginous matrix secretion was observed in both oriented and non-oriented cartilage ECM-derived scaffolds in vivo, which was attributed to the native components of the scaffold. Chondrogenic differentiation was observed by histological and
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FIG. 6. Biochemical characterization and biomechanical properties of oriented and non-oriented TE cartilage and native articular cartilage: (A) Total DNA content, (B) total GAG content, (C) total collagen content, (D) Young's modulus. Gray bars represent oriented TE cartilage, light gray bars represent non-oriented TE cartilage and black bars represent native cartilage. *p b 0.05, significance between the two groups.
immunohistochemical analysis at 4 weeks in vivo and both types of TE cartilages expressed increased amounts of GAG and collagen II. Interconnected macroporous networks in both types of scaffolds enabled uniform distribution of cell-secreted GAG molecules throughout the entire scaffold as observed by histological evaluation. The total GAG content increased with time during the period of implantation, which was consistent with previous studies (19,28). Furthermore, the total collagen content within both types of scaffolds increased with implantation time. Although the biochemical assay did not distinguish the specific collagen type, immunohistological staining (for collagen type II and I) indicated that the cartilage matrix was predominantly collagen type II specific, maintaining the phenotype of differentiated BMSCs on the matrix. These results suggest that the cartilage ECMderived scaffold has good biocompatibility with cells and provides a microenvironment that allows the proliferation and differentiation of chondrogenic BMSCs in vivo. The Young's modulus of the TE cartilage was higher for the oriented scaffold compared with the non-oriented scaffold after implantation for 2 weeks and 4 weeks. However, the total DNA, GAG and collagen contents were similar in the two types of TE cartilages at 2 weeks and 4 weeks. Therefore, it can be concluded that, apart from the biochemical constituents, the structure of constructs is the most important factor determining the Young's modulus of TE cartilage. This indicates that the oriented structure rather than the composition of the scaffold significantly enhances the biomechanical property of TE cartilage in vivo. This may be attributed to the vertically aligned giant collagen bundles in oriented TE cartilage, which provide support for compression in the vertical direction. In this study, the biomechanical property of TE cartilage using a subcutaneous model was shown to differ from those of native articular cartilage, which is consistent with previous reports (18). Bueno et al. reported that the chemical environment and mechanical stimulation are critical for the biochemical and biomechanical
development of chondrogenesis in vivo (29). Therefore, further investigation of oriented TE cartilage and cartilage ECM-derived scaffolds in an animal model of full-thickness articular cartilage defect is required to assess the biomechanical properties and biochemical characteristics in the knee of a living organism over longer time periods. Furthermore, advances in TIPS techniques for fabrication of the oriented architecture are essential to produce an optimal scaffold for the purposes of cartilage tissue engineering. In summary, an oriented cartilage ECM-derived scaffold combined with chondrogenic-induced BMSCs yielded a superior, mechanically functional TE cartilage within 4 weeks in vivo. This demonstrated that the oriented cartilage ECM-derived scaffold not only supported differentiated BMSC proliferation and cartilage-specific ECM secretion, but also enhanced the biomechanical property of TE cartilage through providing an oriented structure, thus indicating the potential application of this strategy for cartilage tissue engineering. Supplementary materials related to this article can be found online at doi:10.1016/j.jbiosc.2011.12.009. ACKNOWLEDGMENT This work was supported by grants from the National Natural Science Foundation of China (No. 31070860). References 1. Buckwalter, J. A. and Mankin, H. J.: Articular cartilage repair and transplantation, Arthritis Rheum., 41, 1331–1342 (1998). 2. Becerra, J., Andrades, J. A., Guerado, E., Zamora-Navas, P., Lopez-Puertas, J. M., and Reddi, A. H.: Articular cartilage: structure and regeneration, Tissue Eng. Part B Rev., 16, 617–627 (2010). 3. Poole, A. R., Pidoux, I., Reiner, A., and Rosenberg, L.: An immunoelectron microscope study of the organization of proteoglycan monomer, link protein, and collagen in the matrix of articular cartilage, J. Cell Biol., 93, 921–937 (1982).
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Oriented cartilage extracellular matrix-derived scaffold for cartilage tissue engineering Shuaijun Jia, 1, † Lie Liu, 2 Weimin Pan, 3 Guolin Meng, 1, † Chunguang Duan, 1, † Laquan Zhang, 4 Zhuo Xiong, 4 and Jian Liu 1,⁎ Institute of Orthopaedics, Xijing Hospital, The Fourth Military Medical University, Xi'an 710032, China, 1 Department of Orthopaedics, Baoji Central Hospital, Baoji 721008, China, 2 Department of Human Movement Studies, Xi'an Physical Education University, Xi'an 710032, China, 3 and Department of Mechanical Engineering, Tsinghua University, Beijing 100084, China 4 Received 14 November 2011; accepted 15 December 2011 Available online 21 January 2012
The structure of a cartilage scaffold is required to mimic native articular cartilage, which has an oriented structure associated with its mechanical function. In this study, an oriented cartilage extracellular matrix (ECM)-derived scaffold was fabricated composed of microtubules arranged in parallel in vertical section. The mechanical property was higher than that of a typical non-oriented scaffold (p b 0.05). Oriented and non-oriented scaffolds were seeded with chondrogenic-induced bone mesenchymal stem cells and cell-scaffold constructs were implanted subcutaneously in the dorsa of nude mice. At 4 weeks, all samples stained positive for safranin O, toluidine blue, and collagen type II, but negative for collagen type I. Oriented-structure constructs contained numerous parallel giant bundles of densely packed collagen fibers with chondrocyte-like cells aligned along the fibers. Total DNA, glycosaminoglycans and collagen contents increased with time and these values were similar in the two groups. Compared with the native articular cartilage, the Young's modulus of the tissue-engineered (TE) cartilage reached 42.9%, 23.0% in oriented and non-oriented scaffolds respectively, at 4 weeks. These results indicate that oriented ECMderived scaffolds enhance the biomechanical property of TE cartilage and thus represent a promising approach to cartilage tissue engineering. © 2011, The Society for Biotechnology, Japan. All rights reserved. [Key words: Oriented scaffold; Extracellular matrix; Bone mesenchymal stem cells; Cartilage tissue engineering; Biomechanical property]
Articular cartilage provides near frictionless motion between articulating surfaces and protects the bones of synovial joints from being damaged when subjected to impact and load bearing (1). Anatomically and functionally, articular cartilage consists of four zones: the superficial, transitional, deep and calcified zones and generally exhibits columnar orientation of cells and anisotropic direction of collagen fibers that run vertically from the tidemark towards the joint surface (2,3). This aligned collagen fiber network is believed to be a critical factor required for the biomechanical properties of articular cartilage (4). Although articular cartilage is highly susceptible to damage, it has limited intrinsic regeneration and self-repair capacity due to its innate avascular nature and low cell-to-matrix ratio (5). Tissue engineering has proved to be the most promising alternative therapy that combines cells, scaffolds and environmental factors for repair of articular cartilage defects (6). The scaffold serves as an extracellular matrix (ECM) providing three-dimensional conformation and orientation to cells and has a vital role in cartilage tissue engineering (7). ⁎ Corresponding author. Tel.: + 86 29 8477 1013; fax: + 86 29 8477 1012. E-mail address: [email protected] (J. Liu). † These authors contributed equally to this work.
The availability of suitable scaffold material is a major component of tissue engineering strategies. Therefore, identification of an optimal matrix for cartilage tissue engineering has led to the development and application of numerous scaffolds derived from both natural and synthetic materials (6). Previous studies have demonstrated that scaffolds derived from naturally decellularized cartilage ECM are devoid of allogeneic or xenogeneic cellular antigens while preserving the majority of ECM structural and functional proteins (8,9). Furthermore, it has been shown that cartilage ECM-derived scaffolds preserve the main constituents of native cartilage including glycosaminoglycan (GAG) and collagen type II, which possess good biocompatibility and provide a natural microenvironment for the support of bone mesenchymal stem cell (BMSC) attachment, proliferation and differentiation into chondrocytes (10). Therefore it is hypothesized that cartilage ECM is a suitable material for use as a cartilage scaffold. More importantly, the designed structure of cartilage scaffolds should mimic native articular cartilage, which has an oriented structure associated with its mechanical and physiological functions (11). Furthermore, the oriented architecture of this scaffold should possess superior biomechanical properties and protect cells from early critical compression prior to secretion of abundant ECM. Therefore, scaffold with a biomimetic-oriented architecture is an important requirement for tissue-engineered (TE) cartilage.
1389-1723/$ - see front matter © 2011, The Society for Biotechnology, Japan. All rights reserved. doi:10.1016/j.jbiosc.2011.12.009
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The source of cells is an additional factor critical to the development of tissue engineering strategies. Although targeted regenerative tissue is formed from autologous chondrocytes, the supply of these cells is very limited. BMSCs, which can be safely harvested by bone marrow biopsy, have been reported as an alternative cell source due to their characteristic capacity for rapid proliferation and multiple differentiation potential (12). Extensive research has demonstrated BMSCs differentiation into chondrocytes by application of transforming growth factor-β (TGF-β) family members in various 3-D scaffolds (13). Therefore, BMSCs represent promising candidates for use in cartilage tissue engineering. The purpose of this study was to fabricate an oriented cartilage ECM-derived scaffold combined with chondrogenic-induced BMSCs for enhancement of the biomechanical property of TE cartilage in vivo. The resultant constructs were cultured for 2 weeks and 4 weeks at subcutaneous sites in nude mice, after which biochemical, histological and biomechanical properties were evaluated. MATERIALS AND METHODS Preparation of the scaffolds Bovine articular cartilage ECM was obtained by decellularization using previously described methods (8). Oriented scaffolds were prepared using a modified temperature gradient-guided thermal-induced phase separation (TIPS) technique followed by freeze-drying (Fig. 1). Briefly, a suspension of cartilage ECM (3%, w/v) was prepared and infused into a cylindrical mould with an inner diameter of 8 mm and a height of 10 mm. The top of the mould was open so that the upper surface of the slurry was maintained at room temperature. The mould was then placed vertically onto a metal plate equilibrated to − 196°C and frozen in liquid nitrogen. This technique allowed orientation of the structure of the scaffold by solvent crystallization under a unidirectional temperature gradient. Frozen samples were lyophilized in a freeze-dryer (Alpha 2–4, Chaist, Germany) for 48 h to form an oriented microtubule structure. Scaffolds were then removed from the mould and cut into cylinders (diameter, 5 mm; thickness, 2 mm) using biopsy punch and scalpel. Typical non-oriented scaffolds were fabricated by simple freeze-drying. Briefly, the suspension was frozen for consecutive periods of 1 h at − 20°C and − 80°C and lyophilized for 48 h. All scaffolds were cross-linked by treatment with genipin solution (0.5%, w/v) (Wako Pure Chemical Industries, Osaka, Japan) for 48 h at room temperature (14) and sterilized by exposure to 20 kGy 60Co radiation prior to cell culture and experimentation. Scaffolds characterization The oriented and non-oriented scaffold samples were sputter-coated with gold at 40 mA for scanning electron microscope analysis (SEM; S-3400N, Hitachi, Tokyo, Japan) at an accelerating voltage of 5.0 kV. The porosity of the two types of scaffold was estimated according to previously reported liquid displacement methods (11). Mechanical property of scaffolds Mechanical property of oriented and non-oriented scaffold was determined by measurement of Young's modulus according to previously reported methods (15). The compressive strength of scaffolds along the longitudinal direction was measured by using a universal material testing machine (Shimadzu, Kyoto, Japan) at a crosshead speed of 1.0 mm/min. Young's modulus was obtained from the initial slopes of the stress–strain curves. Results are expressed as the mean of six independent measurements. Isolation and chondrogenic induction of BMSCs In this study, all experimental procedures involving animals were approved by the Institutional Animal Review Committee of Fourth Military Medical University. BMSCs were obtained from the tibia of New Zealand White rabbits as described previously (16) and cultured to 80% confluence. Adherent cells were released with trypsin (Sigma-Aldrich, St. Louis, MO,
USA) and subcultured in chondrogenic induction medium to induce differentiation of BMSCs. The induction medium contained high glucose Dulbecco's modified Eagle's medium (DMEM; Invitrogen, Camarillo, CA, USA), 10% fetal bovine serum (FBS; HyClone, Utah, USA), 10 ng/ml TGF-β3 (PeproTech, Rocky Hill, NJ, USA), 1% ITS+ premix (BD, Franklin Lakes, NJ, USA), 10− 7 M dexamethasone (Sigma), 50 μg/ml ascorbic acid (Sigma), 1 mM sodium pyruvate (Sigma), 4 mM proline (Sigma), and 1% antibiotics (100 U/ml penicillin, 100 mg/ml streptomycin, Sigma) (8,17). The medium was replaced twice per week and the cells were harvested at day 21 for seeding. Proliferation and morphology of chondrogenic BMSCs on scaffolds Chondrogenic-induced BMSCs were resuspended in culture medium (cell density, 1 × 107 cells/ml) and seeded onto scaffolds (5 × 105 cells/scaffold). Cells were allowed to attach to the scaffolds for 2 h at 37°C prior to the addition of fresh induction medium (1 ml). Cell-scaffold constructs were further incubated at 37°C in an atmosphere of 5% CO2 and 95% humidity and the medium changed twice a week. Cell viability and proliferation within the scaffolds were analyzed by 3-(4,5-Dimethyl-2-thiazolyl)-2, 5diphenyl-2H-tetrazolium bromide (MTT; Sigma) assay (14). Adhesion properties, morphological characteristics and the distribution of the cells within scaffolds were analyzed by SEM (S-3400N, Hitachi, Tokyo, Japan). In vivo implantation Following in vitro culture (1 week), cell-scaffold constructs were implanted subcutaneously in the dorsa of nude mice (aged 4 weeks) (8). Mice were sacrificed at 2 weeks and 4 weeks, and samples of TE cartilage were harvested. Histological and immunohistochemical analysis of TE cartilage Implants were fixed overnight with 4% paraformaldehyde and then embedded in paraffin at 4 weeks after implantation. Sections (thickness, 5 μm) were stained for proteoglycans with safranin O and toluidine blue according to standard protocols (8). For immunohistochemical analysis, sections (thickness, 20 μm) were deparaffinized with xylene, rehydrated in graded ethanol series and washed with PBS. Collagen was detected immunohistochemically using monoclonal antibodies against collagen types I (ab90395; Abcam, Cambridge, MA, USA) and II (433120; Invitrogen, Camarillo, CA, USA) as previously described (18). Rabbit articular cartilage was prepared by the same method described for TE cartilage for use as positive controls. Biochemical analysis of TE cartilage Samples of TE cartilage were harvested at 2 weeks and 4 weeks after implantation for biochemical evaluation and digested by lysis buffer containing 125 μg/ml papain (Sigma), 10 mM cysteine hydrochloride (Sigma), 100 mM sodium phosphate and 10 mM EDTA, for 24 h at 60°C (19). The DNA content was evaluated using the Quant-iT™ PicoGreen® dsDNA Assay Kit (Molecular Probes, Eugene, OR, USA) (20). Fluorescence intensity was measured using a Vicam Series-4 Fluorimeter (Vicam, Milford, MA, USA). A standard curve for the analysis was generated using bacteriophage lambda DNA supplied with the kit. Total GAG content was quantified using a Blyscan™ Sulfated Glycosaminoglycan Assay kit (Biocolor, Carrickfergus, Northern Ireland, UK) and a spectrophotometer (Bio-Tek Instruments, Winooski, VT, USA) at 656 nm, based on dimethylmethylene blue binding (18). A standard curve was established using bovine cartilage chondroitin sulfate (Sigma) (21). Aliquots of the papain-digested sample solution were incubated at 48°C for 48 h in 0.05 M acetic acid containing 0.5 M NaCl (pH 3) and 10 mg/ml pepsin (Sigma). Total collagen was determined by the Sircol™ Soluble Collagen Assay kit (Biocolor) with absorbance at 555 nm measured using a spectrophotometer (BioTek). A standard curve was generated using L-hydroxyproline (Sigma) (22). Six samples from each TE cartilage group were measured. Native rabbit articular cartilage was used as a control. Biomechanical assay of TE cartilage Biomechanical property of the two types of TE cartilage developed during the 4 weeks of implantation was determined by measurement of Young's modulus (E), which was measured by using a universal material testing machine (Shimadzu, Kyoto, Japan). For unconfined compression tests (UCC) (20,23), samples were transferred to a stainless steel dish containing PBS equilibrated to room temperature. The height of each specimen was determined at a compressive force threshold of 0.05 N. Five progressive strain loadings at 4% of the original cartilage height were performed with a test velocity of 0.01 mm/s. Each loading
FIG. 1. Schematic diagram showing the fabrication of oriented scaffolds by thermal-induced phase separation technique: (A) scheme of the cylindrical mould (with an inner diameter of 8 mm and a height of 10 mm) filled cartilage ECM, (B) scheme of oriented scaffold (diameter of the microtubule: 105.2 ± 20.3 μm). TH represents high temperature and TL represents low temperature.
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cycle was followed by a relaxation phase to reach equilibrium for 2000 s. The load and displacement data at the end of each relaxation phase were used to determine Young's modulus, including the information of diameter and height of each cartilage specimen. Six samples from each group were measured. Native rabbit articular cartilage was used as a control. Statistical analysis Statistical analysis was performed using the SPSS 13.0 software package. All values were expressed as mean ± standard deviation. Statistical comparisons of the MTT assay results obtained for oriented and non-oriented scaffolds were performed using independent t-tests. Differences in biochemical content and Young's modulus among groups were analyzed by one-way ANOVA. Values of p b 0.05 were considered statistically significant.
RESULTS Scaffold characterization Following genipin-mediated crosslinking, a scaffold color change was observed (white to dark blue). The microstructure of oriented and non-oriented scaffolds was observed by SEM as shown in Fig. 2. It was noted that the pore structure of the oriented and non-oriented scaffolds was noticeably distinct. Pores within the oriented scaffold were microtubule-like and arranged in parallel in vertical section (Fig. 2A). The microtubules (diameter, 105.2 ± 20.3 μm) were interconnected. In contrast, pores within the non-oriented scaffold were distributed randomly and uniformly in vertical and cross sections (Figs. 2C, D). The diameter of random macropores was 114.3 ± 32.2 μm. There was no significant difference in the porosity between oriented and non-oriented scaffolds (91.6% ± 3.1% and 92.2% ± 3.0% respectively). Mechanical property of scaffolds Young's modulus of oriented and non-oriented scaffolds was 89.35 ± 7.96 kPa and 36.20 ± 4.67 kPa, respectively. The compressive modulus of oriented scaffolds was 2.5-fold higher than that of non-oriented scaffolds (p b 0.05). Proliferation of chondrogenic BMSCs on scaffolds A significant increase in the proliferation of chondrogenic-induced BMSCs seeded on oriented and non-oriented scaffolds was measured by MTT assay (Fig. 3), although proliferation in the oriented scaffold group was higher than in the non-oriented scaffold group from day 3 to day
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9 (p b 0.05). However, there was no significant difference in proliferation between the two groups at day 11 and day 13. Adhesion and morphology of chondrogenic BMSCs on scaffolds SEM micrographs showing adhesion and morphology of cells on scaffolds are presented in Fig. 4. It was observed that chondrogenic-induced BMSCs adhered to the scaffolds and displayed a spherical morphology at day 1 (Figs. 4A, D). Moreover, cells were aligned along the oriented microtubules in the oriented scaffold (Fig. 4A). An improved distribution and level of contact between cells to form larger aggregates on the scaffolds were observed after 3 days in culture (Figs. 4B, E). Most cells exhibited a round or elliptic morphology similar to that of chondrocyte-like cells and were well distributed in the core of pores (Figs. 4B, E). Higher magnification revealed moderate cell adhesion and chondrocyte-like morphology in oriented scaffolds (Fig. 4C, white arrows) and non-oriented scaffolds (Fig. 4F, white arrows). There was no significant difference in the cell morphology between the two types of constructs throughout the period of experiment. Histological and immunohistochemical staining of TE cartilage All samples stained positive for safranin O (Figs. 5A–C), toluidine blue (Figs. 5D–F) and collagen type II (Figs. 5G–I), but negative for collagen type I (Figs. 5J–L) at 4 weeks after implantation. These observations indicated that chondrogenic-induced BMSCs in all implants continued to proliferate and secrete cartilage-specific ECM components to form homogeneous cartilage-like tissue. The staining for sections was uniform in matrices and there were no significant differences in the staining intensity between the two types of TE cartilages. Moreover, the round and elongated chondrocyte-like cells were surrounded by abundant ECM and typical cartilage lacunae were observed (Fig. 5 and Fig. S1, black arrows). In oriented-structure constructs, numerous parallel giant bundles of densely-packed collagen fibers were visible and chondrocyte-like cells were aligned along the oriented collagen fibers (Fig. 5 and Fig. S1, black arrows).
FIG. 2. SEM micrographs of the oriented scaffold in (A) vertical section and (B) cross section, and of the non-oriented scaffold in (C) vertical section and (D) cross section.
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J. BIOSCI. BIOENG., Mechanical property for both experimental groups also increased with time. Compared to native articular cartilage (E of 0.664 ± 0.031 MPa), the Young's modulus of the two types of TE cartilages reached 42.9% and 23.0% in oriented and non-oriented scaffolds respectively, at 4 weeks. DISCUSSION
FIG. 3. Proliferation of chondrogenic-induced BMSCs seeded on oriented scaffold (filled squares) and non-oriented scaffolds (circles). *p b 0.05, significance between the two groups.
Biochemical analysis of TE cartilage Biochemical characterization of the two types of TE cartilages at 2 weeks and 4 weeks after implantation and native cartilage is shown in Figs. 6A–C. The amount of DNA, GAG and collagen in both TE cartilage groups increased in a time-dependent manner (Figs. 6A–C). Total DNA, GAG and collagen contents were similar in the two experimental groups at 2 weeks and 4 weeks (Figs. 6A–C). Total DNA, GAG and collagen in the two experimental groups at both time points were lower compared with native cartilage (p b 0.05) (Figs. 6A–C). Biomechanical assay of TE cartilage The biomechanical properties of the two types of TE cartilages and native rabbit cartilage are shown in Fig. 6D. Constructs formed on oriented scaffolds exhibited superior mechanical property (E of 0.196 ± 0.019 MPa and 0.285 ± 0.021 MPa) than those formed on non-oriented scaffolds (E of 0.091 ± 0.007 MPa and 0.153 ± 0.013 MPa) at 2 weeks and 4 weeks (p b 0.05). It was observed that scaffold structure significantly impacted the Young's modulus of the two experimental groups.
In this study, in vitro and in vivo experiments showed that the oriented cartilage ECM-derived scaffold possessed sufficient mechanical strength to meet the requirements of cartilage regeneration. The value of Young's modulus of the oriented scaffold was 2.5 times higher than that of non-oriented scaffold in vitro (p b 0.05). Seeded chondrogenic-induced BMSCs successfully adhered and distributed onto oriented and non-oriented scaffolds in vitro. Furthermore, implantation of oriented scaffolds combined with differentiated BMSCs in the dorsa of nude mice formed superior biomechanically functional TE cartilage constructs after 4 weeks, and efficient cartilaginous matrix secretion was observed. Oriented scaffolds fabricated from various materials through diverse techniques have been shown to possess superior strength and compression modulus than that of typical non-oriented scaffolds in the dry state in vitro (11,15,24). In this study, a microtubularoriented cartilage scaffold was fabricated using an improved temperature gradient-guided TIPS technique. Due to the existence of a temperature gradient during phase separation, fibrous crystals are formed along the orientation of the temperature gradient with generation of the oriented scaffold resulting in vertical microtubules following solvent removal by freeze-drying (25,26). In accordance with previous studies, the characteristics of this fabricated oriented scaffold included parallel arrangement of microtubules, appropriate pore size and high porosity making these scaffolds suitable for chondrocyte proliferation and differentiation (11,15,25). Furthermore, the compressive modulus of oriented scaffolds was higher than that of non-oriented scaffolds. This may be attributed to an increased
FIG. 4. Adhesion and morphology of chondrogenic-induced BMSCs cultured on (A–C) oriented scaffolds and (D–F) non-oriented scaffold. Cultured cells showed a spherical morphology at day 1 (A, D; magnification is 500×) and a round or elliptic morphology at day 3 (B, E; magnification is 1000 ×). (C, F) Higher magnification of cells adhesion and chondrocyte-like morphology at day 3 (magnification is 3000×). White arrows indicate the locations of the cells.
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FIG. 5. Histological and immunohistochemical staining of oriented and non-oriented TE cartilage at 4 weeks after implantation and native articular cartilage. Black arrows indicate the locations of the chondrocyte-like cells and cartilage lacunae. Scale bars: 100 μm.
capacity to support more compressive stress due to the greater thickness of the microtubule walls (11). Chondrogenic BMSCs successfully adhered and distributed in both oriented and non-oriented cartilage ECM-derived scaffolds in vitro and phenotypic stability of differentiated BMSCs was supported. These observations indicate that chondrogenic-induced BMSCs do not undergo a change in phenotype towards a fibroblast-like shape as observed during monolayer expansion. Moreover, it was shown that the oriented scaffold served as a guide for chondrogenic-induced BMSC adherence and alignment along the orientation of vertical microtubules, thus mimicking the physiological structure of the native cartilage. Although both types of scaffolds supported the proliferation of seeded BMSCs, cell proliferation on oriented scaffold was higher than that on non-oriented scaffold from day 3 to day 9. This result may be attributed to the oriented microtubules structure (congruent alignment and interconnected), which facilitates the transport of
culture medium and the exchange of metabolite during the initial stages of culture. However, this advantage was lost at day 11 and day 13 due to increased cell number and ECM accumulation (27). Following implantation for 4 weeks, the total DNA content of both types of TE cartilages increased with time in vivo, although these values were lower than those of native articular cartilage (p b 0.05). However, the total DNA content was similar in the two experimental groups at 2 weeks and 4 weeks, indicating that the oriented scaffold structure did not significantly affect cell proliferation in vivo after maximum capacity of the scaffold was reached. This demonstrated that the superior mechanical property of oriented TE cartilage may be related to the aligned structure rather than the cell number. Efficient cartilaginous matrix secretion was observed in both oriented and non-oriented cartilage ECM-derived scaffolds in vivo, which was attributed to the native components of the scaffold. Chondrogenic differentiation was observed by histological and
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FIG. 6. Biochemical characterization and biomechanical properties of oriented and non-oriented TE cartilage and native articular cartilage: (A) Total DNA content, (B) total GAG content, (C) total collagen content, (D) Young's modulus. Gray bars represent oriented TE cartilage, light gray bars represent non-oriented TE cartilage and black bars represent native cartilage. *p b 0.05, significance between the two groups.
immunohistochemical analysis at 4 weeks in vivo and both types of TE cartilages expressed increased amounts of GAG and collagen II. Interconnected macroporous networks in both types of scaffolds enabled uniform distribution of cell-secreted GAG molecules throughout the entire scaffold as observed by histological evaluation. The total GAG content increased with time during the period of implantation, which was consistent with previous studies (19,28). Furthermore, the total collagen content within both types of scaffolds increased with implantation time. Although the biochemical assay did not distinguish the specific collagen type, immunohistological staining (for collagen type II and I) indicated that the cartilage matrix was predominantly collagen type II specific, maintaining the phenotype of differentiated BMSCs on the matrix. These results suggest that the cartilage ECMderived scaffold has good biocompatibility with cells and provides a microenvironment that allows the proliferation and differentiation of chondrogenic BMSCs in vivo. The Young's modulus of the TE cartilage was higher for the oriented scaffold compared with the non-oriented scaffold after implantation for 2 weeks and 4 weeks. However, the total DNA, GAG and collagen contents were similar in the two types of TE cartilages at 2 weeks and 4 weeks. Therefore, it can be concluded that, apart from the biochemical constituents, the structure of constructs is the most important factor determining the Young's modulus of TE cartilage. This indicates that the oriented structure rather than the composition of the scaffold significantly enhances the biomechanical property of TE cartilage in vivo. This may be attributed to the vertically aligned giant collagen bundles in oriented TE cartilage, which provide support for compression in the vertical direction. In this study, the biomechanical property of TE cartilage using a subcutaneous model was shown to differ from those of native articular cartilage, which is consistent with previous reports (18). Bueno et al. reported that the chemical environment and mechanical stimulation are critical for the biochemical and biomechanical
development of chondrogenesis in vivo (29). Therefore, further investigation of oriented TE cartilage and cartilage ECM-derived scaffolds in an animal model of full-thickness articular cartilage defect is required to assess the biomechanical properties and biochemical characteristics in the knee of a living organism over longer time periods. Furthermore, advances in TIPS techniques for fabrication of the oriented architecture are essential to produce an optimal scaffold for the purposes of cartilage tissue engineering. In summary, an oriented cartilage ECM-derived scaffold combined with chondrogenic-induced BMSCs yielded a superior, mechanically functional TE cartilage within 4 weeks in vivo. This demonstrated that the oriented cartilage ECM-derived scaffold not only supported differentiated BMSC proliferation and cartilage-specific ECM secretion, but also enhanced the biomechanical property of TE cartilage through providing an oriented structure, thus indicating the potential application of this strategy for cartilage tissue engineering. Supplementary materials related to this article can be found online at doi:10.1016/j.jbiosc.2011.12.009. ACKNOWLEDGMENT This work was supported by grants from the National Natural Science Foundation of China (No. 31070860). References 1. Buckwalter, J. A. and Mankin, H. J.: Articular cartilage repair and transplantation, Arthritis Rheum., 41, 1331–1342 (1998). 2. Becerra, J., Andrades, J. A., Guerado, E., Zamora-Navas, P., Lopez-Puertas, J. M., and Reddi, A. H.: Articular cartilage: structure and regeneration, Tissue Eng. Part B Rev., 16, 617–627 (2010). 3. Poole, A. R., Pidoux, I., Reiner, A., and Rosenberg, L.: An immunoelectron microscope study of the organization of proteoglycan monomer, link protein, and collagen in the matrix of articular cartilage, J. Cell Biol., 93, 921–937 (1982).
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