Conditioned Medium from Chondrocyte/Scaffold Constructs Induced Chondrogenic Differentiation of Bone Marrow Stromal Cells

For the application of bone marrow stromal cells (BMSCs) in cartilage tissue engineering, it is imperative to develop efficient strategies for their chondrogenic differentiation. In this study, the conditioned media derived from chondrocyte/scaffold constructs were used to direct chondrogenic differentiation of BMSCs. The porcine articular chondrocytes were seeded on the PGA/PLA scaffolds to form chondrocyte/scaffold constructs and were cultured to form engineered cartilage in vitro. The culture media were collected as conditioned media and used for chondrogenic induction of BMSC pellets (experimental group, Exp.). The chondrocyte pellets and BMSC pellets were cultured routinely as positive control (PC) and negative control (NC), respectively. After 4 weeks, the wet weight and GAG content in Exp. group and PC group were significantly higher than that in NC group. Histological and immunohistochemical analysis showed that cartilaginous tissue was formed with typical cartilage lacuna structure and positive staining of collagen Type II (Col II) in the peripheral area of the BMSC pellets in Exp. group. Gene expression of Sox9, Col II, and COMP in Exp. group and PC group were significantly higher than that in NC group. The growth factors in the conditioned media derived from human costal chondrocytes‐scaffold constructs were tested by protein microassay. The conditioned media contained low levels of TGF‐β1,2,3, IGF‐1 and high levels of IGF‐2, FGF‐4, and IGFBP4,6, and so forth. The soluble factors derived from the engineered cartilage can induce chondrogenic differentiation of BMSCs independently. Many cytokines may function in chondrogenesis in a coordinated way. Anat Rec, 2012. © 2012 Wiley Periodicals, Inc.     

Potential of 3-D tissue constructs engineered from bovine chondrocytes/silk fibroin-chitosan for in vitro cartilage tissue engineering

The use of cell-scaffold constructs is a promising tissue engineering approach to repair cartilage defects and to study cartilaginous tissue formation. In this study, silk fibroin/chitosan blended scaffolds were fabricated and studied for cartilage tissue engineering. Silk fibroin served as a substrate for cell adhesion and proliferation while chitosan has a structure similar to that of glycosaminoglycans, and shows promise for cartilage repair. We compared the formation of cartilaginous tissue in silk fibroin/chitosan blended scaffolds seeded with bovine chondrocytes and cultured in vitro for 2 weeks. The constructs were analyzed for cell viability, histology, extracellular matrix components glycosaminoglycan and collagen types I and II, and biomechanical properties. Silk fibroin/chitosan scaffolds supported cell attachment and growth, and chondrogenic phenotype as indicated by Alcian Blue histochemistry and relative expression of type II versus type I collagen. Glycosaminoglycan and collagen accumulated in all the scaffolds and was highest in the silk fibroin/chitosan (1:1) blended scaffolds. Static and dynamic stiffness at high frequencies was higher in cell-seeded constructs than non-seeded controls. The results suggest that silk/chitosan scaffolds may be a useful alternative to synthetic cell scaffolds for cartilage tissue engineering.     

A comparison of human umbilical cord matrix stem cells and temporomandibular joint condylar chondrocytes for tissue engineering temporomandibular joint condylar cartilage

The temporomandibular joint (TMJ) presents many problems in modern musculoskeletal medicine. Patients who suffer from TMJ disorders often experience a major loss in quality of life due to the debilitating effects that TMJ disorders can have on everyday activities. Cartilage tissue engineering can lead to replacement tissues that could be used to treat TMJ disorders. In this study, a spinner flask was used for a period of 6 days to seed polyglycolic acid (PGA) scaffolds with either TMJ condylar chondrocytes or mesenchymal-like stem cells derived from human umbilical cord matrix (HUCM). Samples were then statically cultured for 4 weeks either in growth medium containing chondrogenic factors or in control medium. Immunohistochemical staining of HUCM constructs after 4 weeks revealed a strong presence of collagen I and minute amounts of collagen II, whereas TMJ constructs revealed little collagen I and no collagen II. The HUCM constructs were shown to contain more GAGs than the TMJ constructs quantitatively at week 0 and histologically at week 4. Moreover, the cellularity of HUCM constructs was 55% higher at week 0 and nearly twice as high after 4 weeks, despite being seeded at the same density. The increased level of biosynthesis and higher cellularity of HUCM constructs clearly demonstrates that the HUCM stem cells outperformed the TMJ condylar cartilage cells under the prescribed conditions. HUCM stem cells may therefore be an attractive alternative to condylar cartilage cells for TMJ tissue engineering applications. Further, given the availability and ease of obtaining HUCM stem cells, these findings may have far-reaching implications, leading to novel developments in both craniofacial and orthopaedic tissue replacement therapies.     

Repair of articular cartilage defect in non-weight bearing areas using adipose derived stem cells loaded polyglycolic acid mesh

The current study was designed to observe chondrogenic differentiation of adipose derived stem cells (ASCs) on fibrous polyglycolic acid (PGA) scaffold stabilized with polylactic acid (PLA), and to further explore the feasibility of using the resulting cell/scaffold constructs to repair full thickness articular cartilage defects in non-weight bearing area in porcine model within a follow-up of 6 months. Autologous ASCs isolated from subcutaneous fat were expanded and seeded on the scaffold to fabricate ASCs/PGA constructs. Chondrogenic differentiation of ASCs in the constructs under chondrogenic induction was monitored with time by measuring the expression of collagen type II (COL II) and glycosaminoglycan (GAG). The constructs after being in vitro induced for 2 weeks were implanted to repair full thickness articular cartilage defects (8 mm in diameter, deep to subchondral bone) in femur trochlea (the experimental group), while scaffold alone was implanted to serve as the control. Histologically, the generated neo-cartilage integrated well with its surrounding normal cartilage and subchondral bone in the defects of experimental group at 3 months post-implantation, whereas only fibrous tissue was filled in the defects of control group. Immunohistochemical and toluidine blue staining confirmed the similar distribution of COL II and GAG in the regenerated cartilage as the normal one. A vivid remolding process with post-operation time was also witnessed in the neo-cartilage as its compressive moduli increased significantly from 50.55% of the normal cartilage at 3 months to 88.05% at 6 months. The successful repair thus substantiates the potentiality of using chondrogenic induced ASCs and PGA/PLA scaffold for cartilage regeneration.     

Cartilage-like tissue engineering using silk scaffolds and mesenchymal stem cells

Silk fibroin scaffolds were studied as a new biomaterial option for tissue-engineered cartilage-like tissue. Human bone marrow-derived mesenchymal stem cells (MSCs) were seeded on silk, collagen, and crosslinked collagen scaffolds and cultured for 21 days in serum-free chondrogenic medium. Cells proliferated more rapidly on the silk fibroin scaffolds than on the collagen matrices. The total content of glycosaminoglycan deposition was three times higher on silk as compared to collagen scaffolds. Glycosaminoglycan deposition coincided with overexpression of collagen type II and aggrecan genes. Cartilage-like tissue was homogeneously distributed throughout the entire silk scaffolds, while on the collagen and crosslinked collagen systems tissue formation was restricted to the outer rim, leaving a doughnut appearance. Round or angular-shaped cells resided in deep lacunae in the silk systems and stained positively for collagen type II. The aggregate modulus of the tissue-engineered cartilage constructs was more than 2-fold higher than that of the unseeded silk scaffold controls. These results suggest that silk fibroin scaffolds are suitable biomaterial substrates for autologous cartilage tissue engineering in serum-free medium and enable mechanical improvements along with compositional features suitable for durable implants to generate or regenerate cartilage.     

Repair of an articular cartilage defect using adipose-derived stem cells loaded on a polyelectrolyte complex scaffold based on poly(l-glutamic acid) and chitosan

As a synthetic polypeptide water-soluble poly(l-glutamic acid) (PLGA) was designed to fabricate scaffolds for cartilage tissue engineering. Chitosan (CHI) has been employed as a physical cross-linking component in the construction of scaffolds. PLGA/CHI scaffolds act as sponges with a swelling ratio of 760 ± 45% (mass%), showing promising biocompatibility and biodegradation. Autologous adipose-derived stem cells (ASCs) were expanded and seeded on PLGA/CHI scaffolds, ASC/scaffold constructs were then subjected to chondrogenic induction in vitro for 2 weeks. The results showed that PLGA/CHI scaffolds could effectively support ASC adherence, proliferation and chondrogenic differentiation. The ASCs/scaffold constructs were then transplanted to repair full thickness articular cartilage defects (4 mm in diameter, to the depth of subchondral bone) created in rabbit femur trochlea. Histological observations found that articular defects were covered with newly formed cartilage 6 weeks post-implantation. After 12 weeks the regenerated cartilage had integrated well with the surrounding native cartilage and subchondral bone. Toluidine blue and immunohistochemical staining confirmed similar accumulation of glycosaminoglycans and type II collagen in engineered cartilage as in native cartilage 12 weeks post-implantation. The result was further supported by quantitative analysis of extracellular matrix deposition. The compressive modulus of the engineered cartilage increased significantly from 30% of that of normal cartilage at 6 weeks to 83% at 12 weeks. Cyto-nanoindentation also showed analogous biomechanical behavior of the engineered cartilage to that of native cartilage. The results of the present study thus demonstrate the potentiality of PLGA/CHI scaffolds in cartilage tissue engineering.     

Fetal cartilage engineering from amniotic mesenchymal progenitor cells

We determined whether cartilage could be engineered from mesenchymal progenitor cells (MPCs) normally found in amniotic fluid. Mesenchymal amniocytes were isolated from ovine amniotic fluid samples (n = 5) and had their identity confirmed by immunocytochemistry. Cells were expanded and then cultured as micromass pellets (n = 5) in a chondrogenic medium containing transforming growth factor-beta2 (TGF-beta2) and insulin growth factor-1 (IGF-1) for 6-12 weeks. Pellets derived from fetal dermal fibroblasts (n = 4) were cultured under identical conditions. Additionally, expanded mesenchymal amniocytes were seeded onto biodegradable polyglycolic acid scaffolds (n = 5) and maintained in the same chondrogenic medium within a rotating bioreactor for 10-15 weeks. Engineered specimens were analyzed quantitatively and compared with native fetal hyaline cartilage samples (n = 5). Statistical analysis was by the unpaired Student's t-test (p < 0.05). The isolated cells stained positively for vimentin and cytokeratins-8 and -18, but negatively for CD31. Micromass pellets derived from mesenchymal amniocytes exhibited chondrogenic differentiation by both standard and matrix-specific staining. In contrast, these findings could not be replicated in dermal fibroblast-based pellets. The engineered constructs derived from mesenchymal amniocytes similarly displayed histological evidence of chondrogenic differentiation and maintained their original size and three-dimensional architecture. Quantitative assays of the engineered constructs revealed lower concentrations of collagen type II, but similar amounts of glycosaminoglycans, elastin, and DNA, when compared to native fetal hyaline cartilage. We conclude that mesenchymal amniocytes can be used for the engineering of cartilaginous tissue in vitro. Cartilage engineering from the amniotic fluid may become a practical approach for the surgical treatment of select congenital anomalies.     

Tissue engineered cartilage from hTGF beta2 transduced human adipose derived stem cells seeded in PLGA/alginate compound in vitro and in vivo

Chondrogenic potential of human adipose derived stem cells (hASCs) makes them a possible source of seeding cells for cartilage tissue engineering. We aim to examine the chondrogenic differentiation of human transforming growth factor beta2 (hTGF beta2) transduced hASCs seeded in three-dimensional scaffold in vitro and in vivo. In this study, hASCs were isolated from human subcutaneous adipose tissue and transduced with a replication deficient adenovirus carrying hTGF beta2 (Ad5-hTGF beta2), and then the transduced cells were seeded and cultured in PLGA/alginate compounds. RT-PCR analysis revealed that Ad5-hTGF beta2 transduced hASCs produced aggrecan and collagen type II after 7-day induction in vitro and continued throughout the culture period; this was also demonstrated by the positive staining of Alcian blue and immunohistochemistry for collagen type II. For in vivo study, Ad5-hTGF beta2 transduced hASCs seeded in PLGA/alginate compounds were implanted in subcutaneous pockets of nude mice; after 12 weeks, the implants were harvested and examined by haematoxylin and eosin staining, AB-PAS staining, and immunohistochemical analysis, and the results demonstrated the formation of cartilage tissue. As a control, all these were not observed in the constructs with Ad5-EGFP transduced hASCs. In conclusion, our study demonstrates that adenovirus-mediated hTGF beta2 gene transfer is able to induce the hASCs into chondrogenic lineage both in vitro and in vivo. Ad5-hTGF beta2 transduced hASCs combined with three-dimensional PLGA/alginate compound may be a viable method in treating injuries of cartilage.     

Chondrogenic derivatives of embryonic stem cells seeded into 3D polycaprolactone scaffolds generated cartilage tissue in vivo

In spite of recent scientific advances, treatment and repair of cartilage using tissue engineering techniques remains challenging. The major constraint is the limited proliferative capacity of mature autologous chondrocytes used in the tissue engineering approach. This problem can be addressed by using stem cells, which can self-renew with greater proliferative potential. Cartilage tissue engineering using adult mesenchymal stem cells derived from bone marrows has met with limited success. In this study we explored cartilage tissue generation from embryonic stem cells (ESCs). ESCs were induced to differentiate into chondroprogenitors, capable of proliferating and subsequently differentiating into cartilage-producing cells. The chondrogenic cells expressed chondrocyte-specific markers and deposited extracellular matrix proteins, proteoglycans. ESC-derived chondrogenic cells and polycaprolactone scaffolds seeded with these cells implanted in mice (129 SvImJ) generated cartilage tissue in vivo. Postimplant analysis of the retrieved tissues demonstrated cartilage-like tissue formation in 3-4 weeks. The cells of retrieved tissues also expressed the chondrocyte-specific marker collagen II. These findings suggest that ESCs can be used for tissue engineering and cultivation of cartilage tissues.     

Cartilage engineering from ovine umbilical cord blood mesenchymal progenitor cells

We aimed to determine whether three-dimensional (3D) cartilage could be engineered from umbilical cord blood (CB) cells and compare it with both engineered fetal cartilage and native tissue. Ovine mesenchymal progenitor cells were isolated from CB samples (n=4) harvested at 80-120 days of gestation by low-density fractionation, expanded, and seeded onto polyglycolic acid scaffolds. Constructs (n=28) were maintained in a rotating bioreactor with serum-free medium supplemented with transforming growth factor-beta1 for 4-12 weeks. Similar constructs seeded with fetal chondrocytes (n=13) were cultured in parallel for 8 weeks. All specimens were analyzed and compared with native fetal cartilage samples (n=10). Statistical analysis was by analysis of variance and Student's t-test (p<.01). At 12 weeks, CB constructs exhibited chondrogenic differentiation by both standard and matrix-specific staining. In the CB constructs, there was a significant time-dependent increase in extracellular matrix levels of glycosaminoglycans (GAGs) and type-II collagen (C-II) but not of elastin (EL). Fetal chondrocyte and CB constructs had similar GAG and C-II contents, but CB constructs had less EL. Compared with both hyaline and elastic native fetal cartilage, C-II and EL levels were, respectively, similar and lower in the CB constructs, which had correspondingly lower and similar GAG levels than native hyaline and elastic fetal cartilage. We conclude that CB mesenchymal progenitor cells can be successfully used for the engineering of 3D cartilaginous tissue in vitro, displaying select histological and functional properties of both native and engineered fetal cartilage. Cartilage engineered from CB may prove useful for the treatment of select congenital anomalies.     

The dependence of in vivo stable ectopic chondrogenesis by human mesenchymal stem cells on chondrogenic differentiation in vitro

In vivo niche plays an important role in determining the fate of implanted mesenchymal stem cells (MSCs) by directing committed differentiation. An inappropriate in vivo niche can also alter desired ultimate fate of exogenous MSCs even they are in vitro induced to express a specific phenotype before in vivo implantation. Studies have shown that in vitro chondrogenically differentiated MSCs are apt to lose their phenotype and fail to form stable cartilage in subcutaneous environment. We hypothesized that failure of maintaining the phenotype of induced MSCs in subcutaneous environment is due to the insufficient chondrogenic differentiation in vitro and fully differentiated MSCs can retain their chondrocyte-like phenotype and form stable ectopic cartilage. To test this hypothesis, extended in vitro chondrogenic induction and cartilage formation were carried out before implantation. Human bone marrow stem cells (hBMSCs) were seeded onto polylactic acid coated polyglycolic acid scaffolds. The cell-scaffold constructs were chondrogenically induced from 4 to 12 weeks for in vitro chondrogenesis, and then implanted subcutaneously into nude mice for 12 or 24 weeks. The engineered cartilages were evaluated by gross view, glycosaminoglycan content measurement, and histological staining before and after implantation. Histological examination showed typical cartilage structure formation after 8 weeks of induction in vitro. However, part of the constructs became ossified after implantation when in vitro induction lasted 8 weeks or less time. In contrast, those induced for 12 weeks in vitro could retain their cartilage structure after in vivo implantation. These results indicate that a fully differentiated stage achieved by extended chondrogenic induction in vitro is necessary for hBMSCs to form stable ectopic chondrogenesis in vivo.     

Chondrogenic differentiation of adipose-derived adult stem cells by a porous scaffold derived from native articular cartilage extracellular matrix

Adipose-derived adult stem cells (ASCs) have the ability to differentiate into a chondrogenic phenotype in response to specific environmental signals such as growth factors or artificial biomaterial scaffolds. In this study, we examined the hypothesis that a porous scaffold derived exclusively from articular cartilage can induce chondrogenesis of ASCs. Human ASCs were seeded on porous scaffolds derived from adult porcine articular cartilage and cultured in standard medium without exogenous growth factors. Chondrogenesis of ASCs seeded within the scaffold was evident by quantitative RT-PCR analysis for cartilage-specific extracellular matrix (ECM) genes. Histological and immunohistochemical examination showed abundant production of cartilage-specific ECM components-particularly, type II collagen-after 4 or 6 weeks of culture. After 6 weeks of culture, the cellular morphology in the ASC-seeded constructs resembled those in native articular cartilage tissue, with rounded cells residing in the glycosaminoglycan-rich regions of the scaffolds. Biphasic mechanical testing showed that the aggregate modulus of the ASC-seeded constructs increased over time, reaching 150 kPa by day 42, more than threefold higher than that of the unseeded controls. These results suggest that a porous scaffold derived from articular cartilage has the ability to induce chondrogenic differentiation of ASCs without exogenous growth factors, with significant synthesis and accumulation of ECM macromolecules, and the development of mechanical properties approaching those of native cartilage. These findings support the potential for a processed cartilage ECM as a biomaterial scaffold for cartilage tissue engineering. Additional in vivo evaluation is necessary to fully recognize the clinical implication of these observations.     

Engineering cartilage with human nasal chondrocytes and a silanized hydroxypropyl methylcellulose hydrogel

Tissue engineering strategies, based on developing three-dimensional scaffolds capable of transferring autologous chondrogenic cells, holds promise for the restoration of damaged cartilage. In this study, the authors aimed at determining whether a recently developed silanized hydroxypropyl methylcellulose (Si-HPMC) hydrogel can be a suitable scaffold for human nasal chondrocytes (HNC)-based cartilage engineering. Methyltetrazolium salt assay and cell counting experiments first revealed that Si-HPMC enabled the proliferation of HNC. Cell tracker green staining further demonstrated that HNC were able to form nodular structures in this three-dimensional scaffold. HNC phenotype was then assessed by RT-PCR analysis of type II collagen and aggrecan expression as well as alcian blue staining of extracellular matrix. Our data indicated that Si-HPMC allowed the maintenance and the recovery of a chondrocytic phenotype. The ability of constructs HNC/Si-HPMC to form a cartilaginous tissue in vivo was finally investigated after 3 weeks of implantation in subcutaneous pockets of nude mice. Histological examination of the engineered constructs revealed the formation of a cartilage-like tissue with an extracellular matrix containing glycosaminoglycans and type II collagen. The whole of these results demonstrate that Si-HPMC hydrogel associated to HNC is a convenient approach for cartilage tissue engineering.     

Three-dimensional tissue engineering of hyaline cartilage: comparison of adult nasal and articular chondrocytes

Adult chondrocytes are less chondrogenic than immature cells, yet it is likely that autologous cells from adult patients will be used clinically for cartilage engineering. The aim of this study was to compare the postexpansion chondrogenic potential of adult nasal and articular chondrocytes. Bovine or human chondrocytes were expanded in monolayer culture, seeded onto polyglycolic acid (PGA) scaffolds, and cultured for 40 days. Engineered cartilage constructs were processed for histological and quantitative analysis of the extracellular matrix and mRNA. Some engineered constructs were implanted in athymic mice for up to six additional weeks before analysis. Using adult bovine tissues as a cell source, nasal chondrocytes generated a matrix with significantly higher fractions of collagen type II and glycosaminoglycans as compared with articular chondrocytes. Human adult nasal chondrocytes proliferated approximately four times faster than human articular chondrocytes in monolayer culture, and had a markedly higher chondrogenic capacity, as assessed by the mRNA and protein analysis of in vitro-engineered constructs. Cartilage engineered from human nasal cells survived and grew during 6 weeks of implantation in vivo whereas articular cartilage constructs failed to survive. In conclusion, for adult patients nasal septum chondrocytes are a better cell source than articular chondrocytes for the in vitro engineering of autologous cartilage grafts. It remains to be established whether cartilage engineered from nasal cells can function effectively when implanted at an articular site.     

Ectopic neocartilage formation from predifferentiated human adipose derived stem cells induced by adenoviral-mediated transfer of hTGF beta2

Chondrogenic potential of human adipose derived stem cells (hASCs) makes them a possible source of seeding cells for cartilage tissue engineering. In this study, chondrogenic differentiation of hASCs induced by transduction with replication-deficient adenovirus carrying human transforming growth factor beta2 (Ad5-hTGF beta2) was demonstrated by RT-PCR, immunohistochemistry staining, biochemical and western blot analysis. To evaluate if the in vitro differentiated hASCs could keep their chondrocytic phenotype and produce neo-cartilage in vivo, predifferentiated hASCs were seeded in different scaffolds and implanted in subcutaneous pockets on the dorsum of nude mice. After 4 and 12 weeks culture in vivo, specimens were harvested and examined by histological and immunohistochemical analysis, cartilage-like tissue formation was only found in alginate gel and PLGA/alginate compound groups, in PLGA group, fibrous tissues and angiogenesis ingrowth were observed. These findings demonstrated that adenovirus-mediated hTGF beta2 gene transfer could induce hASCs into a chondrogenic lineage in vitro, however, this predifferentiation did not guarantee ectopic cartilage formation in vivo unless appropriate three-dimensional scaffolds were used as the cell carry vehicles.     

Novel Melt-Processable Chitosan–Polybutylene Succinate Fibre Scaffolds for Cartilage Tissue Engineering

Novel chitosan/polybutylene succinate fibre-based scaffolds (C-PBS) were seeded with bovine articular chondrocytes in order to assess their suitability for cartilage tissue engineering. Chondrocytes were seeded onto C-PBS scaffolds using spinner flasks under dynamic conditions, and cultured under orbital rotation for a total of 6 weeks. Non-woven polyglycolic acid (PGA) felts were used as reference materials. Tissue-engineered constructs were characterized by scanning electron microscopy (SEM), hematoxylin–eosin (H&E), toluidine blue and alcian blue staining, immunolocalization of collagen types I and II, and dimethylmethylene blue (DMB) assay for glycosaminoglycans (GAG) quantification at different time points. SEM showed the chondrocytes' typical morphology, with colonization at the surface and within the pores of the C-PBS scaffolds. These observations were supported by routine histology. Toluidine blue and alcian blue stains, as well as immunohistochemistry for collagen types I and II, provided qualitative information on the composition of the engineered extracellular matrix. More pronounced staining was observed for collagen type II than collagen type I. Similar results were observed with constructs engineered on PGA scaffolds. These also exhibited higher amounts of matrix glycosaminoglycans and presented a central region which contained fewer cells and little matrix, a feature that was not detected with C-PBS constructs.     

三维环境下软骨细胞诱导滑膜间充质干细胞向软骨样细胞的分化

BACKGROUND: The co-culture of chondrocytes and synovial mesenchymal stem cells can induce the cartilage differentiation of synovial mesenchymal stem cells in vitro, but the cell differentiation induced by co-culture in vivo is rarely reported. OBJECTIVE: To investigate the chondrogenic differentiation of synovial mesenchymal stem cells co-cultured with chondrocytes on the chitosan/type I collagen composite scaffolds after being transplanted into the subcutaneous layer of Sprague-Dawley rats. METHODS: The synovial mesenchymal stem cells and chondrocytes harvested from the synovial membrane and articular cartilage of Sprague-Dawley rats were obtained by enzyme digestion method and cultured respectively. Passage 3 synovial mesenchymal stem cells and passage 2 chondrocytes, which were divided into four groups: group A (chondrocytes alone), group B (synovial mesenchymal stem cells alone), group C (ratio of synovial mesenchymal stem cells:chondrocytes=1:2) and group D (scaffold material without cells), were cultured on chitosan/type I collagen composite scaffolds and transplanted into the subcutaneous layer of rats followed by morphological observation and immunohistochemical staining at 4 and 8 weeks.   . RESULTS AND CONCLUSION: After 4 and 8 weeks, the discoid-like scaffold was visible. The immunohistochemical staining of type II collagen and the toluidine blue staining of aggrecan were significantly positive in groups A and C. These results show that the co-culture of synovial mesenchymal stem cells and chondrocytes on the scaffold in vivo can form cartilage-like tissues.      

Chondrogenic and osteogenic differentiations of human bone marrow-derived mesenchymal stem cells on a nanofibrous scaffold with designed pore network

The utilization of 3D scaffolds and stem cells is a promising approach to solve the problem of bone and cartilage tissue shortage and to construct osteochondral (cartilage/bone composite) tissues. In this study, 3D highly porous nanofibrous (NF) poly(l-lactic acid) (PLLA) scaffolds fabricated using a phase separation technique were seeded with multi-potent human bone marrow-derived mesenchymal stem cells (hMSCs) and the constructs were induced along osteogenic and chondrogenic development routes in vitro. Histological analysis and calcium content quantification showed that NF scaffolds supported in vitro bone differentiation. SEM observation showed an altered shape for cells cultured on an NF matrix compared with those on smooth films. Consistent with the morphological change, the gene expression of early chondrogenic commitment marker Sox-9 was enhanced on the NF matrix. NF scaffolds were then used to support long-term in vitro 3D cartilaginous development. It was found that in the presence of TGF-β1, cartilage tissue developed on PLLA NF scaffolds, with the cartilage-specific gene expressed, glycosaminoglycan and type II collagen accumulated, and typical cartilage morphology formed. These findings suggest that NF scaffolds can support both bone and cartilage development and are excellent candidate scaffolds for osteochondral defect repair.     

Chondrogenesis in perfusion bioreactors using porous silk scaffolds and hESC-derived MSCs

Tissue engineered cartilage can be grown in vitro with the use of cell-scaffold constructs and bioreactors. The present study was designed to investigate the effects of perfusion bioreactors on the chondrogenic potential of engineered constructs prepared from porous silk fibroin scaffolds seeded with human embryonic stem cell (hESC)-derived mesencyhmal stem cells (MSCs). After four weeks of incubation, constructs cultured in perfusion bioreactors showed significantly higher amounts of glycosaminoglycans (GAGs) (p < 0.001), DNA (p < 0.001), total collagen (p < 0.01), and collagen II (p < 0.01) in comparison to static culture. Mechanical stiffness of constructs increased 3.7-fold under dynamic culture conditions and RT-PCR results concluded that cells cultured in perfusion bioreactors highly expressed (p < 0.001) cartilage-related genes when compared with static culture. Distinct differences were noted in tissue morphology, including polygonal extracellular matrix structure of engineered constructs in thin superficial zones and an inner zone under static and dynamic conditions, respectively. The results suggest that the utility of perfusion bioreactors to modulate the growth of tissue-engineered cartilage and enhance tissue growth in vitro.