Adenovirus-mediated expression of growth and differentiation factor-5 promotes chondrogenesis of adipose stem cells

The repair of articular cartilage injuries is impeded by the avascular and non-innervated nature of cartilage. Transplantation of autologous chondrocytes has a limited ability to augment the repair process due to the highly differentiated state of chondrocytes and the risks of donor-site morbidity. Mesenchymal stem cells can undergo chondrogenesis in the presence of growth factors for cartilage defect repair. Growth and differentiation factor-5 (GDF5) plays an important role in chondrogenesis. In this study, we examined the effects of GDF5 on chondrogenesis of adipose-derived stem cells (ADSCs) and evaluate the chondrogenic potentials of GDF5 genetically engineered ADSCs using an in vitro pellet culture model. Rat ADSCs were grown as pellet cultures and treated with chondrogenic media (CM). Induction of GDF5 by an adenovirus (Ad-GDF5) was compared with exogenous supplementation of GDF5 (100 ng/ml) and transforming growth factor-β (TGF-β1; 10 ng/ml). The ADSCs underwent chondrogenic differentiation in response to GDF5 exposure as demonstrated by production of proteoglycan, and up-regulation of collagen II and aggrecan at the protein and mRNA level. The chondrogenic potential of a one-time infection with Ad-GDF5 was weaker than exogenous GDF5, but equal to that of TGF-β1. Stimulation with growth factors or CM alone induced transient expression of the mRNA for collagen X, indicating a need for optimization of the CM. Our findings indicate that GDF5 is a potent inducer of chondrogenesis in ADSCs, and that ADSCs genetically engineered to express prochondrogenic growth factors, such as GDF5, may be a promising therapeutic cell source for cartilage tissue engineering.     

Chondrogenic differentiation of mesenchymal stem cells isolated from patients in late adulthood: the optimal conditions of growth factors

There is a controversy about the capacity of the mesenchymal stem cells (MSCs) from aged individuals to proliferate and differentiate into cartilage. The purpose of this study was to investigate the optimal condition to culture human MSCs from the aged individuals (>50 years) for cartilage tissue engineering. We tested the hypothesis that effective proliferation and chondrogenesis can be achieved with human MSCs from aged individuals under appropriate conditions. To investigate the best condition for proliferation, MSCs were cultured in medium containing four concentrations subsets (0, 0.05, 0.5, 5 ng/mL) of recombinant human TGF-beta2 and FGF-2, either with or without fetal calf serum. The cell numbers were counted 0, 1, 3, and 7 days after growth factors were given. For the induction of chondrogenesis in 3-dimensional (3-D) culture, cells were cultured in pellets with chondrogenic medium containing combinations of various growth factors. After 4 weeks of culture, the pellets were fixed and evaluated with Safranin-O staining for proteoglycan and immunohistochemical staining for type II collagen. RT-PCR was also performed for the mRNAs of type I collagen, type II collagen, and cartilage oligomeric protein (COMP). In a monolayer culture, TGF-beta2 in concentrations of 0.5 and 5 ng/mL caused significant reduction in cell number irrespective of the presence of serum. FGF-2 of 5 ng/mL most effectively increased cell number even in the absence of serum. In a pellet culture, remarkable chondrocyte-like differentiation of cells was induced around the peripheral areas of a pellet with 5 ng/mL of TGF-beta2, accompanied by increased proteoglycan and type II collagen production. The addition of 100 ng/mL of IGF-I induced notable increase in proteoglycan contents. The results of RT-PCR mirrored those of histological studies. This study shows that an effective proliferation and chondrogenesis may be obtained with proper combinations of growth factors and mesenchymal stem cells from aged individuals.     

An in vitro study of collagen hydrogel to induce the chondrogenic differentiation of mesenchymal stem cells

It is controversial whether a biomaterial itself, rather than addition of any exogenous growth factor, could induce mesenchymal stem cells (MSCs) to differentiate into chondrogenic lineage, further to regenerate cartilage. Previous studies have shown that collagen-based hydrogel could induce MSCs to differentiate into chondrocytes in vivo but the in vitro studies only have a few reports. The evidence that biomaterials could induce chondrogenesis is not adequate. In this study, we tried to address whether type I collagen hydrogel has chondro-inductive capability in vitro and how this scaffold induces MSCs to generate cartilage tissue without exogenous growth factors in the culture medium. We encapsulated neonatal rabbit bone marrow mesenchymal stem cells (BMSCs) in type I collagen hydrogel homogeneously or implanted cell aggregates in hydrogel, and cultured them in nonchondrogenic inductive media. After at least 28 days culture, cells in the homogeneous group were tending to chondrogenic differentiation while cell density was high, and cells in the aggregate group have almost gone through chondrogenesis and formed neo-cartilage tissue with abundant specific extracellular matrix (ECM) deposition. These results indicate collagen hydrogel has inherent inductivity for the chondrogenic differentiation of BMSCs, and the optimum specification and tissue formation were accompanied with local high cell density. This research suggests a feasible strategy to induce the chondro differentiation of BMSCs independent of exogenous growth factors, which may greatly contribute to clinical cartilage regeneration. ? 2012 Wiley Periodicals, Inc. J Biomed Mater Res Part A 100A: 2717-2725, 2012.     

A comparison between the chondrogenic potential of human bone marrow stem cells (BMSCs) and adipose-derived stem cells (ADSCs) taken from the same donors

Cartilage damage has been documented as one of the major problems leading to knee repair procedures worldwide. The low availability of cartilage that can be harvested without causing a negative health impact has led to the focus on the potential of stem cells, which have been transplanted into damaged areas and successfully grown into new healthy tissue. This study aims to compare the chondrogenic potential of two stem cell sources--adipose tissue and bone marrow. Stem cells were isolated from donor-matched adipose tissue and bone marrow, following established protocols. The cells were grown in a chondrogenic cocktail containing transforming growth factor-beta3 (TGF-beta3) up till 28 days, and assessed for expression changes of cartilage markers at the gene and protein level, using qualitative and quantitative methods. Controls were included for every time point. Real-time polymerase chain reaction (PCR) results showed increases in the gene expression of collagen II in both the cell types that received TGF-beta3 treatment. However, histological, immunohistochemical, and glycosaminoglycan (GAG) assay clearly showed that collagen II and proteoglycans (PG) were synthesized only in the growth factor-treated bone marrow stem cells (BMSCs). These findings support the results obtained in our in vivo comparative study done on an animal model, suggesting that BMSCs are more suitable than adipose-derived stem cells (ADSCs) for chondrogenesis.     

IGF-1 and BMP-2 Induces Differentiation of Adipose-Derived Mesenchymal Stem Cells into Chondrocytes-Like Cells

Articular cartilage defects are common, causing significant morbidities. Tissue engineering using pluripotent stem cells is a new promising modality for cartilage repair. In the current study, we investigated the chondrogenesis of rabbit adipose-derived stem cells (ADSCs). We isolated rabbit ADSCs and transfected these cells with constructs encoding human insulin growth like factor 1 (IGF-1) and bone morphogenic protein 2 (BMP-2). We examined the growth and morphology of these transfected cells and their production of type II collagen and MMP-3. We found that IGF-1 and BMP-2 drove the chondrogenesis of ADSCs, which showed mature chondrocyte-like cells and formed cartilage nodules. These cells also produced type II collagen with a reduced production of MMP-3. Our findings suggested that human ADSCs could differentiate into chondrocyte-like cells driven by IGF-1 and BMP-2 and held promises as an abundant and ready source of stem cells for cartilage repair and regeneration.     

The effect of co-culturing costal chondrocytes and dental pulp stem cells combined with exogenous FGF9 protein on chondrogenesis and ossification in engineered cartilage

Dental pulp stem cells (DPSCs), which arise from cranial neural crest cells, are multipotent, making them a candidate for use in tissue engineering that may be especially useful for craniofacial tissues. Costal chondrocytes (CCs) can be easily obtained and demonstrate higher initial cell yields and expansion than articular chondrocytes. CCs have been found to retain chondrogenic capacity that can effectively repair articular defects. In this study, human CCs were co-cultured with human DPSCs, and the results showed that the CCs were able to supply a chondro-inductive niche that promoted the DPSCs to undergo chondrogenic differentiation and to enhance the formation of cartilage. Although CCs alone could not prevent the mineralization of chondro-differentiated DPSCs, CCs combined with exogenous FGF9 were able to simultaneously promote the chondrogenesis of DPSCs and partially inhibit their mineralization. Furthermore, FGF9 may activate this inhibition by binding to FGFR3 and enhancing the phosphorylation of ERK1/2 in DPSCs. Our results strongly suggest that the co-culture of CCs and DPSCs combined with exogenous FGF9 can simultaneously enhance chondrogenesis and partially inhibit ossification in engineered cartilage.     

Engineering of rat articular cartilage on porous sponges: effects of tgf-beta 1 and microgravity bioreactor culture

The objective of this study was to develop an engineered rat hyaline cartilage by culturing articular chondrocytes on three-dimensional (3D) macroporous poly(DL-lactic-co-glycolic acid) (PLGA) sponges under chondrogenic induction and microgravity bioreactor conditions. Experimental groups consisted of 3D static and dynamic cultures, while a single cell monolayer (2D) served as the control. The effect of seeding conditions (static vs. dynamic) on cellularization of the scaffolds was investigated. MTT assay was used to evaluate the number of viable cells in each group at different time points. Formation of a hyaline-like cartilage was evaluated for up to 4 weeks in vitro. While 2D culture resulted in cell sheets with very poor matrix production, 3D culture was in the favor of tissue formation. A higher yield of cell attachment and spatially uniform cell distribution was achieved when dynamic seeding technique was used. Dynamic culture promoted cell growth and infiltration throughout the sponge structure and showed the formation of cartilage tissue, while chondrogenesis appeared attenuated more towards the outer region of the constructs in the static culture group. Medium supplemented with TGF-beta 1 (5 ng/ml) had a positive impact on proteoglycan production as confirmed by histochemical analyses with Alcian blue and Safranin-O stainings. Formation of hyaline-like tissue was demonstrated by immunohistochemistry performed with antibodies against type II collagen and aggrecan. SEM confirmed higher level of cellularization and cartilage tissue formation in bioreactor cultures induced by TGF-beta 1. The data suggest that PLGA sponge inside rotating bioreactor with chondrogenic medium provides an environment that mediates isolated rat chondrocytes to redifferentiate and form hyaline-like rat cartilage, in vitro.     

Is continuous treatment with transforming growth factor-beta necessary to induce chondrogenic differentiation in mesenchymal stem cells?

This study was conducted to determine whether short-term administration of transforming growth factor (TGF)-beta would be as effective for inducing chondrogenesis in human mesenchymal stem cells (hMSCs) as continuous treatment. Four groups of hMSCs were cultured in a monolayer for 3 days followed by a pellet culture for 3 weeks under various conditions: group A, the control group, no growth factors treated; group B, 5 ng/ml of TGF-beta(2) was treated for 3 days in monolayer culture; group C, 5 ng/ml of TGF-beta(2) was treated for 3 days in a monolayer culture and the initial 3 days of pellet culture; group D, 5 ng/ml of TGF-beta(2) was treated for 3 days in a monolayer culture and the initial 10 days of pellet culture; group E, 5 ng/ml of TGF-beta(2) was continuously treated throughout the culture period. Glycosaminoglycan contents significantly increased in group E only. Real-time PCR indicated that expression of Sox-9, type II collagen, type II procollagen B and type X collagen increased with longer duration of TGF-beta(2) treatment. The histological findings showed that longer duration of TGF-beta(2) treatment led to significantly better quality of chondrogenesis. This study demonstrated that longer duration of TGF-beta treatment is necessary for effective chondrogenesis in hMSCs from bone marrow.     

重组hTGF-β1基因转染人脂肪干细胞对其向软骨分化的影响

目的研究重组hTGF-β1腺病毒(AdhTGF-β1)转染人脂肪干细胞(ADSCs)对其向软骨分化的作用。方法重组AdhTGF-β1转染人ADSCs,对照组转染AdLacZ,腺病毒的量以200pfu/细胞计算,体外细胞团聚集连续诱导培养21d,酶联免疫吸附定量检测(ELISA)hTGF-β1蛋白的表达,然后分别从大体观察、组织学和II型胶原蛋白免疫组化的检测对形成组织进行评价。结果 hTGF-β1蛋白量在14d时达最高峰,随后逐渐降低。连续细胞团聚集诱导培养21d,细胞团收缩成近似小球形的组织块,外观成乳白色。HE染色可见细胞团外周为由数层扁平状成纤维样细胞组成的纤维软骨膜,下部区域有巢状软骨样细胞组成,有些区域可见软骨样细胞包埋在软骨陷窝内。Safranin'O染色显示,形成的软骨组织区域有被染成桔红色蛋白多糖类基质分泌。而对照组苏木素-伊红染色观察见无软骨样组织形成或有向软骨分化现象。Ⅱ型胶原免疫组化染色检测显示实验组细胞团出现较明显的阳性染色区域,可见棕黄色的颗粒分布于胞浆内。对照组Ⅱ型胶原免疫组化染色检测显示无明显的阳性染色区。结论重组hTGF-β1腺病毒转染人ADSCs诱导人ADSCs向软骨细胞表型分化形成软骨样组织,为hTGF-β1基因转染的人ADSCs在软骨组织工程应用中奠定了基础。     

Xenogeneic chondrocytes promote stable subcutaneous chondrogenesis of bone marrow-derived stromal cells

The local microenvironment may change the ultimate fate of engineered cartilage differentiated from bone marrow stromal cells (BMSCs) after subcutaneous implantation. Chondrogenically differentiated BMSCs directed by growth factors or low-intensity ultrasound are apt to fibrose or vascularize in the subcutaneous environment, while BMSCs implanted in articular cartilage defects can form stable cartilage. We hypothesized that chondrocytes would provide an ideal chondrogenic environment, and thus promote the maintenance of the chondrocytic phenotype in ectopia. To test this hypothesis, we developed a new method to promote chondrocyte development from BMSCs in a chondrogenic environment produced by xenogeneic chondrocytes and compared the subcutaneous chondrogenesis of BMSCs mediated by xenogeneic chondrocytes with that produced by growth factors. These results indicate that subcutaneous chondrogenesis of BMSCs directed by xenogeneic chondrocytes is more effective than that induced by growth factors. BMSCs induced by xenogeneic chondrocytes formed relatively mature cartilage before or after implantation, following 4 weeks of culture, which reduced the induction time in?vitro and led to maintenance of a stable cartilage phenotype after subcutaneous implantation.     

Effects of co-culturing BMSCs and auricular chondrocytes on the elastic modulus and hypertrophy of tissue engineered cartilage

Co-culture of BMSCs and chondrocytes is considered as a promising strategy to generate tissue engineered cartilage as chondrocytes induce the chondrogenesis of BMSCs and inhibit the hypertrophy of engineered cartilage. Because the tissue specific stem/progenitor cells have been isolated from mature tissues including auricular cartilage, we hypothesized that adding stem cells to auricular chondrocytes in co-culture would also enhance the quality of engineered cartilage. In the present study, using the histological assay, biomechanical evaluation, and quantitative analysis of gene expression, we compared different strategies of auricular chondrocytes, BMSCs induction, and co-culture at different ratios on PGA/PLA scaffolds to construct tissue engineered elastic cartilage in vitro and in vivo. The up-regulation of RUNX2 and down-regulation of SOX9 were found in BMSCs chondrogenic induction group, which might imply a regulatory mechanism for the hypertrophy and potential osteogenic differentiation. Engineered cartilage in co-culture 5:5 group showed the densest elastic fibers and the highest Young's modulus, which were consistent with the expression profile of cartilage matrix-related genes including DCN and LOXL2 genes. Moreover, the better proliferative and chondrogenic potentials of engineered cartilage in co-culture 5:5 group were demonstrated by the stronger expression of Ki67 and Dlk1.     

Chondrocytic differentiation of human adipose-derived adult stem cells in elastin-like polypeptide

Human adipose derived adult stem (hADAS) cells have the ability to differentiate into a chondrogenic phenotype in three-dimensional culture and media containing dexamethasone and TGF-beta. The current study examined the potential of a genetically engineered elastin-like polypeptide (ELP) to promote the chondrocytic differentiation of hADAS cells without exogenous chondrogenic supplements. hADAS cells were cultured in ELP hydrogels in either chondrogenic or standard medium at 5% O2 for up to 2 weeks. By day 14, constructs cultured in either medium exhibited significant increases in sulfated glycosaminoglycan (up to 100%) and collagen contents (up to 420%). Immunolabeling confirmed that the matrix formed consisted mainly of type II and not type I collagen. The composition of the constructs cultured in either medium did not differ significantly. To assess the effect of oxygen tension on the differentiation of the above constructs, samples were cultured in standard medium at either 5% or 20% O2 for 7 days and their gene expression profile was evaluated using real time RT-PCR. In both cases, the hADAS-ELP constructs upregulated SOX9 and type II collagen gene expression, while type I collagen was downregulated. However, constructs cultured in 20% O2 highly upregulated type X collagen, which was not detected in the 5% O2 cultures. The study suggests that ELP can promote chondrogenesis for hADAS cells in the absence of exogenous TGF-beta1 and dexamethasone, especially under low oxygen tension conditions.     

Enhanced in vitro chondrogenesis of primary mesenchymal stem cells by combined gene transfer

Because articular cartilage has a poor regeneration capacity, numerous cell-based approaches to therapy are currently being explored. The present study involved the use of gene transfer as a means to provide sustained delivery of chondrogenic proteins to primary mesenchymal stem cells (MSCs). In previous work, we found that adenoviral-mediated gene transfer of transforming growth factor-beta1 (TGF-beta1) and bone morphogenetic protein 2 (BMP-2), but not insulin-like growth factor 1 (IGF-1), could be used to induce chondrogenic differentiation of MSCs in an aggregate culture system. In the present study, we examined the effects on chondrogenesis of these transgenes when delivered in combination. Cultures of bone marrow-derived MSCs were infected with 2.5 x 10(2) or 2.5 x 10(3) viral particles/cell of each adenoviral vector individually, or in combination, seeded into aggregates, and cultured for 3 weeks in a defined serum-free medium. Levels of transgene product in the medium were initially high, approximately 100 ng/mL TGF-beta1, 120 ng/mL BMP-2, and 80 ng/mL IGF-1 at day 3, and declined thereafter. We found that co-expression of IGF-1 and TGF-beta1, BMP-2, or both at low doses resulted in larger aggregates, higher levels of glycosaminoglycan synthesis, stronger staining for proteoglycans and collagen type II and X, and greater expression of cartilage-specific marker genes than with either transgene alone. Gene-induced chondrogenesis of MSCs using multiple genes that act synergistically may enable the administration of reduced viral doses in vivo and could be of considerable benefit for the development of cell-based therapies for cartilage repair.     

兔脂肪干细胞在含有转化因子和转铁蛋白培养基中向软骨细胞的分化

背景：成软骨的种子细胞的选择是软骨组织工程研究中的关键因素。 目的：观察脂肪干细胞在含有转化因子和转铁蛋白诱导条件下向软骨细胞分化的能力。 方法：取新西兰大白兔颈背部脂肪组织,机械分离及酶消化法获得脂肪干细胞,显微镜下观察细胞黏附及生长情况;加入含有转化因子β1和转铁蛋白的诱导培养基培养2周后以免疫组化方法检测Ⅱ型胶原的表达。 结果与结论：从兔脂肪组织中分离出的干细胞原代培养时24 h贴壁,96 h后达80%融合;于软骨诱导培养基内向软骨诱导7 d后形成软骨结节,14 d后Ⅱ型胶原免疫组化阳性。结果初步表明兔脂肪干细胞经诱导后可以向软骨细胞分化。     

Hyaline cartilage engineered by chondrocytes in pellet culture: histological, immunohistochemical and ultrastructural analysis in comparison with cartilage explants

Cartilage engineering is a strategic experimental goal for the treatment of multiple joint diseases. Based on the process of embryonic chondrogenesis, we hypothesized that cartilage could be engineered by condensing chondrocytes in pellet culture and, in the present study, examined the quality of regenerated cartilage in direct comparison with native cartilage. Chondrocytes isolated from the sterna of chick embryos were cultured in pellets (4 x 10(6) cells per pellet) for 2 weeks. Cartilage explants from the same source were cultured as controls. After 2 weeks, the regenerated cartilage from pellet culture had a disc shape and was on average 9 mm at the longest diameter. The chondrocyte phenotype was stabilized in pellet culture as shown by the synthesis of type II collagen and aggrecan, which was the same intensity as in the explant after 7 days in culture. During culture, chondrocytes also continuously synthesized type IX collagen. Type X collagen was negatively stained in both pellets and explants. Except for fibril orientation, collagen fibril diameter and density in the engineered cartilage were comparable with the native cartilage. In conclusion, hyaline cartilage engineered by chondrocytes in pellet culture, without the transformation of cell phenotypes and scaffold materials, shares similarities with native cartilage in cellular distribution, matrix composition and density, and ultrastructure.     

Glucocorticoids promote chondrogenic differentiation of adult human mesenchymal stem cells by enhancing expression of cartilage extracellular matrix genes

In the adult human, mesenchymal stem cells (hMSCs) resident in the bone marrow retain the capacity to proliferate and differentiate along multiple connective tissue lineages, including cartilage. Glucocorticoids (GCs) are required for chondrogenic differentiation of hMSCs in vitro; however, the exact role of GCs in this process is not known. In this study, we examined the effects of dexamethasone (DEX) on chondrogenic differentiation of hMSCs in the presence or absence of DEX, transforming growth factor-beta (TGF-beta), or DEX plus TGF-beta. GC treatment upregulated gene expression of cartilage matrix components aggrecan, dermatopontin, and collagen type XI; enhanced TGF-beta-mediated upregulation of collagen type II and cartilage oligomeric matrix protein; and increased aggrecan and collagen type II production as well as cartilage matrix-sulfated proteoglycans as assessed by immunohistochemistry and alcian blue staining. Inclusion of an antagonist of GCs inhibited expression of chondrogenic differentiation markers, suggesting that the GC effects during chondrogenesis are mediated by the GC receptor (GR). Steady levels of the major active form of GR, GRalpha, were detected in both undifferentiated and differentiating hMSCs, whereas the dominant-negative isoform GRbeta, present at low levels in undifferentiated hMSCs, was downregulated during chondrogenesis. In the presence of DEX and TGF-beta, expression of a collagen type II gene promoter luciferase reporter construct in hMSCs was upregulated. However, coexpression of GRbeta dramatically inhibited promoter activity, suggesting that GRalpha is required for GC-mediated modulation of chondrogenesis and that GCs may play an important role in the maintenance of cartilage homeostasis.     

The use of de-differentiated chondrocytes delivered by a heparin-based hydrogel to regenerate cartilage in partial-thickness defects

Partial-thickness cartilage defects, with no subchondral bone injury, do not repair spontaneously, thus there is no clinically effective treatment for these lesions. Although the autologous chondrocyte transplantation (ACT) is one of the promising approaches for cartilage repair, it requires in vitro cell expansion to get sufficient cells, but chondrocytes lose their chondrogenic phenotype during expansion by monolayer culture, leading to de-differentiation. In this study, a heparin-based hydrogel was evaluated and optimized to induce cartilage regeneration with de-differentiated chondrocytes. First, re-differentiation of de-differentiated chondrocytes encapsulated in heparin-based hydrogels was characterized in vitro with various polymer concentrations (from 3 to 20 wt.%). Even under a normal cell culture condition (no growth factors or chondrogenic components), efficient re-differentiation of cells was observed with the optimum at 10 wt.% hydrogel, showing the complete re-differentiation within a week. Efficient re-differentiation and cartilage formation of de-differentiated cell/hydrogel construct were also confirmed in vivo by subcutaneous implantation on the back of nude mice. Finally, excellent cartilage regeneration and good integration with surrounding, similar to natural cartilage, was also observed by delivering de-differentiated chondrocytes using the heparin-based hydrogel in partial-thickness defects of rabbit knees whereas no healing was observed for the control defects. These results demonstrate that the heparin-based hydrogel is very efficient for re-differentiation of expanded chondrocytes and cartilage regeneration without using any exogenous inducing factors, thus it could serve as an injectable cell-carrier and scaffold for cartilage repair. Excellent chondrogenic nature of the heparin-based hydrogel might be associated with the hydrogel characteristic that can secure endogenous growth factors secreted from chondrocytes, which then can promote the chondrogenesis, as suggested by the detection of TGF-β1 in both in vitro and in vivo cell/hydrogel constructs.     

Effects of cyclic compressive loading on chondrogenesis of rabbit bone-marrow derived mesenchymal stem cells

The objective of this study was to examine the effects of cyclic compressive loading on chondrogenic differentiation of rabbit bone-marrow mesenchymal stem cells (BM-MSCs) in agarose cultures. Rabbit BM-MSCs were obtained from the tibias and femurs of New Zealand white rabbits. After the chondrogenic potential of BM-MSCs was verified by pellet cultures, cell-agarose constructs were made by suspending BM-MSCs in 2% agarose (10(7) cells/ml) for a cyclic, unconfined compression test performed in a custom-made bioreactor. Specimens were divided into four groups: control; transforming growth factor (TGF-beta) (with TGF-beta1 treatment); loading (with stimulation of cyclic, unconfined compressive loading); and TGF-beta loading (with TGF-beta1 treatment and loading stimulation) groups. In the loading experiment, specimens were subjected to sinusoidal loading with a 10% strain magnitude at a frequency of 1 Hz for 4 hours a day. Experiments were conducted for 3, 7, and 14 consecutive days. While the experimental groups (TGF-beta, loading, and TGF-beta loading) exhibited significantly higher levels of expressions of chondrogenic markers (collagen II and aggrecan) at three time periods, there were no differences among the experimental groups after an extra 5-day culture. This suggests that compressive loading alone induces chondrogenic differentiation of rabbit BM-MSCs as effectively as TGF-beta or TGF-beta plus loading treatment. Moreover, both the compressive loading and the TGF-beta1 treatment were found to promote the TGF-beta1 gene expression of rabbit BM-MSCs. These findings suggest that cyclic compressive loading can promote the chondrogenesis of rabbit BM-MSCs by inducing the synthesis of TGF-beta1, which can stimulate the BM-MSCs to differentiate into chondrocytes.