Induction of superficial zone protein (SZP)/lubricin/PRG 4 in muscle-derived mesenchymal stem/progenitor cells by transforming growth factor-β1 and bone morphogenetic protein-7

Introduction

Articular cartilage (AC) is an avascular tissue with precise polarity and organization. The three distinct zones are: surface, middle and deep. The production and accumulation of the superficial zone protein (SZP), also known as lubricin, by the surface zone is a characteristic feature of AC. To date, there is a wealth of evidence showing differentiation of AC from mesenchymal stem cells. Most studies that described chondrogenic differentiation did not focus on AC with characteristic surface marker SZP/lubricin. The present investigation was initiated to determine the induction of SZP/lubricin in skeletal muscle-derived mesenchymal stem/progenitor cells (MDMSCs) by transforming growth factor-β1 (TGF-β1) and bone morphogenetic protein-7 (BMP-7).

Methods

MDMSCs were cultured as a monolayer at a density of 1 × 10⁵ cells/well in 12-well tissue culture plates. Cell cultures were treated for 3, 7 and 10 days with TGF-β1 and BMP-7. The medium was analyzed for SZP. The cells were used to isolate RNA for RT-PCR assays for SZP expression.

Results

The SZP/lubricin increased in a time-dependent manner on Days 3, 7 and 10 in the medium. As early as Day 3, there was a three-fold increase in response to 3 ng/ml of TGF-β1 and 300 ng/ml of BMP-7. This was confirmed by immunochemical localization of SZP as early as Day 3 after treatment with TGF-β1. The expression of SZP mRNA was enhanced by TGF-β1.

Conclusions

The present investigation demonstrated the efficient and reproducible induction of SZP/lubricin accumulation by TGF-β1 and BMP-7 in skeletal MDMSCs. Optimization of the experimental conditions may permit the utility of MDMSCs in generating surface zone-like cells with phenotypic markers of AC and, therefore, constitute a promising cell source for tissue engineering approaches of superficial zone cartilage.     

Identification of superficial zone articular chondrocyte stem/progenitor cells

Identification of progenitor/stem cell populations that differentiate specifically towards superficial zone articular chondrocytes is an unmet challenge for cartilage tissue engineering. Using fluorescence activated cell sorting (FACS) analysis we found a characteristic pattern of "side population" (SP) stem cells identified by the Hoechst 33342 dye. We established micromass cultures from this population of cells and tested their chondrogeneic potential. Control (untreated) cultures were minimally stained for Alcian blue - a marker of chondrogenesis. However, with BMP-7 treatment, Alcian blue staining was increased. Superficial zone protein - a specific marker for articular cartilage superficial zone chondrocytes - increased with BMP-7 and/or TGF-beta1 treatment in SP micromass cultures. Our results demonstrate the presence of stem/progenitor cells in the SP fraction isolated from the surface zone of bovine cartilage and have the ability to specifically differentiate towards the superficial zone articular chondrocyte.     

Bone morphogenetic protein (BMP) localization in developing human and rat growth plate, metaphysis, epiphysis, and articular cartilage.

We assessed the distribution and relative staining intensity of bone morphogenetic protein (BMP)-1-7 by immunohistochemistry in tibial growth plates, epiphyses, metaphyses, and articular cartilage in one 21-week and one 22-week human fetus and in five 10-week-old Sprague-Dawley rats. In the rats, articular cartilage was also examined. BMP proteins were mostly cytoplasmic, with negligible matrix staining. Highest BMP levels were seen in (a) hypertrophic and calcifying zone chondrocytes of growth plate (BMP-1-7), (b) osteoblasts and/or osteoprogenitor fibroblasts and vascular cells of the metaphyseal cortex and medulla (BMP-1-6), (c) osteoclasts of the metaphysis and epiphysis (BMP-1,-4,-5, and -6), and (d) mid to deep zone articular chondrocytes of weanling rats (BMP-1-7). BMP staining in osteoclasts, an unexpected finding, was consistently strong with BMP-4, -5, and -6 but was variable and dependent on osteoclast location with BMP-2,-3, and -7. BMP-1-7 were moderately to intensely stained in vascular canals of human fetal epiphyseal cartilage by endothelial cells and pericytes. BMP-1,-3,-5,-6, and -7 were localized in hypertrophic chondrocytes adjacent to cartilage canals. We conclude that BMP expression is associated with maturing chondrocytes of growth plate and articular cartilage, and may play a role in chondrocyte differentiation and/or apoptosis. BMP appears to be expressed by osteoclasts and might be involved in the intercellular "cross-talk" between osteoclasts and neighboring osteoprogenitor cells at sites of bone remodeling.     

Superficial zone protein affects boundary lubrication on the surface of mandibular condylar cartilage

We examined the localization and boundary lubricating function of superficial zone protein (SZP) on the surface of mandibular condylar cartilage. Chondrocytes were separated from the surface layer of mandibular condylar cartilage of 6- to 9-month-old female pigs. A cyclic tensile strain of 7% or 21% cell elongation was applied to the cultured chondrocytes. Gene expression levels of cartilage matrix proteins and secretory phospholipase A₂ (sPLA₂) were quantified by real-time polymerase chain reaction analysis. The friction coefficient of the mandibular condylar surface was measured by a friction tester before and after treatment with 0.1 U/ml sPLA₂. Significantly higher mRNA levels of SZP and type I collagen were found in chondrocytes from the superficial layer than in those in the other layers. The SZP mRNA level was up-regulated by cyclic tensile strain of 7% and 21% cell elongation. Cyclic tensile strain of 21% cell elongation up-regulated the sPLA₂ mRNA level. The friction coefficient of the condylar surface was increased significantly by treatment with sPLA₂. The removal of SZP from the surface layer of mandibular condylar cartilage by sPLA₂ resulted in a significant increase in the friction coefficient on the surface of articular cartilage.     

Adverse effects of adenovirus-mediated gene transfer of human transforming growth factor beta 1 into rabbit knees

To examine the effect of transforming growth factor (TGF)-β1 on the regulation of cartilage synthesis and other articular pathologies, we used adenovirus-mediated intra-articular gene transfer of TGF-β1 to both naïve and arthritic rabbit knee joints. Increasing doses of adenoviral vector expressing TGF-β1 were injected into normal and antigen-induced arthritis rabbit knee joints through the patellar tendon, with the same doses of an adenoviral vector expressing luciferase injected into the contralateral knees as the control. Intra-articular injection of adenoviral vector expressing TGF-β1 into the rabbit knee resulted in dose-dependent TGF-β1 expression in the synovial fluid. Intra-articular TGF-β1 expression in both naïve and arthritic rabbit knee joints resulted in significant pathological changes in the rabbit knee as well as in adjacent muscle tissue. The observed changes induced by elevated TGF-β1 included inhibition of white blood cell infiltration, stimulation of glycosaminoglycan release and nitric oxide production, and induction of fibrogenesis and muscle edema. In addition, induction of chondrogenesis within the synovial lining was observed. These results suggest that even though TGF-β1 may have anti-inflammatory properties, it is unable to stimulate repair of damaged cartilage, even stimulating cartilage degradation. Gene transfer of TGF-β1 to the synovium is thus not suitable for treating intra-articular pathologies.     

miR-29b inhibits TGF-β1-induced cell proliferation in articular chondrocytes

Transforming growth factor β1 (TGF-β1) is a known regulator of chondrocyte proliferation and promotes cartilage repair in osteoarthritis (OA). microRNA-29b-3p (miR-29b-3p) is downregulated by TGF-β1 and overexpressed in OA cartilage. However, the ability of miR-29b-3p to mediate the chondrocyte pro-proliferative effects of TGF-β1 is not yet understood. This current study aimed to investigate the effect of miR-29b-3p on TGF-β1-induced cell proliferation in murine articular chondrocytes. The stimulation of chondrocytes by TGF-β1 for 24 h resulted in the downregulation of miR-29b-3p expression. The ratio of G0/G1 phase cells decreased in response to TGF-β1 whereas the ratio of S phase cells was increased. Consistent with this observation, miR-29b-3p overexpression inhibited TGF-β1’s ability to promote the ratio of S phase cells and downregulate the ratio of G0/G1 phase cells. These findings suggest that the downregulation of miR-29b-3p is a likely requirement for TGF-β1-mediated proliferation of murine articular chondrocytes. Furthermore, implying that miR-29b-3p expression may be involved in reduced chondrocyte proliferation in OA.     

SnoN Suppresses Maturation of Chondrocytes by Mediating Signal Cross-talk between Transforming Growth Factor-β and Bone Morphogenetic Protein Pathways

Hypertrophic maturation of chondrocytes is a crucial step in endochondral ossification, whereas abnormally accelerated differentiation of hypertrophic chondrocytes in articular cartilage is linked to pathogenesis of osteoarthritis. This cellular process is promoted or inhibited by bone morphogenetic protein (BMP) or transforming growth factor-β (TGF-β) signaling, respectively, suggesting that these signaling pathways cross-talk during chondrocyte maturation. Here, we demonstrated that expression of Tgfb1 was increased, followed by phosphorylation of Smad2, during BMP-2-induced hypertrophic maturation of ATDC5 chondrocytes. Application of a TGF-β type I receptor inhibitor compound, SB431542, increased the expression of Id1, without affecting the phosphorylation status of Smad1/5/8, indicating that the activated endogenous TGF-β pathway inhibited BMP signaling downstream of the Smad activation step. We searched for TGF-β-inducible effectors that are able to inhibit BMP signaling in ATDC5 cells and identified SnoN. Overexpression of SnoN suppressed the activity of a BMP-responsive luciferase reporter in COS-7 cells as well as expression of Id1 in ATDC5 cells and, subsequently, the expression of Col10a1, a hallmark of hypertrophic chondrocyte maturation. siRNA-mediated loss of SnoN showed opposite effects in BMP-treated ATDC5 cells. In adult mice, we found the highest level of SnoN expression in articular cartilage. Importantly, SnoN was expressed, in combination with phosphorylated Smad2/3, in prehypertrophic chondrocytes in the growth plate of mouse embryo bones and in chondrocytes around the ectopically existing hypertrophic chondrocytes of human osteoarthritis cartilage. Our results indicate that SnoN mediates a negative feedback mechanism evoked by TGF-β to inhibit BMP signaling and, subsequently, hypertrophic maturation of chondrocytes.     

IGF-1 and BMP-7 synergistically stimulate articular cartilage repairing in the rabbit knees by improving chondrogenic differentiation of bone-marrow mesenchymal stem cells

Knee injury is known as a frequently occurred damage related to sports, which may affect the function of cartilage. This study aims to explore whether Insulin-like growth factor 1 (IGF-1) and bone morphogenetic protein-7 (BMP-7)-modified bone-marrow mesenchymal stem cells (BMSCs) affect the repair of cartilage damage found in the knee. Primarily, BMSCs were treated with a series of pEGFP-C1, IGF-1, and BMP-7, followed by determination of IGF-1 and BMP-7 expression. A rabbit cartilage defect model was also established. Afterfward, cell morphology, viability, cartilage damage repair effect, and expression of collagen I and collagen II at the 6th and the 12th week were measured. BMSCs treated with pEGFP-C1/IGF-1, pEGFP-C1/BMP-7, and pEGFP-C1/BMP-7-IGF-1 exhibited elevated expression of BMP-7 and IGF-1. Besides, BMSCs in the P10 generation displayed decreased cell proliferation. Moreover, BMSCs treated with IGF-1, BMP-7, and IGF-1-BMP-7 showed reduced histological score and collagen I expression while elevated collagen II expression, as well as better repair effect, especially in those treated with IGF-1-BMP-7. Collectively, these results demonstrated a synergistic effect of IGF-1 and BMP-7 on the BMSC chondrogenic differentiation on the articular cartilage damage repair in the rabbit knees, highlighting its therapeutic potential for the treatment of articular cartilage damage.     

BMP-7/TGF-β1 signalling in myoblasts: components involved in signalling and BMP-7-dependent blockage of TGF-β-mediated CTGF expression

We and others have recently described the antagonistic role of Bone morphogenetic protein-7 (BMP-7) in TGF-β signalling and myogenic differentiation. To specify the underlying mechanism(s), we here analysed the expression and function of the individual components mediating TGF-β1 and BMP-7 responses. We found that BMP-7 at a concentration of 25 ng/ml induces signalling exclusively via ALK2 and ALK3 leading to the activation of Smad1 and Smad5 and subsequent expression of Id proteins. In contrast, low doses of TGF-β1 (0.1 ng/ml) lead to an exclusive activation of ALK5 and phosphorylation of Smad2 and Smad3 that regulate specific target genes including connective tissue growth factor (CTGF). CTGF is rapidly induced by TGF-β1 already 1h after stimulation and reduced by BMP-7 application. Smad1/Smad5 or Id1/2 overexpression reduced the TGF-β1-mediated expression of CTGF. However, although siRNA-mediated knock down of Alk2/3 or Smad1/5 counteracts the BMP-7 effect on basal CTGF expression there was no consistent reversion of the observed BMP-7 effect on TGF-β1-mediated CTGF expression. Moreover, ALK5 inhibition using the SB431542 inhibitor significantly affected CTGF expression only at later time points whereas ERK1/2 inhibition completely abrogated CTGF expression. These findings point towards a regulatory role of BMP-7 that relies on modulation of Mitogen-activated protein kinases rather than mechanisms that are exclusively driven by differential Smad activation.     

TGF-β-Operated Growth Inhibition and Translineage Commitment into Smooth Muscle Cells of Periodontal Ligament-Derived Endothelial Progenitor Cells through Smad- and p38 MAPK-Dependent Signals

The periodontal ligament (PDL) is a fibrous connective tissue that attaches the tooth to the alveolar bone. We previously demonstrated the ability of PDL fibroblast-like cells to construct an endothelial cell (EC) marker-positive blood vessel-like structure, indicating the potential of fibroblastic lineage cells in PDL tissue as precursors of endothelial progenitor cells (EPCs) to facilitate the construction of a vascular system around damaged PDL tissue. A vascular regeneration around PDL tissue needs proliferation of vascular progenitor cells and the subsequent differentiation of the cells. Transforming growth factor-β (TGF-β) is known as an inducer of endothelial-mesenchymal transition (EndMT), however, it remains to be clarified what kinds of TGF-β signals affect growth and mesenchymal differentiation of PDL-derived EPC-like fibroblastic cells. Here, we demonstrated that TGF-β1 not only suppressed the proliferation of the PDL-derived EPC-like fibroblastic cells, but also induced smooth muscle cell (SMC) markers expression in the cells. On the other hand, TGF-β1 stimulation suppressed EC marker expression. Intriguingly, overexpression of Smad7, an inhibitor for TGF-β-induced Smad-dependent signaling, suppressed the TGF-β1-induced growth inhibition and SMC markers expression, but did not the TGF-β1-induced downregulation of EC marker expression. In contrast, p38 mitogen-activated protein kinase (MAPK) inhibitor SB 203580 suppressed the TGF-β1-induced downregulation of EC marker expression. In addition, the TGF-β1-induced SMC markers expression of the PDL-derived cells was reversed upon stimulation with fibroblast growth factor (FGF), suggesting that the TGF-β1 might not induce terminal SMC differentiation of the EPC-like fibroblastic cells. Thus, TGF-β1 not only negatively controls the growth of PDL-derived EPC-like fibroblastic cells via a Smad-dependent manner but also positively controls the SMC-differentiation of the cells possibly at the early stage of the translineage commitment via Smad- and p38 MAPK-dependent manners.     

Inhibitor of DNA binding/differentiation helix-loop-helix proteins mediate bone morphogenetic protein-induced osteoblast differentiation of mesenchymal stem cells

Bone morphogenetic proteins (BMPs) belong to the TGF-beta superfamily and play an important role in development and in many cellular processes. We have found that BMP-2, BMP-6, and BMP-9 induce the most potent osteogenic differentiation of mesenchymal stem cells. Expression profiling analysis has revealed that the Inhibitors of DNA binding/differentiation (Id)-1, Id-2, and Id-3 are among the most significantly up-regulated genes upon BMP-2, BMP-6, or BMP-9 stimulation. Here, we sought to determine the functional role of these Id proteins in BMP-induced osteoblast differentiation. We demonstrated that the expression of Id-1, Id-2, and Id-3 genes was significantly induced at the early stage of BMP-9 stimulation and returned to basal levels at 3 days after stimulation. RNA interference-mediated knockdown of Id expression significantly diminished the BMP-9-induced osteogenic differentiation of mesenchymal progenitor cells. Surprisingly, a constitutive overexpression of these Id genes also inhibited osteoblast differentiation initiated by BMP-9. Furthermore, we demonstrated that BMP-9-regulated Id expression is Smad4-dependent. Overexpression of the three Id genes was shown to promote cell proliferation that was coupled with an inhibition of osteogenic differentiation. Thus, our findings suggest that the Id helix-loop-helix proteins may play an important role in promoting the proliferation of early osteoblast progenitor cells and that Id expression must be down-regulated during the terminal differentiation of committed osteoblasts, suggesting that a balanced regulation of Id expression may be critical to BMP-induced osteoblast lineage-specific differentiation of mesenchymal stem cells.     

Combined use of adipose derived stem cells and TGF-β3 microspheres promotes articular cartilage regeneration in vivo

We investigated enhancement of articular cartilage regeneration using a combination of human adipose derived stem cells (hADSCs) and TGF-β3 microspheres (MS) in vivo. Poly-lactic-co-glycolic acid (PLGA)MS were prepared using a solid/oil/water emulsion solvent evaporation-extraction method. The morphology of the MS was evaluated by scanning electron microscopy (SEM). The release characteristic of the TGF-β3 MS was evaluated. A New Zealand rabbit model for experimental osteoarthritis (OA) was established using the anterior medial meniscus excision method. Thirty OA rabbits were divided randomly into three groups according to different treatments of the right knee joints on day 7 after surgery: hADSCs/MS group received injection of both hADSCs and TGF-β3 MS; hADSCs group was injected with hADSCs; control group was injected with normal saline. Gross observation, histological staining and RT-PCR for collagen II and aggrecan) were used to assess the severity of OA and for evaluating the effect of combined use of hADSCs and TGF-β3 MS on articular cartilage regeneration in vivo. The MS were spherical with a smooth surface and the average diameter was 28 ± 2.3 µm. The encapsulation efficiency test showed that 73.8 ± 2.9% of TGF-β3 were encapsulated in the MS. The release of TGF- β3 lasted for at least 30 days. At both 6 and 12 weeks after injection, three groups exhibited different degrees of OA. Histological analysis showed that the hADSCs/MS group exhibited less OA than the hADSCs group, and the control group exhibited the most severe OA. Real-time RT-PCR showed that the gene expression of both collagen II and aggrecan were significantly up-regulated in the hADSCs/MS group. At 12 weeks after injection, the hADSCs/MS group also exhibited less OA than the other two groups. Combined use of hADSCs and TGF-β3 MS promoted articular cartilage regeneration in rabbit OA models.     

Transient anabolic effects accompany epidermal growth factor receptor signal activation in articular cartilage in vivo

Introduction

Signals from the epidermal growth factor receptor (EGFR) have typically been considered to provide catabolic activities in articular cartilage, and accordingly have been suggested to have a causal role in osteoarthritis progression. The aim of this study was to determine in vivo roles for endogenous EGFR signal activation in articular cartilage.

Methods

Transgenic mice with conditional, limb-targeted deletion of the endogenous intracellular EGFR inhibitor Mig-6 were generated using CreLoxP (Mig-6-flox; Prx1Cre) recombination. Histology, histochemical staining and immunohistochemistry were used to confirm activation of EGFR signaling in the articular cartilage and joints, and to analyze phenotypic consequences of Mig-6 loss on articular cartilage morphology, proliferation, expression of progenitor cell markers, presence of chondrocyte hypertrophy and degradation of articular cartilage matrix.

Results

The articular cartilage of Mig-6-conditional knockout (Mig-6-cko) mice was dramatically and significantly thicker than normal articular cartilage at 6 and 12 weeks of age. Mig-6-cko articular cartilage contained a population of chondrocytes in which EGFR signaling was activated, and which were three to four times more proliferative than normal Mig-6-flox articular chondrocytes. These cells expressed high levels of the master chondrogenic regulatory factor Sox9, as well as high levels of putative progenitor cell markers including superficial zone protein (SZP), growth and differentiation factor-5 (GDF-5) and Notch1. Expression levels were also high for activated β-catenin and the transforming growth factor beta (TGF-β) mediators phospho-Smad2/3 (pSmad2/3). Anabolic effects of EGFR activation in articular cartilage were followed by catabolic events, including matrix degradation, as determined by accumulation of aggrecan cleavage fragments, and onset of hypertrophy as determined by type × collagen expression. By 16 weeks of age, the articular cartilage of Mig-6-cko knees was no longer thickened and was degenerating.

Conclusions

These results demonstrate unexpected anabolic effects of EGFR signal activation in articular cartilage, and suggest the hypothesis that these effects may promote the expansion and/or activity of an endogenous EGFR-responsive cell population within the articular cartilage.     

Functional properties of cartilaginous tissues engineered from infrapatellar fat pad-derived mesenchymal stem cells

Articular cartilage has a poor intrinsic capacity for self-repair. The advent of autologous chondrocyte implantation has provided a feasible method to treat cartilage defects. However, the associated drawbacks with the isolation and expansion of chondrocytes from autologous tissue has prompted research into alternative cell sources such as mesenchymal stem cells (MSCs) which have been found to exist in the bone marrow as well as other joint tissues such as the infrapatellar fat pad (IFP), synovium and within the synovial fluid itself. In this work we assessed the chondrogenic potential of IFP-derived porcine cells over a 6 week period in agarose hydrogel culture in terms of mechanical properties, biochemical content and histology. It was found that IFP cells underwent robust chondrogenesis as assessed by glycosaminoglycan (1.47±0.22% w/w) and collagen (1.44±0.22% w/w) accumulation after 42 days of culture. The 1 Hz dynamic modulus of the engineered tissue at this time point was 272.8 kPa (±46.8). The removal of TGF-β3 from culture after 21 days was shown to have a significant effect on both the mechanical properties and biochemical content of IFP constructs after 42 days, with minimal increases occurring from day 21 to day 42 without continued supplementation of TGF-β3. These findings further strengthen the case that the IFP may be a promising cell source for putative cartilage repair strategies.     

Experimental study of the synergistic effect and network regulation mechanisms of an applied combination of BMP-2, VEGF,and TGF-β1 on osteogenic differentiation

The study aimed to explore the osteogenic effect induced by the combined use of bone morphogenetic protein-2 (BMP-2), vascular endothelial growth factor (VEGF), and transforming growth factor-β1 (TGF-β1), attain the best combination for osteogenic quality and efficiency, and explore the network regulation mechanisms of induced osteogenesis. MC3T3-E1 cells were cultured in vitro, and BMP-2, VEGF, and TGF β1 were added to osteogenic induction mediums in different combinations to conduct experiments. At 7 and 14 days, the alkaline phosphatase (ALP) and Alizarin Red S (ARS) staining of the applied BMP-2 and VEGF combination were deeper and the quantitative analysis were higher than those of the other groups. After optimizing the time–effect relationship of the combined application, with BMP-2, VEGF, and TGF-β1 adding in the early stage and BMP-2 and VEGF adding in the late, the ALP and ARS staining of these groups were deeper and the quantitative analyses were meaningfully higher than the BMP-2 and VEGF combination group at 7 and 14 days. The expression of the RUNX2 gene and the Smad1 signaling pathway in the optimized combination group was also significantly higher. The results demonstrate that the combination of BMP-2, VEGF, and TGF-β1 applied according to the time–effect relationship can significantly promote osteogenic differentiation mainly through the classical BMP-receptor-Smad signal pathway.     

Platelet-rich plasma promotes the regeneration of cartilage engineered by mesenchymal stem cells and collagen hydrogel via the TGF-β/SMAD signaling pathway

The tissue engineering technique using mesenchymal stem cells (MSCs) and scaffolds is promising. Transforming growth factor-β1 (TGF-β1) is generally accepted as an chondrogenic agent, but immunorejection and unexpected side effects, such as tumorigenesis and heterogeneity, limit its clinical application. Autogenous platelet-rich plasma (PRP), marked by low immunogenicity, easy accessibility, and low-cost, may be favorable for cartilage regeneration. In our study, the effect of PRP on engineered cartilage constructed by MSCs and collagen hydrogel in vitro and in vivo was investigated and compared with TGF-β1. The results showed that PRP promoted cell proliferation and gene and protein expressions of chondrogenic markers via the TGF-β/SMAD signaling pathway. Meanwhile, it suppressed the expression of collagen type I, a marker of fibrocartilage. Furthermore, PRP accelerated cartilage regeneration on defects with engineered cartilage, advantageous over TGF-β1, as evaluated by histological analysis and immunohistochemical staining. Our work demonstrates that autogenous PRP may substitute TGF-β1 as a potent and reliable chondrogenic inducer for therapy of cartilage defect.     

FAK mediates signal crosstalk between type II collagen and TGF-beta 1 cascades in chondrocytic cells

The purpose of this study was to evaluate the mechanism of crosstalk between the type II collagen and TGF-β1 signaling pathways in chondrocytic cells. Articular chondrocytes, isolated from porcine knee cartilage, and the SW1353 cell line were cultured on either type II collagen-coated or -uncoated plates in the presence or absence of TGF-β1. Expression of pSMAD 2, pSMAD 3, pFAK_Y397 and pFAK_Y925 in articular chondrocytes and the SW1353 cell line was analyzed by immunoblotting. Cell proliferation rates and glycosaminoglycan (GAG) content was determined after treatment with type II collagen or/and TGF-β1. For inhibition study, human FAK-specific RNA small interference (siFAK) in SW1353 cell line was performed. In this study, expression of pSMAD 2, pSMAD 3, pFAK_Y397 and pFAK_Y925 were synergistically increased by co-treatment with type II collagen and TGF-β1 in articular chondrocytes. The proliferation of porcine articular chondrocytes and GAG secretion in SW1353 cells were synergistically increased by co-stimulation with type II collagen and TGF-β1. Synergistically increased expression and nuclear translocation of pSMAD 2 and pSMAD 3 and GAG secretion of SW1353 cells were significantly inhibited by siFAK transfection. Therefore, we suggest that FAK-SMAD 2/3 mediates signal crosstalk between type II collagen and TGF-β1 and regulates GAG secretion in chondrocytic cells.     

A growth factor delivery system for chondrogenic induction of infrapatellar fat pad-derived stem cells in fibrin hydrogels

Articular cartilage has a limited capacity for self-renewal and repair. Tissue engineering of cartilage in vitro has been proposed as a solution to this problem; however, this approach is costly and requires a significant amount of time to grow the graft. An alternative approach is to implant chondroprogenitor cells seeded within a growth factor delivery scaffold directly into the defect site to promote tissue regeneration. The objective of this study was to develop a biocompatible growth factor delivery system capable of promoting chondrogenesis of infrapatellar fat pad (IFP)-derived stem cells. Transforming growth factor beta-1 (TGF-β1) was loaded into gelatin microspheres and incorporated into fibrin hydrogels containing IFP-derived stem cells. The release of TGF-β1 was quantified using an enzyme-linked immunosorbent assay, whereas chondrogenesis was demonstrated histologically and by quantifying sulfated glycosaminoglycan production after 21 days of in vitro culture. TGF-β1 loaded into gelatin microspheres appeared to be as effective in promoting chondrogenesis of IFP-derived stem cells as adding TGF-β1 directly to the medium. The influence of different microsphere fabrication parameters and TGF-β1 loading concentrations was also investigated but appeared to only have a small effect on subsequent chondrogenesis. The development of such growth factor delivery systems in combination with IFP-derived stem cells represents a potential new strategy for cartilage defect repair.