Differentiation of human adipose-derived mesenchymal stem cells toward tenocyte by platelet-derived growth factor-BB and growth differentiation factor-6.

Mesenchymal Stem Cells (MSCs) are important in regenerative medicine and tissue engineering and will be a very sensible choice for repair and regeneration of tendon. New biological practices, such as cellular therapy using stem cells, are promising for facilitating or expediting tendon therapy. Before using these cells clinically, it is best to check and confirm the optimal conditions for differentiation of these cells in the laboratory. Hence, in the present study, the impacts of PDGF-BB and GDF-6 supplementation on adipose-derived MSCs (ASCs) culture were studied. The frozen ASC were recovered and expanded in basic culture medium (DMEM with 10%FBS). The cells after passage five (P5) were treated with basic medium containing L-Prolin, Ascorbic Acid and only PDGF-BB or GDF-6 (20 ng/ml) or both of them (mix) as 3 groups for 14 days to investigate efficiency of ASCs differentiation towards tenocytes. The cells culturing in basic medium were used as control group. To validate tenogenic differentiation, H&E and Sirius Red staining were used to assess cell morphology and collagen production, respectively. In addition, mRNA levels of collagen I and III, Scleraxis and Tenomodulin as tenogenic markers were analyzed using qPCR. In all test groups, cells appeared slenderer, elongated cytoplasmic attributes compared to the control cells. The intensity of Sirius Red staining was significantly higher in GDF-6, PDGF-BB alone, than in group without supplements. The optical density was higher in the GDF-6 than PDGF-BB and mix-group. QPCR results showed that Col I and III gene expression was increased in all groups compared to the control. SCX expression was significantly increased only in the PDGF-BB group. TNMD mRNA expression was not significant among groups. In this study, we have corroborated that human ASCs are reactionary to tenogenic induction by GDF-6 and PDGF-BB alone or in combination. These outcomes will help greater insight into GDF-6 and PDGF-BB driven tenogenesis of ASCs and new directions of discovery in the design of ASC-based treatments for tendon healing.

Growth differentiation factor-6 attenuates inflammatory and pain-related factors and degenerated disc-induced pain behaviors in rat model.

Previous studies have indicated that growth differentiation factor 6 (GDF6) is a potential candidate for intervertebral disc (IVD) degeneration (IDD) treatment. Here, we investigated the effect of GDF6 on IDD by examining changes in disc structure and the expression of inflammatory and pain-related factors. A rat posterior disc puncture model of single segments and three consecutive segments was constructed, and GDF6 or phosphate-buffered solution was administered via intradiscal injection 1 or 2 weeks after surgery. Magnetic resonance imaging showed a clear degeneration signal in the punctured disc, which was inhibited by GDF6. Histological staining revealed that GDF6 did not significantly improve the structure of IVDs in rats 8 weeks after puncture surgery, but it had an inhibitory effect on expression of the tumor necrosis factor-alpha (TNF-α) and interleukin (IL)-1ß in the IVD. Furthermore, GDF6 was found to protect the morphology and structure of the IVD 32 weeks after surgery. Mechanical and thermal hyperalgesia tests suggested that GDF6 injection can significantly improve mechanical and thermal-stimulated pain behavior in rats and inhibit the expression of inflammatory factors TNF-α and IL-1ß and the pain factor calcitonin gene-related peptide in the dorsal root ganglion. A rat protein array test indicated that GDF6 could reduce the expression of cytokines IL-6, intercellular cell adhesion molecule-1, matrix metalloproteinase-13, IL-1ß, and TNF-α and increase the expression of tissue inhibitor of metalloproteinases 1, Transforming growth factor-beta 2, IL-10, and resistin in a TNF-α-induced IDD cell model. Thus, our study demonstrates that GDF6 can improve the structure of the IVD, inhibit the expression of inflammatory and pain-related factors, and improve pain behavior in rats. Clinical Significance: To establish further preclinical research and clinical trials, comprehensive data are needed to validate the regenerative properties of GDF6. Ideally, a regenerative agent should also be able to relieve discogenic pain, achieving the best clinical outcomes.

Induction of tenogenic differentiation of equine adipose-derived mesenchymal stem cells by platelet-derived growth factor-BB and growth differentiation factor-6.

Managing tendon healing process is complicated mainly due to the limited regeneration capacity of tendon tissue. Mesenchymal stem cells (MSCs) have potential applications in regenerative medicine and have been considered for tendon repair and regeneration. This study aimed to evaluate the capacity of equine adipose tissue-derived cells (eASCs) to differentiate into tenocytes in response to platelet-derived growth factor-BB (PDGF-BB) and growth differentiation factor-6 (GDF-6) in vitro. Frozen characterized eASCS of 3 mares were thawed and the cells were expanded in basic culture medium (DMEM supplemented with 10% FBS). The cells at passage 5 were treated for 14 days in different conditions including: (1) control group in basic culture medium (CM), (2) induction medium as IM (CM containing L-prolin, and ascorbic acid (AA)) supplemented with PDGF-BB (20 ng/ml), (3) IM supplemented with GDF-6 (20 ng/ml), and (4) IM supplemented with PDGF-BB and GDF-6. At the end of culture period (14th day), tenogenic differentiation was evaluated. Sirius Red staining was used to assess collagen production, and H&E was used for assessing cell morphology. mRNA levels of collagen type 1 (colI), scleraxis (SCX), and Mohawk (MKX), as tenogenic markers, were analyzed using real-time reverse-transcription polymerase chain reaction (qPCR). H&E staining showed a stretching and spindle shape (tenocyte-like) cells in all treated groups compared to unchanged from of cells in control groups. Also, Sirius red staining data showed a significant increase in collagen production in all treated groups compared with the control group. MKX expression was significantly increased in PDGF-BB and mixed groups and COLI expression was significantly increased only in PDGF-BB group. In conclusion, our results showed that PDGF-BB and GDF-6 combination could induce tenogenic differentiation in eASCs. These in vitro findings could be useful for cell therapy in equine regenerative medicine.

ISSLS PRIZE IN BASIC SCIENCE 2018: Growth differentiation factor-6 attenuated pro-inflammatory molecular changes in the rabbit anular-puncture model and degenerated disc-induced pain generation in the rat xenograft radiculopathy model.

PURPOSE: To elucidate the effects of growth differentiation factor-6 (GDF6) on: (i) gene expression of inflammatory/pain-related molecules and structural integrity in the rabbit intervertebral disc (IVD) degeneration model, and (ii) sensory dysfunction and changes in pain-marker expression in dorsal nerve ganglia (DRGs) in the rat xenograft radiculopathy model. METHODS: Forty-six adolescent rabbits received anular-puncture in two non-consecutive lumbar IVDs. Four weeks later, phosphate-buffered saline (PBS) or GDF6 (1, 10 or 100 µg) was injected into the nucleus pulposus (NP) of punctured discs and followed for 4 weeks for gene expression analysis and 12 weeks for structural analyses. For pain assessment, eight rabbits were sacrificed at 4 weeks post-injection and NP tissues of injected discs were transplanted onto L5 DRGs of 16 nude rats to examine mechanical allodynia. The rat DRGs were analyzed immunohistochemically. RESULTS: In GDF6-treated rabbit NPs, gene expressions of interleukin-6, tumor necrosis factor-α, vascular endothelial growth factor, prostaglandin-endoperoxide synthase 2, and nerve growth factor were significantly lower than those in the PBS group. GDF6 injections resulted in partial restoration of disc height and improvement of MRI disc degeneration grades with statistical significance in rabbit structural analyses. Allodynia induced by xenograft transplantation of rabbit degenerated NPs onto rat DRGs was significantly reduced by GDF6 injection. Staining intensities for ionized calcium-binding adaptor molecule-1 and calcitonin gene-related peptide in rat DRGs of the GDF6 group were significantly lower than those of the PBS group. CONCLUSION: GDF6 injection may change the pathological status of degenerative discs and attenuate degenerated IVD-induced pain.

Differentiation of MSC and annulus fibrosus cells on genetically engineered silk fleece-membrane-composites enriched for GDF-6 or TGF-ß3.

Intervertebral disc (IVD) repair is a high-priority topic in our active and increasingly ageing society. Since a high number of people are affected by low back pain treatment options that are able to restore the biological function of the IVD are highly warranted. Here, we investigated whether the feasibility of genetically engineered (GE)-silk from Bombyx mori containing specific growth factors to precondition human bone-marrow derived mesenchymal stem cells (hMSC) or to activate differentiated human annulus fibrosus cells (hAFC) prior transplantation or for direct repair on the IVD. Here, we tested the hypothesis that GE-silk fleece can thrive human hMSC towards an IVD-like phenotype. We aimed to demonstrate a possible translational application of good manufacturing practice (GMP)-compliant GE-silk scaffolds in IVD repair and regeneration. GE-silk with growth and differentiation factor 6 (GDF-6-silk) or transforming growth factor ß3 (TGF-ß3, TGF-ß3-silk) and untreated silk (cSilk) were investigated by DNA content, cell activity assay and glycosaminoglycan (GAG) content and their differentiation potential by qPCR analysis. We found that all silk types demonstrated a very high biocompatibility for both cell types, that is, hMSC and hAFC, as revealed by cell activity, and DNA proliferation assay. Further, analyzing qPCR of marker genes revealed a trend to differentiation toward an NP-like phenotype looking at the Aggrecan/Collagen 2 ratio which was around 10:1. Our results support the conclusion that our GE-silk scaffold treatment approach can thrive hMSC towards a more IVD-like phenotype or can maintain the phenotype of native hAFC. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:1324-1333, 2018.

Growth differentiation factor 6 and transforming growth factor-beta differentially mediate mesenchymal stem cell differentiation, composition, and micromechanical properties of nucleus pulposus constructs.

INTRODUCTION: Currently, there is huge research focus on the development of novel cell-based regeneration and tissue-engineering therapies for the treatment of intervertebral disc degeneration and the associated back pain. Both bone marrow-derived (BM) mesenchymal stem cells (MSCs) and adipose-derived MSCs (AD-MSCs) are proposed as suitable cells for such therapies. However, currently no consensus exists as to the optimum growth factor needed to drive differentiation to a nucleus pulposus (NP)-like phenotype. The aim of this study was to investigate the effect of growth differentiation factor-6 (GDF6), compared with other transforming growth factor (TGF) superfamily members, on discogenic differentiation of MSCs, the matrix composition, and micromechanics of engineered NP tissue constructs. METHODS: Patient-matched human AD-MSCs and BM-MSCs were seeded into type I collagen hydrogels and cultured in differentiating media supplemented with TGF-ß3, GDF5, or GDF6. After 14 days, quantitative polymerase chain reaction analysis of chondrogenic and novel NP marker genes and sulfated glycosaminoglycan (sGAG) content of the construct and media components were measured. Additionally, construct micromechanics were analyzed by using scanning acoustic microscopy (SAM). RESULTS: GDF6 stimulation of BM-MSCs and AD-MSCs resulted in a significant increase in expression of novel NP marker genes, a higher aggrecan-to-type II collagen gene expression ratio, and higher sGAG production compared with TGF-ß or GDF5 stimulation. These effects were greater in AD-MSCs than in BM-MSCs. Furthermore, the acoustic-wave speed measured by using SAM, and therefore tissue stiffness, was lowest in GDF6-stiumlated AD-MSC constructs. CONCLUSIONS: The data suggest that GDF6 stimulation of AD-MSCs induces differentiation to an NP-like phenotype and results in a more proteoglycan-rich matrix. Micromechanical analysis shows that the GDF6-treated AD-MSCs have a less-stiff matrix composition, suggesting that the growth factor is inducing a matrix that is more akin to the native NP-like tissue. Thus, this cell and growth-factor combination may be the ideal choice for cell-based intervertebral disc (IVD)-regeneration therapies.

Effect of growth and differentiation factor 6 on the tenogenic differentiation of bone marrow-derived mesenchymal stem cells.

BACKGROUND: Recent studies showed that bone marrow-derived mesenchymal stem cells (BMSCs) had risk of ectopic bone formation. In this study, we aimed to investigate the effect of growth and differentiation factor 6 (GDF-6) on the tenogenic differentiation of BMSCs in vitro, and then combined with small intestine submucous (SIS) to promote tendon regeneration in vivo. METHODS: The BMSCs were isolated from the green fluorescent protein (GFP) rats, and were characterized by multi-differentiation assays following our previous study protocol. BMSCs cultured with different concentrations of GDF-6, without growth factors served as control. After 2 weeks, mRNA expression and protein expression of tendon specific markers were examined by qRT-PCR and Western blotting to define an optimal concentration of GDF-6. Mann-Whitney U-test was used to compare the difference in relative mRNA expression among all groups; P ≤ 0.05 was regarded as statistically significant. The GDF-6 treated BMSCs combined with SIS were implanted in nude mice and SD rat acute patellar tendon injury model, the BMSCs combined with SIS served as control. After 12 and 4 weeks in nude mice and tendon injury model, the samples were collected for histology. RESULTS: After the BMSCs were treated with different concentration of GDF-6 for 2 weeks, the fold changes of the specific markers (Tenomodulin and Scleraxis) mRNA expression were significantly higher in GDF-6 (20 ng/ml) group (P ≤ 0.05), which was also confirmed by Western blotting result. The BMSCs became parallel in orientation after GDF-6 (20 ng/ml) treatment, but the BMSCs in control group were randomly oriented. The GDF-6 (20 ng/ml) treated BMSCs were combined with SIS, and were implanted in nude mice for 12 weeks, the histology showed neo-tendon formation. In the SD rat patellar tendon window injury model, the histology also indicated the GDF-6 (20 ng/ml) treated BMSCs combined with SIS could promote tendon regeneration. CONCLUSIONS: GDF-6 has tenogenic effect on the tenogenic differentiation of BMSCs, and GDF-6 (20 ng/ml) has better tenogenic effect compared to other concentrations. The GDF-6 (20 ng/ml) treated BMSCs combined with SIS can form neo-tendons and promote tendon regeneration.