Regulatory Mechanisms of Prg4 and Gdf5 Expression in Articular Cartilage and Functions in Osteoarthritis.

Owing to the rapid aging of society, the numbers of patients with joint disease continue to increase. Accordingly, a large number of patients require appropriate treatment for osteoarthritis (OA), the most frequent bone and joint disease. Thought to be caused by the degeneration and destruction of articular cartilage following persistent and excessive mechanical stimulation of the joints, OA can significantly impair patient quality of life with symptoms such as knee pain, lower limb muscle weakness, or difficulty walking. Because articular cartilage has a low self-repair ability and an extremely low proliferative capacity, healing of damaged articular cartilage has not been achieved to date. The current pharmaceutical treatment of OA is limited to the slight alleviation of symptoms (e.g., local injection of hyaluronic acid or non-steroidal anti-inflammatory drugs); hence, the development of effective drugs and regenerative therapies for OA is highly desirable. This review article summarizes findings indicating that proteoglycan 4 (Prg4)/lubricin, which is specifically expressed in the superficial zone of articular cartilage and synovium, functions in a protective manner against OA, and covers the transcriptional regulation of Prg4 in articular chondrocytes. We also focused on growth differentiation factor 5 (Gdf5), which is specifically expressed on the surface layer of articular cartilage, particularly in the developmental stage, describing its regulatory mechanisms and functions in joint formation and OA pathogenesis. Because several genetic studies in humans and mice indicate the involvement of these genes in the maintenance of articular cartilage homeostasis and the presentation of OA, molecular targeting of Prg4 and Gdf5 is expected to provide new insights into the aetiology, pathogenesis, and potential treatment of OA.

Apoptotic Body-Rich Media from Tenocytes Enhance Proliferation and Migration of Tenocytes and Bone Marrow Stromal Cells.

The intrinsic healing following tendon injury is ideal, in which tendon progenitor cells proliferate and migrate to the injury site to directly bridge or regenerate tendon tissue. However, the mechanism determining why and how those cells are attracted to the injury site for tendon healing is not understood. Since the tenocytes near the injury site go through apoptosis or necrosis following injury, we hypothesized that secretions from injured tenocytes might have biological effects on cell proliferation and migration to enhance tendon healing. Tenocyte apoptosis was induced by 24 h cell starvation. Apoptotic body-rich media (T-ABRM) and apoptotic body-depleted media (T-ABDM) were collected from culture media after centrifuging. Tenocytes and bone marrow-derived stem cells (BMDSCs) were isolated and cultured with the following four media: (1) T-ABRM, (2) T-ABDM, (3) GDF-5, or (4) basal medium with 2% fetal calf serum (FCS). The cell activities and functions were evaluated. Both T-ABRM and T-ABDM treatments significantly stimulated the cell proliferation, migration, and extracellular matrix synthesis for both tenocytes and BMDSCs compared to the control groups (GDF-5 and basal medium). However, cell proliferation, migration, and extracellular matrix production of T-ABRM-treated cells were significantly higher than the T-ABDM, which indicates the apoptotic bodies are critical for cell activities. Our study revealed the possible mechanism of the intrinsic healing of the tendon in which apoptotic bodies, in the process of apoptosis, following tendon injury promote tenocyte and stromal cell proliferation, migration, and production. Future studies should analyze the components of the apoptotic bodies that play this role, and, thus, the targeting of therapeutics can be developed.

Dose-Response Tendon-Specific Markers Induction by Growth Differentiation Factor-5 in Human Bone Marrow and Umbilical Cord Mesenchymal Stem Cells.

Mesenchymal stem cells derived from human bone marrow (hBM-MSCs) are utilized in tendon tissue-engineering protocols while extra-embryonic cord-derived, including from Wharton's Jelly (hWJ-MSCs), are emerging as useful alternatives. To explore the tenogenic responsiveness of hBM-MSCs and hWJ-MSCs to human Growth Differentiation Factor 5 (hGDF-5) we supplemented each at doses of 1, 10, and 100 ng/mL of hGDF-5 and determined proliferation, morphology and time-dependent expression of tenogenic markers. We evaluated the expression of collagen types 1 (COL1A1) and 3 (COL3A1), Decorin (DCN), Scleraxis-A (SCX-A), Tenascin-C (TNC) and Tenomodulin (TNMD) noting the earliest and largest increase with 100 ng/mL. With 100 ng/mL, hBM-MSCs showed up-regulation of SCX-A (1.7-fold) at Day 1, TNC (1.3-fold) and TNMD (12-fold) at Day 8. hWJ-MSCs, at the same dose, showed up-regulation of COL1A1 (3-fold), DCN (2.7-fold), SCX-A (3.8-fold) and TNC (2.3-fold) after three days of culture. hWJ-MSCs also showed larger proliferation rate and marked aggregation into a tubular-shaped system at Day 7 (with 100 ng/mL of hGDF-5). Simultaneous to this, we explored the expression of pro-inflammatory (IL-6, TNF, IL-12A, IL-1ß) and anti-inflammatory (IL-10, TGF-ß1) cytokines across for both cell types. hBM-MSCs exhibited a better balance of pro-inflammatory and anti-inflammatory cytokines up-regulating IL-1ß (11-fold) and IL-10 (10-fold) at Day 8; hWJ-MSCs, had a slight expression of IL-12A (1.5-fold), but a greater up-regulation of IL-10 (2.5-fold). Type 1 collagen and tenomodulin proteins, detected by immunofluorescence, confirming the greater protein expression when 100 ng/mL were supplemented. In the same conditions, both cell types showed specific alignment and shape modification with a length/width ratio increase, suggesting their response in activating tenogenic commitment events, and they both potential use in 3D in vitro tissue-engineering protocols.

Intra-articular therapy with recombinant human GDF5 arrests disease progression and stimulates cartilage repair in the rat medial meniscus transection (MMT) model of osteoarthritis.

OBJECTIVE: Investigation of osteoarthritis (OA) risk alleles suggests that reduced levels of growth and differentiation factor-5 (GDF5) may be a precipitating factor in OA. We hypothesized that intra-articular recombinant human GDF5 (rhGDF5) supplementation to the OA joint may alter disease progression. METHODS: A rat medial meniscus transection (MMT) joint instability OA model was used. Animals received either one intra-articular injection, or two or three bi-weekly intra-articular injections of either 30 µg or 100 µg of rhGDF5 beginning on day 21 post surgery after structural pathology had been established. Nine weeks after MMT surgery, joints were processed for histological analysis following staining with toluidine blue. Control groups received intra-articular vehicle injections, comprising a glycine-buffered trehalose solution. OA changes in the joint were evaluated using histopathological end points that were collected by a pathologist who was blinded to treatment. RESULTS: Intra-articular rhGDF5 supplementation reduced cartilage lesions on the medial tibial plateau in a dose-dependent manner when administered therapeutically to intercept OA disease progression. A single 100 µg rhGDF5 injection on day 21 slowed disease progression at day 63. A similar effect was achieved with two bi-weekly injections of 30 µg. Two bi-weekly injections of 100 µg or three bi-weekly injections of 30 µg stopped progression of cartilage lesions. Importantly, three biweekly injections of 100 µg rhGDF5 stimulated significant cartilage repair. CONCLUSIONS: Intra-articular rhGDF5 supplementation can prevent and even reverse OA disease progression in the rat MMT OA model. Collectively, these results support rhGDF5 supplementation as an intra-articular disease modifying OA therapy.

Preparation and Evaluation of Dexamethasone (DEX)/Growth and Differentiation Factor-5 (GDF-5) Surface-Modified Titanium Using ß-Cyclodextrin-Conjugated Heparin (CD-Hep) for Enhanced Osteogenic Activity In Vitro and In Vivo.

The most ideal implant models in the dental and orthopedic fields to minimize the failure rate of implantation involve the improvement of osseointegration with host bone. Therefore, a focus of this study is the preparation of surface-modified titanium (Ti) samples of disc and screw types using dexamethasone (DEX) and/or growth and differentiation factor-5 (GDF-5), as well as the evaluation of their efficacies on bone formation in vitro and in vivo. X-ray photoelectron spectroscopy (XPS), scanning electron microscopy (SEM) and contact angle measurement were used to evaluate the surface chemical composition, surface morphology and wettability, respectively. The results showed that implant surfaces were successfully modified with DEX and/or GDF-5, and had rough surfaces along with hydrophilicity. DEX, GDF-5 or DEX/GDF-5 on the surface-modified samples were rapidly released within one day and released for 28 days in a sustained manner. The proliferation and bone formation of MC3T3-E1 cells cultured on pristine and surface-modified implants in vitro were examined by cell counting kit-8 (CCK-8) assay, as well as the measurements of alkaline phosphatase (ALP) activity and calcium deposition, respectively. MC3T3-E1 cells cultured on DEX/GDF-5-Ti showed noticeable ALP activity and calcium deposition in vitro. Active bone formation and strong osseointegration occurred at the interface between DEX/GDF-5-Ti and host bone, as evaluated by micro computed-tomography (micro CT) analysis. Surface modification using DEX/GDF-5 could be a good method for advanced implants for orthopaedic and dental applications.

Surface Modification of Titanium with BMP-2/GDF-5 by a Heparin Linker and Its Efficacy as a Dental Implant.

In this study, we prepared human bone morphogenetic protein-2 (hBMP-2)/human growth and differentiation factor-5 (hGDF-5)-coated titanium (Ti) disc and screw types for controlled release of the growth factors (GFs). The two growth factors were coated onto Ti with a smooth surface using their specific interaction with heparin, because they have heparin binding sites in their molecular structures. Efficacy of the two growth factor-coated Ti for enhancement of bone formation and osseointegration was compared to pristine Ti, and hBMP-2- and hGDF-5-coated Ti in vivo. The surface chemical composition, surface morphology, and wettability characteristics of the metal samples were determined by X-ray photoelectron spectroscopy (XPS), scanning electron microscopy (SEM), and contact angle measurement, respectively. The initial burst of hBMP-2, hGDF-5, and their combination, occurred within one day of the release study, resulting in 12.5%, 4.5%, and 13.5%/3.2%, and then there was a sustained, even release of these two growth factors from the coated metal for 30 days. In vitro tests revealed that MC3T3-E1 cells cultured on the two growth factor-coated Ti had a higher proliferation rate and a higher activity for alkaline phosphatase (ALP), which led to a larger amount of calcium deposition and larger expressions of type I collagen (COL 1), ALP, and osteocalcin (OCN) mRNAs. In vivo animal tests using ten white New Zealand rabbits showed that the two growth factor-coated Ti enhanced bone formation and osseointegration at the interface between the implants and host bone. In addition, histological evaluation showed that bone remodeling, including bone formation by osteoblasts and bone resorption by osteoclasts, actively occurred between the two growth factor-coated Ti and host bone. Consequently, it is suggested that Ti surface modification with the combination of hBMP-2 and hGDF-5 for the two growth factor-coated Ti implants can improve the clinical properties of implants for orthopedic and dental applications.

[Comparative study of proliferative and periodontal differentiation propensity of induced pluripotent stem cells at different passages].

OBJECTIVE: To compare the proliferative and periodontal specific differentiation abilities of induced pluripotent stem cells (iPSCs) at different passages, and to investigate whether long term culturing would have a negative influence on their proliferation and specific differentiation capacity, thus providing a theoretical basis for further in-depth research on periodontal regeneration and the possible clinical applications of iPSCs. METHODS: IPSCs derived from human gingival fibroblasts at passages 5, 10, 15 and 20 were recovered and cultured in vitro. Their morphology and proliferation rates were observed respectively. We further induced the iPSCs at different passages toward periodontal tissue under the treatment of growth/differentiation factor-5 (GDF-5) for 14 days through the EB routine, then compared the periodontal differentiation propensities between the different passages of iPSCs by detecting their calcified nodules formation by Alizarin red staining and assaying their relative periodontal tissue related marker expressions by qRT-PCR and immunofluorescence staining, including bone related markers: osteocalcin (OCN), bone sialoprotein (BSP); periodontal ligament related markers: periostin, vimentin; and cementum related markers: cementum attachment protein (CAP), cementum protein 1 (CEMP1). The untreated spontaneous differentiation groups were set as negative controls respectively. RESULTS: iPSCs at different passages all showed a high proliferative capacity when cultured in vitro and turned into a spindle-like shape similar to fibroblasts upon periodontal specific differentiation. All iPSCs formed typical calcified nodules upon GDF-5 induction by Alizarin red staining in comparison to their untreated controls. The relative calcium deposition at all passages had been significantly upgraded under the treatment of GDF-5 (P5: t=2.125, P=0.003; P10: t=2.246, P=0.021; P15: t=3.754, P=0.004; P20: t=3.933, P=0.002), but no significant difference in their calcium deposition were detected within passages 5, 10, 15 and 20 (periodontal differentiation: F=2.365, P=0.109; spontaneously differentiation: F=2.901, P=0.067). Periodontal tissue related marker expressions of iPSCs at all passages had also been significantly upgraded under the treatment of GDF-5 (P<0.05), but still, no significant difference in their expression levels of periodontal tissue related proteins were detected within passages (BSP: F=0.926 7, P=0.450; vimentin: F=0.917 1, P=0.455; CEMP1: F=2.129, P=0.1367). CONCLUSION: Our results preliminarily confirmed that long term culturing won't influence the proliferation capacity and periodontal specific differentiation propensity of iPSCs, as they can still proliferate and differentiate toward periodontal cells with high efficiency upon growth factor induction after continuous passaging. Therefore, iPSCs could be recognized as a promising cell source for future possible application in periodontal tissue regeneration.

TGF-ß1, GDF-5, and BMP-2 stimulation induces chondrogenesis in expanded human articular chondrocytes and marrow-derived stromal cells.

Replacement of degenerated cartilage with cell-based cartilage products may offer a long-term solution to halt arthritis' degenerative progression. Chondrocytes are frequently used in cell-based FDA-approved cartilage products; yet human marrow-derived stromal cells (hMSCs) show significant translational potential, reducing donor site morbidity and maintaining their undifferentiated phenotype with expansion. This study sought to investigate the effects of transforming growth factor ß1 (TGF-ß1), growth/differentiation factor 5 (GDF-5), and bone morphogenetic protein 2 (BMP-2) during postexpansion chondrogenesis in human articular chondrocytes (hACs) and to compare chondrogenesis in passaged hACs with that of passaged hMSCs. Through serial expansion, chondrocytes dedifferentiated, decreasing expression of chondrogenic genes while increasing expression of fibroblastic genes. However, following expansion, 10 ng/mL TGF-ß1, 100 ng/mL GDF-5, or 100 ng/mL BMP-2 supplementation during three-dimensional aggregate culture each upregulated one or more markers of chondrogenic gene expression in both hACs and hMSCs. Additionally, in both cell types, the combination of TGF-ß1, GDF-5, and BMP-2 induced the greatest upregulation of chondrogenic genes, that is, Col2A1, Col2A1/Col1A1 ratio, SOX9, and ACAN, and synthesis of cartilage-specific matrix, that is, glycosaminoglycans (GAGs) and ratio of collagen II/I. Finally, TGF-ß1, GDF-5, and BMP-2 stimulation yielded mechanically robust cartilage rich in collagen II and GAGs in both cell types, following 4 weeks maturation. This study illustrates notable success in using the self-assembling method to generate robust, scaffold-free neocartilage constructs using expanded hACs and hMSCs.

In Vitro Osteogenic Differentiation Enhanced by Zirconia Coated with Nano-Layered Growth and Differentiation Factor-5.

Zirconia (Zr) is also known as a biocompatible material with favorable mechanical properties as well as low plaque adhesion. In this study, we examined the efficacy of Zr coated with growth and differentiation factor-5 (GDF-5) bonded via click reaction as a substrate to support osteogenic differentiation of MC3T3-E1 cells. Pristine and surface-modified Zr surfaces were characterized by scanning electron microscopy (SEM) and X-ray photoelectron spectroscopy (XPS), resulting that GDF-5 was successfully coated to the pristine Zr surface. GDF-5 coated to Zr surfaces was released for 28 days in a sustained manner. New bone formation onto GDF-5 coated Zr (Zr/GDF-5) surface was confirmed by in vitro test including cell proliferation, alkaline phosphatase activity and calcium deposition assays, and in vivo test including real-time polymerase chain reaction (qPCR) assay including osterix (OSX), runt-related transcription factor 2 (Runx 2), COL 1 (type I collagen) and osteocalcin (OC). Cell proliferation, alkaline phosphatase activity, and calcium deposition of MC3T3- E1 cells were significantly enhanced when the cells were cultured on Zr/GDF-5. Additionally, the results of qPCR revealed that genes related with osteogenic differentiation were up regulated when the cells were cultured on Zr/GDF-5. Our findings demonstrate that Zr/GDF-5 could be used as a material for enhancing the efficacy of osteogenic differentiation.

The effective mode of growth and differentiation factor-5 in promoting the chondrogenic differentiation of adipose-derived stromal cells.

Our study aimed to find out the most effective mode for chondrogenic differentiation based on time, dose and culture method. ADSCs were cultured and identified by CD44, CD49d, and CD106 immumohistochemical staining method, and their differentiation potential to chondrocyte were detected by Alizarin red staining. ADSCs induced by different concentrations of GDF-5 for chondrogenic differentiation were detected by blue and toluidine blue staining and collagen type II and X immumohistochemical staining. The expression of collagen I, II, X and aggrecan gene in GDF-induced ADSCs cultured in 2- and 3-dimension was identified by real-time PCR. Cell microstructure and proliferation in three-dimensional scaffolds at day 7, 14, 21 and 28 were analyzed by scanning electron microscopy and MTS assay. The ADSCs were successfully identified by CD44 and CD49d, and their differentiation potential was detected by Alizarin red staining. Real-time PCR showed that collagen and aggrecan were expressed at high levels in 100 or 200 ng/mL GDF-5 treated cells. The collagen types (I, II) and aggrecan genes were higher expressed in GDF-5 induced scaffold group than that in monolayer group. MTS showed that the cell counts were not significantly different among different treated time. Both collagen type II and aggrecan gene were highly expressed at day 14, while collagen types I and X gene expressions peaked at day 21 and 28. The 100 ng/mL GDF-5 is effective and cost-effective for chondrogenic differentiation when cultured at day 14 in vitro under three-dimensional culture conditions.

Growth and differentiation factor-5 contributes to the structural and functional maintenance of the intervertebral disc.

Intervertebral disc degeneration (IDD) is a widely recognized contributor to low back pain (LBP). The Prevention or reversal of IDD is a potential treatment for LBP. Unfortunately, current treatments for IDD are aimed at relieving symptoms rather than regenerating disc structure or function. Recently, the injection of growth factors and mesenchymal stem cell (MSC) transplantation have been shown to be promising biological therapies for IDD. Growth factors stimulate the proliferation of and matrix synthesis by intervertebral disc (IVD) cells, leading to the regeneration of degenerative discs. Growth factors, hypoxia and co-culture with nucleus pulposus (NP) cells induce MSCs to differentiate toward an NP-like phenotype, which can increase the number of functional cells in the IVD or enhance the function of endogenous disc cells to facilitate IVD regeneration. Therefore, the emerging roles of growth factors in IVD regeneration have piqued the interest of researchers. Growth factors including transforming growth factor-ß (TGF-ß), fibroblast growth factor (FGF), insulin-like growth factor-1 (IGF-1) and growth and differentiation factor-5 (GDF-5), among others, have been demonstrated to enhance anabolism in IVD cells and to induce NP-like differentiation of MSCs. However, the injection of TGF, IGF and FGF into human IVDs may induce unwanted blood vessel ingrowth, which accelerates the process of IDD, the injection of GDF-5 may not have the same effect. This finding suggests that GDF-5 is a preferable growth factor for use in IDD treatment compared with TGF, IGF and FGF. The GDF-5 gene is one of the few growth factor genes that have been found to be associated with IDD thus far; moreover, the GDF-5 gene defects lead to collagen and proteoglycan abnormalities in discs in mice, suggesting that GDF-5 contributes to the structural and functional maintenance of the IVD. This review is focused on the functions of GDF-5 in the IVD and on the association between GDF-5 and a genetic predisposition to IDD. The effects of GDF-5 on IVD regeneration and on MSC differentiation are also discussed. GDF-5 plays a crucial role in the pathogenesis of IDD and is a promising therapeutic agent for IDD. Additionally, stem cell transplantation has been shown to be a promising biological therapy for IDD.

Increase of homogenous new bone formation using osteoinductive factor rhGDF-5 during sinus floor augmentation in Goettingen Minipigs.

OBJECTIVES: The aim of this study was to test the hypothesis that recombinant human growth and differentiation factor-5 (rhGDF-5) induces an increased and homogenous distribution of new bone formation across the entire volume of sinus floor augmentation in 12 Goettingen Minipigs. MATERIAL AND METHODS: In a randomized split-mouth design, one maxillary sinus was augmented with the bone substitute ß-TCP, whereas a combination of ß-TCP and the osteoinductive growth factor rhGDF-5 was used on the contralateral side. To evaluate the influence of dose and time on the effectiveness of the factor, two different concentrations of rhGDF-5 (400 µg and 800 µg) and healing periods (4 and 12 weeks) were each analysed. RESULTS: After 4 weeks, a homogenous gradient of bone formation could be observed for all dosage groups, with decreasing bone density from the local bone towards the sinus membrane. Both test groups, however, achieved a higher total level of bone formation compared with the control group, which was only significant in the low-dose group (P = 0.0184). After 12 weeks, the influence of the growth factor significantly depends on the region (P = 0.023). In the low-dose group, the new bone formation did not differ significantly within the examined regions of the graft (P = 0.1118), suggesting a homogeneous bone formation over the entire augmentation. The gradient of the high-dose group was similar to the control group with a decrease of local bone development. CONCLUSIONS: rhGDF-5 delivered on a ß-TCP scaffold material leads to an increase in homogeneous new bone formation across the entire volume of the sinus floor augmentation.

Comparison of GDF5 and GDNF as neuroprotective factors for postnatal dopamine neurons in ventral mesencephalic cultures.

Loss of dopamine neurons is associated with the motor deficits that occur in Parkinson's disease. Although many drugs have proven to be useful in the treatment of the symptoms of this disease, none has been shown to have a significant impact on the development of the disease. However, we believe that several neurotrophic factors have the potential to reduce its progression. Glial cell line-derived neurotrophic factor (GDNF), a member of the transforming growth factor-ß superfamily of neurotrophic factors, has been extensively studied in this regard. Less attention has been paid to growth/differentiation factor 5 (GDF5), another member of the same superfamily. This study compares GDNF and GDF5 in dissociated cultures prepared from ventral mesencephalon and in organotypic co-cultures containing substantia nigra, striatum, and neocortex. We report that both GDNF (10-500 ng/ml) and GDF5 (100-500 ng/ml) promoted the survival of dopamine neurons from the substantia nigra of postnatal rats, although GDNF was considerably more potent than GDF5. In contrast, neither factor had any significant effect on the survival of dopamine neurons from the rat ventral tegmental area. Using organotypic co-cultures, we also compared GDF5 with GDNF as chemoattractants for the innervation of the striatum and the neocortex by dopamine neurons from the substantia nigra. The addition of either GDF5 or GDNF (100-500 ng/ml) caused innervation by dopamine neurons into the cortex as well as the striatum, which did not occur in untreated cultures. Our results are consistent with similar findings suggesting that GDF5, like GDNF, deserves attention as a possible therapeutic intervention for Parkinson's disease.

GDF5 reduces MMP13 expression in human chondrocytes via DKK1 mediated canonical Wnt signaling inhibition.

OBJECTIVE: Growth differentiation factor 5 (GDF5) is important for joint formation and associated with osteoarthritis (OA). Its role for the homeostasis of cartilage extracellular matrix (ECM) is, however, unknown. The canonical Wnt signaling pathway is also implemented in OA and activation of the pathway has detrimental effects on the cartilage ECM. The objective of this study was to investigate the effect of GDF5 stimulation on the Wnt signaling pathway and on the expression of known modulators of cartilage ECM. DESIGN: Human chondrocytes were cultured in the pellet mass system and stimulated with increasing concentrations of GDF5. Expression of matrix modulating enzymes and canonical Wnt inhibitors dickkopf 1 (DKK1) and frizzled related protein (FRZB) were measured with quantitative PCR (qPCR). Protein levels of matrix metalloprotease 13 (MMP13), DKK1 and ß-catenin were measured with enzyme-linked immunosorbent assay (ELISA). Canonical Wnt signaling was stimulated with Wnt3a and small molecule CHIR-99021 and DKK1 was blocked with small molecule WAY-262611. RESULTS: In this study, we show that GDF5 stimulation of human chondrocytes inhibits expression of the cartilage ECM degrading enzymes MMP13 and ADAMTS4 and stimulates the expression of cartilage anabolic genes ACAN and SOX9. We further show that the stimulation inhibits the canonical Wnt signaling pathway through expression of the canonical Wnt inhibitors DKK1 and FRZB. Finally we show that inhibition of MMP13 expression through GDF5 stimulation is mediated by DKK1. CONCLUSION: Herein, we provide evidence of a previously unknown link between GDF5 signaling and canonical Wnt signaling that may contribute to the understanding of the molecular mechanisms of OA.

BMP2 and GDF5 induce neuronal differentiation through a Smad dependant pathway in a model of human midbrain dopaminergic neurons.

Parkinson's disease is the second most common neurodegenerative disease, and is characterised by the progressive degeneration of the nigrostriatal dopaminergic (DA) system. Current treatments are symptomatic, and do not protect against the DA neuronal loss. One of the most promising treatment approaches is the application of neurotrophic factors to rescue the remaining population of nigrostriatal DA neurons. Therefore, the identification of new neurotrophic factors for midbrain DA neurons, and the subsequent elucidation of the molecular bases of their effects, are important. Two related members of the bone morphogenetic protein (BMP) family, BMP2 and growth differentiation factor 5 (GDF5), have been shown to have neurotrophic effects on midbrain DA neurons both in vitro and in vivo. However, the molecular (signalling pathway(s)) and cellular (direct neuronal or indirect via glial cells) mechanisms of their effects remain to be elucidated. Using the SH-SH5Y human neuronal cell line, as a model of human midbrain DA neurons, we have shown that GDF5 and BMP2 induce neurite outgrowth via a direct mechanism. Furthermore, we demonstrate that these effects are dependent on BMP type I receptor activation of canonical Smad 1/5/8 signalling.

A comparative study of the effects of growth and differentiation factor 5 on muscle-derived stem cells and bone marrow stromal cells in an in vitro tendon healing model.

PURPOSE: To investigate the ability of muscle-derived stem cells (MDSCs) supplemented with growth and differentiation factor-5 (GDF-5) to improve tendon healing compared with bone marrow stromal cells (BMSCs) in an in vitro tendon culture model. METHODS: Eighty canine flexor digitorum profundus tendons were assigned into 5 groups: repaired tendon (1) without gel patch interposition (no cell group), (2) with BMSC-seeded gel patch interposition (BMSC group), (3) with MDSC-seeded gel patch interposition (MDSC group), (4) with GDF-5-treated BMSC-seeded gel patch interposition (BMSC+GDF-5 group), and (5) with GDF-5-treated MDSC-seeded gel patch interposition (MDSC+GDF-5 group). After culturing for 2 or 4 weeks, the failure strength of the healing tendons was measured. The tendons were also evaluated histologically. RESULTS: The failure strength of the repaired tendon in the MDSC+GDF-5 group was significantly higher than that of the non-cell and BMSC groups. The stiffness of the repaired tendons in the MDSC+GDF-5 group was significantly higher than that of the non-cell group. Histologically, the implanted cells became incorporated into the original tendon in all 4 cell-seeded groups. CONCLUSIONS: Interposition of a multilayered GDF-5 and MDSC-seeded collagen gel patch at the repair site enhanced tendon healing compared with a similar patch using BMSC. However, this increase in vitro was relatively small. In the clinical setting, differences between MDSC and BMSC may not be substantially different, and it remains to be shown that such methods might enhance the results of an uncomplicated tendon repair clinically. CLINICAL RELEVANCE: Muscle-derived stem cell implantation and administration of GDF-5 may improve the outcome of tendon repair.

Repairing cartilage defects with bone marrow mesenchymal stem cells induced by CDMP and TGF-ß1.

The aim of this study was to explore the ability for chondrogenic differentiation of bone marrow mesenchymal stems cells (BMSCs) induced by either cartilage-derived morphogenetic protein 1 (CDMP-1) alone or in the presence of transforming growth factor-ß1 (TGF-ß1) in vivo and in vitro. BMSCs and poly-lactic acid/glycolic acid copolymer (PLGA) scaffold were analyzed for chondrogenic capacity induced by CDMP-1 and TGF-ß1 in vivo and in vitro. Chondrogenic differentiation of BMSCs into chondrocytes using a high density pellet culture system was tested, whether they could be maintained in 3-D PLGA scaffold instead of pellet culture remains to be explored. Under the culture of high-density cell suspension and PLGA frame, BMSCs were observed the ability to repair cartilage defects by either CDMP-1 alone or in the presence of TGF-ß1 in vitro. Then the cell-scaffold complex was implanted into animals for 4 and 8 weeks for in vivo test. The content of collagen type II and proteoglycan appeared to increase over time in the constructs of the induced groups (CDMP in the presence of TGF-ß1), CDMP group and TGF group. However, the construct of the control group did not express them during the whole culture time. At 4 and 8 weeks, the collagen type II expression of the induced group was higher than the sum of TGF group and CDMP group by SSPS17.0 analysis. BMSCs and PLGA complex induced by CDMP-1 and TGF- ß1 can repair cartilage defects more effectively than that induced by CDMP-1 or TGF-ß1 only.

Alveolar ridge augmentation using implants coated with recombinant human growth/differentiation factor -5 (rhGDF-5). Radiographic observations.

OBJECTIVES: Application of growth factors onto dental implant surfaces is being considered to support local bone formation. Bone morphogenetic protein-2 (BMP-2) and BMP-7 have been shown to support local bone formation, but are also associated with adverse events including seroma formation, extensive bone remodeling, and implant displacement captured in the radiographic evaluation. This report presents mineralized tissue formation and associated adverse events following implantation of recombinant human growth/differentiation factor-5 (rhGDF-5) coated onto a purpose-designed titanium porous-oxide implant surface. MATERIAL AND METHODS: Twelve young adult Labrador dogs were used. Three 10-mm titanium implants/jaw quadrant were placed 5 mm into the alveolar ridge in the posterior mandible following surgical extraction of the premolar teeth and reduction of the alveolar ridge. Six animals received implants coated with rhGDF-5 at 30 or 60 µg/implant in contralateral jaw quadrants. Six animals received implants coated with rhGDF-5 at 120 µg/implant or uncoated implants (sham-surgery control) using the same split-mouth design. The mucoperiosteal flaps were advanced, adapted, and sutured to submerge the implants. Radiographic recordings were made immediately postsurgery (baseline), and at week 4 and 8 (end of study). Two masked examiners performed the analysis using computer enhanced radiographic images. RESULTS: rhGDF-5 coated implants displayed mineralized tissue formation significantly exceeding that of the sham-surgery control in a dose-dependent order. The greatest increase was observed for implants coated with rhGDF-5 at 60 µg and 120 µg amounting to approximately 2.2 mm for both groups at 8 weeks. Importantly, none of the implants showed evidence of peri-implant bone remodeling, implant displacement, or seroma formation. The newly formed mineralized tissues assumed characteristics of the resident bone. CONCLUSIONS: rhGDF-5 coated onto a titanium porous-oxide implant surface exhibits a dose-dependent potential to stimulate local mineralized tissue formation. Application of rhGDF-5 appears safe as it is associated with limited, if any, adverse events.

Construction of self-assembled cartilage tissue from bone marrow mesenchymal stem cells induced by hypoxia combined with GDF-5.

It is widely known that hypoxia can promote chondrogenesis of human bone marrow derived mesenchymal stem cells (hMSCs) in monolayer cultures. However, the direct impact of oxygen tension on hMSC differentiation in three-dimensional cultures is still unknown. This research was designed to observe the direct impact of oxygen tension on the ability of hMSCs to "self assemble" into tissue-engineered cartilage constructs. hMSCs were cultured in chondrogenic medium (CM) containing 100 ng/mL growth differentiation factor 5 (GDF-5) at 5% (hypoxia) and 21% (normoxia) O2 levels in monolayer cultures for 3 weeks. After differentiation, the cells were digested and employed in a self-assembly process to produce tissue-engineered constructs under hypoxic and normoxic conditions in vitro. The aggrecan and type II collagen expression, and type X collagen in the self-assembled constructs were assessed by using immunofluorescent and immunochemical staining respectively. The methods of dimethylmethylene blue (DMMB), hydroxyproline and PicoGreen were used to measure the total collagen content, glycosaminoglycan (GAG) content and the number of viable cells in each construct, respectively. The expression of type II collagen and aggrecan under hypoxic conditions was increased significantly as compared with that under normoxic conditions. In contrast, type X collagen expression was down-regulated in the hypoxic group. Moreover, the constructs in hypoxic group showed more significantly increased total collagen and GAG than in normoxic group, which were more close to those of the natural cartilage. These findings demonstrated that hypoxia enhanced chondrogenesis of in vitro, scaffold-free, tissue-engineered constructs generated using hMSCs induced by GDF-5. In hypoxic environments, the self-assembled constructs have a Thistological appearance and biochemical parameters similar to those of the natural cartilage.

Microtissue Culture Provides Clarity on the Relative Chondrogenic and Hypertrophic Response of Bone-Marrow-Derived Stromal Cells to TGF-ß1, BMP-2, and GDF-5.

Chondrogenic induction of bone-marrow-derived stromal cells (BMSCs) is typically accomplished with medium supplemented with growth factors (GF) from the transforming growth factor-beta (TGF-ß)/bone morphogenetic factor (BMP) superfamily. In a previous study, we demonstrated that brief (1-3 days) stimulation with TGF-ß1 was sufficient to drive chondrogenesis and hypertrophy using small-diameter microtissues generated from 5000 BMSC each. This biology is obfuscated in typical large-diameter pellet cultures, which suffer radial heterogeneity. Here, we investigated if brief stimulation (2 days) of BMSC microtissues with BMP-2 (100 ng/mL) or growth/differentiation factor (GDF-5, 100 ng/mL) was also sufficient to induce chondrogenic differentiation, in a manner comparable to TGF-ß1 (10 ng/mL). Like TGF-ß1, BMP-2 and GDF-5 are reported to stimulate chondrogenic differentiation of BMSCs, but the effects of transient or brief use in culture have not been explored. Hypertrophy is an unwanted outcome in BMSC chondrogenic differentiation that renders engineered tissues unsuitable for use in clinical cartilage repair. Using three BMSC donors, we observed that all GFs facilitated chondrogenesis, although the efficiency and the necessary duration of stimulation differed. Microtissues treated with 2 days or 14 days of TGF-ß1 were both superior at producing extracellular matrix and expression of chondrogenic gene markers compared to BMP-2 and GDF-5 with the same exposure times. Hypertrophic markers increased proportionally with chondrogenic differentiation, suggesting that these processes are intertwined for all three GFs. The rapid action, or "temporal potency", of these GFs to induce BMSC chondrogenesis was found to be as follows: TGF-ß1 > BMP-2 > GDF-5. Whether briefly or continuously supplied in culture, TGF-ß1 was the most potent GF for inducing chondrogenesis in BMSCs.