Growth Differentiation Factor 5 Research Articles

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.

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. 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 100µg) was injected into the nucleus pulposus (NP) of punctured discs and followed for 4weeks for gene expression analysis and 12weeks for structural analyses. For pain assessment, eight rabbits were sacrificed at 4weeks 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. 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. GDF6 injection may change the pathological status of degenerative discs and attenuate degenerated IVD-induced pain.

Structural Basis for Growth Differentiation Factor 5 (GDF5) Signaling Inhibition by Repulsive Guidance Molecules (RGMs)

Structural Basis for Growth Differentiation Factor 5 (GDF5) Signaling Inhibition by Repulsive Guidance Molecules (RGMs)

Orthotopic Transplantation of Achilles Tendon Allograft in Rats

The biology and function of orthotopic transplantation of Achilles tendon allograft are unknown. Particularly, the revitalization of Achilles allograft is a clinical concern. Achilles allografts were harvested from donor rats and stored at −80 °C. Subcutaneous adipose tissue was harvested from the would-be allograft recipient rats for isolation of mesenchymal stem cells (MSCs). MSCs were cultured with growth differentiation factor-5 (GDF-5) and applied onto Achilles allografts on the day of transplantation. After the native Achilles tendon was resected from the left hind limb of the rats, Achilles allograft, with or without autologous MSCs, was implanted and sutured with calf muscles proximally and calcaneus distally. Animal gait was recorded presurgery and postsurgery weekly. The animals were sacrificed at week 4, and the transplanted Achilles allografts were collected for biomechanical testing and histology. The operated limbs had altered gait. By week 4, the paw print intensity, stance time, and duty cycle (percentage of the stance phase in a step cycle) of the reconstructed limbs were mostly recovered to the baselines recorded before surgery. Maximum load of failure was not different between Achilles allografts, with or without MSCs, and the native tendons. The Achilles allograft supplemented with MSCs had higher cellularity than the Achilles allograft without MSCs. Deposition of fine collagen (type III) fibers was active in Achilles allograft, with or without MSCs, but it was more evenly distributed in the allografts that were incubated with MSCs. In conclusion, orthotopically transplanted Achilles allograft healed with host tissues, regained strength, and largely restored Achilles function in 4 wk in rats. It is therefore a viable option for the reconstruction of a large Achilles tendon defect. Supplementation of MSCs improved repopulation of Achilles allograft, but large animal models, with long-term follow up and cell tracking, may be required to fully appreciate the functional benefits of MSCs.

Fabrication of a Cu/Zn co-incorporated calcium phosphate scaffold-derived GDF-5 sustained release system with enhanced angiogenesis and osteogenesis properties.

Synthetic scaffolds with multifunctional properties, including angiogenesis and osteogenesis capacities, play an essential role in accelerating bone regeneration. In this study, various concentrations of Cu/Zn ions were incorporated into biphasic calcium phosphate (BCP) scaffolds, and then growth differentiation factor-5 (GDF-5)-loaded poly(lactide-co-glycolide) (PLGA) microspheres were attached onto the ion-doped scaffold. The results demonstrated that with increasing concentration of dopants, the scaffold surface gradually changed from smooth grain crystalline to rough microparticles, and further to a nanoflake film. Additionally, the mass ratio of β-tricalcium phosphate/hydroxyapatite increased with the dopant concentration. Furthermore, GDF-5-loaded PLGA microspheres attached onto the BCP scaffold surface exhibited a sustained release. In vitro co-culture of bone mesenchymal stem cells and vascular endothelial cells showed that the addition of Cu/Zn ions and GDF-5 in the BCP scaffold not only accelerated cell proliferation, but also promoted cell differentiation by enhancing the alkaline phosphatase activity and bone-related gene expression. Moreover, the vascular endothelial growth factor secretion level increased with the dopant concentration, and attained a maximum when GDF-5 was added into the ions-doped scaffold. These findings indicated that BCP scaffold co-doped with Cu/Zn ions exhibited a combined effect of both metal ions, including angiogenic and osteogenic capacities. Moreover, GDF-5 addition further enhanced both the angiogenic and osteogenic capacities of the BCP scaffold. The Cu/Zn co-incorporated BCP scaffold-derived GDF-5 sustained release system produced multifunctional scaffolds with improved angiogenesis and osteogenesis properties.

Tenogenically differentiated adipose-derived stem cells are effective in Achilles tendon repair in vivo

The purpose of this study was to characterize rat adipose-derived stem cells, induce adipose-derived stem cell tenogenesis, and analyze adipose-derived stem cell effects on tendon repair in vivo. Adipose-derived stem cells demonstrated an immunomodulatory, pro-angiogenic, and pro-proliferatory profile in vitro. Tenogenesis was induced for 1, 7, 14, and 21 days with 24 combinations of growth differentiation factor-5, 6, and 7 and platelet-derived growth factor–BB. Adipose-derived stem cells expression of scleraxis and collagen type I increased the most after 14 days of induction with growth differentiation factor-6 and platelet-derived growth factor–BB. Achilles excision defects injected with hydrogel alone (Gp2), with undifferentiated (Gp3) adipose-derived stem cells, or tenogenically differentiated (Gp4) adipose-derived stem cells exhibited improved tissue repair compared with untreated tendons (Gp1). Addition of adipose-derived stem cells improved tissue cytoarchitecture and increased expression of collagen type I and III, scleraxis, and tenomodulin. Adipose-derived stem cells significantly improved biomechanical properties (ultimate load and elastic toughness) over time more than hydrogel alone, while tenogenically differentiated adipose-derived stem cells improved the mean histological score and collagen fiber dispersion range closest to normal tendon. In addition, tendon sections treated with GFP-adipose-derived stem cells exhibited green fluorescence and positive GFP immunostaining on microscopy confirming the in vivo survival of adipose-derived stem cells that were injected into tendon defects to support the effects of adipose-derived stem cells on tissue up to 4.5 weeks post injury.

Ligand-activated BMP signaling inhibits cell differentiation and death to promote melanoma.

Oncogenomic studies indicate that copy number variation (CNV) alters genes involved in tumor progression; however, identification of specific driver genes affected by CNV has been difficult, as these rearrangements are often contained in large chromosomal intervals among several bystander genes. Here, we addressed this problem and identified a CNV-targeted oncogene by performing comparative oncogenomics of human and zebrafish melanomas. We determined that the gene encoding growth differentiation factor 6 (GDF6), which is the ligand for the BMP family, is recurrently amplified and transcriptionally upregulated in melanoma. GDF6-induced BMP signaling maintained a trunk neural crest gene signature in melanomas. Additionally, GDF6 repressed the melanocyte differentiation gene MITF and the proapoptotic factor SOX9, thereby preventing differentiation, inhibiting cell death, and promoting tumor growth. GDF6 was specifically expressed in melanomas but not melanocytes. Moreover, GDF6 expression levels in melanomas were inversely correlated with patient survival. Our study has identified a fundamental role for GDF6 and BMP signaling in governing an embryonic cell gene signature to promote melanoma progression, thus providing potential opportunities for targeted therapy to treat GDF6-positive cancers.

Growth/differentiation factor-5 promotes in vitro/vivo periodontal specific differentiation of induced pluripotent stem cell-derived mesenchymal stem cells.

Mesenchymal stem cells (MSCs) derived from induced pluripotent stem cells (iPSCs) represent a promising alternative source of MSCs for effective periodontal regeneration. Scientific evidence has demonstrated that growth/differentiation factor-5 (GDF-5) supports regeneration of periodontal tissues and has a key role in MSC differentiation. The present study investigated the effects of recombinant human GDF-5 (rhGDF-5) on periodontal specific differentiation of iPSC-derived MSCs (iPSC-MSCs) and bone marrow mesenchymal stem cells (BMSCs). rhGDF-5 treatment in vitro significantly enhanced the expression levels of marker genes associated with osteogenesis (OCN), fibrogenesis (periostin) and cementogenesis (CAP) in the iPSC-MSCs compared with untreated controls (all P<0.05). Interestingly, the rhGDF-5-treated BMSCs failed to exhibit overexpression of periostin and CAP despite highly upregulated expression of OCN. In the presence of rhGDF-5, both the iPSC-MSCs and BMSCs demonstrated marked formation of mineralized nodules. Notably, rhGDF-5 greatly promoted periodontal specific differentiation of the iPSC-MSCs encapsulated in hyaluronic acid (HA) hydrogels in vivo as determined by immunohistochemical and immunofluorescence staining. The majority of the PKH67-labeled iPSC-MSCs implanted with rhGDF-5 exhibited strong expression of OCN, periostin and CAP. In conclusion, iPSC-MSCs demonstrate high periodontal specific differentiation potential in response to rhGDF-5 both in vitro and in vivo. The delivery of iPSC-MSCs and rhGDF-5 with HA hydrogel may have beneficial effects in regenerative periodontal therapy.

MicroRNA-132 upregulation promotes matrix degradation in intervertebral disc degeneration

MicroRNAs (miRNAs) have been shown to be involved in the pathogenesis of intervertebral disc degeneration (IDD). This experiment was designed to study the expression and role of the miRNA, miR-132, in IDD. MiR-132 expression in human nucleus pulposus (NP) tissue was assessed by quantitative real-time PCR. The methylation status of the miR-132 was assessed with methylation-specific PCR and bisulfite sequencing PCR. The regulation of growth differentiation factor5 (GDF5) expression by miR-132 was evaluated by luciferase reporter assay. Moreover, we investigated the function of miR-132 on IDD in vivo using a classic needle-punctured rat tail model. These results showed that miR-132 expression was upregulated during IDD and this upregulation was associated with hypomethylation of its promoter. MiR-132 overexpression led to increased expression of ECM catabolic factors, including MMP13 and ADAMTS4, in NP cells while levels of anabolic proteins, such as type II collagen and aggrecan, were diminished. GDF5 was identified as a direct target of negative regulation by miR-132. MAPK/ERK signaling was also found to be associated with miR-132-induced ECM degradation. In addition, we showed that miR-132 inhibition effectively attenuated NP ECM degradation in IDD in vivo. Our findings demonstrated that miR-132 promotes ECM degradation by human NP cells by direct targeting of GDF5. Hence, miR-132 represents a potential therapeutic target in the treatment of IDD.

Growth Differentiation Factor 5 Accelerates Wound Closure and Improves Skin Quality During Repair of Full-Thickness Skin Defects.

A fast and stable wound closure is important, especially for extended and unstable wounds found after burn injuries. Growth can regulate a variety of cellular processes, including those involved in wound healing. Growth differentiation factor 5 (GDF-5) can accelerate fibroblast cell migration, cell proliferation, and collagen synthesis, which are essential for wound healing. Nevertheless, no standardized evaluation of the effect of GDF-5 on the healing of full-thickness wounds has been published to date. Five full-thickness skin defects were created on the backs of 6 minipigs. Three wounds were treated with GDF-5 in different concentrations with the help of a gelatin-collagen carrier, and 2 wounds served as control group. The first was treated with the gelatin carrier and an Opsite film (Smith & Nephew, Fort Worth, Texas), and the other was treated solely with an Opsite film that was placed above all wounds and renewed every second day. Growth differentiation factor 5 accelerates wound closure (10.91 [SD, 0.99] days) compared with treatment with the carrier alone (11.3 [SD, 1.49] days) and control wounds (13.3 [SD, 0.94] days). Epidermal cell count of wounds treated with GDF-5 revealed a higher number of cells compared with the control group. In addition, mean epidermal thickness was significantly increased in GDF-5-treated wounds compared with the control wounds. Because of its ability to improve skin quality, GDF-5 should be considered when developing composite biomaterials for wound healing.

Signaling by growth/differentiation factor 5 through the bone morphogenetic protein receptor type IB protects neurons against kainic acid-induced neurodegeneration

Growth/differentiation factor-5 (GDF-5), a member of the transforming growth factor-beta (TGF-β) superfamily, has been shown to protect rat dopaminergic neurons against insult both in embryonic neuronal culture and in Parkinson’s disease models. However, whether GDF-5 exerts neuroprotective effects in hippocampal neurons is unclear. Here, we show that both mRNA levels and protein levels of GDF-5 are decreased in the mouse hippocampus upon kainic acid (KA) treatment. KA induced dramatic neuronal loss specifically in the cornu ammonis 1 (CA1) and CA3 areas of the mouse hippocampus, while intracerebral infusion of GDF-5 prevented this neuronal loss. The neuroprotective effects of GDF-5 were recapitulated by constitutively active bone morphogenetic protein type IB receptor (BMPRIB-CA) and could be blocked by BMPRI kinase inhibitor LDN-193189. Furthermore, the neuroprotective effects of GDF-5 were mediated through the prevention of apoptosis, which was indicated by terminal deoxynucleotidyl transferase (TdT) dUTP nick-end labeling (TUNEL) staining and reduced cleaved caspase 3 expression level. Thus, we conclude that GDF-5 protects hippocampal neurons against KA-induced neurodegeneration by signaling through BMPRIB, suggesting a therapeutic potential for GDF-5 in neurodegenerative diseases.

Growth Differentiation Factor 5-Mediated Enhancement of Chondrocyte Phenotype Is Inhibited by Heparin: Implications for the Use of Heparin in the Clinic and in Tissue Engineering Applications.

The highly sulfated glycosaminoglycan (GAG) heparin is widely used in the clinic as an anticoagulant, and researchers are now using it to enhance stem cell expansion/differentiation protocols, as well as to improve the delivery of growth factors for tissue engineering (TE) strategies. Growth differentiation factor 5 (GDF5) belongs to the bone morphogenetic protein family of proteins and is vital for skeletal formation; however, its interaction with heparin and heparan sulfate (HS) has not been studied. We identify GDF5 as a novel heparin/HS binding protein and show that HS proteoglycans are vital in localizing GDF5 to the cell surface. Clinically relevant doses of heparin (≥10 nM), but not equivalent concentrations of HS, were found to inhibit GDF5's biological activity in both human mesenchymal stem/stromal cell-derived chondrocyte pellet cultures and the skeletal cell line ATDC5. We also found that heparin inhibited both GDF5 binding to cell surface HS and GDF5-induced induction of Smad 1/5/8 signaling. Furthermore, GDF5 significantly increased aggrecan gene expression in chondrocyte pellet cultures, without affecting collagen type X expression, making it a promising target for the TE of articular cartilage. Importantly, this study may explain the variable (and disappointing) results seen with heparin-loaded biomaterials for skeletal TE and the adverse skeletal effects reported in the clinic following long-term heparin treatment. Our results caution the use of heparin in the clinic and in TE applications, and prompt the transition to using more specific GAGs (e.g., HS derivatives), with better-defined structures and fewer off-target effects.

Novel homozygous sequence variants in the GDF5 gene underlie acromesomelic dysplasia type‐grebe in consanguineous families

Acromesomelic dysplasia Grebe type (AMDG) is characterized by severe knob like non-functional fingers and short acromesomelic limbs, and is inherited in an autosomal recessive manner. Disease causing sequence variants in the GDF5 (Growth Differentiation Factor 5) gene located on chromosome 20q11.22 are responsible for causing AMDG. In the study, presented here, two consanguineous families with AMDG were clinically and genetically characterized. After establishing linkage in the two families (A and B) to GDF5 gene on chromosome 20q11.22, Sanger DNA sequencing was performed in all available affected and unaffected members. Sequence analysis of the GDF5 gene revealed two novel variants including a duplication (c.157_158dupC, p.Leu53Profs*41) in family A, and a nonsense (p.Trp291*) in family B. Our findings extend the body of evidence that supports the importance of GDF5 in the development of limbs.

HTRA1 and TGF-β1 Concentrations in the Aqueous Humor of Patients With Neovascular Age-Related Macular Degeneration.

The purpose of this study was to evaluate the expression of high-temperature requirement A serine peptidase 1 (HTRA1), TGF-β1, bone morphogenetic protein 4 (BMP4), growth differentiation factor 6 (GDF6), and VEGFA proteins in the aqueous humor of patients with naïve choroidal neovascularization (nCNV) secondary to AMD. We measured by ELISA the concentrations of HTRA1, TGF-β1, BMP4, GDF6, and VEGFA in the aqueous humor of 23 patients affected by nCNV who received three consecutive monthly intravitreal injections of 0.5 mg ranibizumab. Samples were collected at baseline (before the first injection), month 1 (before the second injection), and month 2 (before the third injection). Twenty-three age-matched cataract patients served as controls. Bone morphogenetic protein 4 and GDF6 were not detectable in any samples. Baseline HTRA1 was higher than controls (P < 0.0001) and higher than both the month 1 (P < 0.0001) and the month 2 (P < 0.0001) values. Baseline VEGFA was higher than controls (P < 0.0001), not different from month 1 value (P = 0.0821), but higher than month 2 value (P < 0.0001). Baseline TGF-β1 was higher than controls (P = 0.0015) and not different from month 1 (P = 0.129) and month 2 values (P = 0.5529). No correlation was found in naïve patients between concentrations of HTRA1 and TGF-β1, HTRA 1 and VEGFA, or TGF-β1 and VEGFA. In nCNV patients, HTRA1 and TGF-β1 were significantly higher compared to controls. After treatment, TGF-β1 was persistently elevated, while HTRA1 returned to control levels, suggesting the involvement of TGF-β1 and HTRA1 in neovascular AMD and a VEGFA-independent role for TGF-β1.

The use of heparin chemistry to improve dental osteogenesis associated with implants

In this study, we designed a hybrid Ti by heparin modifying the Ti surface followed by Growth/differentiation factor-5 (GDF-5) loading. After that, products were characterized by physicochemical analysis. Quantitative analysis of functionalized groups was also confirmed. The release behavior of GDF-5 grafted samples was confirmed for up to 21days. The surface modification process was found to be successful and to effectively immobilize GDF-5 and provide for its sustained release behavior. As an in vitro test, GDF-5 loaded Ti showed significantly enhanced osteogenic differentiation with increased calcium deposition under nontoxic conditions against periodontal ligament stem cells (PDLSc). Furthermore, an in vivo result showed that GDF-5 loaded Ti had a significant influence on new bone formation in a rabbit model. These results clearly confirmed that our strategy may suggest a useful paradigm by inducing osseo-integration as a means to remodeling and healing of bone defects for restorative procedures in dentistry.

Association between GDF5 +104T/C polymorphism and knee osteoarthritis in Caucasian and Asian populations: a meta-analysis based on case-control studies.

BackgroundOsteoarthritis (OA) is a degenerative joint disease with a complex genetic background. Variants in growth differentiation factor 5 (GDF5) have been reported to be associated with rheumatoid arthritis (RA) in several ethnic populations. The present study aimed to assess the association between the GDF5 +104T/C polymorphism and the susceptibility of the knee to OA through a meta-analysis of available case-control studies.MethodsThe PubMed and Science Direct citation databases were used to search electronic literature in order to identify studies published between January 2007 and July 2016 that evaluated the association between the GDF5 +104T/C polymorphism and the susceptibility of the knee to OA. Different genetic models were used to assess the pooled and stratified data.ResultsA positive association was found in all pooled studies (OR = 0.808, 95 % CI = 0.754–0.866, p < 0.001). Regarding genotypes, significant associations were found using a dominant model (OR = 0.777, 95 % CI = 0.708–0.852, p < 0.001), a recessive model (OR = 0.723, 95%CI = 0.623–0.839, p < 0.001), and an additive model (CC vs TT OR = 0.648, 95 % CI = 0.552–0.760, p < 0.001; CC vs CT OR = 0.801, 95 % CI = 0.685–0.936, p = 0.005). Meta-analysis data were stratified by ethnicity, and the GDF5 C allele was found to be positively associated with OA of the knee in both Caucasians and Asians, as were the GDF5 TC and CC genotypes. In addition, using an additive model, the CC genotype was found to be significantly associated with OA of the knee in both Caucasians and Asians when comparing CC vs TT genotypes, but not in Caucasians when comparing TT vs CT genotypes.ConclusionsMeta-analysis results indicated that the GDF5 +104T/C polymorphism is a protective factor for OA among Caucasian and Asian populations.

MicroRNA-7 regulates IL-1β-induced extracellular matrix degeneration by targeting GDF5 in human nucleus pulposus cells.

The precise role of interleukin-1 beta (IL-1β)-induced extracellular matrix degeneration in the pathogenesis of intervertebral disc degeneration (IDD) is currently unknown. Recent evidence has revealed that microRNAs (miRNAs) are associated with IDD, but their function in the extracellular matrix degradation of nucleus pulposus (NP) tissues is also poorly understood. The aim of this study was to evaluate the expression and functional role of miR-7 in IL-1β-induced disc degeneration. The expression level of miR-7 was investigated in degenerative NP tissues and in IL-1β-induced NP cells using quantitative reverse transcription-polymerase chain reaction amplification analysis. A dual-luciferase reporter assay was then utilized to determine whether growth differentiation factor 5 (GDF5) is a target of miR-7. Finally, mRNA and protein levels of known matrix components and of matrix degradation enzymes were determined to elucidate the function of miR-7 in IL-1β-induced disc degeneration. In this study, we found that miR-7 is highly expressed in human degenerative NP tissues and in IL-1β stimulated NP cells compared to normal controls. We also determined that GDF5 was a target of miR-7. Functional analysis showed that the overexpression of miR-7 significantly enhanced the IL-1β-induced extracellular matrix degeneration, whereas inhibition of miR-7 function by antagomiR-7 prevented NP cell detrimental catabolic changes in response to IL-1β. Additionally, the prevention of IL-1β-induced NP extracellular matrix degeneration by miR-7 silencing was attenuated by GDF5 siRNA. These findings suggest that miR-7 contributes to an impaired ECM in intervertebral discs through targeting GDF5 and miR-7 might therefore represent a novel therapeutic target for the prevention of IDD.

MiR-132-3p Regulates the Osteogenic Differentiation of Thoracic Ligamentum Flavum Cells by Inhibiting Multiple Osteogenesis-Related Genes.

Ossification of the ligamentum flavum (OLF) is a disorder of heterotopic ossification of spinal ligaments and is the main cause of thoracic spinal canal stenosis. Previous studies suggested that miR-132-3p negatively regulates osteoblast differentiation. However, whether miR-132-3p is involved in the process of OLF has not been investigated. In this study, we investigated the effect of miR-132-3p and its target genes forkhead box O1 (FOXO1), growth differentiation factor 5 (GDF5) and SRY-box 6 (SOX6) on the osteogenic differentiation of ligamentum flavum (LF) cells. We demonstrated that miR-132-3p was down-regulated during the osteogenic differentiation of LF cells and negatively regulated the osteoblast differentiation. Further, miR-132-3p targeted FOXO1, GDF5 and SOX6 and down-regulated the protein expression of these genes. Meanwhile, FOXO1, GDF5 and SOX6 were up-regulated after osteogenic differentiation and the down-regulation of endogenous FOXO1, GDF5 or SOX6 suppressed the osteogenic differentiation of LF cells. In addition, we also found FOXO1, GDF5 and SOX6 expression in the ossification front of OLF samples. Overall, these results suggest that miR-132-3p inhibits the osteogenic differentiation of LF cells by targeting FOXO1, GDF5 and SOX6.

Prospective cohort study of the risk factors for stress fractures in Chinese male infantry recruits.

ObjectiveTo determine potential risk factors that could predict stress fractures over an 8-week basic military training in Chinese male infantry recruits.MethodsRecruits from three infantry units enrolled in this prospective study. At baseline, demographic data, personal history of stress fractures, mean duration of weekly exercise and smoking history were recorded on questionnaires and blood samples taken for analysis of bone turnover biomarkers and genetic factors.ResultsOf the 1516 male recruits who volunteered to participate in the study, 1398 recruits provided data for analysis. In total, 189 stress fracture cases were observed (incidence rate: 13.5%) during the 8-week training period. Recruits with stress fractures had a significantly higher incidence of prior fracture history and lower exercise level prior to enrolment compared with those without stress fractures. A significant difference in both allelic frequency and genotypic distribution of the growth differentiation factor 5 (GDF5) gene rs143383 polymorphism was observed between recruits with and without stress fractures. However, no difference in serum bone turnover biomarkers was detected between groups.ConclusionThis prospective, cohort study indicates that fracture history, lower exercise level and GDF5 rs143383 may be predictive risk factors for stress fractures in Chinese male infantry recruits.

Protection of cultured dopamine neurons from MPP(+) requires a combination of neurotrophic factors.

Parkinson's disease is a progressive neurodegenerative disorder, caused in part by the loss of dopamine (DA) neurons in the substantia nigra (SN). Neurotrophic factors have been shown to increase the basal survival of DA neurons invitro, as well as to protect the neurons from some toxins under certain invitro conditions and in animal models. Although these factors have often been tested individually, they have rarely been studied in combinations. We therefore examined the effect of such combinations after acute exposure to the toxin 1-methyl-4-phenylpyridinium (MPP(+) ) using dissociated postnatal rat midbrain cultures isolated from SN and ventral tegmental area (VTA). We found that significant loss of DA neurons in the SN occurred with an LC50 of between 1 and 10μm, whereas the LC50 of DA neurons from the VTA was approximately 1000-fold higher. We did not observe neuroprotection against MPP(+) by individual exposure to glial cell-line derived neurotrophic factor (GDNF), brain derived neurotrophic factor (BDNF), transforming growth factor beta (TGFβ), basic fibroblast growth factor (FGF-2) or growth/differentiation factor 5 (GDF5) at concentrations of 100 or 500ng/mL. Combinations of two, three or four neurotrophic factors were also ineffective. However, when the SN cultures were exposed to a combination of all five neurotrophic factors, each at a concentration of 100ng/mL, we observed a 30% increase in DA neuron survival in the presence of 10 and 500μm MPP(+) . These results may be relevant to the use of neurotrophic factors as therapeutic treatments for Parkinson's disease.

Mutant GDF5 enhances ameloblast differentiation via accelerated BMP2-induced Smad1/5/8 phosphorylation.

Bone morphogenetic proteins (BMPs) regulate hard tissue formation, including bone and tooth. Growth differentiation factor 5 (GDF5), a known BMP, is expressed in cartilage and regulates chondrogenesis, and mutations have been shown to cause osteoarthritis. Notably, GDF5 is also expressed in periodontal ligament tissue; however, its role during tooth development is unclear. Here, we used cell culture and in vivo analyses to determine the role of GDF5 during tooth development. GDF5 and its associated BMP receptors are expressed at the protein and mRNA levels during postnatal tooth development, particularly at a stage associated with enamel formation. Furthermore, whereas BMP2 was observed to induce evidently the differentiation of enamel-forming ameloblasts, excess GDF5 induce mildly this differentiation. A mouse model harbouring a mutation in GDF5 (W408R) showed enhanced enamel formation in both the incisors and molars, but not in the tooth roots. Overexpression of the W408R GDF5 mutant protein was shown to induce BMP2-mediated mRNA expression of enamel matrix proteins and downstream phosphorylation of Smad1/5/8. These results suggest that mutant GDF5 enhances ameloblast differentiation via accelerated BMP2-signalling.