Growth Differentiation Factor 5 Research Articles

Microparticles for controlled growth differentiation factor 6 delivery to direct adipose stem cell-based nucleus pulposus regeneration.

Currently, there is no effective long‐term treatment for intervertebral disc (IVD) degeneration, making it an attractive candidate for regenerative therapies. Hydrogel delivery of adipose stem cells (ASCs) in combination with controlled release of bioactive molecules is a promising approach to halt IVD degeneration and promote regeneration. Growth differentiation factor 6 (GDF6) can induce ASC differentiation into anabolic nucleus pulposus (NP) cells and hence holds promise for IVD regeneration. Here, we optimised design of novel poly(DL‐lactic acid‐co‐glycolic acid) (PLGA)–polyethylene glycol–PLGA microparticles to control GDF6 delivery and investigated effect of released GDF6 on human ASCs differentiation to NP cells. Recombinant human (rh)GDF6 was loaded into microparticles and total protein and rhGDF6 release assessed. The effect of microparticle loading density on distribution and gel formation was investigated through scanning electron microscopy. ASC differentiation to NP cells was examined after 14 days in hydrogel culture by quantitative polymerase chain reaction, histological, and immunohistochemical staining in normoxic and IVD‐like hypoxic conditions. RhGDF6 microparticles were distributed throughout gels without disrupting gelation and controlled rhGDF6 release over 14 days. Released GDF6 significantly induced NP differentiation of ASCs, with expression comparable with or exceeding media supplemented rhGDF6. Microparticle‐delivered rhGDF6 also up‐regulated sulphated glycosaminoglycan and aggrecan secretion in comparison with controls. In hypoxia, microparticle‐delivered rhGDF6 continued to effectively induce NP gene expression and aggrecan production. This study demonstrates the effective encapsulation and controlled delivery of rhGDF6, which maintained its activity and induced ASC differentiation to NP cells and synthesis of an NP‐like matrix suggesting suitability of microparticles for controlled growth factor release in regenerative strategies for treatment of IVD degeneration.

151 Investigating BMP signaling as a therapeutic target in advanced melanoma

Melanoma is the leading cause of skin cancer death in the United States, with a 5-year survival rate of 20% for patients with advanced disease. The development of targeted therapies and immunotherapy has helped improve survival for some patients, but many others still face significant therapeutic challenges, including resistance to therapy and relapse of their disease. Furthermore, our understanding of the steps involved in melanoma initiation and progression is incomplete. We are interested in uncovering factors involved in melanoma initiation and progression with a focus on identifying those key factors that have therapeutic potential. Previously, we identified a novel melanoma oncogene, growth differentiation factor 6 (GDF6), a bone morphogenetic protein (BMP) ligand that promotes melanoma by suppressing differentiation and apoptosis. We have shown that the dedifferentiation effects are mediated by suppression of MITF, the master regulator of melanocyte differentiation, and the anti-apoptotic effects of GDF6 are mediated by suppression of SOX9, a pro-apoptotic factor in melanoma. These results establish GDF6-activated BMP signaling may play a significant role during melanoma progression. Here, we further establish BMP activity impacts melanocyte development and may have therapeutic value in established disease. We find that GDF6 inhibits embryonic melanocyte differentiation from the neural crest, suggesting that its function in melanoma to maintain a dedifferentiated state is a reiteration (or reawakening) of its role in early development. We further identify novel anti-GDF6 therapeutic antibodies that exhibit anti-melanoma activity in vitro, indicating inhibition of GDF6-activated BMP signaling is a potential therapeutic approach for advanced melanoma. Together, these results indicate the importance of GDF6-activated BMP signaling in the development of the melanocyte lineage and melanoma, and provide evidence for targeting BMP signaling as a therapeutic approach in advanced melanoma.

Identification of a Novel NOG Missense Mutation in a Chinese Family With Symphalangism and Tarsal Coalitions.

BackgroundProximal symphalangism (SYM1) is a rare genetic bone disorder characterized by the fusion of proximal interphalangeal joints in the hands and feet. Genetic studies have identified two genes underlying SYM1 as the noggin (NOG) and the growth differentiation factor 5 (GDF5).Case ReportIn the present report, a 43-year-old gravida at 11 weeks of gestation was referred for evaluation of abnormal fusions of the joints. In the initial diagnosis, physical examination was undertaken. However, traditional radiological examination was not applied due to the need to protect the fetus, making diagnosis results inefficient to determine the exact disease affecting the proband. To acquire alternative clinical evidences, we conducted radiological examinations on two other affected family members. The radiological examination revealed that they carried the symphalangism accompanied with tarsal coalition, a very rare manifestation of SYM1. A combination of whole exome sequencing (WES) and Sanger sequencing revealed a novel heterozygous missense mutation (c.163G > T; p.Asp55Tyr) in the NOG gene, which could be associated with the observed pathogenic SYM1 in the studied family. The p.Asp55Tyr mutation co-segregated with SYM1 through the affected and unaffected family members. In silico structural modeling of the p.Asp55Tyr mutation showed that it abolishes the interaction with the Arg167 residue and causes a change in the electrostatic potential profile of the type II binding site of the noggin protein.ConclusionOur findings indicate that the genetic test based on WES can be useful in diagnosing SYM1 patients, with particular advantages in preventing the fetus from contacting harmful X-ray through the traditional radiography. The novel pathogenic mutation identified would further expand our understanding of the mutation spectrum of NOG in association with SYM1 disease and provide a guidance on how to determine whether the fetus is affected by SYM1 through the prenatal diagnosis.

Long Non-Coding RNA Myoparr Regulates GDF5 Expression in Denervated Mouse Skeletal Muscle.

Skeletal muscle is a highly plastic tissue and decreased skeletal muscle mass (muscle atrophy) results in deteriorated motor function and perturbed body homeostasis. Myogenin promoter-associated long non-coding RNA (lncRNA) Myoparr promotes skeletal muscle atrophy caused by surgical denervation; however, the precise molecular mechanism remains unclear. Here, we examined the downstream genes of Myoparr during muscle atrophy following denervation of tibialis anterior (TA) muscles in C57BL/6J mice. Myoparr knockdown affected the expression of 848 genes. Sixty-five of the genes differentially regulated by Myoparr knockdown coded secretory proteins. Among these 65 genes identified in Myoparr-depleted skeletal muscles after denervation, we focused on the increased expression of growth/differentiation factor 5 (GDF5), an inhibitor of muscle atrophy. Myoparr knockdown led to activated bone morphogenetic protein (BMP) signaling in denervated muscles, as indicated by the increased levels of phosphorylated Smad1/5/8. Our detailed evaluation of downstream genes of Myoparr also revealed that Myoparr regulated differential gene expression between myogenic differentiation and muscle atrophy. This is the first report demonstrating the in vivo role of Myoparr in regulating BMP signaling in denervated muscles. Therefore, lncRNAs that have inhibitory activity on BMP signaling may be putative therapeutic targets for skeletal muscle atrophy.

Regulation of a GDF5 Associated Enhancer During Limb Development

Growth differentiation factor 5 (GDF5) is an anabolic growth factor that plays a critical role in the formation, maintenance and repair of bones and joints. Reduced levels of GDF5 are associated with accelerated degradation of joint cartilage, a disease called osteoarthritis (OA). Identification of factors involved in GDF5 regulation may offer therapeutic targets for treating or preventing OA. A GDF5 Associated Regulatory Region (GARR) has been previously identified as a 900 bp enhancer located 78 kb downstream of the GDF5 locus. GARR contains a number of potential transcription factor binding sites, including sites for odd‐skipped related (Osr) zinc‐finger and Sry box (Sox) transcription factors. As Osr1/2 and Sox11 are important for normal Gdf5 expression, and their expression patterns overlap that of Gdf5, we hypothesized that mutation of OSR and SOX binding sites within GARR would disrupt enhancer activity.To identify whether putative OSR and SOX transcription factor binding sites contribute to GARR activity, we performed site‐directed mutagenesis on both potential OSR binding sites (GARRmutOSR), or on both potential SOX binding sites (GARRmutSOX) in the GARR‐tk‐EGFP reporter construct (GARRcontrol). We used targeted regional electroporation (TREP) to introduce control and mutated reporter constructs into Hamburger‐Hamilton stage 23 chicken limb buds near the developing elbow joint. Transfection efficiency was determined by co‐transfection with a β‐actin promoter‐driven RFP construct. We determined GARR activity by fluorescence microscopy, following 48 hours of incubation after TREP.Comparison of GARRcontrol and GARRmutOSR demonstrated similar GFP fluorescence intensity localized to the elbow joint. In contrast, fluorescence of GARRmutSOX was markedly reduced relative to GARRcontrol, although similarly restricted to the elbow. Though Osr transcription factors are associated with Gdf5 expression, mutation of the OSR binding sites in GARR was insufficient to substantially alter the level of GARR activity. Mutation of SOX binding sites, however, dramatically reduced GARR activity, implicating SOX transcription factors as critical regulators of GDF5 expression.Support or Funding InformationThe research was funded in part by a grant from the LLU Pathology Research Endowment.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Low-intensity pulsed ultrasound combination with induced pluripotent stem cells-derived neural crest stem cells and growth differentiation factor 5 promotes sciatic nerve regeneration and functional recovery.

The treatment of lengthy peripheral nerve defect is challenging in the field of nerve regeneration. Our previous studies have shown that low-intensity pulsed ultrasound (LIPUS) could promote the proliferation, cell viability, and neural differentiation of induced pluripotent stem cells-derived neural crest stem cells (iPSCs-NCSCs) and improve the regeneration of damaged peripheral nerve. In this study, the mechanical signal transduction pathway of LIPUS promoting iPSCs-NCSCs proliferation and differentiation was further explored, and the effects of LIPUS combined with iPSCs-NCSCs, perfluorotributylamine (PFTBA), and growth differentiation factor 5 (GDF5) on the repair of peripheral nerve injury were evaluated. Results showed LIPUS may regulate the proliferation and differentiation of iPSCs-NCSCs through FAK-ERK1/2 signal pathway. PFTBA could supply sufficient oxygen to promote the viability of iPSCs-NCSCs under 5% hypoxia culture condition and provide a favourable microenvironment for nerve regeneration. The addition of GDF5 could promote the neural differentiation of iPSCs-NCSCs in vitro. LIPUS treatment of allogeneic decellularized nerve conduit containing iPSCs-NCSCs, PFTBA, and GDF5 has very good effect on the repair of sciatic nerve injury. Taken together, these results provide functional evidence that LIPUS might be a useful tool to explore alternative approaches in the field of nerve regeneration.

3D printed titanium cages combined with the Masquelet technique for the reconstruction of segmental femoral defects: Preliminary clinical results and molecular analysis of the biological activity of human-induced membranes.

Introduction:Traumatic femoral segmental bone loss is a complex clinical problem, one that often requires extreme solutions. This study examines a new treatment strategy for segmental bone loss using patient-specific 3D printed titanium cages in conjunction with the Masquelet technique.Methods:The study was composed of a clinical observational case series, and a basic science investigation to evaluate the biological activity of the induced membranes using histology, immunohistochemistry (IHC), and gene expression analysis. Eligible patients were: adult; post-traumatic; with segmental femoral defects; minimum follow-up 1 year; managed under a 2-stage protocol, with an interim antibiotic poly (methyl methacrylate) (PMMA) spacer. Definitive reconstruction was completed with exchange to a 3D printed custom titanium cage filled with bone graft, and stabilized with either an intramedullary (IM) nail or a lateral locked plate.Results:Patient-specific 3D printed titanium cages were used in 5 consecutive patients to reconstruct post-traumatic segmental femoral defects. The mean interval between stages was 100.2 days (83–119 days), the mean defect length was 14.0 cm (10.3–18.4 cm), and the mean bone defect volume measured 192.4 cc (114–292 cc). The mean length of follow-up was 21.8 months (12–33 months). There were no deep infections, fractures, nerve injuries, loss of alignment, or nonunions identified during the period of follow-up. All of the patients achieved union clinically and radiographically. Histology and IHC demonstrated a greater number of vessels, cell nuclei, and extensive staining for cluster of differentiation 68 (CD68), platelet and endothelial cell adhesion molecule 1 (PECAM-1), and vascular endothelial growth factor (VEGF) in the induced membranes compared to local fascia controls. Gene expression analysis revealed significant differential regulation of essential genes involved in inflammatory, angiogenic, and osteogenic pathways [interleukin 6 (IL-6), nuclear factor kappa B1 (NF-κB1), receptor activator of nuclear factor kappa-β ligand (RANKL), vascular endothelial growth factor A (VEGFA), angiogenin (ANG), transforming growth factor, beta 1 (TGF-β1), bone morphogenetic protein-2 (BMP-2), growth differentiation factor 5 (GDF-5), growth differentiation factor 10 (GDF-10), and runt-related transcription factor 2 (RUNX-2)] in the induced membranes.Conclusions:This study demonstrates that the use of a patient-specific 3D printed custom titanium cage, inserted into an induced membrane in a 2-stage protocol, can achieve very acceptable clinical outcomes in selected cases of post-traumatic femoral segmental defects. Patient-specific 3D printed titanium cages, used in conjunction with the Masquelet technique, are a promising new treatment option for managing complex trauma patients with femoral bone loss.Level of Evidence:Level IV (observational case series).

3D-bioprinting a genetically inspired cartilage scaffold with GDF5-conjugated BMSC-laden hydrogel and polymer for cartilage repair.

Rationale: Articular cartilage injury is extremely common in congenital joint dysplasia patients. Genetic studies have identified Growth differentiation factor 5 (GDF5) as a shared gene in joint dysplasia and OA progression across different populations. However, few studies have employed GDF5 in biological regeneration for articular cartilage repair.Methods & Results: In the present study, we report identified genetic association between GDF5 loci and hip joint dysplasia with genome-wide association study (GWAS). GWAS and replication studies in separate populations achieved significant signals for GDF5 loci. GDF5 expression was dysregulated with allelic differences in hip cartilage of DDH and upregulated in the repaired cartilage in a rabbit cartilage defect model. GDF5 in vitro enhanced chondrogenesis and migration of bone marrow stem cells (BMSCs), GDF5 was tested in ectopic cartilage generation with BMSCs by GDF5 in nude mice in vivo. Genetically inspired, we further generated functional knee articular cartilage construct for cartilage repair by 3d-bioprinting a GDF5-conjugated BMSC-laden scaffold. GDF5-conjugated scaffold showed better cartilage repairing effects compared to control. Meanwhile, transplantation of the 3D-bioprinted GDF5-conjugated BMSC-laden scaffold in rabbit knees conferred long-term chondroprotection.Conclusions: In conclusion, we report identified genetic association between GDF5 and DDH with combined GWAS and replications, which further inspired us to generate a ready-to-implant GDF5-conjugated BMSC-laden scaffold with one-step 3d-bioprinting for cartilage repair.

Comparison of the effect of growth factors on chondrogenesis of canine mesenchymal stem cells.

Mesenchymal stem cells (MSCs) are proposed to be useful in cartilage regenerative medicine, however, canine MSCs have been reported to show poor chondrogenic capacity. Therefore, optimal conditions for chondrogenic differentiation should be determined by mimicking the developmental process. We have previously established novel and superior canine MSCs named bone marrow peri-adipocyte cells (BM-PACs) and the objective of this study was to evaluate the effects of growth factors required for in vivo chondrogenesis using canine BM-PACs. Spheroids of BM-PACs were cultured in chondrogenic medium containing 10 ng/ml transforming growth factor-β1 (TGF-β1) with or without 100 ng/ml bone morphogenetic protein-2 (BMP-2), 100 ng/ml growth differentiation factor-5 (GDF-5) or 100 ng/ml insulin-like growth factor-1 (IGF-1). Chondrogenic differentiation was evaluated by the quantification of glycosaminoglycan and Safranin O staining for proteoglycan production. The expression of cartilage matrix or hypertrophic gene/protein was also evaluated by qPCR and immunohistochemistry. Spheroids in all groups were strongly stained with Safranin O. Although BMP-2 significantly increased glycosaminoglycan production, Safranin O-negative outer layer was formed and the mRNA expression of COL10 relating to cartilage hypertrophy was also significantly upregulated (P<0.05). GDF-5 promoted the production of glycosaminoglycan and type II collagen without increasing COL10 mRNA expression. The supplementation of IGF-1 did not significantly affect cartilaginous and hypertrophic differentiation. Our results indicate that GDF-5 is a useful growth factor for the generation of articular cartilage from canine MSCs.

Association Between the SNP rs143383 + 104T/C in the GDF5 Gene and the Risk of Knee Osteoarthritis in a Population from Northern Mexico-A Case-Control Study.

Previous studies have found an association between the SNP rs143383 + 104T/C in the growth differentiation factor 5 (GDF5) gene and the risk of developing knee osteoarthritis (KOA) in various populations worldwide. To discover if there is an association between the SNP rs143383 + 104T/C, in the GDF5 gene and the risk of developing KOA in individuals from northern Mexico. We used a case-control study format that included 145 patients with KOA and 145 healthy controls unrelated to patients. A peripheral blood sample was taken for each of them to identify the SNP by means of real-time PCR according to standard protocols. We found a strong association between the TT genotype and the risk of developing KOA (OR = 1.7, 95% CI = 1.12-2.8, p = 0.014), but not in the heterozygous TC state (OR = 1.56, CI 95% = 0.58-4.17, p = 0.367). The TT genotype of the SNP rs143383 increases the susceptibility to KOA in the population of northern Mexico, according to the recessive segregation model.

Growth Differentiation Factor 5 Improves Neurogenesis and Functional Recovery in Adult Mouse Hippocampus Following Traumatic Brain Injury.

The aim of this study was to investigate the therapeutic effect of growth differentiation factor 5 (GDF-5) on traumatic brain injury (TBI) in mice. We utilized a controlled cortical impact to establish a mouse TBI model, and then stereotaxically administered 25 or 100 ng GDF-5 into the bilateral hippocampal dentate gyrus (DG) of each of the animals. Seven days after the injury, some of the animals were sacrificed for immunohistochemical and immunofluorescence examination of 5-bromo-2′-deoxyuridine (BrdU), Sox-2, doublecortin (DCX) and phosphorylated cAMP response element binding protein (p-CREB). Dendrite quantification was also performed using DCX positive cells. Activation of newborn neurons was assessed 35 days after the injury. The remaining animals were subjected to open field, Y maze and contextual fear conditioning tests 2 months after TBI. As a result, we found that post-injury stereotaxical administration of GDF-5 can improve neural stem cell proliferation and differentiation in the DG of the hippocampus, evidenced by the increase in BrdU, Sox-2, and DCX-labeled cells, as well as the improvement in dendrite arborization and newborn neuron activation in response to GDF-5 treatment. Mechanistically, these effects of GDF-5 may be mediated by the CREB pathway, manifested by the recovery of TBI-induced dephosphorylation of CREB upon GDF-5 administration. Behavioral tests further verified the effects of GDF-5 on improving cognitive and behavioral dysfunction after TBI. Collectively, these results reveal that direct injection of GDF-5 into the hippocampus can stimulate neurogenesis and improve functional recovery in a mouse TBI model, indicating the potential therapeutic effects of GDF-5 on TBI.

Association between GDF5 single nucleotide polymorphism rs143383 and lumbar disc degeneration.

The association between growth differentiation factor 5 (GDF5), single nucleotide polymorphism (SNP) rs143383 and lumbar disc degeneration (LDD) was investigated. A total of 210 patients with LDD (observation group) and 320 patients without lumbar diseases (control group) diagnosed in Shanghai General Hospital of Nanjing Medical University from August 2013 to March 2017 were randomly selected. Then, deoxyribonucleic acid (DNA) was extracted from the blood of each patient, and Taq-man fluorescent quantitative polymerase chain reaction (qPCR) technique was used to detect rs143383 in GFD5 gene. The frequency of different genotypes in observation group and control group was counted, and the associations between different SNP genotypes and the incidence of LDD were analyzed. Good genotyping results were found in both LDD patient group and control group. There were no significant differences in distribution frequency of TT and TC genotypes at site rs143383 between LDD patient group and control group (P>0.05), but the distribution frequency of CC genotype at site rs143383 in LDD patient group had a statistically significant difference from that in control group (P<0.05). In dominant models, odds ratio (OR) of (TC+CC/TT) was 1.195 (P=0.532). In recessive models, OR of (CC/TT+TC) was 4.333 (P=0.028). In co-dominant models, ORs of (TC/TT) and (CC/TT) were 0.967 and 4.43, respectively (P=0.99). The differences in 3 genotypes showed no statistical significance among different pathological grades (Grade I to V) (χ2=1.034, P=0.998), and there was no statistically significant difference in T and C (χ2=0.012, P=0.999). Pathological grades in dominant models, recessive models and over dominant models were analyzed, and no statistically significant difference was found (P>0.05). In conclusion, CC mutant type at rs143383 in GDF5 gene has a strong association with the incidence of LDD, and a high prevalence risk, but it has no evident correlation with pathological grades.

MicroRNA-449a upregulation promotes chondrocyte extracellular matrix degradation in osteoarthritis.

Osteoarthritis (OA) is a highly prevalent chronic and degenerative joint disease characterized by the continuous destruction of the articular cartilage. MicroRNAs (miRNAs) have been reported to be strongly involved in the pathogenesis of OA. The aim of this study was to explore whether miR-449a regulates the expression of growth differentiation factor5 (GDF5), which promotes chondrocyte extracellular matrix (ECM) degradation. We found that miR-449a expression was upregulated in OA cartilage compared to that in normal cartilage. Overexpression of miR-449a increased the expression of chondrocyte ECM catabolic factors, such as matrix metalloproteinases and a disintegrin and metalloproteinase with thrombospondin motif, while inhibiting that of anabolic genes, such as type II collagen and aggrecan. In contrast, suppression of miR-449a exerted the opposite effects. Moreover, GDF5 was identified as a direct target of miR-449a, and its expression was significantly suppressed by miR-449a overexpression. In addition, the suppression of chondrocyte ECM degradation induced by miR-449a inhibitor was attenuated by small interfering RNA-mediated knockdown of GDF5. Overall, these results suggest that miR-449a contributes to chondrocyte ECM degradation in OA via directly targeting GDF5, thereby providing insights to a promising therapeutic target for the treatment of human OA.

MicroRNA-665 promotes the proliferation and matrix degradation of nucleus pulposus through targeting GDF5 in intervertebral disc degeneration.

Growing evidences suggested that microRNAs (miRNAs) played important roles in the development of intervertebral disc degeneration (IDD). However, the expression level and function of miR-665 in IDD remain unknown. In this study, we showed that the expression level of miR-665 was upregulated in degenerative human NP samples. In addition, miR-665 expression level gradually increased with the exacerbation of disc degeneration grade. Moreover, miR-665 expression level was positively associated with the Pfirrmann grade. Ectopic expression of miR-665 promoted NP cell growth. Furthermore, miR-665 overexpression decreased aggrecan and Col II expression and ectopic expression of miR-665 increased MMP-3 and MMP-13 expression in NP cell. We identified growth differentiation factor 5 (GDF5) was a direct target gene of miR-665 in NP cell and enforced expression of miR-665 decreased GDF5 expression. Elevated expression of miR-665 enhanced NP cell proliferation and decreased aggrecan and Col II expression. In addition, ectopic expression of miR-665 increased MMP-3 and MMP-13 expression through inhibiting GDF5 expression in NP cells. These results suggested that dysregulated miR-665 expression might act an important role in the development of IDD.

Development of controlled release systems of biological factors for the regeneration of intervertebral disc

Purpose: The degeneration of intervertebral disc (IVD), and notably of its central part the nucleus pulposus (NP), is responsible for 40% of chronic low back pain (LBP). Recent studies have reported the existence of endogenous regenerative cells in the IVD vicinity and within the IVD. These endogenous cells have been characterized as mesenchymal stem cell-like progenitors residing in specific niches. In response to chemokines (stromal derived factor-1 (SDF-1)) these cells can be recruited and migrate to the site of injury and thus contribute to the endogenous repair process. In this context, the development of microbeads-based local delivery systems of factors involved in progenitors recruitment has recently been contemplated for IVD regenerative medicine. Because of their physicochemical and biological properties, microbeads of pullulans (PMBs) have long been investigated as drug carriers. In addition, we recently demonstrated that transforming growth factor-β1 (TGF-β1) and growth differentiation factor5 (GDF5) are potent stimulators of the differentiation of mesenchymal stem cells (MSC) into NP-like cells. In this context, the aim of this work was to develop an intradiscal pullulan microbeads-based delivery system for the controlled release of SDF-1, TGF-β1 and GDF5. This drug delivery system would be able to sequentially contribute to 1) the recruitment and mobilization of resident progenitors, 2) the differentiation of the mobilized progenitors and 3) the subsequent regeneration of NP. Methods: Chemotaxis assays were performed to determine the in vitro cell migration. Human MSCs (1250 cells/μl) were incubated with or without SDF-1 (250 ng/ml) in Transwells for 4h, migratory cells were stained by crystal violet then quantified by spectrophotometry. In parallel, PMBs were prepared by a simultaneous crosslinking protocol coupled to a water-in-oil emulsification process. Freeze-dried PMBs were incubated with GDF-5 and TGF-β1 separately (25 mg of PMBs at final concentration of 1, 2 and 4 μg/mL and in a final volume of 500 μL of PBS) for 24 h at 4°C under rotary stirring at 24 rpm. GDF-5 and TGF-β1 release assays were performed in PBS at 37°C for 21 days and concentrations were measured by ELISA. Results: SDF-1 has improved the in vitro migration of hMSCs, increasing by more than twice the number of migratory cells. GDF-5 and TGF-β1 were successfully adsorbed on PMBs with a 100% efficiency. Release experiments showed a burst release within the 1st h, at 604 ng/h and 50 ng/h for GDF-5 and TGF-β1 respectively, then the release rate decreased during 21 days with 0.6 ng/h and 0.15 ng/h during the last 7 days for GDF-5 and TGF-β1, respectively. At day 21, GDF-5 was entirely released, whereas only 40% of TGF-β1 was released. This different release profiles could be explained by the difference of molecular weight (13 kDa for GDF-5 and 25 kDa for TGF-β1). Conclusions: We have confirmed that SDF-1 improved hMSCs in vitro migration, and that PMBs are suitable microcarriers for the loading and release of GDF-5 and TGFβ-1. The loading and release capability of SDF-1 by PMBs, as well as SDF-1 bioactivity after release will be analyzed, to obtain a fast and massive recruitment of resident progenitors in vivo. Then, we will study the action of GDF-5 and TGFβ1 released from PMBs on in vitro NP differentiation, by using a 3D matrix model to mimic the NP microenvironment. Nucleopulpogenic differentiation will be evaluated by analysis of specific extracellular matrix production and gene expression markers.

Heparin functionalization increases retention of TGF-β2 and GDF5 on biphasic silk fibroin scaffolds for tendon/ligament-to-bone tissue engineering

The tendon/ligament-to-bone transition (enthesis) is a highly specialized interphase tissue with structural gradients of extracellular matrix composition, collagen molecule alignment and mineralization. These structural features are essential for enthesis function, but are often not regenerated after injury. Tissue engineering is a promising strategy for enthesis repair. Engineering of complex tissue interphases such as the enthesis is likely to require a combination of biophysical, biological and chemical cues to achieve functional tissue regeneration. In this study, we cultured human primary adipose-derived mesenchymal stem cells (AdMCs) on biphasic silk fibroin scaffolds with integrated anisotropic (tendon/ligament-like) and isotropic (bone/cartilage like) pore alignment. We functionalized those scaffolds with heparin and explored their ability to deliver transforming growth factor β2 (TGF-β2) and growth/differentiation factor 5 (GDF5). Heparin functionalization increased the amount of TGF-β2 and GDF5 remaining attached to the scaffold matrix and resulted in biological effects at low growth factor doses. We analyzed the combined impact of pore alignment and growth factors on AdMSCs. TGF-β2 and pore anisotropy synergistically increased the expression of tendon/ligament markers and collagen I protein content. In addition, the combined delivery of TGF-β2 and GDF5 enhanced the expression of cartilage markers and collagen II protein content on substrates with isotropic porosity, whereas enthesis markers were enhanced in areas of mixed anisotropic/isotropic porosity. Altogether, the data obtained in this study improves current understanding on the combined effects of biological and structural cues on stem cell fate and presents a promising strategy for tendon/ligament-to-bone regeneration. Statement of significanceRegeneration of the tendon/ligament-to-bone interphase (enthesis) is of significance in the repair of ruptured tendons/ligaments to bone to improve implant integration and clinical outcome. This study proposes a novel approach for enthesis regeneration based on a biomimetic and integrated tendon/ligament-to-bone construct, stem cells and heparin-based delivery of growth factors. We show that heparin can keep growth factors local and biologically active at low doses, which is critical to avoid supraphysiological doses and associated side effects. In addition, we identify synergistic effects of biological (growth factors) and structural (pore alignment) cues on stem cells. These results improve current understanding on the combined impact of biological and structural cues on the multi-lineage differentiation capacity of stem cells for regenerating complex tissue interphases.

A very rare case of cervicothoracic vertebral synostosis spanning eight adjacent segments: congenital vs acquired

Cervical roentgenogram revealed a long-segment cervicothoracicvertebral fusion spanning 8 adjacent spinal levels. The patient wasevaluated with computed tomography (CT) and magnetic resonance(MR) imagings of the spine, electromyography (EMG) and growthdifferentiation factor 6 (GDF6) gene mutation analysis. Imagingfindings were atypical for congenital block vertebrae and therewas no GDF6 mutation. A revision of very old medical records andpatient’s recollections revealed long-term stay in sanatorium forrehabilitation of chronic partially-treated brucella spondylodiscitisduring adolescence. Block vertebrae spanning several levels havepreviously been reported; but, this is the first report of an acquiredcervicothoracic fusion spanning 8 adjacent vertebral bodies.

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.