Growth And Differentiation Factor-5 Research Articles

Injectable Dynamic Hydrogel with Responsive Mechanical Reinforcing Ability Reverses Intervertebral Disc Degeneration by Suppressing Ferroptosis and Restoring Matrix Homeostasis

AbstractIntervertebral disc degeneration (IDD) remains an essential challenge in the clinics due to the ferroptosis of nucleus pulposus cells (NPCs) and the imbalance of extracellular matrix (ECM) metabolism. Here, a dynamic hydrogel (HPGO) based on oxidized hyaluronic acid (OHA) and polyvinyl alcohol (PVA) for the integration of oridonin (Ori)‐loaded NPC membrane‐based nanovesicles is proposed, which is further grafted with growth and differentiation factor 5 (GDF5) on the OHA chain via Schiff base ligation. The precursors can rapidly form mechanically resilience hydrogels after injection into nucleus pulposus (NP) through the formation of various dynamic bonds. Meanwhile, the acidic pH in the degenerated NP can trigger the responsive release of GDF5, which may cooperate with Ori to alleviate inflammation in the microenvironment for restoring the metabolic homeostasis of ECM. In addition, the Schiff base moieties in HPGO hydrogel can scavenge free ferrous ions in the microenvironment to strengthen the dynamic bonds for self‐regulated reinforcement, thus providing mechanical support for the degenerated intervertebral discs (IVDs). Furthermore, HPGO hydrogel can enhance ferroptosis resistance of NPCs by activating the nuclear factor erythoid 2‐related factor 2 signaling pathway. Overall, HPGO hydrogel can mechanically support the damaged IVDs as well as reversing their degenerative status through cooperative ferroptosis inhibition, which provides an approach for the clinical treatment of IDD.

Transcriptome profiling reveals transcriptional regulation of Protegrin-1 on immune defense and development in porcine granulosa cells

Protegrin-1 (PG1) is an antimicrobial peptide (AMP) that has garnered increasing attention due to its potent immune defense activity. Our previous studies demonstrated the ability of PG1 to enhance proliferation and inhibit apoptosis of porcine granulosa cells (GCs) under oxidative stress. GCs play a crucial role in ovary follicular development. However, the specific function and underlying mechanisms of AMP in follicular development still need further elucidation. The present study aimed to comprehensively explore the biological effects of PG1 on porcine GCs using transcriptome profiling by RNA sequencing technology. Isolated GCs were incubated with or without PG1 for 24 h and transcriptome-wide analysis was exerted to identify differentially expressed genes (DEGs). The results of expression analysis revealed 1,235 DEGs, including 242 up-regulated genes and 993 down-regulated genes (|log2 (FoldChange)| > 1; adjusted P-value < 0.05). The expression levels of 7 selected DEGs were validated by quantitative reverse transcription-polymerase chain reaction (RT-qPCR) analysis, which was consistent with the RNA-sequencing data. Among the significant DEGs, several genes associated with GC function and ovarian follicle development were identified, such as estrogen receptor 2 (ESR2), growth and differentiation factor 6 (GDF6), cell division cycle 20 homolog (CDC20), Notch3, ephrin and Eph receptor system, Egl nine homolog 3 (EGLN3), and BCL2 like 14 (BCL2L14). Gene Ontology (GO) analysis revealed that the top three significant GO terms were inflammatory response, defense response, and granulocyte migration. Additionally, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis presented that DEGs were mainly enriched in the immune system, infectious disease, signaling molecules and interaction, and immune disease. Furthermore, Ingenuity Pathway Analysis (IPA) predicted that the top activated pathway was Liver X Receptor (LXR)/ Retinoid X Receptor (RXR) Activation which is known to be associated with female reproduction. Predicted protein–protein interactions (PPIs) analysis identified complement C3 (C3) as the top node with the highest degree of network connection and revealed that DEGs in the sub-networks were involved in cytokine-cytokine receptor interaction, neuroactive ligand-receptor interaction, chemokine signaling pathway, and metabolic process. In conclusion, this study expanded the understanding of the effects of PG1 on porcine GCs at the transcriptomic level and provided a theoretical basis for further investigation into the role of PG1 in immune defense and mammalian ovarian follicular development.

Epigenetics as a mediator of genetic risk in osteoarthritis: role during development, homeostasis, aging, and disease progression.

The identification of genomic loci that are associated with osteoarthritis (OA) has provided a starting point for understanding how genetic variation activates catabolic processes in the joint. However, genetic variants can only alter gene expression and cellular function when the epigenetic environment is permissive to these effects. In this review, we provide examples of how epigenetic shifts at distinct life stages can alter the risk for OA, which we posit is critical for the proper interpretation of genome-wide association studies (GWAS). During development, intensive work on the growth and differentiation factor 5 (GDF5) locus has revealed the importance of tissue-specific enhancer activity in controlling both joint development and the subsequent risk for OA. During homeostasis in adults, underlying genetic risk factors may help establish beneficial or catabolic "set points" that dictate tissue function, with a strong cumulative effect on OA risk. During aging, methylation changes and the reorganization of chromatin can "unmask" the effects of genetic variants. The destructive function of variants that alter aging would only mediate effects after reproductive competence and thus avoid any evolutionary selection pressure, as consistent with larger frameworks of biological aging and its relationship to disease. A similar "unmasking" may occur during OA progression, which is supported by the finding of distinct expression quantitative trait loci (eQTLs) in chondrocytes depending on the degree of tissue degradation. Finally, we propose that massively parallel reporter assays (MPRAs) will be a valuable tool to test the function of putative OA GWAS variants in chondrocytes from different life stages.

Skeletal Muscle-Derived Exosomal miR-146a-5p Inhibits Adipogenesis by Mediating Muscle-Fat Axis and Targeting GDF5-PPARγ Signaling

Skeletal muscle-fat interaction is essential for maintaining organismal energy homeostasis and managing obesity by secreting cytokines and exosomes, but the role of the latter as a new mediator in inter-tissue communication remains unclear. Recently, we discovered that miR-146a-5p was mainly enriched in skeletal muscle-derived exosomes (SKM-Exos), 50-fold higher than in fat exosomes. Here, we investigated the role of skeletal muscle-derived exosomes regulating lipid metabolism in adipose tissue by delivering miR-146a-5p. The results showed that skeletal muscle cell-derived exosomes significantly inhibited the differentiation of preadipocytes and their adipogenesis. When the skeletal muscle-derived exosomes co-treated adipocytes with miR-146a-5p inhibitor, this inhibition was reversed. Additionally, skeletal muscle-specific knockout miR-146a-5p (mKO) mice significantly increased body weight gain and decreased oxidative metabolism. On the other hand, the internalization of this miRNA into the mKO mice by injecting skeletal muscle-derived exosomes from the Flox mice (Flox-Exos) resulted in significant phenotypic reversion, including down-regulation of genes and proteins involved in adipogenesis. Mechanistically, miR-146a-5p has also been demonstrated to function as a negative regulator of peroxisome proliferator-activated receptor γ (PPARγ) signaling by directly targeting growth and differentiation factor 5 (GDF5) gene to mediate adipogenesis and fatty acid absorption. Taken together, these data provide new insights into the role of miR-146a-5p as a novel myokine involved in the regulation of adipogenesis and obesity via mediating the skeletal muscle-fat signaling axis, which may serve as a target for the development of therapies against metabolic diseases, such as obesity.

Comparative in vitro treatment of mesenchymal stromal cells with GDF-5 and R57A induces chondrogenic differentiation while limiting chondrogenic hypertrophy

PurposeHypertrophic cartilage is an important characteristic of osteoarthritis and can often be found in patients suffering from osteoarthritis. Although the exact pathomechanism remains poorly understood, hypertrophic de-differentiation of chondrocytes also poses a major challenge in the cell-based repair of hyaline cartilage using mesenchymal stromal cells (MSCs). While different members of the transforming growth factor beta (TGF-β) family have been shown to promote chondrogenesis in MSCs, the transition into a hypertrophic phenotype remains a problem. To further examine this topic we compared the effects of the transcription growth and differentiation factor 5 (GDF-5) and the mutant R57A on in vitro chondrogenesis in MSCs.MethodsBone marrow-derived MSCs (BMSCs) were placed in pellet culture and in-cubated in chondrogenic differentiation medium containing R57A, GDF-5 and TGF-ß1 for 21 days. Chondrogenesis was examined histologically, immunohistochemically, through biochemical assays and by RT-qPCR regarding the expression of chondrogenic marker genes.ResultsTreatment of BMSCs with R57A led to a dose dependent induction of chondrogenesis in BMSCs. Biochemical assays also showed an elevated glycosaminoglycan (GAG) content and expression of chondrogenic marker genes in corresponding pellets. While treatment with R57A led to superior chondrogenic differentiation compared to treatment with the GDF-5 wild type and similar levels compared to incubation with TGF-ß1, levels of chondrogenic hypertrophy were lower after induction with R57A and the GDF-5 wild type.ConclusionsR57A is a stronger inducer of chondrogenesis in BMSCs than the GDF-5 wild type while leading to lower levels of chondrogenic hypertrophy in comparison with TGF-ß1.

Association of synovial expression of growth and differentiation factor 5 (GDF5) with radiographic severity of knee osteoarthritis

ObjectivesThe growth and differentiation factor 5 (GDF5) gene plays a significant role in facilitating chondrogenesis, and GDF5 polymorphism is a genetic factor contributing to osteoarthritis (OA). However, the role of GDF5 expression in the synovial membrane remains unclear. The aim of this study was to determine the expression of GDF5 in the synovium in patients with primary knee OA. Materials and methodsThirty patients scheduled for total knee arthroplasty were enrolled. Patients were grouped according to the Kellgren and Lawrence classification (KL) as grade 3 (15 patients) and grade 4 (15 patients). Synovial tissue was collected, and the GDF5 expression level was determined by real-time PCR. Ten synovial samples were randomly selected to evaluate the degree of synovitis. ResultsBaseline characteristics did not differ between the two groups. The expression of GDF5 was significantly higher in the KL4 group (median expression 3.50, range 1.45–13.62) than in the KL3 group (median expression 1.81, range 0–9.46) (p value = 0.02). Histological staining of the synovium indicated low-grade synovitis in both groups. ConclusionsGDF5 expression in the synovium was positively associated with the radiographic severity of knee OA. The difference in GDF5 expression between the KL3 and KL4 groups supports the hypothesis that, through GDF5, the synovium may have important roles in cartilage maintenance and homeostasis in primary knee OA.

Current Insights Into the Maintenance of Structure and Function of Intervertebral Disc: A Review of the Regulatory Role of Growth and Differentiation Factor-5.

Intervertebral disc degeneration (IDD), characterized by conversion of genotypic and phenotypic, is a major etiology of low back pain and disability. In general, this process starts with alteration of metabolic homeostasis leading to ongoing inflammatory process, extracellular matrix degradation and fibrosis, diminished tissue hydration, and impaired structural and mechanical functionality. During the past decades, extensive studies have focused on elucidating the molecular mechanisms of degeneration and shed light on the protective roles of various factors that may have the ability to halt and even reverse the IDD. Mutations of GDF-5 are associated with several human and animal diseases that are characterized by skeletal deformity such as short digits and short limbs. Growth and differentiation factor-5 (GDF-5) has been shown to be a promise biological therapy for IDD. Substantial literature has revealed that GDF-5 can decelerate the progression of IDD on the molecular, cellular, and organ level by altering prolonged imbalance between anabolism and catabolism. GDF family members are the central signaling moleculars in homeostasis of IVD and upregulation of their gene promotes the expression of healthy nucleus pulposus (NP) cell marker genes. In addition, GDF signaling is able to induce mesenchymal stem cells (MSCs) to differentiate into NPCs and mobilize resident cell populations as chemotactic signals. This review will discuss the promising critical role of GDF-5 in maintenance of structure and function of IVDs, and its therapeutic role in IDD endogenous repair.

Synthesis and characterizations of sugar-glass nanoparticles mediated protein delivery system for tissue engineering application

The present work focuses on the synthesis and characterization of a sugar-glass nanoparticle (SGnP) based reservoir type protein delivery system pertinent to tissue engineering applications. The SGnP nanocarriers were prepared via inverse micelle of sodium bis(2-ethylhexyl) sulfosuccinate based on an anionic surfactant and subsequent flash-freezing technique. Initially, a total of five different grades of protein-free SGnPs have been prepared to examine the effects of systematic changes in starting concentrations of the aqueous phase, organic solvent, the molar ratio of water, and surfactant in controlling the size, shape, and uniformity of micelles. Evidently, the Fourier transform infrared (FTIR) and scanning electron microscope (SEM) results confirmed that the SGnP can be successfully prepared. Subsequently, SGnP based protein depot has been validated using bovine serum albumin (BSA), horseradish peroxidase (HRP) and growth and differentiation factor-5 (GDF-5). The particle size, morphology, protein encapsulation efficiency and in vitro release kinetics were assessed using SEM, FTIR, UV–visible spectroscopy and Bradford protein assays. Excellent encapsulation efficiency (93%–94%) and sustained release behaviour of BSA (∼22% protein release after 14 d) and GDF-5 proteins (∼29% protein release after 30 d) were exhibited by the optimal grades of SGnP constructs with an average particle size of 266 nm and 93 nm, respectively. Furthermore, FTIR, differential scanning calorimeter (DSC), polyacrylamide gel electrophoresis (PAGE) and NATIVE-PAGE studies results confirm successful encapsulation, stability and preserving the structural integrity of proteins placed into the core of the SGnP constructs. Evidently, a very high (93%) residual HRP enzyme activity signifies the capability of our SGnP system to protect the encapsulated proteins from process-related stresses. In vitro cytotoxicity and fluorescence cell morphology analyses using human adipose-derived mesenchymal stem cells affirmed good cytocompatibility of protein encapsulated SGnP. Overall, the study findings indicate SGnP nanocarrier-mediated protein delivery systems as a promising approach complementary to conventional techniques in tissue engineering and therapeutic applications.

Effect of lentivirus-mediated growth and differentiation factor-5 transfection on differentiation of rabbit nucleus pulposus mesenchymal stem cells

BackgroundIntervertebral disc degeneration (IDD) is a natural progression of age-related processes. Associated with IDD, degenerative disc disease (DDD) is a pathologic condition implicated as a major cause of chronic lower back pain, which can have a severe impact on the quality of life of patients. As degeneration progression is associated with elevated levels of inflammatory cytokines, enhanced aggrecan and collagen degradation, and changes in the disc cell phenotype. The purpose of this study was to investigate the biological and cytological characteristics of rabbit nucleus pulposus mesenchymal stem cells (NPMSCs)—a key factor in IDD—and to determine the effect of the growth and differentiation factor-5 (GDF5) on the differentiation of rabbit NPMSCs transduced with a lentivirus vector.MethodsAn in vitro culture model of rabbit NPMSCs was established and NPMSCs were identified by flow cytometry (FCM) and quantitative real-time PCR (qRT-PCR). Subsequently, NPMSCs were randomly divided into three groups: a transfection group (the lentiviral vector carrying GDF5 gene used to transfect NPMSCs); a control virus group (the NPMSCs transfected with an ordinary lentiviral vector); and a normal group (the NPMSCs alone). FCM, qRT-PCR, and western blot (WB) were used to detect the changes in NPMSCs.ResultsThe GDF5-transfected NPMSCs displayed an elongated shape, with decreased cell density, and significantly increased GDF5 positivity rate in the transfected group compared to the other two groups (P < 0.01). The mRNA levels of Krt8, Krt18, and Krt19 in the transfected group were significantly higher in comparison with the other two groups (P < 0.01), and the WB results were consistent with that of qRT-PCR.ConclusionsGDF5 could induce the differentiation of NPMSCs. The lentiviral vector carrying the GDF5 gene could be integrated into the chromosome genome of NPMSCs and promoted differentiation of NPMSCs into nucleus pulposus cells. Our findings advance the development of feasible and effective therapies for IDD.

Fibroblastic differentiation of mesenchymal stem/stromal cells (MSCs) is enhanced by hypoxia in 3D cultures treated with bone morphogenetic protein 6 (BMP6) and growth and differentiation factor 5 (GDF5).

Culture conditions and differentiation cocktails may facilitate cell maturation and extracellular matrix (ECM) secretion and support the production of engineered fibroblastic tissues with applications in ligament regeneration. The objective of this study is to investigate the potential of two connective tissue-related ligands (i.e., BMP6 and GDF5) to mediate collagenous ECM synthesis and tissue maturation in vitro under normoxic and hypoxic conditions based on the hypothesis that BMP6 and GDF5 are components of normal paracrine signalling events that support connective tissue homeostasis. Human adipose-derived MSCs were seeded on 3D-printed medical-grade polycaprolactone (PCL) scaffolds using a bioreactor and incubated in media containing GDF5 and/or BMP6 for 21days in either normoxic (5% oxygen) or hypoxic (2% oxygen) conditions. Constructs were harvested on Day 3 and 21 for cell viability analysis by live/dead staining, structural analysis by scanning electron microscopy, mRNA levels by RTqPCR analysis, and in situ deposition of proteins by immunofluorescence microscopy. Pro-fibroblastic gene expression is enhanced by hypoxic culture conditions compared to normoxic conditions. Hypoxia renders cells more responsive to treatment with BMP6 as reflected by increased expression of ECM mRNA levels on Day 3 with sustained expression until Day 21. GDF5 was not particularly effective either in the absence or presence of BMP6. Fibroblastic differentiation of MSCs is selectively enhanced by BMP6 and not GDF5. Environmental factors (i.e., hypoxia) also influenced the responsiveness of cells to this morphogen.

GDF6-CD99 Signaling Regulates Src and Ewing Sarcoma Growth

SUMMARYWe report here that the autocrine signaling mediated by growth and differentiation factor 6 (GDF6), a member of the bone morphogenetic protein (BMP) family of cytokines, maintains Ewing sarcoma growth by preventing Src hyperactivation. Surprisingly, Ewing sarcoma depends on the prodomain, not the BMP domain, of GDF6. We demonstrate that the GDF6 prodomain is a ligand for CD99, a transmembrane protein that has been widely used as a marker of Ewing sarcoma. The binding of the GDF6 prodomain to the CD99 extracellular domain results in recruitment of CSK (C-terminal Src kinase) to the YQKKK motif in the intracellular domain of CD99, inhibiting Src activity. GDF6 silencing causes hyperactivation of Src and p21-dependent growth arrest. We demonstrate that two GDF6 prodomain mutants linked to Klippel-Feil syndrome are hyperactive in CD99-Src signaling. These results reveal a cytokine signaling pathway that regulates the CSK-Src axis and cancer cell proliferation and suggest the gain-of-function activity for disease-causing GDF6 mutants.

The GDF-5 mutant M1673 exerts robust anabolic and anti-catabolic effects in chondrocytes.

The growth and differentiation factor 5 (GDF‐5) is known to play a key role in cartilage morphogenesis and homeostasis, and a single‐nucleotide polymorphism in its promoter sequence was found to be associated with osteoarthritis (OA). In addition, GDF‐5 was shown to promote extracellular matrix (ECM) production in healthy chondrocytes, to stimulate chondrogenesis of mesenchymal stem cells (MSCs) and to protect against OA progression in vivo. Therefore, GDF‐5 appears to be a promising treatment for osteoarthritis. However, GDF‐5 also promotes osteogenesis and hypertrophy, limiting its therapeutic utility. To circumvent this, a GDF‐5 mutant with lower hypertrophic and osteogenic properties was engineered: M1673. The present study aimed to evaluate and compare the effects of GDF‐5 and M1673 on primary porcine and human OA chondrocytes. We found that both GDF‐5 and M1673 can robustly stimulate ECM accumulation, type II collagen and aggrecan expression in porcine and human OA chondrocytes in 3D culture. In addition, both molecules also down‐regulated MMP13 and ADAMTS5 expression. These results suggest that M1673 retained the anabolic and anti‐catabolic effects of GDF‐5 on chondrocytes and is an alternative to GDF‐5 for osteoarthritis.

Expression of growth and differentiation factor 5 during joint repair and osteoarthritis

Purpose: Growth and differentiation factor 5 (GDF5) is a key risk locus for osteoarthritis (OA), with single-nucleotide polymorphisms that result in decreased GDF5 expression contributing to OA risk. Gdf5, together with Wnt9a, is well known for its critical roles during joint formation. However, little is known regarding the role of Gdf5 following adult joint tissue damage. Regulatory sequences that control Gdf5 expression are distributed over a hundred kilobases, including regions both upstream and downstream of its coding exons.

Increasing the Medium Osmolarity Reduces the Inflammatory Status of Human OA Chondrocytes and Increases Their Responsiveness to GDF-5.

The environment surrounding chondrocytes changes drastically in osteoarthritis (OA). For instance, the osmolarity in cartilage (ranging from 350 to 460 mOsm in healthy tissue) decreases during the progression of OA, reaching 270 mOsm. The objective of this study was to evaluate how osmolarity influences human OA chondrocytes. For this purpose, the osmolarity of the culture medium (340 mOsm) was increased to 380, 420 or 460 mOsm and its effect on the phenotype, matrix production, protease expression, cytokine release and growth and differentiation factor-5 (GDF-5) receptor expression in human OA chondrocytes was evaluated in a monolayer. Afterwards, the same parameters, as well as the responsiveness to GDF-5, were evaluated in 3D culture at 340 and 380 mOsm. Our results revealed that increasing the medium osmolarity increased matrix production but also reduced cytokine release, type I collagen and protease expression. It was also demonstrated that at 380 mOsm, the response to GDF-5 in 3D culture was more robust than at 340 mOsm. For the first time, it was established that a decreased osmolarity plays a role in sustaining inflammation and catabolic activities in OA chondrocytes and decreases their responsiveness to GDF-5. This indicates that osmolarity is a critical aspect of OA pathobiology.

Regulation of Gdf5 expression in joint remodelling, repair and osteoarthritis

Growth and Differentiation Factor 5 (GDF5) is a key risk locus for osteoarthritis (OA). However, little is known regarding regulation of Gdf5 expression following joint tissue damage. Here, we employed Gdf5-LacZ reporter mouse lines to assess the spatiotemporal activity of Gdf5 regulatory sequences in experimental OA following destabilisation of the medial meniscus (DMM) and after acute cartilage injury and repair. Gdf5 expression was upregulated in articular cartilage post-DMM, and was increased in human OA cartilage as determined by immunohistochemistry and microarray analysis. Gdf5 expression was also upregulated during cartilage repair in mice and was switched on in injured synovium in prospective areas of cartilage formation, where it inversely correlated with expression of the transcriptional co-factor Yes-associated protein (Yap). Indeed, overexpression of Yap suppressed Gdf5 expression in chondroprogenitors in vitro. Gdf5 expression in both mouse injury models required regulatory sequence downstream of Gdf5 coding exons. Our findings suggest that Gdf5 upregulation in articular cartilage and synovium is a generic response to knee injury that is dependent on downstream regulatory sequence and in progenitors is associated with chondrogenic specification. We propose a role for Gdf5 in tissue remodelling and repair after injury, which may partly underpin its association with OA risk.

Cells from a GDF5 origin produce zonal tendon-to-bone attachments following anterior cruciate ligament reconstruction.

Following anterior cruciate ligament (ACL) reconstruction surgery, a staged repair response occurs where cells from outside the tendon graft participate in tunnel integration. The mechanisms that regulate this process, including the specific cellular origin, are poorly understood. Embryonic cells expressing growth and differentiation factor 5 (GDF5) give rise to several mesenchymal tissues in the joint and epiphyses. We hypothesized that cells from a GDF5 origin, even in the adult tissue, would give rise to cells that contribute to the stages of repair. ACLs were reconstructed in Gdf5-Cre;R26R-tdTomato lineage tracing mice to monitor the contribution of Gdf5-Cre;tdTom+ cells to the tunnel integration process. Anterior-posterior drawer tests demonstrated 58% restoration in anterior-posterior stability. Gdf5-Cre;tdTom+ cells within the epiphyseal bone marrow adjacent to tunnels expanded in response to the injury by 135-fold compared with intact controls to initiate tendon-to-bone attachments. They continued to mature the attachments yielding zonal insertion sites at 4 weeks with collagen fibers spanning across unmineralized and mineralized fibrocartilage and anchored to the adjacent bone. The zonal attachments possessed tidemarks with concentrated alkaline phosphatase activity similar to native entheses. This study established that mesenchymal cells from a GDF5 origin can contribute to zonal tendon-to-bone attachments within bone tunnels following ACL reconstruction.

Intradiscal Injection of Induced Pluripotent Stem Cell-Derived Nucleus Pulposus-Like Cell-Seeded Polymeric Microspheres Promotes Rat Disc Regeneration.

Background Cell replacement therapy is an attractive alternative for treating degenerated intervertebral discs (IVDs), which are related to the reduction of nucleus pulposus-like cells (NP-lCs) and the loss of the extracellular matrix. Induced pluripotent stem cells (iPSCs) which resemble embryonic stem cells are considered to be a potential resource for restoring NP-lCs and disc homeostasis. Here, we proposed an efficient two-step differentiation protocol of human iPSCs into NP-lCs and continuously tested their in vivo ability to regenerate IVDs. Methods A polymeric gelatin microsphere (GM) was generated for sustained release of growth and differentiation factor-5 (GDF-5) and as a cell delivery vehicle of NP-lCs. By injecting NP-lC-seeded GDF-5-loaded GMs into the rat coccygeal intervertebral discs, the disc height and water content were examined with the molybdenum target radiographic imaging test and magnetic resonance imaging examination. Histology and immunohistochemistry results were shown with H&E, S-O-Fast Green, and immunohistochemistry staining. Results We demonstrated that the injection of NP-lC-seeded GDF-5-loaded GMs could reverse IDD in a rat model. The imaging examination indicated that disc height recovered and water content increased. Histology and immunohistochemistry results indicated that the NP cells as well as their extracellular matrix were partially restored. Conclusions The results suggest that NP-lC-seeded GDF-5-loaded GMs could partially regenerate degenerated intervertebral discs after transplantation into rat coccygeal intervertebral discs. Our study will help develop a promising method of stem cell-based therapy for IDD.

CD146/MCAM distinguishes stem cell subpopulations with distinct migration and regenerative potential in degenerative intervertebral discs

This study aimed to characterize the mesenchymal stem cell (MSC) subpopulation migrating towards a degenerated intervertebral disc (IVD) and to assess its regenerative potential. Based on initial screening for migration towards C-C motif chemokine ligand 5 (CCL5), the migration potential of CD146+and CD146- mesenchymal stem cells (MSCs) was evaluated invitro and in a degenerated organ culture model (degeneration by high-frequency loading in a bioreactor). Discogenic differentiation potential of CD146+and CD146- MSCs was investigated by invitro pellet culture assay with supplementation of growth and differentiation factor-6 (GDF6). Furthermore, trypsin degenerated IVDs were treated by either homing or injection of CD146+or CD146- MSCs and glycosaminoglycan synthesis was evaluated by Sulphur 35 incorporation after 35 days of culture. Surface expression of CD146 led to a higher number of migrated MSCs both invitro and in organ culture. CD146+and CD146- pellets responded with a similar up-regulation of anabolic markers. A higher production of sulfated glycosaminoglycans (sGAG)/DNA was observed for CD146+pellets, while in organ cultures, sGAG synthesis rate was higher for IVDs treated with CD146- MSCs by either homing or injection. The CD146+MSC subpopulation held greater migration potential towards degenerative IVDs, while the CD146- cells induced a stronger regenerative response in the resident IVD cells. These findings were independent of the application route (injection vs migration). From a translational point of view, our data suggests that CD146+MSCs may be suitable for re-population, while CD146- MSCs may represent the primary choice for stimulation of endogenous IVD cells.

Assessment of structurally and functionally high-risk nsSNPs impacts on human bone morphogenetic protein receptor type IA (BMPR1A) by computational approach

BMPR1A (BMP type 1 receptor) is a transmembrane cell-surface receptor also known as ALK3 (activin-like kinases-3) encodes for a type I serine/threonine kinase receptor and a member of the transforming growth-factor β–receptor (TGF-β) super family. The BMPR1A has a significant interaction with BMP-2 for protein activity and also has a low affinity with growth and differentiation factor 5 (GDF5); positively regulates chondrocyte differentiation. The genetic variations can alter the structure and function of the BMPR1A gene that causes several diseases such as juvenile polyposis syndrome or hereditary cancer-predisposing syndrome. The current study was carried out to identify potential deleterious non-synonymous single nucleotide polymorphisms (nsSNPs) in BMPR1A by implementing different computational algorithms such as SIFT, PolyPhen2, SNAP2, PROVEAN, PhD-SNP, SNPs&GO, nsSNPAnalyzer, and P-Mut. From 205 nsSNPs in BMPR1A, 7 nsSNPs (C76Y, C124R, C124Y, C376Y, R443C, R480W, and W487R) were predicted as deleterious in 8 prediction algorithms. The Consurf analysis showed that selected 7 nsSNPs were present in the highly conserved regions. Molecular dynamics simulation analysis also performed to explore conformational changes in the variant structure with respect to its native structure. According to the MDS result, all variants flexibility and rigidity were unbalanced, which may alter the structural and functional behavior of the native protein. Although, three nsSNPs i.e., C124R, C376Y, and R443C have already been reported in patients associated with JPS, but their structural and functional molecular studies remain uncharacterized. Therefore, the findings of this study can provide a better understanding of uncharacterized nsSNPS and to find their association with disease susceptibility and also facilitate to the researchers for designing or developing the target dependent drugs.

Interaction between GDF5 gene polymorphisms and environment factors increased the risk of knee osteoarthritis: a case-control study.

Using a case–control design, we assessed the association between single nucleotide polymorphisms (SNPs) of growth and differentiation factor 5 (GDF5)/rs143383 gene and interaction with environments and knee osteoarthritis (KOA). We recruited 288 KOA patients from the First Clinical College, Henan University of Chinese Medicine between June 2017 and May 2018. There was significant difference in genotype distribution between case group and control group (χ2 = 22.661, P=0.000). The minor C allele was significantly higher in the case group than that in the control group (20.5 vs 8.1%, P=0.000, odds ratio (OR) = 1.62, 95% confidence interval (CI): 1.29–2.03). Significant differences were also observed in other gene models. For age, all models show significant differences (P<0.05) for those whose age was more than 60 years, and no significant difference was observed for those under 60 years. For non-smoking group, there were significant differences between case group and control group, and for smoker, significance level was found in TT compared with CC and allele gene models. Patients with drinking and Bbody mass index (MI )≥ 24 also showed significant relationship between rs143383 and osteoarthritis (OA) under the following models: TT vs CC (P=0.000, P=0.018), TT/CT vs CC (P=0.043), TT vs CT/CC (P=0.000, P=0.009), and T vs C (P=0.024, P=0.000). Other gene models indicated no significance (P>0.05). Our results revealed a possible genetic association between GDF5 and KOA, and the TT genotype of rs143383 increased the risk of KOA in Chinese Han population. The interaction between GDF5 gene and drinking, smoking, and obesity further increased the risk of KOA.