Stromal cell-derived factor-1α regulates chondrogenic differentiation via activation of the Wnt/β-catenin pathway in mesenchymal stem cells

BACKGROUND Mesenchymal stem cells(MSCs)have been applied to treat degenerative articular diseases,and stromal cell-derived factor-1α(SDF-1α)may enhance their therapeutic efficacy.However,the regulatory effects of SDF-1αon cartilage differentiation remain largely unknown.Identifying the specific regulatory effects of SDF-1αon MSCs will provide a useful target for the treatment of degenerative articular diseases.AIM To explore the role and mechanism of SDF-1αin cartilage differentiation of MSCs and primary chondrocytes.METHODS The expression level of C-X-C chemokine receptor 4(CXCR4)in MSCs was assessed by immunofluorescence.MSCs treated with SDF-1αwere stained for alkaline phosphatase(ALP)and with Alcian blue to observe differentiation.Western blot analysis was used to examine the expression of SRY-box transcription factor 9,aggrecan,collagen II,runt-related transcription factor 2,collagen X,and matrix metalloproteinase(MMP)13 in untreated MSCs,of aggrecan,collagen II,collagen X,and MMP13 in SDF-1α-treated primary chondrocytes,of glycogen synthase kinase 3β(GSK3β)p-GSK3βandβ-catenin expression in SDF-1α-treated MSCs,and of aggrecan,collagen X,and MMP13 in SDF-1α-treated MSCs in the presence or absence of ICG-001(SDF-1αinhibitor).RESULTS Immunofluorescence showed CXCR4 expression in the membranes of MSCs.ALP stain was intensified in MSCs treated with SDF-1αfor 14 d.The SDF-1αtreatment promoted expression of collagen X and MMP13 during cartilage differentiation,whereas it had no effect on the expression of collagen II or aggrecan nor on the formation of cartilage matrix in MSCs.Further,those SDF-1α-mediated effects on MSCs were validated in primary chondrocytes.SDF-1αpromoted the expression of p-GSK3βandβ-catenin in MSCs.And,finally,inhibition of this pathway by ICG-001(5μmol/L)neutralized the SDF-1α-mediated up-regulation of collagen X and MMP13 expression in MSCs.CONCLUSION SDF-1αmay promote hypertrophic cartilage differentiation in MSCs by activating the Wnt/β-catenin pathway.These findings provide further evidence for the use of MSCs and SDF-1αin the treatment of cartilage degeneration and osteoarthritis.

Chondrogenic Differentiation of Mouse Bone Marrow Mesenchymal Stem Cells Induced by Cartilage-derived Morphogenetic Protein-2 In Vitro

To study the cartilage differentiation of mouse mesenchymal stem cells （MSCs） induced by cartilage-derived morphogenetic proteins-2 in vitro, the MSCs were isolated from mouse bone marrow and cultured in vitro. The cells in passage 3 were induced into chondrogenic differentiation with different concentrations of recombinant human cartilage-derived morphogenetic proteins-2 （0, 10, 20, 50 and 100 ng/mL）. After 14 days of induction, morphology of cells was observed under phase-contrast microscope. Collagen Ⅱ mRNA and protein were examined with RT-PCR, Western blotting and immunocytochemistry respectively and the sulfate glycosaminoglycan was measured by Alcian blue staining. RT-PCR showed that CDMP-2 could promote expression of collagen Ⅱ mRNA in an dose-dependant manner, especially at the concentration of 50 ng/mL and 100 ng/mL. Immunocytochemistry and Western blotting revealed a similar change. Alcian blue staining exhibited deposition of typical cartilage extracellular matrix. Our results suggest that mouse bone marrow mesencymal stem cells can differentiate into chondrogenic phonotype with the induction of CDMP-2 in vitro, which provides a basis for further research on the role of CDMP-2 in chondrogenesis.

Tumor necrosis factor-αinhibition restores matrix formation by human adipose-derived stem cells in the late stage of chondrogenic differentiation

BACKGROUND Cartilage tissue engineering is a promising strategy for treating cartilage damage.Matrix formation by adipose-derived stem cells(ADSCs),which are one type of seed cell used for cartilage tissue engineering,decreases in the late stage of induced chondrogenic differentiation in vitro,which seriously limits research on ADSCs and their application.AIM To improve the chondrogenic differentiation efficiency of ADSCs in vitro,and optimize the existing chondrogenic induction protocol.METHODS Tumor necrosis factor-alpha(TNF-α)inhibitor was added to chondrogenic culture medium,and then Western blotting,enzyme linked immunosorbent assay,immunofluorescence and toluidine blue staining were used to detect the cartilage matrix secretion and the expression of key proteins of nuclear factor kappa-B(NF-κB)signaling pathway.RESULTS In this study,we found that the levels of TNF-αand matrix metalloproteinase 3 were increased during the chondrogenic differentiation of ADSCs.TNF-αthen bound to its receptor and activated the NF-κB pathway,leading to a decrease in cartilage matrix synthesis and secretion.Blocking TNF-αwith its inhibitors etanercept(1μg/mL)or infliximab(10μg/mL)significantly restored matrix formation.CONCLUSION Therefore,this study developed a combination of ADSC therapy and targeted anti-inflammatory drugs to optimize the chondrogenesis of ADSCs,and this approach could be very beneficial for translating ADSC-based approaches to treat cartilage damage.

Chondrogenic differentiation of human bone mesenchymal stem cells treated with growth differentiation factor 5 under hypoxia

Objective To explore the feasibility and effectiveness of the self-assembly cartilage tissue engineered with chondrogenically differentiated human bone mesenchymal stem cells (hBMCs) induced by growth differentiation factor-5 (GDF-5)

Ion elemental-optimized layered double hydroxide nanoparticles promote chondrogenic differentiation and intervertebral disc regeneration of mesenchymal stem cells through focal adhesion signaling pathway

Chronic low back pain and dyskinesia caused by intervertebral disc degeneration(IDD)are seriously aggravated and become more prevalent with age.Current clinical treatments do not restore the biological structure and inherent function of the disc.The emergence of tissue engineering and regenerative medicine has provided new insights into the treatment of IDD.We synthesized biocompatible layered double hydroxide(LDH)nanoparticles and optimized their ion elemental compositions to promote chondrogenic differentiation of human umbilical cord mesenchymal stem cells(hUC-MSCs).The chondrogenic differentiation of LDH-treated MSCs was validated using Alcian blue staining,qPCR,and immunofluorescence analyses.LDH-pretreated hUC-MSCs were differentiated prior to transplantation into the degenerative site of a needle puncture IDD rat model.Repair and regeneration evaluated using X-ray,magnetic resonance imaging,and tissue immunostaining 4-12 weeks after transplantation showed recovery of the disc space height and integrated tissue structure.Transcriptome sequencing revealed significant regulatory roles of the extracellular matrix(ECM)and integrin receptors of focal adhesion signaling pathway in enhancing chondrogenic differentiation and thus prompting tissue regeneration.The construction of ion-specific LDH nanomaterials for in situ intervertebral disc regeneration through the focal adhesion signaling pathway provides theoretical basis for clinical transformation in IDD treatment.

Sox9 augments BMP2-induced chondrogenic differentiation by downregulating Smad7 in mesenchymal stem cells(MSCs)

Cartilage injuries caused by arthritis or trauma pose formidable challenges for effective clinical management due to the limited intrinsic proliferative capability of chondrocytes.Autologous stem cell-based therapies and transgene-enhanced cartilage tissue engineering may open new avenues for the treatment of cartilage injuries.Bone morphogenetic protein 2(BMP2)induces effective chondrogenesis of mesenchymal stem cells(MSCs)and can thus be explored as a potential therapeutic agent for cartilage defect repair.However,BMP2 also induces robust endochondral ossification.Although the precise mechanisms through which BMP2 governs the divergence of chondrogenesis and osteogenesis remain to be fully understood,blocking endochondral ossification during BMP2-induced cartilage formation may have practical significance for cartilage tissue engineering.Here,we investigate the role of Sox9-donwregulated Smad7 in BMP2-induced chondrogenic differentiation of MSCs.We find that overexpression of Sox9 leads to a decrease in BMP2-induced Smad7 expression in MSCs.Sox9 inhibits BMP2-induced expression of osteopontin while enhancing the expression of chondrogenic marker Col2a1 in MSCs.Forced expression of Sox9 in MSCs promotes BMP2-induced chondrogenesis and suppresses BMP2-induced endochondral ossification.Constitutive Smad7 expression inhibits BMP2-induced chondrogenesis in stem cell implantation assay.Mouse limb explant assay reveals that Sox9 expands BMP2-stimulated chondrocyte proliferating zone while Smad7 promotes BMP2-intitated hypertrophic zone of the growth plate.Cell cycle analysis indicates that Smad7 induces significant early apoptosis in BMP2-stimulated MSCs.Taken together,our results strongly suggest that Sox9 may facilitate BMP2-induced chondrogenesis by downregulating Smad7,which can be exploited for effective cartilage tissue engineering.

Nanosecond pulsed electric fields prime mesenchymal stem cells to peptide ghrelin and enhance chondrogenesis and osteochondral defect repair in vivo

Mesenchymal stem cells(MSCs) are important cell sources in cartilage tissue development and homeostasis,and multiple strategies have been developed to improve MSCs chondrogenic differentiation with an aim of promoting cartilage regeneration.Here we report the effects of combining nanosecond pulsed electric fields(ns PEFs) followed by treatment with ghrelin(a hormone that stimulates release of growth hormone) to regulate chondrogenesis of MSCs.ns PEFs and ghrelin were observed to separately enhance the chondrogenesis of MSCs,and the effects were significantly enhanced when the bioelectric stimulation and hormone were combined,which in turn improved osteochondral tissue repair of these cells within Sprague Dawley rats.We further found that ns PEFs can prime MSCs to be more receptive to subsequent stimuli of differentiation by upregulated Oct4/Nanog and activated JNK signaling pathway.Ghrelin initiated chondrogenic differentiation by activation of ERK1/2 signaling pathway,and RNA-seq results indicated 243 genes were regulated,and JAK-STAT signaling pathway was involved.Interestingly,the sequential order of applying these two stimuli is critical,with ns PEFs pretreatment followed by ghrelin enhanced chondrogenesis of MSCs in vitro and subsequent cartilage regeneration in vivo,but not vice versa.This synergistic prochondrogenic effects provide us new insights and strategies for future cell-based therapies.

Tetrahedral framework nucleic acids promote the biological functions and related mechanism of synovium-derived mesenchymal stem cells and show improved articular cartilage regeneration activity in situ

Many recent studies have shown that joint-resident mesenchymal stem cells(MSCs)play a vital role in articular cartilage(AC)in situ regeneration.Specifically,synovium-derived MSCs(SMSCs),which have strong chondrogenic differentiation potential,may be the main driver of cartilage repair.However,both the insufficient number of MSCs and the lack of an ideal regenerative microenvironment in the defect area will seriously affect the regeneration of AC.Tetrahedral framework nucleic acids(tFNAs),notable novel nanomaterials,are considered prospective biological regulators in biomedical engineering.Here,we aimed to explore whether tFNAs have positive effects on AC in situ regeneration and to investigate the related mechanism.The results of in vitro experiments showed that the proliferation and migration of SMSCs were significantly enhanced by tFNAs.In addition,tFNAs,which were added to chondrogenic induction medium,were shown to promote the chondrogenic capacity of SMSCs by increasing the phosphorylation of Smad2/3.In animal models,the injection of tFNAs improved the therapeutic outcome of cartilage defects compared with that of the control treatments without tFNAs.In conclusion,this is the first report to demonstrate that tFNAs can promote the chondrogenic differentiation of SMSCs in vitro and enhance AC regeneration in vivo,indicating that tFNAs may become a promising therapeutic for AC regeneration.

Synthesis of photo-reactive poly(vinyl alcohol)and construction of scaffold-free cartilage like pellets in vitro

Photo-reactive poly(vinyl alcohol)(PRPVA)was synthesized by introduction of phenyl azido groups into poly(vinyl alcohol)(PVA)and applied for surface modification.PRPVA was grafted onto cell culture plate surface homogeneously or in a micropattern.Human mesenchymal stem cells(hMSCs)cultured on cell culture plate surface and PVA-modified surface showed different behaviors.Cells adhered and spread well on cell culture plate surface,while they did not adhere on PVA-grafted surface at all.When hMSCs were cultured on PVA-micropatterned surface,they formed a cell micropattern.Cells formed pellets after cultured on PVA homogeneously modified surface in chondrogenic induction medium for 2 weeks.The pellets were positively stained by hematoxylin/eosin,safranin-O/fast green and toluidin blue,and they were also stained brown by Type II collagen and proteoglycan immunohistological staining.Real-time PCR analysis was conducted to investigate the expression of colI,colII,colX,aggrecan and sox9 mRNA.Results of gene expression were in agreement with those of histological and immunohistological observations.These results indicated that hMSCs cultured on PVA-modified surface performed chondrogenic differentiation,and it was possible to construct scaffold-free cartilage like pellets with PVA-modified surface in vitro.