The objectives of this study were to （1） determine the distribution and synthesis of pericellular matrix （PCM） molecules （collagen VI, collagen IV and laminin） in rat temporomandibular joint （TMJ） and （2） in...The objectives of this study were to （1） determine the distribution and synthesis of pericellular matrix （PCM） molecules （collagen VI, collagen IV and laminin） in rat temporomandibular joint （TMJ） and （2） investigate the effects of PCM molecules on chondrocytes against inflammation in osteoarthritis. Four zones （fibrous, proliferating, mature and hypertrophic） of condylar cartilage and three bands （anterior, intermediate and posterior） of disc were analysed by immunohistochemistry for the presence of PCM molecules in rat TMJs. Isolated chondrocytes were pre-treated with PCM molecules before being subjected to interleukin （IL）-II~ treatment to stimulate inflammation. The responses of the chondrocytes were analysed using gene expression, nitric oxide release and matrix metalloproteinase （MMP）-13 production measures. Histomorphometric analyses revealed that the highest areal deposition of collagen VI （67.4%）, collagen IV （45.7%） and laminin （52.4%） was in the proliferating zone of TMJ condylar cartilage. No significant difference in the distribution of PCM molecules was noted among the three bands of the TMJ disc. All three PCM molecules were expressed intracellularly by chondrocytes cultured in the monolayer. Among the PCM molecules, pre-treatment with collagen VI enhanced cellular proliferation, ameliorated IL-lp-induced MMP-3, MMP-9, MMP-13 and inducible nitric oxide synthase gene expression, and attenuated the downregulation of cartilage matrix genes, including collagen I, aggrecan and cartilage oligomeric matrix protein （COMP）. Concurrently, collagen VI pretreatment inhibited nitric oxide and MMP-13 production. Our study demonstrates for the first time the distribution and role of PCM molecules, particularly collagen VI, in the protection of chondrocytes against inflammation.展开更多
Conventional methods of stem cell therapy for tissue regeneration often face challenges,such as poor cell viability and integration posttransplantation.To address this,we proposed transplanting cells within synthetic ...Conventional methods of stem cell therapy for tissue regeneration often face challenges,such as poor cell viability and integration posttransplantation.To address this,we proposed transplanting cells within synthetic microenviron-ments that maintain viability,cell phenotype,support extracellular matrix(ECM)secretion,and promote differentiation to enhance the regeneration of damaged host tissue.This hypothesis was tested in dental tissue regeneration using dental pulp stem cell-laden microcarriers(MCs)mixed in a gelatin methacrylate(GelMA)hydrogel as a delivery system.The combination of MCs and GelMA exhibited similar physical properties and favorable biological properties compared to GelMA alone.Specifically,cell-laden MC mixed into GelMA enhanced cell proliferation and ECM secretion and maintained a normal phenotype.Notably,MC-modified GelMA amplified odontogenic differentiation,mineralization,and vascular endothelial growth factor release.Moreover,the storage of MC-modified GelMA showed no detrimental effects on its injection force,cell viability,and mineralization potential,which demonstrates that the composite hydrogel is a promising injectable vehicle for therapeutic stem cell delivery.This strategy may be broadly applied to various tissues and organ systems,in which the provision and instruction of a cell population to participate in regeneration may be clinically useful.展开更多
Secretome derived from mesenchymal stem cells (MSCs) have profound effects on tissue regeneration, which could become the basis of future MSCs therapies. Hypoxia, as the physiologic environment of MSCs, has great pote...Secretome derived from mesenchymal stem cells (MSCs) have profound effects on tissue regeneration, which could become the basis of future MSCs therapies. Hypoxia, as the physiologic environment of MSCs, has great potential to enhance MSCs paracrine therapeutic effect. In our study, the paracrine effects of secretome derived from MSCs preconditioned in normoxia and hypoxia was compared through both in vitro functional assays and an in vivo rat osteochondral defect model. Specifically, the paracrine effect of total EVs were compared to that of soluble factors to characterize the predominant active components in the hypoxic secretome. We demonstrated that hypoxia conditioned medium, as well as the corresponding EVs, at a relatively low dosage, were efficient in promoting the repair of critical-sized osteochondral defects and mitigated the joint inflammation in a rat osteochondral defect model, relative to their normoxia counterpart. In vitro functional test shows enhancement through chondrocyte proliferation, migration, and matrix deposition, while inhibit IL-1β-induced chondrocytes senescence, inflammation, matrix degradation, and pro-inflammatory macrophage activity. Multiple functional proteins, as well as a change in EVs’ size profile, with enrichment of specific EV-miRNAs were detected with hypoxia preconditioning, implicating complex molecular pathways involved in hypoxia pre-conditioned MSCs secretome generated cartilage regeneration.展开更多
Bone marrow-derived mesenchymal stem cell(MSC)is one of the most actively studied cell types due to its regenerative potential and immunomodulatory properties.Conventional cell expansion methods using 2D tissue cultur...Bone marrow-derived mesenchymal stem cell(MSC)is one of the most actively studied cell types due to its regenerative potential and immunomodulatory properties.Conventional cell expansion methods using 2D tissue culture plates and 2.5D microcarriers in bioreactors can generate large cell numbers,but they compromise stem cell potency and lack mechanical preconditioning to prepare MSC for physiological loading expected in vivo.To overcome these challenges,in this work,we describe a 3D dynamic hydrogel using magneto-stimulation for direct MSC manufacturing to therapy.With our technology,we found that dynamic mechanical stimulation(DMS)enhanced matrix-integrinβ1 interactions which induced MSCs spreading and proliferation.In addition,DMS could modulate MSC biofunctions including directing MSC differentiation into specific lineages and boosting paracrine activities(e.g.,growth factor secretion)through YAP nuclear localization and FAK-ERK pathway.With our magnetic hydrogel,complex procedures from MSC manufacturing to final clinical use,can be integrated into one single platform,and we believe this‘all-in-one’technology could offer a paradigm shift to existing standards in MSC therapy.展开更多
Mesenchymal stem/stromal cells(MSCs)have demonstrated therapeutic efficacy for bone regeneration in animal and clinical studies.Although MSCs were initially thought to differentiate to various cell types to replace th...Mesenchymal stem/stromal cells(MSCs)have demonstrated therapeutic efficacy for bone regeneration in animal and clinical studies.Although MSCs were initially thought to differentiate to various cell types to replace the injured/diseased tissue,it is now accepted that these cells secrete factors to promote tissue repair.1 Among these factors,small extracellular vesicles(sEVs)of size 50–200 nm,which include the exosomes,have been identified as the principal agent mediating the wide-ranging therapeutic efficacy of MSCs.2 Several studies have also reported the therapeutic effects of MSC-sEVs to enhance bone repair in animal models,as recently reviewed.3 However,the cellular processes and mechanisms mediated by MSC-sEVs in bone regeneration remain to be fully elucidated.展开更多
Background There is a difficulty in evaluating the in heterogeneity among cartilage affected by osteoarthritis vivo functionality of individual chondrocytes, and there is much （OA）. In this study, in vitro cultured ...Background There is a difficulty in evaluating the in heterogeneity among cartilage affected by osteoarthritis vivo functionality of individual chondrocytes, and there is much （OA）. In this study, in vitro cultured chondrocytes harvested from varying stages of degeneration were studied as a projective model to further understand the pathogenesis of osteoarthritis. Methods Cartilage of varying degeneration of end-stage OA was harvested, while cell yield and matrix glycosaminoglycan （GAG） content were measured. Cell morphology, proliferation, and gene expression of collagen type 1,‘11, and X, aggrecan, matrix metalloproteinase 13 （MMP-13）, and ADAMTS5 of the acquired chondrocytes were measured during subsequent in vitro culture. Results Both the number of cells and the GAG content increased with increasing severity of OA. Cell spreading area increased and gradually showed spindle-like morphology during in vitro culture. Gene expression of collagen type II, collagen type X as well as GAG decreased with severity of cartilage degeneration, while expression of collagen type I increased. Expression of MMP-13 increased with severity of cartilage degeneration, while expression of ADAMTS-5 remained stable. Expression of collagen type II, X, GAG, and MMP-13 substantially decreased with in vitro culture. Expression of collagen type I increased with in vitro cultures, while expression of ADAMTS 5 remained stable. Conclusions Expression of functional genes such as collagen type Ⅱ and GAG decreased during severe degeneration of OA cartilage and in vitro dedifferentiation. Gene expression of collagen I and MMP-13 increased with severity of cartilage degeneration.展开更多
基金supported by grants from the National University Healthcare System(R221000077733)the National University of Singapore(R221000090112)
文摘The objectives of this study were to （1） determine the distribution and synthesis of pericellular matrix （PCM） molecules （collagen VI, collagen IV and laminin） in rat temporomandibular joint （TMJ） and （2） investigate the effects of PCM molecules on chondrocytes against inflammation in osteoarthritis. Four zones （fibrous, proliferating, mature and hypertrophic） of condylar cartilage and three bands （anterior, intermediate and posterior） of disc were analysed by immunohistochemistry for the presence of PCM molecules in rat TMJs. Isolated chondrocytes were pre-treated with PCM molecules before being subjected to interleukin （IL）-II~ treatment to stimulate inflammation. The responses of the chondrocytes were analysed using gene expression, nitric oxide release and matrix metalloproteinase （MMP）-13 production measures. Histomorphometric analyses revealed that the highest areal deposition of collagen VI （67.4%）, collagen IV （45.7%） and laminin （52.4%） was in the proliferating zone of TMJ condylar cartilage. No significant difference in the distribution of PCM molecules was noted among the three bands of the TMJ disc. All three PCM molecules were expressed intracellularly by chondrocytes cultured in the monolayer. Among the PCM molecules, pre-treatment with collagen VI enhanced cellular proliferation, ameliorated IL-lp-induced MMP-3, MMP-9, MMP-13 and inducible nitric oxide synthase gene expression, and attenuated the downregulation of cartilage matrix genes, including collagen I, aggrecan and cartilage oligomeric matrix protein （COMP）. Concurrently, collagen VI pretreatment inhibited nitric oxide and MMP-13 production. Our study demonstrates for the first time the distribution and role of PCM molecules, particularly collagen VI, in the protection of chondrocytes against inflammation.
基金Faculty of Dentistry,National University of Singapore,Grant/Award Numbers:A-0002938-00-00,A-0006270-00-00。
文摘Conventional methods of stem cell therapy for tissue regeneration often face challenges,such as poor cell viability and integration posttransplantation.To address this,we proposed transplanting cells within synthetic microenviron-ments that maintain viability,cell phenotype,support extracellular matrix(ECM)secretion,and promote differentiation to enhance the regeneration of damaged host tissue.This hypothesis was tested in dental tissue regeneration using dental pulp stem cell-laden microcarriers(MCs)mixed in a gelatin methacrylate(GelMA)hydrogel as a delivery system.The combination of MCs and GelMA exhibited similar physical properties and favorable biological properties compared to GelMA alone.Specifically,cell-laden MC mixed into GelMA enhanced cell proliferation and ECM secretion and maintained a normal phenotype.Notably,MC-modified GelMA amplified odontogenic differentiation,mineralization,and vascular endothelial growth factor release.Moreover,the storage of MC-modified GelMA showed no detrimental effects on its injection force,cell viability,and mineralization potential,which demonstrates that the composite hydrogel is a promising injectable vehicle for therapeutic stem cell delivery.This strategy may be broadly applied to various tissues and organ systems,in which the provision and instruction of a cell population to participate in regeneration may be clinically useful.
基金supported by National Medical Research Council of Singapore(MOH-000371-00)the National Research Foundation,Prime Minister’s Office,Singapore under its Campus for Research Excellence and Technological Enterprise(CREATE)program,through Singapore-MIT Alliance for Research and Technology(SMART):Critical Analytics for Manufacturing Personalized-Medicine(CAMP)Inter-Disciplinary Research Group.YY was supported by NUS Research Scholarship.
文摘Secretome derived from mesenchymal stem cells (MSCs) have profound effects on tissue regeneration, which could become the basis of future MSCs therapies. Hypoxia, as the physiologic environment of MSCs, has great potential to enhance MSCs paracrine therapeutic effect. In our study, the paracrine effects of secretome derived from MSCs preconditioned in normoxia and hypoxia was compared through both in vitro functional assays and an in vivo rat osteochondral defect model. Specifically, the paracrine effect of total EVs were compared to that of soluble factors to characterize the predominant active components in the hypoxic secretome. We demonstrated that hypoxia conditioned medium, as well as the corresponding EVs, at a relatively low dosage, were efficient in promoting the repair of critical-sized osteochondral defects and mitigated the joint inflammation in a rat osteochondral defect model, relative to their normoxia counterpart. In vitro functional test shows enhancement through chondrocyte proliferation, migration, and matrix deposition, while inhibit IL-1β-induced chondrocytes senescence, inflammation, matrix degradation, and pro-inflammatory macrophage activity. Multiple functional proteins, as well as a change in EVs’ size profile, with enrichment of specific EV-miRNAs were detected with hypoxia preconditioning, implicating complex molecular pathways involved in hypoxia pre-conditioned MSCs secretome generated cartilage regeneration.
基金supported by NUS Presidential Young Professorship,MOE Tier 1 grantsupported by the NUS Research Scholarship.
文摘Bone marrow-derived mesenchymal stem cell(MSC)is one of the most actively studied cell types due to its regenerative potential and immunomodulatory properties.Conventional cell expansion methods using 2D tissue culture plates and 2.5D microcarriers in bioreactors can generate large cell numbers,but they compromise stem cell potency and lack mechanical preconditioning to prepare MSC for physiological loading expected in vivo.To overcome these challenges,in this work,we describe a 3D dynamic hydrogel using magneto-stimulation for direct MSC manufacturing to therapy.With our technology,we found that dynamic mechanical stimulation(DMS)enhanced matrix-integrinβ1 interactions which induced MSCs spreading and proliferation.In addition,DMS could modulate MSC biofunctions including directing MSC differentiation into specific lineages and boosting paracrine activities(e.g.,growth factor secretion)through YAP nuclear localization and FAK-ERK pathway.With our magnetic hydrogel,complex procedures from MSC manufacturing to final clinical use,can be integrated into one single platform,and we believe this‘all-in-one’technology could offer a paradigm shift to existing standards in MSC therapy.
基金This work was funded by National University of Singapore(No.R221000114114,R221000134114)National Medical Research Council Singapore(No.R221000123213)SKL and WST are supported by the Agency for Science,Technology and Research under its Health and Biomedical Sciences Industry Alignment Fund Pre-Positioning(No.H19H6a0026).
文摘Mesenchymal stem/stromal cells(MSCs)have demonstrated therapeutic efficacy for bone regeneration in animal and clinical studies.Although MSCs were initially thought to differentiate to various cell types to replace the injured/diseased tissue,it is now accepted that these cells secrete factors to promote tissue repair.1 Among these factors,small extracellular vesicles(sEVs)of size 50–200 nm,which include the exosomes,have been identified as the principal agent mediating the wide-ranging therapeutic efficacy of MSCs.2 Several studies have also reported the therapeutic effects of MSC-sEVs to enhance bone repair in animal models,as recently reviewed.3 However,the cellular processes and mechanisms mediated by MSC-sEVs in bone regeneration remain to be fully elucidated.
基金This work is supported by National Basic Research Program of China （973 Program） （No. 2011CB707500, 2012CB619102） and National Natural Science Foundation of China （No. 30772198, 30970881, 31011140348）.
文摘Background There is a difficulty in evaluating the in heterogeneity among cartilage affected by osteoarthritis vivo functionality of individual chondrocytes, and there is much （OA）. In this study, in vitro cultured chondrocytes harvested from varying stages of degeneration were studied as a projective model to further understand the pathogenesis of osteoarthritis. Methods Cartilage of varying degeneration of end-stage OA was harvested, while cell yield and matrix glycosaminoglycan （GAG） content were measured. Cell morphology, proliferation, and gene expression of collagen type 1,‘11, and X, aggrecan, matrix metalloproteinase 13 （MMP-13）, and ADAMTS5 of the acquired chondrocytes were measured during subsequent in vitro culture. Results Both the number of cells and the GAG content increased with increasing severity of OA. Cell spreading area increased and gradually showed spindle-like morphology during in vitro culture. Gene expression of collagen type II, collagen type X as well as GAG decreased with severity of cartilage degeneration, while expression of collagen type I increased. Expression of MMP-13 increased with severity of cartilage degeneration, while expression of ADAMTS-5 remained stable. Expression of collagen type II, X, GAG, and MMP-13 substantially decreased with in vitro culture. Expression of collagen type I increased with in vitro cultures, while expression of ADAMTS 5 remained stable. Conclusions Expression of functional genes such as collagen type Ⅱ and GAG decreased during severe degeneration of OA cartilage and in vitro dedifferentiation. Gene expression of collagen I and MMP-13 increased with severity of cartilage degeneration.
