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Enamel Matrix Derivative Stimulates Osteogenesis-and chondrogenesis-related Transcription Factors in C3H10T1/2 Cells

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Acta Biochimica et Biophysica Sinica 2007年第1期39卷 1-7页

作者： Masataka NARUKAWA Naoto SUZUKI Tadahiro TAKAYAMA Yasuhiko YAMASHITA Kichibee OTSUKA Koichi ITO Department of Periodontology Nihon University School of DentistryTokyo101-8310Japan Department of Biochemistry Nihon University School of DentustryTokyo 101-8310Japan Division of Functional Morphology Dental Research CenterNihon University School of DentistryTokyo 101-8310Japan Department of Biochemistry Nihon University School of DentistryTokyo101-8310Japan Division of Functional Morphology Dental Research CenterNihon University School of DentistryTokyo101-8310Japan Division of Advanced Dental Treatment Dental Research CenterNihon University School of DentistryTokyo101-8310Japan

Our purpose was to determine how enamel matrix derivative (EMD) affects the expression ofosteogenesis- and chondrogenesis-related transcription factors in undifferentiated mesenchymal cells.C3H10T1/2 cell line,a typical pluripotential mesenchymal cell line,was cultured with or without EMD forup to 7 d.Expression of mRNAs encoding osteogenesis-and chondrogenesis-related transcription factors(Runx2, Osterix,AJ18,Dlx5,Msx2,Sox5,Sox9 and Zfp60) was measured using real-time polymerasechain reaction.Runx2 and Sox9 protein expression and the presence of bone morphogenetic protein (BMP)-6-like molecules in EMD were determined by Western blotting.EMD substantially increased mRNA levels ofosteogenesis-and chondrogenesis-related transcription factors.EMD also induced Runx2 and Sox9 proteinexpression.Western blotting analysis of EMD using anti-BMP-6 antibody revealed immunoreactive bandscorresponding to about 14 kDa and 60 kDa.These results suggest that EMD stimulates osteogenesis- andchondrogenesis-related transcription factors,and these activities may be mediated,at least in part,by BMP-6in EMD.

关键词： enamel matrix derivative osteogenesis chondrogenesis

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Effect of different aged cartilage ECM on chondrogenesis of BMSCs in vitro and in vivo

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Regenerative Biomaterials 2020年第6期7卷 583-595页

作者： Xiuyu Wang Yan Lu Wan Wang Qiguang Wang Jie Liang Yujiang Fan Xingdong Zhang Guangxi Engineering Center in Biomedical Materials for Tissue and Organ Regeneration Guangxi Collaborative Innovation Center for BiomedicineGuangxi Medical UniversityNanning 530021China National Engineering Research Center for Biomaterials Sichuan UniversityWangjiang Road 29Chengdu 610064China

Extracellular matrix(ECM)-based biomaterials are promising candidates in cartilage tissue engineering by simulating the native microenvironment to regulate the chondrogenic differentiation of bone marrow mesenchymal stem cells(BMSCs)without exogenous growth factors.The biological properties of ECM scaffolds are primarily depended on the original source,which would directly influence the chondrogenic effects of the ECM materials.Despite the expanding investigations on ECM scaffolds in recent years,the selection of optimized ECM materials in cartilage regeneration was less reported.In this study,we harvested and compared the articular cartilage ECM from newborn,juvenile and adult rabbits.The results demonstrated the significant differences in the mechanical strength,sulphated glycosaminoglycan and collagen contents of the different aged ECM,before and after decellularization.Consequently,different compositional and mechanical properties were shown in the three ECM-based collagen hydrogels,which exerted age-dependent chondrogenic inducibility.In general,both in vitro and in vivo results suggested that the newborn ECM promoted the most chondrogenesis of BMSCs but led to severe matrix calcification.In contrast,BMSCs synthesized the lowest amount of cartilaginous matrix with minimal calcification with adult ECM.The juvenile ECM achieved the best overall results in promoting chondrogenesis of BMSCs and preventing matrix calcification.Together,this study provides important information to our current knowledge in the design of future ECM-based biomaterials towards a successful repair of articular cartilage.

关键词： cartilage ECM mesenchymal stem cells collagen hydrogel chondrogenesis calcification

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Compound screening platform using human induced pluripotent stem cells to identify small molecules that promote chondrogenesis

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Protein & Cell 2012年第12期3卷 934-942页

作者： Sheng-Lian Yang Erica Harnish Thomas Leeuw Uwe Dietz Erika Batchelder Paul SWright Jane Peppard Paul August Cecile Volle-Challier Francoise Bono Jean-Marc Herbert Juan Carlos Izpisua Belmonte Gene Expression Laboratories The Salk Institute for Biological Studies10010 N.Torrey Pines Rd.La JollaCA 92037USA Sanofi US R&DEarly to Candidate UnitTucson Research Center2090 E.Innovation Park DriveTucsonAZ 85755USA Sanofi Deutschland GmbH R&DTSU Aging Quality of LifeIndustriepark HoechstFrankfurt am Main 65926Germany Sanofi R&D Early to Candidate Unit195Route d'Espagne-BP 13669ToulouseFrance Center of Regeneration Medicine Dr.Aiguader 88Barcelona 08003Spain

Articular cartilage,which is mainly composed of collagen Ⅱ,enables smooth skeletal movement.Degeneration of collagen Ⅱ can be caused by various events,such as injury,but degeneration especially increases over the course of normal aging.Unfortunately,the body does not fully repair itself from this type of degeneration,resulting in impaired movement.Microfracture,an articular cartilage repair surgical technique,has been commonly used in the clinic to induce the repair of tissue at damage sites.Mesenchymal stem cells(MSC)have also been used as cell therapy to repair degenerated cartilage.However,the therapeutic outcomes of all these techniques vary in different patients depending on their age,health,lesion size and the extent of damage to the cartilage.The repairing tissues either form fibrocartilage or go into a hypertrophic stage,both of which do not reproduce the equivalent functionality of endogenous hyaline cartilage.One of the reasons for this is inefficient chondrogenesis by endogenous and exogenous MSC.Drugs that promote chondrogenesis could be used to induce self-repair of damaged cartilage as a non-invasive approach alone,or combined with other techniques to greatly assist the therapeutic outcomes.The recent development of human induced pluripotent stem cell(iPSCs),which are able to self-renew and differentiate into multiple cell types,provides a potentially valuable cell resource for drug screening in a“more relevant”cell type.Here we report a screening platform using human iPSCs in a multi-well plate format to identify compounds that could promote chondrogenesis.

关键词： hESC hiPSC chondrogenesis compound screening platform

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Bone morphogenetic protein 2 promotes transforming growth factor β3-induced chondrogenesis of human osteoarthritic synovium-derived stem cells

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Chinese Medical Journal 2010年第21期123卷 3040-3048页

作者： RUI Yun-feng DU Lin WANG You WANG Yang LUI Pauline po-yee TANG Ting-ting CHAN Kai-ming DAI Ke-rong Department of Orthopaedics Ninth People's Hospital Shanghai Jiao Tong University School of Medicine Shanghai 200011 China Shanghai Key Laboratory of Orthopaedic Implant Ninth People's Hospital Shanghai Jiao Tong University School of Medicine Shanghai 200011 China Department of Orthopaedics and Traumatology Faculty of Medicine The Chinese University of Hong Kong Hong Kong SAR China The Hong Kong Jockey Club Sports Medicine and Health Sciences Centre Faculty of Medicine The Chinese University of Hong Kong Hong Kong SAR China Program of Stern Cell and Regeneration School of Biomedical Science The Chinese University of Hong Kong Hong Kong SAR China

Background Synovium-derived stem cells （SDSCs） with higher chondrogenic potential are attracting considerable attention as a cell source for cartilage regeneration. We investigated the effect of bone morphogenetic protein 2 （BMP-2） on transforming growth factor beta3 （TGF-β3）-induced chondrogenesis of SDSCs isolated from human osteoarthritic synovium in a pellet culture system. Methods The clonogenicity, stem cell marker expression and multi-differentiation potential of isolated SDSCs were determined by colony forming unit assay, flow cytometry and specific staining including alizarin red S, Oil red O and alcian blue staining, respectively. SDSCs pellet was cultured in chondrogenic medium with or without TGF-β3 or/and BMP-2. At day 21, the diameter and the weight of the pellets were measured. Chondrogenic differentiation of SDSCs was evaluated by Safranin O staining, immunohistochemical staining of collagen type Ⅱ, sulfated glycosaminoglycan （sGAG） synthesis and mRNA expression of collagen type Ⅱ, aggrecan, SOX9, link-protein, collagen type X and BMP receptor Ⅱ. Results Cells isolated under the optimized culturing density （104/60 cm2） showed clonogenicity and multi-differentiation potential. These cells were positive （〉99%） for CD44, CD90, CD105 and negative （〈10%） for CD34 and CD71. SDSCs differentiated to a chondrocytic phenotype in chondrogenic medium containing TGF-β3 with or without BMP-2. Safranin O staining of the extracellular matrix was positive and the expression of collagen type Ⅱ was detected. Cell pellets treated with TGF-β3 and BMP-2 were larger in diameter and weight, produced more sGAGs, and expressed higher levels of collagen type Ⅱ and other chondrogenic markers, except COL10A1, than medium with TGF-β3 alone. Conclusions SDSCs could be isolated from human osteoarthritic synovium. Supplementation with BMP-2 significantly promoted the in vitro TGF-β3-induced chondrogenic differentiation of SDSCs.

关键词： chondrogenesis synovial membrane mesenchymal stem cell osteoarthritis bone morphogenetic protein 2 transforming growth factor beta3

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Proteome analysis of human mesenchymal stem cells undergoing chondrogenesis when exposed to the products of various magnesium-based materials degradation

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Bioactive Materials 2019年第1期4卷 168-188页

作者： Adela Helvia Martínez Sánchez Maryam Omidi Marcus Wurlitzer Marceline Manka Fuh Frank Feyerabend Hartmut Schlüter Regine Willumeit-Römer Bérengère J.C.Luthringer Division of Metallic Biomaterials Institute of Material ResearchHelmholtz-Zentrum GeesthachtMax Planck Strasse 121502GeesthachtGermany Institute of Clinical Chemistry and Laboratory Medicine Mass Spectrometric ProteomicsUniversity Medical Center Hamburg-EppendorfMartinistraße 5220246HamburgGermany

Treatment of physeal fractures(15%–30%of all paediatric fractures)remains a challenge as in approximately 10%of the cases,significant growth disturbance may occur.Bioresorbable Magnesium-based implants represent a strategy to minimize damage(i.e.,load support until bone healing without second surgery).Nevertheless,the absence of harmful effects of magnesium-implants and their degradation products on the growth plate should be confirmed.Here,the proteome of human mesenchymal stem cells undergoing chondrogenesis was evaluated when exposed to the products of various Magnesium-based materials degradation.The results of this study indicate that the materials induced regulation of proteins associated with cell chondrogenesis and cartilage formation,which should be beneficial for cartilage regeneration.

关键词： Stem cells differentiation chondrogenesis Proteomics Magnesium biomaterial Biodegradable implant

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Induction of chondrogenesis of Adipose-derived Stem Cells by Novel Recombinant TGF-β3 Fusion Protein

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Journal of Huazhong University of Science and Technology(Medical Sciences) 2013年第4期33卷 536-542页

作者：郑东但洋黄朋夏天杨述华许伟华杨操刘国辉刘先哲冯勇 Department of Orthopedics Union HospitalTongji Medical CollegeHuazhong University of Science and Technology

Summary： A new type of TGF-β3 fusion protein with targeted therapy function was constructed, and its feasibility and target specificity of inducing chondrogenesis were investigated by transfecting LAP-MMP-mTGF-β3 gene into adipose-derived stem cells （ADSCs）. The recombinant pIRES- EGFP-MMP was constructed by inserting the sense and antisense DNA of encoding the amino acid of the synthetic MMP enzyme cutting site into the eukaryotic expression vector pIRES-EGFE LAP and mTGF-β3 fragments were obtained by using RT-PCR and inserted into the upstream and downstream of MMP from pIRES-EGFP-MMP respectively, and the recombinant plasmid of pIRES-EGFP- LAP-MMP-mTGF-β3 was constructed, which was transferred to ADSCs. The ADSCs were cultured and divided in three groups： experimental group （MMP group）, negative control group （no MMP） and non-transfection group. The morphological changes were observed microscopically, and the expression of proteoglycan and type II collagen （Col II） was detected by using Alcian blue staining and immuno- histochemistry staining at 7th, 14th and 21st day after culture. The recombinant plasmid of pIRES-EGFP-LAP-MMP-mTGF-β3 was correctly constructed by methods of enzyme cutting and se- quencing analysis. The mTGF-β3 fusion protein was successfully expressed after transfection, and in the presence of the MMP, active protein mTGF-β3 was generated, which significantly promoted differ- entiation of ADSCs into chondrocytes and the expression of cartilage matrix. The novel fusion protein LAP-MMP-mTGF-β3 can targetedly induce differentiation of ADSCs into chondrocytes, which would open up prospects for target therapy of cartilage damage repair in future.

关键词： adipose-derived stem cells recombinant protein gene clone TGF-β3 chondrogenesis car-tilage damage target therapy

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Systematic review on the use of autologous matrix-induced chondrogenesis for the repair of articular cartilage defects in patients

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World Journal of Orthopedics 2017年第7期8卷 588-601页

作者： Nafisa Shaikh Matthew K T Seah Wasim S Khan SAS Facility Faculty of Science University of TechnologySydney Department of Trauma and Orthopaedics Addenbrookes Hospital University College London Institute of Orthopaedics and Musculoskeletal Sciences Royal National Orthopaedic Hospital

AIM To systematically review the results of studies looking at autologous matrix-induced chondrogenesis(AMIC) in humans. METHODS A literature search was performed, adhering to the PRISMA guidelines, to review any studies using such techniques in humans. Our initial search retrieved 297 articles listed on MEDLINE, Google Scholar, CINHal and EMBASE. From these studies, 15 studies meeting the eligibility criteria were selected and formed the basis of our systematic review.RESULTS The study designs, surgical techniques and outcome measures varied between the studies. Although all studies reported improvements in patient outcome measures, this was not necessarily correlated with magnetic resonance imaging findings. Although there were many additional procedures performed, when AMIC was performed in isolation, the results tended to peak at 24 mo before declining. CONCLUSION Although short-term studies suggest improved patient reported outcomes with a variety of scaffolds, surgical techniques and rehabilitation regimes, the literature remains equivocal on whether the defect size and location, and patient factors affect the outcome. Patientbenefit appears to be maintained in the short-tomedium term but more high level studies with extensive and robust validated outcome measures should be conducted to evaluate the medium-and long-term effect of the AMIC procedure.

关键词： Autologous matrix-induced chondrogenesis Cartilage defects Humans,PRISMA

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The role of Nkx3.2 in chondrogenesis

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Frontiers in Biology 2014年第5期9卷 376-381页

作者： Roshni S. RAINBOW Heenam KWON Li ZENG Department of Integrative Physiology and Pathobiology Tufts University School of Medicine 136 Harrison Avenue Boston MA 02111 USA Program in Cellular Molecular and Developmental Biology Sackler School of Graduate Biomedical Sciences Tufts University 136 Harrison Avenue Boston MA 02111 USA Department of Orthopaedics Tufts Medical Center 800 Washington Street Boston MA 02111 USA

Transcription factor, Nkx3.2, is a member of the NK family of developmental genes and is expressed during embryogenesis in a variety of mammalian model organisms, including chicken and mouse. It was first identified in Drosophila as the Bagpipe （bap） gene, where it has been demonstrated to be essential during formation of the midgut musculature. However, mammalian homolog Nkx3.2 has been shown to play a significant role in axial and limb skeletogenesis; in particular, the human skeletal disease, spondylo-megaepiphyseal-metaphyseal dysplasia （SMMD）, is associated with mutations of the Nkx3.2 gene. In this review, we highlight the role of Nkx3.2 during musculoskeletal development, with an emphasis on the factor＇s role in determining chondrogenic cell fate and its subsequent role in endochondral ossification and chondrocyte survival.

关键词： Nkx3.2 musculoskeletal development chondrogenesis chondrocyte hyperlxophy

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Multifaceted signaling regulators of chondrogenesis:Implications in cartilage regeneration and tissue engineering

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Genes & Diseases 2015年第4期2卷 307-327页

作者： Jordan D.Green Viktor Tollemar Mark Dougherty Zhengjian Yan Liangjun Yin Jixing Ye Zachary Collier Maryam K.Mohammed Rex C.Haydon Hue H.Luu Richard Kang Michael J.Lee Sherwin H.Ho Tong-Chuan He Lewis L.Shi Aravind Athiviraham The University of Chicago Pritzker School of Medicine ChicagoIL 60637USA Department of Orthopaedic Surgery and Rehabilitation Medicine The University of Chicago Medical CenterChicagoIL 60637USA Department of Orthopaedic Surgery The Second Affiliated Hospital of Chongqing Medical UniversityChongqing 400016China School of Bioengineering Chongqing UniversityChongqingChina

Defects of articular cartilage present a unique clinical challenge due to its poor self-healing capacity and avascular nature.Current surgical treatment options do not ensure consistent regeneration of hyaline cartilage in favor of fibrous tissue.Here,we review the current understanding of the most important biological regulators of chondrogenesis and their interactions,to provide insight into potential applications for cartilage tissue engineering.These include various signaling pathways,including fibroblast growth factors(FGFs),transforming growth factor b(TGF-b)/bone morphogenic proteins(BMPs),Wnt/b-catenin,Hedgehog,Notch,hypoxia,and angiogenic signaling pathways.Transcriptional and epigenetic regulation of chondrogenesis will also be discussed.Advances in our understanding of these signaling pathways have led to promising advances in cartilage regeneration and tissue engineering.

关键词： BMPs Cartilage Cell signaling chondrogenesis FGF Regenerative medicine

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Enhancing cartilage repair with optimized supramolecular hydrogel-based scaffold and pulsed electromagnetic field

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Bioactive Materials 2023年第4期22卷 312-324页

作者： Yucong Li Linlong Li Ye Li Lu Feng Bin Wang Ming Wang Haixing Wang Meiling Zhu Yongkang Yang Erik IWaldorff Nianli Zhang Ingmar Viohl Sien Lin Liming Bian Wayne Yuk-Wai Lee Gang Li Department of Orthopaedics&Traumatology Stem Cells and Regenerative Medicine LaboratoryLi Ka Shing Institute of Health SciencesThe Chinese University of Hong KongPrince of Wales HospitalShatinHong Kong Special Administrative Region Research&Clinical Affairs Orthofix Medical Inc.LewisvilleTXUSA Department of Rehabilitation Sciences Hong Kong Polytechnic UniversityHong Kong Special Administrative Region Innovation Centre for Advanced Interdisciplinary Medicine Key Laboratory of Biological Targeting DiagnosisTherapy and Rehabilitation of Guangdong Higher Education InstitutesThe Fifth Affiliated Hospital of Guangzhou Medical UniversityGuangzhouChina The Eighth Affiliated Hospital Sun Yat-Sen UniversityShenzhenPR China School of Biomedical Sciences and Engineering National Engineering Research Center for Tissue Restoration and ReconstructionKey Laboratory of Biomedical Materials and Engineering of the Ministry of EducationSouth China University of TechnologyGuangzhouPR China Department of Orthopaedics and Traumatology SH Ho Scoliosis Research LaboratoryJoint Scoliosis Research Center of the Chinese University of Hong Kong and Nanjing UniversityThe Chinese University of Hong KongHong Kong Special Administrative Region

Functional tissue engineering strategies provide innovative approach for the repair and regeneration of damaged cartilage.Hydrogel is widely used because it could provide rapid defect filling and proper structure support,and is biocompatible for cell aggregation and matrix deposition.Efforts have been made to seek suitable scaffolds for cartilage tissue engineering.Here Alg-DA/Ac-β-CD/gelatin hydrogel was designed with the features of physical and chemical multiple crosslinking and self-healing properties.Gelation time,swelling ratio,biodegradability and biocompatibility of the hydrogels were systematically characterized,and the injectable self-healing adhesive hydrogel were demonstrated to exhibit ideal properties for cartilage repair.Furthermore,the new hydrogel design introduces a pre-gel state before photo-crosslinking,where increased viscosity and decreased fluidity allow the gel to remain in a semi-solid condition.This granted multiple administration routes to the hydrogels,which brings hydrogels the ability to adapt to complex clinical situations.Pulsed electromagnetic fields(PEMF)have been recognized as a promising solution to various health problems owing to their noninvasive properties and therapeutic potentials.PEMF treatment offers a better clinical outcome with fewer,if any,side effects,and wildly used in musculoskeletal tissue repair.Thereby we propose PEMF as an effective biophysical stimulation to be 4th key element in cartilage tissue engineering.In this study,the as-prepared Alg-DA/Ac-β-CD/gelatin hydrogels were utilized in the rat osteochondral defect model,and the potential application of PEMF in cartilage tissue engineering were investigated.PEMF treatment were proven to enhance the quality of engineered chondrogenic constructs in vitro,and facilitate chondrogenesis and cartilage repair in vivo.All of the results suggested that with the injectable self-healing adhesive hydrogel and PEMF treatment,this newly proposed tissue engineering strategy revealed superior clinical potential for cartilag

关键词： Supramolecular hydrogels Pulsed electromagnetic field Cartilage tissue engineering Mesenchymal stem cells chondrogenesis

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