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1.
Plast Reconstr Surg ; 145(2): 409-418, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31985633

RESUMO

BACKGROUND: Irradiated allogeneic costal cartilage is an alternative option of cartilage graft in patients with insufficient autologous cartilage. However, complications can occur during long-term follow-up. This study investigated whether Tutoplast-processed cartilage, one of the irradiated allogeneic costal cartilages, acts as a scaffold for adipose-derived stem cells and chondrogenesis. METHODS: In vitro setting, human adipose-derived stem cells seeded onto Tutoplast-processed cartilage were cultured in chondrogenic medium and observed using a scanning electron microscope. Next, 3 types of irradiated cartilage-including Tutoplast-processed cartilage, undifferentiated stem cells on Tutoplast-processed cartilage (undifferentiated group), and chondrogenic differentiated stem cells on Tutoplast-processed cartilage (chondrogenic group)-were implanted subcutaneously into nude mice. Gross, histologic, and gene expression analyses of Tutoplast-processed cartilages were performed at postoperative weeks 2 and 4. RESULTS: Human adipose-derived stem cells subjected to in vitro three-dimensional culture differentiated into chondrocytes and expressed cartilage-specificgenes. Adipose-derived stem cells seeded onto Tutoplast-processed cartilage were differentiated into chondrocytes in chondrogenic medium. In the chondrogenic group, the chondrogenic-differentiated cells attached to the surface of the Tutoplast-processed cartilage were maintained during the follow-up and were distinct from the existing Tutoplast-processed cartilage. Moreover, the chondrogenic group had higher expression of cartilage-specific genes compared with the undifferentiated group. CONCLUSIONS: Adipose-derived stem cells seeded onto Tutoplast-processed cartilage underwent chondrogenic differentiation, generating new cartilage, which was maintained after implantation without critical complications. The findings are clinically valuable in terms of overcoming the limitations of irradiated allogeneic costal cartilage, and broaden the surgical options for treatments requiring cartilage.


Assuntos
Cartilagem/fisiologia , Condrogênese/fisiologia , Células-Tronco Mesenquimais/fisiologia , Agrecanas/metabolismo , Animais , Biomarcadores/metabolismo , Cartilagem/efeitos da radiação , Diferenciação Celular/fisiologia , Células Cultivadas , Colágeno Tipo X/metabolismo , Feminino , Humanos , Técnicas In Vitro , Injeções Subcutâneas , Músculos Intercostais , Transplante de Células-Tronco Mesenquimais/métodos , Camundongos Nus , Microscopia Eletrônica de Varredura , Pessoa de Meia-Idade , Modelos Animais , Reação em Cadeia da Polimerase em Tempo Real , Transplante Heterólogo , Transplante Homólogo
2.
Cell Prolif ; 53(1): e12727, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31747713

RESUMO

OBJECTIVE: Chondrogenesis and endochondral ossification in mandibular condyle play crucial roles in maxillofacial morphogenesis and function. Circadian regulator brain and muscle arnt-like 1 (BMAL1) is proven to be essential for embryonic and postnatal development. The goal of this study was to define the functions of BMAL1 in the embryonic and postnatal growth of mandibular condylar cartilages (MCC). MATERIALS AND METHODS: Micro-CT, TUNEL staining and EdU assay were performed using BMAL1-deficient mice model, and in vitro experiments were performed using rat chondrocytes isolated from MCC. RNA sequencing in mandibular condyle tissues from Bmal1-/- mice and the age-matched wild-type mice was used for transcriptional profiling at different postnatal stages. RESULTS: The expression levels of BMAL1 decrease gradually in MCC. BMAL1 is proved to regulate sequential chondrocyte differentiation, and its deficiency can result in the impairment of endochondral ossification of MCC. RNA sequencing reveals hedgehog signalling pathway is the potential target of BMAL1. BMAL1 regulates hedgehog signalling and affects its downstream cascades through directly binding to the promoters of Ptch1 and Ihh, modulating targets of hedgehog signalling which is indispensable for endochondral ossification. Importantly, the short stature phenotypes caused by BMAL1 deficiency can be rescued by hedgehog signalling activator. CONCLUSIONS: Collectively, these results indicate that BMAL1 plays critical roles on chondrogenesis and endochondral ossification of MCC, giving a new insight on potential therapeutic strategies for facial dysmorphism.


Assuntos
Fatores de Transcrição ARNTL/metabolismo , Cartilagem/embriologia , Diferenciação Celular/fisiologia , Condrócitos/metabolismo , Ritmo Circadiano/fisiologia , Côndilo Mandibular/embriologia , Transdução de Sinais/fisiologia , Animais , Cartilagem/citologia , Condrócitos/citologia , Condrogênese/fisiologia , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Côndilo Mandibular/citologia , Camundongos , Camundongos Knockout , Receptor Patched-1/genética , Receptor Patched-1/metabolismo
3.
Biomed Pharmacother ; 117: 109146, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31387186

RESUMO

Mesenchymal stem cells and chondrocytes are an important source of the cells for cartilage tissue engineering. Therefore, the culture and expansion methods of these cells need to be improved to overcome the aging of chondrocytes and induced chondrogenic differentiation of mesenchymal stem cells. The aim of this study was to expand the cells for cartilage tissue engineering by combining the advantages of growing cells in co-culture and under a mechanically-stimulated environment. Rabbit chondrocytes and co-cultured cells (bone mesenchymal stem cells and chondrocytes) were subjected to cyclic sinusoidal dynamic tensile mechanical stimulationusing the FX-4000 tension system. Chondrocyte proliferation was assayed by flow cytometry and CFSE labeling. The cell cartilage phenotype was determined by detecting GAG, collagen II and TGF-ß1 protein expression by ELISA and the Col2α1, TGF-ß1 and Sox9 gene expression by RT-PCR. The results show that the co-culture improved both the proliferation ability of chondrocytes and the cartilage phenotype of co-cultured cells. A proper cyclic sinusoidal dynamic tensile mechanical stimulation improved the proliferation ability and cartilage phenotype of chondrocytes and co-cultured cells. These results suggest that the co-culture of mesenchymal stem cells with chondrocytes and proper mechanical stimulation may be an appropriate way to rapidly expand the cells that have an improved cartilage phenotype for cartilage tissue engineering.


Assuntos
Cartilagem/fisiologia , Proliferação de Células/fisiologia , Condrócitos/fisiologia , Células-Tronco Mesenquimais/fisiologia , Animais , Cartilagem/metabolismo , Diferenciação Celular/fisiologia , Células Cultivadas , Condrócitos/metabolismo , Condrogênese/fisiologia , Técnicas de Cocultura/métodos , Colágeno Tipo II/metabolismo , Matriz Extracelular/metabolismo , Matriz Extracelular/fisiologia , Células-Tronco Mesenquimais/metabolismo , Fenótipo , Coelhos , Fatores de Transcrição SOX9/metabolismo , Engenharia Tecidual/métodos , Fator de Crescimento Transformador beta1/metabolismo
4.
Life Sci ; 232: 116625, 2019 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-31276691

RESUMO

AIMS: The chondrogenic differentiation of bone marrow-derived mesenchymal stem cells (BMSCs) is critical for cartilage regeneration. Tissues constructed from BMSCs through cartilage tissue engineering still exhibit some histological, morphological, and biomechanical differences from normal cartilage tissues. Cyclic tensile strain (CTS) can increase chondrogenic gene expression and reduce hypertrophic gene expression in chondrocytes. miR-365 has been identified as a mechanoresponsive microRNA and is an important regulator of both chondrocyte hypertrophy and differentiation. Therefore, we hypothesized that CTS may promote the chondrogenesis of BMSCs by upregulating the expression of miR-365. METHODS: BMSCs were subjected to CTS to investigate the effects and mechanism on chondrogenesis. An Agilent miRNA microarray was used to profile miRNAs in the CTS-treated BMSCs and 3D-cultured control BMSCs. miR-365 was shown to interact with HDAC4 mRNA through a luciferase reporter assay. An animal cartilage defect model was constructed and different groups of BMSCs were implanted to investigate their in vivo effect. KEY FINDINGS: CTS promoted BMSC chondrogenesis. miR-365 was significantly upregulated in CTS-treated cells and played an important role in CTS-mediated chondrogenesis. Luciferase assays showed that HDAC4 is a direct target of miR-365. An in vivo study showed that CTS treatment and miR-365 overexpression could promote cartilage regeneration from BMSCs. SIGNIFICANCE: CTS can promote the expression of miR-365, a crucial mechanosensitive microRNA involved in the chondrogenesis of BMSCs, which directly inhibits the expression of HDAC4, in turn, enhancing the chondrogenesis of BMSCs.


Assuntos
Condrogênese/genética , Células-Tronco Mesenquimais/fisiologia , MicroRNAs/genética , Animais , Medula Óssea/metabolismo , Células da Medula Óssea/citologia , Cartilagem/metabolismo , Diferenciação Celular/fisiologia , Proliferação de Células , Condrócitos/metabolismo , Condrogênese/fisiologia , MicroRNAs/metabolismo , Ratos , Transdução de Sinais , Resistência à Tração/fisiologia , Engenharia Tecidual
5.
Med Sci Monit ; 25: 4041-4050, 2019 May 31.
Artigo em Inglês | MEDLINE | ID: mdl-31147532

RESUMO

BACKGROUND We synthetized a 3D printed poly-ε-caprolactone (PCL) scaffold with polydopamine (PDA) coating and lithium chloride (LiCl) deposition for cartilage tissue engineering and analyzed its effect on promoting rabbit bone marrow mesenchymal stem cells (rBMSC) chondrogenesis in vitro. MATERIAL AND METHODS PCL scaffolds were prepared by 3D printing with a well-designed CAD digital model, then modified by PDA coating to produce PCL-PDA scaffolds. Finally, LiCl was deposited on the PDA coating to produce PCL-PDA-Li scaffolds. The physicochemical properties, bioactivity, and biocompatibility of PCL-PDA-Li scaffolds were accessed by comparing them with PCL scaffolds and PCL-PDA scaffolds. RESULTS 3D PCL scaffolds exhibited excellent mechanical integrity as designed. PDA coating and LiCl deposition improved surface hydrophilicity without sacrificing mechanical strength. Li⁺ release was durable and ion concentration did not reach the cytotoxicity level. This in vitro study showed that, compared to PCL scaffolds, PCL-PDA and PCL-PDA-Li scaffolds significantly increased glycosaminoglycan (GAG) formation and chondrogenic marker gene expression, while PCL-PDA-Li scaffolds showed far higher rBMSC viability and chondrogenesis. CONCLUSIONS 3D printed PCL-PDA-Li scaffolds promoted chondrogenesis in vitro and may provide a good method for lithium administration and be a potential candidate for cartilage tissue engineering.


Assuntos
Cloreto de Lítio/farmacologia , Engenharia Tecidual/métodos , Tecidos Suporte/química , Animais , Materiais Biocompatíveis/química , Medula Óssea , Caproatos/farmacologia , Cartilagem/metabolismo , Condrogênese/fisiologia , Indóis/farmacologia , Lactonas/farmacologia , Células-Tronco Mesenquimais/metabolismo , Osteogênese/efeitos dos fármacos , Poliésteres/química , Polímeros/farmacologia , Impressão Tridimensional , Coelhos , Regeneração/fisiologia
6.
Res Vet Sci ; 124: 393-398, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31077967

RESUMO

Osteoarthritis associated with hip dysplasia is one of the most common orthopedic abnormalities in dogs, with an incidence of up to 40% in some breeds. Tissue therapy of cartilage has received great attention, with use of mesenchymal stromal cells and different types of biomaterials. The present study aimed to evaluate the effect of platelet lysate (PL) on the proliferation and differentiation of canine adipose tissue-derived mesenchymal stromal cells (ASCs), in liquid culture or hydrogels. PL was prepared from blood collected from healthy dogs and submitted to freezing-thawing cycles, and hydrogel was formed with canine thrombin. The effect of PL on the proliferation and differentiation of canine ASCs was evaluated in liquid and hydrogel systems, with microscopy, quantification of dsDNA, histology and quantification of glycosaminoglycans. The addition of 5% or 10% PL to the culture medium induced a greater proliferation rate than the presence of 10% fetal bovine serum. The cultivation of ASCs in PL gel, with normal or chondrogenic medium, resulted in maintenance of proliferation level similar to the conventional 2D cultivation, and induction of chondrogenic differentiation, especially in the presence of the chondrogenesis induction medium.


Assuntos
Tecido Adiposo/fisiologia , Condrogênese/fisiologia , Liases/metabolismo , Células-Tronco Mesenquimais/efeitos dos fármacos , Animais , Plaquetas/enzimologia , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Cães , Relação Dose-Resposta a Droga , Liases/administração & dosagem , Células-Tronco Mesenquimais/fisiologia
7.
Tissue Cell ; 57: 42-48, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30947962

RESUMO

BACKGROUND AND OBJECTIVE: In the field of cartilage repair, use of two or more cell populations such as mesenchymal stem cells with chondrocytes in an in-vitro co-culture synergistic environment has been attempted to evade limitations of monoculture systems and promote/induce chondrogenesis. Articular cartilage-derived chondroprogenitors (CPs), considered to have stem-cell like characteristics have been proposed as a potential contender for neocartilage development. Our objective was to assess whether co-cultures using different ratios of chondrocytes(C) and CPs would demonstrate better results in terms of growth kinetics, surface marker expression, chondrogenic potential, tendency for hypertrophy and glycosaminoglycan deposition than monocultures. STUDY DESIGN: Human chondrocytes and CPs (fibronectin adhesion assay) from the same cartilage source were isolated. Passage 2 cells were subjected to monolayer/pellet cultures and were grown as monocultures and cocultures at the following percentage ratios(C:CP) 80:20, 65:35, 50:50, 35:65 and 20:80. RESULTS: Analysis of data acquired from population doubling, flow cytometry, RT-PCR and Safranin O uptake demonstrated similar results in all monoculture and co-culture groups with no significant inter-group variation, even when reported specific markers of identification (CD54 and CD44:chondrocyte markers) and isolation (CD29 and CD49e: forming heterodimeric fibronectin receptor for CP sorting) were examined. CONCLUSION: In conclusion, this study suggests the need for improved sorting techniques based on a characteristic differentiating biomarker for selection of cells which are true representatives of CPs possessing properties of enhanced chondrogenesis and reduced hypertrophy.


Assuntos
Cartilagem Articular/citologia , Condrócitos/citologia , Condrogênese/fisiologia , Células-Tronco Mesenquimais/citologia , Técnicas de Cultura de Células , Diferenciação Celular/fisiologia , Células Cultivadas , Técnicas de Cocultura , Humanos
8.
Tissue Cell ; 57: 49-56, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30947963

RESUMO

In this study, we report the development of a nanofiber polycaprolactone scaffold that can act as a stem cell carrier to induce chondrogenesis and promote cartilage repair in vivo. Infrapatellar fat pads were obtained from sheep knee and the stem cells were isolated and characterized by flow cytometry. Defects were created in sheep knee, two defects received adipose tissue derived stem cells (ASCs)-polycaprolactone construct, second group received polycaprolactone (PCL), the third group was chosen as the ASCs group and the fourth group was control group. Morphological evaluation showed that defects treated with ASCs-scaffold constructs were completely filled with cartilage-like tissue, while other groups revealed the formation of a thin layer of cartilage-like tissue in the defects. Real-Time RT-PCR showed the increase in collagen type 2 mRNA levels, aggrecan and Sox9 in ASCs/PCL groups in comparison with the other groups. Immunofluorescence and toluidine blue staining results showed the protein expression of collagen type 2 and formation of round and polygonal clusters of chondrocytes in ASCS/PCL group. According to our results nanofiber polycaprolactone promoted the chondrogenesis of infrapatellar adipose tissue derived stem cells in vivo and could offer significant promise in the biological functionality of stem cell tissue engineering in clinical practice.


Assuntos
Cartilagem Articular/fisiologia , Condrogênese/fisiologia , Transplante de Células-Tronco Mesenquimais/métodos , Engenharia Tecidual/métodos , Animais , Nanofibras , Poliésteres , Regeneração , Ovinos , Tecidos Suporte/química
9.
PLoS One ; 14(3): e0213912, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30883580

RESUMO

Glucose metabolism is altered in injured and healing tendons. However, the mechanism by which the glucose metabolism is involved in the pathogenesis of tendon healing process remains unclear. Injured tendons do not completely heal, and often induce fibrous scar and chondroid lesion. Because previous studies have shown that tendon progenitors play roles in tendon repair, we asked whether connective tissue progenitors appearing in injured tendons alter glucose metabolism during tendon healing process. We isolated connective tissue progenitors from the human injured tendons, obtained at the time of primary surgical repair of rupture or laceration. We first characterized the change in glucose metabolism by metabolomics analysis using [1,2-13C]-glucose using the cells isolated from the lacerated flexor tendon. The flux of glucose to the glycolysis pathway was increased in the connective tissue progenitors when they proceeded toward tenogenic and chondrogenic differentiation. The influx of glucose to the tricarboxylic acid (TCA) cycle and biosynthesis of amino acids from the intermediates of the TCA cycle were strongly stimulated toward chondrogenic differentiation. When we treated the cultures with 2-deoxy-D-glucose (2DG), an inhibitor of glycolysis, 2DG inhibited chondrogenesis as characterized by accumulation of mucopolysaccharides and expression of AGGRECAN. Interestingly, 2DG strongly stimulated expression of tenogenic transcription factor genes, SCLERAXIS and MOHAWK under both chondrogenic and tenogenic differentiation conditions. The findings suggest that control of glucose metabolism is beneficial for tenogenic differentiation of connective tissue progenitors.


Assuntos
Glucose/metabolismo , Traumatismos dos Tendões/metabolismo , Traumatismos dos Tendões/patologia , Adulto , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Condrogênese/efeitos dos fármacos , Condrogênese/fisiologia , Células do Tecido Conjuntivo/efeitos dos fármacos , Células do Tecido Conjuntivo/metabolismo , Células do Tecido Conjuntivo/patologia , Desoxiglucose/metabolismo , Desoxiglucose/farmacologia , Humanos , Masculino , Pessoa de Meia-Idade , Células-Tronco/efeitos dos fármacos , Células-Tronco/metabolismo , Células-Tronco/patologia , Traumatismos dos Tendões/fisiopatologia , Tendões/metabolismo , Tendões/patologia , Cicatrização/fisiologia , Adulto Jovem
10.
Mater Sci Eng C Mater Biol Appl ; 98: 1169-1178, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30813000

RESUMO

During the last decade, many cartilage tissue engineering strategies have been developed, being the stem cell-based approach one of the most promising. Transforming Growth Factor-ß3 (TGF-ß3) and Insulin-like Growth Factor-I (IGF-I) are key proteins involved in the regulation of chondrogenic differentiation. Therefore, these two growth factors (GFs) were immobilized at the surface of a single electrospun nanofibrous mesh (NFM) aiming to differentiate human Bone Marrow-derived Mesenchymal Stem Cells (hBM-MSCs). The immobilization of defined antibodies (i.e. anti-TGF-ß3 and anti-IGF-I) allows the selective retrieval of the abovementioned GFs from human platelet lysates (PL). Biochemical assays, involving hBM-MSCs cultured on biofunctional nanofibrous substrates under basal culture medium during 28 days, confirm the biological activity of bound TGF-ß3 and IGF-I. Specifically, the typical spherical morphology of chondrocytes and the immunolocalization of collagen type II confirmed the formation of a cartilaginous ECM. Therefore, the proposed biofunctional nanofibrous substrate is able to promote chondrogenesis.


Assuntos
Condrogênese/efeitos dos fármacos , Condrogênese/fisiologia , Células-Tronco Mesenquimais/citologia , Nanofibras/química , Idoso de 80 Anos ou mais , Plaquetas/metabolismo , Cartilagem/efeitos dos fármacos , Cartilagem/metabolismo , Técnicas de Cultura de Células/métodos , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Condrócitos/citologia , Condrócitos/efeitos dos fármacos , Condrócitos/metabolismo , Colágeno Tipo II/metabolismo , Feminino , Humanos , Fator de Crescimento Insulin-Like I/metabolismo , Células-Tronco Mesenquimais/metabolismo , Engenharia Tecidual/métodos , Fator de Crescimento Transformador beta3/metabolismo
11.
Int J Mol Sci ; 20(5)2019 Mar 12.
Artigo em Inglês | MEDLINE | ID: mdl-30870992

RESUMO

Mammalian telomere lengths are primarily regulated by telomerase, consisting of a reverse transcriptase protein (TERT) and an RNA subunit (TERC). We previously reported the generation of mouse Terc+/- and Terc-/- embryonic stem cells (ntESCs) by somatic cell nuclear transfer. In the present work, we investigated the germ layer development competence of Terc-/-, Terc+/- and wild-type (Terc+/+) ntESCs. The telomere lengths are longest in wild-type but shortest in Terc-/- ntESCs, and correlate reversely with the population doubling time. Interestingly, while in vitro embryoid body (EB) differentiation assay reveals EB size difference among ntESCs of different genotypes, the more stringent in vivo teratoma assay demonstrates that Terc-/- ntESCs are severely defective in differentiating into the mesodermal lineage cartilage. Consistently, in a directed in vitro chondrocyte differentiation assay, the Terc-/- cells failed in forming Collagen II expressing cells. These findings underscore the significance in maintaining proper telomere lengths in stem cells and their derivatives for regenerative medicine.


Assuntos
Diferenciação Celular/fisiologia , Núcleo Celular/fisiologia , Condrócitos/fisiologia , Células-Tronco Embrionárias Murinas/fisiologia , RNA/genética , Telomerase/genética , Animais , Cartilagem/fisiologia , Diferenciação Celular/genética , Núcleo Celular/genética , Células Cultivadas , Condrogênese/genética , Condrogênese/fisiologia , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Técnicas de Transferência Nuclear , Telômero/genética , Homeostase do Telômero/genética , Homeostase do Telômero/fisiologia
12.
Appl Biochem Biotechnol ; 188(4): 942-951, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-30740625

RESUMO

The aim of this study was to investigate the effect of medium harvested from septal cartilage cells on chondrogenic differentiation of adipose stem cells (hASCs) and to compare/contrast its properties to those of a commonly used standard medium formulation in terms of induction and maintenance of chondrogenic hASCs. Differentiation was carried out under three different conditions: septal cartilage medium-SCM, chondrogenic differentiation medium-CM, and 50:50 mixture of CM/SCM. Mesenchymal stem cells (MSCs) markers were determined by flow cytometry. The cytotoxic and apoptotic effects were determined by MTS and Annexin V assay, respectively. The differentiation status of the cells was confirmed by Alcian blue staining, and quantitative real-time flow cytometry showed that hASCs were positive for MSCs, negative for hematopoietic stem cells and endothelial cell surface markers. According to MTS analysis, the first condition was not toxic at any concentration tested. Annexin V assay revealed that the application of different concentrations of SCM did not result in any cell death. The Alcian blue and gene expression analyses showed that the cells in the SCM group underwent the highest cartilage cell formation. The observed increase in chondrogenesis may offer better treatment options for the cartilage defects seen in nasal septum perforation.


Assuntos
Adipócitos/citologia , Cartilagem/citologia , Diferenciação Celular/fisiologia , Condrócitos/citologia , Condrogênese/fisiologia , Cartilagens Nasais/citologia , Células-Tronco/citologia , Diferenciação Celular/genética , Células Cultivadas , Condrogênese/genética , Citometria de Fluxo , Humanos , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/fisiologia , Septo Nasal/citologia , Reação em Cadeia da Polimerase em Tempo Real
13.
Vet Surg ; 48(3): 375-387, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30801754

RESUMO

OBJECTIVE: To determine the chondrogenic potential of cells derived from interzone tissue, the normal progenitor of articular cartilage during fetal development, compared to that of adult bone marrow-derived and adipose-derived mesenchymal cell isolates. The objective of this study was to compare the chondrogenic potential of fetal musculoskeletal progenitor cells to adult cell types, which are currently used therapeutically to facilitate joint cartilage repair in equine clinical practice. The hypothesis tested was that cells derived from interzone tissue have a chondrogenic potential that exceeds that of adult bone marrow-derived and adipose-derived mesenchymal cell isolates. STUDY DESIGN: In vitro study. ANIMALS: Six young adult horses (15-17 months of age) and 6 equine fetuses aged 45-46 days of gestation. METHODS: Three-dimensional pellet cultures were established under chondrogenic conditions with fresh, primary cells isolated from adult (articular cartilage, bone marrow, adipose, dermis) and fetal (interzone, skeletal anlagen cartilage, dermis) tissues. Cellular morphology, pellet architecture, and proteoglycan synthesis were assessed in the pellet cultures. Steady state levels of ACAN (aggrecan core protein), COL2A1 (collagen type II), and COL1A1 (collagen type I) messenger RNA (mRNA) were compared among these cell types as pellet cultures and monolayer cultures. RESULTS: Adult articular chondrocytes, fetal interzone cells, and fetal anlage cells generated the largest pellets under these chondrogenic culture conditions. Pellets derived from adult articular chondrocytes and fetal anlage cells had the highest scores on a neocartilage grading scale. Fetal anlage and adult articular chondrocyte pellets had low steady-state levels of COL1A mRNA but high COL2A1 expression. Anlage chondrocyte pellets also had the highest expression of ACAN. CONCLUSION: Adult articular chondrocytes, fetal interzone cells, and fetal anlage chondrocytes exhibited the highest chondrogenic potential. In this study, adult adipose-derived cells exhibited very limited chondrogenesis, and bone marrow-derived cells had limited and variable chondrogenic potential. CLINICAL SIGNIFICANCE: Additional investigation of the high chondrogenic potential of fetal interzone cells and anlage chondrocytes to advance cell-based therapies in diarthrodial joints is warranted.


Assuntos
Diferenciação Celular/efeitos da radiação , Condrócitos/fisiologia , Condrogênese/fisiologia , Feto/citologia , Feto/fisiologia , Cavalos/embriologia , Animais , Células da Medula Óssea , Cartilagem Articular , Técnicas de Cultura de Células , Cavalos/metabolismo , Humanos , Células-Tronco Mesenquimais
14.
J Orthop Res ; 37(6): 1339-1349, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30667562

RESUMO

People of all backgrounds are susceptible to bone and cartilage damage, and these injuries can be debilitating. Current treatments for bone and cartilage injuries are less than optimal, and we are interested in developing new approaches to treat these diseases, specifically using human muscle-derived stem cells (hMDSCs). Our lab previously demonstrated that sex differences exist between male and female murine MDSCs; thus, this paper sought to investigate whether sex differences also exist in hMDSCs. In the present study, we characterized the chondrogenic and osteogenic sex differences of hMDSCs in vitro and in vivo. We performed in vitro osteogenic and chondrogenic differentiation using hMDSC pellet cultures. As demonstrated by microCT, histology, and immunohistochemistry, male hMDSCs were more chondrogenic and osteogenic than their female counterparts in vitro. No differences were observed based on the sex of hMDSCs in osteogenic and chondrogenic gene expression and cell surface markers. For our in vivo study, we transduced hMDSCs with lenti-BMP2/GFP and transplanted these cells into critical-sized calvarial defects in mice. MicroCT results revealed that male hMDSCs regenerated more bone at 2 weeks and demonstrated higher bone density at 4 and 6 weeks than female hMDSCs. Histology demonstrated that both male and female hMDSCs regenerated functional bone. Clinical relevance: These studies reinforce that stem cells isolated from male and female patients differ in function, and we should disclose the sex of cells used in future studies. Considering sex differences of hMDSCs may help to improve cell-based therapies for autologous cell treatment of bone and cartilage damage. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 37:1339-1349, 2019.


Assuntos
Condrogênese/fisiologia , Músculo Esquelético/citologia , Osteogênese/fisiologia , Transplante de Células-Tronco , Células-Tronco/citologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Regeneração Óssea , Diferenciação Celular , Criança , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos ICR , Caracteres Sexuais
15.
ACS Appl Mater Interfaces ; 11(4): 3781-3799, 2019 Jan 30.
Artigo em Inglês | MEDLINE | ID: mdl-30609898

RESUMO

Osteochondral (OC) regeneration faces several limitations in orthopedic surgery, owing to the complexity of the OC tissue that simultaneously entails the restoration of articular cartilage and subchondral bone diseases. In this study, novel biofunctional hierarchical scaffolds composed of a horseradish peroxidase (HRP)-cross-linked silk fibroin (SF) cartilage-like layer (HRP-SF layer) fully integrated into a HRP-SF/ZnSr-doped ß-tricalcium phosphate (ß-TCP) subchondral bone-like layer (HRP-SF/dTCP layer) were proposed as a promising strategy for OC tissue regeneration. For comparative purposes, a similar bilayered structure produced with no ion incorporation (HRP-SF/TCP layer) was used. A homogeneous porosity distribution was achieved throughout the scaffolds, as shown by micro-computed tomography analysis. The ion-doped bilayered scaffolds presented a wet compressive modulus (226.56 ± 60.34 kPa) and dynamic mechanical properties (ranging from 403.56 ± 111.62 to 593.56 ± 206.90 kPa) superior to that of the control bilayered scaffolds (189.18 ± 90.80 kPa and ranging from 262.72 ± 59.92 to 347.68 ± 93.37 kPa, respectively). Apatite crystal formation, after immersion in simulated body fluid (SBF), was observed in the subchondral bone-like layers for the scaffolds incorporating TCP powders. Human osteoblasts (hOBs) and human articular chondrocytes (hACs) were co-cultured onto the bilayered structures and monocultured in the respective cartilage and subchondral bone half of the partitioned scaffolds. Both cell types showed good adhesion and proliferation in the scaffold compartments, as well as adequate integration of the interface regions. Osteoblasts produced a mineralized extracellular matrix (ECM) in the subchondral bone-like layers, and chondrocytes showed GAG deposition. The gene expression profile was different in the distinct zones of the bilayered constructs, and the intermediate regions showed pre-hypertrophic chondrocyte gene expression, especially on the BdTCP constructs. Immunofluorescence analysis supported these observations. This study showed that the proposed bilayered scaffolds allowed a specific stimulation of the chondrogenic and osteogenic cells in the co-culture system together with the formation of an osteochondral-like tissue interface. Hence, the structural adaptability, suitable mechanical properties, and biological performance of the hierarchical scaffolds make these constructs a desired strategy for OC defect regeneration.


Assuntos
Tecidos Suporte/química , Animais , Fosfatos de Cálcio/química , Condrócitos/fisiologia , Condrogênese/genética , Condrogênese/fisiologia , Técnicas de Cocultura , Matriz Extracelular , Fibroínas/química , Humanos , Osteoblastos/fisiologia , Osteogênese/fisiologia , Engenharia Tecidual/métodos
16.
Arterioscler Thromb Vasc Biol ; 39(3): 432-445, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30626205

RESUMO

Objective- Vascular smooth muscle cell (VSMC) transformation to an osteochondrogenic phenotype is an initial step toward arterial calcification, which is highly correlated with cardiovascular disease-related morbidity and mortality. TLR2 (Toll-like receptor 2) plays a pathogenic role in the development of vascular diseases, but its regulation in calcification of arteries and VSMCs remains unclear. We postulate that TLR2-mediated inflammation participates in mediating atherosclerotic arterial calcification and VSMC calcification. Approach and Results- We found that ApoE-/- Tlr2-/- genotype in mice suppressed high-fat diet-induced atherosclerotic plaques formation during initiation but progressively lost its preventative capacity, compared with ApoE-/- mice. However, TLR2 deficiency prohibited high-fat diet-induced advanced atherosclerotic calcification, chondrogenic metaplasia, and OPG (osteoprotegerin) downregulation in the calcified lesions. Incubation of VSMCs in a calcifying medium revealed that TLR2 agonists significantly increased VSMC calcification and chondrogenic differentiation. Furthermore, TLR2 deficiency suppressed TLR2 agonist-mediated VSMC chondrogenic differentiation and consequent calcification, which were triggered via the concerted actions of IL (interleukin)-6-mediated RANKL (receptor activator of nuclear factor κB ligand) induction and OPG suppression. Inhibition experiments with pharmacological inhibitors demonstrated that IL-6-mediated RANKL induction is signaled by p38 and ERK1/2 (extracellular signal-regulated kinase 1/2) pathways, whereas the OPG is suppressed via NF-κB (nuclear factor κB) dependent signaling mediated by ERK1/2. Conclusions- We concluded that on ligand binding, TLR2 activates p38 and ERK1/2 signaling to selectively modulate the upregulation of IL-6-mediated RANKL and downregulation of OPG. These signaling pathways act in concert to induce chondrogenic transdifferentiation of VSMCs, which in turn leads to vascular calcification during the pathogenesis of atherosclerosis.


Assuntos
Aterosclerose/patologia , Calcinose/metabolismo , Calcinose/patologia , Condrogênese/fisiologia , Interleucina-6/fisiologia , Sistema de Sinalização das MAP Quinases , Músculo Liso Vascular/citologia , Miócitos de Músculo Liso/metabolismo , Osteoprotegerina/biossíntese , Ligante RANK/biossíntese , Receptor 2 Toll-Like/fisiologia , Animais , Doenças da Aorta/etiologia , Doenças da Aorta/genética , Doenças da Aorta/patologia , Doenças da Aorta/prevenção & controle , Apolipoproteínas E/deficiência , Aterosclerose/etiologia , Aterosclerose/genética , Aterosclerose/prevenção & controle , Calcinose/genética , Células Cultivadas , Colesterol na Dieta/toxicidade , Dieta Hiperlipídica/efeitos adversos , Gorduras na Dieta/toxicidade , Regulação da Expressão Gênica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , NF-kappa B/metabolismo , Osteoprotegerina/genética , Ligante RANK/genética , Distribuição Aleatória
17.
Proc Natl Acad Sci U S A ; 116(5): 1569-1578, 2019 01 29.
Artigo em Inglês | MEDLINE | ID: mdl-30647113

RESUMO

Several recent studies have demonstrated that coculture of chondrocytes (CHs) with bone marrow-derived mesenchymal stem cells (MSCs) improves their chondrogenesis. This implies that intercellular communication dictates fate decisions in recipient cells and/or reprograms their metabolic state to support a differentiated function. While this coculture phenomenon is compelling, the differential chondroinductivity of zonal CHs on MSC cocultures, the nature of the molecular cargo, and their transport mechanisms remains undetermined. Here, we demonstrate that juvenile CHs in coculture with adult MSCs promote functional differentiation and improved matrix production. We further demonstrate that close proximity between the two cell types is a prerequisite for this response and that the outcome of this interaction improves viability, chondrogenesis, matrix formation, and homeostasis in the recipient MSCs. Furthermore, we visualized the transfer of intracellular contents from CHs to nearby MSCs and showed that inhibition of extracellular vesicle (EV) transfer blocks the synergistic effect of coculture, identifying EVs as the primary mode of communication in these cocultures. These findings will forward the development of therapeutic agents and more effective delivery systems to promote cartilage repair.


Assuntos
Cartilagem/citologia , Cartilagem/fisiologia , Condrócitos/citologia , Condrócitos/fisiologia , Vesículas Extracelulares/fisiologia , Células-Tronco Mesenquimais/citologia , Animais , Bovinos , Comunicação Celular/fisiologia , Diferenciação Celular/fisiologia , Células Cultivadas , Condrogênese/fisiologia , Técnicas de Cocultura/métodos , Matriz Extracelular/fisiologia , Engenharia Tecidual/métodos , Tecidos Suporte
18.
Cell Mol Life Sci ; 76(9): 1653-1680, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30689010

RESUMO

Adult stem cells, also termed as somatic stem cells, are undifferentiated cells, detected among differentiated cells in a tissue or an organ. Adult stem cells can differentiate toward lineage specific cell types of the tissue or organ in which they reside. They also have the ability to differentiate into mature cells of mesenchymal tissues, such as cartilage, fat and bone. Despite the fact that the balance has been comprehensively scrutinized between adipogenesis and osteogenesis and between chondrogenesis and osteogenesis, few reviews discuss the relationship between chondrogenesis and adipogenesis. In this review, the developmental and transcriptional crosstalk of chondrogenic and adipogenic lineages are briefly explored, followed by elucidation of signaling pathways and external factors guiding lineage determination between chondrogenic and adipogenic differentiation. An in-depth understanding of overlap and discrepancy between these two mesenchymal tissues in lineage differentiation would benefit regeneration of high-quality cartilage tissues and adipose tissues for clinical applications.


Assuntos
Adipogenia/fisiologia , Tecido Adiposo/citologia , Células-Tronco Adultas/citologia , Cartilagem Articular/citologia , Condrogênese/fisiologia , Células-Tronco Mesenquimais/citologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Linhagem da Célula , Senescência Celular/fisiologia , Humanos , Camundongos , Pessoa de Meia-Idade , Ratos , Transdução de Sinais/fisiologia , Adulto Jovem
19.
J Orthop Res ; 37(6): 1368-1375, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30095195

RESUMO

Ex vivo induction of chondrogenesis is a promising approach to improve upon the use of bone marrow mesenchymal stem cells (MSCs) for cartilage tissue engineering. This study evaluated the potential to induce chondrogenesis with days of culture in chondrogenic medium for MSCs encapsulated in self-assembling peptide hydrogel. To simulate the transition from preconditioning culture to implantation, MSCs were isolated from self-assembling peptide hydrogel into an individual cell suspension. Commitment to chondrogenesis was evaluated by seeding preconditioned MSCs into agarose and culturing in the absence of the chondrogenic cytokine transforming growth factor beta (TGFß). Positive controls consisted of undifferentiated MSCs seeded into agarose and cultured in medium containing TGFß. Three days of preconditioning was sufficient to produce chondrogenic MSCs that accumulated ∼75% more cartilaginous extracellular matrix than positive controls by day 17. However, gene expression of type X collagen was ∼65-fold higher than positive controls, which was attributed to the absence of TGFß. Potential induction of immunogenicity with preconditioning culture was indicated by expression of major histocompatibility complex class II (MHCII), which was nearly absence in undifferentiated MSCs, and ∼7% positive for preconditioned cells. These data demonstrate the potential to generate chondrogenic MSCs with days of self-assembling peptide hydrogel, and the ability to readily recover an individual cell suspension that is suited for injectable therapies. However, continued exposure to TGFß may be necessary to prevent hypertrophy indicated by type X collagen expression, while immunogenicity may be a concern for allogeneic applications. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 37:1368-1375, 2019.


Assuntos
Condrogênese/fisiologia , Células-Tronco Mesenquimais/fisiologia , Peptídeos/farmacologia , Engenharia Tecidual/métodos , Animais , Células da Medula Óssea/citologia , Técnicas de Cultura de Células , Colágeno Tipo II/análise , Cavalos , Hidrogéis , Células-Tronco Mesenquimais/citologia , Antígenos Thy-1/análise , Fatores de Tempo , Fator de Crescimento Transformador beta/farmacologia
20.
Cell Prolif ; 52(1): e12532, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30328655

RESUMO

OBJECTIVES: To investigate the role of zyxin-involved actin regulation in expression level of vinculin focal adhesion and collagen production of chondrocyte and its possible underlying mechanism. MATERIALS AND METHODS: Chondrocytes obtained from rabbit articular cartilage were used in this study. The expression of zyxin, actin and vinculin, as well as the extracellular matrix (ECM) protein collagen type I, II and X (COL I, II and X) of chondrocytes were compared between zyxin-knockdown group and negative control group, and between transforming growth factor-ß1 (TGF-ß1) treatment group and non-treatment group, respectively. RESULTS: Knockdown of zyxin increased the ratio of globular actin (G-actin) to filamentous actin (F-actin) of chondrocyte, which further inhibited expression of vinculin and chondrogenic marker COL II as well as hypertrophy marker COL X. On the other hand, chondrocytes treated with TGF-ß1 showed an enhanced expression of F-actin, and a lower expression of zyxin compared to non-treatment group. In response to TGF-ß1-induced actin polymerization, expression of vinculin and COL I was increased, while expression of COL II and aggrecan was decreased. CONCLUSIONS: These results demonstrate supporting evidence that in chondrocytes the level of zyxin is closely associated with the state of actin polymerization. In particular, the change of zyxin and F-actin parallels with the change of COL II and vinculin, respectively, indicating a major role of zyxin-actin interaction in the synthesis of collagen ECM and the remodelling of cytoskeleton-ECM adhesion.


Assuntos
Actinas/metabolismo , Condrócitos/citologia , Condrogênese/fisiologia , Adesões Focais/fisiologia , Vinculina/metabolismo , Zixina/metabolismo , Animais , Cartilagem Articular/citologia , Células Cultivadas , Condrócitos/metabolismo , Regulação da Expressão Gênica/genética , Interferência de RNA , RNA Interferente Pequeno/genética , Coelhos , Fator de Crescimento Transformador beta1/metabolismo , Zixina/genética
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