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1.
Int J Mol Sci ; 22(19)2021 Sep 24.
Artigo em Inglês | MEDLINE | ID: mdl-34638629

RESUMO

Tissue and organ failure has induced immense economic and healthcare concerns across the world. Tissue engineering is an interdisciplinary biomedical approach which aims to address the issues intrinsic to organ donation by providing an alternative strategy to tissue and organ transplantation. This review is specifically focused on cartilage tissue. Cartilage defects cannot readily regenerate, and thus research into tissue engineering approaches is relevant as a potential treatment option. Cells, scaffolds, and growth factors are three components that can be utilized to regenerate new tissue, and in particular recent advances in microparticle technology have excellent potential to revolutionize cartilage tissue regeneration. First, microspheres can be used for drug delivery by injecting them into the cartilage tissue or joint space to reduce pain and stimulate regeneration. They can also be used as controlled release systems within tissue engineering constructs. Additionally, microcarriers can act as a surface for stem cells or chondrocytes to adhere to and expand, generating large amounts of cells, which are necessary for clinically relevant cell therapies. Finally, a newer application of microparticles is to form them together into granular hydrogels to act as scaffolds for tissue engineering or to use in bioprinting. Tissue engineering has the potential to revolutionize the space of cartilage regeneration, but additional research is needed to allow for clinical translation. Microparticles are a key enabling technology in this regard.


Assuntos
Cartilagem Articular/citologia , Engenharia Tecidual/métodos , Animais , Doenças das Cartilagens/terapia , Diferenciação Celular/fisiologia , Condrócitos/citologia , Humanos , Microesferas
2.
Molecules ; 26(19)2021 Sep 22.
Artigo em Inglês | MEDLINE | ID: mdl-34641274

RESUMO

Articular cartilage (AC) damage is quite common, but due to AC's poor self-healing ability, the damage can easily develop into osteoarthritis (OA). To solve this problem, we developed a microsphere/hydrogel system that provides two growth factors that promote cartilage repair: transforming growth factor-ß3 (TGF-ß3) to enhance cartilage tissue formation and ghrelin synergy TGF-ß to significantly enhance the chondrogenic differentiation. The hydrogel and microspheres were characterized in vitro, and the biocompatibility of the system was verified. Double emulsion solvent extraction technology (w/o/w) is used to encapsulate TGF-ß3 and ghrelin into microspheres, and these microspheres are encapsulated in a hydrogel to continuously release TGF-ß3 and ghrelin. According to the chondrogenic differentiation ability of mesenchymal stem cells (MSCs) in vitro, the concentrations of the two growth factors were optimized to promote cartilage regeneration.


Assuntos
Cartilagem Articular/citologia , Grelina/farmacologia , Células-Tronco Mesenquimais/citologia , Fator de Crescimento Transformador beta3/farmacologia , Cartilagem Articular/efeitos dos fármacos , Cartilagem Articular/metabolismo , Técnicas de Cultura de Células , Células Cultivadas , Condrogênese/efeitos dos fármacos , Meios de Cultura/química , Glicosaminoglicanos/metabolismo , Humanos , Hidrogéis , Teste de Materiais , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/metabolismo , Microesferas , Medicina Regenerativa
3.
Int J Mol Sci ; 22(17)2021 Aug 26.
Artigo em Inglês | MEDLINE | ID: mdl-34502123

RESUMO

Cartilage is frequently damaged with a limited capacity for repair. Current treatment strategies are insufficient as they form fibrocartilage as opposed to hyaline cartilage, and do not prevent the progression of degenerative changes. There is increasing interest in the use of autologous mesenchymal stem cells (MSC) for tissue regeneration. MSCs that are used to treat articular cartilage defects must not only present a robust cartilaginous production capacity, but they also must not cause morbidity at the harvest site. In addition, they should be easy to isolate from the tissue and expand in culture without terminal differentiation. The source of MSCs is one of the most important factors that may affect treatment. The infrapatellar fat pad (IPFP) acts as an important reservoir for MSC and is located in the anterior compartment of the knee joint in the extra-synovial area. The IPFP is a rich source of MSCs, and in this review, we discuss studies that demonstrate that these cells have shown many advantages over other tissues in terms of ease of isolation, expansion, and chondrogenic differentiation. Future studies in articular cartilage repair strategies and suitable extraction as well as cell culture methods will extend the therapeutical application of IPFP-derived MSCs into additional orthopedic fields, such as osteoarthritis. This review provides the latest research concerning the use of IPFP-derived MSCs in the treatment of articular cartilage damage, providing critical information for the field to grow.


Assuntos
Tecido Adiposo/citologia , Regeneração Óssea , Cartilagem Articular/citologia , Cartilagem Articular/fisiologia , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Animais , Técnicas de Cultura de Células , Terapia Baseada em Transplante de Células e Tecidos , Humanos , Osteogênese , Cicatrização
4.
J Orthop Surg Res ; 16(1): 410, 2021 Jun 28.
Artigo em Inglês | MEDLINE | ID: mdl-34183035

RESUMO

BACKGROUND: Osteoarthritis (OA) is a joint disease characterized via destruction of cartilage. Chondrocyte damage is associated with cartilage destruction during OA. Long noncoding RNAs (lncRNAs) are implicated in the regulation of chondrocyte damage in OA progression. This study aims to investigate the role and underlying mechanism of lncRNA homeobox antisense intergenic RNA (HOTAIR) in OA chondrocyte injury. METHODS: Twenty-three OA patients and healthy controls without OA were recruited. Chondrocytes were isolated from OA cartilage tissues. HOTAIR, microRNA-107 (miR-107) and C-X-C motif chemokine ligand 12 (CXCL12) levels were measured by quantitative real-time polymerase chain reaction and western blot. Cell proliferation, apoptosis and extracellular matrix (ECM) degradation were measured using cell counting kit-8, flow cytometry and western blot. The target interaction was explored by bioinformatics, luciferase reporter and RNA immunoprecipitation assays. RESULTS: HOTAIR expression was enhanced, and miR-107 level was reduced in OA cartilage samples. HOTAIR overexpression inhibited cell proliferation, but induced cell apoptosis and ECM degradation in chondrocytes. HOTAIR knockdown caused an opposite effect. MiR-107 was sponged and inhibited via HOTAIR, and knockdown of miR-107 mitigated the effect of HOTAIR silence on chondrocyte injury. CXCL12 was targeted by miR-107. CXCL12 overexpression attenuated the roles of miR-107 overexpression or HOTAIR knockdown in the proliferation, apoptosis and ECM degradation. CXCL12 expression was decreased by HOTAIR silence, and restored by knockdown of miR-107. CONCLUSION: HOTAIR knockdown promoted chondrocyte proliferation, but inhibited cell apoptosis and ECM degradation in OA chondrocytes by regulating the miR-107/CXCL12 axis.


Assuntos
Quimiocina CXCL12/genética , Condrócitos/metabolismo , Genes Homeobox/genética , MicroRNAs/genética , Osteoartrite/genética , RNA Longo não Codificante/genética , Apoptose/genética , Cartilagem Articular/citologia , Estudos de Casos e Controles , Proliferação de Células/genética , Células Cultivadas , Matriz Extracelular/genética , Feminino , Técnicas de Silenciamento de Genes , Humanos , Masculino , Pessoa de Meia-Idade , Transdução de Sinais/genética
5.
J Mater Chem B ; 9(24): 4873-4894, 2021 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-34095925

RESUMO

The use of decellularized native allogenic or xenogenic cartilaginous extracellular matrix (ECM) biomaterials is widely expanding in the fields of tissue engineering and regenerative medicine. In this study, we aimed to develop an acellular, affordable, biodegradable, easily available goat conchal cartilaginous ECM derived scaffolding biomaterial for repair and regeneration of osteochondral defects in rabbits. Cartilages harvested from freshly collected goat ears were decellularized using chemical agents, namely, hypotonic-hypertonic (HH) buffer and Triton X-100 solution, separately. The morphologies and ultrastructure orientations of the decellularized cartilages remained unaltered in spite of complete cellular loss. Furthermore, when the acellular cartilaginous ECMs were cultured with murine mesenchymal stem cells (MSCs) (C3H10T1/2 cells), cellular infiltration and proliferation were thoroughly monitored using SEM, DAPI and FDA stained images, whereas the MTT assay proved the biocompatibility of the matrices. The increasing amounts of secreted ECM proteins (collagen and sGAG) indicated successful chondrogenic differentiation of the MSCs in the presence of the treated cartilage samples. In vivo biocompatibility studies showed no significant immune response or tissue rejection in the treated samples but tissue necrosis in control samples after 3 months. Upon implantation of the constructs in rabbits' osteochondral defects for 3 months, the histological and micro-CT evaluation revealed significant enhancement and regeneration of neocartilage and subchondral bony tissues. The IGF-1 loaded cartilaginous constructs showed comparatively better healing response after 3 months. Our results showed that decellularized xenogenic cartilaginous biomaterials preserved the bioactivity and integrity of the matrices that also favored in vitro stem cell proliferation and chondrogenic differentiation and enabled osteochondral regeneration, thus paving a new way for articular cartilage reconstruction.


Assuntos
Cartilagem Articular/citologia , Cartilagem Articular/fisiologia , Condrogênese , Matriz Extracelular/metabolismo , Engenharia Tecidual , Tecidos Suporte/química , Animais , Diferenciação Celular , Células-Tronco Mesenquimais/citologia , Camundongos , Coelhos
6.
Life Sci ; 280: 119728, 2021 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-34144057

RESUMO

AIMS: Progenitor cells-based regenerative strategy has shown promise to repair cartilage, an avascular tissue in which cells experience hypoxia. Hypoxia is known to improve the early chondrogenic differentiation of stem cells. Therefore, this study aimed to determine whether hypoxia preconditioning could be used to enhance the regenerative potential of the combination of buccal fat pad stem cells (BFPSCs) and bilayer chitosan-based hydrogel scaffold for articular cartilage repair. MATERIALS AND METHODS: Human BFPSCs were seeded on the bilayer chitosan-based hydrogel scaffolds in the culture medium. The viability and proliferation of cells on the scaffolds were monitored using scanning electron microscopy (SEM), MTT assay, and DAPI staining. Hypoxia preconditioned BFPSCs-seeded scaffolds were transplanted into rabbit articular cartilage knee defects for 12 weeks. The newly formed tissue was evaluated by cartilage-specific immunohistological analysis and histological staining. KEY FINDINGS: It was found that the chondrogenic differentiation and osteochondral conjunction in articular cartilage defect via BFPSCs-seeded bilayer scaffolds was enhanced by hypoxic preconditioning compared to a normoxic environment. SIGNIFICANCE: Based on our study, the integrity with subchondral bone in osteochondral defect was enhanced by BFPSCs on bilayer scaffold. Thus, this study provides evidence on the design of preconditioned cell-seeded bilayer hydrogels for articular cartilage regeneration.


Assuntos
Cartilagem Articular/citologia , Quitosana/química , Oxigênio/metabolismo , Transplante de Células-Tronco , Células-Tronco/citologia , Tecidos Suporte/química , Animais , Cartilagem Articular/fisiologia , Hipóxia Celular , Células Cultivadas , Condrogênese , Humanos , Masculino , Coelhos , Células-Tronco/metabolismo , Engenharia Tecidual/métodos
7.
Ann Rheum Dis ; 80(9): 1209-1219, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34039624

RESUMO

OBJECTIVES: Circular RNAs (circRNAs) have emerged as significant biological regulators. Herein, we aimed to elucidate the role of an unidentified circRNA (circPDE4B) that is reportedly downregulated in osteoarthritis (OA) tissues. METHODS: The effects of circPDE4B were explored in human and mouse chondrocytes in vitro. Specifically, RNA pull-down (RPD)-mass spectrometry analysis (MS), immunoprecipitation, glutathione-S-transferase (GST) pull-down, RNA immunoprecipitation and RPD assays were performed to verify the interactions between circPDE4B and the RIC8 guanine nucleotide exchange factor A (RIC8A)/midline 1 (MID1) complex. A mouse model of OA was also employed to confirm the role of circPDE4B in OA pathogenesis in vivo. RESULTS: circPDE4B regulates chondrocyte cell viability and extracellular matrix metabolism. Mechanistically, FUS RNA binding protein (FUS) was found to promote the splicing of circPDE4B, while downregulation of circPDE4B in OA is partially caused by upstream inhibition of FUS. Moreover, circPDE4B facilitates the association between RIC8A and MID1 by acting as a scaffold to promote RIC8A degradation through proteasomal degradation. Furthermore, ubiquitination of RIC8A at K415 abrogates RIC8A degradation. The circPDE4B-RIC8A axis was observed to play an important role in regulating downstream p38 mitogen-activated protein kinase (MAPK) signalling. Furthermore, delivery of a circPDE4B adeno-associated virus (AAV) abrogates the breakdown of cartilage matrix by medial meniscus destabilisation in mice, whereas a RIC8A AAV induces the opposite effect. CONCLUSION: This work highlights the function of the circPDE4B-RIC8A axis in OA joints, as well as its regulation of MAPK-p38, suggesting this axis as a potential therapeutic target for OA.


Assuntos
Cartilagem Articular/metabolismo , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/genética , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Osteoartrite/genética , RNA Circular , Regeneração/genética , Animais , Cartilagem Articular/citologia , Cartilagem Articular/fisiologia , Sobrevivência Celular/genética , Condrócitos/metabolismo , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/metabolismo , Humanos , Camundongos , Osteoartrite/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Processamento de Proteína Pós-Traducional , Proteólise , Proteína FUS de Ligação a RNA/genética , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação
8.
ACS Appl Mater Interfaces ; 13(20): 23369-23383, 2021 May 26.
Artigo em Inglês | MEDLINE | ID: mdl-33979130

RESUMO

Articular cartilage (AC) lesions are fairly common but remain an obstacle for clinicians and researchers due to their poor self-healing capacity. Recently, a promising therapy based on the recruitment of autologous mesenchymal stem cells (MSCs) has been developed for the regeneration of full-thickness cartilage defects in the knee joint. In this study, a 3D-bioprinted difunctional scaffold was developed based on aptamer HM69-mediated MSC-specific recruitment and growth factor-enhanced cell chondrogenesis. The aptamer, which can specifically recognize and recruit MSCs, was first chemically conjugated to the decellularized cartilage extracellular matrix and then mixed with gelatin methacrylate to form a photocrosslinkable bioink ready for 3D bioprinting. Together with the growth factor that promoted cell chondrogenic differentiation, the biodegradable polymer poly(ε-caprolactone) was further chosen to impart mechanical strength to the 3D bioprinted constructs. The difunctional scaffold specifically recruited MSCs, provided a favorable microenvironment for cell adhesion and proliferation, promoted chondrogenesis, and thus greatly improved cartilage repair in rabbit full-thickness defects. In conclusion, this study demonstrated that 3D bioprinting of difunctional scaffolds could be a promising strategy for in situ AC regeneration based on aptamer-directed cell recruitment and growth-factor-enhanced cell chondrogenesis.


Assuntos
Aptâmeros de Nucleotídeos/farmacologia , Bioimpressão , Cartilagem Articular , Condrogênese , Engenharia Tecidual/métodos , Animais , Cartilagem Articular/citologia , Cartilagem Articular/metabolismo , Células Cultivadas , Condrócitos/citologia , Condrócitos/efeitos dos fármacos , Condrogênese/efeitos dos fármacos , Condrogênese/fisiologia , Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Masculino , Impressão Tridimensional , Coelhos , Ratos , Tecidos Suporte/química
9.
Int J Mol Sci ; 22(7)2021 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-33916312

RESUMO

Cartilage is a non-innervated and non-vascularized tissue. It is composed of one main cell type, the chondrocyte, which governs homeostasis within the cartilage tissue, but has low metabolic activity. Articular cartilage undergoes substantial stresses that lead to chondral defects, and inevitably osteoarthritis (OA) due to the low intrinsic repair capacity of cartilage. OA remains an incurable degenerative disease. In this context, several dietary supplements have shown promising results, notably in the relief of OA symptoms. In this study, we investigated the effects of collagen hydrolysates derived from fish skin (Promerim®30 and Promerim®60) and fish cartilage (Promerim®40) on the phenotype and metabolism of human articular chondrocytes (HACs). First, we demonstrated the safety of Promerim® hydrolysates on HACs cultured in monolayers. Then we showed that, Promerim® hydrolysates can increase the HAC viability and proliferation, while decreasing HAC SA-ß-galactosidase activity. To evaluate the effect of Promerim® on a more relevant model of culture, HAC were cultured as organoids in the presence of Promerim® hydrolysates with or without IL-1ß to mimic an OA environment. In such conditions, Promerim® hydrolysates led to a decrease in the transcript levels of some proteases that play a major role in the development of OA, such as Htra1 and metalloproteinase-1. Promerim® hydrolysates downregulated HtrA1 protein expression. In contrast, the treatment of cartilage organoids with Promerim® hydrolysates increased the neosynthesis of type I collagen (Promerim®30, 40 and 60) and type II collagen isoforms (Promerim®30 and 40), the latter being the major characteristic component of the cartilage extracellular matrix. Altogether, our results demonstrate that the use of Promerim® hydrolysates hold promise as complementary dietary supplements in combination with the current classical treatments or as a preventive therapy to delay the occurrence of OA in humans.


Assuntos
Condrócitos/efeitos dos fármacos , Osteoartrite/tratamento farmacológico , Cartilagem Articular/citologia , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Senescência Celular/efeitos dos fármacos , Condrócitos/metabolismo , Avaliação Pré-Clínica de Medicamentos , Humanos , Cultura Primária de Células
10.
Aging (Albany NY) ; 13(8): 11433-11454, 2021 04 04.
Artigo em Inglês | MEDLINE | ID: mdl-33839696

RESUMO

Autologous chondrocyte implantation (ACI) is an effective method for treating chronic articular cartilage injury and degeneration; however, it requires large numbers of hyaline chondrocytes, and human hyaline chondrocytes often undergo dedifferentiation in vitro. Moreover, although long non-coding RNAs (lncRNAs) regulate gene expression in many pathological and physiological processes, their role in human hyaline chondrocyte dedifferentiation remains unclear. Here, we examined lncRNA and mRNA expression profiles in human hyaline chondrocyte dedifferentiation using microarray analysis. Among the many lncRNAs and mRNAs that showed differential expression, lncRNA AP001505.9 (ENST00000569966) was significantly downregulated in chondrocytes after dedifferentiation. We next performed gene ontology, pathway, and CNC (coding-non-coding gene co-expression) analyses to investigate potential regulatory mechanisms for AP001505.9. Pellet cultures were then used to redifferentiate dedifferentiated chondrocytes, and AP001505.9 expression was upregulated after redifferentiation. Finally, both in vitro and in vivo experiments demonstrated that AP001505.9 overexpression inhibited dedifferentiation of chondrocytes. This study characterizes lncRNA expression profiles in human hyaline chondrocyte dedifferentiation, thereby identifying new potential mechanisms of chondrocyte dedifferentiation worthy of further investigation.


Assuntos
Cartilagem Articular/crescimento & desenvolvimento , Desdiferenciação Celular/genética , Condrócitos/fisiologia , Condrogênese/genética , RNA Longo não Codificante/metabolismo , Adulto , Idoso , Cartilagem Articular/citologia , Cartilagem Articular/metabolismo , Células Cultivadas , Feminino , Perfilação da Expressão Gênica , Humanos , Masculino , Pessoa de Meia-Idade , Análise de Sequência com Séries de Oligonucleotídeos , Cultura Primária de Células
11.
Clin Immunol ; 227: 108718, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33819576

RESUMO

BACKGROUND: Osteoarthritis (OA) is a common inflammatory disease characterized by articular cartilage degeneration and injury. Circular RNAs (circRNAs) are widely involved in the development of human diseases, including OA. The objective of this study was to investigate the function and functional mechanism of circ_0001103 in OA. METHODS: Cell model of OA was established by treating chondrocytes with interleukin-1ß (IL-1ß). The expression of circ_0001103, miR-375 and sirtuin 1 (SIRT1) mRNA was measured using quantitative real-time polymerase chain reaction (qRT-PCR). Cell viability was assessed using cell counting kit-8 (CCK-8) assay. Cell apoptosis was determined using flow cytometry assay. The expression levels of inflammatory factors were quantified by qRT-PCR. The expression of extracellular matrix (ECM) metabolism-related markers, including Collagen Type II Alpha 1 Chain (COL2A1) and A disintegrin and metalloproteinase with thrombospondin motifs 4 (ADAMTS4), was detected by western blot. Predicted target relationship between miR-375 and circ_0001103 or SIRT1 by the bioinformatics tools was validated by dual-luciferase reporter assay and RNA immunoprecipitation (RIP) assay. RESULTS: Circ_0001103 was downregulated in OA tissues and IL-1ß-induced chondrocytes. Overexpression of circ_0001103 attenuated IL-1ß-induced chondrocyte apoptosis, inflammatory responses and ECM degradation. MiR-375 was targeted by circ_0001103, and miR-375 could bind to SIRT1. Circ_0001103 overexpression increased the expression of SIRT1 by suppressing miR-375. Rescue experiments suggested that miR-375 restoration reversed the effects of circ_0001103 overexpression, and SIRT1 knockdown overturned the effects of miR-375 inhibition. CONCLUSION: Circ_0001103 governed the miR-375/SIRT1 axis to ameliorate IL-1ß-induced chondrocyte injuries, implying that circ_0001103 was a promising biomarker in OA pathogenesis.


Assuntos
Apoptose/genética , Condrócitos/metabolismo , Inflamação/genética , MicroRNAs/genética , Osteoartrite do Joelho/genética , RNA Circular/genética , Sirtuína 1/genética , Cartilagem Articular/citologia , Cartilagem Articular/metabolismo , Estudos de Casos e Controles , Condrócitos/efeitos dos fármacos , Matriz Extracelular/metabolismo , Regulação da Expressão Gênica , Humanos , Inflamação/metabolismo , Interleucina-1beta/farmacologia , MicroRNAs/metabolismo , Osteoartrite do Joelho/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Sirtuína 1/metabolismo
12.
Knee ; 30: 51-62, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-33857741

RESUMO

BACKGROUND: Articular chondroprogenitors are a promising contender for cartilage repair due to their inherent nature which stands primed for chondrogenesis and minimal hypertrophic preponderance. Platelet rich plasma (PRP) has been extensively used for treating cartilage defects and osteoarthritis (OA), due to its chondro-inductive properties and abundant pool of growth factors. The aim of this study was to assess the efficacy of chondroprogenitors injected with PRP versus PRP alone in the healing of experimentally created early OA and osteochondral defects (OCD) in a rabbit model. METHODS: Adult New Zealand White male rabbits were used for cell and PRP isolation. Chondroprogenitors were isolated by fibronectin adhesion assay, labelled with iron oxide, characterized for surface markers, differential potential and expanded. PRP was isolated by double spin centrifugation using a TriCell kit. Study groups included (a) Monosodium iodoacetate induced early OA and (b) critical OCD. Following intervention (test arm: PRP+ chondroprogenitors and control arm: PRP), assessment was performed at 6- and 12-weeks which included histopathological examination and scoring (OARSI and Modified Wakitani score), immunohistochemistry analysis (Collagen type II and X) and synovial fluid S100A12 levels. RESULTS AND CONCLUSION: Comparable, evident healing was noticed in both test and control arms when the OA group samples were assessed at both time points. In the OCD group, PRP alone exhibited significantly better results than the test arm, although repair was notable in both interventions. Further evaluation of chondroprogenitors is required to assess their role as a standalone therapy and in combination with PRP to further cartilage regeneration.


Assuntos
Cartilagem Articular/fisiopatologia , Osteoartrite do Joelho/terapia , Plasma Rico em Plaquetas , Células-Tronco/citologia , Animais , Cartilagem Articular/citologia , Diferenciação Celular , Células Cultivadas , Condrogênese , Colágeno Tipo II/metabolismo , Modelos Animais de Doenças , Masculino , Osteoartrite do Joelho/induzido quimicamente , Coelhos , Proteína S100A12/metabolismo , Células-Tronco/fisiologia , Líquido Sinovial/metabolismo
13.
Aging (Albany NY) ; 13(8): 11646-11664, 2021 04 20.
Artigo em Inglês | MEDLINE | ID: mdl-33879632

RESUMO

OBJECTIVE: To investigate the heterogeneous responses of in vitro expanded chondrocytes, which were cultured in an interleukin (IL)-1ß -induced inflammatory environment. METHOD: Human articular chondrocytes were expanded, in vitro, for 13 days and treated with IL-1ß for 0, 24, and 48 h. Cells were collected and subjected to single-cell RNA sequencing. Multiple bioinformatics tools were used to determine the signatures that define chondrocyte physiology. RESULTS: Two major cell clusters with distinct expression patterns were identified at the initial phase and were with heterogeneous variation that coincides with inflammation progress. They transformed into two terminal cell clusters one of which exhibited OA-phenotype and proinflammatory characteristics through two paths, "response-to-inflammation" and "atypical response-to-inflammation", respectively. The involved cell clusters exhibited intrinsic relationship with cell types within native cartilage from OA patients. Genes controlling cell transformation to OA-phenotype were relating to the tumor necrosis factor (TNF) signaling pathway via NFKB, up-regulated KRAS signaling and the IL2/STAT5 signaling pathway and pathways relating to apoptosis and reactive oxygen species. CONCLUSION: The in vitro expanded chondrocytes under IL-1ß-induced inflammatory progression behave heterogeneously. One of the initial cell clusters could transform into a proinflammatory subpopulation through a termed response-to-inflammation path, which may serve as the core target to alleviate OA progression.


Assuntos
Condrócitos/patologia , Regulação da Expressão Gênica/imunologia , Osteoartrite/imunologia , Transdução de Sinais/genética , Cartilagem Articular/citologia , Células Cultivadas , Criança , Condrócitos/imunologia , Biologia Computacional , Meios de Cultura/metabolismo , Humanos , Interleucina-1beta/metabolismo , Osteoartrite/genética , Osteoartrite/patologia , Cultura Primária de Células , RNA-Seq , Transdução de Sinais/imunologia , Análise de Célula Única
14.
Int J Mol Sci ; 22(7)2021 Mar 31.
Artigo em Inglês | MEDLINE | ID: mdl-33807323

RESUMO

A continuing challenge in cartilage tissue engineering for cartilage regeneration is the creation of a suitable synthetic microenvironment for chondrocytes and tissue regeneration. The aim of this study was to develop a highly tunable hybrid scaffold based on a silk fibroin matrix (SM) and a hyaluronic acid (HA) hydrogel. Human articular chondrocytes were embedded in a porous 3-dimensional SM, before infiltration with tyramine modified HA hydrogel. Scaffolds were cultured in chondropermissive medium with and without TGF-ß1. Cell viability and cell distribution were assessed using CellTiter-Blue assay and Live/Dead staining. Chondrogenic marker expression was detected using qPCR. Biosynthesis of matrix compounds was analyzed by dimethylmethylene blue assay and immuno-histology. Differences in biomaterial stiffness and stress relaxation were characterized using a one-step unconfined compression test. Cell morphology was investigated by scanning electron microscopy. Hybrid scaffold revealed superior chondro-inductive and biomechanical properties compared to sole SM. The presence of HA and TGF-ß1 increased chondrogenic marker gene expression and matrix deposition. Hybrid scaffolds offer cytocompatible and highly tunable properties as cell-carrier systems, as well as favorable biomechanical properties.


Assuntos
Cartilagem Articular/metabolismo , Fibroínas/farmacologia , Engenharia Tecidual/métodos , Idoso , Materiais Biocompatíveis/metabolismo , Cartilagem/citologia , Cartilagem/metabolismo , Cartilagem Articular/citologia , Sobrevivência Celular/fisiologia , Células Cultivadas , Condrócitos/metabolismo , Condrogênese , Fibroínas/metabolismo , Humanos , Ácido Hialurônico/farmacologia , Hidrogéis/metabolismo , Hidrogéis/farmacologia , Pessoa de Meia-Idade , Porosidade , Seda/metabolismo , Tecidos Suporte/química
15.
Proc Natl Acad Sci U S A ; 118(13)2021 03 30.
Artigo em Inglês | MEDLINE | ID: mdl-33758095

RESUMO

Osteoarthritis (OA) is a painful and debilitating condition of synovial joints without any disease-modifying therapies [A. M. Valdes, T. D. Spector, Nat. Rev. Rheumatol. 7, 23-32 (2011)]. We previously identified mechanosensitive PIEZO channels, PIEZO1 and PIEZO2, both expressed in articular cartilage, to function in chondrocyte mechanotransduction in response to injury [W. Lee et al., Proc. Natl. Acad. Sci. U.S.A. 111, E5114-E5122 (2014); W. Lee, F. Guilak, W. Liedtke, Curr. Top. Membr. 79, 263-273 (2017)]. We therefore asked whether interleukin-1-mediated inflammatory signaling, as occurs in OA, influences Piezo gene expression and channel function, thus indicative of maladaptive reprogramming that can be rationally targeted. Primary porcine chondrocyte culture and human osteoarthritic cartilage tissue were studied. We found that interleukin-1α (IL-1α) up-regulated Piezo1 in porcine chondrocytes. Piezo1 expression was significantly increased in human osteoarthritic cartilage. Increased Piezo1 expression in chondrocytes resulted in a feed-forward pathomechanism whereby increased function of Piezo1 induced excess intracellular Ca2+ at baseline and in response to mechanical deformation. Elevated resting state Ca2+ in turn rarefied the F-actin cytoskeleton and amplified mechanically induced deformation microtrauma. As intracellular substrates of this OA-related inflammatory pathomechanism, in porcine articular chondrocytes exposed to IL-1α, we discovered that enhanced Piezo1 expression depended on p38 MAP-kinase and transcription factors HNF4 and ATF2/CREBP1. CREBP1 directly bound to the proximal PIEZO1 gene promoter. Taken together, these signaling and genetic reprogramming events represent a detrimental Ca2+-driven feed-forward mechanism that can be rationally targeted to stem the progression of OA.


Assuntos
Condrócitos/metabolismo , Interleucina-1alfa/metabolismo , Canais Iônicos/genética , Mecanotransdução Celular/imunologia , Osteoartrite/imunologia , Fator 2 Ativador da Transcrição/metabolismo , Animais , Cálcio/metabolismo , Cartilagem Articular/citologia , Cartilagem Articular/imunologia , Cartilagem Articular/patologia , Células Cultivadas , Condrócitos/imunologia , Feminino , Técnicas de Silenciamento de Genes , Humanos , Canais Iônicos/metabolismo , Mecanotransdução Celular/genética , Osteoartrite/genética , Osteoartrite/patologia , Cultura Primária de Células , Regiões Promotoras Genéticas/genética , Sus scrofa , Regulação para Cima/imunologia
16.
J Cell Mol Med ; 25(9): 4204-4215, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33768729

RESUMO

This study aimed to investigate the ability of CD146+ subset of ADSCs to repair cartilage defects. In this study, we prepared CD146+ liposome magnetic beads (CD146+ LMB) to isolate CD146+ ADSCs. The cells were induced for chondrogenic differentiation and verified by cartilage-specific mRNA and protein expression. Then a mouse model of cartilage defect was constructed and treated by filling the induced cartilage cells into the damaged joint, to evaluate the function of such cells in the cartilage microenvironment. Our results demonstrated that the CD146+ LMBs we prepared were uniform, small and highly stable, and cell experiments showed that the CD146+ LMB has low cytotoxicity to the ADSCs. ADSCs isolated with CD146+ LMB were all CD146+ , CD105+ , CD166+ and CD73+ . After chondrogenic induction, the cells showed significantly increased expression of cartilage markers Sox9, collagen Ⅱ and aggrecan at protein level and significantly increased Sox9, collagen Ⅱ and aggrecan at mRNA level, and the protein expression and mRNA expression of CD146+ ADSCs group were higher than those of ADSCs group. The CD146+ ADSCs group showed superior tissue repair ability than the ADSCs group and blank control group in the animal experiment, as judged by gross observation, histological observation and histological scoring. The above results proved that CD146+ LMB can successfully isolate the CD146+ ADSCs, and after chondrogenic induction, these cells successfully promoted repair of articular cartilage defects, which may be a new direction of tissue engineering.


Assuntos
Doenças das Cartilagens/terapia , Cartilagem Articular/citologia , Diferenciação Celular , Lipossomos/química , Transplante de Células-Tronco Mesenquimais/métodos , Células-Tronco Mesenquimais/citologia , Engenharia Tecidual , Animais , Doenças das Cartilagens/etiologia , Doenças das Cartilagens/patologia , Fenômenos Magnéticos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Tecidos Suporte/química
17.
Knee ; 29: 418-425, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33721626

RESUMO

BACKGROUND: Cell based therapy in cartilage repair predominantly involves the use of chondrocytes and mesenchymal stromal cells (MSC). Co-culture systems, due to their probable synergistic effect on enhancement of functional chondrogenesis and reduction in terminal differentiation have also been attempted. Chondroprogenitors, derived from articular cartilage and regarded as MSCs, have recently garnered interest for consideration in cartilage regeneration to overcome limitations associated with use of conventional cell types. The aim of this study was to assess whetherco-culturing bone marrow (BM)-MSCs and chondroprogenitors at different ratios would yield superior results in terms of surface marker expression, gene expression and chondrogenic potential. METHODS: Human BM-MSCs and chondroprogenitors obtained from three osteoarthritic knee joints and subjected to monolayer expansion and pellet cultures (10,000 cells/cm2) as five test groups containing either monocultures or co-cultures (MSC: chondroprogenitors) at three different ratios (75:25, 50:50 and 25:75) were utilized. RESULTS: Data analysis revealed that all groups exhibited a high expression of CD166, CD29 and CD49e. With regard to gene expression, high expression of SOX9, Aggrecan and Collagen type I; a moderate expression of Collagen type X and RUNX2; with a low expression of Collagen type II was seen. Analysis of pellet culture revealed that chondroprogenitor monoculture and chondroprogenitor dominant coculture, exhibited a subjectively larger pellet size with higher deposition of Collagen type II and glycosaminoglycan. CONCLUSION: In conclusion, this study is suggestive of chondroprogenitor monoculture superiority over MSCs, either in isolation or in a coculture system and proposes further analysis of chondroprogenitors for cartilage repair.


Assuntos
Cartilagem Articular/citologia , Técnicas de Cultura de Células/métodos , Células-Tronco Mesenquimais/citologia , Osteoartrite do Joelho/patologia , Agrecanas/genética , Agrecanas/metabolismo , Antígenos CD/genética , Antígenos CD/metabolismo , Biomarcadores/metabolismo , Cartilagem Articular/fisiologia , Diferenciação Celular , Condrogênese/genética , Técnicas de Cocultura , Colágeno Tipo I/metabolismo , Colágeno Tipo II/genética , Colágeno Tipo II/metabolismo , Feminino , Expressão Gênica , Humanos , Articulação do Joelho/citologia , Masculino , Células-Tronco Mesenquimais/fisiologia , Pessoa de Meia-Idade
18.
Knee ; 29: 365-373, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33690017

RESUMO

BACKGROUND: Chondrocyte transplantation to address cartilage damage is an established solution. Because hyaluronic acid (HA) is an essential component for homeostasis of the cartilage, in order to arrive at methodologies to utilize its advantages in cell-based therapies, we compared the HA retention capability of a thermoreversible gelation polymer scaffold-based environment (3D-TGP) with conventional in vitro cell culture methodologies. METHODS: Chondrocytes derived from osteoarthritis-affected knee joint cartilage of elderly patients were used and accomplished in three phases. In Phase I, the levels of HA secreted by chondrocytes were measured in culture supernatant. In Phase II, retention capacity of externally added HA was quantified indirectly by measuring the HA released in culture supernatant, and in Phase III, the expression of CD44 on cells was analysed by immunohistochemistry. RESULTS: In Phase I, the average HA in the 3D supernatant was 3% that of 2D. In phase II, 80% of externally added HA was detected in the 2D on day 7, while in 3D-TGP, only 0.1% was released until day 21. In Phase III, 2D yielded individual cells that started degenerating from the third week; in 3D-TGP cells grew for a longer duration, formed a tissue-like architecture with extracellular matrix with significantly intense staining of CD44 than 2D. CONCLUSION: The capability of the 3D-TGP culture environment to retain HA and support chondrocytes to grow with a tissue-like architecture expressing higher HA content is considered advantageous as it serves as an in vitro culture platform that enables tissue engineering of cartilage tissue with native hyaline phenotype and higher HA expression. The in vitro environment being conducive, based on this data, we also recommend that the TGP be tried as an encapsulation material in clinical studies of chondrocyte implantation for optimal clinical outcome.


Assuntos
Cartilagem Articular/citologia , Condrócitos/metabolismo , Ácido Hialurônico/metabolismo , Osteoartrite do Joelho/metabolismo , Tecidos Suporte , Idoso , Células Cultivadas , Humanos , Receptores de Hialuronatos/metabolismo , Imuno-Histoquímica
19.
Int Immunopharmacol ; 94: 107489, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33774357

RESUMO

The objective of this study is to investigate the role of IL-38 in osteoarthritis (OA). IL-38 levels in serum and synovial fluid (SF) of patients with OA were examined to identify the correlation between IL-38 expression and OA activity and to determine its anti-inflammatory effects in IL-1ß-induced chondrocytes. A total of 75 patients with OA who underwent joint replacement surgery and 25 age- and sex-matched healthy volunteers were recruited. The levels of IL-38 in serum and SF are shown to be significant elevated in OA patients compared with that of healthy controls. Serum and SF IL-38 levels of OA patients are positively correlated with Kellgren-Lawrence (K-L) grades 2 to 3, as well as with pro-inflammatory cytokines IL-6, IL-23, and TNF-α, but are negatively correlated with the anti-inflammatory cytokine IL-10 in K-L grades 3 to 4. Furthermore, overexpression of IL-38 in vitro is shown to attenuate the expression of pro-inflammatory cytokines such as COX-2, IL-6, IL-8, IL-36Ra, IL-36α/ß/γ, iNOS, and TNF-α, as well as matrix degrading enzymes such as MMP3, MMP13, and ADAMTS5, and apoptosis-related indicators Bax/Bcl-2, cleaved caspase 3/pro-caspase 3, and cleaved caspase 9/pro-caspase 9. IL-38 overexpression also reduces expression of the signaling proteins p-p38, p-p65, p-JNK, and RhoA significantly. Taken together, our results show that expression of IL-38 is increased in OA tissues and OA rat chondrocytes, and is positively correlated with early disease activity. This increased IL-38 expression lead to the inactivation of MAPK, NF-κB, JNK, and RhoA signaling pathways, which might have impletion on OA chondrocytes apoptosis, degradation and inflammatory effect. Thus, IL-38 probably serves as a novel therapeutic target for the treatment of OA.


Assuntos
Condrócitos/imunologia , Citocinas/imunologia , Osteoartrite/imunologia , Idoso , Animais , Cartilagem Articular/citologia , Citocinas/sangue , Citocinas/genética , Feminino , Articulação do Quadril , Humanos , Articulação do Joelho , Masculino , Pessoa de Meia-Idade , Proteínas Quinases Ativadas por Mitógeno/imunologia , NF-kappa B/imunologia , Osteoartrite/sangue , Ratos Sprague-Dawley , Transdução de Sinais , Proteína rhoA de Ligação ao GTP/imunologia
20.
Rheumatology (Oxford) ; 60(7): 3048-3057, 2021 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-33630038

RESUMO

The circadian clock is a specialized cell signalling pathway present in all cells. Loss of clock function leads to tissue degeneration and premature ageing in animal models demonstrating the fundamental importance of clocks for cell, tissue and organism health. There is now considerable evidence that the chondrocyte circadian clock is altered in OA. The purpose of this review is to summarize current knowledge regarding the nature of the change in the chondrocyte clock in OA and the implications of this change for disease development. Expression of the core clock component, BMAL1, has consistently been shown to be lower in OA chondrocytes. This may contribute to changes in chondrocyte differentiation and extracellular matrix turnover in disease. Circadian clocks are highly responsive to environmental factors. Mechanical loading, diet, inflammation and oxidative insult can all influence clock function. These factors may contribute to causing the change in the chondrocyte clock in OA.


Assuntos
Cartilagem Articular/metabolismo , Condrócitos/metabolismo , Relógios Circadianos , Osteoartrite/metabolismo , Fatores de Transcrição ARNTL/metabolismo , Proteínas CLOCK/metabolismo , Cartilagem Articular/citologia , Cartilagem Articular/fisiopatologia , Diferenciação Celular , Proliferação de Células , Sobrevivência Celular , Condrogênese , Criptocromos/metabolismo , Dieta , Matriz Extracelular/metabolismo , Humanos , Inflamação , Osteoartrite/fisiopatologia , Estresse Oxidativo , Proteínas Circadianas Period/metabolismo , Núcleo Supraquiasmático/metabolismo , Suporte de Carga
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