Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 68
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
Int J Mol Sci ; 22(15)2021 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-34360947

RESUMO

The distribution of differential extracellular matrix (ECM) in the lateral and medial menisci can contribute to knee instability, and changes in the meniscus tissue can lead to joint disease. Thus, deep proteomic identification of the lateral and medial meniscus cartilage is expected to provide important information for treatment and diagnosis of various knee joint diseases. We investigated the proteomic profiles of 12 lateral/medial meniscus pairs obtained from excess tissue of osteoarthritis patients who underwent knee arthroscopy surgery using mass spectrometry-based techniques and measured 75 ECM protein levels in the lesions using a multiple reaction monitoring (MRM) assay we developed. A total of 906 meniscus proteins with a 1% false discovery rate (FDR) was identified through a tandem mass tag (TMT) analysis showing that the lateral and medial menisci had similar protein expression profiles. A total of 131 ECM-related proteins was included in meniscus tissues such as collagen, fibronectin, and laminin. Our data showed that 14 ECM protein levels were differentially expressed in lateral and medial lesions (p < 0.05). We present the proteomic characterization of meniscal tissue with mass spectrometry-based comparative proteomic analysis and developed an MRM-based assay of ECM proteins correlated with tissue regeneration. The mass spectrometry dataset has been deposited to the MassIVE repository with the dataset identifier MSV000087753.


Assuntos
Proteínas da Matriz Extracelular/metabolismo , Menisco/metabolismo , Osteoartrite/metabolismo , Proteoma/metabolismo , Idoso , Idoso de 80 Anos ou mais , Proteínas da Matriz Extracelular/química , Feminino , Humanos , Masculino , Proteoma/química
2.
Int J Mol Sci ; 22(13)2021 Jun 27.
Artigo em Inglês | MEDLINE | ID: mdl-34199089

RESUMO

The meniscus possesses low self-healing properties. A perfect regenerative technique for this tissue has not yet been developed. This work aims to evaluate the role of hypoxia in meniscal development in vitro. Menisci from neonatal pigs (day 0) were harvested and cultured under two different atmospheric conditions: hypoxia (1% O2) and normoxia (21% O2) for up to 14 days. Samples were analysed at 0, 7 and 14 days by histochemical (Safranin-O staining), immunofluorescence and RT-PCR (in both methods for SOX-9, HIF-1α, collagen I and II), and biochemical (DNA, GAGs, DNA/GAGs ratio) techniques to record any possible differences in the maturation of meniscal cells. Safranin-O staining showed increments in matrix deposition and round-shape "fibro-chondrocytic" cells in hypoxia-cultured menisci compared with controls under normal atmospheric conditions. The same maturation shifting was observed by immunofluorescence and RT-PCR analysis: SOX-9 and collagen II increased from day zero up to 14 days under a hypoxic environment. An increment of DNA/GAGs ratio typical of mature meniscal tissue (characterized by fewer cells and more GAGs) was observed by biochemical analysis. This study shows that hypoxia can be considered as a booster to achieve meniscal cell maturation, and opens new opportunities in the field of meniscus tissue engineering.


Assuntos
Diferenciação Celular , Hipóxia/metabolismo , Menisco/citologia , Menisco/metabolismo , Animais , Biomarcadores , Células Cultivadas , Condrócitos/metabolismo , Expressão Gênica , Glicosaminoglicanos/metabolismo , Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Imuno-Histoquímica , Suínos , Engenharia Tecidual/métodos
3.
FASEB J ; 35(8): e21779, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34314047

RESUMO

The incredible mechanical strength and durability of mature fibrous tissues and their extremely limited turnover and regenerative capacity underscores the importance of proper matrix assembly during early postnatal growth. In tissues with composite extracellular matrix (ECM) structures, such as the adult knee meniscus, fibrous (Collagen-I rich), and cartilaginous (Collagen-II, proteoglycan-rich) matrix components are regionally segregated to the outer and inner portions of the tissue, respectively. While this spatial variation in composition is appreciated to be functionally important for resisting complex mechanical loads associated with gait, the establishment of these specialized zones is poorly understood. To address this issue, the following study tracked the growth of the murine meniscus from its embryonic formation through its first month of growth, encompassing the critical time-window during which animals begin to ambulate and weight bear. Using histological analysis, region specific high-throughput qPCR, and Col-1, and Col-2 fluorescent reporter mice, we found that matrix and cellular features defining specific tissue zones were already present at birth, before continuous weight-bearing had occurred. These differences in meniscus zones were further refined with postnatal growth and maturation, resulting in specialization of mature tissue regions. Taken together, this work establishes a detailed timeline of the concurrent spatiotemporal changes that occur at both the cellular and matrix level throughout meniscus maturation. The findings of this study provide a framework for investigating the reciprocal feedback between cells and their evolving microenvironments during assembly of a mechanically robust fibrocartilage tissue, thus providing insight into mechanisms of tissue degeneration and effective regenerative strategies.


Assuntos
Cartilagem , Colágeno/metabolismo , Matriz Extracelular/metabolismo , Menisco , Animais , Cartilagem/embriologia , Cartilagem/crescimento & desenvolvimento , Cartilagem/metabolismo , Diferenciação Celular , Proliferação de Células , Menisco/embriologia , Menisco/crescimento & desenvolvimento , Menisco/metabolismo , Camundongos , Camundongos Transgênicos
4.
Int J Mol Sci ; 22(7)2021 Mar 26.
Artigo em Inglês | MEDLINE | ID: mdl-33810460

RESUMO

Osteoarthritis (OA) is the most common type of arthritis and is associated with wear and tear, aging, and inflammation. Previous studies revealed that several antimicrobial peptides are up-regulated in the knee synovium of patients with OA or rheumatoid arthritis. Here, we investigated the functional effects of cathelicidin-related antimicrobial peptide (Cramp) on OA pathogenesis. We found that Cramp is highly induced by IL-1ß via the NF-κB signaling pathway in mouse primary chondrocytes. Elevated Cramp was also detected in the cartilage and synovium of mice suffering from OA cartilage destruction. The treatment of chondrocytes with Cramp stimulated the expression of catabolic factors, and the knockdown of Cramp by small interfering RNA reduced chondrocyte catabolism mediated by IL-1ß. Moreover, intra-articular injection of Cramp into mouse knee joints at a low dose accelerated traumatic OA progression. At high doses, Cramp affected meniscal ossification and tears, leading to cartilage degeneration. These findings demonstrate that Cramp is associated with OA pathophysiology.


Assuntos
Peptídeos Catiônicos Antimicrobianos/efeitos adversos , Osteoartrite do Joelho/fisiopatologia , Animais , Peptídeos Catiônicos Antimicrobianos/administração & dosagem , Cartilagem/metabolismo , Cartilagem Articular/metabolismo , Condrócitos/metabolismo , Modelos Animais de Doenças , Progressão da Doença , Injeções Intra-Articulares , Interleucina-1beta/metabolismo , Articulação do Joelho/efeitos dos fármacos , Articulação do Joelho/fisiopatologia , Masculino , Menisco/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , NF-kappa B/metabolismo , Osteoartrite do Joelho/induzido quimicamente , RNA Interferente Pequeno/metabolismo , Transdução de Sinais/efeitos dos fármacos , Membrana Sinovial/metabolismo
5.
J Orthop Surg (Hong Kong) ; 29(1): 23094990211000168, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33729061

RESUMO

PURPOSE: This study aimed to investigate how fibroblastic and chondrocytic properties of human meniscal fibrochondrocytes are affected in culture conditions according to the type of meniscal pathology and localization, and to provide basic information for tissue-engineering studies. METHODS: Primary fibrochondrocyte cultures were prepared from meniscus samples of patients who had either traumatic tear or degeneration due to osteoarthritis. Cultures were compared in terms of mRNA expression levels of COL1A1, COL2A1, COMP1, HIF1A, HIF2A, and SOX9 and secreted total collagen and sulfated sGAG levels according to the type of meniscal pathology, anatomical localization, and the number of subcultures. RESULTS: mRNA expression levels of COL1A1, COMP1, HIF1A, HIF2A, and SOX9 were found to be increased in subsequent subcultures in all specimens. COL1A1 mRNA expression levels of both lateral and medial menisci of patients with traumatic tear were significantly higher than in patients with degenerative pathology, indicating a more fibroblastic character. P1 subculture of lateral and P3 or further subculture of medial meniscus showed more fibroblastic characteristics in patients with degenerative pathology. Furthermore, in patients with degenerative pathology, the subcultures of the lateral meniscus (especially on the inner part) presented more chondrocytic characteristics than did those of medial meniscus. CONCLUSIONS: The mRNA expression levels of the cultures showed significant differences according to the anatomical localization and pathology of the meniscus, indicating distinct chondrocytic and fibroblastic features. This fundamental knowledge would help researchers to choose more efficient cell sources for cell-seeding of a meniscus scaffold, and to generate a construct resembling the original meniscus tissue.


Assuntos
Fibrocartilagem , Articulações/lesões , Menisco , Osteoartrite/patologia , Transcriptoma , Adolescente , Adulto , Idoso , Células Cultivadas , Condrócitos/citologia , Condrócitos/metabolismo , Condrócitos/patologia , Feminino , Fibrocartilagem/citologia , Fibrocartilagem/metabolismo , Fibrocartilagem/patologia , Perfilação da Expressão Gênica , Humanos , Articulações/metabolismo , Articulações/patologia , Masculino , Menisco/citologia , Menisco/lesões , Menisco/metabolismo , Menisco/patologia , Pessoa de Meia-Idade , Osteoartrite/genética , Osteoartrite/metabolismo , Cultura Primária de Células/métodos , Ruptura/genética , Ruptura/metabolismo , Ruptura/patologia , Adulto Jovem
6.
PLoS One ; 16(3): e0248292, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33690647

RESUMO

Low oxygen and mechanical loading may play roles in regulating the fibrocartilaginous phenotype of the human inner meniscus, but their combination in engineered tissues remains unstudied. Here, we investigated how continuous low oxygen ("hypoxia") combined with dynamic compression would affect the fibrocartilaginous "inner meniscus-like" matrix-forming phenotype of human meniscus fibrochondrocytes (MFCs) in a porous type I collagen scaffold. Freshly-seeded MFC scaffolds were cultured for 4 weeks in either 3 or 20% O2 or pre-cultured for 2 weeks in 3% O2 and then dynamically compressed for 2 weeks (10% strain, 1 Hz, 1 h/day, 5 days/week), all with or without TGF-ß3 supplementation. TGF-ß3 supplementation was found necessary to induce matrix formation by MFCs in the collagen scaffold regardless of oxygen tension and application of the dynamic compression loading regime. Neither hypoxia under static culture nor hypoxia combined with dynamic compression had significant effects on expression of specific protein and mRNA markers for the fibrocartilaginous matrix-forming phenotype. Mechanical properties significantly increased over the two-week loading period but were not different between static and dynamic-loaded tissues after the loading period. These findings indicate that 3% O2 applied immediately after scaffold seeding and dynamic compression to 10% strain do not affect the fibrocartilaginous matrix-forming phenotype of human MFCs in this type I collagen scaffold. It is possible that a delayed hypoxia treatment and an optimized pre-culture period and loading regime combination would have led to different outcomes.


Assuntos
Condrócitos , Matriz Extracelular/metabolismo , Menisco , Estresse Mecânico , Engenharia Tecidual , Adulto , Hipóxia Celular , Células Cultivadas , Condrócitos/citologia , Condrócitos/metabolismo , Humanos , Masculino , Menisco/citologia , Menisco/metabolismo
7.
Sci Rep ; 10(1): 21469, 2020 12 08.
Artigo em Inglês | MEDLINE | ID: mdl-33293598

RESUMO

Meniscal degeneration is a very common condition in elderly individuals, but the underlying mechanisms of its occurrence are not completely clear. This study examines the molecular mechanisms of meniscal degeneration. The anterior cruciate ligament (ACL) and lateral collateral ligament (LCL) of the right rear limbs of seven Wuzhishan mini-pigs were resected (meniscal degeneration group), and the left rear legs were sham-operated (control group). After 6 months, samples were taken for gene chip analysis, including differentially expressed gene (DEG) analysis, gene ontology (GO) analysis, clustering analysis, and pathway analysis. The selected 12 DEGs were validated by real time reverse transcription-polymerase chain reaction (RT-PCR). The two groups showed specific and highly clustered DEGs. A total of 893 DEGs were found, in which 537 are upregulated, and 356 are downregulated. The GO analysis showed that the significantly affected biological processes include nitric oxide metabolic process, male sex differentiation, and mesenchymal morphogenesis, the significantly affected cellular components include the endoplasmic reticulum membrane, and the significantly affected molecular functions include transition metal ion binding and iron ion binding. The pathway analysis showed that the significantly affected pathways include type II diabetes mellitus, inflammatory mediator regulation of TRP channels, and AMPK signaling pathway. The results of RT-PCR indicate that the microarray data accurately reflects the gene expression patterns. These findings indicate that several molecular mechanisms are involved in the development of meniscal degeneration, thus improving our understanding of meniscal degeneration and provide molecular therapeutic targets in the future.


Assuntos
Doenças das Cartilagens/genética , Menisco/patologia , Transcriptoma , Animais , Doenças das Cartilagens/metabolismo , Doenças das Cartilagens/patologia , Modelos Animais de Doenças , Feminino , Perfilação da Expressão Gênica , Ontologia Genética , Masculino , Menisco/metabolismo , Redes e Vias Metabólicas , Suínos , Porco Miniatura
8.
Sci Rep ; 10(1): 21658, 2020 12 10.
Artigo em Inglês | MEDLINE | ID: mdl-33303888

RESUMO

Meniscus degeneration is closely related to the progression of knee osteoarthritis (OA). However, there is currently a lack of quantitative and objective metrics to assess OA meniscal cell phenotypes. In this study we investigated the phenotypic markers and chondrogenic potency of avascular and vascular meniscal cells and chondrocytes from medial OA knee joints (n = 10). Flow cytometry results showed that a significantly greater percentage of meniscal cells were positive for CD49b, CD49c and CD166 compared to donor-matched chondrocytes after 14 days in monolayer culture. The integrins, CD49b and CD29, were expressed at a significantly higher level on avascular meniscal cells derived from tissues with a more degenerated inner border than non-degenerate menisci, suggesting that the integrin family may play an important role in meniscus OA pathology. Collagen fibres arranged in a "tree-like" formation within the meniscus appeared to have less blood vessels associated with them in the vascular region of the most degenerate menisci, which may indicate that such structures are involved in the pathological process. We have demonstrated that meniscal cells derived from the lateral meniscus in medial OA patients have chondrogenic capacity in vitro and hence could represent a potential cell source to consider for meniscus tissue engineering.


Assuntos
Diferenciação Celular/efeitos da radiação , Condrócitos/fisiologia , Condrogênese/fisiologia , Articulação do Joelho/citologia , Menisco/citologia , Menisco/fisiologia , Osteoartrite do Joelho/patologia , Fenótipo , Doadores de Tecidos , Idoso , Idoso de 80 Anos ou mais , Antígenos CD/metabolismo , Células Cultivadas , Condrócitos/metabolismo , Colágeno/metabolismo , Feminino , Humanos , Masculino , Menisco/irrigação sanguínea , Menisco/metabolismo , Pessoa de Meia-Idade , Engenharia Tecidual
9.
J Orthop Surg Res ; 15(1): 428, 2020 Sep 18.
Artigo em Inglês | MEDLINE | ID: mdl-32948236

RESUMO

BACKGROUND: The diagnosis, treatment, and efficacy evaluation of anterior cruciate ligament (ACL) partial rupture remains controversial. This research aims to investigate the underlying mechanism of partial ACL injuries to the meniscus degeneration in the rabbit knee. METHODS: Sixty New Zealand white rabbits were randomly divided into three groups including an experimental group, a sham group (n = 6), and a blank control group (n = 6). The experimental group is composed of an anteromedial bundle (AMB) rupture group (n = 24) and a posterolateral bundle (PLB) rupture group (n = 24). Rabbits in the experimental group were subjected to right hind limbs knee surgery to induce ACL part injury under the arthroscopy. Finally, eight rabbits including 6 in the model group and 2 in the control group were sampled randomly on the 2nd, 4th, and 8th weeks respectively. We observed the typical form of the meniscus through HE staining. Expressions of inflammatory factors including interleukin-1ß (IL-1ß) and IL-17 in the knee joint fluid were determined by means of an ELISA. Analysis of the mRNA expressions of matrix metalloproteinases-13(MMP-13) was performed to evaluate the inflammatory mediators in the pathogenesis of the meniscus. RESULTS: HE staining results showed that the surface was rough and the tissues were loose displaying collagen fibers of varying thickness. Both IL-1ß and IL-17 in the synovial fluid and the positive rate of MMP-13 in addition to MMP-13 mRNA showed a demonstrable increase treads from the 2nd to the 8th week. The significant difference was found (P < 0.05) compared to the control group. CONCLUSION: We conclude that the elevated levels of IL-1ß and IL-17, along with increased MMP13 expression, resulted in meniscus degradation in the rabbit knee joint model with partial ACL injury.


Assuntos
Lesões do Ligamento Cruzado Anterior/complicações , Artropatias/etiologia , Artropatias/patologia , Articulação do Joelho/patologia , Menisco/patologia , Animais , Modelos Animais de Doenças , Expressão Gênica , Mediadores da Inflamação/metabolismo , Interleucina-17/metabolismo , Interleucina-1beta/metabolismo , Artropatias/genética , Articulação do Joelho/metabolismo , Metaloproteinase 13 da Matriz/genética , Metaloproteinase 13 da Matriz/metabolismo , Menisco/metabolismo , RNA Mensageiro/metabolismo , Coelhos , Ruptura/complicações , Líquido Sinovial/metabolismo
10.
Int J Biol Macromol ; 162: 1358-1371, 2020 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-32777410

RESUMO

Failure of bioengineered meniscus implant after transplantation is a major concern owing to mechanical failure, lack of chondrogenic capability and patient specific design. In this article, we have, for the first time, fabricated a 3D printed scaffold with carbohydrate based self-healing interpenetrating network (IPN) hydrogels-based monolith construct for load bearing meniscus tissue. 3D printed PLA scaffold was surface functionalized and embedded with self-healing IPN hydrogel for interfacial bonding further characterized by micro CT. Using collagen (C), alginate (A) and oxidized alginate (ADA), we developed self-healing IPN hydrogels with dual crosslinking (Ca2+ based ionic crosslinking and Schiff base (A-A, A-ADA)) capability and studied their physicochemical properties. Further, we studied human stem cells behaviour and chondrogenic differentiation potential within these IPN hydrogels. In-vivo heterotopic implantation confirmed biocompatibility of the monolith showing the feasibility of using carbohydrate based IPN hydrogel embedded in 3D printed scaffold for meniscal tissue development.


Assuntos
Alginatos/química , Condrogênese , Menisco/metabolismo , Células-Tronco Mesenquimais/metabolismo , Impressão Tridimensional , Engenharia Tecidual , Tecidos Suporte/química , Animais , Humanos , Menisco/citologia , Células-Tronco Mesenquimais/citologia , Ratos , Ratos Wistar
11.
Sci Rep ; 10(1): 13789, 2020 08 14.
Artigo em Inglês | MEDLINE | ID: mdl-32796866

RESUMO

Although intra-articular corticosteroid injections (IACI) are commonly used for the treatment of knee osteoarthritis (OA), there is controversy regarding possible deleterious effects on joint structure. In this line, this study investigates the effects of IACI on the evolution of knee OA structural changes and pain. Participants for this nested case-control study were from the Osteoarthritis Initiative. Knees of participants who had received an IACI and had magnetic resonance images (MRI) were named cases (n = 93), and each matched with one control (n = 93). Features assessed at the yearly visits and their changes within the follow-up period were from MRI (cartilage volume, meniscal thickness, bone marrow lesions, bone curvature, and synovial effusion size), X-ray (joint space width), and clinical (Western Ontario and McMaster Universities Osteoarthritis Index [WOMAC] pain score) data. Participants who received IACI experienced a transient and significantly greater rate of loss of the meniscal thickness (p = 0.006) and joint space width (p = 0.011) in the knee medial compartment in the year they received the injection, compared to controls. No significant effect of the IACI was found on the rate of cartilage loss nor on any other knee structural changes or WOMAC pain post-treatment. In conclusion, a single IACI in knee OA was shown to be safe with no negative impact on structural changes, but there was a transient meniscal thickness reduction, a phenomenon for which the clinical relevance is at present unknown.


Assuntos
Corticosteroides/uso terapêutico , Cartilagem Articular/efeitos dos fármacos , Articulação do Joelho/efeitos dos fármacos , Menisco/efeitos dos fármacos , Osteoartrite do Joelho/prevenção & controle , Corticosteroides/administração & dosagem , Idoso , Cartilagem Articular/metabolismo , Cartilagem Articular/patologia , Estudos de Casos e Controles , Progressão da Doença , Feminino , Humanos , Injeções Intra-Articulares , Articulação do Joelho/diagnóstico por imagem , Articulação do Joelho/fisiopatologia , Imageamento por Ressonância Magnética/métodos , Masculino , Menisco/metabolismo , Menisco/patologia , Pessoa de Meia-Idade , Osteoartrite do Joelho/diagnóstico por imagem , Osteoartrite do Joelho/fisiopatologia , Dor/diagnóstico , Dor/diagnóstico por imagem , Dor/prevenção & controle
12.
Clin Biomech (Bristol, Avon) ; 77: 105028, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32422472

RESUMO

BACKGROUND: Proteoglycans contribute to mechanical stiffness in articular cartilage, aiding load transmission. The magnitude of the ionic contribution of proteoglycans to the stiffness of human meniscal tissue has not been established. METHODS: Thirty-six discs of human meniscal tissue were placed within a custom confined compression chamber and bathed in three solutions of increasing ionic concentration. Following a 0.3 N preload, at equilibrium, a 10% ramp compressive strain was followed by a 7200 s hold phase. A nonlinear poroviscoelastic model with strain dependent permeability was fitted to resultant stress relaxation curves. All samples were assayed for proteoglycan content. Model parameters were analysed using multivariate analysis of variance whilst proteoglycan content was compared using a univariate analysis of variance model. FINDINGS: A significant difference (p < .05) was observed in the value of the Young's modulus (E) between samples tested in deionised water compared to those tested in solutions of high ionic concentration. No differences were observed in the zero-strain permeability or the exponential strain dependent stiffening coefficient. Proteoglycan content was not found to differ with solution; but was found to be significantly increased in the middle meniscal region of both menisci. INTERPRETATION: Proteoglycans make a significant ionic contribution to mechanical stiffness of the meniscus, increasing it by 58% in the physiological condition. It is therefore critical that meniscal regeneration strategies attempt to recreate the function of proteoglycans to ensure normal meniscal function.


Assuntos
Fenômenos Mecânicos , Menisco/metabolismo , Proteoglicanas/metabolismo , Fenômenos Biomecânicos , Cartilagem Articular/citologia , Humanos , Menisco/fisiologia , Estresse Mecânico
13.
Lab Invest ; 100(7): 916-927, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32238905

RESUMO

Synovial mesenchymal stem cells (MSCs) are an attractive cell source for transplantation because of their high chondrogenic potential, especially in areas like the meniscus of the knee. A synovial MSC suspension placed onto the meniscus for 10 min promoted healing of repaired meniscal tears that generally do not heal. Here, we quantified the proportion of human synovial MSCs that adhered to a porcine abraded meniscus, clarified their morphological changes, and revealed the mechanism by which the synovial MSCs adhered to the meniscus. The numbers of adhering cells at immediately after 10, 60 min and 6, 24 h after suspension placement were calculated. The meniscus surface was examined by scanning electron microscopy, and 50 cells were randomly selected at each time period, classified, and quantified for each of the six donors. Approximately 28% of the synovial MSCs immediately adhered to the meniscus after placement and the proportion of adhered cells increased further with time. All cells maintained a round shape for 60 min, and then transformed to a mixture of round and semi-flattened cells. By 24 h, flattened cells covered the meniscus. Microspikes were observed in 36% of the floating synovial MSCs and in 76% of the cells on the meniscus shortly after placement on the meniscus, then the proportion of cells with pseudopodia increased. The bleb-dominant cell proportion significantly decreased, and the smooth-dominant cell proportion increased within 60 min. Microspikes or the bodies of synovial MSCs were trapped by meniscal fibers immediately after placement. The proportion of adhered cells increased with time, and the cell morphology changed dynamically for 24 h as the synovial MSCs adhered to the meniscus. The MSCs in the round morphological state had a heterogeneous morphology. The microspikes, and the subsequent development of pseudopodia, may play an important role in adhesion onto the meniscus.


Assuntos
Adesão Celular/fisiologia , Menisco/metabolismo , Células-Tronco Mesenquimais , Membrana Sinovial/citologia , Idoso , Idoso de 80 Anos ou mais , Animais , Células Cultivadas , Feminino , Humanos , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/fisiologia , Pessoa de Meia-Idade , Suínos
14.
Theranostics ; 10(11): 5090-5106, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32308770

RESUMO

Meniscus deficiency, the most common and refractory disease in human knee joints, often progresses to osteoarthritis (OA) due to abnormal biomechanical distribution and articular cartilage abrasion. However, due to its anisotropic spatial architecture, complex biomechanical microenvironment, and limited vascularity, meniscus repair remains a challenge for clinicians and researchers worldwide. In this study, we developed a 3D printing-based biomimetic and composite tissue-engineered meniscus scaffold consisting of polycaprolactone (PCL)/silk fibroin (SF) with extraordinary biomechanical properties and biocompatibility. We hypothesized that the meticulously tailored composite scaffold could enhance meniscus regeneration and cartilage protection. Methods: The physical property of the scaffold was characterized by scanning electron microscopy (SEM) observation, degradation test, frictional force of interface assessment, biomechanical testing, and fourier transform infrared (FTIR) spectroscopy analysis. To verify the biocompatibility of the scaffold, the viability, morphology, proliferation, differentiation, and extracellular matrix (ECM) production of synovium-derived mesenchymal stem cell (SMSC) on the scaffolds were assessed by LIVE/DEAD staining, alamarBlue assay, ELISA analysis, and qRT-PCR. The recruitment ability of SMSC was tested by dual labeling with CD29 and CD90 by confocal microscope at 1 week after implantation. The functionalized hybrid scaffold was then implanted into the meniscus defects on rabbit knee joint for meniscus regeneration, comparing with the Blank group (no scaffold) and PS group. The regenerated meniscus tissue was evaluated by histological and immunohistochemistry staining, and biomechanical test. Macroscopic and histological scoring was performed to assess the outcome of meniscus regeneration and cartilage protection in vivo. Results: The combination of SF and PCL could greatly balance the biomechanical properties and degradation rate to match the native meniscus. SF sponge, characterized by fine elasticity and low interfacial shear force, enhanced energy absorption capacity of the meniscus and improved chondroprotection. The SMSC-specific affinity peptide (LTHPRWP; L7) was conjugated to the scaffold to further increase the recruitment and retention of endogenous SMSCs. This meticulously tailored scaffold displayed superior biomechanics, structure, and function, creating a favorable microenvironment for SMSC proliferation, differentiation, and extracellular matrix (ECM) production. After 24 weeks of implantation, the histological assessment, biochemical contents, and biomechanical properties demonstrated that the polycaprolactone/silk fibroin-L7 (PS-L7) group was close to the native meniscus group, showing significantly better cartilage protection than the PS group. Conclusion: This tissue engineering scaffold could greatly strengthen meniscus regeneration and chondroprotection. Compared with traditional cell-based therapies, the meniscus tissue engineering approach with advantages of one-step operation and reduced cost has a promising potential for future clinical and translational studies.


Assuntos
Cartilagem Articular/citologia , Fibroínas/química , Menisco/citologia , Células-Tronco Mesenquimais/citologia , Poliésteres/química , Impressão Tridimensional/instrumentação , Engenharia Tecidual/métodos , Tecidos Suporte/química , Animais , Fenômenos Biomecânicos , Cartilagem Articular/efeitos dos fármacos , Cartilagem Articular/metabolismo , Diferenciação Celular , Células Cultivadas , Menisco/efeitos dos fármacos , Menisco/metabolismo , Células-Tronco Mesenquimais/metabolismo , Porosidade , Coelhos
15.
Acta Biomater ; 109: 73-81, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32344175

RESUMO

Knee meniscus injury is frequent, resulting in over 1 million surgeries annually in the United States and Europe. Because of the near-avascularity of this fibrocartilaginous tissue and its intrinsic lack of healing, tissue engineering has been proposed as a solution for meniscus repair and replacement. This study describes an approach employing bioactive stimuli to enhance both extracellular matrix content and organization of neomenisci toward augmenting their mechanical properties. Self-assembled fibrocartilages were treated with TGF-ß1, chondroitinase ABC, and lysyl oxidase-like 2 (collectively termed TCL) in addition to lysophosphatidic acid (LPA). TCL + LPA treatment synergistically improved circumferential tensile stiffness and strength, significantly enhanced collagen and pyridinoline crosslink content per dry weight, and achieved tensile anisotropy (circumferential/radial) values of neomenisci close to 4. This study utilizes a combination of bioactive stimuli for use in tissue engineering studies, providing a promising path toward deploying these neomenisci as functional repair and replacement tissues. STATEMENT OF SIGNIFICANCE: This study utilizes a scaffold-free approach, which strays from the tissue engineering paradigm of using scaffolds with cells and bioactive factors to engineer neotissue. While self-assembled neomenisci have attained compressive properties akin to native tissue, tensile properties still require improvement before being able to deploy engineered neomenisci as functional tissue repair or replacement options. In order to augment tensile properties, this study utilized bioactive factors known to augment matrix content in combination with a soluble factor that enhances matrix organization and anisotropy via cell traction forces. Using a bioactive factor to enhance matrix organization mitigates the need for bioreactors used to apply mechanical stimuli or scaffolds to induce proper fiber alignment.


Assuntos
Matriz Extracelular/metabolismo , Fibrocartilagem/metabolismo , Menisco/metabolismo , Engenharia Tecidual/métodos , Aminoácido Oxirredutases/farmacologia , Animais , Bovinos , Condrócitos/metabolismo , Condroitina ABC Liase/farmacologia , Módulo de Elasticidade , Matriz Extracelular/efeitos dos fármacos , Fibrocartilagem/efeitos dos fármacos , Humanos , Lisofosfolipídeos/farmacologia , Teste de Materiais , Menisco/efeitos dos fármacos , Resistência à Tração , Fator de Crescimento Transformador beta1/farmacologia
16.
Biofabrication ; 12(2): 025028, 2020 03 13.
Artigo em Inglês | MEDLINE | ID: mdl-32069441

RESUMO

The meniscus has critical functions in the knee joint kinematics and homeostasis. Injuries of the meniscus are frequent, and the lack of a functional meniscus between the femur and tibial plateau can cause articular cartilage degeneration leading to osteoarthritis development and progression. Regeneration of meniscus tissue has outstanding challenges to be addressed. In the current study, novel Entrapped in cage (EiC) scaffolds of 3D-printed polycaprolactone (PCL) and porous silk fibroin were proposed for meniscus tissue engineering. As confirmed by micro-structural analysis the entrapment of silk fibroin was successful, and all scaffolds had excellent interconnectivity (≥99%). The EiC scaffolds had more favorable micro-structure compared with the PCL cage scaffolds by improving the pore size while keeping the interconnectivity almost the same. When compared with the PCL cage, the entrapment of porous silk fibroin into the PCL cage decreased the high compressive modulus in a favorable matter in the wet state thanks to the silk fibroin's high swelling properties. The in vitro studies with human stem cells or meniscocytes seeded constructs, demonstrated that the EiC scaffolds had superior cell adhesion, metabolic activity, and proliferation compared to the PCL cage scaffolds. Upon subcutaneous implantation of scaffolds in nude mice, all groups were free of adverse incidents, and mildly invaded by inflammatory cells with neovascularization, while the EiC scaffolds showed better tissue infiltration. The results of this work indicated that the EiC scaffolds of PCL and silk fibroin are favorable for meniscus tissue engineering, and the findings are encouraging for further studies using a larger animal model.


Assuntos
Fibroínas/química , Poliésteres/química , Impressão Tridimensional , Engenharia Tecidual/métodos , Tecidos Suporte/química , Animais , Materiais Biocompatíveis/química , Materiais Biocompatíveis/farmacologia , Adesão Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Humanos , Masculino , Menisco/citologia , Menisco/metabolismo , Menisco/transplante , Camundongos , Camundongos Nus , Porosidade , Transplante de Células-Tronco , Células-Tronco/citologia , Células-Tronco/metabolismo
17.
Proc Natl Acad Sci U S A ; 117(6): 3135-3143, 2020 02 11.
Artigo em Inglês | MEDLINE | ID: mdl-31980519

RESUMO

The objective of this study was to examine FoxO expression and FoxO function in meniscus. In menisci from human knee joints with osteoarthritis (OA), FoxO1 and 3 expression were significantly reduced compared with normal menisci from young and old normal donors. The expression of FoxO1 and 3 was also significantly reduced in mouse menisci during aging and OA induced by surgical meniscus destabilization or mechanical overuse. Deletion of FoxO1 and combined FoxO1, 3, and 4 deletions induced abnormal postnatal meniscus development in mice and these mutant mice spontaneously displayed meniscus pathology at 6 mo. Mice with Col2Cre-mediated deletion of FoxO3 or FoxO4 had normal meniscus development but had more severe aging-related damage. In mature AcanCreERT2 mice, the deletion of FoxO1, 3, and 4 aggravated meniscus lesions in all experimental OA models. FoxO deletion suppressed autophagy and antioxidant defense genes and altered several meniscus-specific genes. Expression of these genes was modulated by adenoviral FoxO1 in cultured human meniscus cells. These results suggest that FoxO1 plays a key role in meniscus development and maturation, and both FoxO1 and 3 support homeostasis and protect against meniscus damage in response to mechanical overuse and during aging and OA.


Assuntos
Proteína Forkhead Box O1 , Proteína Forkhead Box O3 , Articulação do Joelho/metabolismo , Menisco/metabolismo , Osteoartrite/metabolismo , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Modelos Animais de Doenças , Feminino , Proteína Forkhead Box O1/análise , Proteína Forkhead Box O1/genética , Proteína Forkhead Box O1/metabolismo , Proteína Forkhead Box O3/análise , Proteína Forkhead Box O3/genética , Proteína Forkhead Box O3/metabolismo , Humanos , Masculino , Menisco/crescimento & desenvolvimento , Camundongos , Camundongos Knockout , Pessoa de Meia-Idade , Adulto Jovem
18.
Sci Rep ; 9(1): 18448, 2019 12 05.
Artigo em Inglês | MEDLINE | ID: mdl-31804577

RESUMO

We report Polarization-resolved Second Harmonic Generation (P-SHG) and directional SHG (forward and backward, F/B) measurements of equine foetal and adult collagen in meniscus, over large field-of-views using sample-scanning. Large differences of collagen structure and fibril orientation with maturation are revealed, validating the potential for this novel methodology to track such changes in meniscal structure. The foetal menisci had a non-organized and more random collagen fibrillar structure when compared with adult using P-SHG. For the latter, clusters of homogeneous fibril orientation (inter-fibrillar areas) were revealed, separated by thick fibers. F/B SHG showed numerous different features in adults notably, in thick fibers compared to interfibrillar areas, unlike foetal menisci that showed similar patterns for both directions. This work confirms previous studies and improves the understanding of meniscal collagen structure and its maturation, and makes F/B and P-SHG good candidates for future studies aiming at revealing structural modifications to meniscus due to pathologies.


Assuntos
Colágenos Fibrilares/metabolismo , Menisco/crescimento & desenvolvimento , Microscopia de Geração do Segundo Harmônico , Animais , Feto , Cavalos , Imageamento Tridimensional , Menisco/diagnóstico por imagem , Menisco/metabolismo
19.
Int J Mol Sci ; 21(1)2019 Dec 19.
Artigo em Inglês | MEDLINE | ID: mdl-31861690

RESUMO

Meniscal injuries, particularly in the avascular zone, have a low propensity for healing and are associated with the development of osteoarthritis. Current meniscal repair techniques are limited to specific tear types and have significant risk for failure. In previous work, we demonstrated the ability of meniscus-derived matrix (MDM) scaffolds to augment the integration and repair of an in vitro meniscus defect. The objective of this study was to determine the effects of percent composition and dehydrothermal (DHT) or genipin cross-linking of MDM bioscaffolds on primary meniscus cellular responses and integrative meniscus repair. In all scaffolds, the porous microenvironment allowed for exogenous cell infiltration and proliferation, as well as endogenous meniscus cell migration. The genipin cross-linked scaffolds promoted extracellular matrix (ECM) deposition and/or retention. The shear strength of integrative meniscus repair was improved with increasing percentages of MDM and genipin cross-linking. Overall, the 16% genipin cross-linked scaffolds were most effective at enhancing integrative meniscus repair. The ability of the genipin cross-linked scaffolds to attract endogenous meniscus cells, promote glycosaminoglycan and collagen deposition, and enhance integrative meniscus repair reveals that these MDM scaffolds are promising tools to augment meniscus healing.


Assuntos
Matriz Extracelular/metabolismo , Iridoides/farmacologia , Menisco/citologia , Engenharia Tecidual/métodos , Animais , Proliferação de Células , Células Cultivadas , Feminino , Menisco/efeitos dos fármacos , Menisco/metabolismo , Resistência ao Cisalhamento , Suínos , Tecidos Suporte
20.
Eur Cell Mater ; 38: 51-62, 2019 08 12.
Artigo em Inglês | MEDLINE | ID: mdl-31402442

RESUMO

Meniscus regeneration is an unmet clinical need as damage to the meniscus is common and causes early osteoarthritis. The aim of the present study was to investigate the feasibility of a one-stage cell-based treatment for meniscus regeneration by augmenting a resorbable collagen-based implant with a combination of recycled meniscus cells and mesenchymal stromal cells (MSCs). Cell communication and fate of the different cell types over time in co-culture were evaluated by connexin 43 staining for gap junctions and polymerase chain reaction (PCR) to discriminate between meniscus cells and MSCs, based on a Y-chromosome gene. To define optimal ratios, human meniscus cells and bone-marrow-derived MSCs were cultured in different ratios in cell pellets and type I collagen hydrogels. In addition, cells were seeded on the implant in fibrin glue by static seeding or injection. Cellular communication by gap junctions was shown in co-culture and a decrease in the amount of MSCs over time was demonstrated by PCR. 20 : 80 and 10 : 90 ratios showed significantly highest glycosaminoglycan and collagen content in collagen hydrogels. The same statistical trend was found in pellet cultures. Significantly more cells were present in the injected implant and cell distribution was more homogenous as compared to the statically seeded implant. The study demonstrated the feasibility of a new one-stage cell-based procedure for meniscus regeneration, using 20 % meniscus cells and 80 % MSCs seeded statically on the implant. In addition, the stimulatory effect of MSCs towards meniscus cells was demonstrated by communication through gap junctions.


Assuntos
Comunicação Celular , Menisco/citologia , Células-Tronco Mesenquimais/citologia , Regeneração , Tecidos Suporte/química , Idoso , Células Cultivadas , Técnicas de Cocultura/métodos , Colágeno/química , Conexina 43/genética , Conexina 43/metabolismo , Feminino , Junções Comunicantes/metabolismo , Glicosaminoglicanos/metabolismo , Humanos , Hidrogéis/química , Masculino , Menisco/metabolismo , Menisco/fisiologia , Células-Tronco Mesenquimais/metabolismo , Pessoa de Meia-Idade , Transplante de Células-Tronco/métodos
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...