RESUMO
Joint cartilage damage affects 10-12% of the world's population. Medical treatments improve the short-term quality of life of affected individuals but lack a long-term effect due to injury progression into fibrocartilage. The use of mesenchymal stem cells (MSCs) is one of the most promising strategies for tissue regeneration due to their ability to be isolated, expanded and differentiated into metabolically active chondrocytes to achieve long-term restoration. For this purpose, human adipose-derived MSCs (Ad-MSCs) were isolated from lipectomy and grown in xeno-free conditions. To establish the best differentiation potential towards a stable chondrocyte phenotype, isolated Ad-MSCs were sequentially exposed to five differentiation schemes of growth factors in previously designed three-dimensional biphasic scaffolds with incorporation of a decellularized cartilage matrix as a bioactive ingredient, silk fibroin and bone matrix, to generate a system capable of being loaded with pre-differentiated Ad-MSCs, to be used as a clinical implant in cartilage lesions for tissue regeneration. Chondrogenic and osteogenic markers were analyzed by reverse transcription-quantitative PCR and cartilage matrix generation by histology techniques at different time points over 40 days. All groups had an increased expression of chondrogenic markers; however, the use of fibroblast growth factor 2 (10 ng/ml) followed by a combination of insulin-like growth factor 1 (100 ng/ml)/TGFß1 (10 ng/ml) and a final step of exposure to TGFß1 alone (10 ng/ml) resulted in the most optimal chondrogenic signature towards chondrocyte differentiation and the lowest levels of osteogenic expression, while maintaining stable collagen matrix deposition until day 33. This encourages their possible use in osteochondral lesions, with appropriate properties for use in clinical patients.
RESUMO
In cartilage tissue engineering, biphasic scaffolds (BSs) have been designed not only to influence the recapitulation of the osteochondral architecture but also to take advantage of the healing ability of bone, promoting the implant's integration with the surrounding tissue and then bone restoration and cartilage regeneration. This study reports the development and characterization of a BS based on the assembly of a cartilage phase constituted by fibroin biofunctionalyzed with a bovine cartilage matrix, cellularized with differentiated autologous pre-chondrocytes and well attached to a bone phase (decellularized bovine bone) to promote cartilage regeneration in a model of joint damage in pigs. BSs were assembled by fibroin crystallization with methanol, and the mechanical features and histological architectures were evaluated. The scaffolds were cellularized and matured for 12 days, then implanted into an osteochondral defect in a porcine model (n = 4). Three treatments were applied per knee: Group I, monophasic cellular scaffold (single chondral phase); group II (BS), cellularized only in the chondral phase; and in order to study the influence of the cellularization of the bone phase, Group III was cellularized in chondral phases and a bone phase, with autologous osteoblasts being included. After 8 weeks of surgery, the integration and regeneration tissues were analyzed via a histology and immunohistochemistry evaluation. The mechanical assessment showed that the acellular BSs reached a Young's modulus of 805.01 kPa, similar to native cartilage. In vitro biological studies revealed the chondroinductive ability of the BSs, evidenced by an increase in sulfated glycosaminoglycans and type II collagen, both secreted by the chondrocytes cultured on the scaffold during 28 days. No evidence of adverse or inflammatory reactions was observed in the in vivo trial; however, in Group I, the defects were not reconstructed. In Groups II and III, a good integration of the implant with the surrounding tissue was observed. Defects in group II were fulfilled via hyaline cartilage and normal bone. Group III defects showed fibrous repair tissue. In conclusion, our findings demonstrated the efficacy of a biphasic and bioactive scaffold based on silk fibroin and cellularized only in the chondral phase, which entwined chondroinductive features and a biomechanical capability with an appropriate integration with the surrounding tissue, representing a promising alternative for osteochondral tissue-engineering applications.
Assuntos
Regeneração Óssea , Engenharia Tecidual/métodos , Animais , Cartilagem , Diferenciação Celular , Condrócitos , Fibroínas , Suínos , Alicerces TeciduaisRESUMO
Articular cartilage injuries remain as a therapeutic challenge due to the limited regeneration potential of this tissue. Cartilage engineering grafts combining chondrogenic cells, scaffold materials, and microenvironmental factors are emerging as promissory alternatives. The design of an adequate scaffold resembling the physicochemical features of natural cartilage and able to support chondrogenesis in the implants is a crucial topic to solve. This study reports the development of an implant constructed with IGF1-transduced adipose-derived mesenchymal stem cells (immunophenotypes: CD105+, CD90+, CD73+, CD14-, and CD34-) embedded in a scaffold composed of a mix of alginate/milled bovine decellularized knee material which was cultivated in vitro for 28 days (3CI). Histological analyses demonstrated the distribution into isogenous groups of chondrocytes surrounded by a de novo dense extracellular matrix with balanced proportions of collagens II and I and high amounts of sulfated proteoglycans which also evidenced adequate cell proliferation and differentiation. This graft also shoved mechanical properties resembling the natural knee cartilage. A modified Bern/O'Driscoll scale showed that the 3CI implants had a significantly higher score than the 2CI implants lacking cells transduced with IGF1 (16/18 vs. 14/18), representing high-quality engineering cartilage suitable for in vivo tests. This study suggests that this graft resembles several features of typical hyaline cartilage and will be promissory for preclinical studies for cartilage regeneration.
RESUMO
There is no therapy currently available for fully repairing articular cartilage lesions. Our laboratory has recently developed a visible light-activatable methacrylated gelatin (mGL) hydrogel, with the potential for cartilage regeneration. In this study, we further optimized mGL scaffolds by supplementing methacrylated hyaluronic acid (mHA), which has been shown to stimulate chondrogenesis via activation of critical cellular signalling pathways. We hypothesized that the introduction of an optimal ratio of mHA would enhance the biological properties of mGL scaffolds and augment chondrogenesis of human bone marrow-derived mesenchymal stem cells (hBMSCs). To test this hypothesis, hybrid scaffolds consisting of mGL and mHA at different weight ratios were fabricated with hBMSCs encapsulated at 20 × 106 cells/ml and maintained in a chondrogenesis-promoting medium. The chondrogenenic differentiation of hBMSCs, within different scaffolds, was estimated after 8 weeks of culture. Our results showed that mGL/mHA at a 9:1 (%, w/v) ratio resulted in the lowest hBMSC hypertrophy and highest glycosaminoglycan production, with a slightly increased volume of the entire construct. The applicability of this optimally designed mGL/mHA hybrid scaffold for cartilage repair was then examined in vivo. A full-thickness cylindrical osteochondral defect was surgically created in the rabbit femoral condyle, and a three-dimensional cell-biomaterial construct was fabricated by in situ photocrosslinking to fully fill the lesion site. The results showed that implantation of the mGL/mHA (9:1) construct resulted in both cartilage and subchondral bone regeneration after 12 weeks, supporting its use as a promising scaffold for repair and resurfacing of articular cartilage defects, in the clinical setting.
Assuntos
Cartilagem Articular/patologia , Reagentes de Ligações Cruzadas/química , Gelatina/química , Ácido Hialurônico/química , Luz , Alicerces Teciduais/química , Cicatrização , Animais , Contagem de Células , Sobrevivência Celular , Condrogênese , Regulação da Expressão Gênica , Glicosaminoglicanos/metabolismo , Humanos , Hidrogéis/química , Hipertrofia , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Metacrilatos/química , CoelhosRESUMO
To investigate the effects of proteins products of endothelial cells (ECs) on the annulus fibrosus (AF) cell metabolism in an in vitro culture. Human AF cells were expanded in monolayer cultures and treated with proteins from the medium of cell line HMEC-1 (Human Microvascular Endothelial Cells) (125µg/ml). After 72h of treatment RNA was isolated from AF cells for analysis of gene expression and the culture medium was collected for protein expression analysis. The qRT-PCR analysis demonstrated increased gene expression of matrix metalloproteinases (MMPs) in AF cells treated with protein products of endothelial cells compared with cells from control group of AF cells: MMP-1 243.10 times (p<0.05), MMP-2 1.37 time (p<0.05), MMP-3 39.83 times (p<0.05) and MMP-13 5.70 times (p<0.05). In contrast, tissue inhibitors of metalloproteinases (TIMPs) were suppressed; TIMP-2 (0.55 time) (p<0.05) and TIMP-3 (0.60 time) (p<0.05) in the exposed groups. The expression of aggrecan gene (0.83 time) (p<0.05), an important extracellular matrix component, was also reduced. MMP-1 and MMP-3 detection was performed, confirming the results of PCR by Western Blot technique. In this study, we observed that the proteins produced by ECs induced the MMPs expression and suppressed the TIMPs as well as the aggrecan in primary cells of the human intervertebral disc, targeting the development of potential treatments for intervertebral disc degeneration and associated discogenic pain.
Analisar o efeito de produtos proteicos de células endoteliais (CEs) sobre o metabolismo de células de ânulo fibroso (AF) em ambiente controlado de cultura celular in vitro. Células de AF humano foram expandidas em camada única e tratadas com proteínas obtidas a partir do meio de cultura de células da linhagem celular HMEC-1 (Human Microvascular Endothelial Cells) (125µg/ml). Após 72h de tratamento, isolou-se RNA das células de AF para análise da expressão gênica e coletou-se meio de cultura para análise de expressão proteica. A análise da qRT-PCR demonstrou aumento da expressão gênica das metaloproteinases de matriz (MMPs) nas células de AF tratadas com produtos proteicos das células endoteliais, em comparação com grupo controle de células de AF: MMP-1 243,10 vezes (p < 0,05), MMP-2 1,37 vezes (p < 0,05), MMP-3 39,83 vezes (p < 0,05) e MMP13 5,70 vezes (p < 0,05). Em contraste, os inibidores teciduais das metaloproteinases (TIMPs) apresentaram supressão da expressão gênica de TIMP-2 (0,55 vezes) (p < 0,05) e TIMP-3 (0,60 vezes) (p < 0,05) nos grupos expostos. A expressão do gene agrecan (0,83 vezes) (p < 0,05), componente importante da matriz extracelular, também estava diminuída. Foi realizada detecção de MMP-1 e MMP-3, confirmando os resultados de PCR através de técnica de Western Blot. Neste estudo observamos que proteínas produzidas pelas CEs induziram a expressão de MMPs e suprimiram a expressão de TIMPs e agrecan nas células primárias do disco intervertebral humano, objetivando desenvolvimento de potenciais terapias no tratamento da degeneração do disco intervertebral e dor discogênica associada.
Analizar el efecto de los productos de proteína de las células endoteliales (CEs) en el metabolismo celular del anillo fibroso (AF) en sistema in vitro de cultivo controlado. Las células del AF humano se ampliaron en monocapa y se las trató con las proteínas obtenidas a partir de los medios de cultivo de la línea de células HMEC-1 (Human Microvascular Endothelial Cells) (125µg/ml). Después de 72h de tratamiento, se aisló el ARN de las células de AF para el análisis de la expresión génica y se recogió el medio de cultivo para el análisis de expresión de la proteína. El análisis de qRT-PCR demostró una mayor expresión génica de las metaloproteinasas de matriz (MMP) en las células tratadas con productos de proteína de AF en las células endoteliales, en comparación con el grupo de control de células AF: MMP-1 243,10 veces (p < 0,05), MMP-2 1,37 veces (p < 0,05), MMP-3 39,83 veces (p < 0,05) y MMP-13 5,70 veces (p < 0,05). En contraste, los inhibidores tisulares de las metaloproteinasas (TIMP), presentaron supresión de la expresión del gen TIMP-2 (0,55 veces) (p < 0,05) y TIMP-3 (0,60 veces) (p < 0,05) en los grupos expuestos. La expresión génica de agrecano (0,83 veces) (p < 0,05), importante componente de la matriz extracelular, también se redujo. La detección de MMP-1 y de MMP-3 fue realizada y se confirmaron los resultados de la PCR mediante la técnica Western Blot. En el presente estudio se observó que las proteínas producidas por las CEs indujeron la expresión de MMP y suprimieron la expresión del TIMP y de agrecano en células primarias del disco intervertebral humano, con el objetivo de desarrollar posibles tratamientos para la degeneración del disco intervertebral y el dolor discogénico asociado.