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1.
Int J Mol Sci ; 22(16)2021 Aug 10.
Artigo em Inglês | MEDLINE | ID: mdl-34445320

RESUMO

Meniscus injuries can be highly debilitating and lead to knee osteoarthritis. Progenitor cells from the meniscus could be a superior cell type for meniscus repair and tissue-engineering. The purpose of this study is to characterize meniscus progenitor cells isolated by differential adhesion to fibronectin (FN-prog). Human osteoarthritic menisci were digested, and FN-prog were selected by differential adhesion to fibronectin. Multilineage differentiation, population doubling time, colony formation, and MSC surface markers were assessed in the FN-prog and the total meniscus population (Men). Colony formation was compared between outer and inner zone meniscus digest. Chondrogenic pellet cultures were performed for redifferentiation. FN-prog demonstrated multipotency. The outer zone FN-prog formed more colonies than the inner zone FN-prog. FN-prog displayed more colony formation and a higher proliferation rate than Men. FN-prog redifferentiated in pellet culture and mostly adhered to the MSC surface marker profile, except for HLA-DR receptor expression. This is the first study that demonstrates differential adhesion to fibronectin for the isolation of a progenitor-like population from the meniscus. The high proliferation rates and ability to form meniscus extracellular matrix upon redifferentiation, together with the broad availability of osteoarthritis meniscus tissue, make FN-prog a promising cell type for clinical translation in meniscus tissue-engineering.


Assuntos
Adesão Celular , Fibronectinas/metabolismo , Menisco/citologia , Células-Tronco Mesenquimais/citologia , Engenharia Tecidual/métodos , Idoso , Idoso de 80 Anos ou mais , Células Cultivadas , Condrócitos/citologia , Condrócitos/metabolismo , Condrócitos/fisiologia , Condrogênese , Humanos , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/fisiologia , Pessoa de Meia-Idade , Tecidos Suporte/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.
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
4.
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
5.
Arch Orthop Trauma Surg ; 141(4): 699-708, 2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-33550482

RESUMO

INTRODUCTION: Traumatic injuries of the triangular fibrocartilage complex (TFCC) are frequent reasons for ulnar wrist pain. The assessment of the extent of articular disc (AD) degeneration is important for the differentiation of acute injuries versus chronic lesions. MATERIALS AND METHODS: The AD of the TFCC of eleven human cadaver wrists was dissected. Degeneration was analyzed according to the grading of Krenn et al. Hematoxylin-eosin was used to determine the tissue morphology. Degeneration was evaluated using the staining intensity of alcian blue, the immunohistochemistry of the proteoglycan versican and the immunoreactivity of NITEGE, an aggrecan fragment. RESULTS: The staining homogeneity of HE decreased with higher degeneration of the AD and basophilic tissue areas were more frequently seen. Two specimens were characterized as degeneration grade 1, five specimens as grade 2, and four specimens as grade 3, respectively. Staining intensity of alcian blue increased with higher degeneration grade of the specimens. Immunoreactivity for NITEGE was detected around tissue fissures and perforations as well as matrix splits. Immunoreactivity for versican was found concentrated in the tissue around matrix fissures and lesions as well as loose connective tissue at the ulnar border of the AD. Specimens with degeneration grade 2 had the strongest immunoreactivity of NITEGE and versican. Cell clusters were observed in specimens with degeneration grade 2 and 3, which were stained by alcian blue and immunoreactive for NITEGE and versican. Increasing age of the cadaver wrists correlated with a higher degree of degeneration (p < 0.0001, r = 0.68). CONCLUSIONS: The fibrocartilage of degenerated ADs contains NITEGE and versican. The amount of the immunoreactivity of these markers allows the differentiation of degenerative changes into three grades. The degeneration of the AD increases with age and emphasizes its important mechanical function.


Assuntos
Menisco , Fibrocartilagem Triangular , Humanos , Artropatias/patologia , Menisco/citologia , Menisco/patologia , Fibrocartilagem Triangular/citologia , Fibrocartilagem Triangular/patologia
6.
Arthroscopy ; 37(1): 252-265, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-32979500

RESUMO

PURPOSE: To identify, characterize, and compare the resident progenitor cell populations within the red-red, red-white, and white-white (WW) zones of freshly harvested human cadaver menisci and to characterize the vascularity of human menisci using immunofluorescence and 3-dimensional (3D) imaging. METHODS: Fresh adult human menisci were harvested from healthy donors. Menisci were enzymatically digested, mononuclear cells isolated, and characterized using flow cytometry with antibodies against mesenchymal stem cell surface markers (CD105, CD90, CD44, and CD29). Cells were expanded in culture, characterized, and compared with bone marrow-derived mesenchymal stem cells. Trilineage differentiation potential of cultured cells was determined. Vasculature of menisci was mapped in 3D using a modified uDisco clearing and immunofluorescence against vascular markers CD31, lectin, and alpha smooth muscle actin. RESULTS: There were no significant differences in the clonogenicity of isolated cells between the 3 zones. Flow cytometry showed presence of CD44+CD105+CD29+CD90+ cells in all 3 zones with high prevalence in the WW zone. Progenitors from all zones were found to be potent to differentiate to mesenchymal lineages. Larger vessels in the red-red zone of meniscus were observed spanning toward red-white, sprouting to smaller arterioles and venules. CD31+ cells were identified in all zones using the 3D imaging and co-localization of additional markers of vasculature (lectin and alpha smooth muscle actin) was observed. CONCLUSIONS: The presence of resident mesenchymal progenitors was evident in all 3 meniscal zones of healthy adult donors without injury. In addition, our results demonstrate the presence of vascularization in the WW zone. CLINICAL RELEVANCE: The existence of progenitors and presence of microvasculature in the WW zone of the meniscus suggests the potential for repair and biologic augmentation strategies in that zone of the meniscus in young healthy adults. Further research is necessary to fully define the functionality of the meniscal blood supply and its implications for repair.


Assuntos
Menisco/irrigação sanguínea , Células-Tronco Mesenquimais/citologia , Cadáver , Diferenciação Celular , Células Cultivadas , Citometria de Fluxo , Humanos , Menisco/citologia , Células-Tronco/citologia , Adulto Jovem
7.
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
8.
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
9.
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
10.
Eur Rev Med Pharmacol Sci ; 24(4): 1645-1655, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-32141530

RESUMO

OBJECTIVE: To investigate the relationship between the meniscal defect area and OA progression and explore the effect and mechanism of SMSCs cell therapy in knee osteoarthritis (OA) rat model. MATERIALS AND METHODS: For animal experiments, knee osteoarthritis (OA) model was constructed in Sprague Dawley (SD) rats by removing the medial meniscus of the right knee. Synovial mesenchymal stem cells (SMSCs) were engrafted by injecting into the right knee cavity. For in vitro experiments, CCK-8 assay was performed to evaluate the proliferation and differentiation of BMSCs and ATDC5 cells after co-cultured with SMSCs. qRT-PCR analysis was performed to detect the expressions of chondrogenic genes in BMSCs and ATDC5 cells after co-cultured with SMSCs. Western blot analysis was conducted to detect the phosphorylations of c-Jun N-terminal kinase (JNK) and extracellular regulated protein kinases (ERK) in MAPK signaling of BMSCs and ATDC5 cells. Enzyme-linked immunosorbent assay (ELISA) was performed to detect the serum levels of interleukin (IL)-1ß, IL-1ß, IL-6, IL-18 and C-reactive protein (CRP). RESULTS: Results showed that meniscus damaged area is positively correlated to serum inflammatory factor levels. In vitro study showed that the proliferation and differentiation of BMSCs and ATDC5 cells were promoted after co-cultured with SMSCs. By co-culturing with SMSCs, the MAPK signaling pathway was activated and the expression of chondrogenic markers such as aggrecan (acan), SRY-related high mobility group-box gene 9 (sox9) and Type II collagen a1 (col2a1), was up-regulated both in BMSCs and ATDC5 cells. In vivo study showed SMSCs cell therapy significantly decreased serum inflammatory factor levels and protected cartilage by upregulating the expression of chondrogenic genes of meniscus chondrocytes derived from OA rats. CONCLUSIONS: For the first time, we found the positive correlation between meniscal defect area and OA progression and demonstrated the effect and mechanism of SMSCs cell therapy in knee osteoarthritis (OA) treatment.


Assuntos
Condrócitos/citologia , Transplante de Células-Tronco Mesenquimais , Osteoartrite do Joelho/terapia , Animais , Diferenciação Celular , Proliferação de Células , Células Cultivadas , Condrócitos/metabolismo , Técnicas de Cocultura , Citocinas/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/antagonistas & inibidores , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Menisco/citologia , Células-Tronco Mesenquimais/metabolismo , Osteoartrite do Joelho/metabolismo , Ratos Sprague-Dawley , Membrana Sinovial/citologia
11.
Am J Sports Med ; 48(6): 1406-1415, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32105507

RESUMO

BACKGROUND: During meniscal tissue repair, the origin of the reparative cells of damaged meniscal tissue remains unclear. HYPOTHESIS: Comparison of the influence between meniscal and synovial tissues on meniscal repair by the in vivo freeze-thaw method would clarify the origin of meniscal reparative cells. STUDY DESIGN: Controlled laboratory study. METHODS: A total of 48 mature Japanese white rabbits were divided into 4 groups according to the tissue (meniscal or synovial) that received freeze-thaw treatment. The meniscus of each group had a 2 mm-diameter cylindrical defect filled with alginate gel. Macroscopic and histologic evaluations of the reparative tissues were performed at 1, 3, and 6 weeks postoperatively. Additional postoperative measurements included cell density, which was the number of meniscal cells in the cut area per cut area (mm2) of meniscus; cell density ratio, which was the cell density of the sample from each group per the average cell density of the intact meniscus; and cell death rate, which was the number of cells stained by propidium iodide per the number of cells stained by Hoechst 33342 of the meniscal tissue adjacent to the defect. RESULTS: The macroscopic and histologic evaluations of the non-synovium freeze-thaw groups were significantly superior to those of the synovium freeze-thaw groups at 3 and 6 weeks postoperatively. Additionally, the meniscal cell density ratio and cell death rate in the freeze-thaw groups were significantly lower than those in the non-meniscal freeze-thaw groups at 3 and 6 weeks postoperatively. CONCLUSION: The freeze-thawed meniscus recovered few cells in its tissue even after 6 weeks. However, the defect was filled with fibrochondrocytes and proteoglycan when the synovium was intact. On the basis of these results, it is concluded that synovial cells are the primary contributors to meniscal injury repair. CLINICAL RELEVANCE: In meniscal tissue engineering, there is no consensus on the best cell source for meniscal repair. Based on this study, increasing the synovial activity and contribution should be the main objective of meniscal tissue engineering. This study can establish the foundation for future meniscal tissue engineering.


Assuntos
Congelamento , Menisco , Regeneração , Membrana Sinovial/citologia , Animais , Menisco/citologia , Menisco/cirurgia , Coelhos , Engenharia Tecidual
12.
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
13.
FASEB J ; 34(4): 5538-5551, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32090374

RESUMO

The presence of intact menisci is imperative for the proper function of the knee joint. Meniscus injuries are often treated by the surgical removal of the damaged tissue, which increases the likelihood of post-traumatic osteoarthritis. Tissue engineering holds great promise in producing viable engineered meniscal tissue for implantation using the patient's own cells; however, the cell source for producing the engineered tissue is unclear. Nasal chondrocytes (NC) possess many attractive features for engineering meniscus. However, in order to validate the use of NC for engineering meniscus fibrocartilage, a thorough comparison of NC and meniscus fibrochondrocytes (MFC) must be considered. Our study presents an analysis of the relative features of NC and MFC and their respective chondrogenic potential in a pellet culture model. We showed considerable differences in the cartilage tissue formed by the two different cell types. Our data showed that NC were more proliferative in culture, deposited more extracellular matrix, and showed higher expression of chondrogenic genes than MFC. Overall, our data suggest that NC produce superior cartilage tissue to MFC in a pellet culture model. In addition, NCs produce higher quality cartilage tissue at higher cell seeding densities during cell expansion.


Assuntos
Condrócitos/citologia , Condrogênese , Matriz Extracelular/fisiologia , Menisco/citologia , Mucosa Nasal/citologia , Engenharia Tecidual , Adolescente , Idoso , Cartilagem/citologia , Células Cultivadas , Criança , Feminino , Humanos , Masculino
14.
Ann Rheum Dis ; 79(3): 408-417, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31871141

RESUMO

OBJECTIVES: The heterogeneity of meniscus cells and the mechanism of meniscus degeneration is not well understood. Here, single-cell RNA sequencing (scRNA-seq) was used to identify various meniscus cell subsets and investigate the mechanism of meniscus degeneration. METHODS: scRNA-seq was used to identify cell subsets and their gene signatures in healthy human and degenerated meniscus cells to determine their differentiation relationships and characterise the diversity within specific cell types. Colony-forming, multi-differentiation assays and a mice meniscus injury model were used to identify meniscus progenitor cells. We investigated the role of degenerated meniscus progenitor (DegP) cell clusters during meniscus degeneration using computational analysis and experimental verification. RESULTS: We identified seven clusters in healthy human meniscus, including five empirically defined populations and two novel populations. Pseudotime analysis showed endothelial cells and fibrochondrocyte progenitors (FCP) existed at the pseudospace trajectory start. Melanoma cell adhesion molecule ((MCAM)/CD146) was highly expressed in two clusters. CD146+ meniscus cells differentiated into osteoblasts and adipocytes and formed colonies. We identified changes in the proportions of degenerated meniscus cell clusters and found a cluster specific to degenerative meniscus with progenitor cell characteristics. The reconstruction of four progenitor cell clusters indicated that FCP differentiation into DegP was an aberrant process. Interleukin 1ß stimulation in healthy human meniscus cells increased CD318+ cells, while TGFß1 attenuated the increase in CD318+ cells in degenerated meniscus cells. CONCLUSIONS: The identification of meniscus progenitor cells provided new insights into cell-based meniscus tissue engineering, demonstrating a novel mechanism of meniscus degeneration, which contributes to the development of a novel therapeutic strategy.


Assuntos
Diferenciação Celular/genética , Menisco/citologia , Células-Tronco/metabolismo , Animais , Progressão da Doença , Células Endoteliais/metabolismo , Humanos , Camundongos , RNA-Seq , Análise de Sequência de RNA , Análise de Célula Única
15.
Ann Biomed Eng ; 48(3): 968-979, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31147805

RESUMO

Decellularized matrix (DCM) derived from native tissues may be a promising supporting material to induce cellular differentiation by sequestered bioactive factors. However, no previous study has investigated the use of human meniscus-derived DCM to re-differentiate human meniscus fibrochondrocytes (MFCs) to form meniscus-like extracellular matrix (ECM). We expanded human MFCs and seeded them upon a cadaveric meniscus-derived DCM prepared by physical homogenization under hypoxia. To assess the bioactivity of the DCM, we used conditions with and without chondrogenic factor TGF-ß3 and set up a cell pellet culture model as a biomaterial-free control. We found that the DCM supported chondrogenic re-differentiation and ECM formation of MFCs only in the presence of exogenous TGF-ß3. Chondrogenic re-differentiation was more robust at the protein level in the pellet model as MFCs on the DCM appeared to favour a more proliferative phenotype. Interestingly, without growth factors, the DCM tended to promote expression of hypertrophic differentiation markers relative to the pellet model. Therefore, the human meniscus-derived DCM prepared by physical homogenization contained insufficient bioactive factors to induce appreciable ECM formation by human MFCs.


Assuntos
Condrócitos/fisiologia , Matriz Extracelular/fisiologia , Menisco/citologia , Adulto , Idoso , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Condrócitos/efeitos dos fármacos , Condrogênese/efeitos dos fármacos , Fator 2 de Crescimento de Fibroblastos/farmacologia , Humanos , Masculino , Tecidos Suporte , Fator de Crescimento Transformador beta1/farmacologia , Fator de Crescimento Transformador beta3/farmacologia , Adulto Jovem
16.
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
17.
Stem Cells Transl Med ; 8(12): 1318-1329, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31638337

RESUMO

Meniscus-derived stem cells (MeSCs) are a potential cell source for meniscus tissue engineering. The stark morphological and structural changes of meniscus tissue during development indicate the complexity of MeSCs at different tissue regions and stages of development. In this study, we characterized and compared postnatal rat meniscus tissue and MeSCs at different tissue regions and stages of development. We observed that the rat meniscus tissue exhibited marked changes in tissue morphology during development, with day 7 being the most representative time point of different developmental stages. All rat MeSCs displayed typical stem cell characteristics. Rat MeSCs derived from day 7 inner meniscus tissue exhibited the highest self-renewal capacity, cell proliferation, differentiation potential toward various mesenchymal lineage and the highest expression levels of chondrogenic genes and proteins. Transplantation of rat MeSCs derived from day 7 inner meniscus tissue promoted neo-tissue formation and effectively protected joint surface cartilage in vivo. Our results demonstrated for the first time that rat MeSCs are not necessarily better at earlier developmental stages, and that rat MeSCs derived from day 7 inner meniscus tissue may be a superior cell source for effective meniscus regeneration and articular cartilage protection. This information could make a significant contribution to human meniscus tissue engineering in the future. Stem Cells Translational Medicine 2019;8:1318&1329.


Assuntos
Condrogênese , Proteínas de Membrana/metabolismo , Menisco/citologia , Transplante de Células-Tronco Mesenquimais/métodos , Células-Tronco Mesenquimais/citologia , Osteoartrite/terapia , Engenharia Tecidual/métodos , Animais , Diferenciação Celular , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Ratos , Ratos Sprague-Dawley , Regeneração
18.
ACS Appl Mater Interfaces ; 11(44): 41626-41639, 2019 Nov 06.
Artigo em Inglês | MEDLINE | ID: mdl-31596568

RESUMO

Regeneration of an injured meniscus continues to be a scientific challenge due to its poor self-healing potential. Tissue engineering provides an avenue for regenerating a severely damaged meniscus. In this study, we first investigated the superiority of five concentrations (0%, 0.5%, 1%, 2%, and 4%) of meniscus extracellular matrix (MECM)-based hydrogel in promoting cell proliferation and the matrix-forming phenotype of meniscal fibrochondrocytes (MFCs). We found that the 2% group strongly enhanced chondrogenic marker mRNA expression and cell proliferation compared to the other groups. Moreover, the 2% group showed the highest glycosaminoglycan (GAG) and collagen production by day 14. We then constructed a hybrid scaffold by 3D printing a wedge-shaped poly(ε-caprolactone) (PCL) scaffold as a backbone, followed by injection with the optimized MECM-based hydrogel (2%), which served as a cell delivery system. The hybrid scaffold (PCL-hydrogel) clearly yielded favorable biomechanical properties close to those of the native meniscus. Finally, PCL scaffold, PCL-hydrogel, and MFCs-loaded hybrid scaffold (PCL-hydrogel-MFCs) were implanted into the knee joints of New Zealand rabbits that underwent total medial meniscectomy. Six months postimplantation we found that the PCL-hydrogel-MFCs group exhibited markedly better gross appearance and cartilage protection than the PCL scaffold and PCL-hydrogel groups. Moreover, the regenerated menisci in the PCL-hydrogel-MFCs group had similar histological structures, biochemical contents, and biomechanical properties as the native menisci in the sham operation group. In conclusion, PCL-MECM-based hydrogel hybrid scaffold seeded with MFCs can successfully promote whole meniscus regeneration, and cell-loaded PCL-MECM-based hydrogel hybrid scaffold may be a promising strategy for meniscus regeneration in the future.


Assuntos
Matriz Extracelular/química , Hidrogéis/química , Menisco/fisiologia , Poliésteres/química , Regeneração , Tecidos Suporte/química , Animais , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Condrócitos/citologia , Condrócitos/metabolismo , Modelos Animais de Doenças , Hidrogéis/farmacologia , Meniscectomia , Menisco/citologia , Menisco/cirurgia , Porosidade , Impressão Tridimensional , Coelhos , Resistência à Tração , Engenharia Tecidual
19.
Biofabrication ; 12(1): 015003, 2019 10 21.
Artigo em Inglês | MEDLINE | ID: mdl-31480031

RESUMO

Employing tissue engineering principles aided by three-dimensional (3D) printing strategies to fabricate meniscus tissue constructs could help patients with meniscus injury regain mobility, improve pain management and reduce the risk of development of knee osteoarthritis. Here we report a 3D printed meniscus scaffold that biomimics the internal and bulk architecture of the menisci. A shear-thinning novel silk fibroin-gelatin-based bioink with high print fidelity was optimized for the fabrication of scaffolds to serve as potential meniscus implants. Physicochemical characterization of the fabricated scaffolds shows optimum swelling, degradation and mechanical properties. Further, the scaffolds were seeded with meniscus fibrochondrocytes to validate their bioactivity. Fibrochondrocytes seeded on the scaffolds maintained their phenotype and proliferation, and enhanced glycosaminoglycan and total collagen synthesis was observed. Gene expression profile, biochemical quantification and histological studies confirmed the ability of the scaffolds to form meniscus-like tissue constructs. The scaffolds were found to possess amenable immunocompatibility in vitro as well as in vivo. Due to their excellent biological and physicochemical characteristics, these 3D printed scaffolds may be fine-tuned into viable alternatives to the present clinical treatment approaches to meniscus repair.


Assuntos
Biomimética/métodos , Bioimpressão/métodos , Fibroínas/química , Menisco/química , Animais , Biomimética/instrumentação , Bioimpressão/instrumentação , Bombyx , Proliferação de Células , Gelatina/química , Humanos , Menisco/citologia , Menisco/lesões , Impressão Tridimensional , Próteses e Implantes , Suínos , Engenharia Tecidual/instrumentação , Engenharia Tecidual/métodos , Tecidos Suporte/química
20.
Int J Nanomedicine ; 14: 5491-5502, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31410000

RESUMO

Purpose: Meniscus is a fibrocartilagenous tissue that cannot effectively heal due to its complex structure and presence of avascular zone. Thus, tissue engineering and regenerative medicine offer an alternative for the regeneration of meniscus tissues using bioscaffolds as a replacement for the damaged one. The aim of this study was to prepare an ideal meniscus bioscaffold with minimal adverse effect on extracellular matrix components (ECMs) using a sonication treatment system. Methods: The decellularization was achieved using a developed closed sonication treatment system for 10 hrs, and continued with a washing process for 5 days. For the control, a simple immersion treatment was set as a benchmark to compare the decellularization efficiency. Histological and biochemical assays were conducted to investigate the cell removal and retention of the vital extracellular matrix. Surface ultrastructure of the prepared scaffolds was evaluated using scanning electron microscope at 5,000× magnification viewed from cross and longitudinal sections. In addition, the biomechanical properties were investigated through ball indentation testing to study the stiffness, residual forces and compression characteristics. Statistical significance between the samples was determined with p-value =0.05. Results: Histological and biochemical assays confirmed the elimination of antigenic cellular components with the retention of the vital extracellular matrix within the sonicated scaffolds. However, there was a significant removal of sulfated glycosaminoglycans. The surface histoarchitecture portrayed the preserved collagen fibril orientation and arrangement. However, there were minor disruptions on the structure, with few empty micropores formed which represented cell lacunae. The biomechanical properties of bioscaffolds showed the retention of viscoelastic behavior of the scaffolds which mimic native tissues. After immersion treatment, those scaffolds had poor results compared to the sonicated scaffolds due to the inefficiency of the treatment. Conclusion: In conclusion, this study reported that the closed sonication treatment system had high capabilities to prepare ideal bioscaffolds with excellent removal of cellular components, and retained extracellular matrix and biomechanical properties.


Assuntos
Menisco/citologia , Ortopedia , Sonicação/métodos , Engenharia Tecidual/métodos , Tecidos Suporte/química , Animais , Fenômenos Biomecânicos , Bovinos , Colágeno/metabolismo , Força Compressiva , Glicosaminoglicanos/metabolismo
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