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1.
Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi ; 38(6): 748-754, 2024 Jun 15.
Artigo em Chinês | MEDLINE | ID: mdl-38918198

RESUMO

Objective: To investigate the construction of a novel tissue engineered meniscus scaffold based on low temperature deposition three-dimenisonal (3D) printing technology and evaluate its biocompatibility. Methods: The fresh pig meniscus was decellularized by improved physicochemical method to obtain decellularized meniscus matrix homogenate. Gross observation, HE staining, and DAPI staining were used to observe the decellularization effect. Toluidine blue staining, safranin O staining, and sirius red staining were used to evaluate the retention of mucopolysaccharide and collagen. Then, the decellularized meniscus matrix bioink was prepared, and the new tissue engineered meniscus scaffold was prepared by low temperature deposition 3D printing technology. Scanning electron microscopy was used to observe the microstructure. After co-culture with adipose-derived stem cells, the cell compatibility of the scaffolds was observed by cell counting kit 8 (CCK-8), and the cell activity and morphology were observed by dead/live cell staining and cytoskeleton staining. The inflammatory cell infiltration and degradation of the scaffolds were evaluated by subcutaneous experiment in rats. Results: The decellularized meniscus matrix homogenate appeared as a transparent gel. DAPI and histological staining showed that the immunogenic nucleic acids were effectively removed and the active components of mucopolysaccharide and collagen were remained. The new tissue engineered meniscus scaffolds was constructed by low temperature deposition 3D printing technology and it had macroporous-microporous microstructures under scanning electron microscopy. CCK-8 test showed that the scaffolds had good cell compatibility. Dead/live cell staining showed that the scaffold could effectively maintain cell viability (>90%). Cytoskeleton staining showed that the scaffolds were benefit for cell adhesion and spreading. After 1 week of subcutaneous implantation of the scaffolds in rats, there was a mild inflammatory response, but no significant inflammatory response was observed after 3 weeks, and the scaffolds gradually degraded. Conclusion: The novel tissue engineered meniscus scaffold constructed by low temperature deposition 3D printing technology has a graded macroporous-microporous microstructure and good cytocompatibility, which is conducive to cell adhesion and growth, laying the foundation for the in vivo research of tissue engineered meniscus scaffolds in the next step.


Assuntos
Menisco , Impressão Tridimensional , Engenharia Tecidual , Alicerces Teciduais , Animais , Engenharia Tecidual/métodos , Alicerces Teciduais/química , Suínos , Ratos , Menisco/citologia , Materiais Biocompatíveis , Ratos Sprague-Dawley , Células Cultivadas , Meniscos Tibiais/citologia , Microscopia Eletrônica de Varredura
2.
Int J Mol Sci ; 22(22)2021 Nov 18.
Artigo em Inglês | MEDLINE | ID: mdl-34830345

RESUMO

Menisci play an essential role in shock absorption, joint stability, load resistance and its transmission thanks to their conformation. Adult menisci can be divided in three zones based on the vascularization: an avascular inner zone with no blood supply, a fully vascularized outer zone, and an intermediate zone. This organization, in addition to the incomplete knowledge about meniscal biology, composition, and gene expression, makes meniscal regeneration still one of the major challenges both in orthopedics and in tissue engineering. To overcome this issue, we aimed to investigate the role of hypoxia in the differentiation of the three anatomical areas of newborn piglet menisci (anterior horn (A), central body (C), and posterior horn (P)) and its effects on vascular factors. After sample collection, menisci were divided in A, C, P, and they were cultured in vitro under hypoxic (1% O2) and normoxic (21% O2) conditions at four different experimental time points (T0 = day of explant; T7 = day 7; T10 = day 10; T14 = day 14); samples were then evaluated through immune, histological, and molecular analyses, cell morpho-functional characteristics; with particular focus on matrix composition and expression of vascular factors. It was observed that hypoxia retained the initial phenotype of cells and induced extracellular matrix production resembling a mature tissue. Hypoxia also modulated the expression of angiogenic factors, especially in the early phase of the study. Thus, we observed that hypoxia contributes to the fibro-chondrogenic differentiation with the involvement of angiogenic factors, especially in the posterior horn, which corresponds to the predominant weight-bearing portion.


Assuntos
Condrócitos/efeitos dos fármacos , Fibroblastos/efeitos dos fármacos , Hipóxia/metabolismo , Meniscos Tibiais/efeitos dos fármacos , Oxigênio/farmacologia , Animais , Animais Recém-Nascidos , Biomarcadores/metabolismo , Caspase 3/genética , Caspase 3/metabolismo , Diferenciação Celular/efeitos dos fármacos , Condrócitos/citologia , Condrócitos/metabolismo , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Colágeno Tipo II/genética , Colágeno Tipo II/metabolismo , Endostatinas/genética , Endostatinas/metabolismo , Fibroblastos/citologia , Fibroblastos/metabolismo , Expressão Gênica , Hipóxia/genética , Meniscos Tibiais/citologia , Meniscos Tibiais/metabolismo , Molécula-1 de Adesão Celular Endotelial a Plaquetas/genética , Molécula-1 de Adesão Celular Endotelial a Plaquetas/metabolismo , Antígeno Nuclear de Célula em Proliferação/genética , Antígeno Nuclear de Célula em Proliferação/metabolismo , Fatores de Transcrição SOX9/genética , Fatores de Transcrição SOX9/metabolismo , Suínos , Técnicas de Cultura de Tecidos
3.
Knee Surg Sports Traumatol Arthrosc ; 29(6): 1701-1708, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32740878

RESUMO

PURPOSE: A histological study of a structure between the posterior horn of the lateral meniscus and the anterior cruciate ligament. METHODS: Bilateral fresh-frozen cadaveric knees of two male donors (age 71 and 76 years) with no history of prior knee injury were examined. All dissections were performed by one experienced orthopaedic surgeon. Haematoxylin and Eosin staining was used to reveal tissue morphology. Goldner trichrome staining was used to evaluate the connective tissue. S100 and PGP 9.5 labelling were used for immunohistochemical analysis. RESULTS: In all cadaveric knees, a structure between the posterior horn of the lateral meniscus and the anterior cruciate ligament was identified. Histological analysis confirmed the ligamentous nature of this structure. Furthermore, Golgi tendon organs were observed within the ligamentous structure. CONCLUSION: This is the first study showing the presence of mechanoreceptors within the ligamentous structure between the posterior horn of the lateral meniscus and the anterior cruciate ligament. The ligamentous structure could contribute to stability of the knee by providing proprioceptive input, while preservation of the ligamentous structure might ensure a better functional outcome after surgery.


Assuntos
Ligamento Cruzado Anterior/citologia , Mecanorreceptores , Meniscos Tibiais/citologia , Idoso , Ligamento Cruzado Anterior/inervação , Lesões do Ligamento Cruzado Anterior/epidemiologia , Cadáver , Humanos , Traumatismos do Joelho/epidemiologia , Articulação do Joelho , Masculino , Meniscos Tibiais/inervação , Propriocepção
4.
Clin Sports Med ; 39(1): 125-163, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31767102

RESUMO

Meniscus injuries are among the most common athletic injuries and result in functional impairment in the knee. Repair is crucial for pain relief and prevention of degenerative joint diseases like osteoarthritis. Current treatments, however, do not produce long-term improvements. Thus, recent research has been investigating new therapeutic options for regenerating injured meniscal tissue. This review comprehensively details the current methodologies being explored in the basic sciences to stimulate better meniscus injury repair. Furthermore, it describes how these preclinical strategies may improve current paradigms of how meniscal injuries are clinically treated through a unique and alternative perspective to traditional clinical methodology.


Assuntos
Meniscos Tibiais/fisiologia , Regeneração , Lesões do Menisco Tibial/cirurgia , Engenharia Tecidual , Alicerces Teciduais , Tecido Adiposo/citologia , Fenômenos Biomecânicos , Células da Medula Óssea , Cartilagem/citologia , Condrócitos/transplante , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Meniscos Tibiais/citologia , Fibrina Rica em Plaquetas , Plasma Rico em Plaquetas , Transplante de Células-Tronco , Membrana Sinovial/citologia
5.
J Ethnopharmacol ; 247: 112261, 2020 Jan 30.
Artigo em Inglês | MEDLINE | ID: mdl-31577939

RESUMO

ETHNOPHARMACOLOGICAL RELEVANCE: Corni Fructus (CF), the red fruit of Cornus officinalis Siebold & Zucc, has been used both as food and medicinal herb in traditional Chinese medicine (TCM). Loganin is a major iridoid glycoside and one of the quality control indexes of CF. In TCM clinical practice, prescription containing CF is commonly used to treat osteoarthritis (OA), but the underlying mechanisms of loganin are not yet utterly understood. AIM OF THE STUDY: The aims of the present study are to confirm the therapeutic effects of loganin in an OA mouse model and to determine the mechanisms involved in the OA protective effects. MATERIALS AND METHODS: The destabilization of the medial meniscus (DMM) procedure was performed on the right knee of 8-week-old C57BL/6 male mice. 30 or 100 µg/ml of loganin was then injected into articular space twice a week for 8 and 12-week. Safranin O/Fast green staining, H&E staining, micro-CT analysis were performed to analyze structural and morphological changes. The protein expression of collagen type II (Col2), metalloproteinase-3 (Mmp3), matrix metalloproteinase 13 (Mmp13) collagen type X (Col10), cryopyrin and caspase-1 were detected by immunochemistry staining. Immuno-fluorescence assay was performed to assess changes in expression of CD31, endomucin, p65 and p-I-κB. RESULTS: Results of histomorphometry showed that loganin delays the progression of OA in the DMM model. In cartilage, loganin decreased the OARSI score, increasing hyaline cartilage (HC) thickness and decreasing calcified cartilage (CC) thickness. Moreover, loganin inhibited osteophyte formation, reduced the bone volume fraction (BV/TV), lowered trabecular thickness (Tb.Th) and increased trabecular separation (Tb.Sp) in subchondral bone. Mechanistically, loganin increased the expressions of Col2, decreases the expression of Mmp3, Mmp13, Col10, cryopyrin and caspase-1 in cartilage. In parallel, loganin inhibited the expression of CD31 and endomucin in subchondral bone. Furthermore, loganin suppressed nuclear translocation of p65 protein, and decreased the amount of p-I-κB in chondrocytes. CONCLUSIONS: In summary, these results uncovered that loganin inhibits NF-κB signaling and attenuates cartilage matrix catabolism and pyroptosis of chondrocytes in articular cartilage. Loganin may serve as a potential therapeutic agent for OA treatment.


Assuntos
Cornus/química , Iridoides/farmacologia , Meniscos Tibiais/efeitos dos fármacos , Osteoartrite do Joelho/tratamento farmacológico , Animais , Condrócitos/efeitos dos fármacos , Condrócitos/patologia , Colágeno Tipo II/metabolismo , Colágeno Tipo X/metabolismo , Modelos Animais de Doenças , Frutas/química , Humanos , Iridoides/isolamento & purificação , Iridoides/uso terapêutico , Masculino , Meniscos Tibiais/citologia , Meniscos Tibiais/patologia , Meniscos Tibiais/cirurgia , Camundongos , NF-kappa B/imunologia , NF-kappa B/metabolismo , Osteoartrite do Joelho/imunologia , Osteoartrite do Joelho/patologia , Piroptose/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/imunologia
6.
Bratisl Lek Listy ; 119(10): 630-635, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30345770

RESUMO

OBJECTIVES: In this study, we sought to investigate the effect of different amounts of Bone Marrow-Derived Mesenchymal Stem Cells (BMSCs), obtained by different BMSCs, on the healing of avascular zone meniscal defects. BACKGROUND: Treating avascular zone meniscal injuries has gained popularity. BMSCs contribute to the healing of avascular zone meniscal defects. The amount of BMSCs derived from different bone marrow stimulation techniques (BMSTs) varies, which could affect the therapeutic efficacy of this treatment. METHODS: Fifty-four skeletally mature female New Zealand White rabbits were used after local ethical committee approval. A full thickness, 1.5 mm diameter defect was produced in the inner two-thirds of the anterior portion of the medial meniscus avascular zone using a biopsy punch. Animals were enrolled into three different groups according to BMST (0.8 mm, 1.5 mm, and 4 mm). Medial menisci were harvested and prepared for histomorphometric, histologic and immune-histologic analyses. RESULTS: Larger bridging tissues across the defect were detected in the 1.5-mm and 4-mm groups at 4 weeks (p < 0.05). The best quality score at the 1-,4- and 12-week endpoints was in 0.8 mm, 4 mm and 0.8 mm, 1.5 mm, respectively (p 0.05)CONCLUSION: The largest amount of BMSCs did not correlate with best quality and largest quantity of bridging tissue at the avascular zone in meniscal defects (Tab. 3, Fig. 4, Ref. 30).


Assuntos
Medula Óssea , Meniscos Tibiais , Células-Tronco Mesenquimais , Animais , Diferenciação Celular , Feminino , Meniscos Tibiais/citologia , Coelhos , Cicatrização
7.
Proc Inst Mech Eng H ; 232(4): 418-422, 2018 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-29475414

RESUMO

Development of meniscus replacements requires in-depth knowledge of the material properties and biomechanical behavior of the native meniscus. The compressive properties are of particular interest in this context, which are often assessed with indentation tests. However, those tests are usually done on isolated tissue specimens ex situ, which could have a significant impact on the results. It was, therefore, the goal of the study to assess the stiffness of the meniscus tissue in situ in porcine specimens and to compare it to that of artificial substitutes. Porcine knees (n = 8) were prepared such that the medial meniscus periphery was exposed and the knees could be mounted in a materials testing machine. The tissue stiffness was than measured on the meniscus periphery using a Shore-A durometer in (1) the unloaded knee, (2) with 500-N tibio-femoral compressive load, and (3) with 500-N tibio-femoral load and the posterior meniscus root detached. The stiffness of the meniscus tissue was significantly increased when tibio-femoral load was applied, while this effect was lost when the meniscus root was cut (average measurements on a 0-100 Shore-A durometer scale: group A, 33.8; group B, 58.4; and group C, 36.2). Polyurethane and collagen meniscus implants showed an inferior stiffness compared to the native meniscus. These findings might be relevant for the material choice in artificial meniscus replacements and the fixation of allografts. Biomechanical testing of isolated tissue specimens could underestimate the effective meniscus tissue stiffness compared to a physiological joint environment.


Assuntos
Fêmur/fisiologia , Fenômenos Mecânicos , Meniscos Tibiais/citologia , Meniscos Tibiais/fisiologia , Animais , Fenômenos Biomecânicos , Teste de Materiais , Suínos , Suporte de Carga
8.
J Cell Mol Med ; 21(11): 3066-3075, 2017 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-28580627

RESUMO

An in-depth knowledge of the native meniscus morphology and biomechanics in its different areas is essential to develop an engineered tissue. Meniscus is characterized by a great regional variation in extracellular matrix components and in vascularization. Then, the aim of this work was to characterize the expression of factors involved in angiogenesis in different areas during meniscus maturation in pigs. The menisci were removed from the knee joints of neonatal, young and adult pigs, and they were divided into the inner, intermediate and outer areas. Vascular characterization and meniscal maturation were evaluated by immunohistochemistry and Western blot analysis. In particular, expression of the angiogenic factor Vascular Endothelial Growth Factor (VEGF) and the anti-angiogenic marker Endostatin (ENDO) was analysed, as well as the vascular endothelial cadherin (Ve-CAD). In addition, expression of Collagen II (COLL II) and SOX9 was examined, as markers of the fibro-cartilaginous differentiation. Expression of VEGF and Ve-CAD had a similar pattern in all animals, with a significant increase from the inner to the outer part of the meniscus. Pooling the zones, expression of both proteins was significantly higher in the neonatal meniscus than in young and adult menisci. Conversely, the young meniscus revealed a significantly higher expression of ENDO compared to the neonatal and adult ones. Analysis of tissue maturation markers showed an increase in COLL II and a decrease in SOX9 expression with age. These preliminary data highlight some of the changes that occur in the swine meniscus during growth, in particular the ensemble of regulatory factors involved in angiogenesis.


Assuntos
Envelhecimento/metabolismo , Colágeno Tipo II/genética , Meniscos Tibiais/metabolismo , Neovascularização Fisiológica/genética , Fator A de Crescimento do Endotélio Vascular/genética , Fatores Etários , Envelhecimento/genética , Animais , Animais Recém-Nascidos , Antígenos CD/genética , Antígenos CD/metabolismo , Caderinas/genética , Caderinas/metabolismo , Condrócitos/citologia , Condrócitos/metabolismo , Colágeno Tipo II/metabolismo , Endostatinas/genética , Endostatinas/metabolismo , Matriz Extracelular/genética , Matriz Extracelular/metabolismo , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Articulações/citologia , Articulações/crescimento & desenvolvimento , Articulações/metabolismo , Meniscos Tibiais/irrigação sanguínea , Meniscos Tibiais/citologia , Fatores de Transcrição SOX9/genética , Fatores de Transcrição SOX9/metabolismo , Suínos , Fator A de Crescimento do Endotélio Vascular/metabolismo
9.
Stem Cells Transl Med ; 6(4): 1237-1248, 2017 04.
Artigo em Inglês | MEDLINE | ID: mdl-28186682

RESUMO

Meniscal cartilage tears are common and predispose to osteoarthritis (OA). Most occur in the avascular portion of the meniscus where current repair techniques usually fail. We described previously the use of undifferentiated autologous mesenchymal stem cells (MSCs) seeded onto a collagen scaffold (MSC/collagen-scaffold) to integrate meniscal tissues in vitro. Our objective was to translate this method into a cell therapy for patients with torn meniscus, with the long-term goal of delaying or preventing the onset of OA. After in vitro optimization, we tested an ovine-MSC/collagen-scaffold in a sheep meniscal cartilage tear model with promising results after 13 weeks, although repair was not sustained over 6 months. We then conducted a single center, prospective, open-label first-in-human safety study of patients with an avascular meniscal tear. Autologous MSCs were isolated from an iliac crest bone marrow biopsy, expanded and seeded into the collagen scaffold. The resulting human-MSC/collagen-scaffold implant was placed into the meniscal tear prior to repair with vertical mattress sutures and the patients were followed for 2 years. Five patients were treated and there was significant clinical improvement on repeated measures analysis. Three were asymptomatic at 24 months with no magnetic resonance imaging evidence of recurrent tear and clinical improvement in knee function scores. Two required subsequent meniscectomy due to retear or nonhealing of the meniscal tear at approximately 15 months after implantation. No other adverse events occurred. We conclude that undifferentiated MSCs could provide a safe way to augment avascular meniscal repair in some patients. Registration: EU Clinical Trials Register, 2010-024162-22. Stem Cells Translational Medicine 2017;6:1237-1248.


Assuntos
Doenças das Cartilagens/terapia , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/fisiologia , Lesões do Menisco Tibial/terapia , Animais , Terapia Baseada em Transplante de Células e Tecidos/métodos , Células Cultivadas , Feminino , Humanos , Técnicas In Vitro , Meniscos Tibiais/citologia , Ovinos , Engenharia Tecidual/métodos , Alicerces Teciduais , Cicatrização/fisiologia
10.
Connect Tissue Res ; 58(6): 562-572, 2017 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-28165810

RESUMO

We previously reported that circulating peripheral blood-borne cells (PBCs) contribute to early-phase meniscal reparative change. Because macrophages and myofibroblasts are important contributors of tissue regeneration, we examined their origin and distribution in the reparative meniscus. Reparative menisci were evaluated at 1, 2, and 4 weeks post-meniscectomy by immunohistochemistry to locate monocytes and macrophages (stained positive for CD68 and CD163), and myofibroblasts (stained positive for αSMA). Of the total number of cells, 13% were CD68+ at 1 week post-meniscectomy, which decreased to 1% by 4 weeks post-meniscectomy; of these, almost half of CD68+ cells (49.4%: 98.8% as PBCs) were green fluorescent protein (GFP)-positive post-meniscectomy (1, 2, and 4 weeks), indicating that the majority of CD68+ cells were derived from PBCs. Of the total cells, 6% were CD163+ at 1 week post-meniscectomy, which decreased to 1% by week 4. Of the CD163+ cells, the majority were GFP-positive (42.5%: 85.0% as PBCs) after 1 week; however, this decreased significantly over time, which indicates that the majority of CD163+ cells are derived from PBCs during the early phase of meniscal reparative change, but are derived from resident cells at later time points. Of the total cells, 38% were αSMA+ at 1 week post-meniscectomy, which decreased to 3% by 4 weeks. The proportion of GFP-positive αSMA+ cells was 2.8% after 1 week, with no significant change over time, which indicates that the majority of αSMA+ cells originated from resident cells. Here, we describe the origin and distribution of macrophages and myofibroblasts during meniscal reparative change.


Assuntos
Antígenos CD/metabolismo , Antígenos de Diferenciação Mielomonocítica/metabolismo , Macrófagos/metabolismo , Meniscos Tibiais/citologia , Menisco/citologia , Receptores de Superfície Celular/metabolismo , Animais , Feminino , Proteínas de Fluorescência Verde/metabolismo , Imuno-Histoquímica/métodos , Meniscectomia/métodos , Ratos Transgênicos , Cicatrização/fisiologia
11.
J Biomech Eng ; 139(3)2017 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-28005131

RESUMO

A nearly avascular tissue, the knee meniscus relies on diffusive transport for nutritional supply to cells. Nutrient transport depends on solute partitioning in the tissue, which governs the amount of nutrients that can enter a tissue. The purpose of the present study was to investigate the effects of mechanical strain, tissue region, and tissue composition on the partition coefficient of glucose in meniscus fibrocartilage. A simple partitioning experiment was employed to measure glucose partitioning in porcine meniscus tissues from two regions (horn and central), from both meniscal components (medial and lateral), and at three levels of compression (0%, 10%, and 20%). Partition coefficient values were correlated to strain level, water volume fraction, and glycosaminoglycan (GAG) content of tissue specimens. Partition coefficient values ranged from 0.47 to 0.91 (n = 48). Results show that glucose partition coefficient is significantly (p < 0.001) affected by compression, decreasing with increasing strain. Furthermore, we did not find a statistically significant effect of tissue when comparing medial versus lateral (p = 0.181) or when comparing central and horn regions (p = 0.837). There were significant positive correlations between tissue water volume fraction and glucose partitioning for all groups. However, the correlation between GAG content and partitioning was only significant in the lateral horn group. Determining how glucose partitioning is affected by tissue composition and loading is necessary for understanding nutrient availability and related tissue health and/or degeneration. Therefore, this study is important for better understanding the transport and nutrition-related mechanisms of meniscal degeneration.


Assuntos
Fibrocartilagem/citologia , Fibrocartilagem/metabolismo , Glucose/metabolismo , Meniscos Tibiais/citologia , Estresse Mecânico , Animais , Fenômenos Biomecânicos , Meniscos Tibiais/metabolismo , Suínos
12.
J Orthop Res ; 35(9): 1966-1972, 2017 09.
Artigo em Inglês | MEDLINE | ID: mdl-27813166

RESUMO

Serious meniscus injuries seldom heal and increase the risk for knee osteoarthritis; thus, there is a need to develop new reparative therapies. In that regard, stimulating tissue regeneration by autologous stem/progenitor cells has emerged as a promising new strategy. We showed previously that migratory chondrogenic progenitor cells (CPCs) were recruited to injured cartilage, where they showed a capability in situ tissue repair. Here, we tested the hypothesis that the meniscus contains a similar population of regenerative cells. Explant studies revealed that migrating cells were mainly confined to the red zone in normal menisci: However, these cells were capable of repopulating defects made in the white zone. In vivo, migrating cell numbers increased dramatically in damaged meniscus. Relative to non-migrating meniscus cells, migrating cells were more clonogenic, overexpressed progenitor cell markers, and included a larger side population. Gene expression profiling showed that the migrating population was more similar to CPCs than other meniscus cells. Finally, migrating cells equaled CPCs in chondrogenic potential, indicating a capacity for repair of the cartilaginous white zone of the meniscus. These findings demonstrate that, much as in articular cartilage, injuries to the meniscus mobilize an intrinsic progenitor cell population with strong reparative potential. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:1966-1972, 2017.


Assuntos
Células-Tronco Adultas/fisiologia , Meniscos Tibiais/citologia , Regeneração , Animais , Bovinos , Movimento Celular , Cabras , Meniscos Tibiais/fisiologia
13.
J Mater Sci Mater Med ; 28(1): 22, 2017 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-28025802

RESUMO

The aim of the current in vitro study was to investigate if tissue surface modification with collagenase and addition of the TGF-ß3 can increase the number of cells present in meniscus tears repaired with the use of newly developed tissue adhesives based on isocyanate-terminated block copolymers. Cylindrical explants were harvested from the inner part of bovine menisci. To simulate a full-thickness tear, the central core of the explants was removed and glued back into the defect, with or without incubation in collagenase solution prior to gluing. The repair constructs were then cultured with or without addition of TGF-ß3, and assessed for their histological appearance. The histological staining of the constructs confirmed that both developed adhesives were not cytotoxic. After 28 days, meniscus cells were present in direct contact with the glues. The addition of TGF-ß3 to the culture medium resulted in the presence of cells that formed a sheath inside the simulated tear and in increased cell numbers at the edges of annulus of the explants. In the group in which the tissue was incubated in collagenase and cultured in medium containing TGF-ß3, thicker layers of cells were observed. These results suggest that repairing the torn meniscus with tissue adhesives after pre-treatment of the tissue with collagenase and stimulation with TGF-ß3 is a very promising treatment method, especially when treating the inner avascular part of the meniscus. Nevertheless, longer-term in vitro and in vivo studies are needed to confirm the beneficial effects of this combination therapy.


Assuntos
Colagenases/química , Lesões do Menisco Tibial/terapia , Adesivos Teciduais/química , Fator de Crescimento Transformador beta3/química , Animais , Materiais Biocompatíveis/química , Bovinos , Movimento Celular , Meios de Cultura , Isocianatos/química , Meniscos Tibiais/citologia , Ruptura/patologia , Espectroscopia de Infravermelho com Transformada de Fourier , Propriedades de Superfície , Engenharia Tecidual/métodos , Cicatrização/efeitos dos fármacos
14.
Cryobiology ; 74: 50-60, 2017 02.
Artigo em Inglês | MEDLINE | ID: mdl-27956221

RESUMO

The development of a long-term storage method for meniscus, a complex tissue of the knee prone to injury, would improve the procedure and outcomes of meniscus transplantation. Cryopreservation uses cryoprotective agents (CPAs) including ethylene glycol (EG) and glycerol to preserve a variety of live tissues, and understanding of the CPA permeation kinetics will be critical in designing a vitrification protocol for meniscus. The purpose of this preliminary study was to understand the loading and unloading behaviours of EG and glycerol in meniscus by observing their efflux. For the main experiment, lateral and medial porcine menisci were incubated with CPA for 24 h at three temperatures (i.e., 4, 22, and 37 °C). Then, the menisci were immersed in 25 ml of X-VIVO™10 and CPA efflux was recorded by monitoring the molality of two consecutive washout solutions at different time points. In a subsequent experiment, menisci were incubated in the CPA solutions for 48 h at 22 °C, and the results were compared to those obtained at 22 °C in the main experiment. Results showed a rapid efflux of CPA from meniscus at the beginning of each wash. With increasing temperature, the amount of CPA efflux (and hence loading) increased. Using 24 h incubation, EG loaded the menisci more completely than glycerol. But after 48 h of incubation, both EG and glycerol achieved approximately the same degree of meniscus loading. This study provides preliminary data that will facilitate future design of experiments aimed at development of meniscus permeation studies.


Assuntos
Criopreservação/métodos , Crioprotetores/farmacologia , Etilenoglicol/farmacologia , Glicerol/farmacologia , Meniscos Tibiais/metabolismo , Vitrificação , Animais , Meniscos Tibiais/citologia , Suínos
15.
Sci Rep ; 6: 28170, 2016 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-27302206

RESUMO

Alginates are important hydrogels for meniscus tissue engineering as they support the meniscal fibrochondrocyte phenotype and proteoglycan production, the extracellular matrix (ECM) component chiefly responsible for its viscoelastic properties. Here, we systematically evaluated four biomedical- and two nonbiomedical-grade alginates for their capacity to provide the best three-dimensional (3-D) microenvironment and to support proteoglycan synthesis of encapsulated human meniscal fibrochondrocytes in vitro. Biomedical-grade, high mannuronic acid alginate spheres (BioLVM, BioMVM) were the most uniform in size, indicating an effect of the purity of alginate on the shape of the spheres. Interestingly, the purity of alginates did not affect cell viability. Of note, only fibrochondrocytes encapsulated in BioMVM alginate produced and retained significant amounts of proteoglycans. Following transplantation in an explant culture model, the alginate spheres containing fibrochondrocytes remained in close proximity with the meniscal tissue adjacent to the defect. The results reveal a promising role of BioMVM alginate to enhance the proteoglycan production of primary human meniscal fibrochondrocytes in a 3-D hydrogel microenvironment. These findings have significant implications for cell-based translational studies aiming at restoring lost meniscal tissue in regions containing high amounts of proteoglycans.


Assuntos
Alginatos/química , Condrócitos/metabolismo , Meniscos Tibiais/citologia , Proteoglicanas/biossíntese , Engenharia Tecidual/métodos , Idoso , Materiais Biocompatíveis , Técnicas de Cultura de Células , Proliferação de Células , Sobrevivência Celular , Ácido Glucurônico/química , Ácidos Hexurônicos/química , Humanos , Pessoa de Meia-Idade , Técnicas de Cultura de Órgãos/métodos
16.
Arthroscopy ; 32(6): 1106-16, 2016 06.
Artigo em Inglês | MEDLINE | ID: mdl-26874799

RESUMO

PURPOSE: To evaluate the effect of 10% human serum (HS), 5% platelet-rich plasma (PRP), and 5% autologous conditioned plasma (ACP) on migration, proliferation, and extracellular matrix (ECM) synthesis of human meniscus cells. METHODS: Cell migration and proliferation on stimulation with HS, PRP, and ACP were assessed by chemotaxis assays and measurement of genomic DNA content. Meniscus cells were cultivated in pellets stimulated with 10% HS, 5% PRP, or 5% ACP. Meniscal ECM formation was evaluated by histochemical staining of collagen type I, type II, and proteoglycans and by analysis of fibrochondrocyte marker gene expression. RESULTS: Human meniscus cells were significantly attracted by all 3 blood-derived products (10% HS and 5% ACP: P = .0001, 5% PRP: P = .0002). Cell proliferation at day 9 was significantly increased on stimulation with 10% HS (P = .0001) and 5% PRP (P = .0002) compared with 5% ACP and controls. Meniscus cell pellet cultures showed the formation of a well-structured meniscal ECM with deposition of collagen type I, type II, and proteoglycans on stimulation with 10% HS, whereas 5% PRP or 5% ACP resulted in the formation of an inhomogeneous and more fibrous ECM. Stimulation with 10% HS and 5% ACP showed a significant induction of fibrochondrocyte marker genes such as aggrecan (HS: P = .0002, ACP: P = .0147), cartilage oligomeric matrix protein (HS: P = .0002, ACP: P = .0005), and biglycan (HS: P = .0002, ACP: P = .0003), whereas PRP showed no inducing effect. CONCLUSIONS: Among all tested blood-derived products, only stimulation with HS showed the formation of a meniscal ECM as well as positive cell proliferating and migrating effects in vitro. Regarding a potential biological repair of nonvascular meniscus lesions, our results may point toward the use of HS as a beneficial augment in regenerative meniscus repair approaches. CLINICAL RELEVANCE: Our findings may suggest that HS might be a beneficial augment for meniscus repair.


Assuntos
Plaquetas/fisiologia , Movimento Celular , Proliferação de Células , Matriz Extracelular/metabolismo , Meniscos Tibiais/citologia , Plasma Rico em Plaquetas/fisiologia , Soro/fisiologia , Idoso , Células Cultivadas , Quimiotaxia , Colágeno Tipo I/metabolismo , Colágeno Tipo II/metabolismo , Feminino , Humanos , Masculino , Meniscos Tibiais/metabolismo , Menisco , Pessoa de Meia-Idade , Proteoglicanas/metabolismo
17.
J Biomech Eng ; 138(3): 4032381, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26720513

RESUMO

Since the meniscus has limited capacity to self-repair, creating a long-lasting meniscus replacement may help reduce the incidence of osteoarthritis (OA) after meniscus damage. As a first step toward this goal, this study evaluated the mechanical integrity of a decellularized, laser drilled (LD) meniscus as a potential scaffold for meniscal engineering. To evaluate the decellularization process, 24 porcine menisci were processed such that one half remained native tissue, while the other half was decellularized in sodium dodecyl sulphate (SDS). To evaluate the laser drilling process, 24 additional menisci were decellularized, with one half remaining intact while the other half was LD. Decellularization did not affect the tensile properties, but had significant effects on the cyclic compressive hysteresis and unconfined compressive stress relaxation. Laser drilling decreased the Young's modulus and instantaneous stress during unconfined stress relaxation and the circumferential ultimate strength during tensile testing. However, the losses in mechanical integrity in the LD menisci were generally smaller than the variance observed between samples, and thus, the material properties for the LD tissue remained within a physiological range. In the future, optimization of laser drilling patterns may improve these material properties. Moreover, reseeding the construct with cells may further improve the mechanical properties prior to implantation. As such, this work serves as a proof of concept for generating decellularized, LD menisci scaffolds for the purposes of meniscal engineering.


Assuntos
Lasers , Fenômenos Mecânicos , Meniscos Tibiais/citologia , Animais , Fenômenos Biomecânicos , Força Compressiva , Teste de Materiais , Estresse Mecânico , Suínos
18.
Int J Mol Sci ; 17(1)2016 Jan 16.
Artigo em Inglês | MEDLINE | ID: mdl-26784189

RESUMO

Despite the susceptibility to frequent intrinsic and extrinsic injuries, especially in the inner zone, the meniscus does not heal spontaneously owing to its poor vascularity. In this study, the effect of platelet-rich plasma (PRP), containing various growth factors, on meniscal mechanisms was examined under normal and post-traumatic inflammatory conditions. Isolated primary meniscal cells of New Zealand white (NZW) rabbits were incubated for 3, 10, 14 and 21 days with PRP(-), 10% PRP (PRP(+)), IL(+) or IL(+)PRP(+). The meniscal cells were collected and examined using reverse-transcription polymerase chain reaction (RT-PCR). Culture media were examined by immunoblot analyses for matrix metalloproteinases (MMP) catabolic molecules. PRP containing growth factors improved the cellular viability of meniscal cells in a concentration-dependent manner at Days 1, 4 and 7. However, based on RT-PCR, meniscal cells demonstrated dedifferentiation, along with an increase in type I collagen in the PRP(+) and in IL(+)PRP(+). In PRP(+), the aggrecan expression levels were lower than in the PRP(-) until Day 21. The protein levels of MMP-1 and MMP-3 were higher in each PRP group, i.e., PRP(+) and IL(+)PRP(+), at each culture time. A reproducible 2-mm circular defect on the meniscus of NZW rabbit was used to implant fibrin glue (control) or PRP in vivo. After eight weeks, the lesions in the control and PRP groups were occupied with fibrous tissue, but not with meniscal cells. This study shows that PRP treatment of the meniscus results in an increase of catabolic molecules, especially those related to IL-1α-induced inflammation, and that PRP treatment for an in vivo meniscus injury accelerates fibrosis, instead of meniscal cartilage.


Assuntos
Desdiferenciação Celular , Metaloproteinase 1 da Matriz/metabolismo , Metaloproteinase 3 da Matriz/metabolismo , Meniscos Tibiais/metabolismo , Plasma Rico em Plaquetas/metabolismo , Agrecanas/genética , Agrecanas/metabolismo , Animais , Condrócitos/citologia , Colágeno/genética , Colágeno/metabolismo , Interleucinas/genética , Interleucinas/metabolismo , Metaloproteinase 1 da Matriz/genética , Metaloproteinase 3 da Matriz/genética , Meniscos Tibiais/citologia , Coelhos
19.
J Orthop Res ; 34(5): 779-90, 2016 05.
Artigo em Inglês | MEDLINE | ID: mdl-26505891

RESUMO

In knee osteoarthritis (OA), concentrations of interleukin (IL)-1ß and tumor necrosis factor (TNF)-α increase in joint tissues and synovial fluid which incite a catabolic cascade and further the progression of OA. Several microRNAs (miRNA) have been associated with apoptosis (miR-16), inflammation (miR-22, miR-146a), and matrix degradation (miR-140, miR-27b) in developed OA or its symptoms. In this study, the time- and concentration-dependent nature of cellular and extracellular miRNAs in synoviocytes, meniscus cells, and chondrocytes as influenced by inflammatory cytokines was investigated. For time-dependent studies, three cell types were stimulated with 10 ng/ml IL-1ß or 50 ng/ml TNF-α for 8, 16, and 24 h. For concentration-dependent studies, chondrocytes were stimulated with a higher level of IL-1ß (20 ng/ml) or TNF-α (100 ng/ml) for 8 h. Cellular and extracellular expressions of miR-22, miR-16, miR-146a, miR-27b, and miR-140 were analyzed by RT-PCR. Time-dependent cellular miRNA expressions were similar across the three cell types with miR-146a significantly up-regulated and miR-27b significantly down-regulated at all time points. However, chondrocytes exhibited a unique extracellular miRNA profile with an increased release rate of miR-27b at 24 h. Our findings support further research into the characterization of miRNAs in synovial fluid for the development of early detection strategies of OA or cartilage injury. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:779-790, 2016.


Assuntos
Condrócitos/metabolismo , Meniscos Tibiais/metabolismo , MicroRNAs/metabolismo , Osteoartrite/metabolismo , Sinoviócitos/metabolismo , Animais , Células Cultivadas , Interleucina-1beta , Meniscos Tibiais/citologia , Suínos , Fator de Necrose Tumoral alfa
20.
Am J Sports Med ; 43(10): 2481-9, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26306780

RESUMO

BACKGROUND: Proteolytic fragments of fibronectin have catabolic effects on cartilage and menisci. Platelet-rich plasma (PRP) is increasingly being used to treat a range of joint conditions, but it is unknown whether PRP influences fibronectin fragment (FN-f) procatabolic activity. HYPOTHESES: The procatabolic activity of FN-f on meniscocytes and articular chondrocytes is attenuated by cotreatment with PRP. STUDY DESIGN: Controlled laboratory study. METHODS: Human meniscocytes were treated with FN-f (30 kDa) with or without PRP coincubation, and gene expression was analyzed by complementary DNA microarray analysis. Validation of altered expression of known and novel chemokine and protease genes was undertaken by real-time polymerase chain reaction (RT-PCR) in articular chondrocytes and meniscocytes. Chemokine release was assayed by enzyme-linked immunosorbent assay, and intracellular pathway signaling was evaluated by Western immunoblotting. RESULTS: Microarray analysis and RT-PCR showed increased expression of matrix metalloproteinase (MMP)1, MMP2, MMP3, MMP9, MMP13, interleukin (IL)-6, IL-8 (CXCL8), CCL5, CCL20, and CXCL10 chemokines in meniscocytes after treatment with FN-f. Upregulation of these genes was significantly attenuated by PRP. Similar results were seen with articular chondrocytes, although no changes in MMP2 or MMP9 levels were identified. PRP-induced suppression of gene expression was associated with activation of Akt and p44/p42. CONCLUSION: PRP treatment attenuates the 30-kDa FN-f-induced expression of a range of proinflammatory chemokines and MMPs, including IL-8, IL-6, CCL20, CCL5, CXCL10, MMP1, MMP3, and MMP13, by both meniscocytes and articular chondrocytes. CLINICAL RELEVANCE: These observations provide support for the use and further trials of PRP in management of cartilage and meniscal injuries.


Assuntos
Cartilagem Articular/metabolismo , Quimiocinas/genética , Condrócitos/metabolismo , Fibronectinas/genética , Regulação da Expressão Gênica , Metaloproteinases da Matriz/genética , Plasma Rico em Plaquetas/fisiologia , Cartilagem Articular/citologia , Quimiocinas/biossíntese , Condrócitos/citologia , DNA/genética , Ensaio de Imunoadsorção Enzimática , Fibronectinas/biossíntese , Humanos , Masculino , Metaloproteinases da Matriz/biossíntese , Meniscos Tibiais/citologia , Meniscos Tibiais/metabolismo , Osteoartrite do Joelho/genética , Osteoartrite do Joelho/metabolismo , Osteoartrite do Joelho/patologia , Reação em Cadeia da Polimerase em Tempo Real
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