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1.
Int J Mol Sci ; 20(18)2019 Sep 11.
Artigo em Inglês | MEDLINE | ID: mdl-31514329

RESUMO

Cartilage repair using tissue engineering is the most advanced clinical application in regenerative medicine, yet available solutions remain unsuccessful in reconstructing native cartilage in its proprietary form and function. Previous investigations have suggested that the combination of specific bioactive elements combined with a natural polymer could generate carrier matrices that enhance activities of seeded stem cells and possibly induce the desired matrix formation. The present study sought to clarify this by assessing whether a chitosan-hyaluronic-acid-based biomimetic matrix in conjunction with adipose-derived stem cells could support articular hyaline cartilage formation in relation to a standard chitosan-based construct. By assessing cellular development, matrix formation, and key gene/protein expressions during in vitro cultivation utilizing quantitative gene and immunofluorescent assays, results showed that chitosan with hyaluronic acid provides a suitable environment that supports stem cell differentiation towards cartilage matrix producing chondrocytes. However, on the molecular gene expression level, it has become apparent that, without combinations of morphogens, in the chondrogenic medium, hyaluronic acid with chitosan has a very limited capacity to stimulate and maintain stem cells in an articular chondrogenic state, suggesting that cocktails of various growth factors are one of the key features to regenerate articular cartilage, clinically.


Assuntos
Tecido Adiposo/citologia , Materiais Biomiméticos/farmacologia , Cartilagem Articular/fisiologia , Quitosana/farmacologia , Condrogênese , Ácido Hialurônico/farmacologia , Células-Tronco/citologia , Cartilagem Articular/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Condrogênese/efeitos dos fármacos , Condrogênese/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Células-Tronco/efeitos dos fármacos , Células-Tronco/ultraestrutura , Tecidos Suporte/química
2.
Biomed Res Int ; 2019: 5141204, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31346519

RESUMO

Patients with bone and cartilage defects due to infection, tumors, and trauma are quite common. Repairing bone and cartilage defects is thus a major problem for clinicians. Autologous and artificial bone transplantations are associated with many challenges, such as limited materials and immune rejection. Bone and cartilage regeneration has become a popular research topic. Inorganic polyphosphate (polyP) is a widely occurring biopolymer with high-energy phosphoanhydride bonds that exists in organisms from bacteria to mammals. Much data indicate that polyP acts as a regulator of gene expression in bone and cartilage tissues and exerts morphogenetic effects on cells involved in bone and cartilage formation. Exposure of these cells to polyP leads to the increase of cytokines that promote the differentiation of mesenchymal stem cells into osteoblasts, accelerates the osteoblast mineralization process, and inhibits the differentiation of osteoclast precursors to functionally active osteoclasts. PolyP-based materials have been widely reported in in vivo and in vitro studies. This paper reviews the current cellular mechanisms and material applications of polyP in bone and cartilage regeneration.


Assuntos
Doenças Ósseas/tratamento farmacológico , Regeneração Óssea/efeitos dos fármacos , Cartilagem/efeitos dos fármacos , Polifosfatos/uso terapêutico , Doenças Ósseas/patologia , Cartilagem/crescimento & desenvolvimento , Cartilagem/patologia , Diferenciação Celular/efeitos dos fármacos , Condrogênese/efeitos dos fármacos , Humanos , Transplante de Células-Tronco Mesenquimais , Osteoblastos/efeitos dos fármacos , Osteogênese/efeitos dos fármacos , Tecidos Suporte/química
3.
Mater Sci Eng C Mater Biol Appl ; 103: 109833, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31349499

RESUMO

The different lineage-specific biological properties of articular cartilage and subchondral bone present a great challenge in the construction of bi-lineage scaffolds for simultaneous osteochondral regeneration. To overcome this challenge, strontium incorporated calcium silicate (Sr-CS) ceramic was prepared for bi-lineage formation of scaffolds in this study. The positive result of Sr-CS in the regeneration of osteochondral defects was first proven by its improved effect on the osteogenesis and chondrogenesis induction of mesenchymal stem cells (MSCs). After that, scaffold-mediated macrophage polarization between classically activated inflammatory macrophages (termed M1Ф) and alternatively activated inflammatory macrophages (termed M2Ф) was assayed to investigate whether the incorporation of Sr into calcium silicate could alter host-to-scaffold immune response. Furthermore, the interactions between Sr-CS pretreated macrophages and MSCs differentiation were performed to prove the enhancement effect of suppressed inflammatory response on osteogenesis and chondrogenesis. In vivo transplantation showed that the Sr-CS scaffolds distinctly improved the regeneration of cartilage and subchondral bone, as compared to the calcium silicate scaffolds. On the one hand, the mechanism attributes to enhancement of strontium on the osteogenic and chondrogenic differentiation of MSCs. On the other hand, the reason can partially be attributed to suppressed synovial inflammatory response, which has improved effects on enhancement of osteogenesis and chondrogenesis. These findings suggest that monophasic Sr-CS scaffolds with a bi-lineage conducive property and an inflammatory response regulatory property represents a viable strategy for simultaneous regeneration of osteochondral defects.


Assuntos
Condrogênese/efeitos dos fármacos , Fatores Imunológicos , Macrófagos/imunologia , Células-Tronco Mesenquimais/imunologia , Osteogênese/efeitos dos fármacos , Estrôncio , Tecidos Suporte/química , Animais , Compostos de Cálcio/química , Compostos de Cálcio/farmacocinética , Compostos de Cálcio/farmacologia , Células Cultivadas , Condrogênese/imunologia , Fatores Imunológicos/química , Fatores Imunológicos/farmacocinética , Fatores Imunológicos/farmacologia , Osteogênese/imunologia , Coelhos , Silicatos/química , Silicatos/farmacocinética , Silicatos/farmacologia , Estrôncio/química , Estrôncio/farmacocinética , Estrôncio/farmacologia
4.
BMC Musculoskelet Disord ; 20(1): 316, 2019 Jul 06.
Artigo em Inglês | MEDLINE | ID: mdl-31279341

RESUMO

BACKGROUND: Synovial mesenchymal stem cells (MSCs) are an attractive cell source for cartilage and meniscus regeneration. The optimum cryopreservation medium has not been determined, but dimethylsulfoxide (DMSO) should be excluded, if possible, because of its toxicity. The purposes of our study were to examine the possible benefits of higher concentrations of serum and the effectiveness of 100% serum (without DMSO) for the cryopreservation of synovial MSCs. METHODS: Human synovium was harvested from the knees of four donors with osteoarthritis during total knee arthroplasty. Synovial MSCs (8 × 105 cells) were suspended in 400 µL medium and used as a Time 0 control. The same number of synovial MSCs was also suspended in 400 µL α-MEM medium containing 10% fetal bovine serum (FBS) (5% DMSO, and 1% antibiotic), 95% FBS (and 5% DMSO), or 100% FBS (no DMSO) and cryopreserved at - 80 °C for 7 days. After thawing, the cell suspensions (1.5 µL; 3 × 103 cells) were cultured in 60 cm2 dishes for 14 days for colony formation assays. Additional 62.5 µL samples of cell suspensions (1.25 × 105 cells) were added to tubes and cultured for 21 days for chondrogenesis assays. RESULTS: Colony numbers were significantly higher in the Time 0 and 95% FBS groups than in the 10% FBS group (n = 24). Colony numbers were much lower in the 100% FBS group than in the other three groups. The cell numbers per dish reflected the colony numbers. Cartilage pellet weights were significantly heavier in the 95% FBS group than in the 10% FBS group, whereas no difference was observed between the Time 0 and the 95% FBS groups (n = 24). No cartilage pellets formed at all in the 100% FBS group. CONCLUSION: Synovial MSCs cryopreserved in 95% FBS with 5% DMSO maintained their colony formation and chondrogenic abilities to the same levels as observed in the cells before cryopreservation. Synovial MSCs cryopreserved in 100% FBS lost their colony formation and chondrogenic abilities.


Assuntos
Condrogênese/efeitos dos fármacos , Criopreservação/métodos , Crioprotetores/farmacologia , Células-Tronco Mesenquimais , Membrana Sinovial/citologia , Idoso , Técnicas de Cultura de Células , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Crioprotetores/química , Dimetil Sulfóxido/química , Dimetil Sulfóxido/farmacologia , Feminino , Humanos , Articulação do Joelho/citologia , Transplante de Células-Tronco Mesenquimais , Osteoartrite/terapia , Soro/química
5.
BMC Complement Altern Med ; 19(1): 155, 2019 Jul 03.
Artigo em Inglês | MEDLINE | ID: mdl-31269947

RESUMO

BACKGROUND: Osteochondral defects mostly occur as a result of trauma or articular degeneration. The poor regenerative ability of articular cartilage remains osteochondral defects are a tricky problem to deal with. The modern treatment strategies mainly focus on cartilage tissue engineering with bioactive materials. In this study, we aimed to develop icariin conditioned serum (ICS) together with hyaluronic acid (HA) and determine their ability in reparing osteochondral tissue in a critical-sized defect in rabbit knees. METHODS: Primary chondrocytes were incubated with serum conditioned with icariin at different concentrations, then cell proliferation rates and glycosaminoglycan (GAG) secretion were detected. Rabbits were treated with intra-articular injection of 0.5 mL normal saline (NS), ICS, HA and ICS + HA in the right knee joint, respectively. ICRS scores were used to assess the macroscopic cartilage regeneration. Histological and immunohistochemical analysis including H&E, Safranin O, toluidine blue and collagen II staining were used to determine the repair of cartilage and the regeneration of chondrocytes. RESULTS: Icariin at a low dose of 0.94 g/kg was identified to have significantly promoted the proliferation of chondrocytes and enhance the secretion of GAG. Femoral condyle from rabbits treated by ICS together with HA was observed to be integrated with native cartilage and more subchondral bone regeneration. ICS together with HA could promote repair of the cartilage defect and increase the neoformation of cartilage. CONCLUSIONS: These results demonstrated the potential of ICS combined with HA to promote reparative response in cartilage defects and the possible application in bioactive material based cartilage regeneration therapies.


Assuntos
Cartilagem Articular/efeitos dos fármacos , Condrogênese/efeitos dos fármacos , Flavonoides/uso terapêutico , Animais , Condrócitos/efeitos dos fármacos , Epimedium , Flavonoides/farmacologia , Ácido Hialurônico/uso terapêutico , Fitoterapia , Coelhos , Soro , Viscossuplementos/uso terapêutico
6.
Int J Nanomedicine ; 14: 4755-4765, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31308656

RESUMO

Background: Many techniques and methods have been used clinically to relieve pain from cartilage repair, but the long-term effect is still unsatisfactory. Purpose: The objective of this study was to form an artificial chondroid tissue gene enhanced tissue engineering system to repair cartilage defects via nanosized liposomes. Methods: Cationic nanosized liposomes were prepared and characterized using transmission electron microscope (TEM) and dynamic laser light scattering (DLS). The rat mesenchymal stem cells (rMSCs) were isolated, cultivated, and induced by SRY (Sex-Determining Region Y)-Box 9 (Sox9) via cationic nanosized liposomes. The induced rMSCs were mixed with a thermo-sensitive chitosan hydrogel and subcutaneously injected into the nude mice. Finally, the newly-formed chondroid tissue obtained in the injection parts, and the transparent parts were detected by HE, collagen II, and safranin O. Results: It was found that the presently prepared cationic nanosized liposomes had the diameter of 85.76±3.48 nm and the zeta potential of 15.76±2.1 mV. The isolated rMSCs proliferation was fibroblast-like, with a cultivated confluence of 90% confluence in 5-8 days, and stained positive for CD29 and CD44 while negative for CD34 and CD45. After transfection with cationic nanosized liposomes, we observed changes of cellular morphology and a higher expression of SOX9 compared with control groups, which indicated that rMSCs could differentiate into chondrocyte in vitro. By mixing transfected rMSCs with the thermo-sensitive hydrogel of chitosan in nude mice, chondroid tissue was successfully obtained, demonstrating that rMSCs can differentiate into chondrogenic cells in vivo. Conclusion: This study explored new ways to improve the quality of tissue engineered cartilage, thus accelerating clinical transformation and reducing patient pain.


Assuntos
Condrogênese , Técnicas de Transferência de Genes , Nanopartículas/química , Engenharia Tecidual/métodos , Animais , Cátions , Diferenciação Celular , Forma Celular , Células Cultivadas , Condrogênese/efeitos dos fármacos , Géis , Lipossomos/química , Lipossomos/ultraestrutura , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Camundongos Nus , Ratos Sprague-Dawley , Fatores de Transcrição SOX9/metabolismo , Temperatura Ambiente
7.
Toxicol Lett ; 314: 18-26, 2019 Oct 10.
Artigo em Inglês | MEDLINE | ID: mdl-31299270

RESUMO

Epidemiological investigations indicate that effects related to prenatal adverse environments on the organs of the offspring could continue to adulthood. This study intends to confirm that prenatal nicotine exposure (PNE) increases the susceptibility of osteoarthritis (OA) in the male offspring, and to explore the potential intrauterine programming mechanism. During pregnancy, rats were divided into a PNE group and a control group. After birth, rats were given a high-fat diet for 6 months and long-distance running for 6 weeks. The rats were euthanized at 18 months after birth (PM18) and on gestational day 20 (GD20), respectively. Knee joints were collected for histochemistry, immunohistochemistry, and quantitative polymerase chain reaction (qPCR) assays. Histological analyses and the Mankin's score showed increased cartilage destruction and accelerated OA progression in adult offspring from the PNE group. Immunohistochemistry results showed decreased expression of transforming growth factor beta (TGFß) signaling pathway. Furthermore, the expression of apoptosis factors (caspase-3 and caspase-8), inflammatory factors [interleukin (IL)-1, IL-6] and matrix degradation enzymes [matrix metalloproteinase (MMP)-3, MMP-13] were also significantly increased. Traced back to the intrauterine period, it was found that the number of chondrocytes and the contents of Col2A1 and aggrecan in the matrix in the PNE group were decreased. And, the expression of the TGFß signaling pathway was inhibited. These results suggested that PNE enhanced the susceptibility of OA in male elderly offspring rats by down-regulating TGFß signaling, which increased articular cartilage local inflammation, matrix degradation, and cell apoptosis. This study confirmed the developmental origin of OA, and clarified the congenital and the living environment impact on the occurrence and development of OA. Our findings provide a theoretical and experimental basis for OA early prevention.


Assuntos
Articulações/efeitos dos fármacos , Nicotina/toxicidade , Agonistas Nicotínicos/toxicidade , Osteoartrite/induzido quimicamente , Efeitos Tardios da Exposição Pré-Natal , Transdução de Sinais , Fator de Crescimento Transformador beta/metabolismo , Fatores Etários , Agrecanas/metabolismo , Animais , Apoptose/efeitos dos fármacos , Caspase 3/metabolismo , Caspase 8/metabolismo , Condrócitos/efeitos dos fármacos , Condrócitos/metabolismo , Condrócitos/patologia , Condrogênese/efeitos dos fármacos , Colágeno Tipo II/metabolismo , Feminino , Idade Gestacional , Interleucina-1/metabolismo , Interleucina-6/metabolismo , Articulações/metabolismo , Articulações/patologia , Masculino , Exposição Materna , Metaloproteinase 13 da Matriz/metabolismo , Metaloproteinase 3 da Matriz/metabolismo , Osteoartrite/metabolismo , Osteoartrite/patologia , Gravidez , Ratos Wistar , Fatores de Risco , Fatores Sexuais
8.
Biofabrication ; 11(4): 044101, 2019 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-31151123

RESUMO

Osteochondral (OC) tissue is a biphasic material comprised of articular cartilage integrated atop subchondral bone. Damage to this tissue is highly problematic, owing to its intrinsic inability to regenerate functional tissue in response to trauma or disease. Further, the function of the tissue is largely conferred by its compartmentalized zonal microstructure and composition. Current clinical treatments fail to regenerate new tissue that recapitulates this zonal structure. Consequently, regenerated tissue often lacks long-term stability. To address this growing problem, we propose the development of tissue engineered biomaterials that mimic the zonal cartilage organization and extracellular matrix composition through the use of a microfluidic printing head bearing a mixing unit and incorporated into an extrusion-based bioprinter. The system is devised so that multiple bioinks can be delivered either individually or at the same time and rapidly mixed to the extrusion head, and finally deposited through a coaxial nozzle. This enables the deposition of either layers or continuous gradients of chemical, mechanical and biological cues and fabrication of scaffolds with very high shape fidelity and cell viability. Using such a system we bioprinted cell-laden hydrogel constructs recapitulating the layered structure of cartilage, namely, hyaline and calcified cartilage. The construct was assembled out of two bioinks specifically formulated to mimic the extracellular matrices present in the targeted tissues and to ensure the desired biological response of human bone marrow-derived mesenchymal stem cells and human articular chondrocytes. Homogeneous and gradient constructs were thoroughly characterized in vitro with respect to long-term cell viability and expression of hyaline and hypertrophic markers by means of real-time quantitative PCR and immunocytochemical staining. After 21 days of in vitro culture, we observed production of zone-specific matrix. The PCR analysis demonstrated upregulated expression of hypertrophic markers in the homogenous equivalent of calcified cartilage but not in the gradient heterogeneous construct. The regenerative potential was assessed in vivo in a rat model. The histological analysis of surgically damaged rat trochlea revealed beneficial effect of the bioprinted scaffolds on regeneration of OC defect when compared to untreated control.


Assuntos
Bioimpressão , Cartilagem Articular/patologia , Hidrogéis/farmacologia , Microfluídica/instrumentação , Impressão Tridimensional , Regeneração , Animais , Cartilagem Articular/efeitos dos fármacos , Condrogênese/efeitos dos fármacos , Humanos , Implantes Experimentais , Tinta , Masculino , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Ratos Wistar , Regeneração/efeitos dos fármacos
9.
Cells ; 8(6)2019 06 25.
Artigo em Inglês | MEDLINE | ID: mdl-31242641

RESUMO

Recent studies highlighting mesenchymal stem cell (MSC) epigenetic memory suggest that a different differentiation medium may be required depending on the tissue of origin. As synovial-derived stem cells (SDSCs) attract interest we aimed to investigate the influence of TGF-ß1, BMP-2 and dexamethasone on SDSC chondrogenesis in vitro. We demonstrate that dexamethasone-free medium led to enhanced chondrogenic differentiation at both the mRNA and matrix level. The greatest COL2A1/COL10A1 ratio was detected in cells exposed to a combination medium containing 10 ng/mL BMP-2 and 1 ng/mL TGF-ß1 in the absence of dexamethasone, and this was reflected in the total amount of glycosaminoglycans produced. In summary, dexamethasone-free medium containing BMP-2 and TGF-ß1 may be the most suitable when using SDSCs for cartilage tissue regeneration.


Assuntos
Proteína Morfogenética Óssea 2/farmacologia , Condrogênese/efeitos dos fármacos , Dexametasona/farmacologia , Células-Tronco/citologia , Membrana Sinovial/citologia , Fator de Crescimento Transformador beta1/farmacologia , Adulto , Colágeno Tipo II/metabolismo , DNA/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Glicosaminoglicanos/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , Adulto Jovem
10.
Int J Mol Sci ; 20(13)2019 Jun 26.
Artigo em Inglês | MEDLINE | ID: mdl-31248037

RESUMO

In recent years, first evidences emerged that sympathetic neurotransmitters influence osteoarthritis (OA) manifestation. Joint-resident stem cells might contribute to cartilage repair, however, their chondrogenic function is reduced. The neurotransmitter norepinephrine (NE) was detected in the synovial fluid of trauma and OA patients. Therefore, the aim of this study was to analyse how NE influences the chondrogenesis of synovial adipose tissue-derived stem cells (sASCs). sASCs were isolated from knee-OA patients synovia. After adrenoceptor (AR) expression analysis, proliferation and chondrogenic differentiation in presence of NE and/or α- and ß-AR antagonist were investigated. Cell count, viability, chondrogenic and hypertophic gene expression, sulfated glycosaminoglycan (sGAG) and type II collagen content were determined. Key AR-dependent signaling (ERK1/2, PKA) was analyzed via western blot. sASC expressed α1A-, α1B-, α2A-, α2B-, α2C-, and ß2-AR in monolayer and pellet culture. NE did not affect proliferation and viability, but 10-7 and 10-6 M NE significantly reduced sGAG and type II collagen content as well as ERK1/2 phosphorylation. These effects were fully reversed by yohimbine (α2-AR antagonist). Our study confirms the important role of NE in sASC chondrogenic function and provides new insights in OA pathophysiology. Future studies might help to develop novel therapeutic options targeting neuroendocrine pathways for OA treatment.


Assuntos
Tecido Adiposo/citologia , Condrogênese/efeitos dos fármacos , Norepinefrina/farmacologia , Receptores Adrenérgicos alfa 2/metabolismo , Células-Tronco/citologia , Células-Tronco/efeitos dos fármacos , Células-Tronco/metabolismo , Membrana Sinovial/citologia , Antagonistas de Receptores Adrenérgicos alfa 2/farmacologia , Biomarcadores , Diferenciação Celular , Expressão Gênica , Humanos , Imuno-Histoquímica , Imunofenotipagem , Sistema de Sinalização das MAP Quinases , Osteoartrite do Joelho/tratamento farmacológico , Osteoartrite do Joelho/etiologia , Osteoartrite do Joelho/metabolismo , Osteoartrite do Joelho/patologia
11.
Int J Biol Macromol ; 136: 616-624, 2019 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-31207331

RESUMO

Cold Atmospheric Plasma (CAP) is used as a promising method in surface modification for immobilization of chondroitin sulfate functional biomacromolecules on PCL nanofibrous substrates for cartilage tissue engineering. The GAG-grafted scaffolds are able to successfully support the attachment and proliferation of mesenchymal stem cells (MSCs). The seeded scaffolds show the chondro-differentiation of MSCs during a 21-days cell culture in a non-differential medium. Expression of SOX9, Collagen10 and Collagen2 proved the chondro-inductive effect of GAG-grafted scaffolds. Besides, no external chondro-genic differential agent was used in the differentiation of MSCs to chondrocyte. The cells passed the last phase of chondrogenesis after 14 days of incubation. Thus, the GAG-fabricated fibrous scaffolds using CAP are potential candidates for cartilage tissue engineering.


Assuntos
Diferenciação Celular/efeitos dos fármacos , Condrogênese/efeitos dos fármacos , Sulfatos de Condroitina/química , Sulfatos de Condroitina/farmacologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Nanofibras/química , Poliésteres/química , Condrócitos/citologia , Condrócitos/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Teste de Materiais , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo
12.
BMC Musculoskelet Disord ; 20(1): 257, 2019 May 29.
Artigo em Inglês | MEDLINE | ID: mdl-31138200

RESUMO

BACKGROUND: Different substances are combined to compensate for each other's drawbacks and create an appropriate biomaterial. A novel Polyvinyl alcohol (PVA)/chitosan (CS) porous hydrogel was designed and applied to the treatment of osteochondral defects. METHODS: Hydrogels of various PVA/CS ratios were tested for physiochemical and mechanical properties in addition to cytotoxicity and biocompatibility. The hydrogels with the best PVA/CS ratio were used in the animal study. Osteochondral defects were created at the articular cartilage of 18 rabbits. They were assigned to different groups randomly (n = 6 per group): the osteochondral defect only group (control group), the osteochondral defect treated with hydrogel group (HG group), and the osteochondral defect treated with hydrogel loaded with bone marrow mesenchymal stem cells (BMSCs) group (HG-BMSCs group). The cartilage was collected for macro-observation and histological evaluation at 12 weeks after surgery. RESULTS: The Hydrogel with PVA/CS ratio of 6:4 exhibited the best mechanical properties; it also showed stable physical and chemical properties with porosity and over 90% water content. Furthermore, it demonstrated no cytotoxicity and was able to promote cell proliferation. The HG-BMSCs group achieved the best cartilage healing. CONCLUSIONS: The novel PVA/CS porous composite hydrogel could be a good candidate for a tissue engineering material in cartilage repair.


Assuntos
Materiais Biocompatíveis/efeitos adversos , Doenças das Cartilagens/terapia , Hidrogéis/efeitos adversos , Transplante de Células-Tronco Mesenquimais , Tecidos Suporte/efeitos adversos , Animais , Doenças das Cartilagens/patologia , Cartilagem Articular/citologia , Cartilagem Articular/lesões , Cartilagem Articular/fisiologia , Proliferação de Células/efeitos dos fármacos , Quitosana/administração & dosagem , Quitosana/efeitos adversos , Condrogênese/efeitos dos fármacos , Terapia Combinada/efeitos adversos , Terapia Combinada/métodos , Modelos Animais de Doenças , Humanos , Hidrogéis/administração & dosagem , Masculino , Teste de Materiais , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/fisiologia , Álcool de Polivinil/administração & dosagem , Álcool de Polivinil/efeitos adversos , Porosidade , Coelhos , Regeneração/efeitos dos fármacos , Testes de Toxicidade , Resultado do Tratamento , Cicatrização/efeitos dos fármacos , Cicatrização/fisiologia
13.
Biomater Sci ; 7(8): 3178-3189, 2019 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-31143889

RESUMO

Cholesterol and lipid metabolism are associated with osteoarthritis (OA) in human cartilage. High cholesterol levels in OA chondrocytes leads to decreased membrane fluidity and blocks the signaling cascade associated with the expression of chondrogenic genes. It is known that bile acid plays a role in regulating cholesterol homeostasis and the digestion of fats in the human body. Tauroursodeoxycholic acid (TUDCA), as a member of the bile acid family, also aids in the transport of cellular cholesterol. In this study, we hypothesized that TUDCA might be able to promote the restoration of OA cartilage by reducing membrane cholesterol levels in OA chondrocytes and by stimulating the chondrogenic signaling cascade. To assess this hypothesis, we investigated the effects of TUDCA on degenerated chondrocytes isolated from patients with OA. Importantly, treatment with TUDCA at sub-micellar concentrations (2500 µM) significantly increased cell proliferation and Cyclin D1 expression compared with the controls. In addition, the expression of chondrogenic marker genes (SOX9, COL2, and ACAN), proteins (SOX9 and COL2), and glycosaminoglycan (Chondroitin sulfate) was much higher in the TUDCA-treated group compared to the controls. We also found that TUDCA treatment significantly reduced the intracellular cholesterol levels in the chondrocytes and increased membrane fluidity. Furthermore, the stability of TGF receptor 1 and activity of focal adhesion proteins were also increased following TUDCA treatment. Together, these results demonstrated that TUDCA could be used as an alternative treatment for the restoration of OA cartilage.


Assuntos
Colesterol/metabolismo , Condrócitos/efeitos dos fármacos , Espaço Intracelular/efeitos dos fármacos , Espaço Intracelular/metabolismo , Fluidez de Membrana/efeitos dos fármacos , Osteoartrite/tratamento farmacológico , Ácido Tauroquenodesoxicólico/farmacologia , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Condrócitos/metabolismo , Condrócitos/patologia , Condrogênese/efeitos dos fármacos , Relação Dose-Resposta a Droga , Adesões Focais/efeitos dos fármacos , Humanos , Osteoartrite/metabolismo , Osteoartrite/patologia , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Transdução de Sinais/efeitos dos fármacos , Ácido Tauroquenodesoxicólico/química , Ácido Tauroquenodesoxicólico/uso terapêutico
14.
ACS Appl Mater Interfaces ; 11(16): 14608-14618, 2019 Apr 24.
Artigo em Inglês | MEDLINE | ID: mdl-30938503

RESUMO

Continuous delivery of growth factors to the injury site is crucial to creating a favorable microenvironment for cartilage injury repair. In the present study, we fabricated a novel sustained-release scaffold, stromal-derived factor-1α (SDF-1α)/transforming growth factor-ß1 (TGF-ß1)-loaded silk fibroin-porous gelatin scaffold (GSTS). GSTS persistently releases SDF-1α and TGF-ß1, which enhance cartilage repair by facilitating cell homing and chondrogenic differentiation. Scanning electron microscopy showed that GSTS is a porous microstructure and the protein release assay demonstrated the sustainable release of SDF-1α and TGF-ß1 from GSTS. Bone marrow-derived mesenchymal stem cells (MSCs) maintain high in vitro cell activity and excellent cell distribution and phenotype after seeding into GSTS. Furthermore, MSCs acquired enhanced chondrogenic differentiation capability in the TGF-ß1-loaded scaffolds (GSTS or GST: loading TGF-ß1 only) and the conditioned medium from SDF-1α-loaded scaffolds (GSTS or GSS: loading SDF-1α only) effectively promoted MSCs migration. GSTS was transplanted into the osteochondral defects in the knee joint of rats, and it could promote cartilage regeneration and repair the cartilage defects at 12 weeks after transplantation. Our study shows that GSTS can facilitate in vitro MSCs homing, migration, chondrogenic differentiation and SDF-1α and TGF-ß1 have a synergistic effect on the promotion of in vivo cartilage forming. This SDF-1α and TGF-ß1 releasing GSTS have promising therapeutic potential in cartilage repair.


Assuntos
Cartilagem , Quimiocina CXCL12 , Condrogênese/efeitos dos fármacos , Fibroínas , Gelatina , Fator de Crescimento Transformador beta1 , Animais , Cartilagem/lesões , Cartilagem/metabolismo , Cartilagem/patologia , Diferenciação Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Quimiocina CXCL12/química , Quimiocina CXCL12/farmacocinética , Quimiocina CXCL12/farmacologia , Preparações de Ação Retardada/química , Preparações de Ação Retardada/farmacocinética , Preparações de Ação Retardada/farmacologia , Fibroínas/química , Fibroínas/farmacocinética , Fibroínas/farmacologia , Gelatina/química , Gelatina/farmacocinética , Gelatina/farmacologia , Masculino , Porosidade , Ratos , Fator de Crescimento Transformador beta1/química , Fator de Crescimento Transformador beta1/farmacocinética , Fator de Crescimento Transformador beta1/farmacologia
15.
Biofabrication ; 11(3): 035016, 2019 05 03.
Artigo em Inglês | MEDLINE | ID: mdl-30943457

RESUMO

One promising strategy to reconstruct osteochondral defects relies on 3D bioprinted three-zonal structures comprised of hyaline cartilage, calcified cartilage, and subchondral bone. So far, several studies have pursued the regeneration of either hyaline cartilage or bone in vitro while-despite its key role in the osteochondral region-only few of them have targeted the calcified layer. In this work, we present a 3D biomimetic hydrogel scaffold containing ß-tricalcium phosphate (TCP) for engineering calcified cartilage through a co-axial needle system implemented in extrusion-based bioprinting process. After a thorough bioink optimization, we showed that 0.5% w/v TCP is the optimal concentration forming stable scaffolds with high shape fidelity and endowed with biological properties relevant for the development of calcified cartilage. In particular, we investigate the effect induced by ceramic nano-particles over the differentiation capacity of bioprinted bone marrow-derived human mesenchymal stem cells in hydrogel scaffolds cultured up to 21 d in chondrogenic media. To confirm the potential of the presented approach to generate a functional in vitro model of calcified cartilage tissue, we evaluated quantitatively gene expression of relevant chondrogenic (COL1, COL2, COL10A1, ACAN) and osteogenic (ALPL, BGLAP) gene markers by means of RT-qPCR and qualitatively by means of fluorescence immunocytochemistry.


Assuntos
Bioimpressão , Calcificação Fisiológica/efeitos dos fármacos , Fosfatos de Cálcio/química , Cartilagem Hialina/fisiologia , Hidrogéis/farmacologia , Modelos Biológicos , Impressão Tridimensional , Engenharia Tecidual/métodos , Condrogênese/efeitos dos fármacos , Proteínas da Matriz Extracelular/metabolismo , Humanos , Cartilagem Hialina/efeitos dos fármacos , Tinta , Células-Tronco Mesenquimais/citologia , Imagem Óptica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Temperatura Ambiente , Tecidos Suporte/química , Viscosidade
16.
Arq. bras. med. vet. zootec. (Online) ; 71(2): 509-520, mar.-abr. 2019. graf, ilus
Artigo em Inglês | LILACS, VETINDEX | ID: biblio-1011276

RESUMO

The aim of this study was to evaluate the effect of concentrations of caffeine on the viability, synthesis activity and gene expression in cultures of chondrocytes. Extracted articular cartilage from the femurs and tibias of 15 Wistar rats at three days old to isolate chondrocytes. Chondrocytes were cultured in chondrogenic medium (control) or supplemented with caffeine (0.5, 1.0, 2.0mM). Cell viability, alkaline phosphatase activity and collagen synthesis were assessed using colorimetric assays at 7, 14, 21 days. The chondrocyte cultures of all groups grown under coverslips were stained with hematoxylin-eosin to determine the percentage of cells/field and with PAS, safranin O, alcian blue to determine the percentage of matrix chondrogenic/field at 21 days. The expressions of gene transcripts for aggrecan, collagen-II, Sox-9, Runx-2 and alkaline phosphatase were also evaluated by RT-PCR at 21 days. The means were compared using Student-Newman-Keuls. Caffeine significantly reduced the conversion of MTT to formazan, percentage of cells/field, collagen synthesis, alkaline phosphatase activity, synthesis of PAS+, safranin O+ and alcian blue+ chondrogenic matrix, and the expression of aggrecan, Sox-9 and II collagen. It is concluded that caffeine at concentrations of 0.5, 1.0, 2.0mM has a direct inhibitory effect on chondrogenesis in cultures of chondrocytes from rats.(AU)


O objetivo deste estudo foi avaliar o efeito direto de concentrações de cafeína sobre a viabilidade, atividade de síntese e expressão gênica em culturas de condrócitos de ratos. As cartilagens dos fêmures e tíbias de 15 ratos Wistar com três dias foram extraídas para isolamento de condrócitos. Os condrócitos foram cultivados em meio condrogênico (controle) ou em meio acrescido de diferentes concentrações de cafeína (0,5, 1,0, 2,0mM). Foram avaliadas a viabilidade celular, a atividade da fosfatase alcalina e a síntese de colágeno por ensaios colorimétricos aos sete, 14 e 21 dias. Condrócitos cultivados sob lamínulas foram corados pela hematoxilina e eosina, para se determinar a porcentagem de células/campo, e pelo PAS, safranina O, alcian Blue, para se determinar a porcentagem de matriz condrogênica/campo aos 21 dias. Foi avaliada a expressão de transcriptos gênicos para Sox-9, Runx-2, agrecano, colágeno-II e fosfatase alcalina por qRT-PCR, aos 21 dias. As médias foram comparadas pelo Student-Newman-Keuls. A cafeína reduziu significativamente o MTT em cristais de formazan, a porcentagem de células/campo, a síntese de colágeno, a atividade da fosfatase alcalina e a síntese de matriz condrogênica PAS+, safranina O+, alcian blue+ e expressão de Sox-9 e colágeno-II. Conclui-se que a cafeína, nas concentrações de 0,5, 1,0, 2,0mM, apresenta efeito inibidor direto sobre a condrogênese em culturas de condrócitos de ratos.(AU)


Assuntos
Animais , Feminino , Ratos , Cafeína , Cartilagem Articular/efeitos dos fármacos , Condrócitos/efeitos dos fármacos , Condrogênese/efeitos dos fármacos
17.
Eur Cell Mater ; 37: 214-232, 2019 03 22.
Artigo em Inglês | MEDLINE | ID: mdl-30900738

RESUMO

Nasal chondrocytes (NCs) have gained increased recognition for cartilage tissue regeneration. To assess NCs as a source for cell therapy treatment of intervertebral disc (IVD) degeneration, tissue-forming properties of NCs under physiological conditions mimicking the degenerated IVD were compared to those of mesenchymal stromal cells (MSCs) and articular chondrocytes (ACs), two cell sources presently used in clinical trials. Cells were cultured in a combination of low glucose, hypoxia, acidity and inflammation for 28 d. Depending on the conditions, cells were either cultured in the absence of instructive growth factors or underwent chondrogenic instructional priming by addition of transforming growth factor ß1 (TGFß1) for the first 7 d. Histology, immunohistochemistry, biochemistry, enzyme-linked immunosorbent assay (ELISA) and quantitative real-time reverse transcriptase-polymerase chain reaction (qRT-PCR) analyses demonstrated limited cell maintenance and accumulation of cartilaginous extracellular matrix for MSCs in IVD conditions. ACs maintained a steady accumulation of glycosaminoglycans (GAGs) throughout all non-acidic conditions, with and without priming, but could not synthesise type II collagen (Col2). NCs accumulated both GAGs and Col2 in all non-acidic conditions, independent of priming, whereas MSCs strongly diminished their GAG and Col2 accumulation in an inflamed environment. Supplementation with inflammatory cytokines or an acidic environment affected NCs to a lower extent than ACs or MSCs. The data, overall indicating that in an inflamed IVD environment NCs were superior to ACs and MSCs, encourage further assessment of NCs for treatment of degenerative disc disease.


Assuntos
Condrócitos/patologia , Degeneração do Disco Intervertebral/patologia , Nariz/patologia , Adolescente , Adulto , Biomarcadores/metabolismo , Cartilagem Articular/efeitos dos fármacos , Cartilagem Articular/patologia , Condrócitos/efeitos dos fármacos , Condrogênese/efeitos dos fármacos , DNA/metabolismo , Matriz Extracelular/efeitos dos fármacos , Matriz Extracelular/metabolismo , Feminino , Glucose/farmacologia , Glicosaminoglicanos/metabolismo , Humanos , Inflamação/patologia , Masculino , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/metabolismo , Pessoa de Meia-Idade , Núcleo Pulposo/patologia , Oxigênio/farmacologia , Receptores de Citocinas/metabolismo , Fator de Crescimento Transformador beta1/farmacologia , Adulto Jovem
18.
Int J Mol Sci ; 20(4)2019 Feb 22.
Artigo em Inglês | MEDLINE | ID: mdl-30813231

RESUMO

Recent osteochondral repair strategies highlight the promise of mesenchymal progenitors, an accessible stem cell source with osteogenic and chondrogenic potential, used in conjunction with biomaterials for tissue engineering. For this, regenerative medicine approaches require robust models to ensure selected cell populations can generate the desired cell type in a reproducible and measurable manner. Techniques for in vitro chondrogenic differentiation are well-established but largely qualitative, relying on sample staining and imaging. To facilitate the in vitro screening of pro-chondrogenic treatments, a 3D micropellet culture combined with three quantitative GAG assays has been developed, with a fourth parallel assay measuring sample content to enable normalisation. The effect of transforming growth factor beta (TGF-ß) used to validate this culture format produced a measurable increase in proteoglycan production in the parallel assays, in both 2D and 3D culture configurations. When compared to traditional micropellets, the monolayer format appeared less able to detect changes in cell differentiation, however in-well 3D cultures displayed a significant differential response. Effects on collagen 2 expression confirmed these observations. Based on these results, a microplate format was optimised for 3D culture, in a high-throughput in-well configuration. This model showed improved sensitivity and confirmed the 3D micropellet in-well quantitative assays as an effective differentiation format compatible with streamlined, high-throughput chondrogenic screens.


Assuntos
Bioensaio/métodos , Diferenciação Celular , Condrogênese , Modelos Biológicos , Células-Tronco/citologia , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Condrogênese/efeitos dos fármacos , Colágeno Tipo II/metabolismo , Genes Reporter , Glucose/farmacologia , Humanos , Células-Tronco/efeitos dos fármacos
19.
Mater Sci Eng C Mater Biol Appl ; 99: 1123-1132, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30889646

RESUMO

Osteonecrosis of the femoral head (ONFH) results in collapse of the femoral head and rapid destruction of the hip joint. The repair of post-collapse articular cartilage and subchondral bone is challenging. We interrupted the blood supply to the femoral head and established a full-thickness articular defect animal model after ONFH was determined via X-ray. Porous tantalum and a Bio-Gide collagen membrane, co-cultured with bone marrow mesenchymal stem cells (BMSCs) in vitro, were implanted into the defect zone to repair the full-thickness articular defect. Hyaline cartilage was detected on top of the tantalum near the edge of the defect 12 weeks post-operatively. Porous tantalum and a Bio-Gide collagen membrane with BMSCs may repair full-thickness articular defects if the blood supply can be reconstructed in the post-collapse stage of ONFH.


Assuntos
Cartilagem Articular/patologia , Colágeno/farmacologia , Necrose da Cabeça do Fêmur/terapia , Membranas Artificiais , Células-Tronco Mesenquimais/citologia , Tantálio/farmacologia , Animais , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Condrócitos/citologia , Condrócitos/efeitos dos fármacos , Condrogênese/efeitos dos fármacos , Técnicas de Cocultura , Necrose da Cabeça do Fêmur/patologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/ultraestrutura , Osteogênese/efeitos dos fármacos , Fenótipo , Coelhos
20.
Mater Sci Eng C Mater Biol Appl ; 99: 1362-1373, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30889670

RESUMO

The regeneration of cartilage is a challenging problem for lack of innate abilities to mount a sufficient healing response. Kartogenin (KGN), an emerging chondroinductive non-protein small molecule, bound to the surface of the ultrasmall super-paramagnetic iron-oxide (USPIO) by innovational one-step technology, followed by being incorporated into the cross-linking collagen/cellulose nanocrystals (Col/CNC) bioactive scaffolds to stimulate an appropriate microenvironment for the growth and differentiation of bone marrow-derived mesenchymal stem cells (BMSCs), thus facilitating the formation of chondrocyte. Herein, USPIO not only served as a carrier for small molecule drugs, but also as MRI contrast agents, which can non-invasively monitor the degradation of the scaffolds and the self-repair capacity of cartilage. In vitro studies showed that the KGN could release from the composite scaffolds in a sustained and stable manner and promote the chondrogenic differentiation of BMSCs based on UV spectrophotometry test, and specific markers analysis. Of note, USPIO labeled composite scaffolds retained their stability without loss of relaxation rate the composite scaffolds can be a promising biomaterials for cartilage repair, with the function of noninvasive visualization and semiquantitative analysis of scaffolds degradation and neocartilage.


Assuntos
Anilidas/farmacologia , Cartilagem/fisiologia , Celulose/síntese química , Colágeno/síntese química , Dextranos/química , Nanopartículas de Magnetita/química , Nanopartículas/química , Ácidos Ftálicos/farmacologia , Regeneração/efeitos dos fármacos , Tecidos Suporte/química , Animais , Materiais Biocompatíveis/farmacologia , Cartilagem/efeitos dos fármacos , Bovinos , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Celulose/química , Condrogênese/efeitos dos fármacos , Condrogênese/genética , Colágeno/química , Liberação Controlada de Fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Imagem por Ressonância Magnética , Nanopartículas/ultraestrutura
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