RESUMO
Osteoarthritis (OA) is a degenerative and heterogeneous disease that affects all types of joint structures. Current clinical treatments are only symptomatic and do not manage the degenerative process in animals or humans. One of the new orthobiological treatment strategies being developed to treat OA is the use of drug delivery systems (DDS) to release bioactive molecules over a long period of time directly into the joint to limit inflammation, control pain, and reduce cartilage degradation. Two vasoactive peptides, endothelin-1 and bradykinin, play important roles in OA pathogenesis. In this study, we investigated the effects of two functionalized nanogels as DDS. We assessed the effect of chitosan functionalized with a type A endothelin receptor antagonist (BQ-123-CHI) and/or hyaluronic acid functionalized with a type B1 bradykinin receptor antagonist (R-954-HA). The biocompatibility of these nanogels, alone or in combination, was first validated on equine articular chondrocytes cultured under different oxic conditions. Further, in an OA equine organoid model via induction with interleukin-1 beta (IL-1ß), a combination of BQ-123-CHI and R-954-HA (BR5) triggered the greatest decrease in inflammatory and catabolic markers. In basal and OA conditions, BQ-123-CHI alone or in equimolar combinations with R-954-HA had weak pro-anabolic effects on collagens synthesis. These new nanogels, as part of a composite DDS, show promising attributes for treating OA.
Assuntos
Cartilagem Articular , Osteoartrite , Animais , Antagonistas dos Receptores da Bradicinina/metabolismo , Antagonistas dos Receptores da Bradicinina/farmacologia , Antagonistas dos Receptores da Bradicinina/uso terapêutico , Cartilagem/metabolismo , Cartilagem Articular/metabolismo , Células Cultivadas , Condrócitos/metabolismo , Endotelina-1/metabolismo , Cavalos , Humanos , Interleucina-1beta/metabolismo , Nanogéis , Organoides/metabolismo , Osteoartrite/metabolismoRESUMO
Osteoarthritis (OA) is a significant cause of pain in both humans and horses with a high socio-economic impact. The horse is recognized as a pertinent model for human OA. In both species, regenerative therapy with allogeneic mesenchymal stem cells (MSCs) appears to be a promising treatment but, to date, no in vivo studies have attempted to compare the effects of different cell sources on the same individuals. The objective of this study is to evaluate the ability of a single blinded intra-articular injection of allogeneic bone-marrow (BM) derived MSCs and umbilical cord blood (UCB) derived MSC to limit the development of OA-associated pathological changes compared to placebo in a post-traumatic OA model applied to all four fetlock joints of eight horses. The effect of the tissue source (BM vs. UCB) is also assessed on the same individuals. Observations were carried out using clinical, radiographic, ultrasonographic, and magnetic resonance imaging methods as well as biochemical analysis of synovial fluid and postmortem microscopic and macroscopic evaluations of the joints until Week 12. A significant reduction in the progression of OA-associated changes measured with imaging techniques, especially radiography, was observed after injection of bone-marrow derived mesenchymal stem cells (BM-MSCs) compared to contralateral placebo injections. These results indicate that allogeneic BM-MSCs are a promising treatment for OA in horses and reinforce the importance of continuing research to validate these results and find innovative strategies that will optimize the therapeutic potential of these cells. However, they should be considered with caution given the low number of units per group.
Assuntos
Artrite Experimental/prevenção & controle , Medula Óssea/crescimento & desenvolvimento , Sangue Fetal/citologia , Células-Tronco Mesenquimais/citologia , Osteoartrite/prevenção & controle , Líquido Sinovial/citologia , Animais , Artrite Experimental/etiologia , Artrite Experimental/patologia , Feminino , Cavalos , Injeções Intra-Articulares , Masculino , Transplante de Células-Tronco Mesenquimais , Osteoartrite/etiologia , Osteoartrite/patologiaRESUMO
Chondrosarcomas are malignant bone tumors. Their abundant cartilage-like extracellular matrix and their hypoxic microenvironment contribute to their resistance to chemotherapy and radiotherapy, and no effective therapy is currently available. MicroRNAs (miRNAs) may be an interesting alternative in the development of therapeutic options. Here, for the first time in chondrosarcoma cells, we carried out high-throughput functional screening using impedancemetry, and identified five miRNAs with potential antiproliferative or chemosensitive effects on SW1353 chondrosarcoma cells. The cytotoxic effects of miR-342-5p and miR-491-5p were confirmed on three chondrosarcoma cell lines, using functional validation under normoxia and hypoxia. Both miRNAs induced apoptosis and miR-342-5p also induced autophagy. Western blots and luciferase reporter assays identified for the first time Bcl-2 as a direct target of miR-342-5p, and also Bcl-xL as a direct target of both miR-342-5p and miR-491-5p in chondrosarcoma cells. MiR-491-5p also inhibited EGFR expression. Finally, only miR-342-5p induced cell death on a relevant 3D chondrosarcoma organoid model under hypoxia that mimics the in vivo microenvironment. Altogether, our results revealed the tumor suppressive activity of miR-342-5p, and to a lesser extent of miR-491-5p, on chondrosarcoma lines. Through this study, we also confirmed the potential of Bcl-2 family members as therapeutic targets in chondrosarcomas.
Assuntos
Antineoplásicos/farmacologia , Apoptose/genética , Neoplasias Ósseas/metabolismo , Condrossarcoma/metabolismo , MicroRNAs/farmacologia , Organoides/metabolismo , Microambiente Tumoral/genética , Autofagia/genética , Neoplasias Ósseas/genética , Ciclo Celular/genética , Hipóxia Celular/genética , Linhagem Celular Tumoral , Proliferação de Células/genética , Condrócitos/metabolismo , Condrossarcoma/genética , Cisplatino/farmacologia , Receptores ErbB/metabolismo , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , MicroRNAs/genética , MicroRNAs/metabolismo , Organoides/citologia , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteína bcl-X/metabolismoRESUMO
BACKGROUND: Avoidance of antigenic stimuli was found to significantly reverse airway obstruction of horses with severe equine asthma (sEA). To date, no published study investigated the influence of steaming hay on lower airway condition of sEA-affected horses. The objectives were to determine the clinical, cytological and cytokine respiratory responses of both sEA and control (CTL) horses experimentally exposed to steamed or dry hay. RESULTS: A cohort of 6 sEA horses and 6 CTL horses was involved in this field study. On day 0, both groups were fed with steamed hay for 5 consecutive days, followed by a wash-out period of 26 days prior to be fed with dry hay for 5 consecutive days. Investigations performed 2 days prior to and 5 days after each challenge included clinical score, tracheal mucus accumulation, and bronchoalveolar lavage fluid (BALF) cytology and cytokine mRNA expression. Feeding steamed hay significantly decreased its mould content (P < 0.001). Mucus score significantly increased when feeding dry hay (P = 0.01). No significant influence of challenge type was found on clinical score. Percentages of neutrophils (P < 0.001) as well as mRNA expression of IL-1ß (P = 0.024), IL-6R (P = 0.021), IL-18 (P = 0.009) and IL-23 (P = 0.036) in BALF of sEA affected horses were significantly increased after both (steamed and dry hay) challenges. Relative mRNA expression of IL-1ß, IL-6R and IL-23 in BALF were also significantly correlated to neutrophil percentages and both clinical and tracheal mucus score. CONCLUSIONS: Steaming significantly decreased mould content but inconsistently influenced the respiratory response of sEA affected horses when fed hay. Based on BALF cytology and cytokine profiles, its relevance might be controversial as a non-medicinal therapy for sEA-affected horses.
Assuntos
Ração Animal , Asma/veterinária , Doenças dos Cavalos/prevenção & controle , Microbiologia do Ar , Ração Animal/efeitos adversos , Animais , Asma/etiologia , Asma/imunologia , Asma/prevenção & controle , Líquido da Lavagem Broncoalveolar/química , Líquido da Lavagem Broncoalveolar/citologia , Citocinas/metabolismo , Feminino , Doenças dos Cavalos/etiologia , Doenças dos Cavalos/imunologia , Cavalos/imunologia , Masculino , Vapor , Traqueia/metabolismoRESUMO
Cartilage engineering is a new strategy for the treatment of cartilage damage due to osteoarthritis or trauma in humans. Racehorses are exposed to the same type of cartilage damage and the anatomical, cellular, and biochemical properties of their cartilage are comparable to those of human cartilage, making the horse an excellent model for the development of cartilage engineering. Human mesenchymal stem cells (MSCs) differentiated into chondrocytes with chondrogenic factors in a biomaterial appears to be a promising therapeutic approach for direct implantation and cartilage repair. Here, we characterized equine umbilical cord blood-derived MSCs (eUCB-MSCs) and evaluated their potential for chondrocyte differentiation for use in cartilage repair therapy. Our results show that isolated eUCB-MSCs had high proliferative capacity and differentiated easily into osteoblasts and chondrocytes, but not into adipocytes. A three-dimensional (3D) culture approach with the chondrogenic factors BMP-2 and TGF-ß1 potentiated chondrogenic differentiation with a significant increase in cartilage-specific markers at the mRNA level (Col2a1, Acan, Snorc) and the protein level (type II and IIB collagen) without an increase in hypertrophic chondrocyte markers (Col10a1 and Mmp13) in normoxia and in hypoxia. However, these chondrogenic factors caused an increase in type I collagen, which can be reduced using small interfering RNA targeting Col1a2. This study provides robust data on MSCs characterization and demonstrates that eUCB-MSCs have a great potential for cartilage tissue engineering.
Assuntos
Diferenciação Celular , Condrócitos/citologia , Condrogênese , Células-Tronco Mesenquimais/citologia , Animais , Proteína Morfogenética Óssea 2/farmacologia , Cartilagem/fisiologia , Células Cultivadas , Condrócitos/metabolismo , Colágeno/genética , Colágeno/metabolismo , Sangue Fetal/citologia , Cavalos , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/metabolismo , Regeneração , Fator de Crescimento Transformador beta1/farmacologiaRESUMO
Articular cartilage is a tissue characterized by its poor intrinsic capacity for self-repair. This tissue is frequently altered upon trauma or in osteoarthritis (OA), a degenerative disease that is currently incurable. Similar musculoskeletal disorders also affect horses and OA incurs considerable economic loss for the equine sector. In the view to develop new therapies for humans and horses, significant progress in tissue engineering has led to the emergence of new generations of cartilage therapy. Matrix-associated autologous chondrocyte implantation is an advanced 3D cell-based therapy that holds promise for cartilage repair. This study aims to improve the autologous chondrocyte implantation technique by using equine mesenchymal stem cells (MSCs) from bone marrow differentiated into chondrocytes that can be implanted in the chondral lesion. The optimized protocol relies on culture under hypoxia within type I/III collagen sponges. Here, we explored three parameters that influence MSC differentiation: culture times, growth factors and RNA interference strategies. Our results suggest first that an increase in culture time from 14 to 28 or 42 days lead to a sharp increase in the expression of chondrocyte markers, notably type II collagen (especially the IIB isoform), along with a concomitant decrease in HtrA1 expression. Nevertheless, the expression of type I collagen also increased with longer culture times. Second, regarding the growth factor cocktail, TGF-ß3 alone showed promising result but the previously tested association of BMP-2 and TGF-ß1 better limits the expression of type I collagen. Third, RNA interference targeting Col1a2 as well as Col1a1 mRNA led to a more significant knockdown, compared with a conventional strategy targeting Col1a1 alone. This chondrogenic differentiation strategy showed a strong increase in the Col2a1:Col1a1 mRNA ratio in the chondrocytes derived from equine bone marrow MSCs, this ratio being considered as an index of the functionality of cartilage. These data provide evidence of a more stable chondrocyte phenotype when combining Col1a1 and Col1a2 siRNAs associated to a longer culture time in the presence of BMP-2 and TGF-ß1, opening new opportunities for preclinical trials in the horse. In addition, because the horse is an excellent model for human articular cartilage disorders, the equine therapeutic approach developed here can also serve as a preclinical step for human medicine.
Assuntos
Diferenciação Celular/genética , Condrócitos/metabolismo , Colágeno Tipo I/genética , Células-Tronco Mesenquimais/metabolismo , RNA Interferente Pequeno/genética , Fatores de Crescimento Transformadores/genética , Animais , Técnicas de Cultura de Células/métodos , Células Cultivadas , Condrócitos/citologia , Condrogênese/genética , Cavalos , Humanos , Células-Tronco Mesenquimais/citologia , Osteoartrite/terapia , Fenótipo , Interferência de RNA , Engenharia Tecidual/métodosRESUMO
As in humans, osteoarthritis (OA) causes considerable economic loss to the equine industry. New hopes for cartilage repair have emerged with the matrix-associated autologous chondrocyte implantation (MACI). Nevertheless, its limitation is due to the dedifferentiation occurring during the chondrocyte amplification phase, leading to the loss of its capacity to produce a hyaline extracellular matrix (ECM). To enhance the MACI therapy efficiency, we have developed a strategy for chondrocyte redifferentiation, and demonstrated its feasibility in the equine model. Thus, to mimic the cartilage microenvironment, the equine dedifferentiated chondrocytes were cultured in type I/III collagen sponges for 7 days under hypoxia in the presence of BMP-2. In addition, chondrocytes were transfected by siRNA targeting Col1a1 and Htra1 mRNAs, which are overexpressed during dedifferentiation and OA. To investigate the quality of the neo-synthesized ECM, specific and atypical cartilage markers were evaluated by RT-qPCR and Western blot. Our results show that the combination of 3D hypoxia cell culture, BMP-2 (Bone morphogenetic protein-2), and RNA interference, increases the chondrocytes functional indexes (Col2a1/Col1a1, Acan/Col1a1), leading to an effective chondrocyte redifferentiation. These data represent a proof of concept for this process of application, in vitro, in the equine model, and will lead to the improvement of the MACI efficiency for cartilage tissue engineering therapy in preclinical/clinical trials, both in equine and human medicine.
Assuntos
Proteína Morfogenética Óssea 2/metabolismo , Diferenciação Celular , Condrócitos/citologia , Condrócitos/metabolismo , Interferência de RNA , Animais , Biomarcadores , Proteína Morfogenética Óssea 2/farmacologia , Cartilagem Articular/citologia , Cartilagem Articular/metabolismo , Técnicas de Cultura de Células , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Hipóxia Celular/genética , Condrócitos/efeitos dos fármacos , Colágeno Tipo I/metabolismo , Colágeno Tipo III/metabolismo , Matriz Extracelular/metabolismo , Cavalos , Fenótipo , RNA Interferente Pequeno/genética , Engenharia TecidualRESUMO
Umbilical cord blood (UCB) is an attractive alternative to bone marrow for isolation of mesenchymal stem cells (MSCs) to treat articular cartilage defects. Here, we set out to determine the growth factors (bone morphogenetic protein 2 (BMP-2) and transforming growth factor-ß (TGF-ß1)) and oxygen tension effects during chondrogenesis of human UCB-MSCs for cartilage engineering. Chondrogenic differentiation was induced using 3D cultures in type I/III collagen sponges with chondrogenic factors in normoxia (21% O2) or hypoxia (<5% O2) for 7, 14 and 21 days. Our results show that UCB-MSCs can be committed to chondrogenesis in the presence of BMP-2+TGF-ß1. Normoxia induced the highest levels of chondrocyte-specific markers. However, hypoxia exerted more benefit by decreasing collagen X and matrix metalloproteinase-13 (MMP13) expression, two chondrocyte hypertrophy markers. However, a better chondrogenesis was obtained by switching oxygen conditions, with seven days in normoxia followed by 14 days in hypoxia, since these conditions avoid hypertrophy of hUCB-MSC-derived chondrocytes while maintaining the expression of chondrocyte-specific markers observed in normoxia. Our study demonstrates that oxygen tension is a key factor for chondrogenesis and suggests that UBC-MSCs 3D-culture should begin in normoxia to obtain a more efficient chondrocyte differentiation before placing them in hypoxia for chondrocyte phenotype stabilization. UCB-MSCs are therefore a reliable source for cartilage engineering.
Assuntos
Diferenciação Celular , Condrogênese , Colágeno Tipo III/metabolismo , Colágeno Tipo I/metabolismo , Sangue Fetal/citologia , Hipóxia/metabolismo , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Biomarcadores , Proteína Morfogenética Óssea 2/metabolismo , Proteína Morfogenética Óssea 2/farmacologia , Cartilagem Articular/metabolismo , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Linhagem da Célula/genética , Células Cultivadas , Condrócitos/metabolismo , Condrogênese/efeitos dos fármacos , Condrogênese/genética , Matriz Extracelular , Expressão Gênica , Humanos , Hipóxia/genética , Oxigênio/metabolismo , Fenótipo , Fator de Crescimento Transformador beta1/metabolismo , Fator de Crescimento Transformador beta1/farmacologiaRESUMO
BACKGROUND: Articular cartilage defects are a veritable therapeutic problem because therapeutic options are very scarce. Due to the poor self-regeneration capacity of cartilage, minor cartilage defects often lead to osteoarthritis. Several surgical strategies have been developed to repair damaged cartilage. Autologous chondrocyte implantation (ACI) gives encouraging results, but this cell-based therapy involves a step of chondrocyte expansion in a monolayer, which results in the loss in the differentiated phenotype. Thus, despite improvement in the quality of life for patients, reconstructed cartilage is in fact fibrocartilage. Successful ACI, according to the particular physiology of chondrocytes in vitro, requires active and phenotypically stabilized chondrocytes. SCOPE OF REVIEW: This review describes the unique physiology of cartilage, with the factors involved in its formation, stabilization and degradation. Then, we focus on some of the most recent advances in cell therapy and tissue engineering that open up interesting perspectives for maintaining or obtaining the chondrogenic character of cells in order to treat cartilage lesions. MAJOR CONCLUSIONS: Current research involves the use of chondrocytes or progenitor stem cells, associated with "smart" biomaterials and growth factors. Other influential factors, such as cell sources, oxygen pressure and mechanical strain are considered, as are recent developments in gene therapy to control the chondrocyte differentiation/dedifferentiation process. GENERAL SIGNIFICANCE: This review provides new information on the mechanisms regulating the state of differentiation of chondrocytes and the chondrogenesis of mesenchymal stem cells that will lead to the development of new restorative cell therapy approaches in humans. This article is part of a Special Issue entitled Matrix-mediated cell behaviour and properties.
Assuntos
Cartilagem Articular/fisiologia , Diferenciação Celular , Condrócitos/citologia , Matriz Extracelular/metabolismo , Engenharia Tecidual , Animais , Cartilagem Articular/citologia , Condrócitos/transplante , Condrogênese , HumanosRESUMO
BACKGROUND: The benefit of better ballistic and higher efficiency of carbon ions for cancer treatment (hadron-therapy) is asserted since decades, especially for unresectable or resistant tumors like sarcomas. However, hadron-therapy with carbon ions stays underused and raises some concerns about potential side effects for patients. Chondrosarcoma is a cartilaginous tumor, chemo- and radiation-resistant, that lacks reference models for basic and pre-clinical studies in radiation-biology. Most studies about cellular effects of ionizing radiation, including hadrons, were performed under growth conditions dramatically different from human homeostasis. Tridimensional in vitro models are a fair alternative to animal models to approach tissue and tumors microenvironment. METHODS: By using a collagen matrix, standardized culture conditions, physiological oxygen tension and a well defined chondrosarcoma cell line, we developed a pertinent in vitro 3D model for hadron-biology studies. Low- and high-Linear Energy Transfer (LET) ionizing radiations from GANIL facilities of ~1 keV/µm and 103 ± 4 keV/µm were used respectively, at 2 Gy single dose. The impact of radiation quality on chondrosarcoma cells cultivated in 3D was analyzed on cell death, cell proliferation and DNA repair. RESULTS: A fair distribution of chondrosarcoma cells was observed in the whole 3D scaffold. Moreover, LET distribution in depth, for ions, was calculated and found acceptable for radiation-biology studies using this kind of scaffold. No difference in cell toxicity was observed between low- and high-LET radiations but a higher rate of proliferation was displayed following high-LET irradiation. Furthermore, 3D models presented a higher and longer induction of H2AX phosphorylation after 2 Gy of high-LET compared to low-LET radiations. CONCLUSIONS: The presented results show the feasibility and usefulness of our 3D chondrosarcoma model in the study of the impact of radiation quality on cell fate. The observed changes in our tissue-like model after ionizing radiation exposure may explain some discrepancies between radiation-biology studies and clinical data.
Assuntos
Técnicas de Cultura de Células , Condrossarcoma/patologia , Técnicas In Vitro , Animais , Linhagem Celular Tumoral , Proliferação de Células/efeitos da radiação , Sobrevivência Celular/efeitos da radiação , Condrossarcoma/radioterapia , Reparo do DNA , Histonas/metabolismo , Humanos , Camundongos , Doses de Radiação , Radiação Ionizante , Radioterapia/métodos , Radioterapia/normasRESUMO
Transcriptional mechanisms regulating type I collagen genes expression in physiopathological situations are not completely known. In this study, we have investigated the role of nuclear factor-κB (NF-κB) transcription factor on type I collagen expression in adult normal human (ANF) and scleroderma (SF) fibroblasts. We demonstrated that NF-κB, a master transcription factor playing a major role in immune response/apoptosis, down-regulates COL1A1 expression by a transcriptional control involving the -112/-61 bp sequence. This 51-bp region mediates the action of two zinc fingers, Sp1 (specific protein-1) and Sp3, acting as trans-activators of type I collagen expression in ANF and SF. Knockdown of each one of these trans factors by siRNA confirmed the trans-activating effect of Sp1/Sp3 and the p65 subunit of NF-κB trans-inhibiting effect on COL1A1 expression. Despite no existing κB consensus sequence in the COL1A1 promoter, we found that Sp1/Sp3/c-Krox and NF-κB bind and/or are recruited on the proximal promoter in chromatin immunoprecipitation (ChIP) assays. Attempts to elucidate whether interactions between Sp1/Sp3/c-Krox and p65 are necessary to mediate the NF-κB inhibitory effect on COL1A1 in ANF and SF were carried out; in this regard, immunoprecipitation assays revealed that they interact, and this was validated by re-ChIP. Finally, the knockdown of Sp1/Sp3/c-Krox prevents the p65 inhibitory effect on COL1A1 transcription in ANF, whereas only the siRNAs targeting Sp3 and c-Krox provoked the same effect in SF, suggesting that particular interactions are characteristic of the scleroderma phenotype. In conclusion, our findings highlight a new mechanism for COL1A1 transcriptional regulation by NF-κB, and these data could allow the development of new antifibrotic strategies.
Assuntos
Colágeno Tipo I/biossíntese , Proteínas de Ligação a DNA/metabolismo , Derme/metabolismo , Fibroblastos/metabolismo , Elementos de Resposta , Esclerodermia Localizada/metabolismo , Fator de Transcrição Sp1/metabolismo , Fator de Transcrição Sp3/metabolismo , Fator de Transcrição RelA/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Gênica , Adulto , Criança , Pré-Escolar , Colágeno Tipo I/genética , Cadeia alfa 1 do Colágeno Tipo I , Proteínas de Ligação a DNA/genética , Derme/patologia , Fibroblastos/patologia , Regulação da Expressão Gênica/genética , Humanos , Masculino , Esclerodermia Localizada/patologia , Fator de Transcrição Sp1/genética , Fator de Transcrição Sp3/genética , Fator de Transcrição RelA/genética , Fatores de Transcrição/genéticaRESUMO
The burden of osteoarthritis (OA), one of the major causes of functional disabilities in humans and animals, continues to increase worldwide while no disease-modifying OA drugs (DMOADs) that either slow down or reverse disease progression have been made available. Here, we provide a brief overview of recent advances in: designing new OA drug delivery approaches, focusing on lubrication-based biomaterials and drug delivery systems, such as hydrogels, liposomes, dendrimers, micro- and nanoparticles; using either large (horse) or small (zebrafish) relevant animal models to evaluate new therapeutic strategies; and OA in vitro modeling, focusing on 3D (organoid) models of cartilage regarding the Replace, Reduce and Refine (3R) principle of animal experimentation.
Assuntos
Osteoartrite , Peixe-Zebra , Humanos , Animais , Cavalos , Osteoartrite/tratamento farmacológico , Sistemas de Liberação de MedicamentosRESUMO
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMO
Osteoarthritis (OA) remains incurable in humans or horses and mesenchymal stromal/stem cells (MSCs) represent an attractive solution for producing a neocartilage substitute. However, the best MSC source still needs to be identified. This study compared the chondrogenic potential of equine MSCs derived from bone marrow (BM) and umbilical cord blood (UCB), at their undifferentiated status to check if one cell source is better proned, and after chondrogenic-induced differentiation. Chondrogenesis was induced by culture in collagen scaffold with BMP-2 + TGF-ß1 in hypoxia or normoxia. MSCs chondrogenic potential was evaluated using the mRNA and corresponding protein levels for osteogenic, hypertrophic and chondrogenic markers. MSCs characterization demonstrated that BM- and UCB-MSCs differ in proliferation and tripotencies. At undifferentiated status, they also showed differences in their expression of osteogenic, chondrogenic and hypertrophic markers. Upon chondrogenesis induction, both MSCs sources exhibited increased chondrogenic expression and produce an extracellular matrix (ECM) of better quality in hypoxia, although collagen I remained expressed. UCB-MSCs produced higher amounts of collagen II, particularly its IIB isoform, than BM-MSCs, but also collagen I and Htra1, regardless of the oxygen condition. Finally, immunohistochemistry revealed that the BM-MSCs synthesized an ECM of higher quality, regarding the more homogenous distribution of type IIB collagen, compared to UCB-MSCs. Considering collagen I as the major undesirable component in the neo-synthesis of in vitro cartilage, we recommend using BM-MSCs for horse cartilage engineering.
Assuntos
Diferenciação Celular/genética , Condrogênese/genética , Sangue Fetal/citologia , Osteoartrite/terapia , Animais , Proteína Morfogenética Óssea 2/metabolismo , Cartilagem/crescimento & desenvolvimento , Cartilagem/metabolismo , Hipóxia Celular/genética , Proliferação de Células/genética , Condrócitos/metabolismo , Colágeno Tipo I/genética , Colágeno Tipo II/genética , Matriz Extracelular/genética , Sangue Fetal/transplante , Serina Peptidase 1 de Requerimento de Alta Temperatura A/genética , Cavalos , Humanos , Transplante de Células-Tronco Mesenquimais/métodos , Células-Tronco Mesenquimais/metabolismo , Osteoartrite/patologia , Osteogênese/genética , RNA Mensageiro/genética , Engenharia Tecidual , Fator de Crescimento Transformador beta1/metabolismoRESUMO
Osteoarthritis is a common cause of pain and economic loss in both humans and horses. The horse is recognized as a suitable model for human osteoarthritis, because the thickness, structure, and mechanical properties of equine articular cartilage are highly comparable to those of humans. Although a number of equine experimental osteoarthritis models have been described in the literature, these cases generally involve the induction of osteoarthritis in just one joint of each animal. This approach necessitates the involvement of large numbers of horses to obtain reliable data and thus limits the use of this animal model, for both economic and ethical reasons. This study adapts an established equine model of post-traumatic osteoarthritis to induce osteoarthritis-associated lesions in all 4 fetlock joints of the same horse in order to reduce the number of animals involved and avoid individual variability, thus obtaining a more reliable method to evaluate treatment efficacy in future studies. The objectives are to assess the feasibility of the procedure, evaluate variability of the lesions according to interindividual and operated-limb position and describe the spontaneous evolution of osteoarthritis-associated pathological changes over a twelve-week period. The procedure was well tolerated by all 8 experimental horses and successfully induced mild osteoarthritis-associated changes in the four fetlock joints of each horse. Observations were carried out using clinical, radiographic, ultrasonographic, and magnetic resonance imaging methods as well as biochemical analyses of synovial fluid and postmortem microscopic and macroscopic evaluations of the joints. No significant differences were found in the progression of osteoarthritis-associated changes between horses or between the different limbs, with the exception of higher synovial effusion in hind fetlocks compared to front fetlocks and higher radiographic scores for left fetlocks compared to the right. This model thus appears to be a reliable means to evaluate the efficacy of new treatments in horses, and may be of interest for translational studies in human medicine.
Assuntos
Articulação Metatarsofalângica/patologia , Osteoartrite/patologia , Animais , Modelos Animais de Doenças , Cavalos , Imageamento por Ressonância Magnética , Ossos do Metatarso/patologia , Articulação Metatarsofalângica/diagnóstico por imagem , Articulação Metatarsofalângica/cirurgia , Osteoartrite/diagnóstico por imagem , Osteoartrite/metabolismo , Índice de Gravidade de Doença , Líquido Sinovial/químicaRESUMO
Osteoarthritis is a significant and costly cause of pain for both humans and horses. The horse has been identified as a suitable model for human osteoarthritis. Regenerative therapy with allogeneic mesenchymal stem cells (MSCs) is a promising treatment, but the safety of this procedure continues to be debated. The aim of this study is to evaluate the safety of intra-articular injections of allogeneic MSCs on healthy joints by comparing two different dosages and two different tissue sources, namely, bone marrow and umbilical cord blood, with a placebo treatment on the same individuals. We also assessed the influence of autologous versus allogeneic cells for bone marrow-derived MSC treatment. Twelve clinically sound horses were subjected to injections in their 4 fetlock joints. Each of the three fetlocks was administered a different MSC type, and the remaining fetlock was injected with phosphate-buffered saline as a control. Six horses received 10 million cells per joint, and the 6 other horses received 20 million cells per joint. Clinical and ultrasound monitoring revealed that allogeneic bone marrow-derived MSCs induced significantly more synovial effusion compared to umbilical cord blood-derived MSCs but no significant difference was noted within the synovial fluid parameters. The administration of 10 million cells in horses triggered significantly more inflammatory signs than the administration of 20 million cells. Mesenchymal stem cell injections induced mild to moderate local inflammatory signs compared to the placebo, with individual variability in the sensitivity to the same line of MSCs. Understanding the behavior of stem cells when injected alone is a step towards the safer use of new strategies in stem cell therapy, where the use of either MSC secretome or MSCs combined with biomaterials could enhance their viability and metabolic activity.
RESUMO
Umbilical cord blood mesenchymal stromal/stem cells (UCB-MSCs) and umbilical cord matrix MSCs (UCM-MSCs) have chondrogenic potential and are alternative sources to standard surgically derived bone marrow or adipose tissue collection for cartilage engineering. However, the majority of comparative studies explore neonatal MSCs potential only on ISCT benchmark assays accounting for some bias in the reproducibility between in vitro and in clinical studies. Therefore, we characterized equine UCB-MSCs and UCM-MSCs and investigated with particular attention their chondrogenesis potential in 3D culture with BMP-2 + TGF-ß1 in normoxia or hypoxia. We carried out an exhaustive characterization of the extracellular matrix generated by both these two types of MSCs after the induction of chondrogenesis through evaluation of hyaline cartilage, hypertrophic and osteogenic markers (mRNA, protein and histology levels). Some differences in hypoxia sensitivity and chondrogenesis were observed. UCB-MSCs differentiated into chondrocytes express an abundant, dense and a hyaline-like cartilage matrix. By contrast, despite their expression of cartilage markers, UCM-MSCs failed to express a relevant cartilage matrix after chondrogenic induction. Both MSCs types also displayed intrinsic differences at their undifferentiated basal status, UCB-MSCs expressing higher levels of chondrogenic markers whereas UCM-MSCs synthesizing higher amounts of osteogenic markers. Our results suggest that UCB-MSCs should be preferred for ex-vivo horse cartilage engineering. How those results should be translated to in vivo direct cartilage regeneration remains to be determined through dedicated study.
Assuntos
Condrogênese/fisiologia , Sangue Fetal/citologia , Cordão Umbilical/citologia , Animais , Proteína Morfogenética Óssea 2/metabolismo , Cartilagem/metabolismo , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Condrócitos/metabolismo , Condrogênese/genética , Colágeno Tipo I/metabolismo , Matriz Extracelular/química , Matriz Extracelular/metabolismo , Sangue Fetal/fisiologia , Cavalos , Cartilagem Hialina/metabolismo , Células-Tronco Mesenquimais/fisiologia , Osteogênese/efeitos dos fármacos , Regeneração/efeitos dos fármacos , Reprodutibilidade dos Testes , Engenharia Tecidual/métodos , Fator de Crescimento Transformador beta1/metabolismo , Cordão Umbilical/fisiologiaRESUMO
Mesenchymal stem cells (MSCs) hold promise for cartilage engineering. Here, we aimed to determine the best culture conditions to induce chondrogenesis of MSCs isolated from bone marrow (BM) of aged osteoarthritis (OA) patients. We showed that these BM-MSCs proliferate slowly, are not uniformly positive for stem cell markers, and maintain their multilineage potential throughout multiple passages. The chondrogenic lineage of BM-MSCs was induced in collagen scaffolds, under normoxia or hypoxia, by BMP-2 and/or TGF-ß1. The best chondrogenic induction, with the least hypertrophic induction, was obtained with the combination of BMP-2 and TGF-ß1 under hypoxia. Differentiated BM-MSCs were then transfected with siRNAs targeting two markers overexpressed in OA chondrocytes, type I collagen and/or HtrA1 protease. siRNAs significantly decreased mRNA and protein levels of type I collagen and HtrA1, resulting in a more typical chondrocyte phenotype, but with frequent calcification of the subcutaneously implanted constructs in a nude mouse model. Our 3D culture model with BMP-2/TGF-ß1 and COL1A1/HtrA1 siRNAs was not effective in producing a cartilage-like matrix in vivo. Further optimization is needed to stabilize the chondrocyte phenotype of differentiated BM-MSCs. Nevertheless, this study offers the opportunity to develop a combinatory cellular therapy strategy for cartilage tissue engineering.