RESUMO
In vitro models aim to recapitulate the in vivo situation. To more closely mimic the knee joint environment, current in vitro models need improvements to reflect the complexity of the native tissue. High molecular weight hyaluronan (hMwt HA) is one of the most abundant bioactive macromolecules in healthy synovial fluid, while shear and dynamic compression are two joint-relevant mechanical forces. The present study aimed at investigating the concomitant effect of joint-simulating mechanical loading (JSML) and hMwt HA-supplemented culture medium on the chondrogenic differentiation of primary human bone-marrow-derived mesenchymal stem cells (hBM-MSCs). hBM-MSC chondrogenesis was investigated over 28 d at the gene expression level and total DNA, sulphated glycosaminoglycan, TGF-ß1 production and safranin O staining were evaluated. The concomitant effect of hMwt HA culture medium and JSML significantly increased cartilage-like matrix deposition and sulphated glycosaminoglycan synthesis, especially during early chondrogenesis. A stabilisation of the hBM-MSC-derived chondrocyte phenotype was observed through the reduced upregulation of the hypertrophic marker collagen X and an increase in the chondrogenic collagen type II/X ratio. A combination of JSML and hMwt HA medium better reflects the complexity of the in vivo synovial joint environment. Thus, JSML and hMwt HA medium will be two important features for joint-related culture models to more accurately predict the in vivo outcome, therefore reducing the need for animal studies. Reducing in vitro artefacts would enable a more reliable prescreening of potential cartilage repair therapies.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Condrogênese/efeitos dos fármacos , Ácido Hialurônico/farmacologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Idoso , Cartilagem/efeitos dos fármacos , Cartilagem/metabolismo , Células Cultivadas , Condrócitos/efeitos dos fármacos , Condrócitos/metabolismo , Colágeno Tipo II/metabolismo , Colágeno Tipo X/metabolismo , DNA/metabolismo , Feminino , Humanos , Masculino , Células-Tronco Mesenquimais/metabolismo , Pessoa de Meia-Idade , Líquido Sinovial/efeitos dos fármacos , Líquido Sinovial/metabolismo , Engenharia Tecidual/métodos , Alicerces Teciduais/químicaRESUMO
The repair of focal cartilage defects remains one of the foremost issues in the field of orthopaedics. Chondral defects may arise from a variety of joint pathologies and left untreated, will likely progress to osteoarthritis. Current repair techniques, such as microfracture, result in short-term clinical improvements but have poor long-term outcomes. Emerging scaffold-based repair strategies have reported superior outcomes compared to microfracture and motivate the development of new biomaterials for this purpose. In this study, unique composite implants consisting of a base porous reinforcing component (woven poly(ε-caprolactone)) infiltrated with 1 of 2 hydrogels (self-assembling peptide or thermo-gelling hyaluronan) or bone marrow aspirate were evaluated. The objective was to evaluate cartilage repair with composite scaffold treatment compared to the current standard of care (microfracture) in a translationally relevant large animal model, the Yucatan minipig. While many cartilage-repair studies have shown some success in vivo, most are short term and not clinically relevant. Informed by promising 6-week findings, a 12-month study was carried out and those results are presented here. To aid in comparisons across platforms, several structural and functionally relevant outcome measures were performed. Despite positive early findings, the long-term results indicated less than optimal structural and mechanical results with respect to cartilage repair, with all treatment groups performing worse than the standard of care. This study is important in that it brings much needed attention to the importance of performing translationally relevant long-term studies in an appropriate animal model when developing new clinical cartilage repair approaches.
Assuntos
Cartilagem Articular , Animais , Materiais Biocompatíveis , Cartilagem Articular/cirurgia , Modelos Animais de Doenças , Ácido Hialurônico , Suínos , Porco MiniaturaRESUMO
Ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) is the main source of extracellular pyrophosphate. Along with tissue-nonspecific alkaline phosphatase (TNAP), ENPP1 plays an important role in balancing bone mineralisation. Although well established in pre-osteoblasts, the regulating mechanisms of ENPP1 in osteoblasts and osteocytes remain largely unknown. Using bioinformatic methods, osterix (Osx), an essential transcription factor in osteoblast differentiation and osteocyte function, was found to have five predicted binding sites on the ENPP1 promoter. ENPP1 and Osx showed a similar expression profile both in vitro and in vivo. Over-expression of Osx in MC3T3-E1 and MLO-Y4 cells significantly up-regulated the expression of ENPP1 (p < 0.05). The consensus Sp1 sequences, located in the proximal ENPP1 promoter, were identified as Osx-regulating sites using promoter truncation experiments and chromatin immunoprecipitation (ChIP) assays. The p38-mitogen-activated protein kinase (MAPK) signalling pathway was demonstrated to be responsible for ENPP1 promoter activation by Osx. Runt-related transcription factor 2 (Runx2) was confirmed to have synergistic effects with Osx in activating ENPP1 promoter. Taken together, these results provided evidence of the regulating mechanisms of ENPP1 transcription in osteoblasts and osteocytes.
Assuntos
Osteoblastos/metabolismo , Osteócitos/metabolismo , Diester Fosfórico Hidrolases/genética , Pirofosfatases/genética , Fator de Transcrição Sp7/metabolismo , Ativação Transcricional/genética , Animais , Linhagem Celular , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Feminino , Perfilação da Expressão Gênica , Humanos , Sistema de Sinalização das MAP Quinases , Masculino , Camundongos Endogâmicos C57BL , Osteogênese/genética , Diester Fosfórico Hidrolases/metabolismo , Regiões Promotoras Genéticas , Pirofosfatases/metabolismo , Fator de Transcrição Sp7/genética , Transfecção , Regulação para Cima/genética , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Mesenchymal stem cells (MSCs) can be induced towards chondrogenesis through the application of chondrogenic stimuli such as transforming growth factor-ß (TGF-ß) or by multiaxial mechanical load. Previous work has showed that the chondrogenic effect of multiaxial load on MSCs is mediated by the endogenous production of TGF-ß1 by stimulated cells. This work compared the effects of TGF-ß1 stimulation and multiaxial mechanical load on the secretomes of stimulated cells. MSCs were seeded into fibrin-poly(ester-urethane) scaffolds and chondrogenically stimulated with either TGF-ß1 or mechanical load. The culture media was collected and analysed for 174 proteins using a cytokine antibody array. The results of the secretome analysis were then confirmed at a gene expression level by real-time PCR. As results implicated nitric oxide (NO), the media nitrite content was also determined as an indirect measurement of media NO levels. Results showed that TGF-ß1 stimulation and mechanical load lead to similar changes in factors such as BLC, VEGF and MMP13, whilst differences in detected levels were seen for factors including leptin, MDC, MIP3α and LAP. Gene expression analysis confirmed significant changes in four factors: angiopoietin 2, GROα, MMP13 and osteoprotegerin. After one week in culture the media nitrite content was significantly higher in loaded groups than both control and TGF-ß1 stimulated groups, suggesting this may be a major therapeutic target. These data show that despite clear similarities, TGF-ß1 stimulation and load have distinct effects on MSCs and are not analogous. This study has identified a number of potentially novel targets for tissue engineering, these data may also be useful for improving rehabilitation protocols e.g. after microfracture.
Assuntos
Condrogênese/fisiologia , Células-Tronco Mesenquimais/metabolismo , Estresse Mecânico , Estresse Fisiológico/fisiologia , Fator de Crescimento Transformador beta1/farmacologia , Adulto , Idoso , Técnicas de Cultura de Células , Diferenciação Celular , Células Cultivadas , Condrócitos/metabolismo , Citocinas/análise , Citocinas/metabolismo , Humanos , Óxido Nítrico/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Transdução de Sinais , Engenharia Tecidual/métodos , Alicerces Teciduais , Adulto JovemRESUMO
New regenerative materials and approaches need to be assessed through reliable and comparable methods for rapid translation to the clinic. There is a considerable need for proven in vitro assays that are able to reduce the burden on animal testing, by allowing assessment of biomaterial utility predictive of the results currently obtained through in vivo studies. The purpose of this multicentre review was to investigate the correlation between existing in vitro results with in vivo outcomes observed for a range of biomaterials. Members from the European consortium BioDesign, comprising 8 universities in a European multicentre study, provided data from 36 in vivo studies and 47 in vitro assays testing 93 different biomaterials. The outcomes of the in vitro and in vivo experiments were scored according to commonly recognised measures of success relevant to each experiment. The correlation of in vitro with in vivo scores for each assay alone and in combination was assessed. A surprisingly poor correlation between in vitro and in vivo assessments of biomaterials was revealed indicating a clear need for further development of relevant in vitro assays. There was no significant overall correlation between in vitro and in vivo outcome. The mean in vitro scores revealed a trend of covariance to in vivo score with 58 %. The inadequacies of the current in vitro assessments highlighted here further stress the need for the development of novel approaches to in vitro biomaterial testing and validated pre-clinical pipelines.
Assuntos
Materiais Biocompatíveis/farmacologia , Regeneração Óssea/efeitos dos fármacos , Teste de Materiais/métodos , Animais , Humanos , Camundongos , RatosRESUMO
Despite the high innate regenerative capacity of bone, large osseous defects fail to heal and remain a clinical challenge. Healing such defects requires the formation of large amounts of bone in an environment often rendered hostile to osteogenesis by damage to the surrounding soft tissues and vasculature. In recent years, there have been intensive research efforts directed towards tissue engineering and regenerative approaches designed to overcome this multifaceted challenge. In this paper, we describe and critically evaluate the state-of-the-art approaches to address the various components of this intricate problem. The discussion includes (i) the properties of synthetic and natural scaffolds, their use in conjunction with cell and growth factor delivery, (ii) their vascularisation, (iii) the potential of gene therapies and (iv) the role of the mechanical environment. In particular, we present a critical analysis of where the field stands, and how it can move forward in a coordinated fashion.
Assuntos
Regeneração Óssea/fisiologia , Osso e Ossos/patologia , Engenharia Tecidual/métodos , Animais , Sistemas de Liberação de Medicamentos , Terapia Genética , Humanos , Alicerces Teciduais/químicaRESUMO
Runt-related transcription factor 2 (RUNX2) is a transcription factor closely associated with the osteoblast phenotype. While frequently referred to, the complexity of its regulation and its interactions within the osteoblast differentiation pathway are often overlooked. This review aims to summarise the knowledge of its regulation at the transcriptional, translational and post-translational level. In addition, the regulation of RUNX2 by factors commonly used during osteogenic studies will be discussed.
Assuntos
Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Osteoblastos/metabolismo , Osteogênese , Animais , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Osteoblastos/citologiaRESUMO
Stem cells have become the fundamental element in regenerative medicine due to their inherent potential to differentiate into various cell types, and the ability to produce various bioactive molecules, including growth factors, cytokines and extracellular matrix molecules. In vivo, the secretion of tropic factors is modulated by chemotactic and inflammatory factors. In this study, we analysed the influence of a 2 h stimulation of mesenchymal stem cells (MSCs) with interleukin-1ß (IL1ß), granulocyte-colony stimulating factor (GCSF), stromal cell-derived factor 1 (SDF1) and stem cell factor (SCF). Our results demonstrated that this short stimulation exerts pronounced effects on the expression of multiple cytokine genes and proteins in MSC cells 48 and 72 h later. IL1ß strongly promoted the secretion of a wide range of proteins with chemotactic, proinflammatory and angiogenic properties, whereas SCF regulated the expression of proteins involved in proliferation, chondrogenesis and ECM regulation. This demonstrates that the changes in secretome can be directed towards a desired final functional outcome by selection of the most appropriate cytokine. Moreover, the expression pattern of Wnt signalling pathway components suggested the differential regulation of this pathway by IL1ß and SCF. Altogether, the robust paracrine action of MSCs can be achieved within a just 2 h treatment, which would be feasible within the operating theatre during a single surgical procedure. These results suggest that integrating inflammatory modulation in bone tissue engineering, by modifying the MSC secretome by way of a short stimulus, would provide a more targeted approach than administering unmodified MSCs alone.
Assuntos
Regeneração Óssea , Quimiocinas/metabolismo , Células-Tronco Mesenquimais/metabolismo , Adolescente , Adulto , Idoso , Proteínas Morfogenéticas Ósseas/genética , Proteínas Morfogenéticas Ósseas/metabolismo , Proliferação de Células , Quimiocinas/genética , Quimiocinas/farmacologia , Condrogênese , Meios de Cultivo Condicionados/farmacologia , Feminino , Humanos , Masculino , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Pessoa de Meia-Idade , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Wnt/genética , Proteínas Wnt/metabolismo , Via de Sinalização WntRESUMO
An AO Foundation (Davos, Switzerland) sponsored workshop "Cell Therapy in Cartilage Repair" from the Symposium "Where Science meets Clinics" (September 5-7, 2013, Davos) gathered leaders from medicine, science, industry, and regulatory organisations to debate the vision of cell therapy in articular cartilage repair and the measures that could be taken to narrow the gap between vision and current practice. Cell-based therapy is already in clinical use to enhance the repair of cartilage lesions, with procedures such as microfracture and articular chondrocyte implantation. However, even though long term follow up is good from a clinical perspective and some of the most rigorous randomised controlled trials in the regenerative medicine/orthopaedics field show beneficial effect, none of these options have proved successful in restoring the original articular cartilage structure and functionality in patients so far. With the remarkable recent advances in experimental research in cell biology (new sources for chondrocytes, stem cells), molecular biology (growth factors, genes), biomaterials, biomechanics, and translational science, a combined effort between scientists and clinicians with broad expertise may allow development of an improved cell therapy for cartilage repair. This position paper describes the current state of the art in the field to help define a procedure adapted to the clinical situation for upcoming translation in the patient.
Assuntos
Cartilagem Articular/fisiologia , Regeneração Tecidual Guiada/tendências , Regeneração , Animais , Cartilagem Articular/cirurgia , Regeneração Tecidual Guiada/métodos , HumanosRESUMO
Research in orthopaedic tissue engineering has intensified over the last decade and new protocols continue to emerge. The clinical translation of these new applications, however, remains associated with a number of obstacles. This report highlights the major issues that impede the clinical translation of advanced tissue engineering concepts, discusses strategies to overcome these barriers, and examines the need to increase incentives for translational strategies. The statements are based on presentations and discussions held at the AO Foundation-sponsored symposium "Where Science meets Clinics 2013" held at the Congress Center in Davos, Switzerland, in September, 2013. The event organisers convened a diverse group of over one hundred stakeholders involved in clinical translation of orthopaedic tissue engineering, including scientists, clinicians, healthcare industry professionals and regulatory agency representatives. A major point that emerged from the discussions was that there continues to be a critical need for early trans-disciplinary communication and collaboration in the development and execution of research approaches. Equally importantly was the need to address the shortage of sustained funding programs for multidisciplinary teams conducting translational research. Such detailed discussions between experts contribute towards the development of a roadmap to more successfully advance the clinical translation of novel tissue engineering concepts and ultimately improve patient care in orthopaedic and trauma surgery.
Assuntos
Regeneração Tecidual Guiada/métodos , Ortopedia/métodos , Pesquisa Translacional Biomédica/economia , Pesquisa Translacional Biomédica/métodosRESUMO
The process of bone formation, remodelling and healing involves a coordinated action of various cell types. Advances in understanding the biology of osteoblast cells during these processes have been enabled through the use of various in vitro culture models from different origins. In an era of intensive bone tissue engineering research, these cell models are more and more often applied due to limited availability of primary human osteoblast cells. While they are a helpful tool in developing novel therapies or biomaterials; concerns arise regarding their phenotypic state and differences in relation to primary human osteoblast cells. In this review we discuss the osteoblastic development of some of the available cell models; such as primary human, rat, mouse, bovine, ovine and rabbit osteoblast cells; as well as MC3T3-E1, MG-63 and SaOs-2 cell lines, together with their advantages and disadvantages. Through this, we provide suggestions on the selection of the appropriate and most relevant osteoblast model for in vitro studies, with specific emphasis on cell-material based studies.
Assuntos
Osso e Ossos/citologia , Osteoblastos/citologia , Animais , Osso e Ossos/fisiologia , Bovinos , Diferenciação Celular , Linhagem Celular , Humanos , Camundongos , Modelos Biológicos , Osteoblastos/fisiologia , Cultura Primária de Células , Coelhos , Ratos , Ovinos , Especificidade da Espécie , Engenharia TecidualRESUMO
There is great interest in how bone marrow derived stem cells make fate decisions. Numerous studies have investigated the role of individual growth factors on mesenchymal stem cell differentiation, leading to protocols for cartilage, bone and adipose tissue. However, these protocols overlook the role of biomechanics on stem cell differentiation. There have been various studies that have applied mechanical stimulation to constructs containing mesenchymal stem cells, with varying degrees of success. One critical fate decision is that between cartilage and bone. Articular motion is a combination of compressive, tensile and shear deformations; therefore, one can presume that compression alone is unlikely to be a sufficient mechanical signal to generate a cartilage-like tissue in vitro. Within this study, we aimed to determine the role of shear on the fate of stem cell differentiation. Specifically, we investigated the potential enhancing effect of surface shear, superimposed on cyclic axial compression, on chondrogenic differentiation of human bone marrow-derived stem cells. Using a custom built loading device we applied compression, shear or a combination of both stimuli onto fibrin/polyurethane composites in which human mesenchymal stem cells were embedded, while no exogenous growth-factors were added to the culture medium. Both compression or shear alone was insufficient for the chondrogenic induction of human mesenchymal stem cells. However, the application of shear superimposed upon dynamic compression led to significant increases in chondrogenic gene expression. Histological analysis detected sulphated glycosaminoglycan and collagen II only in the compression and shear group. The results obtained may provide insight into post-operative care after cell therapy involving mesenchymal stromal cells.
Assuntos
Condrogênese , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/fisiologia , Estresse Mecânico , Adolescente , Idoso , Reatores Biológicos , Células da Medula Óssea/citologia , Células da Medula Óssea/fisiologia , Diferenciação Celular/fisiologia , Células Cultivadas , Matriz Extracelular , Feminino , Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Masculino , Células-Tronco Mesenquimais/metabolismo , Pessoa de Meia-Idade , Engenharia Tecidual , Alicerces TeciduaisRESUMO
The aim of this work is to investigate the capability of PRP as an adjuvant therapy to autologous chondrocyte implantation (ACI) in combination with multi-axial load with respect to cartilage regeneration. Articular cartilage shows poor repair capacity and therapies for cartilage defects are still lacking. Well-established operative treatments include ACI, and growing evidence shows the beneficial effects of PRP. Platelets contain numerous growth factors, among them transforming growth factor beta (TGF-ß). Dynamic mechanical loading is known to be essential for tissue formation, improving extracellular matrix (ECM) production. For our ACI model monolayer expanded human chondrocytes were seeded into polyurethane scaffolds and embedded in fibrin (hChondro), in PRP-Gel (PRP), or in fibrin with platelet lysate (PL), which was added to the media once a week with a concentration of 50 vol%. The groups were either exposed to static conditions or multi-axial forces in a ball-joint bioreactor for 1 h per day over 2 weeks, mimicking ACI under physiological load. The culture medium was collected and analyzed for glycosaminoglycan (GAG), nitrite and transforming growth factor beta 1 (TGF-ß1) content. The cell-scaffold constructs were collected for DNA and GAG quantification; the expression of chondrogenic genes, TGF-ß and related receptors, as well as inflammatory genes, were analyzed using qPCR. Loading conditions showed superior chondrogenic differentiation (upregulation of COL2A1, ACAN, COMP and PRG4 expression) than static conditions. PRP and PL groups combined with mechanical loading showed upregulation of COL2A1, ACAN and COMP. The highest amount of total TGF-ß1 was quantified in the PL group. Latent TGF-ß1 was activated in all loaded groups, while the highest amount was found in the PL group. Load increased TGFBR1/TGFBR2 mRNA ratio, with further increases in response to supplements. In general, loading increased nitrite release into the media. However, over time, the media nitrite content was lower in the PL group compared to the control group. Based on these experiments, we conclude that chondrogenic differentiation is strongest when simulated ACI is performed in combination with dynamic mechanical loading and PRP-gel or PL supplementation. An inflammatory reaction was reduced by PRP and PL, which could be one of the major therapeutic effects. Loading presumably can enhance the action of TGF-ß1, which was predominantly activated in loaded PL groups. The combination of load and PRP represents an effective and promising synergy concerning chondrocyte-based cartilage repair.
Assuntos
Fatores Biológicos/farmacologia , Plaquetas/química , Condrócitos/citologia , Plasma Rico em Plaquetas/fisiologia , Técnicas de Cultura de Células , Células Cultivadas , Condrócitos/efeitos dos fármacos , Condrócitos/metabolismo , Condrócitos/transplante , Condrogênese , Meios de Cultura/química , Glicosaminoglicanos/metabolismo , Humanos , Modelos Biológicos , Nitritos/metabolismo , Estresse Mecânico , Alicerces Teciduais , Fator de Crescimento Transformador beta1/metabolismo , Transplante AutólogoRESUMO
In vitro monolayer culture of human primary osteoblasts (hOBs) often shows unsatisfactory results for extracellular matrix deposition, maturation and calcification. Nevertheless, monolayer culture is still the method of choice for in vitro differentiation of primary osteoblasts. We believe that the delay in mature ECM production by the monolayer cultured osteoblasts is determined by their state of cell maturation. A functional relationship between the inhibition of osteoblast proliferation and the induction of genes associated with matrix maturation was suggested within a monolayer culture model for rat calvarial osteoblasts. We hypothesize, that a pellet culture model could be utilized to decrease initial proliferation and increase the transformation of osteoblasts into a more mature phenotype. We performed pellet cultures using hOBs and compared their differentiation potential to 2D monolayer cultures. Using the pellet culture model, we were able to generate a population of cuboidal shaped central osteoblastic cells. Increased proliferation, as seen during low-density monolayer culture, was absent in pellet cultures and monolayers seeded at 40,000 cells/cm2. Moreover, the expression pattern of phenotypic markers Runx2, osterix, osteocalcin, col I and E11 mRNA was significantly different depending on whether the cells were cultured in low density monolayer, high density monolayer or pellet culture. We conclude that the transformation of the osteoblast phenotype in vitro to a more mature stage can be achieved more rapidly in 3D culture. Moreover, that dense monolayer leads to the formation of more mature osteoblasts than low-density seeded monolayer, while hOB cells in pellets seem to have transformed even further along the osteoblast phenotype.
Assuntos
Técnicas de Cultura de Células/métodos , Osteoblastos/citologia , Diferenciação Celular , Divisão Celular , Células Cultivadas , Matriz Extracelular/metabolismo , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Osteoblastos/metabolismo , Fenótipo , RNA Mensageiro/metabolismoRESUMO
OBJECTIVES: To devise a macroscopic, radiological, and histological scale for assessing pathological changes associated with medication-related osteonecrosis of the jaw in a minipig model. MATERIALS AND METHODS: Medication-related osteonecrosis of the jaw was induced in Göttingen minipigs by weekly intravenous administration of bisphosphonate (zoledronic acid) combined with a tooth extraction procedure. Controls either did not receive zoledronic acid or did not undergo tooth extraction. After 20 weeks, minipigs were euthanized and underwent computed tomography and micro-computed tomography scanning. The mandible underwent additional histological examination. RESULTS: The most consistent macroscopic findings in animals that had developed bisphosphonate-related osteonecrosis of the jaw (BRONJ) were necrotic, denuded bone, and formation of fistula and pus. Under radiological examination, impaired extraction socket healing, decrease in attenuation of bone beneath the extraction site, and periosteal reaction were observed. Under histological examination, demineralization of the extracellular bone matrix, denuding of bone, and osteonecrosis were recorded. CONCLUSION: These parameters were used to develop a scoring system for grading BRONJ.
Assuntos
Osteonecrose da Arcada Osseodentária Associada a Difosfonatos , Animais , Conservadores da Densidade Óssea , Difosfonatos , Suínos , Porco Miniatura , Extração Dentária , Microtomografia por Raio-XRESUMO
Based upon the powerful bridging and charge-masking properties of lanthanide cations (Ln3+), we have investigated their use to improve the transduction efficiency of adenovirus vectors. Using a luciferase marker gene, it was possible to increase transgene expression by the murine mesenchymal stem cell line C3H10T(1/2) by up to four log orders when using very low multiplicities of infection in conjunction with Ln3+; La3+ was superior to Gd3+, Y3+ and Lu3+ in this regard. All Ln3+ were more effective than Ca2+. Flow cytometry, using a green fluorescent protein marker gene, confirmed that La3+ increased both the percentage of transduced cells and the level of transgene expression per cell. Transduction of primary cultures of a variety of different mesenchymal cells from human, rabbit, bovine and rat sources, as well as gene transfer to synovium and muscle in vivo, was also greatly enhanced. Our findings suggest that this lanthanide-based method holds much promise for expediting both experimental and clinical applications of gene transfer with adenoviral vectors.
Assuntos
Adenoviridae/genética , Terapia Genética/métodos , Vetores Genéticos/genética , Elementos da Série dos Lantanídeos/farmacologia , Transdução Genética/métodos , Animais , Cátions , Bovinos , Linhagem Celular , Expressão Gênica , Engenharia Genética , Proteínas de Fluorescência Verde/genética , Humanos , Proteína Antagonista do Receptor de Interleucina 1/genética , Luciferases/genética , Masculino , Células-Tronco Mesenquimais/metabolismo , Camundongos , Coelhos , Ratos , Ratos Wistar , Transgenes , Ítrio/farmacologiaRESUMO
Articular cartilage is commonly described as a tissue that is made of up to 80% water, is devoid of blood vessels, nerves, and lymphatics, and is populated by only one cell type, the chondrocyte. At first glance, an easy tissue for clinicians to repair and for scientists to reproduce in a laboratory. Yet, chondral and osteochondral defects currently remain an open challenge in orthopedics and tissue engineering of the musculoskeletal system, without considering osteoarthritis. Why do we fail in repairing and regenerating articular cartilage? Behind its simple and homogenous appearance, articular cartilage hides a heterogeneous composition, a high level of organisation and specific biomechanical properties that, taken together, make articular cartilage a unique material that we are not yet able to repair or reproduce with high fidelity. This review highlights the available therapies for cartilage repair and retraces the research on different biomaterials developed for tissue engineering strategies. Their potential to recreate the structure, including composition and organisation, as well as the function of articular cartilage, intended as cell microenvironment and mechanically competent replacement, is described. A perspective of the limitations of the current research is given in the light of the emerging technologies supporting tissue engineering of articular cartilage. STATEMENT OF SIGNIFICANCE: The mechanical properties of articular tissue reflect its functionally organised composition and the recreation of its structure challenges the success of in vitro and in vivo reproduction of the native cartilage. Tissue engineering and biomaterials science have revolutionised the way scientists approach the challenge of articular cartilage repair and regeneration by introducing the concept of the interdisciplinary approach. The clinical translation of the current approaches are not yet fully successful, but promising results are expected from the emerging and developing new generation technologies.
Assuntos
Materiais Biocompatíveis , Cartilagem Articular/fisiologia , Engenharia Tecidual , Animais , Fenômenos Biomecânicos , Cartilagem Articular/crescimento & desenvolvimento , Humanos , RegeneraçãoRESUMO
Autologous chondrocyte implantation for cartilage repair represents a challenge because strongly limited by chondrocytes' poor expansion capacity in vitro. Mesenchymal stem cells (MSCs) can differentiate into chondrocytes, while mechanical loading has been proposed as alternative strategy to induce chondrogenesis excluding the use of exogenous factors. Moreover, MSC supporting material selection is fundamental to allow for an active interaction with cells. Here, we tested a novel thermo-reversible hydrogel composed of 8% w/v methylcellulose (MC) in a 0.05 M Na2SO4 solution. MC hydrogel was obtained by dispersion technique and its thermo-reversibility, mechanical properties, degradation and swelling were investigated, demonstrating a solution-gelation transition between 34 and 37 °C and a low bulk degradation (<20%) after 1 month. The lack of any hydrogel-derived immunoreaction was demonstrated in vivo by mice subcutaneous implantation. To induce in vitro chondrogenesis, MSCs were seeded into MC solution retained within a porous polyurethane (PU) matrix. PU-MC composites were subjected to a combination of compression and shear forces for 21 days in a custom made bioreactor. Mechanical stimulation led to a significant increase in chondrogenic gene expression, while histological analysis detected sulphated glycosaminoglycans and collagen II only in loaded specimens, confirming MC hydrogel suitability to support load induced MSCs chondrogenesis.
Assuntos
Materiais Biocompatíveis , Técnicas de Cultura de Células , Diferenciação Celular , Condrogênese , Hidrogéis , Células-Tronco Mesenquimais/citologia , Metilcelulose , Animais , Materiais Biocompatíveis/química , Biomarcadores , Reatores Biológicos , Diferenciação Celular/genética , Condrogênese/genética , Perfilação da Expressão Gênica , Humanos , Teste de Materiais , Células-Tronco Mesenquimais/metabolismo , CamundongosRESUMO
Biocompatibility studies are carried out either in two dimensional monolayer culture or in animal studies. Bone organ cultures are therefore required in order to reduce the number of animal studies performed, while at the same time ensuring a more natural environment than that provided by monolayer culture of isolated cells. Due to the three dimensional nature of bone explants, assays that determine the distribution of viable cells are required, however dense mineralised bone is not easily penetrated by soluble factors. We sought to compare a number of non-radioactive viability methods in order to assess their suitability for use with cancellous bone. Fluorescent live/dead staining, MTT activity and lactate dehydrogenase detection were all investigated on either whole bone explants (9.5 mm in diameter, 5 mm high) or on sections of explants. All these assays are routinely used in 2 dimensional cell culture systems, yet each required modifications to be suitable for use with cancellous bone. Factors such as penetration of reagent, incubation time, assay temperature and ease of determining viable cells were all compared. It was demonstrated that penetration of the reagents into whole cores was a major problem which easily led to artefacts that could be overcome by preparing 250 mum unfixed sections. Fluorescent live/dead staining had extra complications caused by the autofluorescence of the bone generating a high signal to noise ratio, making assessment of osteocyte viability impossible. MTT staining was difficult to interpret due to the punctate nature of the stain. We found that lactate dehydrogenase staining of 250 mum thick unfixed sections led to excellent viability determination of osteocytes within the mineralised matrix. It also maintained marrow structure and enabled marrow viability to be assessed as a factor of volume occupied by viable marrow. Decreasing the viscosity of the LDH assay solution used in published methods led to a greatly improved penetration into the calcified matrix. Quantification of thick sections is aided by using the autofluorescence of the bone to highlight the darkly stained osteocytes against the fluorescing bone.