RESUMO
Whenever the topic of re-growing human limbs is posed for discussion, it is often argued that 'if a newt can do it, then so can we'. This notion, albeit promising, is somewhat like watching a science-fiction film; the individual components are currently available but we are far from realizing the complete picture. Today's reality is that if we are faced with a limb-severing injury, any regenerative attempt would endeavour to accelerate the pace at which the tissue heals to a clinically relevant/functional state. The science of limb regeneration can be approached from three different angles, developmental biology; regenerative medicine; and tissue engineering. This opinion piece describes how each approach can be used to understand the concepts behind regeneration, how far each approach has advanced and the hurdles faced by each of the approaches.
Assuntos
Extremidades/fisiologia , Regeneração , Humanos , Medicina Regenerativa , Engenharia TecidualRESUMO
Tissue engineering aims to regenerate tissues that can successfully take over the functions of the native tissue when it is damaged or diseased. In most tissues, collagen makes up the bulk component of the extracellular matrix, thus, there is great emphasis on its accurate quantification in tissue engineering. It has already been reported that pepsin digestion is able to solubilize the collagen deposited within the cell layer for accurate quantification of collagen content in cultures, but this method has drawbacks when cultured cells are hyperconfluent. In this condition, Pepsin digestion will result in fragments of the cell layers that cannot be completely resolved. These fragments of the undigested cell sheet are visible to the naked eye, which can bias the final results. To the best of our knowledge, there has been no reported method to accurately quantify the collagen content in hyperconfluent cell sheet. Therefore, this study aims to illustrate that sonication is able to aid pepsin digestion of hyperconfluent cell layers of fibroblasts and bone marrow mesenchymal stem cells, to solubilize all the collagen for accurate quantification purposes.
Assuntos
Técnicas de Cultura de Células , Células Cultivadas/química , Colágeno/análise , Animais , Células da Medula Óssea/citologia , Células da Medula Óssea/metabolismo , Fibroblastos/citologia , Fibroblastos/metabolismo , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Coelhos , SonicaçãoRESUMO
The depletion of chondroitin sulfates (CSs) within the intervertebral disc (IVD) during degenerative disc disease (DDD) results in a decrease in tissue hydration, a loss of fluid movement, cell apoptosis, a loss of nerve growth inhibition and ultimately, the loss of disc function. To date, little is known with regards to the structure and content of chondroitin sulfates (CSs) during IVD ageing. The behavior of glycosaminoglycans (GAGs), specifically CSs, as well as xylosyltransferase I (XT-I) and glucuronyltransferase I (GT-I), two key enzymes involved in CS synthesis as a primer of glycosaminoglycan (GAG) chain elongation and GAG synthesis in the nucleus pulposus (NP), respectively, were evaluated in a bovine ageing IVD model. Here, we showed significant changes in the composition of GAGs during the disc ageing process (6-month-old, 2-year-old and 8-year-old IVDs representing the immature to mature skeleton). The CS quantity and composition of annulus fibrosus (AF) and NP were determined. The expression of both XT-I and GT-I was detected using immunohistochemistry. A significant decrease in GAGs was observed during the ageing process. CSs are affected at both the structural and quantitative levels with important changes in sulfation observed upon maturity, which correlated with a decrease in the expression of both XT-I and GT-I. A progressive switch of the sulfation profile was noted in both NP and AF tissues from 6 months to 8 years. These changes give an appreciation of the potential impact of CSs on the disc biology and the development of therapeutic approaches for disc regeneration and repair.
RESUMO
Cellular morphology has recently been indicated as a powerful indicator of cellular function. The analysis of cell shape has evolved from rudimentary forms of microscopic visual inspection to more advanced methodologies that utilize high-resolution microscopy coupled with sophisticated computer hardware and software for data analysis. Despite this progress, there is still a lack of standardization in quantification of morphometric parameters. In addition, uncertainty remains as to which methodologies and parameters of cell morphology will yield meaningful data, which methods should be utilized to categorize cell shape, and the extent of reliability of measurements and the interpretation of the resulting analysis. A large range of descriptors has been employed to objectively assess the cellular morphology in two-dimensional and three-dimensional domains. Intuitively, simple and applicable morphometric descriptors are preferable and standardized protocols for cell shape analysis can be achieved with the help of computerized tools. In this review, cellular morphology is discussed as a descriptor of cellular function and the current morphometric parameters that are used quantitatively in two- and three-dimensional environments are described. Furthermore, the current problems associated with these morphometric measurements are addressed. Copyright © 2015 John Wiley & Sons, Ltd.
Assuntos
Forma Celular/fisiologia , Processamento de Imagem Assistida por Computador/métodos , Medicina Regenerativa/métodos , Animais , HumanosRESUMO
Cell-sheet tissue engineering retains the benefits of an intact extracellular matrix (ECM) and can be used to produce scaffold-free constructs. Adipose tissue-derived stem cells (ASCs) are multipotent and more easily obtainable than the commonly used bone marrow-derived stem cells (BMSCs). Although BMSC cell sheets have been previously reported to display multipotentiality, a detailed study of the development and multilineage potential of ASC cell sheets (ASC-CSs) is non-existent in the literature. The aims of this study were to temporally profile: (a) the effect of hyperconfluent culture duration on ASC-CSs development; and (b) the multipotentiality of ASC-CSs by differentiation into the osteogenic, adipogenic and chondrogenic lineages. Rabbit ASCs were first isolated and cultured until confluence (day 0). The confluent cells were then cultured in ascorbic acid-supplemented medium for 3 weeks to study cell metabolic activity, cell sheet thickness and early differentiation gene expressions at weekly time points. ASC-CSs and ASCs were then differentiated into the three lineages, using established protocols, and assessed by RT-PCR and histology at multiple time points. ASC-CSs remained healthy up to 3 weeks of hyperconfluent culture. One week-old cell sheets displayed upregulation of early differentiation gene markers (Runx2 and Sox9); however, subsequent differentiation results indicated that they did not necessarily translate to an improved phenotype. ASCs within the preformed cell sheet groups did not differentiate as efficiently as the non-hyperconfluent ASCs, which were directly differentiated. Although ASCs within the cell sheets retained their differentiation capacity and remained viable under prolonged hyperconfluent conditions, future applications of ASC-CSs in tissue engineering should be considered with care. Copyright © 2016 John Wiley & Sons, Ltd.
Assuntos
Tecido Adiposo/metabolismo , Diferenciação Celular , Células-Tronco Mesenquimais/metabolismo , Tecido Adiposo/citologia , Animais , Antígenos de Diferenciação/biossíntese , Subunidade alfa 1 de Fator de Ligação ao Core/biossíntese , Células-Tronco Mesenquimais/citologia , Coelhos , Fatores de Transcrição SOX9/biossíntese , Regulação para CimaRESUMO
Low back pain is generally attributed to intervertebral disc (IVD) degeneration. This is a multifactorial disease induced by genetic and environmental factors and that progresses with aging. Disc degeneration is characterized by a limited ability of IVD cells to produce functional matrix while producing abnormal amounts of matrix-degrading enzymes. The prolonged imbalance between anabolism and catabolism in degenerative discs alters their composition and hydration. In turn, this results in increased angiogenesis and the loss of the disc's ability to maintain its aneural condition. Inflammation in the IVD, in particular the presence of pro-inflammatory cytokines, was found to favor innervation and also sensitization of the nociceptive pathways, thereby exacerbating degenerative symptoms. In this review, we discuss anti-inflammatory approaches to encounter disc catabolism, potential treatments to lower discogenic pain and pro-anabolic approaches in the form of protein delivery, gene therapy and cell delivery, to trigger regeneration in the IVD.
Assuntos
Produtos Biológicos/administração & dosagem , Terapia Baseada em Transplante de Células e Tecidos/tendências , Terapia Genética/métodos , Degeneração do Disco Intervertebral/terapia , Materiais Biocompatíveis/administração & dosagem , Terapia Baseada em Transplante de Células e Tecidos/métodos , Terapia Genética/tendências , Humanos , Degeneração do Disco Intervertebral/metabolismo , Regeneração/fisiologiaRESUMO
BACKGROUND CONTEXT: Intervertebral discs (IVDs) are attractive targets for local drug delivery because they are avascular structures with limited transport. Painful IVDs are in a chronic inflammatory state. Although anti-inflammatories show poor performance in clinical trials, their efficacy treating IVD cells suggests that sustained, local drug delivery directly to painful IVDs may be beneficial. PURPOSE: The purpose of this study was to determine if genipin cross-linked fibrin (FibGen) with collagen Type I hollow spheres (CHS) can serve as a drug-delivery carrier for infliximab, the anti-tumor necrosis factor α (TNFα) drug. Infliximab was chosen as a model drug because of the known role of TNFα in increasing downstream production of several pro-inflammatory cytokines and pain mediators. Genipin cross-linked fibrin was used as drug carrier because it is adhesive, injectable, and slowly degrading hydrogel with the potential to seal annulus fibrosus (AF) defects. CHS allow simple and nondamaging drug loading and could act as a drug reservoir to improve sustained delivery. STUDY DESIGN/SETTING: This is a study of biomaterials and human AF cell culture to determine drug release kinetics and efficacy. METHODS: Infliximab was delivered at low and high concentrations using FibGen with and without CHS. Gels were analyzed for structure, drug release kinetics, and efficacy treating human AF cells after release. RESULTS: Fibrin showed rapid infliximab drug release but degraded quickly. CHS alone showed a sustained release profile, but the small spheres may not remain in a degenerated IVD with fissures. Genipin cross-linked fibrin showed steady and low levels of infliximab release that was increased when loaded with higher drug concentrations. Infliximab was bound in CHS when delivered within FibGen and was only released after enzymatic degradation. The infliximab released over 20 days retained its bioactivity as confirmed by the sustained reduction of interleukin (IL)-1ß, IL-6, IL-8, and TNFα concentrations produced by AF cells. CONCLUSIONS: Direct mixing of infliximab into FibGen was the simplest drug-loading protocol capable of sustained release. Results show feasibility of using drug-loaded FibGen for delivery of infliximab and, in the context with the literature, show potential to seal AF defects and partially restore IVD biomechanics. Future investigations are required to determine if drug-loaded FibGen can effectively deliver drugs, seal AF defects, and promote IVD repair or prevent further IVD degeneration in vivo.
Assuntos
Antirreumáticos/administração & dosagem , Portadores de Fármacos/efeitos adversos , Adesivo Tecidual de Fibrina/efeitos adversos , Infliximab/administração & dosagem , Disco Intervertebral/efeitos dos fármacos , Iridoides/efeitos adversos , Antirreumáticos/farmacologia , Antirreumáticos/uso terapêutico , Células Cultivadas , Portadores de Fármacos/química , Adesivo Tecidual de Fibrina/química , Humanos , Infliximab/farmacologia , Iridoides/químicaRESUMO
Most studies on the intervertebral disc (IVD) focus on the regeneration of the nucleus pulposus (NP). However, without a proper strategy to regenerate the damaged annulus fibrosus (AF), the NP replacements are bound to fail. Therefore the objective of this study was to investigate whether the use of bone marrow-derived mesenchymal stem cells (BMSCs) to form cell sheets, and incorporating them onto silk scaffolds, has the potential to regenerate the annulus fibrosus. The BMSC cell sheets and silk scaffolds were wrapped around a silicone NP substitute to form a simulated IVD-like assembly. The simulated IVD-like assembly was cultured for 4 weeks in static conditions and it was shown that the BMSC cell sheets remained viable, with no significant change in cell numbers. Histological analysis showed that the BMSC cell sheets adhered well onto the silk scaffolds and glycosaminoglycans (GAGs) were detected within the extracellular matrix (ECM). The ratio of collagen type I to collagen type II within the ECM of the BMSC cell sheets also decreased significantly over the period of culture. The results suggested that extensive remodelling of the ECM occurred within the simulated IVD-like assembly, and it is suitable for the regeneration of the inner AF.
Assuntos
Células da Medula Óssea/citologia , Simulação por Computador , Disco Intervertebral/fisiologia , Células-Tronco Mesenquimais/citologia , Regeneração/fisiologia , Seda/química , Alicerces Teciduais/química , Animais , Sobrevivência Celular , Colágeno Tipo I/metabolismo , Colágeno Tipo II/metabolismo , Módulo de Elasticidade , Eletroforese em Gel de Poliacrilamida , Óxido de Etileno/química , Matriz Extracelular/metabolismo , Imuno-Histoquímica , Disco Intervertebral/citologia , Teste de Materiais , Coelhos , Silicones/química , Seda/ultraestrutura , Esterilização , Sus scrofaRESUMO
STUDY DESIGN: The aim of this study was to develop a tissue engineering approach in regenerating the annulus fibrosus (AF) as part of an overall strategy to produce a tissue-engineered intervertebral disc (IVD) replacement. OBJECTIVE: To determine whether a rehabilitative simulation regime on bone marrowderived mesenchymal stem cell cell-sheet is able to aid the regeneration of the AF. SUMMARY OF BACKGROUND DATA: No previous study has used bone marrowderived mesenchymal stem cell cell-sheets simulated by a rehabilitative regime to regenerate the AF. METHODS: The approach was to use bone marrowderived stem cells to form cell-sheets and incorporating them onto silk scaffolds to simulate the native lamellae of the AF. The in vitro experimental model used to study the efficacy of such a system was made up of the tissue engineering AF construct wrapped around a silicone disc to form a simulated IVD-like assembly. The assembly was cultured within a custom-designed bioreactor that provided a compressive mechanical stimulation onto the silicone disc. The silicone nucleus pulposus would bulge radially and compress the simulated AF to mimic the physiological conditions. The simulated IVD-like assembly was compressed using a rehabilitative regime that lasted for 4 weeks at 0.25 Hz, for 15 minutes each day. RESULTS: With the rehabilitative regime, the cell-sheets remained viable but showed a decrease in cell numbers and viability. Gene expression analysis showed significant upregulation of IVD-related genes and there was an increased ratio of collagen type II to collagen type I found within the extracellular matrix. CONCLUSION: The results suggested that a rehabilitative regime caused extensive remodeling to take place within the simulated IVD-like assembly, producing extracellular matrix similar to that found in the inner AF.
Assuntos
Células da Medula Óssea/fisiologia , Células-Tronco Mesenquimais/fisiologia , Regeneração , Engenharia Tecidual/métodos , Alicerces Teciduais , Animais , Fenômenos Biomecânicos , Reatores Biológicos , Técnicas de Cultura de Células , Sobrevivência Celular , Células Cultivadas , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Colágeno Tipo II/genética , Colágeno Tipo II/metabolismo , Matriz Extracelular/genética , Matriz Extracelular/metabolismo , Regulação da Expressão Gênica , Imuno-Histoquímica , Coelhos , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Silicones/química , Seda/química , Estresse Mecânico , Fatores de TempoRESUMO
Bone-marrow-derived mesenchymal stem cells (BMSCs) are a promising source of cells for tissue engineering due to their multilineage mesenchymal differentiation potential. Their ability to proliferate and differentiate into the osteogenic, chondrogenic, and adipogenic lineage makes them an attractive cell source as compared to the terminally differentiated cells. In tissue engineering, use of cell sheet technology is gaining popularity. It is based on culturing cells until hyperconfluence, and it has resulted in the reduction of the number of cells lost when seeding onto scaffolds. Thus, formation of cell sheets with multipotent cells, such as BMSCs, would be a promising alternative to the conventional method of cell seeding, that is, single-cell suspension. However, the multilineage potential of BMSC cell sheets has yet to be verified. Therefore, the aim of this study was to characterize the formation of a hyperconfluent BMSC cell sheet as well as the effects of the hyperconfluent culture conditions on the multipotentiality of BMSCs. Our results showed that the BMSC cell sheets remained viable. The cell sheets were rich with type I collagen and were shown to have retained their multipotentiality. Hence, the use of BMSC cell sheets for tissue engineering application seems promising.