RESUMO
Activation of the canonical Wnt signaling pathway is an attractive anabolic therapeutic strategy for bone. Emerging data suggest that activation of the Wnt signaling pathway promotes bone mineral accrual in osteoporotic patients. The effect of Wnt stimulation in fracture healing is less clear as Wnt signaling has both stimulatory and inhibitory effects on osteogenesis. Here, we tested the hypothesis that transient Wnt stimulation promotes the expansion and osteogenesis of a Wnt-responsive stem cell population present in human bone marrow. Bone marrow mononuclear cells (BMMNCs) were isolated from patients undergoing hip arthroplasty and exposed to Wnt3A protein. The effect of Wnt pathway stimulation was determined by measuring the frequency of stem cells within the BMMNC populations by fluorescence-activated cell sorting and colony forming unit fibroblast (CFU-F) assays, before determining their osteogenic capacity in in vitro differentiation experiments. We found that putative skeletal stem cells in BMMNC isolates exhibited elevated Wnt pathway activity compared with the population as whole. Wnt stimulation resulted in an increase in the frequency of skeletal stem cells marked by the STRO-1(bright) /Glycophorin A(-) phenotype. Osteogenesis was elevated in stromal cell populations arising from BMMNCs transiently stimulated by Wnt3A protein, but sustained stimulation inhibited osteogenesis in a concentration-dependent manner. These results demonstrate that Wnt stimulation could be used as a therapeutic approach by transient targeting of stem cell populations during early fracture healing, but that inappropriate stimulation may prevent osteogenesis.
Assuntos
Células da Medula Óssea/metabolismo , Leucócitos Mononucleares/metabolismo , Osteogênese , Células-Tronco/metabolismo , Via de Sinalização Wnt , Proteína Wnt3A/metabolismo , Idoso , Idoso de 80 Anos ou mais , Artroplastia de Quadril , Células da Medula Óssea/citologia , Feminino , Citometria de Fluxo , Humanos , Leucócitos Mononucleares/citologia , Masculino , Pessoa de Meia-Idade , Células-Tronco/citologia , Células Estromais/citologia , Células Estromais/metabolismoRESUMO
Skeletal stem cells confer to bone its innate capacity for regeneration and repair. Bone regeneration strategies seek to harness and enhance this regenerative capacity for the replacement of tissue damaged or lost through congenital defects, trauma, functional/esthetic problems, and a broad range of diseases associated with an increasingly aged population. This review describes the state of the field and current steps to translate and apply skeletal stem cell biology in the clinic and the problems therein. Challenges are described along with key strategies including the isolation and ex vivo expansion of multipotential populations, the targeting/delivery of regenerative populations to sites of repair, and their differentiation toward bone lineages. Finally, preclinical models of bone repair are discussed along with their implications for clinical translation and the opportunities to harness that knowledge for musculoskeletal regeneration.
Assuntos
Regeneração Óssea/fisiologia , Músculo Esquelético/transplante , Transplante de Células-Tronco/métodos , Células-Tronco/citologia , Animais , Diferenciação Celular , Humanos , Músculo Esquelético/citologia , Medicina Regenerativa/métodos , Engenharia TecidualRESUMO
There is currently an unmet need for the supply of autologous, patient-specific stem cells for regenerative therapies in the clinic. Mesenchymal stem cell differentiation can be driven by the material/cell interface suggesting a unique strategy to manipulate stem cells in the absence of complex soluble chemistries or cellular reprogramming. However, so far the derivation and identification of surfaces that allow retention of multipotency of this key regenerative cell type have remained elusive. Adult stem cells spontaneously differentiate in culture, resulting in a rapid diminution of the multipotent cell population and their regenerative capacity. Here we identify a nanostructured surface that retains stem-cell phenotype and maintains stem-cell growth over eight weeks. Furthermore, the study implicates a role for small RNAs in repressing key cell signalling and metabolomic pathways, demonstrating the potential of surfaces as non-invasive tools with which to address the stem cell niche.
Assuntos
Diferenciação Celular , Células-Tronco Mesenquimais/citologia , Células-Tronco Multipotentes/citologia , Fenótipo , Linhagem da Célula , Proliferação de Células , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Multipotentes/metabolismo , Propriedades de SuperfícieRESUMO
The combination of progenitor cells, cell-friendly scaffold, and a three-dimensional culture system has been investigated for the culture of cartilage tissue. We have assessed chondrogenesis of alginate-chitosan-encapsulated STRO-1-isolated human mesenchymal progenitor cells. In addition, ATDC-5 cells and human articular chondrocytes were also evaluated. We have used a novel 3D bioreactor system that enabled perfusion of the capsules with culture medium up to 28 days. Results from culturing all cell types indicated chondrogenesis, both in static and bioreactor culture. The expression of SOX-9 and type II collagen was examined as a marker of differentiation. ATDC-5s expressed both SOX-9 and type II collagen under perfused and static culture conditions. In monolayer cell culture, human articular chondrocytes did not express either SOX-9 or type II collagen and STRO-1 expressed alkaline phosphatase, indicating osteogenesis. However, when these cells were encapsulated in alginate-chitosan, both expressed SOX-9 under static and perfused cultures, but type II collagen was only expressed under perfused culture conditions. We have also noted that the perfusion rates used were too low to ensure a significant advantage over static culture, but that use of the bioreactor eliminated the need for manual feeding and intervention of the cells over the 28-day period.
Assuntos
Alginatos/química , Reatores Biológicos , Técnicas de Cultura de Células/instrumentação , Técnicas de Cultura de Células/métodos , Quitosana/química , Condrócitos/citologia , Animais , Antígenos de Superfície/metabolismo , Linhagem Celular , Células Cultivadas , Colágeno Tipo II/metabolismo , Meios de Cultura , Ácido Glucurônico/química , Ácidos Hexurônicos/química , Humanos , Células-Tronco Mesenquimais/citologia , Camundongos , Perfusão , Fatores de Transcrição SOX9/metabolismo , Engenharia Tecidual , Alicerces Teciduais/químicaRESUMO
Articular cartilage functions as a shock absorber and facilitates the free movement of joints. Currently, there are no therapeutic drugs that promote the healing of damaged articular cartilage. Limitations associated with the two clinically relevant cell populations, human articular chondrocytes and mesenchymal stem cells, necessitate finding an alternative cell source for cartilage repair. Human embryonic stem cells (hESCs) provide a readily accessible population of self-renewing, pluripotent cells with perceived immunoprivileged properties for cartilage generation. We have developed a robust method to generate 3D, scaffold-free, hyaline cartilage tissue constructs from hESCs that are composed of numerous chondrocytes in lacunae, embedded in an extracellular matrix containing Type II collagen, sulphated glycosaminoglycans and Aggrecan. The elastic (Young's) modulus of the hESC-derived cartilage tissue constructs (0.91 ± 0.08 MPa) was comparable to full-thickness human articular cartilage (0.87 ± 0.09 MPa). Moreover, we have successfully scaled up the size of the scaffold-free, 3D hESC-derived cartilage tissue constructs to between 4.5 mm and 6 mm, thus enhancing their suitability for clinical application.
Assuntos
Cartilagem Articular/crescimento & desenvolvimento , Células-Tronco Embrionárias Humanas/metabolismo , Engenharia Tecidual/métodos , Agrecanas/metabolismo , Cartilagem/metabolismo , Cartilagem Articular/metabolismo , Diferenciação Celular , Células Cultivadas , Condrócitos/metabolismo , Condrogênese , Colágeno Tipo II/metabolismo , Matriz Extracelular/metabolismo , Glicosaminoglicanos/metabolismo , Regeneração Tecidual Guiada/métodos , Células-Tronco Embrionárias Humanas/transplante , Humanos , Células-Tronco Mesenquimais/metabolismoRESUMO
HOXA10 is necessary for embryonic patterning of skeletal elements, but its function in bone formation beyond this early developmental stage is unknown. Here we show that HOXA10 contributes to osteogenic lineage determination through activation of Runx2 and directly regulates osteoblastic phenotypic genes. In response to bone morphogenic protein BMP2, Hoxa10 is rapidly induced and functions to activate the Runx2 transcription factor essential for bone formation. A functional element with the Hox core motif was characterized for the bone-related Runx2 P1 promoter. HOXA10 also activates other osteogenic genes, including the alkaline phosphatase, osteocalcin, and bone sialoprotein genes, and temporally associates with these target gene promoters during stages of osteoblast differentiation prior to the recruitment of RUNX2. Exogenous expression and small interfering RNA knockdown studies establish that HOXA10 mediates chromatin hyperacetylation and trimethyl histone K4 (H3K4) methylation of these genes, correlating to active transcription. HOXA10 therefore contributes to early expression of osteogenic genes through chromatin remodeling. Importantly, HOXA10 can induce osteoblast genes in Runx2 null cells, providing evidence for a direct role in mediating osteoblast differentiation independent of RUNX2. We propose that HOXA10 activates RUNX2 in mesenchymal cells, contributing to the onset of osteogenesis, and that HOXA10 subsequently supports bone formation by direct regulation of osteoblast phenotypic genes.
Assuntos
Diferenciação Celular , Proteínas de Homeodomínio/metabolismo , Osteoblastos/citologia , Osteoblastos/metabolismo , Osteogênese , Fenótipo , Animais , Sequência de Bases , Células Cultivadas , Subunidade alfa 1 de Fator de Ligação ao Core/deficiência , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Regulação da Expressão Gênica , Proteínas Homeobox A10 , Proteínas de Homeodomínio/genética , Camundongos , Camundongos Knockout , Regiões Promotoras Genéticas/genética , Interferência de RNA , Transcrição Gênica/genéticaRESUMO
Skeletal stem and progenitor populations provide a platform for cell-based tissue regeneration strategies. Optimized conditions for ex vivo expansion will be critical and use of serum-free culture may allow enhanced modelling of differentiation potential. Maintenance of human foetal femur-derived cells in a chemically defined medium (CDM) with activin A and fibroblast growth factor-2 generated a unique undifferentiated cell population in comparison to basal cultures, with significantly reduced amino acid depletion, appearance and turnover, reduced alkaline phosphatase (ALP) activity and loss of type I and II collagen expression demonstrated by fluorescence immunocytochemistry. Microarray analysis demonstrated up-regulation of CLU, OSR2, POSTN and RABGAP1 and down-regulation of differentiation-associated genes CRYAB, CSRP1, EPAS1, GREM1, MT1X and SRGN as validated by quantitative real-time polymerase chain reaction. Application of osteogenic conditions to CDM cultures demonstrated partial rescue of ALP activity. In contrast, the addition of bone morphogenetic protein-2 (BMP-2) resulted in reduced ALP levels, increased amino acid metabolism and, strikingly, a marked shift to a cobblestone-like cellular morphology, with expression of SOX-2 and SOX-9 but not STRO-1 as shown by immunocytochemistry, and significantly altered expression of metabolic genes (GFPT2, SC4MOL and SQLE), genes involved in morphogenesis (SOX15 and WIF1) and differentiation potential (C1orf19, CHSY-2,DUSP6, HMGCS1 and PPL). These studies demonstrate the use of an intermediary foetal cellular model for differentiation studies in chemically defined conditions and indicate the in vitro reconstruction of the mesenchymal condensation phenotype in the presence of BMP-2, with implications therein for rescue studies, screening assays and skeletal regeneration research.
Assuntos
Proteína Morfogenética Óssea 2/metabolismo , Diferenciação Celular , Meios de Cultura Livres de Soro , Ativinas/metabolismo , Sobrevivência Celular , Meios de Cultura Livres de Soro/metabolismo , Feminino , Fator 2 de Crescimento de Fibroblastos/metabolismo , Idade Gestacional , Humanos , Imuno-Histoquímica/métodos , Osteogênese , Fenótipo , Gravidez , Regeneração , Fatores de TempoRESUMO
Coherent anti-Stokes Raman scattering (CARS) and second harmonic generation (SHG) are non-linear techniques that allow label-free, non-destructive and non-invasive imaging for cellular and tissue analysis. Although live-imaging studies have been performed previously, concerns that they do not cause any changes at the molecular level in sensitive biological samples have not been addressed. This is important especially for stem cell differentiation and tissue engineering, if CARS/SHG microscopy is to be used as a non-invasive, label-free tool for assessment of the developing neo-tissue. In this work, we monitored the differentiation of human fetal-femur derived skeletal cells into cartilage in three-dimensional cultures using CARS and SHG microscopy and demonstrate the live-imaging of the same developing neo-tissue over time. Our work conclusively establishes that non-linear label-free imaging does not alter the phenotype or the gene expression at the different stages of differentiation and has no adverse effect on human skeletal cell growth and behaviour. Additionally, we show that CARS microscopy allows imaging of different molecules of interest, including lipids, proteins and glycosaminoglycans, in the bioengineered neo-cartilage. These studies demonstrate the label-free and truly non-invasive nature of live CARS and SHG imaging and their value and translation potential in skeletal research, regeneration medicine and tissue engineering.
Assuntos
Cartilagem/diagnóstico por imagem , Imagem Molecular/métodos , Imagem Multimodal/métodos , Engenharia Tecidual/métodos , Cartilagem/metabolismo , Diferenciação Celular , Condrogênese/genética , Fêmur/citologia , Fêmur/embriologia , Expressão Gênica , Glicosaminoglicanos/análise , Humanos , Imagem Molecular/instrumentação , Imagem Multimodal/instrumentação , Proteínas/análise , Análise Espectral Raman/métodos , Técnicas de Cultura de Tecidos/métodosRESUMO
In this chapter, we describe techniques for the isolation and characterisation of skeletal stem cells from human bone marrow. The methods for enrichment of STRO-1+ and STRO-4+ cells using magnetic activated cell sorting are described and we also detail techniques for establishing and characterizing osteogenic, adipogenic, and chondrogenic cultures from these cells. Finally, we present methods for studying the ability of these cells to produce bone in vivo using diffusion chambers which have been implanted subcutaneously into mice.
Assuntos
Células da Medula Óssea/fisiologia , Diferenciação Celular , Osteogênese , Cultura Primária de Células/métodos , Coloração e Rotulagem/métodos , Animais , Transplante de Medula Óssea/instrumentação , Transplante de Medula Óssea/métodos , Separação Celular/instrumentação , Separação Celular/métodos , Células Cultivadas , Meios de Cultura/metabolismo , Humanos , Camundongos , Camundongos Nus , Cultura Primária de Células/instrumentação , Coloração e Rotulagem/instrumentação , Células Estromais/fisiologiaRESUMO
Stem cell differentiation is controlled intrinsically by dynamic networks of interacting lineage-specifying and multipotency genes. However, the relationship between internal genetic dynamics and extrinsic regulation of internal dynamics is complex and, in the case of skeletal progenitor cell differentiation, incompletely understood. In this study we elucidate a set of candidate markers of multipotency in human skeletal progenitor cells by systematic study of the relationships between gene expression and environmental stimulus. We used full genome cDNA microarrays to explore gene expression profiles in skeletal progenitor enriched populations derived from adult human bone marrow, minimally cultured in basal, osteogenic, chondrogenic, and adipogenic lineage-specifying culture conditions. We then used a variety of statistical clustering procedures to identify a small subset of genes which are related to these stromal lineages but are specific to none. For a selection of 11 key genes, conclusions of the microarray study were confirmed using quantitative real-time PCR.
Assuntos
Osso e Ossos/citologia , Condrogênese/genética , Células-Tronco Multipotentes/fisiologia , Osteogênese/genética , Antígenos de Superfície/genética , Técnicas de Cultura de Células , Citometria de Fluxo , Marcadores Genéticos , Genoma Humano , Humanos , Células-Tronco Multipotentes/citologia , Análise de Sequência com Séries de Oligonucleotídeos , EsqueletoRESUMO
BAG-1, an anti-apoptotic protein, was identified by its ability to bind to BCL-2, HSP70-family molecular chaperones and nuclear hormone receptor family members. Two BAG-1 isoforms, BAG-1L (50 kDa) and BAG-1S (32 kDa) were identified in mouse cells and BAG-1 expression was reported in murine growth plate and articular chondrocytes. The present study aimed to elucidate the role of BAG-1 in the regulation of molecular mechanisms governing chondrocyte differentiation and turnover during endochondral ossification. In long bones of skeletally immature mice, we observed expression of BAG-1 in the perichondrium, osteoblasts, osteocytes in the bone shaft, bone marrow, growth plate and articular chondrocytes. Monolayer cultures of murine chondrocytic ATDC5 cells, which exhibited robust expression of both BAG-1 isoforms and the Bag-1 transcript, were utilized as an in vitro model to delineate the roles of BAG-1. Overexpression of BAG-1L in ATDC5 cells resulted in downregulation of Col2a1 expression, a gene characteristically downregulated at the onset of hypertrophy, and an increase in transcription of Runx-2 and Alkaline phosphatase, genes normally expressed at the onset of chondrocyte hypertrophy and cartilage mineralization in the process of endochondral ossification. We also demonstrated the anti-apoptotic role of BAG-1 in chondrocytes as overexpression of BAG-1 protected ATDC5 cells, which were subjected to heat-shock at 48 degrees C for 30 min, against heat-shock-induced apoptosis. Overexpression of the SOX-9 protein in ATDC5 cells resulted in increased Bag-1 gene expression. To further investigate the regulation of Bag-1 gene expression by SOX-9, CHO cells were co-transfected with the human Bag-1 gene promoter-Luciferase reporter construct and the human pSox-9 expression vector. Activity of the Bag-1 promoter was significantly enhanced by the SOX-9 protein. In conclusion, a novel finding of this study is the role of BAG-1 as a transcriptional regulator of genes involved in chondrocyte hypertrophy and cartilage mineralization during the process of endochondral ossification. Additionally, we have demonstrated for the first time the regulation of Bag-1 gene expression by SOX-9 and the anti-apoptotic role of BAG-1 in chondrocytic cells. Modulation of Bag-1 expression can therefore mediate chondrocyte differentiation and turnover, and offer further insight into the molecular regulation of endochondral ossification.
Assuntos
Diferenciação Celular , Condrócitos/citologia , Condrócitos/metabolismo , Proteínas de Ligação a DNA/metabolismo , Osteogênese , Fatores de Transcrição/metabolismo , Transcrição Gênica/genética , Envelhecimento/fisiologia , Animais , Linhagem Celular , Sobrevivência Celular , Proteínas de Ligação a DNA/genética , Regulação da Expressão Gênica , Proteínas de Grupo de Alta Mobilidade/genética , Proteínas de Grupo de Alta Mobilidade/metabolismo , Humanos , Camundongos , Fatores de Transcrição SOX9 , Fatores de Transcrição/genéticaRESUMO
Advances in our knowledge of the biology of skeletal stem cells, together with an increased understanding of the regeneration of normal tissue offer exciting new therapeutic approaches in musculoskeletal repair. Skeletal stem cells from various adult tissues such as bone marrow can be identified and isolated based on their expression of a panel of markers associated with smooth muscle cells, pericytes and endothelial cells. Thus, skeletal stem cell-like populations within bone marrow may share a common perivascular stem cell niche within the microvascular network. To date, the environmental niche that nurtures and maintains the stromal stem cell at different anatomical sites remains poorly understood. However, an understanding of the osteogenic and perivascular niches will inform identification of the key growth factors, matrix constituents and physiological conditions that will enhance the ex vivo amplification and differentiation of osteogenic stem cells to mimic native tissue critical for tissue repair. This review will examine skeletal stem cell biology, the advances in our understanding of the skeletal and perivascular niche and interactions therein and the opportunities to harness that knowledge for musculoskeletal regeneration.
Assuntos
Meio Ambiente , Músculo Esquelético/citologia , Regeneração , Células-Tronco/citologia , Humanos , Fenótipo , Engenharia TecidualRESUMO
Articular cartilage defects arising from trauma or degenerative diseases fail to repair spontaneously. We have adopted a non-viral gene delivery and tissue engineering strategy, in which Sox-9 transfected human mesenchymal progenitors have been encapsulated within alginate/chitosan polysaccharide capsules to promote chondrogenesis. Human bone marrow stromal cells and articular chondrocytes were transfected with flag-tagged Sox-9 plasmid and after 7 days in static culture, large regions of cell-generated matrix containing cartilage proteoglycans were observed as confirmed by positive Alcian blue staining and Sox-9 immunohistochemistry. Further, after 28 days, in vitro and in vivo, samples encapsulated with Sox-9 transfected cells demonstrated large regions of cartilaginous matrix as confirmed by positive Alcian blue staining, Sox-9 and type-II collagen immunohistochemistry, absent in samples encapsulated with untransfected cells. Extracted protein from in vivo constructs was further assessed by western blot analysis and positive expression of Sox-9 and type-II collagen was observed in Sox-9 transfected constructs which was absent in untransfected cells. Regions of cartilage-like matrix were significantly increased in Sox-9 constructs in comparison with untransfected constructs, confirming Sox-9 gene delivery enhances chondrogenesis in targeted cell populations, outlining the potential to promote cartilaginous construct formation with therapeutic implications for regeneration of human articular cartilage tissue defects.
Assuntos
Condrogênese/genética , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Polissacarídeos/metabolismo , Alginatos/química , Medula Óssea/metabolismo , Cápsulas , Diferenciação Celular , Células Cultivadas , Matriz Extracelular/metabolismo , Genes Reporter/genética , Ácido Glucurônico , Ácidos Hexurônicos , Proteínas de Grupo de Alta Mobilidade/genética , Proteínas de Grupo de Alta Mobilidade/metabolismo , Humanos , Engenharia de Proteínas , Fatores de Transcrição SOX9 , Células Estromais/metabolismo , Temperatura , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , TransfecçãoRESUMO
The understanding of cellular response to the shape of their environment would be of benefit in the development of artificial extracellular environments for potential use in the production of biomimetic surfaces. Specifically, the understanding of how cues from the extracellular environment can be used to understand stem cell differentiation would be of special interest in regenerative medicine. In this paper, the genetic profile of mesenchymal stem cells cultured on two osteogenic nanoscale topographies (pitted surface versus raised islands) are compared with cells treated with dexamethasone, a corticosteroid routinely used to stimulate bone formation in culture from mesenchymal stem cells, using 19k gene microarrays as well as 101 gene arrays specific for osteoblast and endothelial biology. The current studies show that by altering the shape of the matrix a cell response (genomic profile) similar to that achieved with chemical stimulation can be elicited. Here, we show that bone formation can be achieved with efficiency similar to that of dexamethasone with the added benefit that endothelial cell development is not inhibited. We further show that the mechanism of action of the topographies and dexamethasone differs. This could have an implication for tissue engineering in which a simultaneous, targeted, development of a tissue, such as bone, without the suppression of angiogenesis to supply nutrients to the new tissue is required. The results further demonstrate that perhaps the shape of the extracellular matrix is critical to tissue development.
Assuntos
Células-Tronco Mesenquimais/fisiologia , Nanoestruturas , Osteogênese/fisiologia , Engenharia Tecidual/métodos , Materiais Biomiméticos , Diferenciação Celular/fisiologia , Perfilação da Expressão Gênica , Humanos , Células-Tronco Mesenquimais/citologia , Análise em Microsséries , Osteogênese/genéticaRESUMO
The rise in the incidence of musculoskeletal diseases is attributed to an increasing ageing population. The debilitating effects of musculoskeletal diseases, coupled with a lack of effective therapies, contribute to huge financial strains on healthcare systems. The focus of regenerative medicine has shifted to pluripotent stem cells (PSCs), namely, human embryonic stem cells and human-induced PSCs, due to the limited success of adult stem cell-based interventions. PSCs constitute a valuable cell source for musculoskeletal regeneration due to their capacity for unlimited self-renewal, ability to differentiate into all cell lineages of the three germ layers and perceived immunoprivileged characteristics. This review summarizes methods for chondrogenic, osteogenic, myogenic and adipogenic differentiation of PSCs and their potential for therapeutic applications.
RESUMO
The unique properties of skeletal stem cells have attracted significant attention in the development of strategies for skeletal regeneration. However, there remains a crucial unmet need to develop quantitative tools to elucidate skeletal cell development and monitor the formation of regenerated tissues using non-destructive techniques in 3D. Label-free methods such as coherent anti-Stokes Raman scattering (CARS), second harmonic generation (SHG) and two-photon excited auto-fluorescence (TPEAF) microscopy are minimally invasive, non-destructive, and present new powerful alternatives to conventional imaging techniques. Here we report a combination of these techniques in a single multimodal system for the temporal assessment of cartilage formation by human skeletal cells. The evaluation of bioengineered cartilage, with a new parameter measuring the amount of collagen per cell, collagen fibre structure and chondrocyte distribution, was performed using the 3D non-destructive platform. Such 3D label-free temporal quantification paves the way for tracking skeletal cell development in real-time and offers a paradigm shift in tissue engineering and regenerative medicine applications.
Assuntos
Osso e Ossos/embriologia , Cartilagem/fisiologia , Condrogênese/fisiologia , Imageamento Tridimensional , Engenharia Tecidual/métodos , Engenharia Biomédica , Diferenciação Celular , Condrócitos , Colágeno/química , Perfilação da Expressão Gênica , Humanos , Processamento de Imagem Assistida por Computador , Microscopia de Fluorescência , Oxirredução , Medicina Regenerativa , Análise Espectral Raman , Células-Tronco/citologia , Fatores de TempoRESUMO
Bioacoustofluidics can be used to trap and levitate cells within a fluid channel, thereby facilitating scaffold-free tissue engineering in a 3D environment. In the present study, we have designed and characterised an acoustofluidic bioreactor platform, which applies acoustic forces to mechanically stimulate aggregates of human articular chondrocytes in long-term levitated culture. By varying the acoustic parameters (amplitude, frequency sweep, and sweep repetition rate), cells were stimulated by oscillatory fluid shear stresses, which were dynamically modulated at different sweep repetition rates (1-50 Hz). Furthermore, in combination with appropriate biochemical cues, the acoustic stimulation was tuned to engineer human cartilage constructs with structural and mechanical properties comparable to those of native human cartilage, as assessed by immunohistology and nano-indentation, respectively. The findings of this study demonstrate the capability of acoustofluidics to provide a tuneable biomechanical force for the culture and development of hyaline-like human cartilage constructs in vitro.
Assuntos
Cartilagem/citologia , Técnicas Analíticas Microfluídicas/instrumentação , Engenharia Tecidual/instrumentação , Engenharia Tecidual/métodos , Alicerces Teciduais , Acústica , Fenômenos Biomecânicos , Reatores Biológicos , Condrócitos/citologia , HumanosRESUMO
Bag1 transcribes a multifunctional protein that participates in many important biological processes such as cell apoptosis, proliferation, differentiation and embryo development. Despite numerous published studies, the role of Bag1 in the context of embryonic stem (ES) cells, has not been explored. To investigate the function of Bag1 in ES cells, we generated mutant Bag1-/- ES cells using the CRISPR/Cas9 system. We established that the Bag1 double knockout ES cell line maintained their pluripotency, possessed a normal karyotype and the ability to differentiate into all three germ layers.
Assuntos
Sistemas CRISPR-Cas , Proteínas de Ligação a DNA/deficiência , Homozigoto , Células-Tronco Embrionárias Murinas/metabolismo , Fatores de Transcrição/deficiência , Animais , Linhagem Celular , Camundongos , Camundongos Knockout , Células-Tronco Embrionárias Murinas/citologiaRESUMO
Given articular cartilage has a limited repair potential, untreated osteochondral lesions of the ankle can lead to debilitating symptoms and joint deterioration necessitating joint replacement. While a wide range of reparative and restorative surgical techniques have been developed to treat osteochondral lesions of the ankle, there is no consensus in the literature regarding which is the ideal treatment. Tissue engineering strategies, encompassing stem cells, somatic cells, biomaterials, and stimulatory signals (biological and mechanical), have a potentially valuable role in the treatment of osteochondral lesions. Mesenchymal stem cells (MSCs) are an attractive resource for regenerative medicine approaches, given their ability to self-renew and differentiate into multiple stromal cell types, including chondrocytes. Although MSCs have demonstrated significant promise in in vitro and in vivo preclinical studies, their success in treating osteochondral lesions of the ankle is inconsistent, necessitating further clinical trials to validate their application. This review highlights the role of MSCs in cartilage regeneration and how the application of biomaterials and stimulatory signals can enhance chondrogenesis. The current treatments for osteochondral lesions of the ankle using regenerative medicine strategies are reviewed to provide a clinical context. The challenges for cartilage regeneration, along with potential solutions and safety concerns are also discussed.
Assuntos
Articulação do Tornozelo/fisiopatologia , Doenças das Cartilagens/terapia , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/citologia , Animais , Células Cultivadas , Humanos , CamundongosRESUMO
AIM: To fabricate PEGylated liposomes which preserve the activity of hydrophobic Wnt3A protein, and to demonstrate their efficacy in promoting expansion of osteoprogenitors from human bone marrow. METHODS: PEGylated liposomes composed of several synthetic lipids were tested for their ability to preserve Wnt3A activity in reporter and differentiation assays. Single-molecule microspectroscopy was used to test for direct association of protein with liposomes. RESULTS: Labeled Wnt3A protein directly associated with all tested liposome preparations. However, Wnt3A activity was preserved or enhanced in PEGylated 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) liposomes but not in PEGylated 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) liposomes. PEGylated Wnt3A liposomes associated with skeletal stem cell populations in human bone marrow and promoted osteogenesis. CONCLUSION: Active Wnt protein-containing PEGylated liposomes may have utility for systemic administration for bone repair.