RESUMO
Cells express several G-protein-coupled receptors (GPCRs) at their surfaces, transmitting simultaneous extracellular hormonal and chemical signals into cells. A comprehensive understanding of mechanisms underlying the integrated signaling response induced by distinct GPCRs is thus required. Here we found that the ß2-adrenergic receptor, which induces a short cAMP response, prolongs nuclear cAMP and protein kinase A (PKA) activation by promoting endosomal cAMP production in parathyroid hormone (PTH) receptor signaling through the stimulatory action of G protein Gßγ subunits on adenylate cyclase type 2.
Assuntos
Endossomos/metabolismo , Subunidades beta da Proteína de Ligação ao GTP/metabolismo , Subunidades gama da Proteína de Ligação ao GTP/metabolismo , Receptor Tipo 1 de Hormônio Paratireóideo/metabolismo , Receptores Adrenérgicos beta 2/metabolismo , Transdução de Sinais , Células Cultivadas , Células HEK293 , Humanos , Subunidades Proteicas/metabolismoRESUMO
PURPOSE: Biodegradable polymeric scaffolds have been used for tissue engineering approaches and can be used to regenerate temporomandibular joint (TMJ) tissues. Synthetic acellular polymeric poly(glycerol sebacate) (PGS) scaffolds and natural scaffolds made from gelatin are polymeric scaffold sponges that could provide a substrate for cell infiltration and remodeling. The authors studied the regenerative potential of these 2 scaffolds in addition to a bioactive signal, magnesium (Mg), in a novel fibrocartilage defect model in the goat mandibular condylar cartilage (MCC). Furthermore, in a departure from the pig model, the authors have started to develop the goat as a repeatable surgical model with easy access into the joint space in skeletally mature animals. MATERIALS AND METHODS: Bilateral osteochondral defects were created in the mandibular condyle of mature female Spanish Boer goats. A 1-mm diameter drill was used to create a trough defect on the articular surface. Four groups were evaluated: 1) an empty control without an implant, 2) PGS with Mg ions, 3) gelatin with Mg ions, and 4) gelatin with Mg ions and trimagnesium phosphate (TMP) powder. Goats were allowed to heal for 3 months, and then the tissues were harvested. RESULTS: The empty control group showed a thin fibrous layer growing within the defect. The PGS and gelatin sponge groups showed a cartilage layer with glycosaminoglycan and collagen type II and robust regeneration of the fibrous layer as exhibited by cell infiltration and collagen in the defect. TMP in the gelatin did not degrade and seemed to hamper healing. CONCLUSION: These results suggest that synthetic and natural sponges can provide a template for new tissue growth in the MCC of the TMJ. Furthermore, this study is the first to attempt to develop the goat as an in vivo TMJ tissue regeneration model.
Assuntos
Decanoatos/farmacologia , Glicerol/análogos & derivados , Côndilo Mandibular/cirurgia , Polímeros/farmacologia , Tampões de Gaze Cirúrgicos , Articulação Temporomandibular/cirurgia , Engenharia Tecidual/métodos , Alicerces Teciduais/química , Animais , Colágeno/farmacologia , Modelos Animais de Doenças , Feminino , Gelatina/farmacologia , Glicerol/farmacologia , Cabras , Compostos de Magnésio/farmacologia , Fosfatos/farmacologia , Propriedades de Superfície , Cicatrização/fisiologiaRESUMO
The objectives of this study were to compare the chondrogenic potential of cells derived from different layers of Mandibular condyle cartilage and to gain further understanding of the impact of chondrogenic cues when embedded into a novel hydrogel scaffold (PGH, a polymer blend of poly (ethylene glycol), gelatin, and heparin) compared to a gelatin hydrogel scaffold (GEL). Cartilage layer cells (CLCs) and fibroblastic superficial layer cells (SLCs) were harvested from the mandibular condyle of boer goats obtained from a local abattoir. After expansion, cells were seeded into PGH and GEL hydrogels and cultured in chondrogenic media for 3 weeks. Scaffolds were harvested at 0, 1, and 3 week(s) and processed for gross appearance, histochemical, biochemical, and mechanical assays. In terms of chondrogenesis, major differences were observed between scaffold materials, but not cell types. Glycosaminoglycan (GAG) staining showed GEL scaffolds deposited GAG during the 3 week period, which was also confirmed with the biochemical testing. Moreover, GEL scaffolds had significantly higher compressive modulus and peak stress than PGH scaffolds at all time points with the largest difference seen in week 3. It can be concluded that GEL outperformed PGH in chondrogenesis. It can also be concluded that materials play a more important role in the process of chondrogenesis than the tested cell populations. Fibroblastic SLCs were shown to have similar chondrogenic potential as CLCs cells, suggesting a rich pool of progenitor cells in the superficial fibroblastic layer capable of undergoing chondrogenesis given appropriate physical and chemical cues.
Assuntos
Cartilagem , Condrogênese , Gelatina , Cabras , Hidrogéis , Articulação Temporomandibular , Hidrogéis/química , Gelatina/química , Animais , Cartilagem/citologia , Articulação Temporomandibular/citologia , Alicerces Teciduais/química , Polimerização , Côndilo Mandibular/citologia , Côndilo Mandibular/fisiologia , Processos Fotoquímicos , Glicosaminoglicanos/química , Glicosaminoglicanos/metabolismoRESUMO
The ezrin-radixin-moesin-binding phosphoprotein 50 (EBP50) is a PDZ-containing scaffolding protein that regulates a variety of physiological functions. In the vasculature, EBP50 promotes neointima formation following arterial injury. In this study the role of EBP50 on vascular smooth muscle cell (VSMC) migration was characterized. The spreading and motility of primary VSMC isolated from EBP50 knockout (KO) mice were significantly reduced compared to wild-type (WT) cells. EBP50-null VSMC had fewer and larger focal adhesions than wild-type cells. Assembly and disassembly of focal adhesion-assessed by live-cell total internal reflection fluorescence imaging-in response to epidermal growth factor (EGF) were significantly reduced in KO cells. Immunoprecipitation experiments showed that EBP50 interacts with EGF receptor via the PDZ2 domain and with focal adhesion kinase (FAK) via the C-terminal ERM domain. EBP50 promoted the formation of a complex containing both EGF receptor and FAK. Phosphorylation of Tyr-925 of FAK in response to EGF was significantly reduced in KO cell compared to WT cells. The residence time of FAK in focal adhesions-determined by fluorescence recovery after photobleaching-was increased in WT cells. Collectively, these studies indicate that EBP50, by scaffolding EGF receptor and FAK, facilitates activation of FAK, focal adhesion turnover, and migration of VSMC.
Assuntos
Vasos Sanguíneos/metabolismo , Movimento Celular , Adesões Focais/metabolismo , Miócitos de Músculo Liso/metabolismo , Fosfoproteínas/metabolismo , Trocadores de Sódio-Hidrogênio/metabolismo , Animais , Linhagem Celular , Movimento Celular/efeitos dos fármacos , Movimento Celular/genética , Fator de Crescimento Epidérmico/farmacologia , Receptores ErbB/metabolismo , Quinase 1 de Adesão Focal/metabolismo , Adesões Focais/efeitos dos fármacos , Adesões Focais/genética , Camundongos , Camundongos Knockout , Miócitos de Músculo Liso/efeitos dos fármacos , Fosfoproteínas/genética , Fosforilação/efeitos dos fármacos , Ligação Proteica , Trocadores de Sódio-Hidrogênio/genéticaRESUMO
The present study utilizes image-based computational methods and indirect solid freeform fabrication (SFF) technique to design and fabricate porous scaffolds, and then computationally estimates their elastic modulus and yield stress with experimental validation. 50:50 Poly (lactide-co-glycolide acid) (50:50 PLGA) porous scaffolds were designed using an image-based design technique, fabricated using indirect SFF technique, and characterized using micro-computed tomography (micro-CT) and mechanical testing. Micro-CT data was further used to non-destructively predict the scaffold elastic moduli and yield stress using a voxel-based finite element (FE) method, a technique that could find application in eventual scaffold quality control. Micro-CT data analysis confirmed that the fabricated scaffolds had controlled pore sizes, orthogonally interconnected pores and porosities which were identical to those of the designed files. Mechanical tests revealed that the compressive modulus and yield stresses were in the range of human trabecular bone. The results of FE analysis showed potential stress concentrations inside of the fabricated scaffold due to fabrication defects. Furthermore, the predicted moduli and yield stresses of the FE analysis showed strong correlations with those of the experiments. In the present study, we successfully fabricated scaffolds with designed architectures as well as predicted their mechanical properties in a nondestructive manner.
Assuntos
Osso e Ossos , Simulação por Computador , Ácido Láctico/química , Ácido Láctico/síntese química , Ácido Poliglicólico/química , Ácido Poliglicólico/síntese química , Engenharia Tecidual/métodos , Alicerces Teciduais , Substitutos Ósseos/síntese química , Substitutos Ósseos/química , Osso e Ossos/fisiologia , Força Compressiva , Análise de Elementos Finitos , Humanos , Teste de Materiais , Modelos Biológicos , Concentração Osmolar , Copolímero de Ácido Poliláctico e Ácido Poliglicólico , Porosidade , Desenho de Prótese/métodos , Estresse Mecânico , Alicerces Teciduais/químicaRESUMO
The ideal combination of hydrogel components for regeneration of cartilage and cartilaginous interfaces is a significant challenge because control over differentiation into multiple lineages is necessary. Stabilization of the phenotype of stem cell derived chondrocytes is needed to avoid undesired progression to terminal hypertrophy and tissue mineralization. A novel ternary blend hydrogel composed of methacrylated poly(ethylene glycol) (PEG), gelatin, and heparin (PGH) was designed to guide chondrogenesis by bone marrow derived mesenchymal stem cells (BMSCs) and maintenance of their cartilaginous phenotype. The hydrogel material effects on chondrogenic and osteogenic differentiation by BMSCs were evaluated in comparison to methacrylated gelatin hydrogel (GEL), a conventional bioink used for both chondrogenic and osteogenic applications. PGH and GEL hydrogels were loaded with goat BMSCs and cultured in chondrogenic and osteogenic mediums in vitro over six weeks. The PGH showed no sign of mineral deposition in an osteogenic environment in vitro. To further evaluate material effects, the hydrogels were loaded with adult human BMSCs (hBMSCs) and transforming growth factor ß-3 and grown in subcutaneous pockets in mice over eight weeks. Consistent with the in vitro results, the PGH had greater potential to induce chondrogenesis by BMSCs in vivo compared to the GEL as evidenced by elevated gene expression of chondrogenic markers, supporting its potential for stable cartilage engineering. The PGH also showed a greater percentage of GAG positive cells compared to the GEL. Unlike the GEL, the PGH hydrogel exhibited anti-osteogenic effects in vivo as evidenced by negative Von Kossa staining and suppressed gene expression of hypertrophic and osteogenic markers. By nature of their polymer composition alone, the PGH and GEL regulated BMSC differentiation down different osteochondral lineages. Thus, the PGH and GEL are promising hydrogels to regenerate stratified cartilaginous interfacial tissues in situ, such as the mandibular condyle surface, using undifferentiated BMSCs and a stratified scaffold design.
Assuntos
Cartilagem/metabolismo , Condrogênese/fisiologia , Hidrogéis/química , Células-Tronco Mesenquimais/citologia , Osteogênese/fisiologia , Células-Tronco/metabolismo , Fator de Crescimento Transformador beta3/metabolismo , Animais , Células da Medula Óssea/citologia , Bovinos , Diferenciação Celular , Linhagem da Célula , Células Cultivadas , Condrócitos , Reagentes de Ligações Cruzadas/química , Gelatina/química , Regulação da Expressão Gênica , Humanos , Espectroscopia de Ressonância Magnética , Camundongos , Fenótipo , Polietilenoglicóis , Polímeros/química , Regeneração/efeitos dos fármacos , Alicerces TeciduaisRESUMO
Aquaporins (AQPs) are membrane channels that provide for transport of water and other small molecules across the lipid bilayer of cells. Their function is essential for physiologic processes such as cell volume regulation, chondrocyte hypertrophy during appendicular skeletal growth, water reabsorption in the kidney tubules, and water excretion by the salivary glands. The ten AQP isoforms show tissue specificity and are involved in different pathologies and inflammatory diseases. This study addresses the hypothesis that arthritis, periodontitis, and temporomandibular joint disorders (TMDs) can be influenced by variation in the AQP genes at 12q13.12 locus. Salivary samples of 688 individuals were obtained from the Dental Registry and DNA Repository project at the University of Pittsburgh. Ten polymorphisms in four AQP genes (AQP1, 2, 5, and 6) were genotyped and correlated to disease status as reported by patients. Associations were found between the single nucleotide polymorphism (SNP) rs467323 in AQP2 and TMD in both genotypic (p = 0.03) and recessive (p = 0.02) models, and between rs1996315 in AQP6 and periodontitis (p = 0.05). Combined analysis of TMD and periodontitis showed an association with rs3741559 in AQP2 (p = 0.02). When conducting haplotype analysis of rs467323 and rs10875989 in AQP2, the haplotype CT showed an association with the TMD phenotype (p = 0.007). Our results suggest that the aquaporin locus at 12q13.12 may contribute to the pathogenesis of inflammatory conditions such as periodontitis and TMD. Thus, oral and skeletal health are correlated and potential susceptibility screening strategies may be developed.
Assuntos
Aquaporinas/genética , Loci Gênicos/genética , Predisposição Genética para Doença/genética , Periodontite/complicações , Transtornos da Articulação Temporomandibular/complicações , Transtornos da Articulação Temporomandibular/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Haplótipos , Humanos , Masculino , Pessoa de Meia-Idade , Polimorfismo de Nucleotídeo Único , Adulto JovemRESUMO
Mesenchymal stem cells (MSCs) respond to a variety of differentiation signal provided by their local environments. A large portion of these signals originate from the extracellular matrix (ECM). At the same time, MSCs secrete various matrix-altering agents, including proteases, that alter ECM-encoded differentiation signals. Here we investigated the interactions between MSC and ECM produced by endothelial cells (EC-matrix), focusing not only on the differentiation signals provided by EC-matrix, but also on MSC-alteration of these signals and the resultant affects on MSC differentiation. MSCs were cultured on EC-matrix modified in one of three distinct ways. First, MSCs cultured on native EC-matrix underwent endothelial cell (EC) differentiation early during the culture period and smooth muscle cell (SMC) differentiation at later time points. Second, MSCs cultured on crosslinked EC-matrix, which is resistant to MSC modification, differentiated towards an EC lineage only. Third, MSCs cultured on EC-matrix pre-modified by MSCs underwent SMC-differentiation only. These MSC-induced matrix alterations were found to deplete the factors responsible for EC-differentiation, yet activate the SMC-differentiation factors. In conclusion, our results demonstrate that the EC-matrix contains factors that support MSC differentiation into both ECs and SMCs, and that these factors are modified by MSC-secreted agents. By analyzing the framework by which EC-matrix regulates differentiation in MSCs, we have uncovered evidence of a feedback system in which MSCs are able to alter the very matrix signals acting upon them.
Assuntos
Diferenciação Celular , Células Endoteliais/citologia , Endotélio Vascular/citologia , Matriz Extracelular/fisiologia , Células-Tronco Mesenquimais/citologia , Técnicas de Cultura de Células , Linhagem Celular , Células Endoteliais/ultraestrutura , Retroalimentação Fisiológica , Humanos , Miócitos de Músculo Liso/citologiaRESUMO
Mesenchymal stem cells (MSCs) are thought to occupy a perivascular niche where they are exposed to signals originating from vascular cells. This study focused on the effects of endothelial cell (EC)-derived signals on MSC differentiation toward vascular cell lineages. Upon co-culture with two types of ECs, macrovascular (macro) ECs and microvascular (micro) ECs, the former caused MSCs to increase expression of both EC and smooth muscle cell (SMC) markers, while the latter induced expression of EC markers only. These marker changes in MSCs were linked to the extracellular matrixes secreted by the ECs (EC-matrix) rather than soluble EC-secreted factors. Beyond enhanced marker expression, EC-matrix also induced functional changes in MSCs indicative of development of a genuine vascular cell phenotype. These included enhanced incorporation into vessels and cytoskeletal localization of vascular SMC-specific contractile elements. The bioactivity of EC-matrix was sensitive to EDTA washes and required sulfated glycosaminoglycans. However, neither soluble VEGF nor substrate surfaces coated with fibronectin, collagen type IV, or laminin recreated the effects of EC-matrix on MSC vascular differentiation. In conclusion, these results identified EC-matrix as a critical regulator of vascular cell differentiation of MSCs. Elucidating these MSC-EC-matrix interactions and identifying the specific EC-matrix components involved will shed light on the perivascular signals seen by MSCs in vivo.
Assuntos
Comunicação Celular , Diferenciação Celular , Células Endoteliais/fisiologia , Endotélio Vascular/citologia , Células-Tronco Mesenquimais/citologia , Movimento Celular , Técnicas de Cocultura , Matriz Extracelular/metabolismo , Humanos , Músculo Liso Vascular/citologia , Miócitos de Músculo Liso/citologia , Nicho de Células-Tronco/citologiaRESUMO
Human monocytes have been grouped into classical (CD14++CD16-), non-classical (CD14dimCD16++), and intermediate (CD14++CD16+) subsets. Documentation of normal function and variation in this complement of subtypes, particularly their differentiation potential to dendritic cells (DC) or macrophages, remains incomplete. We therefore phenotyped monocytes from peripheral blood of healthy subjects and performed functional studies on high-speed sorted subsets. Subset frequencies were found to be tightly controlled over time and across individuals. Subsets were distinct in their secretion of TNFα, IL-6, and IL-1ß in response to TLR agonists, with classical monocytes being the most producers and non-classical monocytes the least. Monocytes, particularly those of the non-classical subtype, secreted interferon-α (IFN-α) in response to intracellular TLR3 stimulation. After incubation with IL-4 and GM-CSF, classical monocytes acquired monocyte-derived DC (mo-DC) markers and morphology and stimulated allogeneic T cell proliferation in MLR; intermediate and non-classical monocytes did not. After incubation with IL-3 and Flt3 ligand, no subset differentiated to plasmacytoid DC. After incubation with GM-CSF (M1 induction) or macrophage colony-stimulating factor (M-CSF) (M2 induction), all subsets acquired macrophage morphology, secreted macrophage-associated cytokines, and displayed enhanced phagocytosis. From these studies we conclude that classical monocytes are the principal source of mo-DCs, but all subsets can differentiate to macrophages. We also found that monocytes, in particular the non-classical subset, represent an alternate source of type I IFN secretion in response to virus-associated TLR agonists.
Assuntos
Diferenciação Celular , Monócitos/citologia , Monócitos/metabolismo , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Células Dendríticas/citologia , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Humanos , Imunofenotipagem , Interleucina-1beta/metabolismo , Interleucina-6/metabolismo , Fator Estimulador de Colônias de Macrófagos/farmacologia , Macrófagos/citologia , Macrófagos/imunologia , Macrófagos/metabolismo , Microscopia de Fluorescência , Fagocitose/efeitos dos fármacos , Fenótipo , Receptores Toll-Like/agonistas , Receptores Toll-Like/metabolismo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Imaging of Förster resonance energy transfer (FRET) is a powerful tool for examining cell biology in real-time. Studies utilizing FRET commonly employ two-dimensional (2D) culture, which does not mimic the three-dimensional (3D) cellular microenvironment. A method to perform quenched emission FRET imaging using conventional widefield epifluorescence microscopy of cells within a 3D hydrogel environment is presented. Here an analysis method for ratiometric FRET probes that yields linear ratios over the probe activation range is described. Measurement of intracellular cyclic adenosine monophosphate (cAMP) levels is demonstrated in chondrocytes under forskolin stimulation using a probe for EPAC1 activation (ICUE1) and the ability to detect differences in cAMP signaling dependent on hydrogel material type, herein a photocrosslinking hydrogel (PC-gel, polyethylene glycol dimethacrylate) and a thermoresponsive hydrogel (TR-gel). Compared with 2D FRET methods, this method requires little additional work. Laboratories already utilizing FRET imaging in 2D can easily adopt this method to perform cellular studies in a 3D microenvironment. It can further be applied to high throughput drug screening in engineered 3D microtissues. Additionally, it is compatible with other forms of FRET imaging, such as anisotropy measurement and fluorescence lifetime imaging (FLIM), and with advanced microscopy platforms using confocal, pulsed, or modulated illumination.
Assuntos
Transferência Ressonante de Energia de Fluorescência/métodos , Hidrogéis , Microscopia de Fluorescência , Imagem ÓpticaRESUMO
UNLABELLED: Craniosynostosis, premature fusion of the skull bones at the sutures, is the second most common human birth defect in the skull. Raman microspectroscopy was used to examine the composition, relative amounts, and locations of the mineral and matrix produced in mouse skulls undergoing force-induced craniosynostosis. Raman imaging revealed decreased relative mineral content in skulls undergoing craniosynostosis compared with unloaded specimens. INTRODUCTION: Raman microspectroscopy, a nondestructive vibrational spectroscopic technique, was used to examine the composition, relative amounts, and locations of the mineral and matrix produced in mouse skulls undergoing force-induced craniosynostosis. Craniosynostosis, premature fusion of the skull bones at the sutures, is the second most common birth defect in the face and skull. The calvaria, or flat bones that comprise the top of the skull, are most often affected, and craniosynostosis is a feature of over 100 human syndromes and conditions. MATERIALS AND METHODS: Raman images of the suture, the tips immediately adjacent to the suture (osteogenic fronts), and mature parietal bones of loaded and unloaded calvaria were acquired. Images were acquired at 2.6 x 2.6 microm spatial resolution and ranged in a field of view from 180 x 210 microm to 180 x 325 microm. RESULTS AND CONCLUSIONS: This study found that osteogenic fronts subjected to uniaxial compression had decreased relative mineral content compared with unloaded osteogenic fronts, presumably because of new and incomplete mineral deposition. Increased matrix production in osteogenic fronts undergoing craniosynostosis was observed. Understanding how force affects the composition, relative amounts, and location of the mineral and matrix provides insight into musculoskeletal disease in general and craniosynostosis in particular. This is the first report in which Raman microspectroscopy was used to study musculoskeletal disease. These data show how Raman microspectroscopy can be used to study subtle changes that occur in disease.
Assuntos
Densidade Óssea , Matriz Óssea/metabolismo , Craniossinostoses/metabolismo , Análise Espectral Raman/métodos , Animais , Matriz Óssea/fisiopatologia , Calcificação Fisiológica , Suturas Cranianas/metabolismo , Suturas Cranianas/patologia , Suturas Cranianas/fisiopatologia , Craniossinostoses/fisiopatologia , Análise Fatorial , Camundongos , Camundongos Endogâmicos CBA , Doenças Musculoesqueléticas/metabolismo , Doenças Musculoesqueléticas/fisiopatologia , Osso Parietal/metabolismo , Osso Parietal/fisiopatologia , Estresse MecânicoRESUMO
Tissue engineering has provided an alternative to traditional strategies to repair cartilage damaged by injury or degenerative disease. A successful strategy to engineer osteochondral tissue will mimic the natural contour of the articulating surface, achieve native mechanical properties and functional load-bearing ability, and lead to integration with host cartilage and underlying subchondral bone. Image-based design (IBD) and solid free-form (SFF) fabrication can be used to generate scaffolds that are load bearing and match articular geometry. The objective of this study was to utilize materials and biological factors in an integrated approach to regenerate a multitissue interface. Biphasic composite scaffolds manufactured by IBD and SFF fabrication were used to simultaneously generate bone and cartilage in discrete regions and provide for the development of a stable interface between cartilage and subchondral bone. Poly-L-lactic acid/hydroxyapatite composite scaffolds were differentially seeded with fibroblasts transduced with an adenovirus expressing bone morphogenetic protein 7 (BMP-7) in the ceramic phase and fully differentiated chondrocytes in the polymeric phase. After subcutaneous implantation into mice, the biphasic scaffolds promoted the simultaneous growth of bone, cartilage, and a mineralized interface tissue. Within the ceramic phase, the pockets of tissue generated included blood vessels, marrow stroma, and adipose tissue. This combination of IBD and SFF-fabricated biphasic scaffolds with gene and cell therapy is a promising approach to regenerate osteochondral defects.
Assuntos
Materiais Biocompatíveis/química , Condrócitos/fisiologia , Condrócitos/transplante , Osteócitos/fisiologia , Osteócitos/transplante , Engenharia Tecidual/métodos , Transplantes , Animais , Órgãos Bioartificiais , Técnicas de Cultura de Células/instrumentação , Técnicas de Cultura de Células/métodos , Diferenciação Celular/fisiologia , Células Cultivadas , Condrócitos/citologia , Condrócitos/diagnóstico por imagem , Condrogênese/fisiologia , Fibroblastos/citologia , Fibroblastos/fisiologia , Humanos , Teste de Materiais , Camundongos , Osteócitos/citologia , Osteócitos/diagnóstico por imagem , Osteogênese/fisiologia , Radiografia , Propriedades de Superfície , Suínos , Engenharia Tecidual/instrumentaçãoRESUMO
Tissue-engineering strategies to stimulate bone regeneration may offer an alternative approach to conventional orthopaedic and maxillofacial surgical therapies. Over the last decade, significant advances have been accomplished in developing biomimetic matrices, growth factors, cell transplantation and gene delivery therapeutics to support new bone growth. However, it is not known if tissue-engineered bone recapitulates the biology of normal skeletal tissue in response to physiologic cues. Here, we report that bone formed by the differentiation of transplanted murine bone marrow stromal cells (BMSCs) responds to a systemically delivered calciotropic hormone. Ectopic ossicles in mice exposed to catabolic doses of parathyroid hormone (PTH) had increased numbers of tartrate-resistant acid phosphatase (TRAP)-positive osteoclasts as compared to control mice. In contrast, treatment with anabolic doses of PTH promoted a marked increase in trabecular bone mass as analyzed by microcomputed tomography and histomorphometry. Our findings demonstrate that bone formed from transplanted BMSCs is responsive to normal physiologic signals, and can be augmented by the addition of a systemic anabolic agent. Because multiple and distinct ossicles can be generated in a single animal, this versatile system may be used to: (a) elucidate cellular/molecular mechanisms in bone regeneration; (b) study cell-to-cell interactions in the bone marrow microenvironment in health and disease; and (c) evaluate the efficacy of osteotropic agents that modulate bone turnover in vivo.
Assuntos
Osso e Ossos , Homeostase , Esqueleto , Engenharia Tecidual , Fosfatase Ácida/metabolismo , Animais , Células da Medula Óssea/citologia , Células da Medula Óssea/efeitos dos fármacos , Transplante de Medula Óssea , Osso e Ossos/citologia , Osso e Ossos/diagnóstico por imagem , Contagem de Células , Diferenciação Celular , Relação Dose-Resposta a Droga , Isoenzimas/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Osteoclastos/citologia , Osteoclastos/metabolismo , Osteogênese , Hormônio Paratireóideo/administração & dosagem , Radiografia , Células Estromais/citologia , Células Estromais/efeitos dos fármacos , Células Estromais/transplante , Fosfatase Ácida Resistente a TartaratoRESUMO
OBJECTIVE: Traumatic impacts on the articular joint surface in vitro are known to lead to degeneration of the cartilage. The main objective of this study was to develop a spring-loaded impact device that can be used to deliver traumatic impacts of consistent magnitude and rate and to find whether impacts cause catabolic activities in articular cartilage consistent with other previously reported impact models and correlated with the development of osteoarthritic lesions. In developing the spring-loaded impactor, the operating hypothesis is that a single supraphysiologic impact to articular cartilage in vitro can affect cartilage integrity, cell viability, sulfated glycosaminoglycan and inflammatory mediator release in a dose-dependent manner. DESIGN: Impacts of increasing force are delivered to adult bovine articular cartilage explants in confined compression. Impact parameters are correlated with tissue damage, cell viability, matrix and inflammatory mediator release, and gene expression 24 hours postimpact. RESULTS: Nitric oxide release is first detected after 7.7 MPa impacts, whereas cell death, glycosaminoglycan release, and prostaglandin E2 release are first detected at 17 MPa. Catabolic markers increase linearly to maximal levels after ≥36 MPa impacts. CONCLUSIONS: A single supraphysiologic impact negatively affects cartilage integrity, cell viability, and GAG release in a dose-dependent manner. Our findings showed that 7 to 17 MPa impacts can induce cell death and catabolism without compromising the articular surface, whereas a 17 MPa impact is sufficient to induce increases in most common catabolic markers of osteoarthritic degeneration.
RESUMO
INTRODUCTION: Mesenchymal stem cells (MSCs) offer promise for intervertebral disc (IVD) repair and regeneration because they are easily isolated and expanded, and can differentiate into several mesenchymal tissues. Notochordal (NC) cells contribute to IVD development, incorporate into the nucleus pulposus (NP), and stimulate mature disc cells. However, there have been no studies investigating the effects of NC cells on adult stem cell differentiation. The premise of this study is that IVD regeneration is more similar to IVD development than to IVD maintenance, and we hypothesize that soluble factors from NC cells differentiate MSCs to a phenotype characteristic of nucleus pulposus (NP) cells during development. The eventual clinical goal would be to isolate or chemically/recombinantly produce the active agent to induce the therapeutic effects, and to use it as either an injectable therapy for early intervention on disc disease, or in developing appropriately pre-differentiated MSC cells in a tissue engineered NP construct. METHODS: Human MSCs from bone marrow were expanded and pelleted to form high-density cultures. MSC pellets were exposed to either control medium (CM), chondrogenic medium (CM with dexamethasone and transforming growth factor, (TGF)-beta3) or notochordal cell conditioned medium (NCCM). NCCM was prepared from NC cells maintained in serum free medium for four days. After seven days culture, MSC pellets were analyzed for appearance, biochemical composition (glycosaminoglycans and DNA), and gene expression profile (sox-9, collagen types-II and III, laminin-beta1 and TIMP1(tissue inhibitor of metalloproteinases-1)). RESULTS: Significantly higher glycosaminoglycan accumulation was seen in NCCM treated pellets than in CM or TGFbeta groups. With NCCM treatment, increased gene expression of collagen III, and a trend of increasing expression of laminin-beta1 and decreased expression of sox-9 and collagen II relative to the TGFbeta group was observed. CONCLUSIONS: Together, results suggest NCCM stimulates mesenchymal stem cell differentiation toward a potentially NP-like phenotype with some characteristics of the developing IVD.