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Tissue engineering and cell therapy for regenerative medicine have great potential to treat chronic disorders. In musculoskeletal disorders, mesenchymal stromal cells (MSCs) have been identified as a relevant cell type in cell and regenerative strategies due to their multi-lineage potential, although this is likely to be a result of their trophic and immunomodulatory effects on other cells. This PRISMA systematic review aims to assess whether the age of the patient influences the chondrogenic potential of MSCs in regenerative therapy. We identified a total of 3027 studies after performing a search of four databases, including Cochrane, Web of Science, Medline, and PubMed. After applying inclusion and exclusion criteria, a total of 14 papers were identified that were reviewed, assessed, and reported. Cell surface characterization and proliferation, as well as the osteogenic, adipogenic, and chondrogenic differentiation, were investigated as part of the analysis of these studies. Most included studies suggest a clear link between aged donor MSCs and diminished clonogenic and proliferative potential. Our study reveals a heterogeneous and conflicting range of outcomes concerning the chondrogenic, osteogenic, and adipogenic potential of MSCs in relation to age. Further investigations on the in vitro effects of chronological age on the chondrogenic potential of MSCs should follow the outcomes of this systematic review, shedding more light on this complex relationship.
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Células Madre Mesenquimatosas , Humanos , Anciano , Células Madre Mesenquimatosas/metabolismo , Diferenciación Celular , Osteogénesis , Adipogénesis , Ingeniería de Tejidos , Células Cultivadas , CondrogénesisRESUMEN
Mechanical loading exerts a profound influence on bone density and architecture, but the exact mechanism is unknown. Our study shows that expression of the neurological transcriptional factor zinc finger of the cerebellum 1 (ZIC1) is markedly increased in trabecular bone biopsies in the lumbar spine compared with the iliac crest, skeletal sites of high and low mechanical stress, respectively. Human trabecular bone transcriptome analyses revealed a strong association between ZIC1 mRNA levels and gene transcripts characteristically associated with osteoblasts, osteocytes and osteoclasts. This supposition is supported by higher ZIC1 expression in iliac bone biopsies from postmenopausal women with osteoporosis compared with age-matched control subjects, as well as strongly significant inverse correlation between ZIC1 mRNA levels and BMI-adjusted bone mineral density (BMD) (Z-score). ZIC1 promoter methylation was decreased in mechanically loaded vertebral bone compared to unloaded normal iliac bone, and its mRNA levels correlated inversely with ZIC1 promoter methylation, thus linking mechanical stress to epigenetic control of gene expression. The findings were corroborated in cultures of rat osteoblast progenitors and osteoblast-like cells. This study demonstrates for the first time how skeletal epigenetic changes that are affected by mechanical forces give rise to marked alteration in bone cell transcriptional activity and translate to human bone pathophysiology.
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Osteoporosis Posmenopáusica , Animales , Densidad Ósea/genética , Epigénesis Genética , Femenino , Humanos , Ilion/metabolismo , Vértebras Lumbares/metabolismo , Osteoporosis Posmenopáusica/genética , Osteoporosis Posmenopáusica/patología , ARN Mensajero/genética , Ratas , Estrés Mecánico , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Eight novel silicate, phosphate and borate glass compositions (coded as NCLx, where x = 1 to 8), containing different oxides (i.e. MgO, MnO2, Al2O3, CaF2, Fe2O3, ZnO, CuO, Cr2O3) were designed and evaluated alongside apatite-wollastonite (used as comparison material), as potential biomaterials for bone tissue repair and regeneration. Glass frits of all the formulations were processed to have particle sizes under 53 µm, with their morphology and dimensions subsequently investigated by scanning electron microscopy (SEM). In order to establish the nature of the raw glass powders, X-ray diffraction (XRD) analysis was also performed. The sintering ability of the novel materials was determined by using hot stage microscopy (HSM). Ionic release potential was assessed by inductively coupled plasma optical emission spectroscopy (ICP-OES). Finally, the cytotoxic effect of the novel glass powders was evaluated for different glass concentrations via a colorimetric assay, on which basis three formulations are considered promising biomaterials.
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The lack of an in vitro real-time osteoclast (OC) activity assay has hampered mechanistic studies of bone resorption. Such an assay is developed, employing a hydroxyapatite matrix impregnated with alkyl-capped silicon nanocrystals, which is capable of monitoring the time-course of resorption by single osteoclasts. Resorption of the matrix by OC releases the nanocrystals, which are internalized by the cell and detected as an increase in OC luminescence. This particular choice of nanocrystals is motivated by their bright pH-independent luminescence, proportional to concentration, and by their rapid uptake without cytotoxicity. In this in vitro assay, OCs are inhibited by calcitonin (CT) and methyl-ß-cyclodextrin (MCD), and stimulated by receptor activator of nuclear factor kappa-B ligand (RANKL) in the expected manner. The kinetics of the assay exhibit a lag phase representing cell attachment and commencement of resorption processes, followed by a growth of cell luminescence intensity, and the whole time-course is satisfactorily described by the logistic equation.
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Bioensayo , Nanopartículas , Osteoclastos/citología , Silicio/química , Animales , Línea Celular , Durapatita , RatonesRESUMEN
A transcriptome analysis compared gene expression in human bone biopsy samples taken from lumbar spine and iliac crest, sites that experience high and low levels of mechanical stress, respectively. The analysis revealed that the zinc finger protein of cerebellum (Zic) family member transcription factor Zic1 was the most up-regulated gene in the lumbar spine (202-fold; P<10(-7)) in comparison with the iliac crest. Software analysis of differential gene expression in the biopsy samples identified the ciliary-related proteins PATCH1 and GLI-Kruppel family members Gli1 and Gli3 as part of a potential molecular network associated with Zic1. RT-PCR confirmed the expression of Zic1, Gli1, and Gli3 and other related key signaling mediators in osteoblastic cells and osteocytes in vitro. Zic1 was immunolocalized in the cytosol and nucleus of the murine osteocyte cell line MLO-Y4 and osteoblast-like cells MC3T3-E1 and in primary rat osteoblasts. MLO-Y4 cells subjected to prolonged oscillatory fluid flow showed increased localization of Zic1 in the nucleus with diminished levels in the cytosol, but no such changes were seen in MC3T3-E1 cells. A shear stress-induced increase in T-cell factor/lymphoid enhancer factor transcriptional activity was abolished by Zic1 gene silencing. These results suggest that Zic1, perhaps together with Gli1 and Gli3, may act as a link between mechanosensing and Wnt signaling. We conclude that Zic1, a neural developmental transcription factor, plays an important role in shear flow mechanotransduction in osteocytes.
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Huesos/metabolismo , Mecanotransducción Celular , Osteocitos/metabolismo , Factores de Transcripción/fisiología , Animales , Línea Celular , Cilios , Perfilación de la Expresión Génica , Factores de Transcripción de Tipo Kruppel/genética , Factores de Transcripción de Tipo Kruppel/fisiología , Ratones , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/fisiología , Ratas , Estrés Mecánico , Proteína con Dedos de Zinc GLI1 , Proteína Gli3 con Dedos de ZincRESUMEN
BACKGROUND: The interfacial molecular mechanisms that regulate mammalian cell growth and differentiation have important implications for biotechnology (production of cells and cell products) and medicine (tissue engineering, prosthetic implants, cancer and developmental biology). We demonstrate here that engineered protein motifs can be robustly displayed to mammalian cells in vitro in a highly controlled manner using a soluble protein scaffold designed to self assemble on a gold surface. RESULTS: A protein was engineered to contain a C-terminal cysteine that would allow chemisorption to gold, followed by 12 amino acids that form a water soluble coil that could switch to a hydrophobic helix in the presence of alkane thiols. Bioactive motifs from either bone morphogenetic protein-2 or osteopontin were added to this scaffold protein and when assembled on a gold surface assessed for their ability to influence cell function. Data demonstrate that osteoblast adhesion and short-term responsiveness to bone morphogenetic protein-2 is dependent on the surface density of a cell adhesive motif derived from osteopontin. Furthermore an immobilised cell interaction motif from bone morphogenetic protein supported bone formation in vitro over 28 days (in the complete absence of other osteogenic supplements). In addition, two-dimensional patterning of this ligand using a soft lithography approach resulted in the spatial control of osteogenesis. CONCLUSION: These data describe an approach that allows the influence of immobilised protein ligands on cell behaviour to be dissected at the molecular level. This approach presents a durable surface that allows both short (hours or days) and long term (weeks) effects on cell activity to be assessed. This widely applicable approach can provide mechanistic insight into the contribution of immobilised ligands in the control of cell activity.
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Proteína Morfogenética Ósea 2/metabolismo , Adhesión Celular/fisiología , Diferenciación Celular/fisiología , Modelos Moleculares , Osteoblastos/fisiología , Osteopontina/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Análisis de Varianza , Animales , Proteína Morfogenética Ósea 2/genética , Escherichia coli , Técnica del Anticuerpo Fluorescente , Oro/metabolismo , Técnicas In Vitro , Ligandos , Osteopontina/genética , Ingeniería de Proteínas/métodosRESUMEN
Increased membrane type-1 matrix metalloproteinase (MT1-MMP) expression in osteosarcoma is predictive of poor prognosis and directs bone metastasis in prostate carcinoma. MT1-MMP subcellular localisation varies with oxygen tension, and, therefore, the aim of the present study was to assess protein interactions between MT1-MMP and the hypoxia inducible factors (HIF-1α and HIF-2α). MT1-MMP protein expression was investigated across a panel of cancer cell lines, including a positive and negative control. The hypoxia-induced alteration in subcellular location of MT1-MMP, HIF-1α and HIF-2α in the U2OS osteosarcoma cell line was assessed using subcellular fractionation. A proximity ligation assay was utilised to assess protein to protein interactions in the osteosarcoma U2OS and prostate carcinoma PC3 cell lines. U2OS and PC3 cells exhibited a significantly increased intra-nuclear interaction between MT1-MMP and HIF-2α in response to hypoxia. The role of this warrants further investigation as it may unveil novel opportunities to target MT1-MMP, which is of particular significance for osteosarcoma since current treatment options are limited.
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There is currently an interest in "active" implantable biomedical devices that include mechanical stimulation as an integral part of their design. This paper reports the experimental use of a porous scaffold made of interconnected networks of slender ferromagnetic fibers that can be actuated in vivo by an external magnetic field applying strains to in-growing cells. Such scaffolds have been previously characterized in terms of their mechanical and cellular responses. In this study, it is shown that the shape changes induced in the scaffolds can be used to promote osteogenesis in vitro. In particular, immunofluorescence, gene and protein analyses reveal that the actuated networks exhibit higher mineralization and extracellular matrix production, and express higher levels of osteocalcin, alkaline phosphatase, collagen type 1α1, runt-related transcription factor 2 and bone morphogenetic protein 2 than the static controls at the 3-week time point. The results suggest that the cells filling the inter-fiber spaces are able to sense and react to the magneto-mechanically induced strains facilitating osteogenic differentiation and maturation. This work provides evidence in support of using this approach to stimulate bone ingrowth around a device implanted in bone and can pave the way for further applications in bone tissue engineering.
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BACKGROUND: Engineered living materials (ELMs) are an exciting new frontier, where living organisms create highly functional materials. In particular, protein ELMs have the advantage that their properties can be manipulated via simple molecular biology. Caf1 is a protein ELM that is especially attractive as a biomaterial on account of its unique combination of properties: bacterial cells export it as a massive, modular, non-covalent polymer which is resistant to thermal and chemical degradation and free from animal material. Moreover, it is biologically inert, allowing the bioactivity of each 15 kDa monomeric Caf1 subunit to be specifically engineered by mutagenesis and co-expressed in the same Escherichia coli cell to produce a mixture of bioactive Caf1 subunits. RESULTS: Here, we show by gel electrophoresis and transmission electron microscopy that the bacterial cells combine these subunits into true mosaic heteropolymers. By combining two separate bioactive motifs in a single mosaic polymer we demonstrate its utility by stimulating the early stages of bone formation by primary human bone marrow stromal cells. Finally, using a synthetic biology approach, we engineer a mosaic of three components, demonstrating that Caf1 complexity depends solely upon the variety of monomers available. CONCLUSIONS: These results demonstrate the utility of engineered Caf1 mosaic polymers as a simple route towards the production of multifunctional biomaterials that will be useful in biomedical applications such as 3D tissue culture and wound healing. Additionally, in situ Caf1 producing cells could create complex bacterial communities for biotechnology.
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Porous coatings on prosthetic implants encourage implant fixation. Enhanced fixation may be achieved using a magneto-active porous coating that can deform elastically in vivo on the application of an external magnetic field, straining in-growing bone. Such a coating, made of 444 ferritic stainless steel fibres, was previously characterised in terms of its mechanical and cellular responses. In this work, co-cultures of human osteoblasts and endothelial cells were seeded into a novel fibrin-based hydrogel embedded in a 444 ferritic stainless steel fibre network. Albumin was successfully incorporated into fibrin hydrogels improving the specific permeability and the diffusion of fluorescently tagged dextrans without affecting their Young's modulus. The beneficial effect of albumin was demonstrated by the upregulation of osteogenic and angiogenic gene expression. Furthermore, mineralisation, extracellular matrix production, and formation of vessel-like structures were enhanced in albumin-enriched fibrin hydrogels compared to fibrin hydrogels. Collectively, the results indicate that the albumin-enriched fibrin hydrogel is a promising bio-matrix for bone tissue engineering and orthopaedic applications.
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The Hedgehog (Hh) signalling pathway plays important roles during embryonic development and in adult tissue homeostasis, for example cartilage, where its deregulation can lead to osteoarthritis (OA). microRNAs (miRNAs) are important regulators of gene expression, and have been implicated in the regulation of signalling pathways, including Hh, thereby impacting upon development and disease. Our aim was to identify the function of miRNAs whose expression is altered in OA cartilage. Here we identified an increase in miR-324-5p expression in OA cartilage and hypothesised that, as in glioma, miR-324-5p would regulate Hh signalling. We determined that miR-324-5p regulates osteogenesis in human mesenchymal stem cells (MSCs) and in mouse C3H10T1/2 cells. Luciferase reporter assays demonstrated that miR-324-5p directly regulated established targets GLI1 and SMO in human but not in mouse, suggesting species-dependent mechanism of Hh pathway regulation. Stable Isotope Labelling with Amino acids in Cell culture (SILAC), mass spectrometry and whole genome transcriptome analysis identified Glypican 1 (Gpc1) as a novel miR-324-5p target in mouse, which was confirmed by real-time RT-PCR, immunoblotting and 3'UTR-luciferase reporters. Knockdown of Gpc1 reduced Hh pathway activity, and phenocopied the effect of miR-324-5p on osteogenesis, indicating that miR-324-5p regulates Hh signalling in mouse via direct targeting of Gpc1. Finally, we showed that human GPC1 is not a direct target of miR-324-5p. Importantly, as well as identifying novel regulation of Indian Hedgehog (Ihh) signalling, this study demonstrates how a miRNA can show conserved pathway regulation in two species but by distinct mechanisms and highlights important differences between human diseases and mouse models.
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Cartílago/metabolismo , Glipicanos/genética , Proteínas Hedgehog/genética , MicroARNs/genética , Osteoartritis/genética , Receptor Smoothened/genética , Proteína con Dedos de Zinc GLI1/genética , Regiones no Traducidas 3' , Adulto , Animales , Cartílago/patología , Línea Celular , Modelos Animales de Enfermedad , Regulación de la Expresión Génica , Genes Reporteros , Glipicanos/antagonistas & inhibidores , Glipicanos/metabolismo , Proteínas Hedgehog/metabolismo , Humanos , Luciferasas/genética , Luciferasas/metabolismo , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/metabolismo , Ratones , MicroARNs/metabolismo , Osteoartritis/metabolismo , Osteoartritis/patología , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Transducción de Señal , Receptor Smoothened/metabolismo , Especificidad de la Especie , Proteína con Dedos de Zinc GLI1/metabolismoRESUMEN
It is envisaged that the creation of cellular environments at multiple length scales, that recapitulate in vivo bioactive and structural roles, may hold the key to creating functional, complex tissues in the laboratory. This review considers recent advances in biofabrication and bioprinting techniques across different length scales. Particular focus is placed on 3D printing of hydrogels and fabrication of biomaterial fibres that could extend the feature resolution and material functionality of soft tissue constructs. The outlook from this review discusses how one might create and simulate microenvironmental cues in vitro. A fabrication platform that integrates the competencies of different biofabrication technologies is proposed. Such a multi-process, multiscale fabrication strategy may ultimately translate engineering capability into an accessible life sciences toolkit, fulfilling its potential to deliver in vitro disease models and engineered tissue implants.
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This paper presents an investigation of how different culture media (i.e. basal and osteogenic media) affect the nanomechanical properties and microstructure of the mineralized matrix produced by the human mesenchymal stem cell line Y201, from both an experimental and theoretical approach. A bone nodule (i.e. mineralized matrix) cultured from basal medium shows a more anisotropic microstructure compared to its counterpart cultured from an osteogenic medium. As confirmed by finite element simulations, this anisotropic microstructure explains the bimodal distribution of the corresponding mechanical properties very well. The overall nanomechanical response of the bone nodule from the osteogenic medium is poorer compared to its counterpart from the basal medium. The bone nodules, from both basal and osteogenic media, have shown reverse aging effects in terms of mechanical properties. These are possibly due to the fact that cell proliferation outcompetes the mineralization process.
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Técnicas de Cultivo de Célula/métodos , Matriz Extracelular/metabolismo , Modelos Biológicos , Nanoestructuras/química , Técnicas de Cultivo de Célula/instrumentación , Diferenciación Celular , Línea Celular , Módulo de Elasticidad , Matriz Extracelular/química , Matriz Extracelular/ultraestructura , Análisis de Elementos Finitos , Humanos , Células Madre Mesenquimatosas/citología , Osteogénesis , Propiedades de SuperficieRESUMEN
The multi-domain neutral endopeptidase, ADAMTS-1 (a disintegrin and metalloprotease with thrombospondin repeats) is induced by parathyroid hormone (PTH) in rat osteoblasts and has therefore been suggested to be involved in initiation of bone remodeling. However, its function(s) in bone cells have not been studied. Here, we first establish that ADAMTS-1 protein is rapidly and transiently produced by human primary osteoblasts in response to PTH (1-34). We also show that ADAMTS-1 is specifically in close proximity to collagen fibrils in bone tissue using ultrastructural immunolabeling. To study the consequence(s) of ADAMTS-1 metalloprotease production in osteoblastic cells, human osteosarcoma cells (SaOS-2), were forced to express either wild-type (wtATS) or a point-mutated (pmATS) metalloprotease dead ADAMTS-1. SaOS-2 cells expressing wtATS had a growth advantage and increased collagenolytic activity when seeded inside a collagen type I gel but exhibited a reduced migration in a scratch wound assay. Immunolabeling of moving cells shows ADAMTS-1 to be located towards the direction of cellular migration. Finally, Western analysis demonstrated excess accumulation of mature collagen type I alpha1 species in the extracellular matrix together with increased release of distinct small collagen fragments into the conditioned media, by cultures of wtATS cells compared to pmATS cells. These results show that ADAMTS-1 has both the opportunity in bone and capability in vitro to induce collagen type I processing, together with a positive influence on osteoblastic three-dimensional growth. Although it is not clear at present if ADAMTS-1 promotes collagen degradation directly or indirectly, it shows that ADAMTS-1 activity can have a profound influence on the osteoblast phenotype, inhibiting migration on a planar substrate but enhancing growth in a collagen scaffold. These findings further establish ADAMTS-1 as a potentially important protein in PTH induced bone remodeling.
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Proteínas ADAM/metabolismo , Colágeno Tipo I/metabolismo , Osteoblastos/fisiología , Proteínas ADAM/genética , Proteína ADAMTS1 , Animales , Movimiento Celular/fisiología , Proliferación Celular , Células Cultivadas , Colágeno Tipo I/ultraestructura , Humanos , Microscopía Inmunoelectrónica , Osteoblastos/ultraestructura , Osteosarcoma , Hormona Paratiroidea/metabolismo , Ratas , Técnicas de Cultivo de TejidosRESUMEN
This article reports on the use of the binder jetting three-dimensional printing process combined with sintering to process bioceramic materials to form micro- and macroporous three-dimensional structures. Three different glass-ceramic formulations, apatite-wollastonite and two silicate-based glasses, have been processed using this route to create porous structures which have Young's modulus equivalent to cortical bone and average bending strengths in the range 24-36 MPa. It is demonstrated that a range of macroporous geometries can be created with accuracies of ±0.25 mm over length scales up to 40 mm. Hot-stage microscopy is a valuable tool in the definition of processing parameters for the sintering step of the process. Overall, it is concluded that binder jetting followed by sintering offers a versatile process for the manufacture of load-bearing bioceramic components for bone replacement applications.
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Materiales Biocompatibles , Cerámica , Impresión Tridimensional , Andamios del Tejido , Ensayo de Materiales , Porosidad , Temperatura , Soporte de PesoRESUMEN
Bone cell interaction with extracellular matrix (ECM) microenvironment is of critical importance when engineering surface interfaces for bone regeneration. In this work layer-by-layer films of type I collagen (coll), the major constituent of bone ECM, and heparin (hep), a glycosaminoglycan, were assembled on poly(l-lactic acid) (PLLA) substrates to evaluate the impact of the biomacromolecular coating on cell activity. The surface modification of PLLA demonstrated that the hep/coll multilayer is stable after 10 bilayers (confirmed by contact angle, infrared spectroscopy, and morphological analysis). This simple approach provided novel information on the effect of heparin on type I collagen hierarchical organization and subsequent cell response of osteoblast-like (MC3T3-E1) and human bone marrow-derived mesenchymal stem cells (hMSCs). Interestingly, the number of deposited heparin layers (1 or 10) appeared to play an important role in the self-assembly of collagen into fibrils, stabilizing the fibrous collagen layer, and potentially impacting hMSCs activity.
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Colágeno/química , Animales , Huesos , Diferenciación Celular , Heparina , Humanos , Células Madre Mesenquimatosas , Ratones , OsteoblastosRESUMEN
The objective of this study was to investigate the effect of combining two biomaterials on osteoblast proliferation, differentiation and mineralised matrix formation in vitro. The first biomaterial has a well-defined architecture and is known as PolyHIPE polymer (PHP). The second biomaterial is a biologically inspired self-assembling peptide hydrogel (RAD16-I, also called PuraMatrix) that produces a nanoscale environment similar to native extracellular matrix (ECM). Our work investigates the effect of combining RAD16-I with two types of PHP (HA (Hydroxyapatite)-PHP and H (Hydrophobic)-PHP) and evaluates effects on osteoblast growth and differentiation. Results demonstrated successful incorporation of RAD16-I into both types of PHP. Osteoblasts were observed to form multicellular layers on the combined biomaterial surface and also within the scaffold. Dynamic cell seeding and culturing techniques were compared to static seeding methods and produced a more even distribution of cells throughout the constructs. Cells were found to penetrate the scaffold to a maximum depth of 3 mm after 35 days in culture. There was a significant increase in cell number in H-PHP constructs coated with RAD16-I compared to H-PHP alone. Our results show that RAD16-I enhances osteoblast differentiation and indicates that the incorporation of this peptide provides a more permissive environment for osteoblast growth. We have developed a microcellular polymer containing a nanoscale environment to enhance cell: biomaterial interactions and promote osteoblast growth in vitro.
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Diferenciación Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Hidrogeles/farmacología , Oligopéptidos/farmacología , Osteoblastos/efectos de los fármacos , Polímeros/farmacología , Estirenos/farmacología , Fosfatasa Alcalina/metabolismo , Animales , Animales Recién Nacidos , Calcificación Fisiológica/efectos de los fármacos , Durapatita/química , Hidrogeles/química , Microscopía Electrónica de Rastreo , Oligopéptidos/síntesis química , Oligopéptidos/química , Osteoblastos/citología , Osteoblastos/metabolismo , Osteopontina , Polímeros/química , Ratas , Sialoglicoproteínas/metabolismo , Estirenos/químicaRESUMEN
Yersina pestis, the bubonic plague bacterium, is coated with a polymeric protein hydrogel for protection from host defences. The protein, which is robust and non-stick, resembles structures found in many eukaryotic extracellular-matrix proteins. Cells grown on the natural polymer cannot adhere and grow poorly; however, when cell-adhesion motifs are inserted into the protein, the cells proliferate.
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Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Materiales Biomiméticos/química , Adhesión Celular/fisiología , Proliferación Celular/fisiología , Proteínas de la Matriz Extracelular/química , Ingeniería de Tejidos/métodos , Adhesividad , Animales , Proteínas Bacterianas/genética , Materiales Biomiméticos/metabolismo , Proteínas de la Matriz Extracelular/genética , Proteínas de la Matriz Extracelular/metabolismo , Humanos , Ensayo de Materiales , Ratones , Células 3T3 NIH , Ingeniería de Proteínas/métodos , Proteínas Recombinantes de Fusión/química , Relación Estructura-ActividadRESUMEN
Substrate topography influences cell adhesion, proliferation, and differentiation. In this study, poly (ε-caprolactone) (PCL) films with a well-defined honeycomb structure of porosity 3-4, 5-6, 10-11, or 15-16 µm were contrasted with flat surfaces for their ability to support primary rat osteoblast adhesion and mineralized extracellular matrix deposition in vitro. Immunofluorescent visualization of vinculin and rhodamine phalloidin binding of actin were used to investigate cell adhesion and morphology. Localization of the alkaline phosphatase activity and Alizarin Red staining were performed to assess the osteoblast activity and deposition of a mineralized matrix. Scanning electron microscopy together with energy-dispersive X-ray spectroscopy was used to provide morphological analysis of cell-film interactions, the deposited matrix, and elemental analysis of the mineralized structures. After 24 h of culture, there were no differences in cell numbers on porous or flat PCL surfaces, but there were changes in cell morphology. Osteoblasts on honeycomb films had a smaller surface area and were less circular than cells on flat PCL. Analysis of cells cultured for 35 days under osteogenic conditions revealed that osteoblasts on all substrates acquired alkaline phosphatase activity, but levels of mineralized matrix were increased on films with 3-4-µm pore sizes. The bone-like matrix with a Ca:P ratio of 1.69±0.08 could be identified in larger areas often aligning with substrate topography. In addition, smaller spherical deposits (0.5-1 µm in diameter) with a Ca:P ratio of 1.3±0.08 were observed at the surface and particularly within the pores of the PCL film. Localization of vinculin showed significant decreases in the number of focal adhesion structures per unit cell area on 5-6, 10-11, and 15-16-µm surfaces compared to flat PCL, while focal complexes with a smaller area (0-2 µm(2)) were more abundant on 3-4 and 5-6-µm surfaces. Observation of cell interaction with these surfaces identified cytoplasmic protrusions that extended into and sealed the pores of these PCL films creating an extracellular space in which, the conditions could influence the deposition and formation of the mineralized matrix.
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Caproatos/química , Lactonas/química , Osteoblastos/citología , Osteogénesis/fisiología , Poliésteres/química , Animales , Células Cultivadas , Inmunohistoquímica , Microscopía Electrónica de Rastreo , RatasRESUMEN
OBJECTIVE: To investigate in vivo simulation of the microenvironment in which osteoarthritis (OA) chondrocytes are cultured in vitro. METHODS: Human articular chondrocytes were cultured under normoxic and hypoxic conditions. Cells were cultured on standard culture plastic or a porous polyHEMA surface that closely resembles the in vivo cartilage microarchitecture. Morphological changes to the cells were demonstrated by fluorescent staining with DAPI and vinculin. Proteoglycan and type II collagen protein levels were assessed using established techniques. Matrix metalloproteinase-1 (MMP-1) production was assessed by ELISA. The gene expression of type II collagen and SOX9 was measured using real-time polymerase chain reaction. RESULTS: Cells grown on culture plastic were seen to be flat and hexagonal. Cells cultured on the porous polyHEMA surface exhibited morphology in keeping with the in vivo microenvironment. Glycosaminoglycan release in hypoxia was high from cells cultured on standard culture plastic. Transcriptional expression of type II collagen was upregulated in hypoxia and by culture on the polyHEMA surface. Transcriptional expression of SOX9 in hypoxia was upregulated compared to normoxia; no significant effect was seen by varying the culture surface. Translational expression of type II collagen was upregulated at 20% oxygen on the polyHEMA surface compared to culture plastic and this was related to MMP-1 expression. CONCLUSION: Culture of chondrocytes in hypoxia and on a porous surface simulates the in vivo microenvironment and illustrates the molecular mechanisms of OA.