RESUMEN
Large bone defects naturally regenerate via a highly vascularized tissue which progressively remodels into cartilage and bone. Current approaches in bone tissue engineering are restricted by delayed vascularization and fail to recapitulate this stepwise differentiation toward bone tissue. Here, we use the morphogen Sonic Hedgehog (Shh) to induce the in vitro organization of an endothelial capillary network in an artificial tissue. We show that endogenous Hedgehog activity regulates angiogenic genes and the formation of vascular lumens. Exogenous Shh further induces the in vitro development of the vasculature (vascular lumen formation, size, distribution). Upon implantation, the in vitro development of the vasculature improves the in vivo perfusion of the artificial tissue and is necessary to contribute to, and enhance, the formation of de novo mature bone tissue. Similar to the regenerating callus, the artificial tissue undergoes intramembranous and endochondral ossification and forms a trabecular-like bone organ including bone-marrow-like cavities. These findings open the door for new strategies to treat large bone defects by closely mimicking natural endochondral bone repair.
Asunto(s)
Huesos/metabolismo , Proteínas Hedgehog/metabolismo , Ingeniería de Tejidos/métodos , Animales , Prótesis Vascular , Células de la Médula Ósea/citología , Diferenciación Celular , Matriz Extracelular/metabolismo , Células Endoteliales de la Vena Umbilical Humana , Humanos , Ratones , Neovascularización Patológica , Osteogénesis , Medicina Regenerativa/métodos , Factores de TiempoRESUMEN
Electrospun fiber meshes are patterned at length scales comparable to or lower than their fiber diameter. Simple nano- and microgrooves and closed geometric shapes are imprinted in different tones using a fast imprint process at physiological temperatures. Human mesenchymal stromal cells cultured on patterned scaffolds show differences in cellular morphology and cytoskeleton organization. Microgrooved electrospun fibers support upregulation of alkaline phosphatase and bone morphogenetic protein-2 gene expression when cells are cultured in osteogenic medium.
Asunto(s)
Impresión Molecular/métodos , Nanotecnología/métodos , Temperatura , Andamios del Tejido/química , Núcleo Celular/efectos de los fármacos , Núcleo Celular/metabolismo , Citoesqueleto/efectos de los fármacos , Citoesqueleto/metabolismo , Humanos , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/efectos de los fármacos , Células Madre Mesenquimatosas/ultraestructura , Polímeros/farmacología , Coloración y EtiquetadoRESUMEN
Electrospun polymeric scaffolds are used for various tissue engineering applications. In this study, we applied a biomimetic coating method to provide electrospun scaffolds from a block copolymer-poly(ethylene oxide terephthalate)-poly(buthylene terephthalate), with a calcium phosphate layer to improve their bioactivity in bone tissue engineering. The in vitro studies with human mesenchymal stem cells demonstrated cell proliferation on both uncoated and coated samples. No significant effect of calcium phosphate coating was observed on the expression of alkaline phosphatase in vitro. Implantation of scaffold-goat mesenchymal stem cells constructs subcutaneously in nude mice resulted in bone formation in the calcium phosphate coated samples, in contrast to the uncoated ones, where no new bone formation was observed. The results of this study showed that the biomimetic method can successfully be used to coat electrospun scaffolds with a calcium phosphate layer, which improved the in vivo bioactivity of the polymer.
Asunto(s)
Biomimética , Huesos/química , Fosfatos de Calcio/química , Materiales Biocompatibles Revestidos/química , Nanofibras/química , Ingeniería de Tejidos , Electroquímica , Humanos , Ensayo de Materiales , Células Madre Mesenquimatosas/citología , Tamaño de la Partícula , Polímeros/química , Propiedades de SuperficieRESUMEN
Despite recent advances in the development of biomaterials intended to replace natural bone grafts for the regeneration of large, clinically relevant defects, most synthetic solutions that are currently applied in the clinic are still inferior to natural bone grafts with regard to regenerative potential and are limited to non-weight-bearing applications. From a materials science perspective, we always face the conundrum of the preservation of bioactivity of calcium phosphate ceramics in spite of better mechanical and handling properties and processability of polymers. Composites have long been investigated as a method to marry these critical properties for the successful regeneration of bone and, indeed, have shown a significant improvement when used in combination with cells or growth factors. However, when looking at this approach from a clinical and regulatory perspective, the use of cells or biologicals prolongs the path of new treatments from the bench to the bedside. Applying 'smart' synthetic materials alone poses the fascinating challenge of instructing tissue regeneration in situ, thereby tremendously facilitating clinical translation. In the journey to make this possible, and with the aim of adding up the advantages of different biomaterials, combinations of fabrication technologies arise as a new strategy for generating instructive three-dimensional (3D) constructs for bone regeneration. Here we provide a review of recent technologies and approaches to create such constructs and give our perspective on how combinations of technologies and materials can help in obtaining more functional bone regeneration.
Asunto(s)
Regeneración Ósea , Sustitutos de Huesos/química , Sustitutos de Huesos/uso terapéutico , Animales , HumanosRESUMEN
Combining technologies to engineer scaffolds that can offer physical and chemical cues to cells is an attractive approach in tissue engineering and regenerative medicine. In this study, we have fabricated polymer-ceramic hybrid scaffolds for bone regeneration by combining rapid prototyping (RP), electrospinning (ESP) and a biomimetic coating method in order to provide mechanical support and a physico-chemical environment mimicking both the organic and inorganic phases of bone extracellular matrix (ECM). Poly(ethylene oxide terephthalate)-poly(buthylene terephthalate) (PEOT/PBT) block copolymer was used to produce three dimensional scaffolds by combining 3D fiber (3DF) deposition, and ESP, and these constructs were then coated with a Ca-P layer in a simulated physiological solution. Scaffold morphology and composition were studied using scanning electron microscopy (SEM) coupled to energy dispersive X-ray analyzer (EDX) and Fourier Tranform Infrared Spectroscopy (FTIR). Bone marrow derived human mesenchymal stromal cells (hMSCs) were cultured on coated and uncoated 3DF and 3DF + ESP scaffolds for up to 21 d in basic and mineralization medium and cell attachment, proliferation, and expression of genes related to osteogenesis were assessed. Cells attached, proliferated and secreted ECM on all the scaffolds. There were no significant differences in metabolic activity among the different groups on days 7 and 21. Coated 3DF scaffolds showed a significantly higher DNA amount in basic medium at 21 d compared with the coated 3DF + ESP scaffolds, whereas in mineralization medium, the presence of coating in 3DF+ESP scaffolds led to a significant decrease in the amount of DNA. An effect of combining different scaffolding technologies and material types on expression of a number of osteogenic markers (cbfa1, BMP-2, OP, OC and ON) was observed, suggesting the potential use of this approach in bone tissue engineering.
Asunto(s)
Huesos/citología , Cerámicas Modificadas Orgánicamente/química , Ingeniería de Tejidos/métodos , Andamios del Tejido/química , Regeneración Ósea , Proliferación Celular , Células Cultivadas , Humanos , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/fisiología , Microscopía Electrónica de Rastreo , Osteogénesis , Medicina Regenerativa/métodos , Ingeniería de Tejidos/instrumentaciónRESUMEN
The rationale for the use of polymer-ceramic composites for bone regeneration stems from the natural composition of bone, with collagen type I and biological apatite as the main organic and inorganic constituents, respectively. In the present study composite materials of PolyActive™ (PA), a poly(ethylene oxide terephthalate)/poly(butylene terephtalate) co-polymer, and hydroxyapatite (HA) at a weight ratio of 85:15 were prepared by rapid prototyping (RP) using two routes. In the first approach pre-extruded composite filaments of PA-HA were processed using three-dimensional fibre deposition (3DF) (conventional composite scaffolds). In the second approach PA scaffolds were fabricated using 3DF and combined with HA pillars produced inside stereolithographic moulds that fitted inside the pores of the PA three-dimensional structure (assembled composite scaffolds). Analysis of calcium and phosphate release in a simulated physiological solution, not containing calcium or phosphate, revealed significantly higher values for the HA pillars compared with other scaffolds. Release in simulated body fluid saturated with respect to HA did not show significant differences among the different scaffolds. Human mesenchymal stromal cells were cultured on polymer (3DF), conventional composite (3DF-HA) and assembled composite (HA assembled in 3DF) scaffolds and assessed for morphology, metabolic activity, DNA amount and gene expression of osteogenic markers using real time quantitative polymerase chain reaction (PCR). Scanning electron microscopy images showed that the cells attached to and infiltrated all the scaffolds. Assembled composites had a higher metabolic activity compared with 3DF-HA scaffolds while no significant differences were observed in DNA amounts. Gene expression of osteopontin in the assembled composite was significantly higher compared with the conventional composites. The strategy of composite fabrication by assembly appears to be a promising alternative to the conventional composite fabrication route for scaffolds for bone regeneration.
Asunto(s)
Regeneración Ósea/efectos de los fármacos , Cerámica/farmacología , Polímeros/farmacología , Biomarcadores/metabolismo , Calcio/análisis , Proliferación Celular/efectos de los fármacos , Forma de la Célula/efectos de los fármacos , Células Cultivadas , Módulo de Elasticidad/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/efectos de los fármacos , Células Madre Mesenquimatosas/metabolismo , Células Madre Mesenquimatosas/ultraestructura , Minerales/análisis , Osteogénesis/efectos de los fármacos , Osteogénesis/genética , Fosfatos/análisis , Reacción en Cadena de la Polimerasa , Andamios del Tejido/químicaRESUMEN
Layer-by-layer biofabrication represents a novel strategy to create three-dimensional living structures with a controlled internal architecture, using cell micromanipulation technologies. Laser assisted bioprinting (LAB) is an effective printing method for patterning cells, biomolecules, and biomaterials in two dimensions. "Biopapers," made of thin polymer scaffolds, may be appropriate to achieve three-dimensional constructs and to reinforce mechanical properties of printed materials. The aim of this work was to evaluate the effect of the tridimensional organization of cells and biomaterials on cell proliferation in vitro and in vivo. The experimental LAB setup was comprised of an infrared laser, focused onto a glass ribbon coated with an absorbing layer of gold. The cell bioink was made of MG63 cells (50 millions cells/mL in culture medium and 1% alginate), transduced with Luciferase gene for tracking and quantification. The printing substrate was a 100-µm-thick polycaprolacton (PCL) electrospun scaffold. The building sequence comprised sequential layers of cells and PCL scaffolds stacked using two different tridimensional arrangements, which were compared in this study (layer-by-layer vs. seeding on a single locus of the scaffolds). Then the cell-seeded materials were cultured in vitro or implanted in vivo in NOD-SCID mice. The qualitative follow-up involved scanning electron microscopy (SEM) observations, live-dead assays, and histology. The cell amount was quantified by photon imager during 21 days in vitro and 2 months in vivo. Live- dead assay and SEM revealed that the cells survived after printing and spread onto PCL membranes. Circle-shaped patterns were maintained in vitro during the first week but they were no longer observable after 2 weeks, due to cell proliferation. Luciferase tracking displayed that the cell amount was increased in vitro and in vivo when the materials and the cells where stacked layer by layer. Histological sections of the in vivo samples revealed a thicker fibrous tissue in the layer-by-layer samples. We have demonstrated in this study that PCL electrospun biopapers can act as a shock-absorbing mattress for cell printing and could further support cell proliferation. The layer-by-layer printing provided an appropriate 3D environment for cell survival and enhanced cell proliferation in vitro and in vivo.
Asunto(s)
Técnicas de Cultivo de Célula , Ingeniería de Tejidos/métodos , Animales , Materiales Biocompatibles/química , Línea Celular Tumoral , Proliferación Celular , Supervivencia Celular , Humanos , Ensayo de Materiales , Ratones , Ratones SCID , Microscopía Electrónica de Rastreo/métodos , Microtecnología , Regeneración , Andamios del TejidoRESUMEN
Electrospun scaffolds are widely used for various biomedical applications. In this study, we prepared electrospun bioactive composite scaffolds combining hydroxyapatite, collagen (Col) and a synthetic polymer-PolyActive™-to mimic naturally occurring extracellular matrix for in situ bone regeneration. Human mesenchymal stem cells (hMSCs) adhered and proliferated on these scaffolds. Cells on all scaffold types showed an increased metabolic activity with time. On day 4, the metabolic activity of cells cultured on PolyActive™ (PA)-hydroxyapatite (HA)-Col in 1,1,1,3,3,3-hexafluoro-2-propanolhexafluoro-2-propanol (HFIP) was significantly higher than that of cells grown on PA-Col samples. Furthermore, on day 6, cells on PA-HA-Col in HFIP showed significantly higher metabolic activity than those on PA and PA-Col scaffolds. Quantitative PCR analysis for a panel of osteogenic genes showed statistically significant differences between scaffolds. Cells cultured on PA-HA scaffolds had a significantly higher osteonectin and RunX2 expression compared to those on PA-HA-Col scaffolds. Cells on PA-HA-Col in HFIP scaffolds had significantly higher expression of alkaline phosphatase (ALP) and Col 1 compared to PA and PA-Col scaffolds respectively. The bone morphogenetic protein-2 and S100A4 expression of PA-Col and PA-HA-Col constructs was significantly lower than the basal level expression of cells on PA scaffolds. Although not statistically significant in all cases, cells cultured on PA-HA-Col in HFIP and PA-HA scaffolds had the highest expression for most of the genes analysed. The results of the study demonstrate that bioactive composite scaffolds prepared by electrospinning could find potential use in bone regeneration applications.