RESUMEN
Guided bone regeneration (GBR) is a widely used procedure that prevents the fast in-growth of soft tissues into bone defect. Among the different types of membranes, the use of collagen membranes is the gold standard. However, these membranes are implanted in tissue location where a severe acute inflammation will occur and can be negatively affected. The aim of this study was to develop a collagen-based membrane for GBR that incorporated alginate-hydroxyapatite microparticles. Membranes were manufactured using collagen type I and gelatin and alginate-hydroxyapatite microparticles. Membranes were assessed in terms of topography by scanning electron microscopy and confocal microscopy; stability by swelling after an overnight incubation in saline and enzymatic degradation against collagenase and mechanical properties by tensile tests. Furthermore, the biological response was assessed with SaOs-2 cells and THP-1 macrophages to determine alkaline phosphatase activity and inflammatory cytokine release. Our results showed that the incorporation of different percentages of these microparticles could induce changes in the surface topography. When the biological response was analyzed, either membranes were not cytotoxic to THP-1 macrophages or to SaOs-2 cells and they did not induce the release of pro-inflammatory cytokines. However, the different surface topographies did not induce changes in the macrophage morphology and the release of pro- and anti-inflammatory cytokines, suggesting that the effect of surface roughness on macrophage behavior could be dependent on other factors such as substrate stiffness and composition. Collagen-gelatin membranes with embedded alginate-hydroxyapatite microparticles increased ALP activity, suggesting a positive effect of them on bone regeneration, remaining unaffected the release of pro- and anti-inflammatory cytokines.
Asunto(s)
Alginatos , Regeneración Ósea , Durapatita , Inflamación , Osteoblastos , Regeneración Ósea/efectos de los fármacos , Humanos , Osteoblastos/efectos de los fármacos , Osteoblastos/citología , Durapatita/química , Durapatita/farmacología , Alginatos/química , Inflamación/patología , Propiedades de Superficie , Gelatina/química , Macrófagos/metabolismo , Regeneración Tisular Dirigida/métodos , Membranas Artificiales , Tamaño de la Partícula , Citocinas/metabolismo , Colágeno Tipo I/química , Colágeno Tipo I/metabolismo , Colágeno Tipo I/farmacología , Ácido Glucurónico/química , Ácido Glucurónico/farmacología , Ácidos Hexurónicos/química , Ácidos Hexurónicos/farmacologíaRESUMEN
The methods described herein allow for the isolation and expansion of fibroblastic-like ovine Wharton's jelly-derived mesenchymal stromal cells (oWJ-MSC) that, similarly to their human counterparts, adhere to standard plastic surfaces in culture; show a mesenchymal profile for specific surface antigens (i.e., positive for CD44 and CD166); and lack expression of endothelial (CD31) and hematopoietic (CD45) markers as well as major histocompatibility complex (MHC) class-II. Homogeneous cell cultures result from a two-phase bioprocess design that starts with the isolation of mesenchymal stromal cells (MSC) from the Wharton's jelly of ovine umbilical cords up to a first step of cryopreservation. The second phase allows for further expansion of ovine WJ-MSC up to sufficient numbers for further studies. Overall, this methodology encompasses a 2-week bioprocess design that encompasses two cell culture passages ensuring sufficient cells for the generation of a Master Cell Bank. Further thawing and scale expansion results in large quantities of oWJ-MSC that can be readily used in proof of efficacy and safety studies in the preclinical development stage of the development of cell-based medicines. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Isolation and expansion of ovine mesenchymal stromal cells from Wharton's jelly of the umbilical cord Basic Protocol 2: Characterization of ovine mesenchymal stromal cells Basic Protocol 3: Growth profile determination of ovine mesenchymal stromal cells from Wharton's jelly.
Asunto(s)
Células Madre Mesenquimatosas , Gelatina de Wharton , Animales , Técnicas de Cultivo de Célula , Diferenciación Celular , Humanos , Ovinos , Cordón UmbilicalRESUMEN
BACKGROUND: Orthopaedic diseases are one of the major targets for regenerative medicine. In this context, Wharton's jelly (WJ) is an alternative source to bone marrow (BM) for allogeneic transplantation since its isolation does not require an invasive procedure for cell collection and does not raise major ethical concerns. However, the osteogenic capacity of human WJ-derived multipotent mesenchymal stromal cells (MSC) remains unclear. METHODS: Here, we compared the baseline osteogenic potential of MSC from WJ and BM cell sources by cytological staining, quantitative real-time PCR and proteomic analysis, and assessed chemical and biological strategies for priming undifferentiated WJ-MSC. Concretely, different inhibitors/activators of the TGFß1-BMP2 signalling pathway as well as the secretome of differentiating BM-MSC were tested. RESULTS: Cytochemical staining as well as gene expression and proteomic analysis revealed that osteogenic commitment was poor in WJ-MSC. However, stimulation of the BMP2 pathway with BMP2 plus tanshinone IIA and the addition of extracellular vesicles or protein-enriched preparations from differentiating BM-MSC enhanced WJ-MSC osteogenesis. Furthermore, greater outcome was obtained with the use of conditioned media from differentiating BM-MSC. CONCLUSIONS: Altogether, our results point to the use of master banks of WJ-MSC as a valuable alternative to BM-MSC for orthopaedic conditions.