Comparison of Phenotypic Characterization between Differentiated Osteoblasts from Stem Cells and Calvaria Osteoblasts In vitro (original) (raw)
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Biomaterials, 2004
A sufficient amount of easily obtained and well-characterized osteoblastic cells is a useful tool to study biomaterial/cell interactions essential for bone tissue engineering. Osteoblastic cells were derived from adult and fetal rat via different isolation techniques. The isolation and in vitro proliferation of primary cultures were compared. The osteogenic potential of subcultures was studied by culturing them in osteogenic medium and compared with respect to alkaline phosphatase activity, nodule formation and mineralization potential. Calvaria cells were easier to obtain and the amount of cells released by enzymatic isolation was higher than for the long bone cells. The expansion of the cells in primary culture was highest for fetal calvaria cells compared to fetal and adult long bone cells. All cultures expressed high alkaline phosphatase activity except for calvaria cells obtained by spontaneous outgrowth. Enzymatic isolation of fetal calvaria and long bone cells favoured the osteogenic differentiation. Enzymatically isolated calvaria cells formed well-defined three-dimensional nodules which mineralized restricted to this area. On the contrary, cultures derived from fetal as well as adult long bones mineralized in ill-defined deposits throughout the culture and only formed occasionally nodular-like structures. The mineral phase of all osteoblastic cultures was identified as a carbonate-containing apatite. The present study demonstrates that considering the isolation method, proliferation capacity and the osteogenic potential, the enzymatically released fetal calvaria cells are most satisfactory to study cell/biomaterial interactions. r
Osteogenic Potentiation of Human Adipose???Derived Stem Cells in a 3-Dimensional Matrix
Annals of Plastic Surgery, 2006
Adipose-derived stem cells (ADSCs) hold promise for use in tissue engineering. Despite growing enthusiasm for use of ADSCs, there is limited research that has examined their behavior in different in vitro and in vivo systems. The purpose of our study was to evaluate the effect of the extracellular matrix structure and composition on osteogenic differentiation by comparing the osteogenic marker expression of ADSCs grown under 2-dimensional or 3-dimensional cell culture conditions. Group 1 (2-D) included ADSCs raised under conventional cell culture conditions (cells in a 2-D monolayer configuration) (n ϭ 24), and group 2 (3-dimensional) included ADSCs seeded in a collagen gel (cells within a 3-dimensional, biologically active environment) (n ϭ 24). Comparison of ADSC behavior between the 2 groups was analyzed during a 14-day time frame. Osteogenic marker expression (CBFA-1, alkaline phosphatase, osteonectin, osteopontin, Collagen I, and JNK2) was quantified by real-time PCR, and histologic analysis was performed. Histologically, group 1 (2-D) showed cell spreading and deposition of a calcified extracellular matrix. Group 2 (3-dimensional) assumed a disorganized state in the collagen gel, with extension of pseudopodia throughout the matrix. Expression of CBFA-1 was up-regulated immediately in both groups. However, cells in group 2 (3-dimensional) had a more rapid and greater overall expression compared with cells in group 1 (2-D) (250-fold greater at 4 days). At day 14, cells in group 2 (3-dimensional) showed greater expression of all other osteogenic markers than cells in group 1 (2-D) (2.3-fold greater expression of alkaline phosphatase ͓P Ͻ 0.05͔, 8.4-fold greater expression of osteonectin ͓P Ͻ 0.05͔, 6.4-fold greater expression of osteopontin ͓P Ͻ 0.05͔, 2.9-fold greater expression of collagen I ͓P Ͻ 0.05͔, and 2.5-fold greater expression of JNK2 ͓P Ͻ 0.05͔). Our data showed there was a progressive stimulatory effect on ADSCs with regard to osteogenesis when cultured in a 3-dimensional gel compared with a 2-D monolayer. FIGURE 1. Three-dimensional collagen gel seeded with ADSCs following polymerization within the microdistractor. K-wires were placed through the microdistractor into the polyethylene bars to prevent gel contraction.
BioResearch Open Access, 2015
Natural bone healing following fractures is initiated by osteoblasts (OBs) and mesenchymal stem cells (MSCs), a cell combination with possible potential in tissue engineering techniques for bony defects. The aim of the study was to investigate MSC/OB-crosstalk, in order to determine optimal cell culture conditions for osteogenic differentiation. Human OBs and MSCs interactions were investigated in an in vitro trans-well co-culture study over a time period of 28 days. Calcification was determined by optical density (OD) at 450 nm and Alizarin red staining. Messenger RNA expression was assessed by quantitative PCR. Osteogenic medium containing 1% fetal bovine serum resulted in superior levels of calcification in MSCs in co-culture with OBs compared to 2% or 5% fetal bovine serum ( p < 0.05). Comparing MSCs and OBs alone with the MSC/OB co-culture, calcification, as measured by OD 450 nm, increased over time in all groups. The highest values were recorded in the co-culture ( p < 0.05). Osteogenic differentiation potential showed significant interindividual differences. In order to predict differentiation potential, OD 450 nm measurements and mRNA expression of alkaline phosphatase were correlated with the population doubling rate during the expansion period. For OBs and MSCs, statistically significant associations of proliferation and differentiation potential were found (p < 0.001). The addition of transforming growth factor beta resulted in up-regulation of collagen type I and Sp7 mRNA, and down-regulation of alkaline phosphatase mRNA. The results suggest the idea of soluble paracrine factors being secreted by OBs to induce osteogenic differentiation of MSCs.
Cell and Tissue Research, 2009
One of the most important issues in orthopaedic surgery is the loss of bone resulting from trauma, infections, tumours or congenital deficiency. In view of the hypothetical future application of mesenchymal stem cells isolated from human adipose tissue in regenerative medicine, we have analysed and characterized adiposederived stem cells (ASCs) isolated from adipose tissue of rat, rabbit and pig. We have compared their in vitro osteogenic differentiation abilities for exploitation in the repair of critical osteochondral defects in autologous preclinical models. The number of pluripotent cells per millilitre of adipose tissue is variable and the yield of rabbit ASCs is lower than that in rat and pig. However, all ASCs populations show both a stable doubling time during culture and a marked clonogenic ability. After exposure to osteogenic stimuli, ASCs from rat, rabbit and pig exhibit a significant increase in the expression of osteogenic markers such as alkaline phosphatase, extracellular calcium deposition, osteocalcin and osteonectin. However, differences have been observed depending on the animal species and/ or differentiation period. Rabbit and porcine ASCs have been differentiated on granules of clinical grade hydroxyapatite (HA) towards osteoblast-like cells. These cells grow and adhere to the scaffold, with no inhibitory effect of HA during osteo-differentiation. Such in vitro studies are necessary in order to select suitable pre-clinical models to validate the use of autologous ASCs, alone or in association with proper biomaterials, for the repair of critical bone defects.
Calvarial bone cells: The search for stem cells Jorge E. Villanueva PhD Dissertation University of Southern California 0 (1993) Dispersed calvarial bone cells from rat fetuses were cultured suspended in agarose and some 30% of them showed deposition of metachromic extracellular matrix. Anchorage independence was a requirement for calvarial cells to express type II collagen Cells recovered from densities 1.04 g percoll/ml or higher were able to mature like chondrocytes. This chondrogenic potential in agarose was affected by subcultivation in monolayers: fresh calvarial cells after 1-2 doubling population in monolayer produced type II collagen less than 15% of that synthesized by freshly suspended and after 3-4 doubling population in monolayer he type II collagen synthesis abolished. Also, monolayer sub-cultivation impaired the ability of calvarial cells to produce bone when implanted in rats while inside demineralized bone matrix (DBM) chambers. Moreover, both in vivo and in vitro chondrogenic potential of these cells were inversely depending upon the cell concentration. Bone development is tightly associated with vascularization. Endothelial cells enhanced bone formation by calvarial cells when implanted together in Millipore diffusion chambers. In chambers with calvarial cells alone cartilage was often seen. The calcium deposition was 70 times higher in chambers containing cell mixture than chambers containing either endothelial or calvarial cells alone. DBM-powder did not mimic similar effect on calvarial cell osteogenesis. It is not well known how angiogenesis may promote osteogenesis and a critical step is to define these events occurring between endothelial and bone cells under controlled genotype backgrounds. A cationic liposome was used for the introduction of E1A-gene to isolated rat endothelial and calvarial cell lines. Because of a low growth rate of the former cells, an improved media was designed to success in transforming these cells. Geneticin resistant and E1A-oncoprotein producing endothelial cell lines were established. In addition, a collection of drug-resistant calvaria cell lines were isolated of which morphological appearance, although similar to primary calvaria cell cultures, was very stable after extensive growth. Finally, the design of an alternative approach for the characterization of bone stem cells is proposed by using transient immortalization with a temperature sensitive oncogenic agent and scrutinizing among others the ability to mature like chondrocytes. (Copies available exclusively from Micrographics Department, Doheny Library, USC, Los Angeles, CA, 90089-0182.) Keywords: Biological sciences, Molecular biology, bone cells
Journal of Biomedical Materials Research Part B: Applied Biomaterials, 2011
A brushite-forming calcium phosphate cement (CPC) was mechanically stabilized by addition of poly (l-lactid-co-glycolide; PLGA) fibers (10% w/w). It proved highly biocompatible and its fiber component enhanced bone formation in a sheep lumbar vertebroplasty model. However, possible effects on the osteogenic differentiation of resident mesenchymal stem cells (MSCs) remained unexplored. The present study used a novel approach, simultaneously analyzing the influence of a solid CPC scaffold and its relatively low PLGA proportion (a mimicry of natural bone) on osteogenic, chondrogenic, and adipogenic differentiation, as well as the pluripotency of human adipose tissuederived mesenchymal stem cells (hASCs). hASCs were cultured on CPC discs with/without PLGA fibers (5% and 10%) in the absence of osteogenic medium for 3, 7, and 14 d. Gene expression of osteogenic markers (Runx2, osterix, alkaline phosphatase, collagen I, osteonectin, osteopontin, osteocalcin), chondrogenic markers (collagen II, Sox9, aggrecan), adipogenic markers (PPARG, Leptin, and FABP4), and pluripotency markers (Nanog, Tert, Rex) was analyzed by RT-PCR. The ability of hASCs to synthesize alkaline phosphatase was also evaluated. Cell number and viability were determined by fluorescein diacetate/propidium iodide staining. Compared to pure CPC, cultivation of hASCs on fiber-reinforced CPC transiently induced the gene expression of Runx2 and osterix (day 3), and long-lastingly augmented the expression of alkaline phosphatase (and its enzyme activity), collagen I, and osteonectin (until day 14). In contrast, augmented expression of all chondrogenic, adipogenic, and pluripotency markers was limited to day 3, followed by significant downregulation. Cultivation of hASCs on fiber-reinforced CPC reduced the cell number, but not the proportion of viable cells (viability>95%). The PLGA component of fiber-reinforced, brushite-forming CPC supports long-lasting osteogenic differentiation of hASCs, whereas chondrogenesis, adipogenesis, and pluripotency are initially augmented, but subsequently suppressed. In view of parallel animal results, PLGA fibers may represent an interesting clinical target for future improvement of CPC-based bone regeneration.
Nowadays high accident rates, fractures leading to permanent bone disorders and the impossibility of bone transplant have made scientists to look for new methods of repairing injured bones. Considering the application of stem cells in bone tissue engineering, there exists the necessity to investigate various culture methods and suitable fields and scaffolds. Thus, we decided to induce adipose-derived stem cells into osteoblast cells in two systems of pellet culture and monolayer and compare osteogenic markers. Methods: Stem cells have been separated via mechanical and enzymatic methods and cultured in monolayer and pellet culture models with osteogenic medium. Then, RNA was separated from differentiated cells, complementary DNA (cDNA) was synthesized and amplified. Polymerase chain reaction (PCR) product was transferred to electrophoresis gel. The intensity of the bands was measured by Image-J software and analyzed by SPSS. Results: average osteopontin, osteocalcin and Runx2 genes in differentiated cells in the two culture systems showed a significant difference. The expression of osteocalcin, osteopontin and Runx2 gense in pellet system were more than monolayer systems in 21 days. Conclusion: This study indicated that pellet and monolayer culture systems are appropriate for bone engineering but osteocalcin, osteopontin and Runx2 genes expressions were different in the two culture system.
Journal of Tissue Engineering and Regenerative Medicine, 2012
potential were found in new sources of cells, such as adipose tissue. This source of stem cells has a promising future in tissue-engineering applications, considering the abundance of this tissue in the human body, the easy harvesting and the high number of stem cells that are available from such a small amount of tissue. The isolation of the adipose stem cells is generally performed by means of enzymatic digestion of the tissues, followed by a natural selection of the stem cells based on their capacity to adhere to the culture flasks, leading to a quite heterogeneous population. This constitutes a major drawback for the use of these cells, since the heterogeneity of the cell culture obtained can compromise their proliferation and differentiation potential. In the present study we have analysed the in vitro and in vivo behaviour of two selected subpopulations with high osteogenic potential. For this purpose, ASCs CD29+ and ASCs STRO−1+ subpopulations were isolated and in vitro cultured onto a biodegradable polymeric scaffold, using osteogenic medium, before implantation in a nude mice model. The biodegradable polymeric scaffold used is a fibre-mesh structure based on a blend of starch and polycaprolatone (SPCL) that has been successfully used in several bone tissue-engineering studies. The implanted ASCs-scaffold constructs promoted the formation of new bone tissue in nude mice. However, the results obtained show differences in the behaviour of the two ASCs subpopulations under study, particularly regarding their potential to differentiate into the osteogenic lineage, and allowed the indentification of ASCs STRO−1+ as the best subpopulation for bone tissue-engineering applications.