Early osteogenic signal expression of rat bone marrow stromal cells is influenced by both hydroxyapatite nanoparticle content and initial cell seeding density in biodegradable nanocomposite scaffolds (original) (raw)
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Biomacromolecules, 2009
The intercellular signaling mechanisms among a transplanted cell population are largely determined by the cell population itself as well as the surrounding environment. Changes in cellto-cell paracrine signaling distance can be obtained by altering cell density, and signal expression of growth factors can be enhanced by auto/paracrine signal transduction. To examine these relationships, we investigated the effect of cell seeding density on viability, proliferation, differentiation, and the endogenous osteogenic signal expression among rat bone marrow stromal cells (BMSCs) cultured on a 2D disk. Rat BMSCs were isolated from rats and then cultured for 8 days on biodegradable poly(propylene fumarate) disks with three different seeding densities (0.06, 0.15, and 0.30 million cells/disk). At day 1, 4, and 8, viability by Live/Dead fluorescent staining, DNA amount, osteogenic differentiation by alkaline phosphatase and osteocalcin mRNA expression, calcium deposition, and osteogenic growth factor mRNA expression were assayed. Osteogenic signal expression was evaluated using quantitative reverse transcriptase-polymerase chain reaction, and signals of interest include bone morphogenetic protein-2, transforming growth factor-β 1 , fibroblast growth factor-2, and platelet-derived growth factor-A. The results from this study demonstrate that rat BMSCs were viable over 8 days without being affected by cell density as well as cell proliferation rate and early osteogenic differentiation were stimulated by lower cell seeding density. Most importantly, this study has demonstrated for the first time that the temporal gene expression profiles of endogenous growth factors can be controlled by altering the initial cell seeding density on poly(propylene fumarate) disks. Therefore, our result suggest that changes in the paracrine signal distance by altering cell seeding density may be a useful strategy to optimize the cell-biomaterial construct microenvironments to enhance the osteogenic signal expression.
Journal of Tissue Engineering and Regenerative Medicine, 2009
In this study, bone marrow stromal cells (BMSCs) were differentiated on cyclic acetal composites containing hydroxyapatite (HA) particles (110 or 550 nm). These composites were evaluated for their role in influencing osteogenic signalling by encapsulated BMSCs. While a number of factors exert influence on osteogenic signalling during the production of an osteogenic matrix, we hypothesize that HA particles may upregulate bone growth factor expression due to enhanced BMSC adhesion. To this end, fluorescence-activated cell sorting (FACS) analysis was performed for the evaluation of BMSC surface marker expression after culture on two-dimensional (2D) cyclic acetal/HA composites. Three-dimensional (3D) composites were then fabricated by incorporating 110 or 550 nm HA particles at 5, 10 and 50 ng/ml concentrations. Bone growth factor molecules (TGFβ1, FGF-2 and PDGFa), bone biomarker molecules (ALP, OC, OPN and OCN) and extracellular matrix-related molecules (FN, MMP-13, Dmp1 and aggrecan) were selected for evaluation of osteogenic signalling mechanisms when in presence of these composites. FACS results at day 0 demonstrated that BMSCs were a heterogeneous population with a small percentage of cells staining positive for CD29, CD90 and CD51/61, while staining negative for CD34 and CD45. At day 3, a significant enrichment of cells staining strongly for CD29, CD90 and CD51/61 was achieved. Gene expression patterns for bone growth factors and extracellular matrix molecules were found to be largely dependent upon the size of HA particles. Bone marker molecules, except OCN, had unaltered expression patterns in response to the varied size of HA particles. Overall, the results indicate that larger-sized HA particles upregulate PDGF and these groups were also associated with the most significant increase in osteodifferentiation markers, particularly ALP. Our results suggest that endogenous signalling is dependent upon material properties. Furthermore, we propose that studying gene expression patterns induced by the surrounding biomaterials environment is a fundamental step in the creation of engineered tissues.
Tissue Engineering Part A, 2013
Due to differing compositions, synthetic scaffolds developed for bone regeneration vary widely in efficacy. To quantify the impact of such differences on osteoinductivity, numerous parameters were examined. Absorbable collagen sponge (ACS), three ceramic-based carriers (#1-3) of varying compositions, mineralized allograft chips, and an experimental phosphoserine-rich nanofiber scaffold [S(P) gel] were compared in their ability to promote cell adhesion, proliferation/survival, growth factor binding/release, and osteogenic gene expression. Human preosteoblasts were found to adhere most efficiently to the S(P) gel, and the growth/survival was greatest on the S(P) and ACS scaffolds, with minimal growth seen on the allograft and Ceramic #3. In bone morphogenetic protein-2 (BMP-2) binding/release assays, ACS demonstrated a burst release pattern, whereas the allograft and the ceramics inefficiently released BMP-2. The S(P) gel showed the most ideal rates of growth factor binding and release. QPCR analyses showed significant differences in the CXCL12, CXCR4, and RANKL transcripts among the cells grown on these various scaffolds. Although some scaffolds showed an advantage over others in individual parameters, the nanofiber gel appears to provide the optimal balance in the factors important to osteoinductivity evaluated here.
Tissue engineering. Part C, Methods, 2016
One approach to the development of an artificial graft material could rely on uniform coverage of a resorbable biomaterial with bone extracellular matrix (ECM). To achieve this on the surface of poly(propylene fumarate) scaffolds, we selected a growth factor regime of basic fibroblast growth factor (FGF-2) (5 ng/ml), platelet derived growth factor (PDGF-bb) (40 ng/ml) and epidermal growth factor (EGF) (20 ng/ml) to stimulate proliferation of bone marrow derived mesenchymal stem cells (BM-hMSC's). Bone morphogenetic protein (BMP) 4 (50 ng/ml), 6 (50 ng/ml) and 7 (27 ng/ml) in the presence of the following osteogenic substances: dexamethasone (10-7 M), β-glycerophosphate (10 mM) and ascorbic acid (50 µg/ml) were chosen to induce differentiation of BM-hMSC's into ECM-secreting osteoblasts. These growth factors were also studied at 10X concentration to determine dose effect. Proliferation was analyzed by MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide] assay, S...
Pharmacy Education, 2023
The percentage of ageing individuals above 65 years old is expected to increase by 2040 as speculated by the Department of Statistics, Malaysia (DOSM) (Rashid et al, 2016). The elderly population in Malaysia is expected to be 15% of the total population (Tengku, 2015). Hence, an osteoporotic fracture will be a significant threat due to the increasing number of ageing populations. The osteoporosis-associated bone disorder caused by trauma, severe infection, tumour resection, and skeleton abnormalities may cause the formation of critical-size bone defects. These may require bone grafting transplantation as an intervention technique. Autograft involves harvesting the patient's bone and transplanting it to the fracture sites while allograft involves harvesting bone from one individual and transplanting it into another individual within the same species. Both techniques pose some limitations such as a high risk of immunological reactions, the transmission of infection, and donor-site injury (Oryan et al., 2014). This limitation has led to the development of engineered bone tissue as a potential alternative to the conventional use of bone grafts due to their unlimited supply and their ability in hindering disease transmission. Engineered bone tissue involves the development of new functional bone to induce regeneration via a synergistic combination of the biomaterial scaffold, cells, and biochemical factors (Bouet et al, 2015). A scaffold acts as a vital part of engineered bone tissue to mimic the structure and function of the natural bone Keywords Bone Bone morphogenetic protein-2 (BMP-2) Hydroxyapatite (HA) Synergistic effect Vascular endothelial growth factor (VEGF)
Journal of Functional Biomaterials, 2012
Synthetic tissue scaffolds have a high potential impact for patients experiencing osteogenesis imperfecta. Using electrospinning, tissue scaffolds composed of hydroxyapatite/polycaprolactone (HAp/PCL) composite nanofibers were fabricated with two different HAp concentrations-1% and 10% of the solid scaffold weight. After physico-chemical scaffold characterization, rat bone marrow stromal cells were cultured on the composite scaffolds in maintenance medium and then in osteogenic medium. Quantitative PCR, colorimetric assays, immunofluorescent labeling, and electron microscopy measured osteogenic cell responses to the HAp/PCL scaffolds. In maintenance conditions, both Hap/PCL scaffolds and control scaffolds supported cell colonization through seven days with minor differences. In osteogenic conditions, the 10% HAp scaffolds exhibited significantly increased ALP assay levels at week 3, consistent with previous reports. However, qPCR analysis demonstrated an overall decrease in bone matrix-associated genes on Hap/PCL scaffolds. Osteopontin and osteocalcin immunofluorescent microscopy revealed a trend that both mineralized scaffolds had greater amounts of both proteins, though qPCR results indicated the opposite trend for osteopontin. Additionally, type I collagen expression decreased on HAp scaffolds. These results indicate that cells are OPEN ACCESS J. Funct. Biomater. 2012, 3 777 sensitive to minor changes in mineral content within nanofibers, even at just 1% w/w, and elucidating the sensing mechanism may lead to optimized osteogenic scaffold designs.
European Cells and Materials, 2010
The basic aspects of bone tissue engineering include chemical composition and geometry of the scaffold design, because it is very important to improve not only cell attachment and growth but especially osteodifferentiation, bone tissue formation, and vascularization. Geistlich Bio-Oss ® (GBO) is a xenograft consisting of deproteinized, sterilized bovine bone, chemically and physically identical to the mineral phase of human bone. In this study, we investigated the growth behaviour and the ability to form focal adhesions on the substrate, using vinculin, a cytoskeletal protein, as a marker. Moreover, the expression of bone specific proteins and growth factors such as type I collagen, osteopontin, bone sialoprotein, bone morphogenetic protein-2 (BMP-2), BMP-7 and de novo synthesis of osteocalcin in normal human osteoblasts (NHOst) seeded on xenogenic GBO were evaluated. Our observations suggest that after four weeks of culture in differentiation medium, the NHOst showed a high affinity for the three dimensional biomaterial; in fact, cellular proliferation, migration and colonization were clearly evident. The osteogenic differentiation process, as demonstrated by morphological, histochemical, energy dispersive X-ray microanalysis and biochemical analysis was mostly obvious in the NHOst grown on threedimensional inorganic bovine bone biomaterial. Functional studies displayed a clear and significant response to calcitonin when the cells were differentiated. In addition, the presence of the biomaterial improved the response, suggesting that it could drive the differentiation of these cells towards a more differentiated osteogenic phenotype. These results encourage us to consider GBO an adequate biocompatible three-dimensional biomaterial, indicating its potential use for the development of tissue-engineering techniques.
Cell Biology International, 2013
Bone regeneration ability of a scaffold strongly depends on its structure and the size of its components. In this study, a nanostructured scaffold was designed for bone repair using nano hydroxyapatite (nHA) (8-16 nm 9 50-80 nm) and gelatin (GEL) as main components. In vitro investigations of calcium matrix deposition and gene expression of the seeded cells for this scaffold, demineralized bone matrix (DBM), scaffold plus DBM, and the control group were carried out. Bone regeneration in rat calvarium with critical defect size after 1, 4, and 8 weeks post implantation was investigated. The calcium matrix depositions by the osteoblast and RUNX2, ALP, osteonectin, and osteocalcin gene expression in scaffold were more significant than in other groups. Histomorphometry analysis confirmed in vitro results. In vitro and in vivo bone regeneration were least in scaffold plus DBM group. Enhanced effects in scaffold could be attributed to the shape and size of nHA particles and good architecture of the scaffold. Reduction of bone regeneration might be due to tight bonding of BMPs and nHA particles in the third group. Results obtained from this study confirmed that nano-scale size of the main components and the scaffold architecture (pore diameter, interconnectivity pores, etc.) have significant effects on bone regeneration ability of the scaffold and are important parameters in designing a temporary bone substitute.
Bone matrix consists of two major phases at the nanoscale: organic and hydroxyapatite. Nanotechnology as a diverse and interdisciplinary area of research has the capacity to revolutionise many areas of applications such as bone tissue engineering. Nanohydroxyapatite/gelatin composite has higher osteoblast attachment and proliferation than micro-sized ones, and shorter culturing period and lower cell seeding density compared to pure gelatin. A nanostructured scaffold was fabricated by three methods for bone repair using nanohydroxyapatite and gelatin as the main components. Its biocompatibility, alizarin red test on the 14th and 21st days, gene expression on the 21st day in in vitro using and histomorphometry after 4 and 8 weeks postimplantation in the rat were investigated. Cultured unrestricted somatic stem cells used for in vitro study showed an excellent level of cell attachment to the scaffold. Cells induced more osteoblast differentiation on the scaffold than in 2D cell culture. Osteoblast differentiation and bone regeneration results of in vitro and in vivo investigation on scaffold were extremely significant, better than control and treatment groups. These effects could be attributed to the shape and size of nanoHA particles and good architecture of the scaffold. The results confirm the feasibility of bone regeneration using synthesised scaffold as a temporary bone substitute.