Cartilage tissue can be engineered by starting from a diversity of cell sources, including stem-cell based and primary cell-based platforms. Selecting an appropriate cell source for the process of cartilage tissue engineering or repair is critical and challenging due to the variety of cell options available. In this study, cellular responses of isolated human chondrocytes, human embryonic stem cells and mesenchymal stem cells (MSCs) derived from three sources, human embryonic stem cells, bone marrow and adipose tissue, were assessed for chondrogenic potential in 3D culture. All cell sources were characterized by FACS analysis to compare expression of some surface markers. The cells were differentiated in two different biomaterial matrices, silk and chitosan scaffolds, in the presence and absence of bone morphogenetic protein 6 (BMP-6) along with the standard chondrogenic differentiating factors. Embryonic stem cells derived MSCs showed unique characteristics with preserved chondrogenic phenotype in both scaffolds with regard to chondrogenesis, as determined by real time RT-PCR, histological and microscopic analyses. After 4 weeks of cultivation, embryonic stem cells derived MSCs were promising for chondrogenesis, particularly in the silk scaffolds with BMP-6. The results suggest that cell source differences are important to consider with regard to chondrogenic outcomes and with the variables addressed here, the human embryonic stem cells derived MSCs were the preferred cell source.
In this study, the influence of degree of deacetylation (DD) and composition on some structural and biological properties of chitosan scaffolds were examined in vitro. 3D chitosan scaffolds of 2% (w/v) and 3% (w/v) composition in different DDs i.e. 75-85% and >85% were prepared by freeze-drying method at -80 degrees C. We noticed that >85% deacetylated chitosan scaffolds of 2% (w/v) composition has a highly interconnected morphological structure having approximately 100 mum pore size with 0.0917 N/mm(2) compression modulus. L929 fibroblastic cells were cultured on chitosan scaffolds in order to evaluate their biocompatibilities. Cell culture studies demonstrated that fibroblastic cell attachment and proliferation is affected by DD. The higher deacetylated chitosan scaffolds strongly supported the attachment and proliferation when compared with the lower deacetylated scaffolds. MTT assay indicated that >85% deacetylated chitosan scaffolds of 2% (w/v) composition, having the highest specific growth rate 0.017 h(-1) of all, was found to be the most suitable for cell culture studies and a potential candidate for tissue engineering with enhanced biostability and good biocompatibility.
A scaffold containing growth factors promoting regeneration may be a useful device to maintain periodontal regeneration when applied with appropriate cells. The aim of this study is to evaluate the convenience of chitosan and hydroxyapatite (HA)-chitosan scaffolds loaded with basic fibroblast growth factor (bFGF) for periodontal tissue engineering applications. Scaffolds were fabricated by freeze-drying technique using 2 and 3% chitosan gel in the absence or presence of HA particles. Addition of HA beads to chitosan gels produced a novel scaffold in which the pore sizes and interconnectivity were preserved. The scaffolds were loaded with 100 ng bFGF by embedding technique. HA-chitosan scaffolds provide better controlled release kinetics for bFGF compared with chitosan scaffolds and total release continued up to 168 h. Cell culture studies were carried out with periodontal ligament (PDL) cells and cementoblasts. Both 3-[4,5-dimethylthiazol-2-yl]-diphenyltetrazolium bromide (MTT) assay and confocal laser scanning microscope analysis revealed cells proliferating inside the scaffolds. The results demonstrated that bFGF-loaded HA-chitosan scaffolds provide a suitable three-dimensional environment supporting the cellular structure, proliferation, and mineralization.
Abstract:The purpose of this study is to investigate the convenience of bone morphogenetic protein-6 (BMP-6)-loaded chitosan scaffolds with preosteoblastic cells for bone tissue engineering. MC3T3-E1 cells were seeded into three different groups: chitosan scaffolds, BMP-6-loaded chitosan scaffolds, and chitosan scaffolds with free BMP-6 in culture medium. Tissue-engineered constructs were characterized by 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide assay, scanning electron microscopy (SEM), mineralization assay (von Kossa), alkaline phosphatase (ALP) activity, and osteocalcin (OCN) assays. BMP-6-loaded chitosan scaffolds supported proliferation of the MC3T3-E1 mouse osteogenic cells in a similar pattern as the unloaded chitosan scaffolds group and as the chitosan scaffolds with free BMP-6 group. SEM images of the cell-seeded scaffolds revealed significant acceleration of extracellular matrix synthesis in BMP-6-loaded chitosan scaffolds. Both levels of ALP and OCN were higher in BMP-6-loaded chitosan scaffold group compared with the other two groups. In addition, BMP-6-loaded scaffolds showed strong staining in mineralization assays. These findings suggest that BMP-6-loaded chitosan scaffold supports cellular functions of the osteoblastic cells; therefore, this scaffold is considered as a new promising vehicle for bone tissue engineering applications.
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