ABSTRACT
Periodontal ligament cells play a crucial role in the regeneration of periodontal tissues and an undifferentiated mesenchymal cell subset is thought to exist within this population. The aim of this study was to assess the osteogenic differentiation potential of human periodontal ligament fibroblasts (hPDLFs) in three dimensional (3D)-osteogenic culture environment following encapsulation in chitosan-hydroxyapatite (C/HA) microspheres with the size range of 350-450 microm. Human PDLF cultures were established and three experimental groups were formed: (i) two-dimensional (2D)-culture as single cell monolayer, (ii) 3D-static culture of C/HA encapsulated hPDLFs, and (iii) 3D-dynamic culture of C/HA encapsulated hPDLFs in a rotating wall vessel bioreactor. The cells were cultured in standard culture medium supplemented with beta-glycerophosphate, dexamethasone, and ascorbic acid. After 21 days, immunohistochemistry was performed using antibodies against osteonectin, osteopontin, bone-sialoprotein, and osteocalcin as osteogenic differentiation markers. Phase-contrast and scanning electron microscopy observations were used for histological and morphological evaluation. The combined effects of osteoinductive medium and HA-containing composite microsphere material on encapsulated hPDLFs resulted in the transformation of a considerable portion of the cells into osteoblastic lineage at the end of the experiments. Results demonstrate the ability of hPDLFs to undergo osteogenic differentiation upon induction in vitro, both under 2D and 3D culture conditions. C/HA microspheres in microgravity bioreactor may serve as a suitable 3D environment to support the osteogenic differentiation of human PDLFs, in vitro.
Subject(s)
Biocompatible Materials , Chitosan , Durapatite , Periodontal Ligament/cytology , Bioreactors , Cell Differentiation , Cells, Cultured , Fibroblasts/cytology , Fibroblasts/metabolism , Humans , Integrin-Binding Sialoprotein , Materials Testing , Microscopy, Electron, Scanning , Microspheres , Osteocalcin/metabolism , Osteogenesis , Osteonectin/metabolism , Osteopontin/metabolism , Periodontal Ligament/metabolism , Phenotype , Sialoglycoproteins/metabolismABSTRACT
This study introduces chitosan-based matrices as cell substrates for bovine chromaffin cell attachment in transplantation procedures. Chitosan ([1-->4] linked 2-amino-2-deoxy-beta-D-glucopyranose), having structural similarity to glycosaminoglycans, was modified using several proteins (collagen, albumin and gelatin) to increase surface area and improve biocompatibility. In vitro, collagen-blended chitosan (CC) matrices were found to attach more readily to chromaffin cells than to gelatin- or albumin-blended matrices. Morphological evidence showed that the chromaffin cells attached to CC substrates integrated well with the hydrogel matrix and survived for at least two weeks, under in vivo culture conditions. The chromaffin cells within chitosan scaffolds also survived for at least two weeks in vitro and after subarachnoid grafting to rats.
