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.

In this study, the osteogenic potential of rat bone marrow mesenchymal stem cells (rBM-MSCs) on a biomimetic poly(ε-caprolactone)/β-tricalcium phosphate (PCL/β-TCP) composite scaffold composed of parallel concentric fibrous membranes was evaluated in vitro and in vivo. PCL/β-TCP composite membranes were prepared by electrospinning and characterized by x-ray diffraction, differential scanning calorimetry, Fourier transform-infrared spectroscopy, and scanning electron microscopy (SEM). rBM-MSCs were seeded on three-dimensional multispiral scaffolds prepared by the assembly of composite membranes. The cell-scaffold constructs were cultured in osteogenic medium for 4 weeks. Histochemical studies and biochemical assays confirmed the osteogenic differentiation of rBM-MSCs inside the scaffold by documenting the dense mineralized extracellular matrix formation starting from the second week of culture. In the in vivo part of the study, cell-scaffold constructs precultured for 7 days were implanted subcutaneously into the epigastric groin fascia of Wistar rats for a duration of 6 months. Ectopic bone-tissue like formation was documented by using computerized tomography, confocal laser microscopy, SEM, and histochemistry. In vivo findings indicated that the biomimetic multispiral scaffold seeded with rBM-MSCs supports the ectopic formation of new bone tissue in Wistar rats.

The objective of this study was to develop an engineered rat hyaline cartilage by culturing articular chondrocytes on three-dimensional (3D) macroporous poly(DL-lactic-co-glycolic acid) (PLGA) sponges under chondrogenic induction and microgravity bioreactor conditions. Experimental groups consisted of 3D static and dynamic cultures, while a single cell monolayer (2D) served as the control. The effect of seeding conditions (static vs. dynamic) on cellularization of the scaffolds was investigated. MTT assay was used to evaluate the number of viable cells in each group at different time points. Formation of a hyaline-like cartilage was evaluated for up to 4 weeks in vitro. While 2D culture resulted in cell sheets with very poor matrix production, 3D culture was in the favor of tissue formation. A higher yield of cell attachment and spatially uniform cell distribution was achieved when dynamic seeding technique was used. Dynamic culture promoted cell growth and infiltration throughout the sponge structure and showed the formation of cartilage tissue, while chondrogenesis appeared attenuated more towards the outer region of the constructs in the static culture group. Medium supplemented with TGF-beta 1 (5 ng/ml) had a positive impact on proteoglycan production as confirmed by histochemical analyses with Alcian blue and Safranin-O stainings. Formation of hyaline-like tissue was demonstrated by immunohistochemistry performed with antibodies against type II collagen and aggrecan. SEM confirmed higher level of cellularization and cartilage tissue formation in bioreactor cultures induced by TGF-beta 1. The data suggest that PLGA sponge inside rotating bioreactor with chondrogenic medium provides an environment that mediates isolated rat chondrocytes to redifferentiate and form hyaline-like rat cartilage, in vitro.

Bone tissue is dependent on an efficient blood supply to ensure delivery of nutrients and oxygen. One method to acquire a vascular-engineered bone tissue could be the use of an angiogenic gene-activated scaffold. In the current study, porous chitosan/hydroxyapatite (C/HA) scaffolds were fabricated via freeze-drying with desired pore size, and then combined with the adenoviral vector encoding vascular endothelial growth factor and green fluorescence protein (Ad-VEGF). Human osteoblasts were cultured and seeded on characterized scaffolds. The attachment, proliferation, and differentiation of cells on gene-activated and unactivated C/HA scaffolds were evaluated in vitro and in vivo by histo- and immunohistochemistry. Findings confirmed that human osteoblasts cultured on gene-activated C/HA scaffold secreted vascular endothelial growth factor, besides maintaining its characteristic phenotype with specific extracellular matrix production. In vivo experiments indicated that scaffolds were tissue biocompatible, and that gene-activated scaffold provided a suitable environment for neovessel formation by recruiting host endothelial cells into the newly forming ectopic bone-like tissue. This study revealed that the Ad-VEGF-activated C/HA composite scaffold has potential for vascular bone regeneration applications.

Mesenchymal stem cells (MSCs) are multipotent progenitor cells with the ability to differentiate into osteoblasts, chondroblasts, myocytes, and adipocytes. They have potential for bone tissue engineering by the utilization of in vitro expanded cells with osteogenic capacity and their delivery to the appropriate sites via biomaterial scaffolds. The objective was to evaluate the potential of rat bone marrow MSCs to form 3D bone-like tissue by the use of mineralized poly(DL-lactic-co-glycolic acid) (PLGA) foam and osteoinductive medium under rotating culture conditions. PLGA foams were prepared by solvent casting and particulate leaching, then mineralized by incubating in simulated body fluid. MSCs isolated from the bone marrow of young Wistar rats were expanded and seeded on the mineralized scaffolds. The cell-polymer constructs were then cultured in a slow turning lateral vessel-type rotating bioreactor for 4 weeks under the effect of osteogenic inducers, β-glycerophosphate, ascorbic acid and dexamethasone. Mineralization was evaluated using FT-IR and increases in dry mass; morphology changes of the mineralized foams and cell adhesion was characterized by SEM; cell viability was monitored by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide). Osteogenic differentiation was determined by using immunohistochemistry (anti-osteopontin). Results indicate the feasibility of bone tissue engineering with MSCs and mineralized PLGA scaffolds supporting cell adhesion, viability and osteogenic differentiation properties of cells in hybrid structures under appropriate bioreactor conditions.