The effect of polyethylenglycol gel on the delivery and osteogenic differentiation of homologous tooth germ-derived stem cells in a porcine model.

OBJECTIVES: The aim of this study was to investigate if bone regeneration can be promoted by homologous transplantation of STRO-1 sorted (STRO-1+) porcine tooth germ mesenchymal stem cells (TGSCs) with the combination of polyethylenglycol (PEG)-based hydrogel and biphasic calcium phosphate (BCP) scaffolds. MATERIAL AND METHODS: TGSCs were isolated from impacted third molars of domestic pigs. Nine critical-sized defects were created as (1) untreated defect; filled with (2) autogenous bone; (3) BCP + PEG; (4) BCP + PEG + unsorted TGSCs; (5) BCP + unsorted TGSCs; (6) BCP + PEG + STRO-1-sorted TGSCs; (7) BCP + STRO-1-sorted TGSCs; (8) BCP + PEG + osteogenic induced unsorted TGSCs; and (9) BCP + PEG + osteogenic induced STRO-1-sorted TGSCs in 20 domestic pigs. CM-DiI labelling was used to track cells in vivo. Histomorphometric assessment of new bone formation was achieved by toluidine blue O staining and microradiography after 1, 2, 4 and 12 weeks posttransplantation. RESULTS: Complete healing was achieved in all defects although defects with PEG hydrogel presented better bone formation while STRO-1+ and unsorted TGSCs showed similar ability to form new bone after 12 weeks. Transplanted cells were seen in defects where PEG hydrogel was used as carriers in contrast to defects treated with cells and only bone grafts. CONCLUSIONS: PEG hydrogel is an efficient carrier for homologous stem cell transplantation. TGSCs are capable of promoting bone healing in critical-sized defects in combination with bone graft and PEG hydrogel. CLINICAL RELEVANCE: This study provides information about the importance of the delivery vehicle for future translational stem cell delivery approaches.

Influence of co-culture on osteogenesis and angiogenesis of bone marrow mesenchymal stem cells and aortic endothelial cells.

Co-culture of bone forming cells and endothelial cells to induce pre-vascularization is one of the strategies used to solve the insufficient vascularization problem in bone tissue engineering attempts. In the study, primary cells isolated from 2 different tissues of the same animal, rat bone marrow stem cells (RBMSCs) and rat aortic endothelial cells (RAECs) were co-cultured to study the effects of co-culturing on both osteogenesis and angiogenesis. The formation of tube like structure in 2D culture was observed for the first time in the literature by the co-culture of primary cells from the same animal and also osteogenesis and angiogenesis were investigated at the same time by using this co-culture system. Co-cultured cells mineralized and formed microvasculature beginning from 14days of incubation. After 28days of incubation in the osteogenic medium, expression of osteogenic genes in co-cultures was significantly upregulated compared to RBMSCs cultured alone. These results suggest that the co-culture of endothelial cells with mesenchymal stem cells induces both osteogenesis and angiogenesis.

Evaluation of natural gum-based cryogels for soft tissue engineering.

In this study, three natural biomaterials, Locust bean gum (LBG), Xanthan gum (XG), and Mastic gum (MG), were combined to form cryogel scaffolds. Thermal and chemical characterizations revealed the successful blend formation from LBG-XG (LX) and LBG-XG-MG (LXM) polymers. All blends resulted in macro-porous scaffolds with interconnected pore structures under the size of 400 µm. The swollen cryogels had similar mechanical properties compared with other polysaccharide-based cryogels. The mean tensile and compressive modulus values of the wet cryogels were in the range of 3.5-11.6 kPa and 82-398 kPa, respectively. The sustained release of the small molecule Kartogenin from varying concentrations and ratios of cryogels was in between 32 and 66% through 21 days of incubation. Physical, mechanical, and chemical properties make LX and LXM polysaccharide-based cryogels promising candidates for cartilage and other soft tissue engineering, and drug delivery applications.

Investigation of Vasculogenesis Inducing Biphasic Scaffolds for Bone Tissue Engineering.

Vascularization is the main obstacle for the bone tissue engineering strategies since the defect size is generally large. Incorporation of angiogenic factors is one of the strategies employed in order to accelerate vascularization and improve bone healing. In this study, a biphasic scaffold consisting of fibrous poly(lactide-co-glycolide) (PLGA) and poly(lactide-co-glycolide)-block-poly(ethylene glycol)-block-poly(lactide-co-glycolide) (PLGA-PEG-PLGA) hydrogel loaded with vascular endothelial growth factor-A (VEGF) inducer, GS4012, was constructed. Mesenchymal stem cells isolated from rat bone marrow (rBMSCs) were used for differentiation into osteogenic cells, and endothelial cells isolated from rat peripheral blood (rPBECs) were used to test the in vitro endothelial cell recruitment. The biphasic scaffold was tested for cell proliferation, ALP expression, VEGF induction, expression of osteogenic genes by rBMSCs, and recruitment of rPBECs in vitro and for improved bone healing and vascularization in vivo on critical size rat cranial defects. Endothelial migration through porous insert and VEGF induction were obtained in vitro in response to GS4012 as well as the upregulation of ALP, Runx2, Col I, and OC gene expressions. The biphasic scaffold was also shown to be effective in improving endothelial cell recruitment, vascularization, and bone healing in vivo. Thus, the proposed design has a great potential for the healing of critical size bone defect in tissue engineering studies according to both in vitro and in vivo investigations.

Fibrous bone tissue engineering scaffolds prepared by wet spinning of PLGA.

Having a self-healing capacity, bone is very well known to regenerate itself without leaving a scar. However, critical size defects due to trauma, tumor, disease, or infection involve bone graft surgeries in which complication rate is relatively at high levels. Bone tissue engineering appears as an alternative for grafting. Fibrous scaffolds are useful in tissue engineering applications since they have a high surface-to-volume ratio, and adjustable, highly interconnected porosity to enhance cell adhesion, survival, migration, and proliferation. They can be produced in a wide variety of fiber sizes and organizations. Wet spinning is a convenient way to produce fibrous scaffolds with consistent fiber size and good mechanical properties. In this study, a fibrous bone tissue engineering scaffold was produced using poly(lactic-co-glycolic acid) (PLGA). Different concentrations (20%, 25%, and 30%) of PLGA (PLA:PGA 75:25) (Mw = 66,000-107,000) were wet spun using coagulation baths composed of different ratios (75:25, 60:40, 50:50) of isopropanol and distilled water. Scanning electron microscopy (SEM) and in vitro degradation studies were performed to characterize the fibrous PLGA scaffolds. Mesenchymal stem cells were isolated from rat bone marrow, characterized by flow cytometry and seeded onto scaffolds to determine the most appropriate fibrous structure for cell proliferation. According to the results of SEM, degradation studies and cell proliferation assay, 20% PLGA wet spun in 60:40 coagulation bath was selected as the most successful condition for the preparation of wet-spun scaffolds. Wet spinning of different concentrations of PLGA (20%, 25%, 30%) dissolved in dichloromethane using different isopropanol:distilled water ratios of coagulation baths (75:25, 60:40, 50:50) were shown in this study.

Role of STRO-1 sorting of porcine dental germ stem cells in dental stem cell-mediated bone tissue engineering.

Stem cells of dental origin emerged as a new source for the regeneration of tissues with advantages mainly including non-invasive collection procedures and lack of ethical contraversies with their harvest or use. In this study, porcine TGSCs (pTGSCs) were isolated from mandibular third molar tooth germs of 6-month-old domestic pigs. This is the first study that reports the isolation and characterization of TGSCs from porcine third molars and their differentiation depending on STRO-1 expression. PTGSCs were sorted according to their STRO-1 expression as STRO-1(+) and STRO-1(-). Sorted and unsorted heterogenous cells (US) were characterized by their osteogenic, chondrogenic and adipogenic differentiation capabilities. STRO-1(+) cells exhibited a higher proliferation rate owing to their clonogenic properties. All three groups of cells were found differentiated into osteogenic lineage as shown by ALP activity, calcium deposition assay, detection of osteogenic mRNAs and, proteins and mineralization staining. According to differentiation analysis, STRO-1(+) cells did not show a better performance for osteogenesis compared to STRO-1(-) and US cells. This might indicate that STRO-1(+) cells might require a heterogeneous population of cells including STRO-1(-) in their niche to perform their proposed role in osteogenesis.

Bone Formation from Porcine Dental Germ Stem Cells on Surface Modified Polybutylene Succinate Scaffolds.

Designing and providing a scaffold are very important for the cells in tissue engineering. Polybutylene succinate (PBS) has high potential as a scaffold for bone regeneration due to its capacity in cell proliferation and differentiation. Also, stem cells from 3rd molar tooth germs were favoured in this study due to their developmentally and replicatively immature nature. In this study, porcine dental germ stem cells (pDGSCs) seeded PBS scaffolds were used to investigate the effects of surface modification with fibronectin or laminin on these scaffolds to improve cell attachment, proliferation, and osteogenic differentiation for tissue engineering applications. The osteogenic potentials of pDGSCs on these modified and unmodified foams were examined to heal bone defects and the effects of fibronectin or laminin modified PBS scaffolds on pDGSC differentiation into bone were compared for the first time. For this study, MTS assay was used to assess the cytotoxic effects of modified and unmodified surfaces. For the characterization of pDGSCs, flow cytometry analysis was carried out. Besides, alkaline phosphatase (ALP) assay, von Kossa staining, real-time PCR, CM-Dil, and immunostaining were applied to analyze osteogenic potentials of pDGSCs. The results of these studies demonstrated that pDGSCs were differentiated into osteogenic cells on fibronectin modified PBS foams better than those on unmodified and laminin modified PBS foams.