A Technique for Salvaging an Existing Four-Implant-Supported Mandibular Fixed Complete Prostheses by Using it as a Surgical Guide after an Implant Failure.

This technique article describes the use of an existing four implant supported fixed complete prosthesis both as a surgical template for reimplantation and for a permanent prosthesis after one of the supporting implants fails. This method offers a reliable and low-cost solution for the patient without the necessity of a new prosthesis.

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.

Investigation of the effects of semaphorin 3A on new bone formation in a rat calvarial defect model.

PURPOSE: This study investigates the effects of semaphorin 3A on new bone formation in an experimental rat model. MATERIALS AND METHODS: Cortical bone defects, 5 mm, were created in the calvaria of 40 Wistar rats, which were then separated into three groups: empty defect (control) group, collagen group, collagen + semaphorin 3A group. The bone blocks were harvested after 4 and 8 weeks. New bone formation was assessed by micro-computed tomography (micro-CT), histology, histomorphometry, transmission electron microscope (TEM) and immunohistochemistry. RESULTS: Increased bone formation was observed in collagen + semaphorin 3A groups both histologically and with micro-CT. In the histomorphometic analysis, the control group had significantly less bone formation compared to both the collagen and collagen + semaphorin 3A group at 4 weeks (p = 0.0001) and 8 weeks (p = 0.0001). The collagen group had significantly less bone formation compared to collagen + semaphorin 3A group both at 4 weeks (p = 0.002) and 8 weeks (p = 0.005). Immunohistochemical analysis revealed that semaphorin 3A inhibited receptor activator of nuclear factor-kB ligand (RANKL) expression and increased the expressions of osteoblastic bone markers at 4 weeks. In TEM analysis, the collagen + semaphorin 3A group had an increased proliferation and bone formation rate at 4 weeks, whereas bone quantity and maturation were enhanced at 8 weeks. CONCLUSION: Locally applied semaphorin 3A increases callus formation at 4 weeks and bone formation at 8 weeks. Semaphorin 3A prevents bone resorption by inhibiting osteoclasts and increases bone formation by inducing osteoblasts.

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.

Influence of STRO-1 selection on osteogenic potential of human tooth germ derived mesenchymal stem cells.

Mesenchymal stem cells derived from the human tooth germ (hTGSCs) are a heterogeneous cell population that can differentiate into osteogenic, neurogenic, and adipogenic lineages. The aim of this study was to compare the osteogenic differentiation capacity of STRO-1 positive (STRO-1+) hTGSCs and unsorted heterogeneous hTGSCs and to establish if STRO-1+ cells are more committed to osteogenic differentiation. HTGSCs were isolated from impacted third molar tooth germ tissues of adolescents, and a subpopulation of STRO-1+ hTGSCs was obtained by fluorescence-activated cell sorting. STRO-1+, STRO-1 negative (STRO-1-), and unsorted cells were cultured in osteogenic and standard culture media to compare their capacity to differentiate towards osteoblastic lineage. Cells were tested for proliferation rates, alkaline phosphatase activity, and amounts of accumulated calcium. Gene expression levels of the RUNX2, osteocalcin, and osteonectin genes were analyzed with real time PCR. Mineralization and osteogenic protein expression were examined by using von Kossa staining and confocal microscopy. Our results indicated that osteogenically induced cell populations showed greater mineralization capacity than non-induced cells. However, expression levels of early and late osteogenic markers were not significantly different between STRO-1+ and unsorted cells. In conclusion, the selection by STRO-1 expression does not yield cells with osteogenic capacity higher than that of the heterogeneous hTGSC population. Cell sorting using osteogenic markers other than STRO-1 might be beneficial in obtaining a more sensitive osteogenic sub-population from unsorted heterogenous hTGSCs.

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.

Bone response to biomimetic implants delivering BMP-2 and VEGF: an immunohistochemical study.

This animal study evaluated bone healing around titanium implant surfaces biomimetically coated with bone morphogenic protein-2 (BMP-2) and/or vascular endothelial growth factor (VEGF) by examining bone matrix proteins and mineralisation. Five different implant surfaces were established: acid-etched surface (AE), biomimetic calcium phosphate surface (CAP), BMP-2 loaded CAP surface, VEGF loaded CAP surface and dual BMP-2 + VEGF loaded CAP surface. The implants were inserted into calvariae of adult domestic pigs. For the comparison of osteoconductive capacity of each surface, bone mineral density and expression of bone matrix proteins (collagen I, BMP-2/4, osteocalcin and osteopontin) inside defined chambers around the implant were assessed using light microscopy and microradiography and immunohistochemical analysis at 1, 2 and 4 weeks. In the both groups delivering BMP-2, the bone mineral density was significantly enhanced after 2 weeks and the highest value was measured for the group BMP + VEGF. In the group VEGF, collagen I and BMP-2/4 expressions were significantly up-regulated at the first and second weeks. The percentage of BMP-2/4 positive cells in the group BMP + VEGF was significantly enhanced compared with the groups AE and CAP at the second week. Although the highest osteocalcin and osteopontin expression values were observed for the group BMP + VEGF after 2 weeks, no statistically significant difference in osteocalcin and osteopontin expressions was found between all groups at any time. It was concluded that combined delivery of BMP-2 and VEGF favoured bone mineralisation and expression of important bone matrix proteins that might explain synergistic interaction between both growth factors.

The effect of combined delivery of recombinant human bone morphogenetic protein-2 and recombinant human vascular endothelial growth factor 165 from biomimetic calcium-phosphate-coated implants on osseointegration.

OBJECTIVES: The delivery of growth factors for enhanced osseointegration depends on the effectiveness of the carrier systems at the bone-implant interface. This study evaluated the effect of solo and dual delivery of recombinant human bone morphogenetic protein-2 (rhBMP-2) and recombinant human vascular endothelial growth factor (rhVEGF(165) ) from biomimetically octacalcium phosphate-coated implants on osseointegration. MATERIALS AND METHODS: Biomimetic implants, bearing either a single growth factor (BMP or VEGF) or their combination (BMP+VEGF), were established, and compared with acid-etched (AE, control) and biomimetic implants without growth factor (CAP). Implants were placed into frontal skulls of nine domestic pigs. The quality of osseointegration was evaluated using microradiographic and histomorphometric analysis of bone formation inside four defined bone chambers of the experimental implant at 1, 2 and 4 weeks. RESULTS: Biomimetic implants, either with or without growth factor, showed enhanced bone volume density (BVD) values after 2 and 4 weeks. This enhancement was significant for the BMP and BMP+VEGF group compared with the control AE group after 2 weeks (P<0.05). All biomimetic calcium-phosphate (Ca-P) coatings exhibited significantly enhanced bone-implant contact (BIC) rates compared with the uncoated control surface after 2 weeks (P<0.05). However, the combined delivery of BMP-2 and VEGF did not significantly enhance BIC at the final observation period. CONCLUSION: It was concluded that the combined delivery of BMP-2 and VEGF enhances BVD around implants, but not BIC. Therefore, it may be assumed that changes in the surface characteristics should be considered when designing growth factor-delivering surfaces.

Human tooth germ stem cells preserve neuro-protective effects after long-term cryo-preservation.

The use of mesenchymal stem cells (MSCs) has been shown to be promising in chronic disorders such as diabetes, Alzheimer's dementia, Parkinson's disease, spinal cord injury and brain ischemia. Recent studies revealed that human tooth germs (hTG) contain MSCs which can be easily isolated, expanded and cryo-preserved. In this report, we isolated human tooth germ stem cells (hTGSCs) with MSC characteristics from third molar tooth germs, cryo-preserved them at -80( degrees )C for 6 months, and evaluated for their surface antigens, expression of pluri-potency associated genes, differentiation capacity, karyotype, and proliferation rate. These characteristics were compared to their non-frozen counterparts. In addition, neuro-protective effects of cryo-preserved cells on neuro-blastoma SH-SY5Y cells were also assessed after exposure to stress conditions induced by hydrogen-peroxide (oxidative stress) and paclitaxel (microtubule stabilizing mitotic inhibitor). After long term cryo-preservation hTGSCs expressed surface antigens CD29, CD73, CD90, CD105, and CD166, but not CD34, CD45 or CD133, which was typical for non-frozen hTGSCs. Cryo-preserved hTGSCs were able to differentiate into osteo-, adipo- and neuro-genic cells. They also showed normal karyotype after high number of population doublings and unchanged proliferation rate. On the other hand, cryo-preserved cells demonstrated a tendency for lower level of pluri-potency associated gene expression (nanog, oct4, sox2, klf4, c-myc) than non-frozen hTGSCs. hTGSCs conditioned media increased survival of SH-SY5Y cells exposed to oxidative stress or paclitaxel. These findings confirm that hTGSCs preserve their major characteristics and exert neuro-protection after long-term cryo-preservation, suggesting that hTGSCs, harvested from young individuals and stored for possible use later as they grow old, might be employed in cellular therapy of age-related degenerative disorders.

Comparison and optimisation of transfection of human dental follicle cells, a novel source of stem cells, with different chemical methods and electro-poration.

INTRODUCTION: Human dental follicle cells (HDFCs) derived from human impacted third molars (wisdom teeth) have been shown to be a significant source of adult stem cells. Generation of mesenchymal stem cell-like cells from dental follicles causes minimal surgical stress. In vitro and in vivo reports showed that HDFCs can be utilized in gene and cell therapy applications which make them an attractive alternative source for different gene-cell therapy applications. However, there are currently no systematic comparative studies on transfection potential of HDFC cells using different chemical and electro-poration techniques. METHODS: Stem cells from impacted third tooth molars were isolated, and analyzed for expression of surface markers. Transfection efficiencies of four commercially available transfection reagents (Transfast, Escort V, Superfect and FuGene HD) and electro-poration on isolated stem cells were compared. RESULTS: Isolated HDFCs were stained positive for CD105, CD90, CD73, CD166, and negative for CD34, CD45, and CD133. Among the chemical transfection reagents used in this study, FuGene HD was the most efficient in transfecting HDFCs, even in the presence of 10% serum. CONCLUSION: Electro-poration of HDFCs yield relatively high transfection rates and cell viability when compared to chemical transfection techniques. Our observations might be useful for developing gene and cell therapy applications using dental follicle stem cells.