Kyoo-Sung Cho

Isolation and characterization of human periodontal ligament (PDL) stem cells (PDLSCs) from the inflamed PDL tissue: in vitro and in vivo evaluations.

OBJECTIVES Mesenchymal stem cells (MSC) could be isolated from healthy periodontal ligaments (PDL). The aims of this study were to isolate and characterize human PDL stem cells (hPDLSCs) from inflamed PDL tissue, and to evaluate their regenerative potential. MATERIALS AND METHODS Inflamed hPDLSCs (ihPDLSCs) were isolated from the inflamed PDL tissue obtained from intra-bony defects during flap surgery, and characterized by immunohistochemical staining, colony-forming unit assay, fluorescence-activated cell sorting, and mRNA expression in comparison with healthy hPDLSCs obtained from extracted teeth for orthodontic purpose. The proliferative potential and migratory potential was evaluated, and compared with healthy hPDLSCs. Regenerative potential was assessed by an in vivo ectopic transplantation model. RESULTS ihPDLSCs were successfully isolated and characterized as MSCs. Both ihPDLSCs and hPDLSCs were successfully differentiated under osteogenic/cementogenic and adipogenic microenvironment. The proliferative potential did not differ between healthy hPDLSCs and ihPDLSCs, while the migratory capacity was significantly increased in ihPDLSCs (p<0.05). Both groups exhibited new cementum-like tissue and related PDL fibre regeneration in an in vivo transplantation model. CONCLUSION ihPDLSCs could be successfully isolated from inflamed PDL tissue, and they retained the regenerative potential for cementum and related PDL tissues.

The induction of bone formation in rat calvarial defects and subcutaneous tissues by recombinant human BMP-2, produced in Escherichia coli

We investigated the ability of recombinant human bone morphogenetic protein-2, produced from Escherichia coli (ErhBMP-2), to form orthotopic and ectopic bone in rat models. BMP-2 was expressed in E. coli and extracted from the inclusion bodies. Critical-sized calvarial defects and subcutaneous pouches were created in rats, and an absorbable collagen sponge (ACS) was loaded with different doses of ErhBMP-2 for implantation. ACS alone and sham surgery controls were also included. Implant sites were evaluated by histological and/or histometric analyses following a 2- or 8-week healing interval. In the calvarial defect model, enhanced bone formation was observed with all doses of ErhBMP-2, while only limited amounts of new bone were found in controls. In the ectopic subcutaneous implant model, bone formation was clearly observed in all animals treated with ErhBMP-2 at 2 weeks. However, at 8 weeks, less new bone formation was detected than at 2 weeks. Nevertheless, the remaining new bone showed an advanced degree of bone remodeling and more maturity than that observed at 2 weeks. These results showed that ErhBMP-2 was osteoinductive under controlled in vivo conditions. Thus, ErhBMP-2 has definite potential as an alternative to rhBMP-2 produced in a eukaryotic system for clinical use.

Spontaneous healing capacity of rabbit cranial defects of various sizes

Purpose This study evaluated the spontaneous healing capacity of surgically produced cranial defects in rabbits with different healing periods in order to determine the critical size defect (CSD) of the rabbit cranium. Methods Thirty-two New Zealand white rabbits were used in this study. Defects of three sizes (6, 8, and 11 mm) were created in each of 16 randomly selected rabbits, and 15-mm defects were created individually in another 16 rabbits. The defects were analyzed using radiography, histologic analysis, and histometric analysis after the animal was sacrificed at 2, 4, 8, or 12 weeks postoperatively. Four samples were analyzed for each size of defect and each healing period. Results The radiographic findings indicated that defect filling gradually increased over time and that smaller defects were covered with a greater amount of radiopaque substance. Bony islands were observed at 8 weeks at the center of the defect in both histologic sections and radiographs. Histometrical values show that it was impossible to determine the precise CSD of the rabbit cranium. However, the innate healing capacity that originates from the defect margin was found to be constant regardless of the defect size. Conclusions The results obtained for the spontaneous healing capacity of rabbit cranial defects over time and the underlying factors may provide useful guidelines for the development of a rabbit cranial model for in vivo investigations of new bone materials.

Periodontal wound healing/regeneration following implantation of recombinant human growth/differentiation factor-5 (rhGDF-5) in an absorbable collagen sponge carrier into one-wall intrabony defects in dogs: A dose-range study

AIM Recombinant human growth/differentiation factor-5 (rhGDF-5) is being evaluated as a candidate therapy in support of periodontal regeneration. The objective of this study was to evaluate cementum and alveolar bone formation, and aberrant healing events following surgical implantation of rhGDF-5 in an absorbable collagen sponge (ACS) carrier using an established periodontal defect model. MATERIALS AND METHODS Bilateral 4 x 5 mm (width x depth), one-wall, critical-size, intrabony periodontal defects were surgically created at the mandibular second and fourth pre-molar teeth in 15 Beagle dogs. Five animals received 1 microg/defect and five animals 20 microg/defect rhGDF-5 in unilateral defect sites. Contralateral sites received treatments reported elsewhere. Five animals received rhGDF-5/ACS with 0 (buffer control) and 100 microg/defect rhGDF-5 in contralateral defect sites. The animals were euthanized at 8 weeks post-surgery for histologic and histometric evaluation. RESULTS Surgical implantation of rhGDF-5 stimulated significant periodontal regeneration. Cementum formation was significantly enhanced in sites implanted with rhGDF-5 (1 and 100 microg) compared with control (p<0.05). Similarly, bone formation height was significantly greater in sites receiving rhGDF-5 (1 and 100 microg) compared with control (p<0.05). There were no significant or remarkable differences in bone and cementum formation within the selected dose interval (1, 20 and 100 microg rhGDF-5). None of the control or the rhGDF-5 sites exhibited root resorption, ankylosis, or other aberrant tissue reactions. CONCLUSION Surgical implantation of rhGDF-5/ACS may be used safely to support periodontal wound healing/regeneration in intrabony periodontal defects without complications.

Osteoinductive activity of biphasic calcium phosphate with different rhBMP-2 doses in rats.

OBJECTIVE The aim of the current study was to determine whether a hydroxyapatite (HA)/beta-tricalcium phosphate (β-TCP) ratio of 20/80 impregnated with recombinant human bone morphogenetic protein (rhBMP-2) enhances new bone formation and to evaluate the dose-dependent response of rhBMP-2. STUDY DESIGN Critical-sized calvarial defects were made in rats, and biphasic calcium phosphate (BCP) with different rhBMP-2 doses was loaded into rat calvarial defects. The animals were allowed to heal for either 2 or 8 weeks. RESULTS The percentages of new bone after 2 and 8 weeks of healing were significantly greater in the rhBMP-2-treated groups (at all doses) than in the control groups. The percentage of remaining BCP was significantly lower at 8 weeks than at 2 weeks in all groups that included BCP. CONCLUSIONS rhBMP-2 administered using a BCP carrier significantly induces new bone formation. A dose-dependent response was not shown in the present study.

Histologic and clinical evaluation for maxillary sinus augmentation using macroporous biphasic calcium phosphate in human.

OBJECTIVES This study evaluated both the clinical and histological aspects of bone formation in maxillary sinus augmentation using MBCP as the bone-grafting material. MATERIAL AND METHODS MBCP was used as a primary bone substitute for maxillary sinus augmentation. Fifty-two patients were selected after a medical and dental examination, and were divided into the following three groups: those augmented with MBCP only; MBCP combined with irradiated cancellous bone; and MBCP combined with intraoral autogenous bone. After a healing period (average 6.78 months after surgery), bone cores were harvested for a histological evaluation and the implant fixtures were installed. These bone cores were evaluated via light microscope and implants were followed up for at least six months after loading. RESULTS Four to ten months after surgery, new vital bone surrounding the MBCP particles was observed in 18 bone biopsies. Two out of the 130 implants installed were explanted due to a failure of osseointegration before the prosthetic procedure. All the remaining implants were functioning for 6 to 27 months (average 12.96 months). The cumulative survival rate of the implants was 98.46%. CONCLUSION These results show that MBCP can be used as a grafting material for sinus floor augmentation, whether combined with other bone graft materials or not, and lead to a predictable prognosis for dental implants in the posterior maxillary area where there is insufficient vertical height for fixture installation.

Volumetric bone regenerative efficacy of biphasic calcium phosphate‐collagen composite block loaded with rhBMP‐2 in vertical bone augmentation model of a rabbit calvarium

Block-type biphasic calcium phosphate (BCP) carriers are more effective at delivering recombinant human bone morphogenetic protein-2 (rhBMP-2) in various clinical situations than are particle-type carriers, due to their potential for highly successful three-dimensional bone regeneration. The aim of this study was to confirm the bone-regenerative capabilities of three-dimensional BCP blocks with a low hydroxyapatite/β-tricalcium phosphate ratio (20/80) combined with collagen (10% wt) as an rhBMP-2 delivery system in a craniofacial vertical bone augmentation model. BCP blocks and BCP-collagen blocks (with average macropore sizes of 296 and 390 μm, respectively) with or without rhBMP-2 were fixed with osteosynthesis screws to the calvarial surface of rabbits. After 8 weeks, histologic and histomorphometric analyses were performed to evaluate the resulting new bone area, augmented area, bone density, and degree of integration. The area of new bone was significantly greater in specimens containing rhBMP-2 than in the control group (p < 0.05). Moreover, the area fractions of newly formed bone within the augmented area and a degree of integration between the regenerative bone and the calvarium were both significantly greater in the BCP-collagen/rhBMP-2 group than in the BCP/rhBMP-2 group (p < 0.05), whereas the two carrier systems exhibited similar rhBMP-2 release profiles, with sustained and linear release. The BCP and BCP/rhBMP-2 blocks exhibited excellent structural integrity, with large fragments of residual ceramic. In conclusion, the BCP-collagen composite block exhibited enhanced osteoinductive potential and could be a good candidate as a carrier of rhBMP-2 due to its characteristics of favorable volumetric stability, ease of handling, and excellent remodeling properties.

Bone formation of block and particulated biphasic calcium phosphate lyophilized with Escherichia coli-derived recombinant human bone morphogenetic protein 2 in rat calvarial defects.

The objective of this study was to evaluate bone formation in rat calvarial defects after surgical implantation of block or particulated biphasic calcium phosphate (BCP) lyophilized with Escherichia coli-derived recombinant human bone morphogenetic protein 2 (ErhBMP-2). Critical-size calvarial osteotomy defects were created in 5 groups of Sprague-Dawley rats. Each group received one of the following: 1) sham surgery control; 2) biphasic calcium phosphate particles (CPP); 3) biphasic calcium phosphate block (CPB); 4) ErhBMP-2-coated CPP; or 5) ErhBMP-2-coated CPB. ErhBMP was coated on BCP by a stepwise lyophilizing protocol. The new bone formation was significantly greater in ErhBMP-2-treated groups compared with the untreated group. In particular, the ErhBMP-2/CPB group showed stability of augmented areas during the period of healing, due to relevant space-providing capacity. Thus, it can be concluded that CPP and CPB lyophilized with ErhBMP-2 enhance the formation of new bone, and CPB appears to be a suitable carrier for ErhBMP-2 in which a 3-dimensional structural integrity is an important consideration factor.

Treatment of FGF-2 on stem cells from inflamed dental pulp tissue from human deciduous teeth

OBJECTIVE The purposes of this study were to isolate and characterize stem cells from inflamed pulp tissue of human functional deciduous teeth (iSHFD) and to evaluate the influence of fibroblastic growth factor-2 (FGF-2) on the regenerative potential. MATERIALS AND METHODS We successfully isolated mesenchymal stem cells (MSCs) from the inflamed dental pulp tissue of human deciduous teeth and demonstrated that their regenerative potential could be enhanced by the application of FGF-2 (20 ng ml(-1)) during ex vivo expansion. Isolated stem cells expanded in FGF-2 were characterized using a colony-forming assay, proliferation, migration, in vitro differentiation, in vivo ectopic transplantation assay, and gene expression profiling. RESULTS MSCs isolated from the inflamed pulp tissue of functional deciduous teeth potentially possess the qualities of those from human exfoliated deciduous teeth. FGF-2 applied to iSHFD during expansion enhanced the colony-forming efficiency of these cells, increased their proliferation and migration potential, and reduced their differentiation potential in vitro. However, the ectopic transplantation of iSHFD/FGF-2 in vivo increased the formation of dentin-like material. CONCLUSION FGF-2 expansion of stem cells from inflamed pulp tissues of human deciduous teeth can be a good source of stem cells for future clinical applications and a novel way of using discarded inflamed tissues.

Enhanced adipogenic differentiation and reduced collagen synthesis induced by human periodontal ligament stem cells might underlie the negative effect of recombinant human bone morphogenetic protein-2 on periodontal regeneration.

BACKGROUND AND OBJECTIVE Recombinant human bone morphogenetic protein-2 (rhBMP-2) is a potent inducer for the regeneration of mineralized tissue, but has a limited effect on the regeneration of cementum and periodontal ligament (PDL). The aim of the present study was to determine the effects of rhBMP-2 on the in vitro and in vivo biologic activity of well-characterized human PDL stem cells (hPDLSCs) and to elucidate the underlying mechanism of minimal periodontal regeneration by rhBMP-2. MATERIAL AND METHODS hPDLSCs were isolated and cultured, and then transplanted into an ectopic subcutaneous mouse model using a carrier treated either with or without rhBMP-2. Comprehensive histologic, histometric and immunohistochemical analyses were performed after an 8-wk healing period. The effects of rhBMP-2 on the adipogenic and osteogenic/cementogenic differentiation of hPDLSCs were also evaluated. The effect of rhBMP-2 on both soluble and insoluble collagen synthesis was analyzed, and the expression of mRNA and protein for collagen types I, II, III and V was assessed. RESULTS In the present study, rhBMP-2 promoted both adipogenic and osteogenic/cementogenic differentiation of hPDLSCs in vitro, and the in vivo potential of hPDLSCs to form mineralized cementum and organized PDL tissue was down-regulated following treatment with rhBMP-2. Collagen synthesis, which plays a crucial role in the regeneration of cementum and the periodontal attachment, was significantly reduced, with associated modification of the relevant mRNA and protein expression profiles. CONCLUSION In summary, the findings of the present study suggest that enhanced adipogenic differentiation and inhibition of collagen synthesis by hPDLSCs appear to be partly responsible for the minimal effect of rhBMP-2 on cementum and PDL tissue regeneration by hPDLSCs.