Takehiko Yoshimoto

Regenerative effect of basic fibroblast growth factor on periodontal healing in two‐wall intrabony defects in dogs

AIM The aim of the present study was to evaluate the effect of a basic fibroblast growth factor (bFGF) candidate treatment on periodontal healing in two-wall intrabony defects in dogs. MATERIALS AND METHODS Two-wall intrabony defects (5 x 5 x 5 mm) were created surgically on the distal and mesial sides of bilateral mandibular second and fourth premolars in four Beagle dogs. bFGF, enamel matrix derivative (EMD) and platelet-derived growth factor with beta-tricalcium phosphate (PDGF/beta-TCP) treatments, and sham-surgery (OFD) were rotated among the four defects in each animal, EMD and PDGF/beta-TCP serving as benchmark controls. The animals were euthanized for radiographic and histologic evaluation at 8 weeks. RESULTS Bone formation was significantly greater in the bFGF group (4.11 +/- 0.77 mm) than in the EMD (3.32 +/- 0.71 mm; p<0.05) and OFD (3.09 +/- 0.52 mm; p<0.01) groups. The EMD (4.59 +/- 1.19 mm) and PDGF/beta-TCP (4.66 +/- 0.7 mm) groups exhibited significantly greater cementum regeneration with periodontal ligament-like tissue than the OFD group (2.96 +/- 0.69 mm; p<0.01). No significant differences were observed between the bFGF and the PDGF/beta-TCP groups in any of the histometric parameters. CONCLUSIONS The candidate bFGF treatment supported periodontal regeneration comparable with that of established benchmarks: EMD and PDGF/beta-TCP.

Involvement of the endocannabinoid system in periodontal healing.

Endocannabinoids including anandamide (AEA) and 2-arachidonoylglycerol (2-AG) are important lipid mediators for immunosuppressive effects and for appropriate homeostasis via their G-protein-coupled cannabinoid (CB) receptors in mammalian organs and tissues, and may be involved in wound healing in some organs. The physiological roles of endocannabinoids in periodontal healing remain unknown. We observed upregulation of the expression of CB1/CB2 receptors localized on fibroblasts and macrophage-like cells in granulation tissue during wound healing in a wound-healing model in rats, as well as an increase in AEA levels in gingival crevicular fluid after periodontal surgery in human patients with periodontitis. In-vitro, the proliferation of human gingival fibroblasts (HGFs) by AEA was significantly attenuated by AM251 and AM630, which are selective antagonists of CB1 and CB2, respectively. CP55940 (CB1/CB2 agonist) induced phosphorylation of the extracellular-regulated kinases (ERK) 1/2, p38 mitogen-activated protein kinase (p38MAPK), and Akt in HGFs. Wound closure by CP55940 in an in-vitro scratch assay was significantly suppressed by inhibitors of MAP kinase kinase (MEK), p38MAPK, and phosphoinositol 3-kinase (PI3-K). These findings suggest that endocannabinoid system may have an important role in periodontal healing.

Co-stimulation with bone morphogenetic protein-9 and FK506 induces remarkable osteoblastic differentiation in rat dedifferentiated fat cells.

Dedifferentiated fat (DFAT) cells, which are isolated from mature adipocytes using the ceiling culture method, exhibit similar characteristics to mesenchymal stem cells, and possess adipogenic, osteogenic, chondrogenic, and myogenic potentials. Bone morphogenetic protein (BMP)-2 and -9, members of the transforming growth factor-β superfamily, exhibit the most potent osteogenic activity of this growth factor family. However, the effects of BMP-2 and BMP-9 on the osteogenic differentiation of DFAT remain unknown. Here, we examined the effects of BMP-2 and BMP-9 on osteoblastic differentiation of rat DFAT (rDFAT) cells in the presence or absence of FK506, an immunosuppressive agent. Co-stimulation with BMP-9 and FK506 induced gene expression of runx2, osterix, and bone sialoprotein, and ALP activity compared with BMP-9 alone, BMP-2 alone and BMP-2+FK506 in rDFAT cells. Furthermore, it caused mineralization of cultures and phosphorylation of smad1/5/8, compared with BMP-9 alone. The ALP activity induced by BMP-9+FK506 was not influenced by addition of noggin, a BMP antagonist. Our data suggest that the combination of BMP-9 and FK506 potently induces osteoblastic differentiation of rDFAT cells.

Injectable growth/differentiation factor‐5–recombinant human collagen composite induces endochondral ossification via Sry‐related HMG box 9 (Sox9)expression and angiogenesis in murine calvariae

BACKGROUND AND OBJECTIVE The types of collagens available today as biomaterials are purified from animal tissues. A major growing concern, however, is their safety, since there are risks of viral and prion contamination and of unknown and potentially zoonotic infectious diseases. The present study aimed to assess, using immunohistochemistry, the effects of recombinant human growth/differentiation factor-5 (rhGDF-5) combined with recombinant human collagen I (rhCI) on bone formation in murine calvariae. MATERIAL AND METHODS Composite rhGDF-5-rhCI or rhCI alone was injected subcutaneously into murine calvariae. After 3, 7 or 14 days, tissues were examined radiologically, histologically and immunohistochemically. The production of vascular endothelial growth factor (VEGF) by primary osteoblasts, periosteal cells and connective tissue fibroblasts isolated enzymatically from neonatal murine calvariae was also assessed. RESULTS A protrusion was observed on the calvariae at the site injected with rhGDF-5/rhCI composite. Its mineral density was shown to be different from that of the existing bone by two-dimensional microcomputed tomography. Type II collagen-positive staining was restricted to newly formed tissues. Thus, the newly formed tissues seemed to be bone- and cartilage-like tissues. A number of vessels with positively stained cells for Von Willebrand factor were detected in the newly formed tissues. The rhGDF-5 enhanced VEGF production in cultured connective tissue fibroblasts. Sry-related HMG box 9 (Sox9)-positive cells were detected in the hypertrophic periosteum, and penetrated into the newly formed tissues. CONCLUSIONS These results suggest that rhCI seems to allow the release of rhGDF-5 and that rhGDF-5-rhCI composite induces endochondral ossification via Sox9 expression and angiogenesis in murine calvariae.

Effect of bone swaging with calcium phosphate bone cement on periodontal regeneration in dogs

OBJECTIVES The present study evaluated the effects of bone swaging (BS) with injectable calcium phosphate bone cement (CPC) on periodontal healing in 1-wall intrabony defects in dogs. STUDY DESIGN Surgically created 1-wall intrabony defects were treated with BS, CPC, BS with CPC (BS/CPC), or open-flap debridement (OFD). The animals were humanely killed for histologic evaluation at 8 weeks. RESULTS New bone formation occurred from the host bone toward the coronal region of the defects in the BS/CPC group. The height of new bone formation was significantly higher in the BS/CPC group than in the other groups (P < .05). New cementum formation was most notable in the BS/CPC group, where it was significantly greater than in the OFD and CPC groups (P < .05). CONCLUSIONS Within the limitations of the present study, it was concluded that BS/CPC treatment effectively promoted new bone and cementum formation in 1-wall intrabony defects in dogs.

Effects of enamel matrix derivative and basic fibroblast growth factor with μ-tricalcium phosphate on periodontal regeneration in one-wall intrabony defects: an experimental study in dogs.

This study evaluated the effects of enamel matrix derivative (EMD) and basic fibroblast growth factor (bFGF) with μ-tricalcium phosphate (μ-TCP) on periodontal healing in intrabony defects in dogs. One-wall intrabony defects created in dogs were treated with μ-TCP alone (μ-TCP), EMD with μ-TCP (EMD/μ-TCP), bFGF with μ-TCP (bFGF/μ-TCP), and a combination of each (EMD/bFGF/μ-TCP). The amount of new bone formation was not significant for any group. The EMD/bFGF/μ-TCP group induced significantly greater new cementum formation than the μ-TCP and bFGF/μ-TCP groups and, although not significantly, formed more new cementum than the EMD/μ-TCP group. These findings indicate that EMD/bFGF/μ-TCP treatment is effective for cementum regeneration.

Bone formation using β-tricalcium phosphate/carboxymethyl-chitin composite scaffold in rat calvarial defects

OBJECTIVE The aim of the present study was to evaluate the effects of β-tricalcium phosphate/carboxymethyl-chitin material (β-TCP/CM-chitin) on bone formation in rat calvarial defects. STUDY DESIGN Eighteen animals surgically received 2 calvarial defects (5 mm) bilaterally in each parietal bone. β-TCP/CM-chitin was implanted in one side of each defect, and the contralateral side of the defect was left empty as a control. The animals were humanely killed at 4, 8, and 12 weeks after surgery for histologic evaluation. RESULTS New bone formation in the β-TCP/CM-chitin group was significantly greater than that in the control group throughout the healing periods (P < .05). β-TCP/CM-chitin was remarkably resorbed 12 weeks after surgery. CONCLUSIONS These results indicate that β-TCP/CM-chitin is useful as a scaffold for bone formation.

Effects of EMD in combination with bone swaging and calcium phosphate bone cement on periodontal regeneration in one‐wall intrabony defects in dogs

BACKGROUND AND OBJECTIVE Although the application of EMD is a widely accepted periodontal-regenerative therapy, its effects on noncontained intrabony defects are unpredictable because of the lack of a space-making property. The combined use of EMD and autogenous bone grafts reportedly stimulates significant periodontal regeneration in intrabony defects. The aim of the present study was to evaluate the effects of EMD in combination with bone swaging (BS) and injectable calcium phosphate bone cement (CPC), which was placed into the spaces between the grafted swaged bone and the proximal host bone, on periodontal healing in one-wall intrabony defects in dogs. MATERIAL AND METHODS One-wall intrabony defects (3 mm wide and 5 mm deep) were surgically created on the mesial and distal sides of the bilateral mandibular premolars in four dogs. The 16 defects were assigned to one of the following treatments: EMD only, BS only, EMD with BS (EMD + BS), or EMD with BS and CPC (EMD + BS + CPC). The animals were killed 8 wk after surgery for histologic evaluation. RESULTS The height of newly formed bone was significantly greater in the EMD + BS + CPC group (3.73 ± 0.30 mm) than in the BS-only (2.74 ± 0.33 mm; p < 0.05) and EMD + BS (2.88 ± 0.98 mm; p < 0.05) groups. The area of newly formed bone was significantly larger in the EMD + BS + CPC group (5.68 ± 1.66 mm(2)) than in the EMD-only (3.68 ± 0.33 mm(2); p < 0.05), BS-only (3.48 ± 1.26 mm(2); p < 0.05) and EMD + BS (3.38 ± 1.37 mm(2); p < 0.05) groups. The EMD-only (4.63 ± 0.42 mm), EMD + BS (4.67 ± 0.30 mm) and EMD + BS + CPC (4.78 ± 0.54 mm) groups showed significantly greater cementum formation than did the BS-only group (3.93 ± 0.56 mm; p < 0.05). CONCLUSION These results indicate that treatment with EMD + BS + CPC promotes favorable periodontal healing in one-wall intrabony defects in dogs.