Isao Ishikawa

Cementum-periodontal ligament complex regeneration using the cell sheet technique

BACKGROUND AND OBJECTIVEnIn the present study we evaluated if a multilayered human periodontal ligament cell sheet could reconstruct the physiological architecture of a periodontal ligament-cementum complex.nnnMATERIAL AND METHODSnHuman periodontal ligament cells were isolated and then cultured in dishes coated with a temperature-responsive polymer to allow cell detachment as a cell sheet. In the control group, human periodontal ligament cells were cultured in Dulbeccos modified Eagles minimal essential medium containing 10% fetal bovine serum and 1% antibiotics. In the experimental group, human periodontal ligament cells were cultured in Dulbeccos modified Eagles minimal essential medium and osteodifferentiation medium containing dexamethasone, ascorbic acid and beta-glycerophosphate. After 3 wk, scanning electron microscopy was carried out, in addition to staining for alkaline phosphatase activity and for calcium (using the Von Kossa stain). Then human periodontal ligament cell sheets were multilayered and placed onto dentin blocks. The constructs were transplanted subcutaneously into the back of immunodeficient rats. At 1 and 6 wk after transplantation, the animals were killed. Demineralized tissue sections were stained using hematoxylin and eosin, and Azan, and then analyzed.nnnRESULTSnAfter 3 wk of culture in osteodifferentiation medium, human periodontal ligament cells produced mineral-like nodules and also showed positive staining for alkaline phosphatase, calcium (Von Kossa) and mRNA expression of type I collagen. By contrast, in the control group only weak alkaline phosphatase staining was observed, the Von Kossa stain was negative and there was no mRNA expression of type I collagen. Six weeks after transplantation with human periodontal ligament cells cultured in osteodifferentiation medium, most of the dentin surfaces showed a newly immature cementum-like tissue formation and periodontal ligament with perpendicular orientation inserted into the newly deposited cementum-like tissue.nnnCONCLUSIONnThis study suggests that the multilayered temperature-responsive culture system can be used as a novel strategy for periodontal regeneration. The human periodontal ligament cell sheet technique may be applicable for regeneration of the clinical periodontal ligament-cementum complex.

Er:YAG laser therapy for peri-implant infection: a histological study.

The purpose of this study was to evaluate the effects of Er:YAG laser on degranulation and implant surface debridement in peri-implant infection. The peri-implant infection was experimentally induced in dogs, and the treatment was performed using an Er:YAG laser or a plastic curet. Animals were sacrificed after 24xa0weeks, and undecalcified histological sections were prepared and analyzed. Degranulation and implant surface debridement were obtained effectively and safely by Er:YAG laser. Histologically, a favorable formation of new bone was observed on the laser-treated implant surface, and the laser group showed a tendency to produce greater bone-to-implant contact than the curet group. These results indicate that the Er:YAG laser therapy has promise in the treatment of peri-implantitis.

Low-level Er:YAG laser irradiation enhances osteoblast proliferation through activation of MAPK/ERK.

Although the use of high-level Er:YAG laser irradiation has been increasing in periodontal and peri-implant therapy, the effects of low-level Er:YAG laser on surrounding tissues and cells remain unclear. In the present study, the effects of low-level Er:YAG laser irradiation on osteoblast proliferation were investigated. Cells of the osteoblastic cell line MC3T3-E1 were treated with low-level Er:YAG laser irradiation with various combinations of laser settings (fluence 0.7–17.2xa0J/cm2) and in the absence or presence of culture medium during irradiation. On day 1 and/or day 3, cell proliferation and death were determined by cell counting and by measurement of lactate dehydrogenase (LDH) levels. Further, the role of mitogen-activated protein kinase (MAPK) pathways in laser-enhanced cell proliferation was investigated by inhibiting the MAPK pathways and then measuring MAPK phosphorylation by Western blotting. Higher proliferation rates were found with various combinations of irradiation parameters on days 1 and 3. Significantly higher proliferation was also observed in laser-irradiated MC3T3-E1 cells at a fluence of approximately 1.0–15.1xa0J/cm2, whereas no increase in LDH activity was observed. Further, low-level Er:YAG irradiation induced the phosphorylation of extracellular signal-regulated protein kinase (MAPK/ERK) 5 to 30xa0min after irradiation. Although MAPK/ERK 1/2 inhibitor U0126 significantly inhibited laser-enhanced cell proliferation, activation of stress-activated protein kinases/Jun N-terminal kinase (SAPK/JNK) and p38 MAPK was not clearly detected. These results suggest that low-level Er:YAG laser irradiation increases osteoblast proliferation mainly by activation of MAPK/ERK, suggesting that the Er:YAG laser may be able to promote bone healing following periodontal and peri-implant therapy.

Processing of Ameloblastin by MMP-20

Ameloblastin (AMBN) cleavage products are the most abundant non-amelogenin proteins in the enamel matrix of developing teeth. AMBN N-terminal cleavage products accumulate in the sheath space between enamel rods, while AMBN C-terminal products localize within rods. We tested the hypothesis that MMP-20 is the protease that cleaves AMBN. Glycosylated recombinant porcine AMBN (rpAMBN) was expressed in human kidney 293F cells, and recombinant porcine enamelysin (rpMMP-20) was expressed in bacteria. The purified proteins were incubated together at an enzyme:substrate ratio of 1:100. N-terminal sequencing of AMBN digestion products determined that rpMMP-20 cleaved rpAMBN after Pro2, Gln130, Gln139, Arg170, and Ala222. This shows that MMP-20 generates the 23-kDa AMBN starting at Tyr223, as well as the 17-kDa (Val1-Arg170) and 15-kDa (Val1-Gln130) AMBN cleavage products that concentrate in the sheath space during the secretory stage. We conclude that MMP-20 processes ameloblastin in vitro and in vivo.

Current and future periodontal tissue engineering

Periodontitis is an inflammatory disease that leads to the loss of tooth-supporting tissues. Conventional periodontal treatment is generally unable to promote regeneration of the damaged periodontal structures. Recently, several studies have investigated the use of tissue engineering to facilitate predictable periodontal regeneration. This article reviews various technologies related to periodontal tissue engineering. These include bone grafting, guided tissue regeneration, enamel matrix protein derivative, growth factors, stem cell therapy, cell sheet engineering and laser treatment. Studies carried out by this group, and available clinical data, together with the authors own clinical experiences, are discussed. In addition, possible new directions that need to be exploited to make periodontal tissue engineering a clinical success are discussed herein.

Twist negatively regulates osteoblastic differentiation in human periodontal ligament cells

Periodontal ligament (PDL) is a thin fibrous connective tissue located between two mineralized tissues, alveolar bone and cementum, which maintains a constant width physiologically. The mechanisms by which PDL resists mineralization are not well understood. Twist is a basic helix loop helix protein that plays a central role in regulation of early osteogenesis. We investigated the localization of Twist in PDL and compared the expression of Twist and osteoblast‐related genes in PDL cells with those in osteoblast‐like cells in the presence or absence of recombinant human bone morphogenetic protein (BMP)‐2. Histochemical analysis showed that Twist was expressed along alveolar bone surface in PDL. PDL cells constitutively expressed Twist gene and the expression level was higher than that in osteoblast‐like cells. In osteoblast‐like cell culture, BMP‐2 enhanced osteoblast‐related gene expression, while Twist expression was slightly decreased. In contrast, BMP‐2 increased runt‐related transcription factor (Runx)‐2, but failed to enhance alkaline phosphatase (ALP) and osteocalcin (OCN) gene expression in PDL cells. Interestingly, unlike in osteoblast‐like cells, Twist expression was upregulated by BMP‐2 in PDL cells. We transiently knocked down Twist gene in PDL cells using a short interference RNA expression vector (siTwist) and found that ALP, osteopontin (OPN), bone sialoprotein (BSP) genes expression and basal level of ALP activity were slightly increased, whereas Runx2 and OCN genes were not affected. Collectively, these results suggest that Twist may act as a negative regulator of osteoblastic differentiation in PDL cells. J. Cell. Biochem. 100: 303–314, 2007.

The effect of chemical and/or mechanical conditioning on the Er:YAG laser-treated root cementum: analysis of surface morphology and periodontal ligament fibroblast attachment.

This study compared the surface morphology as well as the biocompatibility of dental root cementum treated with Er:YAG laser irradiation alone and with the laser irradiation followed by chemical and/or mechanical conditioning.

Twist is required for establishment of the mouse coronal suture

Cranial sutures are the growth centres of the skull, enabling expansion of the skull to accommodate rapid growth of the brain. Haploinsufficiency of the human TWIST gene function causes the craniosynostosis syndrome, Saethre–Chotzen syndrome (SCS), in which premature fusion of the coronal suture is a characteristic feature. Previous studies have indicated that Twist is expressed in the coronal suture during development, and therefore that it may play an important role in development and maintenance of the suture. The Twist‐null mouse is lethal before the onset of osteogenesis, and the heterozygote exhibits coronal suture synostosis postnatally. In this study we investigated the function of Twist in the development of the mouse coronal suture, by inhibiting Twist synthesis using morpholino antisense oligonucleotides in calvarial organ culture. Decreased Twist production resulted in a narrow sutural space and fusion of bone domains within 48 h after the addition of the morpholino oligonucleotides. Proliferation activity in the sutural cells was decreased, and the expression of osteogenic marker genes such as Runx2 and Fgfr2 was up‐regulated in the developing bone domain within 4 h. These results suggest that during establishment of the suture area, Twist is required for the regulation of sutural cell proliferation and osteoblast differentiation.

Comparison of characteristics of periodontal ligament cells obtained from outgrowth and enzyme-digested culture methods

OBJECTIVEnPeriodontal ligament (PDL) cells have an important role in periodontal regeneration. The unique characteristics of PDL cells, mainly outgrown cells derived from PDL tissue, have been investigated. Recently, mesenchymal stem cells have been obtained from PDL tissue using enzyme digestion. The differences in properties of those PDL cells cultured by the two methods (outgrowth and enzyme digestion) are unclear. The objective of this study was to investigate the characteristics of PDL cells obtained by these methods.nnnMETHODSnPDL cells from extracted tooth were cultured using outgrowth and enzyme digest methods. Cell proliferation, colony-forming activity and differentiation capacity to osteoblast, adipocyte and chondrocyte were compared. Gene expressions for PDL cells, mesenchymal stem cells and fibroblasts were also investigated by reverse transcription polymerase chain reaction. Procollagen type I c-peptide (PIP) production was measured using an enzyme-linked immunosorbent assay (ELISA) kit.nnnRESULTSnPDL cells cultured by enzyme digest methods showed a higher proliferation rate, colony-forming activity and differentiation capacity into osteoblast, adipocyte and chondrocyte than those in PDL cells by outgrowth method. CD166, one of the mesenchymal stem cell markers, was slightly higher in enzyme-digested PDL than in outgrowth PDL, whilst gene expressions for type 1 collagen alpha 1 and type 3 collagen were higher in outgrown PDL cells. Moreover, outgrowth PDL exhibited higher PIP production than enzyme-digested PDL cells.nnnCONCLUSIONnPDL cells obtained by outgrowth and enzyme digestion showed different characteristics. The enzyme digestion method yielded cells with higher proliferation rate and mesenchymal stem cell-like properties, whereas cells with fibroblast-like properties were collected in the outgrowth method. PDL cell properties by different culture methods may provide information for inventing new therapeutic uses of PDL cells.

Protein kinase-A-dependent osteoprotegerin production on interleukin-1 stimulation in human gingival fibroblasts is distinct from periodontal ligament fibroblasts

Periodontitis, a chronic inflammatory disease, is characterized by increased expression of interleukin (IL)‐1 and other inflammatory mediators resulting in extensive osteoclast formation and bone loss. Expression of receptor activator of nuclear factor kappa B ligand (RANKL) and its decoy receptor, osteoprotegerin (OPG), by osteoblasts is important to regulate osteoclast differentiation. The aim of the present study was to investigate the regulatory effects of IL‐1 on RANKL and OPG production by mesenchymal fibroblasts in periodontal tissue. Human gingival fibroblasts (HGF) and periodontal ligament fibroblasts (PDL) were stimulated with IL‐1α with or without protein synthesis inhibitor cycloheximide (CHX), protein kinase A (PKA) inhibitors, protein kinase C (PKC) inhibitors and prostaglandin E2 (PGE2) inhibitor. In some experiments, the cultured cells were directly stimulated with either PKA or PKC activators. In HGF, IL‐1α‐stimulated OPG mRNA expression was high and could be reduced by CHX. PKA inhibitor completely abrogated IL‐1α‐induced OPG mRNA expression and OPG production. Endogenous PGE2 further enhanced IL‐1α‐induced OPG production in HGF. In PDL, RANKL mRNA expression was greatly augmented by IL‐1α. IL‐1α induced OPG mRNA expression and protein production. PKC inhibitor partially reduced IL‐1α‐induced OPG production and PKC activator enhanced OPG production in PDL. The IL‐1α‐stimulated OPG mRNA expression in HGF was greater than PDL. These results provide new evidence for the possible osteoclastogenesis‐inhibitory function of HGF through PKA activity pathway. PDL utilized PKC for OPG production. Thus, we emphasize that HGF and PDL have different characteristics of host defence mechanism against inflammatory process.