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Platelet-rich fibrin (PRF) is a fibrin matrix enriched with platelets. The PRF matrix is thought to form a steep gradient of platelet density around the region corresponding to the buffy coat in anticoagulated blood samples. However, this phenomenon has not yet been proven. To visualize platelet distribution in PRF in a non-invasive manner, we utilized near-infrared (NIR) imaging technology. In this study, four types of PRF matrices, bio-PRF, advanced-PRF (A-PRF), leukocyte-rich PRF (L-PRF), and concentrated growth factors (CGF) were compared. Blood samples collected from healthy, non-smoking volunteers were immediately centrifuged using four different protocols in glass tubes. The fixed PRF matrices were sagittally divided into two equal parts, and subjected to modified immunohistochemical examination. After probing with NIR dye-conjugated secondary antibody, the CD41+ platelets were visualized using an NIR imager. In L-PRF and CGF, platelets were distributed mainly on and below the distal surface, while in bio-PRF and A-PRF, platelet distribution was widespread and homogenous. Among three regions of the PRF matrices (upper, middle, and lower), no significant differences were observed. These findings suggest that platelets aggregate on polymerizing fibrin fibers and float up as a PRF matrix into the plasma fraction, amending the current &ldquo, gradient&rdquo, theory of platelet distribution.

We have previously demonstrated that multilayered periosteal sheets prepared from the explant culture of alveolar periosteum serve as a promising osteogenic grafting material in periodontal tissue regeneration. For the preparation of more potent periosteal sheets, we examined the applicability of stem-cell culture media. Compared to the control medium (Medium 199+10% FBS), periosteal sheets expanded with MesenPRO-RS™ medium exhibited these features: Cells grew three-dimensionally and deposited collagen in the extracellular spaces to form thicker multilayers of cells. Chondrocytic markers were not significantly upregulated. Contractile force was generated in proportion with the increased thickness of the periosteal sheets and the formation of cytoplasmic α-smooth muscle actin fibers. However, myofibroblastic markers were not significantly upregulated. The surface marker CD146 was substantially upregulated, while both CD73 and CD105 were downregulated. Alkaline phosphatase, a representative osteoblastic marker, was not upregulated by osteogenic induction. However, these expanded periosteal sheets exhibited substantially stronger osteogenic differentiation when implanted in nude mice. Therefore, despite our reservations, MesenPRO medium effectively expanded the cells contained in periosteal sheets to promote the formation of thicker multilayers of cells in vitro, and these enhanced periosteal sheets expressed increased osteogenic potential at implantation sites in vivo. In conjunction with data indicating that CD146-positive cells were notably expanded and the recently proposed concept that CD146 is a marker for osteogenic progenitor cells found in the bone marrow stroma, our findings suggest that MesenPRO medium improves the preparation of highly osteogenic periosteal sheets suitable for clinical application largely through the induction of CD146-positive cells.

Upon clinical application, thick platelet-rich fibrin (PRF) is usually compressed to fit the implantation site. However, it is speculated that the preservation of platelets and plasma content depends on the compression methods used. To accurately evaluate the clinical outcome of PRF, the preparation protocol should be standardized. Freshly prepared PRF clots were compressed into a thin membrane by our novel PRF compression device. The localization of platelets was examined by SEM and immunostaining. Growth factor levels were evaluated by bioassays and cytokine-antibody array techniques. The angiogenic activity was examined by the chick chorioallantoic membrane assay and the scratch assay using HUVEC cultures. Platelets were concentrated on the surface of the region adjacent to the red thrombus and this region was subjected to the experiments. Compared to the PRF membrane compressed by dry gauze (G-PRF), the preservation of the plasma content, 3D-fibrin meshwork, and platelets was more intact in the compressor-prepared PRF membrane (C-PRF). Among the growth factors tested, C-PRF contained PDGF isoforms at higher levels, and significantly stimulated cell proliferation and neovascularization. C-PRF may be useful for grafting while minimizing the loss of bioactive factors. This C-PRF preparation protocol is proposed as a standardized protocol for PRF membrane preparation.

In ongoing clinical research into the use of cultured autogenous periosteal cells (CAPCs) in alveolar bone regeneration, CAPCs were grafted into 33 sites (15 for alveolar ridge augmentation and 18 for maxillary sinus lift) in 25 cases. CAPCs were cultured for 6weeks, mixed with particulate autogenous bone and platelet-rich plasma, and then grafted into the sites. Clinical outcomes were determined from high-resolution three-dimensional computed tomography (3D-CT) images and histological findings. No serious adverse events were attributable to the use of grafted CAPCs. Bone regeneration was satisfactory even in cases of advanced atrophy of the alveolar process. Bone biopsy after bone grafting with CAPCs revealed prominent recruitment of osteoblasts and osteoclasts accompanied by angiogenesis around the regenerated bone. 3D-CT imaging suggested that remodeling of the grafted autogenous cortical bone particles was faster in bone grafting with CAPCs than in conventional bone grafting. The use of CAPCs offers cell-based bone regeneration therapy, affording complex bone regeneration across a wide area, and thus expanding the indications for dental implants. Also, it enables the content of particulate autogenous bone in the graft material to be reduced to as low as 40%, making the procedure less invasive, or enabling larger amounts of graft materials to be prepared. It may also be possible to dispense with the use of autogenous bone altogether in the future. The results suggest that CAPC grafting induces bone remodeling, thereby enhancing osseointegration and consequently reducing postoperative waiting time after dental implant placement.

We previously demonstrated that human periosteal sheets prepared on culture dishes function as an osteogenic "graft material" applicable to periodontal regenerative therapy. However, a lower level of initial adhesion of the excised periosteum tissue segments to culture dishes was a critical point that compromised the successful preparation of functional periosteal sheets. To improve on this weakness, we developed a transparent, biodegradable poly(L-lactide-co-ɛ-caprolactone) (LCL) film and tested its function as a scaffold and carrier of periosteal sheets.Human periosteum tissue segments excised from alveolar bone of healthy donors were cultured on type I atelocollagen-coated LCL films. Initial adhesion was examined by simple agitation. Cell outgrowth and in vitro mineralization were cytohistochemically examined. Osteogenic activity was histochemically examined in an animal implantation model using nude mice.Surface collagen-coating modified the hydrophobic nature of LCL and substantially improved the initial adhesion. Compared to cultures in plastic dishes, the growth rate was delayed in non-coated films, but not in collagen-coated films. In the trimming process for animal implantation, periosteal sheets were frequently detached from non-coated films, but not from collagen-coated films. Regardless of collagen-coating, LCL films did not cause any significant infiltration of inflammatory cells, or negatively impact mineralized tissue formation.Collagen-coating improved the initial adhesion of periosteum segments, which facilitated cell outgrowth and also handling efficiency on implantation. Therefore, we believe that once evaluated in human studies, our collagen-coated LCL film will contribute to improving the periodontal regenerative methodology with the application of cultured autologous periosteal sheets.

The aim of the present controlled clinical study was to compare the clinical response of human cultured periosteum (HCP) sheets in combination with platelet-rich plasma (PRP) and porous hydroxyapatite (HA) granules to a mixture of PRP and HA in the treatment of human infrabony periodontal defects.Thirty interproximal infrabony osseous defects in 30 healthy, non-smoking subjects diagnosed with chronic periodontitis were included in this study. The subjects were randomly assigned to the test group (HCP sheets combined with PRP and HA) or the control group (PRP with HA). Clinical and radiographic measurements were made at baseline and the 12-month post-surgical evaluation.Compared to baseline, the 12-month results indicated that both treatment modalities resulted in statistically significant changes (P0.01) in the gingival index, bleeding on probing, probing depth, clinical attachment level, and radiographic infrabony defect depth. Compared to the control group, the test group exhibited a statistically significantly more favorable change in clinical attachment gain (3.9 +/- 1.6 mm versus 2.7 +/- 1.3 mm; P0.05), vertical relative attachment gain (83.5% +/- 31.7% versus 55.0% +/- 21.9%; P0.05), and radiographic infrabony defect fill (4.9 +/- 1.2 mm versus 3.2 +/- 1.1 mm; P0.01).Compared to PRP with HA, treatment with a combination of HCP sheets, PRP, and HA led to a significantly more favorable clinical improvement in infrabony periodontal defects. A factor likely contributing to these favorable clinical results is the presence of osteogenic cells in the HCP sheets, which provided greater regeneration potential.

Periodontitis, similar to many other known inflammatory diseases, is thought to increase adenosine triphosphate (ATP) levels in extracellular spaces. Extracellular ATP acts on specific receptors to modulate the proliferation of various cell types. Platelet-rich plasma (PRP) contains high levels of ectonucleotidase activity capable of degrading ATP. The aim of this study was to investigate the effects of ATP on the proliferation of human periodontal ligament (PDL) cells and how these effects are altered by ectonucleotidases.PDL cells were derived from healthy young volunteers. ATP content and DNA synthesis were quantified by a bioluminescence and an enzyme-linked immunosorbent assay, respectively. CD39 and p21(WAF1/cip1) expression was analyzed by Western blot. Apoptosis was evaluated by caspase-3/7 activity, terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine triphosphate nick end-labeling (TUNEL) activity, annexin-V-binding, and DNA fragmentation.CD39 and ectonucleotidase-like activity were found in PDL cells and serum, respectively. Because less CD39 is expressed in freshly plated cells, both exogenous ATP and ATPgammaS, a slowly hydrolysable analog, inhibited cell proliferation under low serum conditions. ATP upregulated p21(WAF1/cip1), an inhibitor of cell-cycle progression, whereas ATPgammaS induced caspase-dependent apoptosis. Either upregulation of CD39 or added serum rescued cells from the cytostatic actions of exogenous ATP.In PDL cells expressing low CD39 levels, both ATP and ATPgammaS inhibited proliferation but by different mechanisms. ATP-induced growth arrest suggests that periodontal tissue regeneration is often suppressed at the site of injury. Furthermore, added ectonucleotidases protected PDL cells from ATP's cytostatic actions, suggesting that ectonucleotidase-rich PRP augments the regenerative actions of its constituent growth factors by protecting against exogenous ATP at clinical sites.

The aim of the present controlled clinical study was to compare platelet-rich plasma (PRP) combined with a biodegradable ceramic, porous hydroxyapatite (HA) with a mixture of HA and saline in the treatment of human intrabony defects.Seventy interproximal intrabony osseous defects in 70 healthy, non-smoking subjects diagnosed with chronic periodontitis were included in this study. Thirty-five subjects each were randomly assigned to either the test group (PRP and HA) or control group (HA with saline). Clinical and radiographic measurements were determined at baseline and the 12-month evaluation.When compared to baseline, the 12-month results indicated that, while both treatment modalities resulted in significant changes in all clinical parameters (gingival index, bleeding on probing, probing depth, clinical attachment level, and intrabony defect fill; P0.001), the test group exhibited statistically significant changes compared to the control sites in probing depth reduction: 4.7 +/- 1.6 mm versus 3.7 +/- 2.0 mm (P0.05); clinical attachment gain: 3.4 +/- 1.7 mm versus 2.0 +/- 1.2 mm (P0.001); and vertical relative attachment gain: 70.3% +/- 23.4% versus 45.5% +/- 29.4% (P0.001).Treatment with a combination of PRP and HA compared to HA with saline led to a significantly more favorable clinical improvement in intrabony periodontal defects.

Objectives: The purpose of the present study was to determine the effect of smoking cessation on gingival blood flow (GBF) and gingival crevicular fluid (GCF). Material and Methods: Sixteen male smokers (aged 22–39 (25.3±4.0) years), with no clinical signs of periodontal and systemic diseases, were recruited. The experiment was performed before (baseline) and at 1, 3 and 5 days, and at 1, 2, 4 and 8 weeks after smoking cessation. The status of smoking and smoking cessation was verified by exhaled carbon monoxide (CO) concentration, and by serum nicotine and cotinine concentrations. A laser Doppler flowmeter was used to record relative blood flow continuously, on three gingival sites of the left maxillary central incisor (mid-labial aspect of the gingival margin and bilateral interdental papillae). The GCF was collected at the mesio- and disto-labial aspects of the left maxillary central incisor and the volume was calculated by the Periotron 6000® system. The same measurements except for the GBF were performed on 11 non-smoking controls (four females and seven males), aged 23–27 (24.4±1.2) years. Results: Eleven of 16 smokers successfully completed smoking cessation for 8 weeks. At 1 day after smoking cessation, there was a significantly lower CO concentration than at baseline (p

Platelet-rich plasma (PRP) contains several growth factors, including platelet-derived growth factor (PDGF) and transforming growth factor-beta (TGF-beta), at high levels. We have demonstrated the PRP functions like TGF-beta to modulate cell proliferation in a cell-type specific manner. In addition, PRP forms gel-like materials in several, but not all, cell cultures tested. This study was designed to investigate PRP's action on extracellular matrix production in periodontal ligament (PDL) and osteoblastic MG63 cell cultures.PRP was prepared from the plasma obtained from autologous blood of healthy volunteers and stored at -20 degrees C until used. Cells treated with PRP (0.5% to 2%) were immunocytochemically stained for type I collagen and fibrin and the viscosity of the culture media was visually evaluated. Fibrinogen in PRP was detected by immunodot-blotting while endogenous thrombin expression in cells was detected by a modified enzyme-linked immunosorbent assay.Gel-like material rapidly (30 minutes) formed in cultures of either PDL or osteoblastic MG63 cell cultures after addition of PRP (or = 0.5%). PRP changed cell shape and up-regulated type I collagen at 24 hours. Fibrinogen was detected in the PRP preparations and insoluble fibrin networks were found in the newly formed gel-like material. PRP's action on collagen synthesis was mimicked by purified fibrinogen and blocked by thrombin inhibitor. Thrombin was expressed both in PDL and MG63 cells.These findings demonstrated that the gel-like material formed in cell cultures of either PDL or MG63 cells is fibrin clot that is capable of up-regulating collagen synthesis in the extracellular matrix. Our data suggest the possibility that fibrinogen, converted to fibrin, in combination with growth factors present in PRP might effectively promote wound healing at sites of injury in periodontal tissue.

Platelet-rich fibrin (PRF) was developed as an advanced form of platelet-rich plasma to eliminate xenofactors, such as bovine thrombin, and it is mainly used as a source of growth factor for tissue regeneration. Furthermore, although a minor application, PRF in a compressed membrane-like form has also been used as a substitute for commercially available barrier membranes in guided-tissue regeneration (GTR) treatment. However, the PRF membrane is resorbed within 2 weeks or less at implantation sites; therefore, it can barely maintain sufficient space for bone regeneration. In this study, we developed and optimized a heat-compression technique and tested the feasibility of the resulting PRF membrane. Freshly prepared human PRF was first compressed with dry gauze and subsequently with a hot iron. Biodegradability was microscopically examined in vitro by treatment with plasmin at 37°C or in vivo by subcutaneous implantation in nude mice. Compared with the control gauze-compressed PRF, the heat-compressed PRF appeared plasmin-resistant and remained stable for longer than 10 days in vitro. Additionally, in animal implantation studies, the heat-compressed PRF was observed at least for 3 weeks postimplantation in vivo whereas the control PRF was completely resorbed within 2 weeks. Therefore, these findings suggest that the heat-compression technique reduces the rate of biodegradation of the PRF membrane without sacrificing its biocompatibility and that the heat-compressed PRF membrane easily could be prepared at chair-side and applied as a barrier membrane in the GTR treatment.

One-year data after autologous grafting of infrabony periodontal defects with human cultured periosteum sheets in combination with platelet-rich plasma and hydroxyapatite granules have shown favorable clinical and radiographic results. A 5-year follow-up evaluation of 22 selected patients indicated that treated infrabony defects remained stable. Radiographically, there was an increase in osseous radiopacity and bone trabeculation suggesting further bone maturation. This novel tissue-engineered periodontal treatment approach has resulted in significant clinical improvement, and defects remained stable after 5 years.

We previously published an investigation indicating freeze-dried platelet-rich plasma (PRP)-coated polyglactin mesh was a promising wound-dressing material. However, one of its disadvantages was the inflammatory nature due to degradation of the polyglactin. Therefore, in this study, we investigated the use of a collagen sponge as the carrier for PRP. When implanted subcutaneously in nude mice, the PRP-coated sponge alone rapidly induced angiogenesis and infiltration of surrounding connective tissue without inducing appreciable inflammation. Moreover, addition of periosteal fibroblastic cells substantially augmented the angiogenic response. With in vitro studies, the PRP-coated sponge provided various major growth factors at high levels to stimulate the proliferation of cells cultured on plastic dishes, but did not stimulate the proliferation of cells inoculated into the PRP-coated sponge. Cells were embedded in the fibrin mesh and maintained their spherical shape without stretching. The atomic force microscopic analysis demonstrated that the fibrin gel formed on the PRP-coated sponge was much softer (approx. 22 kPa) than the cross-linked collagen that formed the sponge base (appox. 1.9 MPa). Because insoluble matrices have recently and increasingly been considered important regulatory factors of cellular behavior, as are soluble growth factors, it is suggested that this soft fibrin mesh possibly suppresses cell survival. Overall, our investigation has successfully demonstrated improved wound-healing and regenerative potential of the PRP-coated mesh by combining it with the collagen sponge. In the clinical setting, this PRP-coated collagen sponge is a promising material for connective tissue regenerative therapy, such as periodontal therapy, burn victim treatment and in cosmetic or plastic surgery.

Cultured human periosteal sheets constitute a promising grafting material for periodontal tissue regenerative therapy. However, preparation of these sheets usually requires six weeks or longer, and this lengthy commitment and delay limits both clinical applicability and availability. The aim of this study is to develop an efficient, practical, cost-effective cryopreservation method for periosteal tissue segments (PTSs). Human PTSs were aseptically excised from alveolar bone and pre-cultured in Medium 199 + 10% fetal bovine serum (FBS) for the indicated number of days before they were slowly frozen down to −75 °C in a commercial freezing vessel using medium containing 10% dimethyl sulfoxide (Me 2 SO) and various concentrations of FBS. After fast-thawing at 37 °C, PTSs were again cultured, and their growth and responses to standard osteogenic induction were evaluated (vs. freshly excised PTSs). Proliferating cells were obtained at the highest levels from cryopreserved PTSs that were pre-cultured for 14 days before freezing. When a concentration of 50% or more FBS was included in the cryopreservation solution, cells migrated out more actively and grew faster. Importantly, osteoinduction up-regulated alkaline phosphatase (ALP) activity and osteoblastic marker mRNAs in cryopreserved PTS-derived sheets just as effectively as it did in native PTS-derived ones. These data suggest that pre-conditioned PTSs can be efficiently cryopreserved in a freezing solution containing high FBS by traditional manual cryopreservation methods without aid of a program freezer or more elaborate equipment.

A superporous (85%) hydroxyapatite (HA) block was recently developed to improve osteoconductivity, but it was often not clinically successful when used to treat periodontal osseous defects. The primary purpose of this study is to develop a clinically applicable tissue-engineered bone substitute using this HA block and human alveolar periosteum-derived cells.Commercially available superporous HA blocks were acid treated and subjected to a three-dimensional (3D) culture for periosteal cell cultivation. Cells in the pore regions of the treated HA block were observed on the fracture surface by scanning electron microscopy. After osteogenic induction, the cell-HA complexes were implanted subcutaneously in nude mice. Osteoid formation was histologically evaluated.Acid treatment enlarged the interconnections among pores, resulting in the deep penetration of periosteal cells. Under these conditions, cells were maintained for2 weeks without appreciable cell death in the deep pore regions of the HA block. The cell-HA complexes that received in vitro osteogenic induction formed osteoids in pore regions of the treated HA blocks in vivo. In contrast, most pore regions in the non-pretreated, cell-free HA blocks that were evaluated in vivo remained cell free.Our findings suggest that an acid-treated HA block could function as a better scaffold for the 3D high-density culture of human periosteal cells in vitro, and this cell-HA complex had significant osteogenic potential at the site of implantation in vivo. Compared with the cell-free HA block, our cell-HA complex using periosteal cells, which are the most accessible for clinical periodontists, showed promising results as a bone substitute in periodontal regenerative therapy.

The purpose of this study was to determine the microbiological efficacy of an adjunctive minocycline periodontal formulation delivered subgingivally. Subjects were systemically healthy but exhibited severe periodontitis; i.e., probing depths greater than 6 mm. The two study groups included individuals who received minocycline or a placebo periodontal formulation after root planing. Subgingival plaque samples were obtained at baseline; prior to treatment; and at 1, 3, and 6 months. Plaque was evaluated by darkfield microscopy and further analyzed for total dark-pigmented Bacteroides species, P. intermedia, P. gingivalis and Streptococcus, Actinomyces, Eikenella, Actinobacillus, Capnocytophaga, and Fusobacterium species using cultivable flora techniques. In addition, plaque was evaluated for yeast on a selective agar medium. When compared to the placebo, the minocycline group had significantly lower proportions of spirochetes at 1 and 3 months and lower proportions of motile rods at 3 months. Furthermore, when compared to the placebo group, the minocycline patients had lower mean proportions of dark-pigmented Bacteroides spp. and P. intermedia at 1 and 3 months as well as lower proportions of E. corrodens at 1 month. The minocycline group had significant decreases in proportions of spirochetes at 1 and 3 months, motile rods at 1 and 3 months, and increases in cocci at 1, 3, and 6 months when compared to baseline. In the placebo group, root planing was also effective at decreasing spirochetes at 1, 3, and 6 months, but with significant differences seen only at 3 and 6 months. However, the degree of reduction in spirochete proportions was greater in the minocycline group when compared with the placebo group.(ABSTRACT TRUNCATED AT 250 WORDS)

Our animal implantation studies have demonstrated that, after osteogenic processing, cultured human periosteal sheets form osteoid tissue ectopically without the aid of conventional scaffolding materials. To improve the osteogenic activity of these periosteal sheets, we have tested the effects of including a scaffold made of salmon collagen-coated ePTFE mesh. Periosteal sheets were produced with minimal manipulation without enzymatic digestion. Outgrown cells penetrated into the coated mesh fiber networks to form complex multicellular layers and increased expression of alkaline phosphatase activity in response to the osteoinduction. In vitro mineralization was notably enhanced in the original tissue segment regions, but numerous micro-mineral deposits were also formed on the coated-fiber networks. When implanted subcutaneously into nude mice, periosteal sheets efficiently form osteoid around the mineral deposits. These findings suggest that the intricate three-dimensional mesh composed of collagen-coated fibers substantially augmented the osteogenic activity of human periosteal sheets both in vitro and in vivo.

Our recent clinical studies have demonstrated that autologous implantation of human cultured periosteal (hCP) sheets in combination with porous hydroxylapatite (HAp) particles at the site of periodontal bone defects strikingly facilitates tissue regeneration. To better understand how the hCP sheet functions at the implantation site, we have now examined its biochemical and morphological characteristics in vitro and its ectopic osteoinductivity in nude mice. Cultured human periosteal tissue segments produced periosteal cells that migrated out from the central region within 4-8 days and grew more rapidly with longer culture times. Alkaline phosphatase activity increased in parallel with actual osteoblastic induction. Cytokine array assays demonstrated that osteoblastic induction downregulated IL-6 and thrombopoietin, but upregulated IL-8, IL-13, IGF-I and IGFBP-2 in hCP sheets. When differentiated hCP sheets were implanted alone, areas of osteoid and mineralized tissue were formed within 2 weeks, but non-induced, immature hCP sheets did not produce much mineralization. These findings suggest that mature hCP sheets potentially function not only as seeds of ectopic bone formation without the need of synthetic tissue scaffolds, but also as living drug-delivery systems, to influence cells near implantation sites by producing several important cytokines. These two major characteristics indicate that a mature hCP sheet is a promising osteoinductive biomaterial, even without conventional scaffolds for periodontal regenerative therapy.

Cultured gingival dermal substitute (CGDS), composed of gingival fibroblasts and matrix and fabricated using tissue-engineering techniques, has been used for root coverage procedures. Fourteen sites from four patients withor = 2 mm of Miller Class I or II facial gingival tissue recession were treated. The autologous CGDS sheet, prepared prior to surgical treatment, was grafted over the teeth with gingival recession and then covered with a coronally positioned flap. Vertical and horizontal recession was measured at baseline (prior to the surgical procedure) and 13 to 40 weeks (average: 30.7 +/- 9.6 weeks) after surgery. The average vertical and horizontal root coverage after surgery was 79.1% +/- 25.7% and 75.2% +/- 31.4%, respectively. Moreover, there was a significant increase of keratinized and attached gingival tissue at the final clinical evaluation compared with preoperative measurements (P.05). These results demonstrate CGDS as a promising grafting material for use with root coverage procedures in periodontal therapy.

Background: In addition to its prominent role in liver regeneration, hepatocyte growth factor (HGF) is now generally thought to be produced by mesenchymal cells to promote the regeneration of epithelial tissue by a paracrine mechanism. However, it is not known how or if HGF could be involved in the regeneration of periodontal tissues. The purpose of this study was to characterize the ability of normal human periodontal ligament (PDL) cells to produce or respond to HGF. Methods: PDL cells derived from healthy young volunteers were used from passages four through 10. HGF receptors were detected both by immunocytochemical staining and Western-blotting analysis. Both DNA synthesis (by bromo-deoxyuridine [BrdU]-incorporation) and secreted HGF were quantified by enzyme-linked immunosorbent assays. Mitogen-activated protein kinase (MAPK) phosphorylation was also analyzed by Western blot. Results: Despite the immunocytochemical demonstration of HGF receptor protein in the cytoplasm and on the plasma membrane of PDL cells, exogenous recombinant human HGF did not exert the mitogenic effects expected. As reported for other mesenchymal cells, PDL cells were found to secrete HGF. Treatments with neutralizing anti-HGF antibody significantly suppressed constitutive PDL cell proliferation and sustained the receptor protein at higher levels than in non-treated cells. Under these conditions, exogenous HGF rapidly phosphorylated extracellular signal-regulated kinase (ERK), an action linked to the cell proliferation and downregulation of cell-surface receptors. Conclusions: Unlike other known mesenchymal or epithelial cells, these findings suggest that normal PDL cells from young donors possess a constitutive HGF/receptor autocrine loop that normally regulates their replacement self-proliferation but reduces sensitivity to exogenously applied HGF by acute receptor downregulation.

It has been suggested that human cultured gingival epithelial sheets may serve as a possible grafting material. The purpose of this study was to examine the biological characteristics of human cultured gingival epithelial sheets by epithelial differentiation and proliferation markers. Immunohistochemical localization of cytokeratin 19, involucrin and proliferating cell nuclear antigen (PCNA) were examined in human cultured gingival epithelial sheets samples from twenty patients. Cytokeratin 19-immunopositive cells were scattered mainly in the suprabasal layer. Immunoreactivity for involucrin was observed in all layers except for the basal layer. The majority of proliferating cell nuclear antigen-immunopositive cells was found in the basal layer. These results suggested that the cultured human gingival epithelial sheets were biologically active and in proliferative condition, which implies that this biological product may be a potential grafting material.

Recent clinical studies have investigated postural sway characteristics in anterior cruciate ligament (ACL)-deficient knees, but the relative contributions of vision and ACL remain unclear. In the current study, we measured and compared postural sway during one-leg standing with eyes open and closed to assess the difference between legs with and without ACL injury, and we discuss the contribution of the ligament relative to vision and to postural sway in patients. We examined 32 patients (17 males, 15 females) with ACL injury before surgery from March 2001 through January 2004. None presented obvious dysfunction in the lower limbs or central nervous system. Using a gravicorder, we measured locus length per time (LG) and environmental area (AR) as the factors of postural sway during two-leg and one-leg standing with eyes open or closed. In the ACL-injured knee, the amount of postural sway increased significantly during injured leg standing with eyes closed (LG, P < 0.0001; AR, P < 0.0001), but it did not increase significantly with eyes open. There were no significant differences with respect to sex or general joint laxity. There was no correlation between postural sway and the anterior translation of the tibia measured by arthrometer KT2000 or between the muscle strength around the knee. We concluded that the amount of postural sway in the ACL-injured knee increased significantly on injured leg standing with eyes closed, and that vision appears to be dominant in compensating for the decreased contribution of the injured ACL.

Radiographic and computed tomography features of unilateral intra-articular calcaneal fractures after open reduction and internal fixation were compared with late functional outcomes to identify prognostic factors. Sixty-one of 67 patients treated between 1997 and 2002 could walk painlessly postoperatively (average, 19.7 months). Functional results (Laasonen's criteria) were excellent or good in 92.5%. Factors associated with better functional results were lower age (p = .0227), greater Bohler angle at the time of injury (p < .0001), lower Sanders' grade (p = .0497), increased height of fractured regions (p = .0249), better reduction of the posterior facet (p = .0126), and better reduction of the calcaneocuboid joint (p = .0023). Only the latter two were found to be surgical prognostic factors.

We evaluated the ability of spinally administered nitric oxide (NO) synthase inhibitor to modulate antinociceptive action of intrathecal (i.t.) morphine in rats by measuring the early and late phases of flinching and licking/biting in the formalin test. To determine the contribution of spinal NO and glutamate, we measured the release of NO metabolites (nitrite/nitrate) and glutamate from the spinal cord in rats, using a microdialysis probe placed in the lumbar space. The i.t. administration of NG-nitro l -arginine methyl ester ( l -NAME) produced a dose-dependent reduction in the number of flinches during the late phase, whereas there were no significant alterations in the late phase licking/biting, and early phase flinching and licking/biting. Spinal administration of morphine at low doses produced a significant antinociceptive activity in the early and late phases of the flinching behaviour, whereas higher doses of morphine were required to obtain a significant effect in the licking/biting behaviour during both phases. Combination of l -NAME with morphine resulted in an enhanced reduction in the early and late phase flinching. Enhanced antinociceptive activity was observed in the late phase licking/biting by i.t. combined administration of l -NAME (400 nmol) and morphine (1.25 nmol). In the present study, we have confirmed our prior results that injection of formalin (5.0%) into the plantar surface of the paw evoked a biphasic spinal release of nitrite/nitrate and a transient release of glutamate. Formalin-evoked release of nitrite/nitrate and glutamate was also reduced markedly by i.t. combined administration of l -NAME and morphine. These behavioural and biochemical results suggest that i.t. administered l -NAME may enhance morphine-induced antinociception through an increased inhibition of nitrite/nitrate and glutamate releases evoked by formalin injection at the spinal cord level.

849 Background: Platelet-rich plasma (PRP) is a fraction of plasma, in which platelet-derived growth factor (PDGF) and transforming growth factor-β (TGF-β) are thought to be concentrated. It is plausible that topically-applied PRP up-regulates cellular activity and subsequently promotes periodontal regeneration in vivo. However, the concentrations of these growth factors in PRP have not been specifically determined and the biological effects of PRP at the cellular and molecular levels have not been determined. Methods: PRP obtained from 20 healthy subjects was prepared from plasma by centrifugation. These PRP preparations were immediately subjected to an evaluation for PDGF-AB and TGF-β1 using enzyme-linked immunosorbent assay (ELISA) kits. The biological effects of the PRP preparations were evaluated on osteoblastic, epithelial, fibroblastic, and periodontal ligament cells. Cellular mitogenic activity was evaluated by counting cell numbers or evaluating 5-bromodeoxyuridine (BrdU) incorporation. Expression of alkaline phosphatase (ALP) was immunocytochemically evaluated. Results: In the PRP preparations, platelets were concentrated up to 70.9 × 10 4 cells/µl (283.4% of the unconcentrated plasma). The levels of PDGFAB and TGF-β1 were also concentrated up to 182.0 ng/ml (440.6%) and 140.9 ng/ml (346.6%), respectively. Scatter plots revealed significant correlations between platelet counts and levels of these growth factors. PRP stimulated osteoblastic DNA synthesis and cell division (138% of control), with simultaneous down-regulation of ALP, but suppressed epithelial cell division (80% of control). PRP also stimulated DNA synthesis in gingival fibroblasts and periodontal ligament cells. Conclusions: These data demonstrated that both PDGF-AB and TGF-β1 were highly concentrated in the PRP preparations. It is suggested PRP modulates cell proliferation in a cell type-specific manner similar to what has been observed with TGF-β1. Since synchronized behavior of related cell types is thought to be required for successful periodontal regeneration, it is further suggested these cell type-specific actions may be beneficial for periodontal regenerative therapy. J Periodontol 2003;74:849-857.

The aim of the present study was to analyze the levels of osteocalcin, deoxypyridinoline (Dpd) and interleukin-1beta as markers of bone metabolism in peri-implant crevicular fluid (PICF) from peri-implantitis patients. PICF was sampled from a total of 34 endosseous titanium implants from 16 patients; nine females (mean age 52.8, range 40-62 years) and seven males (mean age 56.0, range 36-66 years). The implants had been in place for a period of 9-112 months (mean; 35.8 months) since the loading. These sites were categorized as six peri-implantitis, eight peri-implant mucositis and 20 healthy implant. PICF volume from peri-implantitis sites was significantly higher than mucositis and healthy implant sites (P0.01). Osteocalcin levels in PICF from mucositis sites were significantly higher than healthy implants (P0.05), whereas peri-implantitis sites were not significantly different from either mucositis or healthy implant sites. Dpd could not be detected in any of the samples examined. IL-1beta levels in PICF from peri-implantitis sites were significantly higher than levels from peri-implant mucositis (P0.05) and healthy implant sites (P0.01). In conclusion, osteocalcin in PICF may reflect increased local bone turnover around implants. Further, IL-1beta should be a useful marker for peri-implant inflammation.

Previous research has demonstrated that intrathecal i.t. morphine in a dose of 60.0 nmol into the spinal subarachnoid space of mice can evoke nociceptive behavioral responses consisting of a severe hindlimb scratching directed toward the flank followed by biting/licking of the hindpaw. The present study was undertaken to examine the involvement of spinal N-methyl-D-aspartate (NMDA) and opioid receptors on the behavioral responses evoked by high-dose i.t. morphine. Pretreatment with naloxone, an opioid receptor antagonist (1.0 and 4.0 mg/kg, s.c.), failed to reverse the morphine-evoked behavioral response, suggesting that the morphine effect is not mediated through the opioid receptors in the spinal cord. The morphine-induced behavior was dose-dependently inhibited by i.t. co-administration of the competitive NMDA receptor antagonists, D(-)-2-amino-5-phosphonovaleric acid (D-APV) (6.25-50.0 pmol) and 3-((+)-2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid (CPP) (3.125-25.0 pmol). The characteristic behavior was also reduced by co-administration of (5R,10S)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cycloheptene-5,10-imine maleate (MK-801) (74.1-250 pmol), an NMDA ion-channel blocker. Ifenprodil, a competitive antagonist of the polyamine recognition site of NMDA receptor ion channel complex, produced a dose-related inhibitory effect on the behavioral response to i.t. morphine with less potency than the competitive and non-competitive antagonists examined. High doses of (+)-HA-966, a glycine/NMDA antagonist, induced a dose-dependent inhibition of morphine-induced response. The effective dose of i.t. 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), a non-NMDA receptor antagonist, needed to reduce the morphine-induced response, was approximately 10-fold greater than that of D-APV. These results suggest that spinal NMDA receptors, but not non-NMDA receptors, may be largely involved in elicitation of the behavioral episode following i.t. injection of morphine in mice.

It has been demonstrated that human cultured epithelial sheets prepared by tissue engineering techniques provide useful graft material for wound healing and tissue regeneration. However, limited information is available with regard to biological effects such as release of growth factors from human cultured gingival epithelial sheets (HCGES). The purpose of this study was to measure the levels of growth factors released from HCGES into culture medium Methods: Twenty patients aged 44 to 71 years with generalized chronic periodontitis were recruited, and their gingival tissues obtained during periodontal flap surgery. The levels of vascular endothelial growth factor (VEGF), transforming growth factor-α and -βl (TGF-α and -βl), and epidermal growth factor (EGF) released into the culture medium were determined using enzyme-linked immunosorbent assay at the just confluent (Tl) and the adequate stratification (T2) culture stages. The medium without cells was collected as a control (TO) statistical tests were performed by analysis of variance and sheffé multiple range test among TO, T1, and T2.Significantly higher levels of VEGF and TGF-α were observed at T1 and T2 compared to To (P0.001). In addition, there was a significant difference in the TGF-α levels between T2 and T1 (P0.001). TGF-βl at T1 was significantly higher in comparison to T0 (P0.0l). EGF had been released only in a small amount at T2.This study indicates that meaningful amounts of VEGF and TGF-α and -βl are released from HCGES, which suggests potential for promoting wound healing and tissue regeneration after grafting. J Periodontol 2002;73:748-753.

There is limited information available from clinical trials regarding the performance of enamel matrix derivative (EMD) in the treatment of periodontal intrabony defects. This randomized, double-blind, placebo-controlled, split-mouth study was designed to compare the clinical and radiographical effects of EMD treatment to that of placebo-controlled treatment for intrabony defects.Sixteen patients were included, each of whom had 1 or 2 pairs of intrabony defects located contralaterally in the same arch. Thirty-six intrabony defects were randomly assigned treatment with flap surgery plus EMD or flap surgery plus placebo. At baseline and at the 12-month follow-up evaluation visit, clinical and radiographic measurements were determined. Data were statistically analyzed using the Wilcoxon-signed rank test (alpha = 0.05).At the 12-month visit, bleeding on probing for the EMD group was 0.11 +/- 0.32 compared to the placebo group, 0.61 +/- 0.50 (P0.05). Probing depth reduction was greater in the EMD group (3.00 +/- 0.97 mm) compared to the placebo group (2.22 +/- 0.81 mm) (P0.05). Mean values for clinical attachment gain in the EMD and the placebo groups were 1.72 +/- 1.07 mm and 0.83 +/- 0.86 mm, respectively (P0.05). Vertical relative attachment gain was 38.5 +/- 22.6% in the EMD group and 21.4 +/- 25.2% in the placebo group (P0.05). Radiographic bone density gain was greater in the EMD (20.2 +/- 16.6%) compared to the placebo group (-3.94 +/- 23.3%) (P0.01).Treatment with flap surgery and EMD, compared to flap surgery with placebo, produced a significantly more favorable clinical improvement in intrabony periodontal defects.

The effect of topically applied transforming growth factor beta1 (TGF-beta1) on the rat gingival wound healing process after flap surgery was evaluated by immunohistochemistry for extracellular matrix molecules (ECM), such as tenascin, heparan sulfate proteoglycan (HSPG) and type IV collagen, and for proliferating cell nuclear antigen (PCNA) in fibroblasts. TGF-beta1 solution was applied to the surgical wound experimental sites. Two microg/microl were applied at the time of the operation, and 1 microg/microl at days 1 and 2 after surgery, with contralateral control sites receiving the vehicle alone. Periodontal tissues were histologically examined at 3 and 7 days post-surgery. Tenascin was found to be more strongly stained in the granulation tissue from experimental sites at 3 days post-surgery. At 7 days postsurgery, HSPG-positive areas in granulation tissue had become smaller and there was a prominent proliferation of PCNA-positive fibroblast-like cells and type IV collagen-positive blood vessels. These results suggest that TGF-beta1 applied to surgical wounds influences early proliferation of gingival fibroblast-like cells, the formation of blood vessels, and ECM remodelling. In conclusion, TGF-beta1 application appears to promote granulation tissue formation in periodontal wound healing.

OBJECTIVE To examine the role of central mechanisms on the production and release of an ouabain-like factor, the effects of intracerebroventricular injections of 6-hydroxydopamine on the tissue content and on the plasma level of the ouabain-like factor were determined in rats. METHODS The vehicle (0.1% ascorbic acid in 0.9% saline) and 6- hydroxydopamine (250 micrograms/rat) were injected into the left lateral ventricle in ether-anaesthetized Wistar rats. Hypothalamus, pituitary, adrenal and venous blood was sampled 24h and 7 days later. The procedure was repeated using another rat group 7 days later. Characteristics of immunoreactive ouabain-like factor were determined by a combination of high-performance liquid chromatography and a highly sensitive enzyme-linked immunosorbent assay for ouabain. The level of the ouabain-like factor in these tissues and in plasma extracts measured by the enzyme-linked immunosorbent assay was compared between the two groups receiving 6-hydroxydopamine and the vehicle. RESULTS Twenty-four hours after the intracerebroventricular injections of 6-hydroxydopamine, the ouabain-like factor level in the pituitary, hypothalamus and plasma had decreased significantly, whereas the ouabain-like factor level in the adrenal had not changed. The content of noradrenaline in the hypothalamus was also decreased markedly 7 days later and the content of ouabain-like factor in the pituitary remained low. On liquid chromatography the elution pattern of the ouabain-like factor in plasma and in tissue extracts coincided with that of authentic ouabain. CONCLUSIONS Intracerebroventricular treatments with 6-hydroxydopamine elicited decreases in ouabain-like factor contents in the pituitary, the hypothalamus and the plasma. These results suggest that the production and release of ouabain-like factor are closely associated with the brain, particularly the hypothalamus-pituitary axis, and that noradrenergic or dopaminergic neurons, or both, play a key role in this mechanism.

Microflora from root canals and periodontal pockets of periodontally affected teeth were compared in order to elucidate the as yet unknown relationship between pulpal and periodontal disease. Caries-free teeth affected with advanced periodontitis and diagnosed as clinically dead by electric pulp testing were selected. The root canals and periodontal pockets were sampled, and the bacterial flora examined by both culture and interference microscopy. The results indicated that the aerobe/anaerobe ratio in the periodontal pocket was 0.23, while it was 0.0022 in the root canal, the large predominance of obligate anaerobes reflecting the anaerobic environment found in the root canal. Morphological classification obtained from interference microscopy showed similar proportions of morphotypes in the two sites. Results of anaerobic culture demonstrated a significantly higher rate of detection of facultative Streptococcus bacteria in the periodontal pocket than in the root canal. The predominant bacterial species common to both regions were Streptococcus, Peptostreptococcus, Eubacterium, Bacteroides, and Fusobacterium for obligate anaerobes. As for facultative anaerobes, Actinomyces and Streptococcus were detected predominantly in the periodontal pocket. The occurrence of micro-organisms common to both sites in this study suggests that the periodontal pocket may be a possible source of root canal infections.

4～8mmの歯周ポケットを有する9名め成人性歯周炎罹患者について, プラークコントロール, 歯石除去及びルートプレーニングを施した。その前後にポケット底部より採取した細菌叢の変動を, グラム染色, 暗視野顕微鏡, 嫌気培養法を用いて検討した。その結果, 歯周初期治療後にグラム陽性球菌の増加とグラム陰性桿菌, 糸状菌の減少が認められた。また, 術前, プラークコントロール, 歯石除去とルートプレーニングの3時期において Fusobacterium 属は6.8%, 4.8%, 1.4%, Bacteroides 属は11.0%, 3.1%, 0.4%, Spirochetes は11.0%, 9.5%, 0.5%の変動を示した。black pigmented Bacteroides は7被検部位中3部位で, Fusobacterium necrophorum は7被検部位中5部位で検出された。Bacteroides はポケットの深さと, Spirochetes はポケットの深さ, 歯肉炎指数, プラークスコアとの間で各々有意な相関性が認められた。