In vitro and in vivo evaluation of surface functionalization of titanium with H2 O2 hydrothermal treatment and FGF-2.

The goals of the study were to investigate the effects on bone bioactivity of a titanium dioxide layer formed by hydrothermal oxidation of a titanium surface with hydrogen peroxide (H2 O2 ) and loading with fibroblast growth factor-2 (FGF-2) in vitro and in vivo. Ti-6Al-4V discs were hydrothermally oxidized with H2 O2 and then loaded with FGF-2. After cytotoxicity testing, Ti-6Al-4V mini-implants were subjected to the same treatment, and their osteogenic potential was evaluated histologically in a rat model. H2 O2 hydrothermal oxidation resulted in a dense porous network structure and hydrophilic changes, which improved retention of FGF-2. Morphologically, the cell density was higher, cell elongation was more pronounced, and the cell adhesion area was significantly higher in FGF-2-loaded cells than in those without FGF-2. In a cell proliferation assay using mouse osteoblast-like cells, absorbance tended to increase over time, especially in the FGF-2 group after 7 and 14 days, and in a bone differentiation assay based on ALP activity, there was a significant increase in the FGF-2 group after 14 days. In the rat model, H2 O2 hydrothermal oxidation and FGF-2 loading both resulted in more laminar bone tissue in the bone marrow around the mini-implant. These results suggest that titanium surface functionalization by H2 O2 hydrothermal oxidation and FGF-2 may promote initial cell adhesion, proliferation, and osteodifferentiation, and enhance bone bioactivity. These effects all contribute to early bonding of an implant with the surrounding bone.

Bioactivity Evaluation of Biphasic Hydroxyapatite Bone Substitutes Immersed and Grown with Supersaturated Calcium Phosphate Solution.

Recently, the frequency of use of bone substitute materials for the purpose of bone augmentation has increased in implant treatment, but bone formation with bone substitute materials alone is limited. Calcification of bone in the body progresses as Ca2+, H2PO4-, and HPO42- in the body form hydroxyapatite (HA) crystals. In this study, therefore, we prepared a biphasic bone substitute with biological activity to promote bone formation by inducing precipitation and growth of HA crystals on the surface of a bone substitute and evaluated it. Biphasic bone substitute granules were prepared by immersing HA granules in a supersaturated calcium phosphate solution prepared by mixing five medical infusion solutions, the precipitate was analyzed, and the biological activities of biphasic HA granules were evaluated in vitro and in vivo. As a result, the precipitated calcium phosphate crystals were identified as low crystalline HA. On the surface of the HA granules, low-crystalline HA grew markedly as needle-shaped crystals and significantly promoted cell proliferation and bone differentiation. In animal experiments, biphasic HA granules had a significantly higher bone mineral density, new bone volume ratio, and new bone area ratio. Therefore, it suggests that biphasic hydroxyapatite is a useful bone substitute for bone augmentation in dental implant treatment.

Relationship between hyposalivation and oxidative stress in aging mice.

The increase in oxidative stress that accompanies aging has been implicated in the abnormal advance of aging and in the onset of various systemic diseases. However, the details of what effects the increase in oxidative stress that accompanies aging has on saliva secretion are not known. In this study, naturally aging mice were used to examine the stimulated whole saliva flow rate, saliva and serum oxidative stress, antioxidant level, submandibular gland H-E staining, and immunofluorescence staining to investigate the effect of aging on the volume of saliva secretion and the relationship with oxidative stress, as well as the effect of aging on the structure of salivary gland tissue. The stimulated whole saliva flow rate decreased significantly with age. Also, oxidative stress increased significantly with age. Antioxidant levels, however, decreased significantly with age. Structural changes of the submandibular gland accompanying aging included atrophy of parenchyma cells and fatty degeneration and fibrosis of stroma, and the submandibular gland weight ratio decreased. These results suggest that oxidative stress increases with age, not just systemically but also locally in the submandibular gland, and that oxidative stress causes changes in the structure of the salivary gland and is involved in hyposalivation.

In vitro/in vivo evaluation of the efficacy of gatifloxacine-loaded PLGA and hydroxyapatite composite for treating osteomyelitis.

Molten 10 wt% gatifloxacine (GLFX-loaded poly(lactide-co-glycolide) (PLGA) was introduced into three-dimensionally interconnected pores and onto the surfaces of hydroxyapatite (HA) granules. The composite granules exhibited clinically sufficient bactericidal activities against Streptococcus milleri and Bacteroides fragilis from 3 h to 10 days. The composite granules were implanted in bone defects created by debridement of osteomyelitis lesions in rabbit mandibles. After 4-week implantation, inflammation in the composite granule-implanted group was significantly smaller than that in the debridement group (p<0.05). Moreover, newly formed bone was observed in the pores and on the surface of HA granules of the composite. These findings show that GFLX/HA composite controls bacterial infection and supports bone regeneration for osteomyelitis treatment.

Astaxanthin affects oxidative stress and hyposalivation in aging mice.

Oral dryness, a serious problem for the aging Japanese society, is induced by aging-related hyposalivation and causes dysphagia, dysgeusia, inadaptation of dentures, and growth of oral Candida albicans. Oxidative stress clearly plays a role in decreasing saliva secretion and treatment with antioxidants such astaxanthin supplements may be beneficial. Therefore, we evaluated the effects of astaxanthin on the oral saliva secretory function of aging mice. The saliva flow increased in astaxanthin-treated mice 72 weeks after administration while that of the control decreased by half. The plasma d-ROMs values of the control but not astaxanthin-treated group measured before and 72 weeks after treatment increased. The diacron-reactive oxygen metabolites (d-ROMs) value of astaxanthin-treated mice 72 weeks after treatment was significantly lower than that of the control group was. The plasma biological antioxidative potential (BAP) values of the control but not astaxanthin-treated mice before and 72 weeks after treatment decreased. Moreover, the BAP value of the astaxanthin-treated group 72 weeks after treatment was significantly higher than that of the control was. Furthermore, the submandibular glands of astaxanthin-treated mice had fewer inflammatory cells than the control did. Specifically, immunofluorescence revealed a significantly large aquaporin-5 positive cells in astaxanthin-treated mice. Our results suggest that astaxanthin treatment may prevent age-related decreased saliva secretion.

Effects of gatifloxaine content in gatifloxacine-loaded PLGA and ß-tricalcium phosphate composites on efficacy in treating osteomyelitis.

Composites of gatifloxacin (GFLX)-loaded poly (lactic-co-glycolic) acid (PLGA) and ß-tricalcium phosphate (ßTCP) containing 0, 1, and 10 wt % GFLX (0, 1, and 10 wt % GFLX composites), and GFLX-loaded PLGA containing 1, 5, and 10 wt % GFLX (1, 5, and 10wt % GFLX-PLGA) as controls were fabricated and characterized in vitro and in vivo. On in vitro evaluation, the 10 wt % GFLX composite released GFLX over at least 28 days in Hanks' balanced solution and exhibited clinically sufficient bactericidal activities against Streptococcus milleri and Bacteroides fragilis from 1 h to 10 days. The 0, 1, and 10 wt % GFLX composites and 10 wt % GFLX-PLGA were implanted in bone defects created by debridement of osteomyelitis lesions induced by S. milleri and B. fragilis in the mandible of rabbits (n = 5). Four weeks after implantation of the 10 wt % GFLX composite, inflammation in the debrided area disappeared in all the rabbits, while inflammation remained in all the rabbits after implantation of the 0 wt % GFLX composite and 10 wt % GFLX-PLGA, and in three rabbits after implantation of the 1 wt % GFLX composite. Bone formation appears to be less intense for the 10 wt % GFLX composite than for the 1 wt % GFLX composite probably owing to the rapid degradation of the 10 wt % GFLX composite. These findings show that the GFLX composite is effective for the local treatment of osteomyelitis.

Controlled release of simvastatin from biodegradable hydrogels promotes odontoblastic differentiation.

The objective of this study was to investigate the odontoblastic differentiation of dental pulp stem cells (DPSC) by biodegradable hydrogels incorporating simvastatin micelles, both in vitro and in vivo. Simvastatin (ST) was incorporated into the micelles of gelatin grafted with L-lactic acid oligomers (LAo) to allow water-solubilization. The simvastatin-LAo-grafted gelatin (LAo-g-gelatin) micelles were mixed with gelatin, followed by chemical crosslinking to form gelatin hydrogels (ST Mi/GH). The ST Mi were released from the gelatin hydrogel granules (GH) through enzymatic degradation. The ST Mi enhanced alkaline phosphatase activity, calcium deposition, and bone morphogenic protein-2 secretion of DPSC. When implanted subcutaneously into mice, the ST Mi/GH treated group exhibited increased dentin sialoprotein and calcium deposition, compared with those treated with GH plus free ST. It is possible to achieve odontoblastic differentiation of DPSC through the controlled release of ST from GH.

Anti-inflammatory effects of astaxanthin in the human gingival keratinocyte line NDUSD-1.

Oral lichen planus is a chronic inflammatory disease that affects the mucous membrane of the oral cavity and can contribute to the development of other diseases. Inflammation in oral lichen planus is a T-cell-mediated autoimmune disease that acts through cytotoxic CD8(+) T cells to trigger apoptosis of keratinocytes. However, the specific cause of oral lichen planus remains unknown and no effective medical treatment has yet been established. Astaxanthin is a carotenoid pigment with capacity for anti-inflammatory and anti-oxidant activities. In this study, we evaluated whether astaxanthin could be used to improve the pathology of oral lichen planus by reducing inflammation. In particular, the anti-inflammatory effects of astaxanthin on the chronic inflammation caused by lipopolysaccharide derived from Escherichia coli O55 in human gingival keratinocytes (NDUSD-1) were evaluated. Following astaxanthin treatment, localization of nuclear factor κB/p65 and the level of inflammatory cytokines (interleukin-6, tumor necrosis factor-α) tended to decrease, and cell proliferation significantly increased in vitro. These results suggest that astaxanthin could be useful for improving chronic inflammation such as that associated with oral lichen planus.

Enhanced bone regeneration by gelatin-ß-tricalcium phosphate composites enabling controlled release of bFGF.

The objective of this study was to investigate the feasibility of biodegradable gelatin-ß-tricalcium phosphate (ß-TCP) composites as a cell scaffold and controlled-release carrier of basic fibroblast growth factor (bFGF) suitable for inducing bone regeneration at a segmental bone defect. The composite of gelatin sponge and ß-TCP granules had an interconnected pore structure with an average size of 340 µm. The composite provided the controlled release of bFGF over 2 weeks. Segmental, critical-sized, bone defects of 20 mm length were created in the ulnas of New Zealand white rabbits and the gelatin-ß-TCP composites, with or without incorporated bFGF, were implanted into the defects. Bone regeneration and ß-TCP resorption profiles were evaluated by microcomputed tomography scanner analysis and haematoxylin and eosin staining. The composites incorporating bFGF promoted significantly higher bone regeneration at the defect site as compared to the bFGF-free composites. The controlled release of biologically active bFGF from the composites may possibly be achieved through the biodegradation of the composites, resulting in the promotion of bone regeneration. We conclude that the biodegradable gelatin-ß-TCP composite is a promising scaffold for bone regeneration that enables the controlled release of bFGF.

Preparation of thermoplastic poly(L-lactic acid) membranes for guided bone regeneration.

PURPOSE: The objective of this study was to evaluate the feasibility of application of thermoplastic poly-L lactic acid (PLLA) membranes for guided bone regeneration in rabbit parietal bone. MATERIALS AND METHODS: PLLA membranes with a molecular weight of 100,000 (PLLA-100,000) and a molecular weight of 380,000 (PLLA-380,000) were dissolved in chloroform to prepare concentrations of 8% by weight and 4% by weight, respectively. The compression strength, temperature, and time to prepare each formulation were measured. Moreover, the pH was noted and cytotoxicity of the membrane was determined by monotetrazolium assay. In vivo experiments were performed to measure the volume of newly formed bone tissue in hematoxylin and eosin-stained tissue sections 4 and 12 weeks after implantation. RESULTS: The membrane prepared from PLLA-380,000 showed excellent thermoplasticity at 75°C to 80°C and the compressive strength was equal to that of titanium mesh, in contrast to that of PLLA-100,000 and poly(lactic acid-co-glycolic acid). There was a significant change in the pH of an aqueous solution in which the PLLA-380,000 membrane was placed, but there was no cytotoxic activity. The membrane made of PLLA-380,000 induced new bone formation in a dome shape without any membrane deformation. CONCLUSION: Thermoplastic PLLA membrane shows promise for guided bone regeneration in vertical bone augmentation.

In vitro evaluation of H2O2 hydrothermal treatment of aged titanium surface to enhance biofunctional activity.

Surface modification of titanium has been extensively investigated in implant science and technology in an effort to improve its osteoconductivity. The rate of protein adsorption on titanium surfaces is known to vary depending on the chemistry, structure, morphology, and titanium-specific biological aging of the surface. It is thus desirable to modify smooth titanium surfaces of miniimplants used as orthodontic anchors immediately prior to use. In this study, we have developed a simple surface modification of titanium alloy that improves its biofunctional activity. The surface of a Ti-6Al-4V disk was modified by applying 3% H(2)O(2) hydrothermal treatment using an autoclave. A nanostructured porous network TiO(2) was observed on the treated surface. Treated surfaces exhibited higher hydrophilicity, protein adsorption, and cell proliferation than untreated surfaces. 3% H(2)O(2) hydrothermal treatment is thought to provide biofunctional activity for aged titanium surface.

Gatifloxacine-loaded PLGA and ß-tricalcium phosphate composite for treating osteomyelitis.

Gatifloxacine (GFLX)-containing poly(lactide-co-glycolide) (PLGA) was introduced to the pores and surfaces of porous ß-tricalcium phosphate (ßTCP) granules by melt compounding whereby no toxic solvent was used. The granular composite of GFLX-loaded PLGA and ßTCP released GFLX for 42 days in Hanks' balanced solution and exhibited sufficient in vitro bactericidal activity against Streptococcus milleri and Bacteroides fragilis for at least 21 days. For in vivo evaluation, the granular composite was implanted in the dead space created by the debridement of osteomyelitis lesion induced by S. milleri and B. fragilis in rabbit mandible. After a 4-week implantation, the inflammation area within the debrided area was markedly reduced accompanied with osteoconduction and vascularization in half of the rabbits, and even disappeared in one of the six rabbits without any systemic administration of antibiotics. Outside the debrided area, inflammation and sequestrum were observed but the largest of such affected areas amounted to only 0.125 times of the originally infected and debrided area. These findings showed that the granular composite was effective for the local treatment of osteomyelitis as well as an osteoconductive scaffold which supported and encouraged vascularization.

Effect of an injectable 3D scaffold for osteoblast differentiation depends on bead size.

The objective of this study was to evaluate the effect of beta-tricalcium phosphate (beta-TCP) bead size on the behavior of KUSA/A1 mouse osteoblasts when the beta-TCP beads are used as the solid phase of a scaffold in which alginate was used as the gel phase. KUSA/A1 cells were loaded onto a three-dimensional (3D) scaffold fabricated from beta-TCP beads with diameters ranging from 300 to 500 microm (small beads), 500-700 microm (medium beads) and 700-850 microm (large beads); cells were cultured for 3, 7 and 14 days. Scanning electron microscope observations showed that each bead was connected in a network consisting of the alginate gel and KUSA/A1 cellular matrix that was tightly bonded to form a 3D structure. After 3 days, cells in the 3D scaffold with medium beads had a significantly higher alkaline phosphatase activity (ALP) than cells in the other scaffolds. However, by 7 and 14 days in culture there was no significant difference in DNA levels, ALP activity or osteocalcin expression. At 8 weeks, only the composite containing small beads and KUSA/A1 cells had turned completely into bone in vivo. Thus, bead size may influence the success of bone formation in this context.

Antibiotic-loaded poly-epsilon-caprolactone and porous beta-tricalcium phosphate composite for treating osteomyelitis.

A composite of poly-epsilon-caprolactone (PCL) loaded with gatifloxacine (GFLX), an antibiotic, and a beta-tricalcium phosphate (betaTCP) porous ceramic body was prepared by a solvent-free process in which no toxic solvent was used. GFLX mostly retained its bactericidal property after the processing. The composite of GFLX-loaded PCL and betaTCP ceramic released GFLX for 4 weeks in Hanks' balanced solution, and had sustained bactericidal activity against Streptococcus milleri and Bacteroides fragilis for at least 1 week. The composite of the GFLX-loaded PCL and betaTCP ceramic was implanted in an osteomyelitis lesion induced by S. milleri and B. fragilis in the rabbit mandible. The osteomyelitis lesion expanded in the mesial-distal direction when no composite was implanted or when the lesion was treated with debridement only. The composite of GFLX-loaded PCL and betaTCP showed efficacy in controlling infection at the bone defect formed by debridement, and supported bone tissue reconstruction at the bone defect. Twelve and 50 weeks after the implantation, the inflammation even disappeared. New bone formation was observed on the surface of the composite after 4 weeks. After 50 weeks, ingrowth of bone tissues with vascular channels was observed along the PCL and betaTCP interface, which indicated degradation of PCL and/or betaTCP ceramic at the ceramic/polymer interface followed by replacement by bone tissues. The GFLX concentrations in the serum and soft tissues were very low. Therefore, the composite of GFLX-loaded PCL and betaTCP ceramic would help arrest osteomyelitis when it is used in addition to intravenous antibiotic administration, and help new bone formation and osteoconduction.

Preparation of injectable 3D-formed beta-tricalcium phosphate bead/alginate composite for bone tissue engineering.

A novel, injectable bone tissue engineering material was developed that consisted of beta-tricalcium phosphate (beta-TCP) beads as the solid phase and alginate as the gel phase. To prepare the instantaneously formed composite scaffold, an aqueous calcium chloride solution was dried on the surface of beta-TCP beads and crosslinked with an alginic acid sodium solution, thereby forming stable beta-TCP beads and alginate gel which were injectable via a syringe. This biodegradable composite was a three-dimensional (3D) material that could be used as an injectable scaffold for bone tissue engineering. In particular, the composite with 2.0 wt% alginate concentration exhibited a compressive strength of 69 kPa in dry conditions, which was significantly higher than that exhibited by 1.0 wt%. Furthermore, mesenchymal stem cells (MSC) were 3D-cultured within the composite and then investigated for osteogenic markers. MSC-loaded composite was subjected to scanning electron microscope (SEM) examination and implanted subcutaneously for in vivo experiment. Results showed that the scaffold provided support for osteogenic differentiation. In light of the encouraging results obtained, this novel injectable composite material may be useful for bone tissue engineering.

Fibronectin-calcium phosphate composite layer on hydroxyapatite to enhance adhesion, cell spread and osteogenic differentiation of human mesenchymal stem cells in vitro.

Fibronectin (Fn) and type I collagen (Col) were immobilized on a surface of a hydroxyapatite (HAP) ceramic by coprecipitation with calcium phosphate in a supersaturated calcium phosphate solution prepared by mixing clinically approved infusion fluids. These proteins and the calcium phosphate precipitate formed a composite surface layer. As a result, the proteins were immobilized firmly as not to be released completely for 3 d in a physiological salt solution. When human mesenchymal stem cells (hMSCs) were cultured on a HAP ceramic in a differentiation medium supplemented with dexamethasone, beta-glycerophosphate and ascorbic acid, hMSCs spread well within 1 h. The alkaline phosphatase (ALP) activity of hMSCs cultured on the Fn-calcium phosphate composite layer significantly increased compared with that of hMSCs cultured on the untreated HAP ceramic. On the other hand, Col did not increase the ALP activity of hMSCs and no synergy between Fn and Col was observed. Therefore, the Fn-calcium phosphate composite layer formed on the HAP is useful for the enhancement of the spreading and osteogenic differentiation of hMSCs in vitro.

Development of beta-tricalcium phosphate/collagen sponge composite for bone regeneration.

Synthetic biomaterials have been developed and used for bone grafting. Here, we developed a biodegradable sponge composite for bone tissue engineering by combining beta-tricalcium phosphate (beta-TCP) and collagen. In addition, we sought to determine the optimal beta-TCP granules/collagen ratio by evaluating and bone formation in vivo. Porous beta-TCP granules were mixed with atelocollagen hydrochloride solution at various ratios--0.02, 0.05, 0.1, and 0.2 g/mL. The resultant mixtures were freeze-dried and subjected to dehydrothermal treatment in vacuo. The final composites obtained were designated beta-TCP/collagen sponge composites (beta-TCP/CS). Through compression testing, it was found that the stress values for beta-TCP/CS (0.2 g/mL) were higher than those of the other three composites over the whole strain range. Histological evaluation at four weeks after implantation revealed that the collagen sponge had degraded and newly formed bone was present on the surface of the beta-TCP granules. At 12 weeks, the beta-TCP granules were completely degraded and remodeling of the lamellar bone was observed.

Quantitative detection of hepatitis C virus (HCV) RNA in saliva and gingival crevicular fluid of HCV-infected patients.

The search for hepatitis C virus (HCV) in body fluids other than blood is important when assessing possible nonparenteral routes of viral transmission. However, the role of oral fluids in HCV transmission remains controversial. Here we quantitatively determined HCV RNA in saliva and gingival crevicular fluid (GCF) of anti-HCV-positive patients. Most patients (14 of 18; 78%) whose saliva specimens were negative had HCV RNA in their GCF. Most patients (20 of 26; 77%) had higher HCV RNA levels in their GCF than in their saliva. Although there was not a statistically significant correlation between the serum viral load and HCV level in saliva or GCF, patients with low serum HCV loads were less likely to have detectable HCV in their saliva. These findings have important implications for medical personnel and suggest that epidemiological studies designed to understand the significance of the oral route of transmission of HCV are warranted.

In situ tissue engineering of periodontal tissues by seeding with periodontal ligament-derived cells.

The feasibility of an in situ tissue-engineering method employing cell-based therapy with autologous periodontal ligament-derived cells was investigated. Periodontal ligament cells were obtained from six beagle dogs. Periodontal fenestration defects (6 x 4 mm) were created bilaterally at a location 6 mm apical to the marginal alveolar crest in the maxillary canines. Alkaline phosphatase-positive periodontal ligament cells (3 x 10(5) cells) were seeded onto a collagen sponge scaffold just before implantation. One defect was filled with the cell-scaffold construct, and another was left empty as the control. All animals were killed 4 weeks after surgery, and specimens were evaluated histomorphometrically. All the histomorphometrical data were analyzed by three-way analysis of variance with the Bonferroni multiple comparisons test. Regeneration of apical tissue was faster than that of coronal and isolated tissues on the control side (apical > coronal > isolated; p < 0.0001). On the other hand, on the cell-seeded side, regeneration of the cementum was observed uniformly on the root surface. Our data suggest that the seeded cells induced cementum regeneration on the root surface, indicating the potential of in situ tissue engineering using autologous cells for the regeneration of periodontal tissues.