In vivo evaluation of composites of PLGA and apatite with two different levels of crystallinity.

The cortical bone response towards poly(lactide-co-glycolide) (70/30) (PLGA) (70/30)/apatite complex scaffolds with different levels of crystallinity was investigated. Apatite with different levels of crystallinity, Ca-deficient hydroxyapatite (CDHA), which has a low crystallinity, and a mixture of carbonated hydroxyapatite (CHA) and CDHA, which has a higher crystallinity, were prepared from an aqueous mixture of Ca-EDTA complex, H(2)O(2), H(3)PO(4), and NH(4)OH. Two porous PLGA(70/30)/apatite composite scaffolds, composite scaffold A (containing low crystallinity CDHA) and composite scaffold B (containing the higher crystallinity CHA/CDHA mixture), were prepared. Afterwards, pure porous PLGA and the two composite scaffolds were implanted into the cortical bone of rabbit tibiae for 12 weeks. High-resolution microfocus X-ray computed tomography and histological examinations revealed a better bone response for composite scaffold A compared with PLGA and composite scaffold B. For composite scaffold A, the original bone defect was almost filled with new bone. Quantitative analysis revealed that composite scaffold A produced a significantly greater amount of new bone. The present study demonstrated that the level of apatite crystallinity influences bone response. A PLGA/apatite porous composite with a low level of apatite crystallinity shows promise as a bone substitute or scaffold material for bone tissue engineering.

Molecular precursor method for thin carbonate-containing apatite coating on dental implants.

The molecular precursor method is an easy and simple method for coating thin carbonate-containing apatite (CA) films onto titanium surfaces. A molecular precursor solution containing ethanol, calcium-EDTA complex, and phosphate salt was dropped onto a titanium surface and then heated at 600°C for 2 h. An adherent thin CA coating was achieved. Animal implantation experiments showed that CA-coated implants had significantly higher bone-to-implant values than non-coated implants (p<0.05). The molecular precursor method was also used to coat three-dimensional titanium webs (TWs). Thin CA films could be coated inside the center area, as well as the surface of the TW, with excellent bone formation inside the CA-coated TW. Furthermore, the molecular precursor method was used to coat partially stabilized zirconia with CA. Better bone response was observed for CA-coated zirconia. From this, it is concluded that the molecular precursor method is useful for producing thin CA coatings on implant materials.

Characterization and Bone Response of Carbonate-Containing Apatite-Coated Titanium Implants Using an Aqueous Spray Coating.

We performed thin carbonate-containing apatite (CA) coating on titanium (Ti) by an aqueous spray coating (ASC) method that consisted of a Ca-CO3-PO4 complex. Two different CA coatings were produced by two different spray amounts and were heat-treated after spraying. We evaluated three-dimensional structures, adhesiveness to Ti, and durability of the CA film. In addition, we performed immersion experiments in simulated body fluid (SBF), and bone responses were evaluated after implantation into a femoral bone defect in rats. The bonding ability of ASC-coated implant into the bone was examined by push-in tests. Unique network structures with small particles were identified on CA coatings. Although heat treatment produced no significant difference in surface morphology, scratch tests revealed that heat treatment improved the adhesion of CA coatings to Ti. Crystal formation progressed on CA-coated specimens, and the sample placement direction influenced crystal formation and growth in SBF immersion. Animal implantation experiments revealed significantly greater bone-to-implant contact ratio and bone mass in both cortical and bone marrow, respectively, four weeks after implantation. Push-in tests suggested that the bonding of the CA coating to Ti is clinically acceptable. Therefore, we conclude that CA coating to Ti by the ASC method would be possible for clinical applications, including dentistry.

Effect of YM529 on a model of mandibular invasion by oral squamous cell carcinoma in mice.

PURPOSE: This study examined the mechanisms of osteoclast-mediated bone invasion in a model of oral squamous cell carcinoma (OSCC). C3H/HeN mice were inoculated with SCC VII cells into the masseter region to establish an animal model of mandibular invasion by OSCC. EXPERIMENTAL DESIGN: The mice were divided into three groups: a control group, given daily s.c. injections of saline; group 1, given 2 microg per mouse per day of the bisphosphonate YM529; and group 2, given 10 microg per mouse per day of YM529. After 3 weeks of treatment, the lesions were studied by micro-computed tomography. After tartrate-resistant acid phosphatase (TRAP) staining, the osteoclasts were easily identified, and the percentages of the area occupied by osteoclasts were calculated by computer for each sample. The tumors were analyzed by RT-PCR to determine the mRNA expression of interleukin-6 (IL-6), parathyroid hormone-related protein (PTHrP), tumor necrosis factor-alpha (TNF-alpha), receptor activator of nuclear factor-kappaB (RANK), RANK ligand (RANKL), and osteoprotegerin. RESULTS: SCC VII cells rapidly multiplied in the masseter muscle of the mice. Bone invasion was evident only in the control group on micro-computed tomography. On TRAP-stained slices, the percentages of osteoclasts in groups 1 and 2 were significantly lower than that in the control group. The mRNA expressions of IL-6, PTHrP, THF-alpha, and RANK decreased as the concentration of YM529 increased. CONCLUSIONS: We conclude that various cancer-derived cytokines play important roles in the invasion of bone by OSCC. YM529, a third-generation bisphosphonate, can suppress osteoclast-mediated bone invasion by OSCC. The mechanism of this effect might involve inhibition of cytokines such as IL-6, PTHrP, TNF-alpha, and RANK by YM529.

Trabecular bone response to titanium implants with a thin carbonate-containing apatite coating applied using the molecular precursor method.

PURPOSE: The influence of thin carbonate-containing apatite (CA) coating on the trabecular bone response to titanium implants was investigated. MATERIALS AND METHODS: Thin CA coatings were deposited by a new method known as the molecular precursor method. Using a precursor solution composed of an EDTA-calcium complex, a CA film was deposited on the titanium surfaces. Uncoated and CA-coated titanium were placed in the trabecular bone of the left and right femoral condyles of 16 rabbits. After implantation periods of 2, 4, 8, and 12 weeks, the bone-implant interface was evaluated histologically and histomorphometrically. RESULTS: Histologic evaluation revealed new bone formation around the uncoated and CA-coated implant surfaces after only 4 weeks of implantation. After 12 weeks, mature trabecular bone surrounded all implants. At 4 and 8 weeks of implantation, no difference existed in bone contact between uncoated and CA-coated implants. After 12 weeks of implantation, the CA-coated implant group showed a significantly higher percentage of bone contact than the uncoated implant group. DISCUSSION AND CONCLUSION: The present study demonstrated that thin CA coatings applied using the molecular precursor method showed greater bone-to-implant contact during the healing phase than uncoated controls. The results were similar to those observed with implants with calcium phosphate coatings deposited with a physical vapor deposition technique.

Development of a biointegrated mandibular reconstruction device consisting of bone compatible titanium fiber mesh scaffold.

Coating biomaterials with a thin hydroxyapatite (HA) was proven effective in enhancing bone compatibility. Segmental bone defects are considered as the most difficult defect to repair in bone regeneration therapy. We developed submicron-thin HA-coated titanium fiber mesh scaffolds to reconstruct immediately loaded segmental mandibular defects and evaluated their bone compatibility in vitro and in vivo. Human osteoblasts attachment, proliferation, and osteocalcin expression in non- and HA-coated scaffolds were evaluated. A 10-mm long segmental bone defect in a rabbit mandibular bone was reconstructed with non- or HA-coated scaffolds, which were removed at 9 and 21 weeks, to evaluate the mechanical strength of the bone-scaffold connection and the bone formation around the scaffold. Expression of osteocalcin was greater in HA-coated scaffolds. In vivo bone formation in HA-coated scaffolds was greater than that in non-coated scaffolds at 21 weeks. Newly formed bone in HA-coated scaffolds mostly restored bone continuity. Scanning electron microscopy identified strong integration of the bone and HA-coated scaffolds. The mechanical strength of the bone-scaffold connection was 3-fold greater in HA-coated scaffolds than that in non-coated scaffolds. These results suggest that a thin HA-coated titanium fiber mesh scaffold is a bone-compatible mandibular reconstruction device in immediately loaded segmental defects.

Influence of thin carbonate-containing apatite coating with molecular precursor method to zirconia on osteoblast-like cell response.

The influence of a thin carbonate-containing hydroxyapatite (CA) coating to tetragonal zirconia polycrystal (TZP) on osteoblastlike cell response was investigated. TZP disks were subjected to blasting and acid etching. Thin CA coatings were deposited by the molecular precursor method (TZP-CA). Initial cell adhesion of mouse osteoblast-like cells MC3T3-E1 was enhanced, and marked progress of actin filaments was observed on TZP-CA compared to on TZP. After 3, 5 or 7 days, cell proliferation on TZP-CA was significantly higher than that on TZP. Alkaline phosphatase activity was slightly lower on TZP-CA than on TZP at 7 days, and no difference was observed at 14 or 21 days. At 28 days incubation, collagenous fibers with mineral precipitants accompanied by phosphorous and amino groups were observed. These results indicate that thin CA coating with molecular precursor method offers promise as a means of enhancing cell response, particularly initial adhesion and proliferation of MC3T3-E1 cells.

Bone responses to zirconia implants with a thin carbonate-containing hydroxyapatite coating using a molecular precursor method.

Thin carbonate-containing hydroxyapatite (CA) films coating partially stabilized zirconia (Y-TZP) were prepared (CA-Y-TZP) to establish a metal-free implant system. CA was coated using a molecular precursor method. The CA film was deposited on the surface of Y-TZP using a precursor solution, which was a mixture of a calcium-ethylenediaminetetraacetic acid (EDTA) complex and phosphate compounds. The deposited CA film was characterized by X-ray diffraction, Fourier transform infrared spectroscopy, and energy dispersive X-ray spectroscopy measurements. A focus ion beam system technique revealed that the thickness of the CA film was less than 1.0 µm. Biological evaluations of CA-Y-TZP were performed by immersion experiments in simulated body fluid (SBF) and implantation experiments in the tibiae and femoral condyles of rabbits. In the SBF immersion experiment, apatite deposition progressed more on CA-Y-TZP at the early stage of immersion than on Y-TZP without the CA coating. Animal experiments revealed that bone formation on CA-Y-TZP was similar with than on Y-TZP. Histomorphometrical evaluations showed a significantly higher bone-to-implant contact ratio and bone mass on CA-Y-TZP after implantation into the femoral trabecular bone of rabbits. Therefore, CA-Y-TZP appears to be applicable as a metal-free implant.

Hydroxyapatite coating for titanium fibre mesh scaffold enhances osteoblast activity and bone tissue formation.

This study investigated the bone regeneration properties of titanium fibre mesh as a tissue engineering material. A thin hydroxyapatite (HA) coating on the titanium fibre web was created using the developed molecular precursor method without losing the complex interior structure. HA-coated titanium fibre mesh showed apatite crystal formation in vitro in a human osteoblast culture. Titanium fibre mesh discs with or without a thin HA coating were implanted into rat cranial bone defects, and the animals were killed at 2 and 4 weeks. The in vivo experience revealed that the amount of newly formed bone was significantly higher in the HA-coated titanium fibre mesh than in the non-coated titanium fibre mesh 2 weeks after implantation. These results suggest that thin HA coating enhances osteoblast activity and bone regeneration in the titanium fibre mesh scaffold. Thin HA-coating improved the ability of titanium fibre mesh to act as a bone regeneration scaffold.

The effect of hydroxyapatite-coated titanium fiber web on human osteoblast functional activity.

PURPOSE: The aim of this study was to evaluate human osteoblast activity on thin hydroxyapatite (HA)-coated three-dimensional scaffolds made of titanium fiber web. MATERIALS AND METHODS: A thin HA film was coated on a titanium fiber web by the molecular precursor method. Human osteoblasts were disseminated onto the uncoated and HA-coated titanium fiber web, and osteoblast activity was observed at days 3, 7, 14, and 21 of culture. RESULTS: Proliferation activities of osteoblasts were significantly higher in the uncoated titanium fiber web. Osteoblasts in the uncoated titanium fiber web showed a typical expression pattern, but those in the HA-coated titanium fiber web showed rapid osteocalcin expression and calcification at an early stage of culture. Moreover, osteocalcin expression per osteoblast was significantly higher in the HA-coated group. CONCLUSION: These results suggest that HA coating with the molecular precursor method accelerated osteoblast functional activity.

The effect of composition of calcium phosphate composite scaffolds on the formation of tooth tissue from human dental pulp stem cells.

Different approaches towards making 3-dimensional (3-D) bioengineered tooth for future replacement therapy have been developed including scaffold-based tooth regeneration. However, selection of optimal scaffold for future clinical application remains a challenge. In the present study, we tested biocompatibility of four different types of 3-D scaffolds for tooth-tissue regeneration, including a pure poly(lactide-co-glycolide) (PLGA) (70/30, mol/mol) scaffold and three types of calcium phosphate contained composites scaffolds that were 50 wt% of PLGA combined with 50 wt% of hydroxyapatite (HA), tricalcium phosphate (TCP) or calcium carbonate hydroxyapatite (CDHA) respectively. These scaffolds were fabricated by the particle leaching in combination with phase separation technology. Surface modification of these scaffolds was further performed by an ammonia plasma treatment and anchorage of collagen technology. Effect of composition of the composite scaffolds on proliferation of human dental pulp stem cells (DPSCs) was accessed using in vitro MTT assay and in vivo BrdU labeling. Differentiation capability of the DPSCs in the scaffolds was analyzed by measurement of the levels of calcified tissue formation and ALP activity. Our results showed that while the calcium phosphate contained compound is able to support regeneration of tooth tissue effectively, the PLGA/TCP scaffold is more appropriate for the proliferation and differentiation of DPSCs. Furthermore, seeding of dissociated 4-dpn rat tooth bud cells on the PLGA/TCP scaffold generated dentin- and pulp-like tissues. Our results demonstrate that the PLGA/TCP scaffold is superior to the other three scaffolds for tooth-tissue regeneration, especially for dentin formation.

Crystallinity control of apatite through Ca-EDTA complexes and porous composites with PLGA.

Apatite compounds with different levels of crystallinity were prepared using Ca-EDTA complexes. Ca-deficient hydroxyapatite (CDHA) with low crystallinity was synthesized by ultrasonic stirring of a mixture of Ca-EDTA complex, phosphoric acid, and ammonium hydroxide in hydrogen peroxide aqueous solution. Mixtures of carbonate hydroxyapatite (HA) and CDHA with higher crystallinity were also prepared from a solution involving the same complex. The porous composites with lower or higher crystallinity apatite with a copolymer of poly(L-lactide-co-glycilide)(70/30) (PLGA(70/30)) were fabricated by a solution-casting/particles leaching method. The apatites and porous composites were characterized, and it was found that the degradation of composites of apatite with a low level of crystallinity was fastest in phosphate-bufferd saline (PBS) solution compared with other apatite composites with higher levels of crystallinity; however, the rate was smaller than that of PLGA alone. Plasma treatment influenced the degradation of composites in PBS and apatite precipitation in simulated body fluid (SBF). Hydroxyapatite deposition on the PLGA composite with the low crystallinity occurred six times faster than that on PLGA alone after immersion in SBF. The incorporation of apatite into the PLGA matrix did not cause any adverse effects on cell attachment in an assay employing human gingival fibroblasts. This study suggested that the current apatite and PLGA porous composite will be a promising scaffold material for tissue engineering.

Multi-institutional phase II trial of S-1 in patients with oral squamous cell carcinoma.

The aim of this study was to investigate the efficacy and safety of an oral fluoropyrimidine anticancer agent, S-1, in patients with oral squamous cell carcinoma. Patients with pathologically confirmed squamous cell carcinoma and at least one measurable lesion were enrolled. Oral administration of S-1 (40 mg/m2 twice daily) for 28 days was followed by a 14-day rest period. A total of 41 consecutive eligible patients were enrolled in the study between October 2002 and August 2004. The sites of the primary tumor were the gingiva (n=18), the tongue (n=12), the palate (n=5), the oral floor (n=4), the buccal mucosa (n=1), and the labial mucosa (n=1). A median of two cycles of treatment (range, 1-5) was administered. A complete response was achieved in nine patients and a partial response in eight patients, for an overall response rate of 41.5% (95% confidence interval, 26.4-56.5%). The 3-year survival rate was 76.4% (95% confidence interval, 62.8-90.0%). Although grade 3 anemia and anorexia occurred in two patients each (4.9%), and grade 3 neutropenia, thrombocytopenia, nausea, vomiting, stomatitis, and diarrhea in one patient each (2.4%), no grade 4 toxicities were observed. S-1 exhibits definite antitumor activity in patients with oral squamous cell carcinoma and is well tolerated.

Effect of thin carbonate-containing apatite (CA) coating of titanium fiber mesh on trabecular bone response.

The influence of thin carbonate-containing apatite (CA) coating on trabecular bone response to cylindrical titanium fiber mesh (porosity of 85%, pore size of 200-300 microm, 2.8 mm diameter x 6 mm length) implants was investigated. Thin CA coatings were deposited by the so-called molecular precursor method. Molecular precursor solution was obtained by adding dibutylammonium diphosphate salt to Ca-EDTA/amine ethanol solution by adjusting Ca/P = 1.67. Sintered cylindrical titanium fiber mesh was immersed into molecular precursor solution and then tempered at 600 degrees C for 2 h. The immersion and tempering process was repeated three times. An adherent thin CA film could be deposited on the inside of titanium fiber mesh. After the immersion of a CA-coated titanium fiber mesh in simulated body fluid, apatite crystals precipitated on the titanium fiber mesh. Uncoated and CA-coated titanium fiber mesh was inserted into the trabecular bone of the left and right femoral condyles of rabbits. Histological and histomorphometrical evaluation revealed a significantly greater amount of bone formation inside the porous area of the CA-coated titanium fiber mesh after 12 weeks of implantation. The present results suggested that a thin CA-coated titanium mesh has better osteoconductivity and will be useful for a three-dimensional scaffold.

Constitutive expression of a bacterial pattern recognition receptor, CD14, in human salivary glands and secretion as a soluble form in saliva.

Saliva contains a number of proteins and glycoproteins that protect oral tissues, but little is known about the role of human saliva in innate immunity. Here we showed that human major salivary gland cells constitutively expressed a bacterial pattern recognition receptor, CD14, by immunohistochemistry. Human salivary gland cells in culture express CD14 mRNA and a 55-kDa CD14 protein in, but not on the cells, and secrete a soluble form with the same molecular mass. Human whole saliva contains a 55-kDa CD14, and the concentration of parotid saliva was 10-fold higher than whole saliva, which is comparable to that of serum CD14. Levels of CD14 in unstimulated whole and parotid saliva were unchanged before and after a meal and between unstimulated and stimulated saliva, indicating that saliva CD14 is constitutively secreted into the oral cavity. In contrast, lipopolysaccharide (LPS)-binding protein was below the detectable level. The saliva CD14 is functionally active in that it mediated the activation of CD14-lacking intestinal epithelial cells by LPS in a Toll-like receptor 4-dependent manner. These results suggested that saliva CD14 is important for the maintenance of oral health and possibly intestinal homeostasis.

Mechanism for high stability of liposomal glucose oxidase to inhibitor hydrogen peroxide produced in prolonged glucose oxidation.

Glucose oxidase (GO) was encapsulated in the liposomes composed of POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) to increase the enzyme stability through its decreased inhibition because of hydrogen peroxide (H(2)O(2)) produced in the glucose oxidation. The GO-containing liposomes (GOLs) were completely free of the inhibition even in the complete conversion of 10 mM glucose at 25 degrees C because the H(2)O(2) concentration was kept negligibly low both outside and inside liposomes throughout the reaction. It was interestingly revealed that the H(2)O(2) produced was decomposed not only by a slight amount of catalase originally contained in the commercially available GO but also by the lipid membranes of GOL. As compared to the GOL-catalyzed reaction, the free GO-catalyzed reaction more highly accumulated H(2)O(2) because of the more rapid glucose conversion despite containing free catalase, leading to the completely inhibited GO before reaching a sufficient glucose conversion. This suggested that only the liposomal catalase could continue to catalyze the H(2)O(2) decomposition. The effect of the glucose oxidation rate, i.e., the H(2)O(2) production rate on the liposomal GO inhibition, was also examined employing the various GOLs with different permeabilities to glucose present in their external phase. It was concluded that the liposomal GO free of the inhibition could be obtained when the GOL-catalyzed H(2)O(2) formation rate was limited by such a suitable lipid bilayer as POPC membrane so that the rate was well-balanced with the sum of the above two H(2)O(2) decomposition rates. The highly stable GOL obtained in the present paper was shown to be a useful biocatalyst for the prolonged glucose oxidation.

Possible role of stromal-cell-derived factor-1/CXCR4 signaling on lymph node metastasis of oral squamous cell carcinoma.

We examined the role of chemokine signaling on the lymph node metastasis of oral squamous cell carcinoma (SCC) using lymph node metastatic (HNt and B88) and nonmetastatic oral SCC cells. Of 13 kinds of chemokine receptors examined, only CXCR4 expression was up-regulated in HNt and B88 cells. CXCR4 ligand, stromal-cell-derived factor-1alpha (SDF-1alpha; CXCL12), induced characteristic calcium fluxes and chemotaxis only in CXCR4-expressing cells. CXCR4 expression in metastatic cancer tissue was significantly higher than that in nonmetastatic cancer tissue or normal gingiva. Although SDF-1alpha was undetectable in either oral SCC or normal epithelial cells, submandibular lymph nodes expressed the SDF-1alpha protein, mainly in the stromal cells, but occasionally in metastatic cancer cells. The conditioned medium from lymphatic stromal cells promoted the chemotaxis of B88 cells, which was blocked by the CXCR4 neutralization. SDF-1alpha rapidly activated extracellular signal-regulated kinase (ERK)1/2 and Akt/protein kinase B (PKB), and their synthetic inhibitors attenuated the chemotaxis by SDF-1alpha. SDF-1alpha also activated Src family kinases (SFKs), and its inhibitor PP1 diminished the SDF-1alpha-induced chemotaxis and activation of both ERK1/2 and Akt/PKB. These results indicate that SDF-1/CXCR4 signaling may be involved in the establishment of lymph node metastasis in oral SCC via activation of both ERK1/2 and Akt/PKB induced by SFKs.