Binding of collagen gene products with titanium oxide.

Titanium is the only metal to which osteoblasts can adhere and on which they can grow and form bone tissue in vivo, resulting in a strong bond between the implant and living bone. This discovery provides the basis for the universal medical application of Ti. However, the biochemical mechanism of bond formation is still unknown. We aimed to elucidate the mechanism of bond formation between collagen, which constitutes the main organic component of bone, and TiO2, of which the entire surface of pure Ti is composed. We analysed the binding between the soluble collagen and TiO2 by chromatography with a column packed with Ti beads of 45 µm, and we explored the association between collagen fibrils and TiO2 (anatase) powders of 0.2 µm. We ran the column of chromatography under various elution conditions. We demonstrated that there is a unique binding affinity between Ti and collagen. This binding capacity was not changed even in the presence of the dissociative solvent 2M urea, but it decreased after heat denaturation of collagen, suggesting the contribution of the triple-helical structure. We propose a possible role of periodically occurring polar amino acids and the collagen molecules in the binding with TiO2.

Phosphorylated chitin increased bone formation when implanted into rat calvaria with the Ti-device.

BACKGROUND: Previously we found that a group of phosphorylated proteins (SIBLINGs) in bone binds with the Ti-device, and increases the early bone formation around the Ti-implants remarkably. From these results, we explained the biochemical mechanism of a strong bond between living bone and Ti, which was discovered by Brånemark and colleagues. For the clinical application of our findings, we need a large amount of these proteins or their substitutes. OBJECTIVE: We aimed to create a new molecule that equips with essential functions of SIBLINGs, Ti-binding, and bone enhancement around the Ti implant. METHODS: We chemically phosphorylated chitin and obtained a soluble form of phosphorylated chitin (P-chitin). In this solution, we immersed the Ti-devices of web-form (TW) which we previously developed and obtained the P-chitin coated TWs. Then we tested the P-chitin coated TWs for their calcification ability in vitro, and bone enhancing ability in vivo, by implanting them into rat calvaria. We compared the P-chitin coated TW and the non-coated TW in regard to their calcification and bone enhancing abilities. RESULTS: Ti-devices coated with phosphorylated-chitin induced a ten times higher calcification in vitro at 20 days, and four times more elevated amount of bone formation in vivo at two weeks than the uncoated Ti-device. CONCLUSIONS: Phosphorylated chitin could be a partial substitute of bone SIBLING proteins and are clinically applicable to accelerate bone formation around the Ti implants, thereby achieving the strong bond between living bone and Ti.

Bone enhancing effect of titanium-binding proteins isolated from bovine bone and implanted into rat calvaria with titanium scaffold.

Based on our previous finding that a chromatography with titanium beads selectively binds phosphoproteins, including caseins, phosvitin and dentin phosphoproteins, we investigated whether bone phosphoproteins also bind to titanium. Bovine bone matrix proteins were extracted with 2 M urea/PBS after demineralization. The 2 M urea extract was directly applied to the titanium chromatography column as reported. The chromatogram showed an initial large peak at breakthrough position (non-binding fraction) and a smaller second peak eluted later (titanium-binding fraction). Both peaks were analyzed by SDS polyacrylamide gel electrophoresis. Stains-all staining which preferentially identifies phospho-proteins revealed that the first peak contained no positively stained band, while the second peak showed 4 or 5 distinctive bands indicative of bone phosphoproteins. To investigate the biological functions of the titanium-binding bone proteins (TiBP), we implanted them into calvaria of rats, combined with titanium web (TW), a highly porous titanium scaffold of thin titanium-fibers. Bone TiBP induced significantly enhanced bone formation, and new bone appeared connected directly to titanium fibers, accompanied by active blood vessel formations. Control TW alone did not induce bone formation within the titanium framework. These results demonstrate that the bone titanium-binding proteins include phosphoproteins which enhance bone formation when implanted into bone with titanium.

Implantation with new three-dimensional porous titanium web for treatment of parietal bone defect in rabbit.

Titanium net (meshes) with excellent mechanical properties can promote bone compatibility and has been used as a repairing material for bone defects in clinical settings. In the present study, using spiral computed tomography (CT) and histomorphological techniques, we investigated the effect of a novel kind of titanium web with a three-dimensional (3D) porous structure on bone formation in rabbit skull (os parietal) defect. The images from the spiral CT scan demonstrate that the titanium web is completely fused with the surrounding bone tissue, even at the first month after implantation. The histomorphological findings show that different cells and tissues, including osseous tissue, connective tissue, and adipose cells, can easily grow into the 3D scaffold meshes of the titanium web, even in the center of the web and combine together as a whole body, suggesting that the titanium web possesses a very good biocompatibility, which is beneficial to the growth of bone tissue and promotes healing of the defected rabbit skull.

Periodontal regeneration following application of basic fibroblast growth factor-2 in combination with beta tricalcium phosphate in class III furcation defects in dogs.

The aim of this study was to investigate the effect of tunnel structured ß-tricalcium phosphate (ß-TCP) on the regenerative potential of basic fibroblast growth factor-2 (bFGF-2) in class III furcation defects in dogs. The furcations of 30 mandibular premolar teeth received: 1) 0.3% bFGF-2 solution in conjunction with ß-TCP; 2) 0.3% bFGF-2 solution; and 3) no implant material (Control group). The dogs were sacrificed 8 weeks post-surgery, and healing was evaluated histologically. New bone formation was significantly greater in the bFGF-2/ß-TCP group compared to the bFGF-2 solution and Control groups (p<0.01). New cementum formation in the bFGF-2/ß-TCP and bFGF-2 solution groups was significantly greater than that in the Control group (p<0.01). These findings suggested that bFGF-2 alone enhances connective tissue attachment in a manner similar to the combination of bFGF-2 and ß-TCP. Furthermore, this combination enhances bone formation up to the fornix in class III furcation defects.

Interaction between titanium and phosphoproteins revealed by chromatography column packed with titanium beads.

The biochemical mechanism behind the strong binding between titanium and living bone has not been fully elucidated, in spite of worldwide clinical application of this phenomenon. We hypothesized that one of the core mechanisms may reside in the interaction between certain proteins in the host tissues and the implanted titanium. To verify the interaction between titanium and proteins, we chose the technique of chromatography in that titanium spherical beads (45 µm) were packed into a column to obtain a bed volume of 16×50 mm, which was eluted with phosphate buffered saline (PBS) and a straight gradient system made by using PBS and 25 mM NaOH. Fetal calf serum, albumin, lysozyme, casein, phosvitin and dentin phosphoprotein (phosphophoryn) were applied to the column. Most part of albumin and lysozyme eluted with the breakthrough peak, indicating practically no affinity to titanium. Fetal bovine serum also eluted mostly as the breakthrough peak, but distinct retained peak was observed. On the other hand, α-casein, phosvitin and phosphophoryn exhibited a distinct retained peak separated from the breakthrough peak. We proposed that phosphate groups (phosphoserines) in the major phosphoproteins, α-casein, phosvitin and phosphophoryn may be involved in the binding of these proteins with titanium.

Periodontal repair following implantation of beta-tricalcium phosphate with different pore structures in Class III furcation defects in dogs.

The aim of this study was to investigate the effect of the pore characteristics of ß-tricalcium phosphate (ß-TCP) on periodontal healing in class III furcation defects in dogs. Two types of ß-TCP were prepared for grafting; 1) a tunnel pipe structure with an inner diameter of 300 µm, and 2) continuous pore structure with interconnected macropores. The furcations of thirty mandibular premolar teeth were implanted with each type of ß-TCP or were left untreated as control. The dogs were sacrificed 8 weeks post-surgery, and healing was evaluated histologically. Downgrowth of junctional epithelium in the tunnel structure group was significantly less than that in the other two groups (p<0.01). There was significantly more new bone formation and new cementum formation in the tunnel structure group than that in the other two groups (p<0.01). These findings suggested that ß-TCP with a tunnel pipe structure promotes periodontal healing in class III furcation defects.

Three-dimensional geometry of honeycomb collagen promotes higher beating rate of myocardial cells in culture.

Myocardial cells were isolated from newborn rats, cultured on a novel three-dimensional (3-D) honeycomb collagen scaffold (HC) and their morphology and beating rates compared with ones on conventional plastic dishes. On the first day, the cells attached to HC had already started beating. As time went on, the rate of beating increased as the pores of HC gradually filled with the cells, which integrated to form the cell-matrix complex. At day 8, beating reached the highest frequency of 162 beats per minute, which was twice that of the control cells on plastic dishes. It was concluded that 3-D geometry of the HC is conducive to functional growth of the myocardial tissues, and will potentially be useful for tissue engineering of myocardial regeneration.

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.

Honeycomb form ß-tricalcium phosphate induces osteogenesis by geometrical property with BMSC.

To establish an effective method for bone augmentation, we introduced a new honeycomb-like ß-tricalcium phosphate (H-ß-TCP) with BMP-2 as a scaffold, whose unique geometrical properties induce osteoblastic differentiation of autologous bone marrow mesenchymal stem cells (BMSCs). A total of six beagle dogs from 6 to 7 years old were used for this study. BMSCs were cultured with autologous serum and BMP-2 on H-ß-TCP. Differentiation to osteoblasts was demonstrated in vitro and exo vivo. Scanning electron microscopy revealed formation and calcification of a matrix-like structure within the H-ß-TCP tunnels in BMSC culture. Moreover, treatment of BMP-2 promoted osteoblastic differentiation of BMSCs in H-ß-TCP in a diffusion chamber. These results indicated that H-ß-TCP may be a useful tool for construction of functional artificial bone.

Chromatography of carbon nanotubes separated albumin from other serum proteins: a method for direct analysis of their interactions.

Chromatography technology was employed to clarify the mechanism of interaction between multi-wall carbon nanotubes (MWCNT) and proteins. A column (16x100 mm) was packed with purified MWCNT, and various proteins were eluted with phosphate buffered saline (PBS) with and without gradient systems. It was found that albumin in bovine serum was eluted immediately from the column without any adsorption to MWCNT. Conversely, the non-albumin proteins, including a protein of 85 kDa molecular mass and a group of proteins with molecular masses higher than 115 kDa, exhibited considerably high affinity towards MWCNT. A sample of pure bovine serum albumin was also eluted immediately from the column, while lysozyme did not elute as a peak with PBS, but eluted with 0.6 M NaCl. Fundamentally, carbon nanotubes are devoid of any electrical charge. Therefore, other forces including the hydrogen bonds, hydrophilic interactions, and van der Waals forces were most probably responsible for the differential elution behaviors. In conclusion, this chromatographic method provided a simple and direct analysis of the interactions between carbon nanotubes and the various proteins.

Biodistribution imaging of magnetic particles in mice: X-ray scanning analytical microscopy and magnetic resonance imaging.

Nano-sized particles have received much attention in view of their varied application in a wide range of fields. For example, magnetite (Fe(3)O(4)) nanoparticles have been investigated for various medical applications. In this study, we visualized the distribution of administered magnetic nanoparticles in mice using both X-ray scanning analytical microscopy (XSAM) and magnetic resonance imaging (MRI). After administration, the nanoparticles were rapidly dispersed via the blood circulation, and reached the liver, kidney and spleen. Using the XSAM and MRI methods in a complementary fashion, the biodistribution of nano-sized magnetite particles was successfully visualized.

In vivo internal diffusion of several inorganic microparticles through oral administration.

We observed the internal diffusion behavior of inorganic micro/nano particles through oral administration. By oral exposure, the fed particles were absorbed through the digestive system then reached some organs after internal diffusion in the body. For example, TiO2 particles fed to mice were detected in the lung, liver, and spleen after 10 days of feeding. Whereas, the absorption efficiency was extremely low compared with intravenous injection. In a comparison of the simple amount of administration, oral exposure required 102 times or more amount by intravenous injection for detection by an X-ray scanning analytical microscope. During dental treatment, micro/nano particles from tooth or dental materials would generate in the oral cavity, and some of the particles had a possibility to be swallowed, absorbed through the digestive system, and then diffuse into the body. However, our results suggest that biocompatible microparticles that are naturally taken orally affect animals only rarely because of the low absorption efficiency.

Interaction of collagen triple-helix with carbon nanotubes: Geometric property of rod-like molecules.

The interactions between carbon nanotubes and important biomolecules, above all collagen molecules, have not been studied in detail. This situation is partly due to the fact that CNT are solid entities, while most of the biomolecules can be prepared in solution. We used turbidity as a means of evaluating the interaction between CNT and collagen molecules. To a stable suspension of CNT (10 ppm in 0.1% Triton), collagen solution was added to obtain a final concentration of 25 ppm. The degree of aggregation was evaluated by measuring the turbidity of the suspension at 660 nm. It was found that native collagen induced distinct aggregation with CNT, while denaturation of this protein at 60 degrees C for 1 hr deprived the molecules of their ability to aggregate with CNT. Also other globular molecules, albumin and lysozyme, did not induce aggregation of CNT. These results indicate that the rigid rod-like structure of the native collagen triple helix is essential for interaction with CNT to cause aggregation. The mechanisms are considered to be dependent upon geometric properties of rod-like collagen molecules. The findings in this paper will open a new avenue to clarify the detailed mechanism of the interaction between collagen molecules and CNT.

Bone regeneration using titanium nonwoven fabrics combined with fgf-2 release from gelatin hydrogel microspheres in rabbit skull defects.

The objective of this study was to modify titanium nonwoven fabrics (Ti) with a hydroxyapatite (HA)-like coating and fibroblast growth factor (FGF)-2 combination, and evaluate the bone regeneration potential of the modified Ti. Biodegradable gelatin hydrogel microspheres (GM) were prepared as a carrier matrix for the controlled release of FGF-2. Ti, HA-coated Ti (Ti-HA), and Ti-HA incorporating GM (Ti-HA-GM) infused FGF-2 were applied to skull defects of rabbits. Then osteointegration in the Ti was evaluated by alkaline phosphatase activity, Ca(2+) content, and histological observation, and the hemoglobin content was assessed for angiogenic measurement. Ti-HA-GM promoted bone regeneration to a significantly greater extent than Ti, Ti-HA, or mixed Ti-HA and free FGF-2 6 weeks after application, and it also enhanced the hemoglobin content. It is concluded that the combination of HA-like coating and FGF-2 release promotes Ti induction of bone regeneration.

The axonal regeneration across a honeycomb collagen sponge applied to the transected spinal cord.

We developed a honeycomb-shaped lyophilized Type I atelocollagen (Honeycomb Collagen: HC) with different pore sizes, and the effectiveness of the honeycomb shape on nerve regeneration was examined. We analyzed neurite outgrowth of dorsal root ganglion (DRG) explants on HC, both in vitro and, with direct implantation of HC into the defects of adult rat spinal cords, in vivo. The neurites of DRGs on HC extended linearly through the pores. HC with a 400 microm-pore size enhanced neurite extension, and YIGSR laminin peptide coating to the HC extended more neurites than fibronectin coating. The HC scaffolds coated with YIGSR were implanted into 2 mm-defects of spinal cords at the level of T8-9. Four weeks after implantation, the implants had degraded and been replaced with self-tissues, repairing the injured site. Neurofilament-positive fibers were observed in the implantation area and passed the borders between the HC and spinal cord stumps. Functionally, a motor-evoked potential was observed in the quadriceps femoris muscle 10 weeks after implantation. The electrophysiological examination showed reconstruction of axon tracts over the implant. This result indicates that our developed honeycomb shape is advantageous for host spinal cord compared to the random pored sponge shape, and that it promotes axonal regeneration after spinal cord injury.

Alveolar ridge augmentation using various bone substitutes--a web form of titanium fibers promotes rapid bone development.

Alveolar ridge augmentation with beta-tricalcium phosphate (beta-TCP) granules, calcium phosphate cement (CPC) powder and web form of titanium fibers (TW) added to Platelet-Rich-Plasma (PRP) was histologically observed for 5 months by the experiments using the maxilla of rabbits. As a result, TW was thus found to promote the rapid bone development within a period of less than 5 months, because TW had an appropriate gap between the titanium fibers for new bone progression. However, titanium fibers remained intact within the maxilla. The CPC powder was transformed into dense cement and also remained intact in the bone. These residual materials must be obstacles to perform the placement of dental implants. On the other hand, beta-TCP granulations were gradually replaced by the new bone, but this process took about 5 months to be completed. Consequently, for an ideal alveolar bone substitute, there needs to be a suitable gap formation within the substitute, such as TW, and properties of total bone replacement, such as that seen in beta-TCP granules.

Differentiation of mesenchymal stem cells into osteoblasts on honeycomb collagen scaffolds.

Tissue engineering using living cells is emerging as an alternative to tissue or organ transplantation. The adult mesenchymal stem cells can be differentiated into multilineage cells, such as adipocytes, chondrocytes, or osteoblasts when cultured with specific growth factors. In the present investigation, we have studied the effect of honeycomb collagen scaffolds for the adhesion, differentiation and proliferation of bone marrow-derived mesenchymal stem cells into osteoblasts. Mesenchymal stem cells were isolated from 6-week old albino rat femur bone marrow, and cultured in alpha-MEM medium without beta-glycerophosphate and dexamethasone. Honeycomb collagen discs were prepared from bovine dermal atelocollagen, cross-linked by UV-irradiation and sterilized by heat. The honeycomb discs were placed on the culture dishes before seeding the stem cells. The cells attached quickly to the honeycomb collagen scaffold, differentiated and proliferated into osteoblasts. The differentiated osteoblasts were characterized by morphological examination and alkaline phosphatase activity. The osteoblasts also synthesized calcium-deficient hydroxyapatite (pseudo-hydroxyapatite) crystals in the culture. The mineralization was confirmed by Von Kossa staining and the crystals were analyzed by X-ray diffraction. Light microscopy and DNA measurements showed that the differentiated osteoblasts multiplied into several layers on the honeycomb collagen scaffold. The results demonstrated that the honeycomb collagen sponge is an excellent scaffold for the differentiation and proliferation of mesenchymal stem cells into osteoblasts. The data further proved that honeycomb collagen is an effective substrate for tissue engineering applications, and is very useful in the advancing field of stem cell technology and cell-based therapy.

Enhanced cementum formation in experimentally induced cementum defects of the root surface with the application of recombinant basic fibroblast growth factor in collagen gel in vivo.

BACKGROUND: Therapies using biologically active, soluble factors such as growth factors or cytokines have been investigated for potential clinical use in regenerating lost periodontal tissue due to periodontitis. Basic fibroblast growth factor (bFGF, FGF-2) is a multifunctional growth factor that has a variety of effects including induction of proliferation and morphogenesis in a wide range of cells and tissues including periodontal ligament tissue. METHODS: In this study, we examined the effects of bFGF on the regeneration of cementum and periodontal ligament in experimentally induced partial defects in a beagle dog model. bFGF in a collagen gel was applied to the defects and root surfaces, and the teeth were replanted. RESULTS: Eight weeks post-surgery, formation of cementum on denuded dentin was enhanced by application of 0.1, 1, or 5 microg of bFGF in a collagen gel compared to collagen gel containing vehicle. Histological analyses revealed that at 4 weeks post-surgery, random periodontal ligament fibers had bound to dentin, but were attached only to denuded dentin to which 0.1, 1, or 5 microg of bFGF in collagen gel had been applied. At 8 weeks post-surgery, we observed the formation of dense fibers bound to alveolar bone and newly synthesized cementum in teeth treated with 1 microg of bFGF. CONCLUSION: These results suggest that basic fibroblast growth factor in a collagen gel is a suitable therapy for damaged periodontal ligament and could lead to readily achievable methods of treatment for periodontal disease.

Failure to induce supracrestal bone growth between and around partially inserted titanium implants using bone morphogenetic protein (BMP): an experimental study in dogs.

The effect of bone morphogenetic protein on supracrestal bone growth around partially inserted implants in a dog model is described. The lower premolar teeth (P1, P2, P3 and P4) were extracted on both sides of the mandible in six dogs. At a surgical exposure 12 weeks later, two 10-mm turned titanium implants were partially inserted, approximately 15 mm apart, in the areas of the P1 and P3 in each side of the mandible, allowing five threads to protrude from the bone crest. A titanium mesh was fastened to the coronal aspect of the two fixtures and the space beneath the mesh was filled with bone morphogenetic protein (S300 BMP) in combination with an insoluble bone matrix carrier, or with the carrier alone. The mesh was covered with an ePTFE membrane. Thus, a space for potential bone formation was created between the two implants. The surgical flaps were coronally positioned and secured with vertical mattress sutures. After 16 weeks of healing, biopsy specimens were retrieved and examined histologically. Bone was not formed around the protruding implants or in the created space between the implants in any case. The carrier was incompletely resorbed. We conclude that supracrestal bone growth beyond the crestal limit with or without BMP in such a large space as in this experimental design may not be possible.