Static and dynamic guided bone regeneration using a shape-memory polyethylene terephthalate membrane: An experimental study in rabbit mandible.

BACKGROUND: Periosteal expansion (PEO) results in the formation of new bone in the space created between existing bone by expanding the periosteum. PEO has already been performed on rabbit parietal bone and effective new bone formation has been demonstrated. In this study, the utility of a polyethylene terephthalate (PET) membrane as an activator was evaluated in the more complex morphology of the mandible. METHODS: A PET membrane coated with hydroxyapatite (HA)/gelatine was placed in the rabbit mandibular bone at lower margin of mandibular molar region underneath periosteum, and screw-fixed. In the experimental group, the membrane was bent and screw-fixed along the lateral surface of the bone, with removal of the outer screw after 7 days followed by activation of the membrane. The experimental group was divided into two subgroups: with and without a waiting period for activation. Three animals were euthanized at 3 weeks and another three at 5 weeks postoperatively. Bone formation was assessed using micro-CT as well as histomorphometric and histological methods. RESULTS: No PET membrane-related complications were observed. The area of newly formed bone and the percentage of new bone in the space created by the stretched periosteum did not significantly differ between the control and experimental groups. However, in the experimental group a greater volume was present after 5 weeks than after 3 weeks. Histologically, bone formation occurred close to the site of cortical bone perforation, with many sinusoidal vessels extending through the perforations in the new bone into the overlying fibrous tissue. Inflammatory cells were not seen in the bone.

In vivo bioresorbability and bone formation ability of sintered highly pure calcium carbonate granules.

Calcium carbonate-based bone substitutes derived from natural coral exoskeleton (aragonite) are resorbed and remodeled faster than calcium phosphate-based substitutes. However, coral species with structures appropriate for use as bone substitutes are very limited. Therefore, it is important to evaluate potential of artificial calcium carbonate ceramics as a bone substitute. In this study, calcium carbonate granules with various porosities and pore sizes were prepared by sintering a highly pure (>99.98%) calcium carbonate powder (calcite), and their resorption properties and bone formation abilities were examined in vivo for the first time. The sintered calcium carbonate was resorbed faster than ß-tricalcium phosphate, which has a similar structure. However, sintered calcium carbonate did not promote new bone formation during long-term implantation. Furthermore, both resorption and new bone formation were affected by the pore structure. The optimal structures of the artificially sintered calcium carbonate bone substitute were also discussed.

In vivo behaviors of highly flexible paper consisting of ultralong hydroxyapatite nanowires.

This study was conducted in order to investigate biological compatibility of a thin and flexible hydroxyapatite (HAP) paper which consists of ultralong hydroxyapatite nanowires. Circular-shaped cranial bone defects with a diameter of 8.8 mm were prepared to expose the dura maters in Wistar rats. The similar-sized, circular-shaped HAP paper was placed at the bottom of the bone defects. After 2, 4, and 8 weeks, the rats were sacrificed, and the experimental sections were examined by micro-CT scanning and histological observation. The HAP paper covered with fibrous tissues showed no inflammatory cell infiltration, and their thicknesses decreased over time. Tartrate-resistant acid phosphatase-positive osteoclast-like cells were induced around the edges of the HAP paper along with the exfoliation of the HAP paper. The newly-formed bones were observed in the bone-defected areas, either with a direct contact with the HAP paper or through thin fibrous tissues. The HAP paper-induced osteoblast differentiation was confirmed since the alkaline phosphatase activities were detected on the surfaces of the HAP paper. These results indicated that the HAP paper may induce osteogenesis without causing any harmful effects. The highly flexible HAP paper can contribute to further development of bone regenerative therapy.

Periosteal expansion osteogenesis using an innovative, shape-memory polyethylene terephthalate membrane: An experimental study in rabbits.

Periosteal expansion osteogenesis (PEO) results in the formation of new bone in the gap between periosteum and original bone. The purpose of this study is to evaluate the use of a polyethylene terephthalate (PET) membrane as an activation device. A dome-shaped PET membrane coated with hydroxyapatite/gelatin on the inner side was inserted between the elevated periosteum and bone at the rabbit calvaria. In the experimental group, the membrane was pushed, bent, and attached to the bone surface and fixed with a titanium screw. In control group, the membrane was only inserted and fixed with titanium screw at original shape under the periosteum. After 7 days, the screw was removed and the mesh was activated in the experimental group. Three animals per group with or without setting a latency period for activation were sacrificed at 3 and 5 weeks after surgery. Bone formation was evaluated via micro-computed tomography and determined by histomorphometric methods and histological evaluation. No PET membrane-associated complications were observed during this study. The quantitative data by the area and the occupation of newly formed bone indicated that the experimental group had a higher volume of new bone than the control group at 3 weeks after surgery. Histologically, bone formation progressed to areas adjacent to the cortical perforations; many sinusoidal vessels ran from the perforations to overlying fibrous tissue via the new bone. No bone or obvious inflammatory cells were observed over the membrane. The PET membrane has biocompatible device for PEO that induces a natural osteogenic response at the gap between the original bone and periosteum.

Enhanced bone formation in the vicinity of porous ß-TCP scaffolds exhibiting slow release of collagen-derived tripeptides.

It has been experimentally proven that orally ingested collagen-derived tripeptides (Ctp) are quickly absorbed in the body and effectively promote the regeneration of connective tissues including bone and skin. Ctp are capable to activate osteoblasts and fibroblasts, which eventually promotes tissue regeneration. Based on these findings, a hypothesis was formulated in this study that direct delivery of Ctp to bone defect would also facilitate tissue regeneration as well as oral administration. To test the hypothesis, we prepared a bone augmentation material with the ability to slowly release Ctp, and investigated its in vivo bone regeneration efficacy. The implant material was porous ß-tricalcium phosphate (ß-TCP) scaffold which was coated with a co-precipitated layer of bone-like hydroxyapatite and Ctp. The ß-TCP was impregnated with approximately 0.8%(w/w) Ctp. Then, the Ctp-modified ß-TCP was implanted into bone defects of Wistar rats to evaluate in vivo efficacy of Ctp directly delivered from the material to the bone defects. The control was pristine porous ß-TCP. In vitro tests showed that Ctp were steadily released from the co-precipitated layer for approximately two weeks. The Ctp-modified scaffolds significantly promoted new bone formation in vivo in their vicinity as compared with pristine ß-TCP scaffolds; 6 weeks after the implantation, Ctp-modified scaffolds promoted twice as much bone formation as the control implants. Consequently, we achieved the slow and steady release of Ctp, and found that direct delivery of Ctp from implant materials was effective for bone regeneration as well as oral administration. A ß-TCP scaffold capable of slowly releasing bone-enhancing substances significantly promoted bone formation.

Effect of the up-front heat treatment of gelatin particles dispersed in calcium phosphate cements on the in vivo material resorption and concomitant bone formation.

Calcium phosphate cements (CPCs), consisting of a mixture of calcium phosphate powders and setting liquid, have been widely used in orthopedic applications. One of the drawbacks of CPCs is their poor resorbability in the living body, which hinders substitution with natural bones. One of the strategies to facilitate the resorption of CPCs is the incorporation of bioresorbable or water-soluble pore-generating particles (porogens), such as gelatin, in the CPC matrices. In spite of numerous reports, however, little is known about the effect of the dissolution/resorption rate of the porogens on concomitant bone regeneration. In the present study, we prepared preset CPCs dispersed with 10 mass% of low-endotoxin gelatin particles 200-500 µm in diameter having different heat-treatment histories, therefore exhibiting different dissolution rate, and then the obtained CPC/gelatin composites were evaluated for in vivo resorption and concomitant in vivo bone formation behaviors. As the results, the dispersion of gelatin particles markedly promoted in vivo resorption of CPC, and enhanced concomitant bone formation, connective tissue formation, osteoblast proliferation, and vascularization. The dissolution/resorption rate was able to be controlled by changing the up-front heat-treatment temperature. In particular, when CPC/gelatin composites were implanted in distal metaphysis of rabbits, the optimum dissolution/resorption was attained by heat-treating gelatin particles at 383 K for 24 h before dispersing in CPC. Quick resorption of calcium phosphate cement and concomitant bone formation by dispersing properly heat-treated with gelatin particles.

Enhancement of mechanical strength and in vivo cytocompatibility of porous ß-tricalcium phosphate ceramics by gelatin coating.

BACKGROUND: In an attempt to prepare scaffolds with porosity and compressive strength as high as possible, we prepared porous ß-tricalcium phosphate (TCP) scaffolds and coated them with regenerative medicine-grade gelatin. The effects of the gelatin coating on the compressive strength and in vivo osteoblast compatibility were investigated. METHODS: Porous ß-TCP scaffolds were prepared and coated with up to 3 mass% gelatin, and then subjected to thermal cross-linking. The gelatin-coated and uncoated scaffolds were then subjected to compressive strength tests and implantation tests into bone defects of Wistar rats. RESULTS: The compressive strength increased by one order of magnitude from 0.45 MPa for uncoated to 5.1 MPa for gelatin-coated scaffolds. The osteoblast density in the internal space of the scaffold increased by 40 % through gelatin coating. CONCLUSIONS: Coating porous bone graft materials with gelatin is a promising measure to enhance both mechanical strength and biomedical efficacy at the same time.

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.

Promotion of osteogenic differentiation of stem cells and increase of bone-bonding ability in vivo using urease-treated titanium coated with calcium phosphate and gelatin.

Because of its excellent biocompatibility and low allergenicity, titanium has been widely used for bone replacement and tissue engineering. To produce a desirable composite with enhanced bone response and mechanical strength, in this study bioactive calcium phosphate (CaP) and gelatin composites were coated onto titanium (Ti) via a novel urease technique. The cellular responses to the CaP/gelatin/Ti (CaP/gel/Ti) and bone bonding ability were evaluated with proliferation and osteogenic differentiation of mesenchymal stem cells (MSCs) on CaP/gel/Ti and CaP/Ti in vitro. The results showed that the optical density values, alkaline phosphatase expression and genes expression of MSCs on CaP/gel/Ti were similar to those on CaP/Ti, yet significantly higher than those on pure Ti (p < 0.05). CaP/gel/Ti and CaP/Ti rods (2 mm in diameter, 10 mm in length) were also implanted into femoral shaft of rabbits and pure Ti rods served as control (n = 10). Histological examination, scanning electron microscopy (SEM) and energy dispersive spectroscopy (EDS) measurements were performed at 4 and 8 weeks after the operation. The histological and SEM observations demonstrated clearly that more new bone formed on the surface of CaP/gel/Ti than in the other two groups at each time point. The CaP/gel/Ti bonded to the surrounding bone directly with no intervening soft tissue layer. An interfacial layer, containing Ti, Ca and P, was found to form at the interface between bone and the implant on all three groups by EDS analysis. However, the content of Ca, P in the surface of CaP/gel/Ti implants was more than in the other two groups at each time point. The CaP/gel/Ti modified by the urease method was not only beneficial for MSCs proliferation and osteogenic differentiation, but also favorable for bone bonding ability on Ti implants in vivo, suggesting that Ti functionalized with CaP and gelatin might have a great potential in clinical joint replacement or dental implants.

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.

Deposition of bone-like hydroxyapatite on the surface of silk cloth with the aid of immobilized urease.

Continuous layers of hydroxyapatite were deposited on silk cloth from aqueous solutions by using urease as the precipitant supplier. Silk cloth was surface-modified with urease and was immersed in an aqueous solution containing Ca2+, PO4(3-) , and urea. As urea was hydrolyzed to form ammonia with the aid of the immobilized urease, hydroxyapatite precipitated predominantly on the surface of the silk cloth. It took only a few hours to form continuous layers of hydroxyapatite on the silk cloth. The resultant hydroxyapatite was found to be bone-like apatite because it had low crystallinity, contained carbonate ion in the lattice, and had a calcium-deficient composition.