Junichi Tazaki

BMP-2 release and dose-response studies in hydroxyapatite and β-tricalcium phosphate

The purpose of this study is to compare in vivo retention of BMP-2 and bone induction in HAp (porosity: 60-80%, pore size: 100-600 mum, sintering temperature: 800 degrees C, surface area: 1 m(2)/g) and beta-TCP (porosity: 75%, pore size: 100-400 mum, sintering temperature: 1050 degrees C, surface area: 4 m(2)/g). We estimated the in vivo release profile of (125)I-labeled BMP-2 and bone induction of hard tissues histologically. The amount of BMP-2 remaining in the beta-TCP at 1 day after implantation was 49.6%, while the amount was 34.0% in the HAp. Furthermore, the HAp and beta-TCP containing 0.0, 0.05, 0.1, 0.3, 0.5, 1.0, 5.0 microg of BMP-2 were implanted into the back subcutis of 4-week old Wistar rats. At 3 weeks after implantation, the ceramics were explanted and evaluated histologically. The HAp/BMP-2 (5.0 microg) system showed 3.0% in the total volume of bone at 3 weeks, while only in the beta-TCP/BMP-2 (5.0 microg) system showed 32.5%. These results indicate that the absorbable beta-TCP block may be an effective bioceramic for bone induction to deliver BMP-2 to the site of action.

Acid-insoluble human dentin as carrier material for recombinant human BMP-2 †

The aim of this study is to estimate the increase of bone-inductive potency by human demineralized dentin matrix (DDM) with recombinant human bone morphogenetic protein-2 (BMP-2). Human teeth were crushed, completely demineralized in 0.6M HCl, and freeze-dried. The tooth-derived material is called DDM. The shape of DDM was a particle type and its size varied from 0.4 to 0.8 mm. The BMP-2 dose-dependent study in the rat subcutaneous tissues demonstrated that the volume of induced bone and marrow increased at a dose-dependent manner. The time-course study of bone induction by the BMP-2 (5.0 μg)/DDM (70 mg) was estimated histologically and biochemically. Histological findings showed that the BMP-2/DDM increased bone and marrow sequentially between the DDM particles. Calcium content in the BMP-2/DDM-induced tissue was compatible to the histological findings. ALP activity in the BMP-2/DDM showed a maximal value at 1 week and gradually decreased. The morphometric analysis demonstrated that the BMP-2/DDM showed 66.9%, 79.0% in the volume of bone and marrow, and 32.4%, 21.0% in that of DDM at 8, 32 weeks, respectively. We confirmed that BMP-2 significantly accelerated bone formation in the acid-insoluble human-dentin carriers. These results indicate that human DDM should be an effective carrier for delivering BMP-2 and superior scaffold for bone-forming cells.

BMP-2 Dose-Response and Release Studies in Functionally Graded HAp

Hydroxyapatite (HAp) has been used as a biomaterial for hard tissues. Critical characteristics of biomaterials will include surface geometry, hydrophobicity and hydrophilicity, crystallinity, biodegradation rates, and release pharmacokinetics (PK) of incorporated molecules such as BMP-2. Optimizing BMP-2 for clinical application may be dependent on localized sustained release from biomaterials. We forcused on in vivo local BMP-2 PK and bone induction in two ceramics systems, based on different surface structures. The functionally graded apatites (fg-HAp) was designed by the step-wise calcinations and partial dissolution-precipitation methods. We estimated the in vivo release profile of 125I-labeled BMP-2 from fg-HAp and the dose response of bone induction by BMP-2 in the back subcutis histologically. Bulk-HAp (b-HAp) by only the step-wise calcinations was prepared as a control. The amount of BMP-2 remaining in the fg-HAp at 1 day after implantation was 83.8%, while that was 34.6% in the b-HAp. Moreover, ectopic bone formation were found surely in the fg-HAp/BMP-2 (0.5μg) system at 3 weeks, not in the b-HAp/BMP-2 system. By using fg-HAp, it is likely that an extremely low dose of BMP-2 is enough to enhance bone induction if BMP-2 is appropriately delivered to the site of action.

Surface Structure Design and Characterization of Bioabsorbable and Functionally Graded Apatites Originated from Bovine Bone

Abstract. Bioabsorbable and functionally graded apatites (fg-HAp) ceramics were designed using calcined bovine bone (b-HAp) by the partial dissolution-precipitation methods. The fg-HAp ceramics with micro-pores of 10-160 nm had larger specific surface areas (30-40 m2･g-1) than the b-HAp ceramics, although the two HAp ceramics exhibited same macro-pore sizes of 100-600 µm and porosities of 60-80 %. Surface structure of these ceramics was modified by soaking at 309.5 K for 1-90 days in a simulated body fluid (SBF). At 8 days after the soaking, microstructure of the fg-HAp changed from small grains to dense cocoon-like ones by rapid precipitation of HAp microcrystals, while at 14 days, that of the b-HAp was porous urchin-like grains, suggesting that the fg-HAp had higher bone-bonding ability than the b-HAp.

Behavior of Human Blood Adsorption to Biomimetic Functionally Graded Hydroxyapatite

Our approach to the design of biological material scaffolds for bone regeneration is the creation of cell environments that mimic natural tissues. Recently, we confirmed hitologically that the material surfaces of conventional, nonabsorbable ceramics shed body fluid. For bone engineering, it is known that ideal scaffolds should be bioabsorbable, mimetic, and hydrophilic materials that allow for the permeation of liquid components, such as blood and/or extracellular fluid. In our previous study showed functionally graded hydroxyapatite (fg-HAp) absorbed body fluid including albumin. In this study, we investigated the behavior of human blood adsorption to the fg-HAp by using the scanning electron microscope (SEM). The adsorption of the platelets and the formation of the fibrinous network were observed in the fg-HAp group incubated 20 minutes.

Simultaneous Implantation of Dental Implants and Autogenous Human Dentin

In our previous clinical study, autogenous demineralized dentin matrices (DDM) prepared from the functional vital teeth (#38, #41) of thirty-five-year-old female were grafted on the bone defect, using newly developed mill, and then received to the host without troubles. In this study, we implanted the human tooth dentin adjusted previously and the dental implants into the regions of missing tooth simultaneously. Fifty-seven-year-old female presented with missing teeth (#35-#37, #45-#47). First, a non-functional vital tooth (#18) were extracted and cryopreserved immediately. 11 months after extraction, the tooth was crushed by newly developed auto-crash mill using ZrO2 vessel and ZrO2 blade for 1 minute. The crushed granules were demineralized completely in 2% HNO3 solution, rinsed in cold distilled water and lyophilized (granule size: 0.5-2.0mm). The bacteria-free of the DDM were confirmed by the bacteriological examination before use. Drilling of the prospective implant beds were then performed according to the manufacture’s protocol and a screw-type rough surface implants (Nobel Biocare® Mk III) were placed. The adjusted DDM granules were implanted into the bone defect (#45). There are no postoperative complications at 3 years after implantation. This case indicates that the preserved autogenous DDM can be used as collagenous biomaterials with osteoinductive potency.

Multinucleated Giant Cells for Biomaterials - Ceramics and Dentin Collagen -

Bone and dentin consist of hydroxyapatite, collagen and body fluid. From biological points of view, we have been focusing on HAp and collagen materials for bone regeneration. The aim of this study is to estimate the appearance of multinuclear giant cells for non-organic (functionally graded HAp: fg-HAp) and organic materials (demineralized dentin matrix: DDM), histologically. The fg-HAp ceramic: Biomimetic fg-HAp was designed by using the partial dissolution-precipitation methods. The fg-HAp with micro-pores of 10-160 nm had larger specific surface areas (30-40 m2･g-1) than the synthetic HAp. Acid- insoluble dentin matrix (DDM): Human teeth were crushed under the cooling, completely demineralized in 0.026N HNO3 solution, and dried. The materials were implanted into the subcutaneous tissues (Wistar rats, 4 week-old, male), and removed at 1 and 4 weeks after the operations. Multinucleated giant cells were counted in the H-E sections. Giant cells predominantly appeared on the biodegradable micro-crystals at 1 week. The number of giant cells was more numerous in fg-HAp than in DDM. There was a significant difference in the cell number between fg-HAp and DDM. The absorption mechanism of fg-HAp should be predominantly cellular phagocytosis, while that of DDM might be predominantly enzymatic digestion. These data support the hypothesis that the biological HAp crystals may function as mineral signal in the recruitment and differentiation of multinucleated giant cells.

Comparison of HAp and β-TCP in BMP-2 Dose-Response and Release Study

The aim of this study is to investigate in vivo local BMP-2 PK and bone induction in two bioceramics blocks (HAp, β-TCP), based on different composition and surface structures. We estimated the in vivo release profile of 125I–labeled BMP-2 and bone induction of hard tissues histologically. β-TCP is more effective for both BMP-2 retention and bone induction, compared to HAp, in the ectopic model.

Blood Permeability of a Novel Ceramic Scaffold for BMP-2

Introduction Living bone consists of approximately 10% body fluid, 20% organic materials, and 70% minerals. Cells never live in dry conditions, and implanted biomaterials necessarily contact body fluid and cells in the body [1,2]. The hydrophilic condition and absorption of the biomaterials are, therefore, highly preferable in tissue regeneration. In addition, when degradation of the biomaterials is too slow, bone formation is inhibited by the material walls (Fig. 1). Bone morphogenetic proteins (BMPs), discovered from bone, are strong accelerating factors of bone formation [3,4,5]. We hypothesized that ideal scaffolds should be bioabsorbable and mimetic materials that allow for the permeation of body fluid and bone formation and can be replaced by new bone ingrowth. We decided to re-form the surface and bulk of bovine-derived and sintered spongy bone and fabricate an artificial surface. The aim of this study is to confirm the permeation of body fluid into inside of a new HAp and to evaluate the efficacy of the HAp as an absorbable scaffold for BMP-2 in the subcutaneous tissue of rats.

Characteristics of Surface Behavior and Osteoinductivity of Biomimetic Ceramic Scaffold

We previously reported functionally graded hydroxyapatite (fg-HAp) with the characteristics of blood permeability into the bulk and osteoinduction by adding low dose of bone morphogenetic protein-2 (BMP-2). In this study, we evaluated the bioactivity of the obtained dense HAp bodies in a simulated body fluid (SBF) and the osteoinductive activity with or without SBF treatment in vivo. The fg-HApSBF was prepared by immersing the fg-HAp in the SBF solution at 14 days and the surface structure was observed by SEM. The fg-HApSBF showed fine bone-like crystal on the surface of the HAp. Ectopic bone formation occurred in the fg-HAp/BMP-2 (1.0, 0.5μg) system at 3 weeks, while only in the fg-HApSBF/BMP-2 (1.0μg), bone induction was found. The histological finding showed body fluid permeation into the fg-HApSBF bulk and bone formation were obserbed.