Jun Hino

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.

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.

Bone Induction in Porous HAp Block Modified by Partial Dissolution-Precipitation Technique with Supersonic Treatment in Rat Scalp

Microcracks and trabecular fractures can be observed in physiological bone. Biomimetic hydroxyapatite (HAp) scaffolds have been strongly needed in bone regenerative medicine. We have been developing the combination method of the partial dissolution-precipitation techniques involving the stirring-supersonic treatment in 1.7×10-2 N HNO3 solution containing Ca2＋and PO43- ions to improve the surface and the bulk of commercially available synthetic HAp block (82.5% in porosity, 50-300µm in macropore). The modified HAp was named as a partially dissolved and precipitated HAp (PDP-HAp). The aims of this study are to characterize the PDP-HAp and to observe cell response for the ceramics in rat scalp tissue. The PDP-HAp exhibited the macropore sizes of 50-200µm, the porosities of 85-90%, and the specific surface areas of 1.0-2.0 m2･g-1, with many micro-cracks. Twenty rats were divided into 2 groups. At 9 months, bone induction occurred inside the many pores in the PDP-HAp group, while bone and cartilage were not found in the HAp group. We believe that osteoinduction by the PDP-HAp is different from the process of BMP-loaded HAp-induced bone formation. The PDP-HAp might be applied as potential ceramics with osteoinductive properity and excellent biocompatibility in difficult bone regenerative cases.

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.

BMPs as Adsorptive Proteins for Ceramic Scaffolds

Body fluid permeability and blood compatibility of biomaterials are especially critical properties for regenerative bone therapy [1, 2]. To have a role in bone repair, biomaterials must have the adsorptive performance of various bone growth factors. The bone-inductive property of rabbit dentin was discovered in 1967 [3]. In our previous study, we have been researching the autograft of human demineralized dentin matrices (DDM) as a clinical study [4]. DDM is an acid-insoluble collagenous material. On the other hand, hydroxyapatite (HAp) is a mineralized material. Commercially available HAp block (APACERAM-AX®,85%-porosity with micropore) has been used as the artificial biomaterial in bone therapy [5]. Bone morphogenetic proteins (BMPs) are the strong hard tissue-inducing factors [6]. In this study, we investigated the existence of BMP-2 and -7, among proteins adsorbed to DDM and HAp, using immunoblottong analyses. The DDM granules and HAp blocks (64mm3) were implanted subcutaneously in 8-week-old Wistar rats, and sacrificed at 2 days after the implantation. Explanted DDM and HAp were homogenized by the ultrasonic procedure in phosphate-buffered saline (PBS) and the adsorbed proteins were separated on a 5-20% sodium dodecyl sulphate (SDS) polyacrylamide gradient gel by electrophoresis. For Western blotting, proteins in the gel were transferred to a polyvinylidene difluoride membrane and detected by anti-BMP-2 monoclonal antibody and anti-BMP-7 monoclonal antibody. BMP-2 and BMP-7 were detected as a major band at 50 kDa among proteins collected from the in vivo implanted DDM and HAp. BMP-2 was detected the second major band at 125 kDa in HAp and both BMP-2 and BMP-7 were detected the some minor bands in DDM and HAp. The bands of BMP-2 were stronger than those of BMP-7 in all. The DDM and HAp adsorbed BMP-2 and BMP-7. These results indicate that DDM is a useful bone substitute as much as HAp, adsorbed to the bone-inducing factors, in the bone engineering field.

Human Dentin Transplantation for Bone Engineering

The aims of this study are to confirm the capacity of hard tissue induction by human demineralized dentin matrics (DDM) and calcified dentin matrics (CDM) in subcutaneous tissues of nude mice, and to report a human pioneering trial following the autotransplantation of DDM for bone augmentation in a case of the atrophied upper jaw. Human DDM and CDM particles were prepared from adult, extracted vital teeth, and were implanted into the subcutaneous tissues of 4-week-old nude mice. DDM induced bone and cartilage independently at 4 weeks after implantation, while CDM did not induce a hard tissue formation. Autogenous DDM prepared from the non-functional second molar of a 58-year-old female were grafted on the atrophied jaw and received to the host. Human dentin can be recycled as autogenous biomaterials for local bone engineering.