Toshiyuki Akazawa

In vitro proliferation and chondrogenic differentiation of rat bone marrow stem cells cultured with gelatin hydrogel microspheres for TGF-beta1 release.

The objective of this study was to evaluate the proliferation and chondrogenic differentiation of rat bone marrow-derived mesenchymal stem cells (MSCs) cultured with gelatin hydrogel microspheres of cell scaffold which can release transforming growth factor-β1 (TGF-β1). Gelatin was dehydrothermally cross-linked in different conditions in a water-in-oil emulsion state to obtain gelatin hydrogel microspheres with different water content. The microspheres functioned not only as the scaffold of MSC, but also the carrier matrix of TGF-β1 release. The MSC proliferation depended on the water content of microspheres. Higher MSC proliferation was observed for the gelatin microspheres with lower water content. When cultured with the gelatin hydrogel microspheres, MSC formed their aggregates, in contrast to culturing with hydrogel sheets. The cell viability was significantly high compared with that of the hydrogel sheet. The production of sulfated glycosaminaglycan (sGAG) from MSC was examined as a measure of chondrogenic differentiation, after their culturing in a normal and chondrogenic differentiation media. For both the cultures, the amount of sGAG produced was significantly higher for MSC cultured with the gelatin microspheres than that of the gelatin sheet. Stronger differentiation of MSC was achieved in culture with the microspheres incorporating TGF-β1 than that of MSC cultured in the medium containing the same amount of TGF-β1. It is concluded that the gelatin hydrogel microspheres function well as both the scaffold of MSC and the matrix of TGF-β1 release, resulting in enhanced MSC aggregation and the consequent promotion of cell proliferation and differentiation.

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.

Tooth-derived bone graft material

With successful extraction of growth factors and bone morphogenic proteins (BMPs) from mammalian teeth, many researchers have supported development of a bone substitute using tooth-derived substances. Some studies have also expanded the potential use of teeth as a carrier for growth factors and stem cells. A broad overview of the published findings with regard to tooth-derived regenerative tissue engineering technique is outlined. Considering more than 100 published papers, our team has developed the protocols and techniques for processing of bone graft material using extracted teeth. Based on current studies and studies that will be needed in the future, we can anticipate development of scaffolds, homogenous and xenogenous tooth bone grafts, and dental restorative materials using extracted teeth.

Human Dentin as Novel Biomaterial for Bone Regeneration

Human dentin autograft was reported in 2003 as a first clinical case (Murata et al., 2003), while human bone autograft was done in 1820. There was a long-long time lag between the autografts of dentin and bone. In 2009, Korea Tooth Bank was established in Seoul for the processing of the tooth-derived materials in Seoul, and an innovative medical service has begun for bone regeneration. Recently, the tooth-derived materials have been becoming a realistic alternative to bone grafting. The regeneration of lost-parts of the skeleton has been generally carried out with fresh, autogenous bone as a gold standard. To obviate the need for harvesting of grafts and thus, to avoid morbidity resulting from it, the researches for bone substitutes (Kuboki et al., 1995; Asahina et al., 1997; Takaoka et al., 1991; Artzi et al., 2004; Kim et al., 2010) or bone production via bio-engineering have begun (Wozney et al., 1988; Wang et al., 1990; Murata et al., 1999). In the regenerative field, there is a medical need for biomaterials that both allow for bone formation and also gradually absorb as to be replaced by bone. Non-absorbable materials are never replaced by bone and thus, reveal chronic inflammation in tissues as foreign bodies. As bone and dentin consist of fluid (10%), collagen (20%) and hydroxyapatite (70%) in weight volume, our attention for biomaterials is collagenous and ceramic materials (Murata et al., 2000; Murata et al., 2002; Akazawa et al., 2006; Murata et al., 2007). Generally, extracted teeth have been discarded as infective medical dusts in the world. We have thought the non-functional teeth as native resource for self and family (Fig. 1). Therefore, we noticed on bone-inductive, absorbable properties of dentin, and have been studying a medical recycle of human teeth as a novel graft material for bone regeneration in Japan and Korea (Akazawa et al. 2007; Kim et al. 2010). Biomaterial science should support and develop the advanced regenerative therapy using enamel and dentin matrix for patients in the near future.

Characterization of albumin- and lysozyme–adsorption evaluated on two differently prepared apatites

Ca2+-deficient hydroxyapatite (r-HAp) originated from cattle bones and stoichiometric hydroxyapatite (s-HAp) derived from reagents were prepared by wet syntheses. The adsorption characteristics of albumin (BSA) and lysozyme (LSZ) on the two HAp surfaces were compared by changing the heating temperature of the powders at 273–1073 K in a stream of water vapour. The saturated amount of adsorption (ASA(B) for BSA and ASA(L) for LSZ) on these HAp powders changed little at 273–673 K (Region I), but at 673–1073 K (Region II), clearly increased with crystallite size growth and transformation of crystal morphology. As far as the surface proportions of HAp for P- and C-adsorption sites (the ratios ASA(L)/ASA(B)) are concerned, r-HAp gave no change in Region I and decreased in Region II, whereas those for s-HAp were kept constant through all regions. The heats of LSZ adsorption, QL, for r-HAp and s-HAp, respectively, increased and decreased in Region II. These differences could be a result of the Ca2+-deficient structure of r-HAp with the OH−-vacancy and loosening surface structure due to segregation of impurities in Region II. r-HAp exhibited a 157% higher heat of BSA adsorption, QB, and a 60% lower QL in Region I than s-HAp. Conclusively, r-HAp can be used as an excellent adsorbent, rather than s-HAp, because of its chromatographic characteristics for the separation of acidic and basic proteins.

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.

Improved liquid chromatographic separation of different proteins by designing functional surfaces of cattle bone-originated apatite

Spherical particles of cattle bone-originated hydroxyapatite (r-HAp) were prepared by dissolution-precipitation, spray-drying using a two fluid-nozzle apparatus, and subsequent heat treatment. The product had effective pore structures for liquid chromatographic separation of albumin, myoglobin, ribonuclease, lysozyme and cytochrome c. The activated surfaces of the r-HAp particles were easily prepared with desired proportions of P- and C-sites and appropriate acid-basic strength for selective protein adsorption by optimizing the synthesis conditions. Liquid chromatography columns packed with the particles exhibited high resolution and durability in protein separation, reflecting stable distribution of pore size.

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.

Autograft of Dentin Materials for Bone Regeneration

While human bone autograft was done in 19th century, human dentin autograft for bone augmentation was reported in IADR 2003. The first clinical case was a sinus lifting using au‐ to-dentin for bone augmentation (Murata, 2003). Dentin is acellular matrix, while bone in‐ clude osteocytes. Very interestingly, biochemical components in dentin and bone are almost simillar. They consist of body fluid (10%), collagen (18%), non-collagenous proteins (NCPs: 2%) and hydroxyapatite (HAp: 70%) in weight volume (Fig. 1). Demineralized dentin matrix (DDM) and demineralized bone matrix (DBM) are mainly type I collagen with growth fac‐ tors such as bone morphogenetic proteins (BMPs) (Urist, 1965) and fibroblast growth factors (FGFs) (Fig. 2) (Butler et al.,1977; Murata et al, 2010a,b).

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.