Hydroxyapatite formed on/in agarose gel induces activation of blood coagulation and platelets aggregation.

We reported earlier that hydroxyapatite (HA) formed on/in agarose gels (HA/agarose) produced by alternate soaking process is a bone-filling material possessing osteoconductive and hemostatic effects. This process could allow us to make bone-like apatite that was formed on/in organic polymer hydrogel matrices. Here, we investigated the mechanism of hemostasis induced by HA/agarose and found that HA/agarose, but not agarose or HA powder, significantly shortened activated partial thromboplastin time (APTT). While HA/agarose did not show significant platelet aggregation, it markedly enhanced adenosine diphosphate (ADP)-induced platelet aggregation. Moreover, Western blot analysis revealed selective adsorption of vitronectin onto HA/agarose. We also observed marked differences between HA powder and HA/agarose in their XRD patterns. The crystallinity of HA powder was much higher compared to that of HA/agarose. Furthermore, 50-100 nm of tube-form aggregations was observed in HA powder on the other hand 100-200 nm of particles was observed in HA/agarose by SEM observation. Thus 100-200 nm of low crystallized particles on the surface structure of HA/agarose may play an important role in hemostasis. Our results demonstrated a crucial role of HA/agarose in the mechanism of hemostasis and suggested a potential role for HA/agarose as a bone-grafting material.

Controlled release based on the dissolution of a calcium carbonate layer deposited on hydrogels.

It is possible that inorganic materials conjugated to suitable organic materials may induce unique mechanical, optical and other functional properties. Therefore, artificial conjugation of organic and inorganic components is attractive for preparing novel functional materials. Recently, we developed an alternate soaking process for calcium salt formation on/in polymer materials. In this study, a poly(vinyl alcohol) (PVA) hydrogel-calcium carbonate (CaCO(3)) composite was prepared by the aforementioned process as a controlled release support. Brilliant blue FCF (Mw = 794), FITC labeled BSA (Mw = 6.6 x 10(4)), FITC labeled dextran-10 k (Mw = 9.5 x 10(3)) and FITC labeled dextran-40 k (Mw = 4.3 x 10(4)) were loaded into the composite as model drugs. CaCO(3) dissolution and model drug release rates increased with a decrease in buffer pH. In addition, model drug release rates increased with a decrease in model drug molecular weight. These results show that CaCO(3) layers on hydrogels behave as capping layers for model drug release; the release rate of model drugs can be controlled by the dissolution rate of CaCO(3) and the molecular weight of the drug.

Apatite formed on/in agarose gel as a bone-grafting material in the treatment of periodontal infrabony defect.

The present study was designed to evaluate the effects of a hydroxyapatite/agarose (HA/agarose) composite gel formed by a novel alternate soaking process for the treatment of periodontal infrabony defects in three dogs. After creating two-wall infrabony periodontal defects on the medial aspect of the maxillary and mandibular second and forth premolars, the defects were implanted with temporary dental filling material (stopping) to induce inflammatory periodontal disease. Two months later, the mucoperiosteal flaps were raised, and after debridement, the infrabony defects were filled with one of the following three materials: (a) HA/agarose, (b) Bone ject (True-Bone Ceramic-collagen combined bone graft material, Koken, Japan), or (c) no material implantation (negative control). The animals were then randomly scheduled for sacrifice at 1, 2, and 6 months, and samples were taken for histological examination. In the HA/agarose gels, the 2-month postoperative cavities exhibited regeneration to new attachments with the apposition of a new cementum and well-oriented fibers. The neocementum was narrow and acellular, and the new bone apposition was limited. Six months postoperatively, newly formed bone was predominantly observed. The neocementum was wider and cellular. In the negative control, the 2-month postoperative cavities exhibited no regeneration of the cementum, nor any formation of periodontal pockets. The six-month postoperative cavities were nearly the same as the 2-month cavities. The Bone ject, 2-month postoperative cavities exhibited no regeneration of the periodontal tissue, nor any formation of periodontal pockets. Six months postoperatively, inflammatory granulation tissue was observed around the particles. The present study suggests that HA/agarose gels may play an important role in the regeneration of lost periodontal tissue.

Bioinspired organic-inorganic composite materials prepared by an alternate soaking process as a tissue reconstitution matrix.

Poly(acrylic acid) (PAAc) grafted poly(ethylene) (PE) (PAAc-g-PE) film-apatite or calcium carbonate (CaCO3) composite materials were prepared by an alternate soaking process, which simply forms apatite or CaCO3 on the polymer materials by alternate soaking in Ca(2+)- and PO(3-)4- or CO(3)2- -containing solutions. X-ray diffraction analysis of the composite films indicated the presence of hydroxyapatite or CaCO3 on the film. Scanning electron microscopic observation revealed that the whole surface of the film was covered by the apatite or CaCO3. Cell compatibility tests of the apatite- or CaCO3-coated film suggested that the greater number of cells adhered on the films and that the cell proliferation properties were extremely greater on the films.

Osteoconductive and hemostatic properties of apatite formed on/in agarose gel as a bone-grafting material.

The biologic behavior of hydroxyapatite formed on/in agarose (HA/agarose) gels with the use of a novel alternate soaking process was compared with commercially available Bone Ject (True-Bone ceramic-collagen combined bone-graft material, Koken, Japan) as a filler for the tooth-extraction sockets of six adult monkeys (Macasa fascicularis). After the monkeys' first premolars were extracted, the defects created were replaced with one of the following materials: (a). HA/agarose created by 12 soaking cycles, (b). HA/agarose created by 9 soaking cycles, (c). Bone Ject, and (d). no material implantation (control). The time of hemostasis in each extraction site was estimated, and the samples were then studied histologically. In the controls, the time of hemostasis was about 5 min. The Bone Ject particles were easily washed out by bleeding, and the time of hemostasis was about 15 min. The HA/agarose gel was densely packed into the bony defect. The hemorrhage from the defects stopped within a few seconds after graft placement. This hemostasis was most likely related to the compactibility and adhesiveness of the material. After 12 weeks of implantation, free Bone Ject particles surrounded by inflammatory cells were observed. The bony defect filled with the HA/agarose gels was completely absorbed and replaced by newly formed bone possessing bone marrow. There was no difference in the biologic behavior of HA/agarose gels created by 9 versus 12 soaking cycles. The present study suggests that HA/agarose gels may play an important role as an alternative biodegradable bone-graft material for autogenous bone in humans.