Yoshitake Takahashi

Controlled release by biodegradable hydrogels enhances the ectopic bone formation of bone morphogenetic protein.

The objective of this study is to develop a carrier for the controlled release of bone morphogenetic protein-2 (BMP-2) suitable for enhancement of the bone regeneration activity. Hydrogels with different water contents were prepared through glutaraldehyde crosslinking of gelatin with an isoelectric point of 9.0 under varied reaction conditions. Following subcutaneous implantation of the gelatin hydrogels incorporating 125I-labeled BMP-2 into the back of mice, the in vivo retention period of BMP-2 prolonged with a decrease in the water content of hydrogels used, although every time period was much longer than that of BMP-2 solution injection. Ectopic bone formation studies demonstrated that the alkaline phosphatase (ALP) activity and osteocalcin content around the implanted site of BMP-2-incorporated gelatin hydrogels were significantly high compared with those around the injected site of BMP-2 solution. The values became maximum for the gelatin hydrogel incorporating BMP-2 with a middle period of BMP-2 retention, while bone formation was histologically observed around the hydrogel incorporating BMP-2. The ALP activity was significantly higher than that of the collagen sponge incorporating BMP-2. We concluded that the controlled release technology of BMP-2 for a certain time period was essential to induce the potential activity for bone formation.

Effect of the fiber diameter and porosity of non-woven PET fabrics on the osteogenic differentiation of mesenchymal stem cells

The proliferation and osteogenic differentiation of mesenchymal stem cells (MSCs) was investigated in three-dimensional non-woven fabrics prepared from polyethylene terephthalate (PET) fiber with different diameters. When seeded into the fabrics of cell scaffold, more MSC attached in the fabric of thicker PET fibers than that of thinner ones, irrespective of the fabric porosity. The morphology of cells attached became more spreaded with an increase in the fiber diameter of fabrics. The rate of MSC proliferation depended on the PET fiber diameter and porosity of fabrics: the bigger the fiber diameter of fabrics with higher porosity, the higher their proliferation rate. When the alkaline phosphatase (ALP) activity and osteocalcin content of MSC cultured in different types of fabrics was measured to evaluate the ostegenic differentiation, they became maximum for the non-woven fabrics with a fiber diameter of 9.0 μm, although the values of low-porous fabrics were significantly high compared with those of high porous fabrics. We concluded that the attachment, proliferation and bone differentiation of MSC was influenced by the fiber diameter and porosity of non-woven fabrics as the scaffold.

Homogeneous seeding of mesenchymal stem cells into nonwoven fabric for tissue engineering

Mesenchymal stem cells (MSCs) were isolated from the bone marrow of rats and seeded into a nonwoven fabric of polyethylene terephtalate (PET) by agitation and static methods. MSC attachment was investigated in terms of the number of cells attached to the fabric, their distribution inside the fabric, and cell damage. The number of MSCs attached was greater for the agitation seeding method than for the static seeding method. The higher the rotating speed in the agitation seeding method, the greater the number of cells attached. When the cell suspension was seeded into the fabric in culture medium volumes of 50 and 200 microL per well of the culture plate or per culture tube, the best cell attachment was observed for the tube culture group at the larger volume. These cells attached more homogeneously throughout the fabric in greater numbers than was the case for the other culture groups. It is possible that agitation of the cell suspension allows cells to infiltrate uniformly inside the fabric, resulting in a homogeneous distribution of the cells in the fabric. A biochemical study revealed that neither the agitation nor static seeding method damaged cells, irrespective of the medium volume and the type of culture vessel. We conclude that the agitation seeding method is a promising method by which to formulate a homogeneous construct of fabric and MSCs.

Combination of BMP-2-releasing gelatin/β-TCP sponges with autologous bone marrow for bone regeneration of X-ray-irradiated rabbit ulnar defects

The objective of this study is to evaluate the feasibility of gelatin sponges incorporating β-tricalcium phosphate (β-TCP) granules (gelatin/β-TCP sponges) to enhance bone regeneration at a segmental ulnar defect of rabbits with X-ray irradiation. After X-ray irradiation of the ulnar bone, segmental critical-sized defects of 20-mm length were created, and bone morphogenetic protein-2 (BMP-2)-releasing gelatin/β-TCP sponges with or without autologous bone marrow were applied to the defects to evaluate bone regeneration. Both gelatin/β-TCP sponges containing autologous bone marrow and BMP-2-releasing sponges enhanced bone regeneration at the ulna defect to a significantly greater extent than the empty sponges (control). However, in the X-ray-irradiated bone, the bone regeneration either by autologous bone marrow or BMP-2 was inhibited. When combined with autologous bone marrow, the BMP-2 exhibited significantly high osteoinductivity, irrespective of the X-ray irradiation. The bone mineral content at the ulna defect was similar to that of the intact bone. It is concluded that the combination of bone marrow with the BMP-2-releasing gelatin/β-TCP sponge is a promising technique to induce bone regeneration at segmental bone defects after X-ray irradiation.

Design of an osteoinductive biodegradable cell scaffold based on controlled release technology of bone morphogenetic protein

Biodegradable gelatin sponges with or without 50 wt% of s-tricalcium phosphate (s-TCP) incorporation were fabricated to design an osteoinductive scaffold that is capable of the controlled release of bone morphogenetic protein (BMP)-2. The sponges prepared had an interconnected pore structure with an average pore size of 200 μm, irrespective of the s-TCP incorporation. When seeded into the sponge by an agitated method, mesenchymal stem cells (MSC) isolated from rat bone marrow were homogeneously distributed throughout the sponge. Osteogenic differentiation experiments demonstrated that the in vitro differentiation and proliferation of MSC was enhanced by s-TCP incorporation. The in vivo osteoinduction activity of gelatin or s-TCP-incorporated gelatin sponges containing BMP-2 was studied in terms of histological and biochemical examinations following the implantation into the back subcutis of rats. As a result, homogeneous bone formation was histologically observed inside the sponge implanted, although the gelatin sponge exhibited significantly higher osteoinduction activity than that of the gelatin sponge incorporating s-TCP. The in vivo release test revealed that BMP-2 was released from the sponge in vivo, in a similar time period, whether or not s-TCP was incorporated. BMP-2 was retained for a time period longer than 28 days. These results suggest that the gelatin sponge with an ability to release BMP-2 is a promising cell scaffold for osteoinduction in vivo, although the effect of s-TCP incorporation on the osteoinductivity of sponges was different between the in vitro and in vivo systems.

Bone Induction by Controlled Release of BMP-2 from a Biodegradable Hydrogel in Various Animal Species - From Mouse to Non-Human Primate -

The objective of this study is to examine feasibility of biodegradable gelatin hydrogels incorporating bone morphogenetic protein (BMP)-2 in inducing bone regeneration at a bone defect of non-human primates and rabbits considering their BMP-2 release profiles. As a result, controlled release by the hydrogel system enabled BMP-2 to induce successful bone regeneration in non-human primates even at the BMP-2 dose as low as that for rabbit case (0.034 mg of BMP-2/cm3 of hydrogel).