Repair of segmental radial defects in dogs using tailor-made titanium mesh cages with plates combined with calcium phosphate granules and basic fibroblast growth factor-binding ion complex gel.

Repair of large segmental defects of long bones are a tremendous challenge that calls for a novel approach to supporting immediate weight bearing and bone regeneration. This study investigated the functional and biological characteristics of a combination of a tailor-made titanium mesh cage with a plate (tTMCP) with tetrapod-shaped alpha tricalcium phosphate granules (TB) and basic fibroblast growth factor (bFGF)-binding ion complex gel (f-IC gel) to repair 20-mm segmental radial defects in dogs. The defects were created surgically in 18 adult beagle dogs and treated by implantation of tTMCPs with TB with (TB-gel group) or without (TB group) f-IC gel. Each tTMCP fitted the defect well, and all dogs could bear weight on the affected limb immediately after surgery. Dogs were euthanized 4, 8 and 24 weeks after implantation. Histomorphometry showed greater infiltration of new vessels and higher bone union rate in the TB-gel group than in the TB group. The lamellar bone volume and mineral apposition rate did not differ significantly between the groups, indicating that neovascularization may be the primary effect of f-IC gel on bone regeneration. This combination method which is tTMCP combined with TB and f-IC gel, would be useful for the treatment of segmental long bone defects.

Sugar-based crosslinker forms a stable atelocollagen hydrogel that is a favorable microenvironment for 3D cell culture.

We created sugar-based crosslinkers for precise chemical crosslinking of atelocollagen to form a stable hydrogel microenvironment for three-dimensional (3D) cell culture. Crosslinkers were synthesized by partially oxidizing glucose, fructose, maltose, sucrose, or raffinose with periodate. The partial oxidization of sugar generated multiple aldehydes in the molecule, which acted as multifunctional crosslinkers, allowing atelocollagen to form chemical hydrogels. The crosslink reaction of atelocollagen was competitively suppressed by the addition of amino acid-rich medium, enabling flexible control of 3D molecular density in the acquired hydrogel. To evaluate structural stability, ultrasonic stress was loaded onto the acquired hydrogel and sequential measurement of water content showed that the crosslinking considerably stabilized the hydrogel structure. The effectiveness of the atelocollagen hydrogel as a 3D culture scaffold was analyzed by embedding and culturing endothelial cells or cardiomyocytes in it. Endothelial cells formed 3D capillary-like structures at 12 h, and cardiomyocytes formed a beating 3D netlike structure by 7 days. These findings suggest that crosslinked atelocollagen hydrogel effectively functions as a stable scaffold in 3D culture.

Bone regeneration by the combined use of tetrapod-shaped calcium phosphate granules with basic fibroblast growth factor-binding ion complex gel in canine segmental radial defects.

The effect of tetrapod-shaped alpha tricalcium phosphate granules (Tetrabones(®) [TB]) in combination with basic fibroblast growth factor (bFGF)-binding ion complex gel (f-IC gel) on bone defect repair was examined. Bilateral segmental defects 20-mm long were created in the radius of 5 dogs, stabilized with a plate and screws and implanted with 1 of the following: TB (TB group), TB and bFGF solution (TB/f group), and TB and f-IC gel (TB/f-IC group). Dogs were euthanized 4 weeks after surgery. Radiographs showed well-placed TB granules in the defects and equal osseous callus formation in all the groups. Histomorphometry revealed that the number of vessels and volume of new bone in the TB/f-IC group were significantly higher than those in the other groups. However, no significant differences in neovascularization and new bone formation were observed between the TB/f and TB groups. Furthermore, no significant difference in the lamellar bone volume or rate of mineral apposition was observed among groups. These results suggest that increased bone formation might have been because of the promotion of neovascularization by the f-IC gel. Therefore, the combinatorial method may provide a suitable scaffold for bone regeneration in large segmental long bone defects.

Repair of rabbit segmental femoral defects by using a combination of tetrapod-shaped calcium phosphate granules and basic fibroblast growth factor-binding ion complex gel.

The effect of tetrapod-shaped alpha tricalcium phosphate granules (TB) as a scaffold combined with basic fibroblast growth factor (bFGF)-binding ion complex gel (f-IC gel) on neovascularization and bone regeneration was evaluated in segmental femoral defects of rabbits. The defects were stabilized using a plate with a polypropylene mesh cage (PMC) containing one of the following: PMC alone (PMC group), TB (TB group), TB and bFGF (TB/f group), TB and IC gel (TB/IC group), or TB and f-IC gel (TB/f-IC group). Four rabbits from each group were euthanized at 2 and 4 weeks after surgery. Histomorphometry showed that the number of vessels and the volume of new bone in the TB/f-IC group were significantly higher than those in the other groups at all time points. There were no differences in the extent of neovascularization and new bone formation between the TB and TB/f groups. These findings suggest that the combination of TB and f-IC gel facilitated both neovascularization and new bone formation in segmental femoral defects of rabbits. This combination may be of considerable use for treating segmental long bone defects.

A calcium-cross-linked hydrogel based on alginate-modified atelocollagen functions as a scaffold material.

We have developed a scaffold material consisting of a covalently-bonded structure of alginate and atelocollagen (AtCol). Addition of calcium ions caused the material to form a hydrogel (alginate-modified AtCol gel). The condition of the alginate-modified AtCol gel could be controlled by the feed ratio of alginate and the activating reagent. Measurement of temporal stability in culture medium suggested that covalent bonding between alginate and AtCol might contribute to the structural stability of the alginate-modified AtCol gel. Culture with endothelial cells indicated that cell adhesiveness on the alginate-modified AtCol gel was similar to that on native collagen. Collagenase digestion revealed that the alginate-modified AtCol gel had considerable ability to retain basic fibroblast growth factor. Additionally, active cell migration into alginate-modified AtCol was detected by in vitro assay using endothelial cells. These findings indicate that this gel material can be expected to function as a scaffold for inducing vascular in-growth.

The growth of a vascular network inside a collagen-citric acid derivative hydrogel in rats.

Three-dimensional regenerative tissue with a certain bulk cannot survive without sufficient blood perfusion in vivo, so construction of a vascular system in regenerative tissue is a key technology in tissue engineering. In order to construct such a vascular system, we attempted to create a scaffold material that induces neovascular growth from the recipient bed into the material. This material, an ion complex gel matrix (IC gel) consisting of collagen and a citric acid derivative, enabled it to associate with basic fibroblast growth factor (bFGF). The IC gel was implanted in the subfascial space of the rat rectus muscle and excised 5 days later. Cross-sections of the excised samples were stained for von Willebrand factor, and then neovascular development into the gel was observed and also quantified by image analysis. These data showed that the IC gel markedly induced growth of vascular-rich tissue into the inside of the gel by day 5, which surpassed that after implantation of Matrigel or gelated collagen. Further, combination with bFGF significantly enhanced the vascularization ability of IC gel. These findings suggest that IC gel functioned as a scaffold material for neovascular ingrowth and a reservoir of bFGF.

Fabrication of nanofibrous inhaler device.

It has been proven that administration by inhalation is one of the alternative and effective approaches to dose biomedicines such as insulin for diabetics. Here, we introduce a new approach to fabricate the inhaler, posing the merits of not using volatile solvent, having selectable size of drug particles, and being simple and low cost compared with conventional devices. Creatine particles were prepared by spray-drying, and simultaneously a nanofiber mat of crosslinked poly(vinyl pyrrolidone) (PVP) was used to collect the graduated creatine nanoparticles. The scanning electron microscopy (SEM) photos indicated that the nanoparticles were individually dispersed without any agglomerate on the PVP nanofibers and released after the nitrogen gas flow (10 L/min) passed through the PVP nanofiber mat. This approach is unique and universal for nonagglomerate dispersing of nanoparticles and releasing without using any dispersing solvent.