Gelatin powders accelerate the resorption of calcium phosphate cement and improve healing in the alveolar ridge.

The aim of this study was to show the effectiveness of combining calcium phosphate cement and gelatin powders to promote bone regeneration in the canine mandible. We mixed gelatin powders with calcium phosphate cement to create a macroporous composite. In four beagle dogs, two saddle-type bone defects were created on each side of the mandible, and calcium phosphate cement alone or calcium phosphate cement containing composite gelatin powders was implanted in each of the defects. After a healing period of six months, mandibles were removed for µCT and histological analyses. The µCT and histological analyses showed that at experimental sites at which calcium phosphate cement alone had been placed new bone had formed only around the periphery of the residual calcium phosphate cement and that there had been little or no ingrowth into the calcium phosphate cement. On the other hand, at experimental sites at which calcium phosphate cement containing composite gelatin powders had been placed, we observed regenerated new bone in the interior of the residual calcium phosphate cement as well as around its periphery. The amount of resorption of calcium phosphate cement and bone regeneration depended on the mixing ratio of gelatin powders to calcium phosphate cement. New bone replacement was significantly better in the sites treated with calcium phosphate cement containing composite gelatin powders than in those treated with calcium phosphate cement alone.

Recent developments of functional scaffolds for craniomaxillofacial bone tissue engineering applications.

Autogenous bone grafting remains a gold standard for the reconstruction critical-sized bone defects in the craniomaxillofacial region. Nevertheless, this graft procedure has several disadvantages such as restricted availability, donor-site morbidity, and limitations in regard to fully restoring the complicated three-dimensional structures in the craniomaxillofacial bone. The ultimate goal of craniomaxillofacial bone reconstruction is the regeneration of the physiological bone that simultaneously fulfills both morphological and functional restorations. Developments of tissue engineering in the last two decades have brought such a goal closer to reality. In bone tissue engineering, the scaffolds are fundamental, elemental and mesenchymal stem cells/osteoprogenitor cells and bioactive factors. A variety of scaffolds have been developed and used as spacemakers, biodegradable bone substitutes for transplanting to the new bone, matrices of drug delivery system, or supporting structures enhancing adhesion, proliferation, and matrix production of seeded cells according to the circumstances of the bone defects. However, scaffolds to be clinically completely satisfied have not been developed yet. Development of more functional scaffolds is required to be applied widely to cranio-maxillofacial bone defects. This paper reviews recent trends of scaffolds for crania-maxillofacial bone tissue engineering, including our studies.

Bone regeneration by polyhedral microcrystals from silkworm virus.

Bombyx mori cypovirus is a major pathogen which causes significant losses in silkworm cocoon harvests because the virus particles are embedded in micrometer-sized protein crystals called polyhedra and can remain infectious in harsh environmental conditions for years. But the remarkable stability of polyhedra can be applied on slow-release carriers of cytokines for tissue engineering. Here we show the complete healing in critical-sized bone defects by bone morphogenetic protein-2 (BMP-2) encapsulated polyhedra. Although absorbable collagen sponge (ACS) safely and effectively delivers recombinant human BMP-2 (rhBMP-2) into healing tissue, the current therapeutic regimens release rhBMP-2 at an initially high rate after which the rate declines rapidly. ACS impregnated with BMP-2 polyhedra had enough osteogenic activity to promote complete healing in critical-sized bone defects, but ACS with a high dose of rhBMP-2 showed incomplete bone healing, indicating that polyhedral microcrystals containing BMP-2 promise to advance the state of the art of bone healing.

Evaluation of guided bone regeneration with poly(lactic acid-co-glycolic acid-co-ε-caprolactone) porous membrane in lateral bone defects of the canine mandible.

PURPOSE: The aim of this study was to qualitatively evaluate a poly(lactic acid-co-glycolic acid-co-ε-caprolactone) (PLGC) membrane as a barrier for guided bone regeneration in the canine mandible and to compare it to a nonresorbable polytetrafluoroethylene (PTFE) membrane. MATERIALS AND METHODS: Two wedge-shaped bone defects were created bilaterally in the mandibles of 12 beagle dogs. The bone defects in the left mandible were divided into three groups and treated as follows: PLGC membrane alone, PLGC membrane plus autogenous cortical bone chips, and titanium-reinforced expanded PTFE (TR-PTFE) membrane. The bone defects in the right mandible of each animal were left without membranes as a control. Computed tomography (CT) was performed at 3 and 6 months postoperative to evaluate bone regeneration. After a healing period of 6 months, the mandibles were removed en bloc for micro-CT and histologic analyses. RESULTS: CT analyses at 3 and 6 months showed that there was significantly more bone augmentation at all experimental sites than at the control sites. The volume of bone at defect sites covered with TR-PTFE was significantly greater than at defect sites covered with PLGC membrane with or without autogenous cortical bone. Micro-CT measurements showed that the volume of new bone formed at sites covered with TR-PTFE was significantly greater than at sites covered with PLGC membrane. However, the density of new bone was significantly higher at sites covered with PLGC membrane, with or without cortical bone, than at sites covered with TR-PTFE. Histologic analysis verified the presence of well-vascularized loose connective tissue in the pores of the PLGC membrane. CONCLUSIONS: Compared to TR-PTFE, the macroporous bioresorbable PLGC membrane did not significantly increase the amount of new bone in defect sites, but it facilitated the regeneration of mature bone.

Alveolar bone regeneration using poly-(lactic acid-co-glycolic acid-co-ε-caprolactone) porous membrane with collagen sponge containing basic fibroblast growth factor: an experimental study in the dog.

The aim of this study was to evaluate the effects of combining porous poly-lactic acid-co-glycolic acid-co-ε-caprolactone (PLGC) as a barrier membrane and collagen sponge containing basic fibroblast growth factor (bFGF) to promote bone regeneration in the canine mandible. In six beagle dogs, two lateral bone defects per side were created in the mandible. The lateral bone defects on the left side were treated with a PLGC membrane plus a collagen sponge containing bFGF. In half of these, the collagen sponge contained 50 µg of bFGF. In the other half, it contained 250 µg of bFGF. As a control, we treated the right-side bone defects in each animal with the same PLGC membrane but with a collagen sponge containing phosphate buffered saline. Computed tomography (CT) images were recorded at 3 and 6 months post-op to evaluate regeneration of the bone defects. After a healing period of 6 months, whole mandibles were removed for micro-CT and histological analyses. The post-op CT images showed that more bone had formed at all experimental sites than at control sites. At 3 months post-op, the volume of bone at defect sites covered with PLGC membrane plus 250 µg of bFGF was significantly greater than it was at defect sites covered with PLGC membrane plus 50 µg of bFGF. At 6 months post-op, however, this difference was smaller and not statistically significant. Micro-CT measurement showed that the volume of new bone regenerated at bone-defect sites, covered with PLGC membrane plus bFGF, was significantly greater than that of control sites. However, the presence or absence of bFGF in the collagen sponge did not significantly affect the bone density of new bone. These results suggest that the macroporous bioresorbable PLGC membrane plus collagen sponge containing bFGF effectively facilitates healing in GBR procedures.

Enhanced regeneration of critical bone defects using a biodegradable gelatin sponge and beta-tricalcium phosphate with bone morphogenetic protein-2.

We examine the osteogenicity of a sponge biomaterial consisting of a biodegradable mixture of gelatin and beta-tricalcium phosphate (betaTCP) that bound bone morphogenetic protein 2 (BMP-2) in critical-sized bone defects in rats. Gelatin-betaTCP sponges containing either phosphate buffered saline or incorporating BMP-2 are implanted into 5 mm diameter bone defects created in rat mandibles. We assess the defects biweekly for 8 weeks following implantation. There is significantly higher osteoinductive activity and significantly more Gla-osteocalcin content at bone-defect healing sites treated with gelatin-betaTCP sponges incorporating BMP-2 than there is in those treated with sponges that did not contain BMP-2. Histologically, new bone that contains bone marrow and that is connected to the original bone almost entirely replaces the regenerated bone. These results show that biodegradable gelatin-betaTCP incorporating BMP-2 is osteogenic enough to promote healing in large bone defects.

Regional differences in D/L aspartic acid ratios in the human mandible as a possible indicator of the bone remodeling rate.

In slowly metabolizing tissue such as tooth, bone and ocular lens, D-amino acids converted from L-amino acids accumulate with age and thus reflect the tissue turnover rate. To investigate whether D-amino acids play a role in determining the bone remodeling rate, we measured the accumulation of D-aspartic acid, which has the fastest rate of racemization, in various areas of the mandible. The level of D-aspartic acid was higher in the ramus than in the body (P < 0.01), and within the body, the level was higher in the basal area than in the alveolar area (P < 0.01). Within the alveolar area, the level of D-aspartic acid was higher in the molar region than in the incisal region (P: 0.05-0.01). No correlation was found between the accumulated level of D-aspartic acid with age, because all the specimens were obtained from elderly people with only a few years difference in age. There was also no correlation between D-aspartic acid and sex. In conclusion, we suggest that accumulation of D-aspartic acid in the mandibular bone reflects the differences in remodeling associated with occlusion. The incisal portion of the alveolar area of the mandible (above the mandibular canal), shows the greatest evidence of active remodeling.

Age-dependent changes in the racemisation ratio of aspartic acid in human alveolar bone.

We investigated the racemisation ratio of aspartic acid (Asp) in alveolar bone. In addition, we designed and created a new column to detect Asp in a short period of time, which allowed us to detect d-Asp and l-Asp separately from each other within 5min. Comparing identical ages, the racemisation ratio of alveolar bone was generally lower than that of other bones reported so far. This result suggests that alveolar bone is metabolically more active than other bones, as expected. The rate constant for the racemisation reaction (k(y)) of alveolar bone was calculated to be 0.000338 in males and 0.000084 in females. The rate constants in males and females were each similar to the respective ratios of the femur. This result suggests that the age-dependent reduction in metabolic turnover in alveolar bone proceeds similarly to that in the femur, although those changes proceed more slowly in females than in males. The correlation coefficient between the racemisation ratio of alveolar bone and chronological age was 0.660. It was high in males (r=0.912) and low in females (r=0.527), and this gender difference was statistically significant (P: 0.01-0.001), as in the femur.

Cationized gelatin delivery of a plasmid DNA expressing small interference RNA for VEGF inhibits murine squamous cell carcinoma.

Double-stranded RNA (dsRNA) plays a major role in RNA interference (RNAi), a process in which segments of dsRNA are initially cleaved by the Dicer into shorter segments (21-23 nt) called small interfering RNA (siRNA). These siRNA then specifically target homologous mRNA molecules causing them to be degraded by cellular ribonucleases. RNAi down regulates endogenous gene expression in mammalian cells. Vascular endothelial growth factor (VEGF) is a key molecule in vasculogenesis as well as in angiogenesis. Tumor growth is an angiogenesis-dependent process, and therapeutic strategies aimed at inhibiting angiogenesis are theoretically attractive. To investigate the feasibility of using siRNA for VEGF in the specific knockdown of VEGF mRNA, thereby inhibiting angiogenesis, we have performed experiments with a DNA vector based on a siRNA system that targets VEGF (siVEGF). It almost completely inhibited the expression of three different isoforms (VEGF120, VEGF164 and VEGF188) of VEGF mRNA and the secretion of VEGF protein in mouse squamous cell carcinoma NRS-1 cells. The siVEGF released from cationized gelatin microspheres suppressed tumor growth in vivo. A marked reduction in vascularity accompanied the inhibition of a siVEGF-transfected tumor. Fluorescent microscopic study showed that the complex of siVEGF with cationized gelatin microspheres was still present around the tumor 10 days after injection, while free siVEGF had vanished by that time. siVEGF gene therapy increased the fraction of vessels covered by pericytes and induced expression of angiopoietin-1 by pericytes. These data suggest that cationized-gelatin microspheres containing siVEGF can be used to normalize tumor vasculature and inhibit tumor growth in a NRS-1 squamous cell carcinoma xenograft model.

Proliferation and differentiation of rat bone marrow stromal cells on poly(glycolic acid)-collagen sponge.

We studied the effects of dexamethasone (Dex) and basic fibroblast growth factor (bFGF) on proliferation and differentiation of rat bone marrow stromal cells (RBMSCs), using three scaffolds: collagen sponge, poly(glycolic acid) (PGA)-collagen sponge, and PGA-collagen (UV) sponge. RBMSCs were seeded into the sponges, and cultured in primary medium, primary medium with Dex, and primary medium with bFGF and Dex. Three weeks after cultivation, we examined alkaline phosphatase (ALP) activity and cell number in the sponges, and also performed macroscopic, light microscopic, and scanning electron microscopic (SEM) observations. Collagen sponge shrank considerably, but PGA-collagen and PGA-collagen (UV) sponges maintained most of their original shape. PGA-collagen (UV) sponge supplemented with bFGF and Dex together had the highest ALP activity and cell number, followed by PGA-collagen sponge. Although collagen sponge showed cell proliferation only on the surface, the other two sponges showed cell proliferation in the interior. SEM showed the best cell attachment to PGA-collagen (UV) sponge in the presence of bFGF and Dex, followed by PGA-collagen sponge. In conclusion, PGA-collagen (UV) and PGA-collagen sponges proved to be much more useful as scaffolding for bone regeneration when combined with bFGF and Dex.

[Tissue engineering for jawbone].

One goal of jawbone reconstruction is to regenerate bone that is strong enough to support dentures or dental implants. Here we describe mandibular reconstructions using bioabsorbable poly (L-lactic acid) (PLLA) mesh and particulate cancellous bone and marrow (PCBM). PLLA mesh is made of monofilaments spun from molecular weight 20.5 x 10(4) that is elongated and woven into mesh. Mandibles of 62 patients with tumors, cysts, or alveolar atrophy were reconstructed with PLLA mesh trays/sheets and PCBM. The operative procedures were as follows: 1) a heat-softened PLLA mesh sheet or tray was cut and bent to conform to the shape and size of the bone defect; 2) PLLA mesh was fixed to the residual bone with stainless steel wire; and 3) the tray was filled with PCBM harvested from the ilium. Clinical evaluations six months after surgery showed that in 35 cases (56.5%) the results were judged to be excellent (bone formation range: beyond 2/3), in 17 cases (27.4%) they were good (bone formation range under 2/3, but re-operation unnecessary), and in 10 cases (16.1%) they were poor (cases not falling into either category above). Long-term observations showed that in 32 cases (80.0%) bone resorption was less than 10% of the regenerated bone. Among the patients wearing early dentures or dental implants, bone resorption was less. There is a good success rate for jawbone reconstruction surgery using PLLA mesh and PCBM, and patients can expect to have mandibles strong enough to support dentures and dental implants. In addition, there is an advantage of regenerating in the donor site as well. However, we need to develop therapies that combine bioactive factors to promote angiogenesis and osteogenesis or hybrid artificial bone for patients with poor regional blood circulation and for elderly patients who have too few osteoprogenitor cells.