Bone bonding strength of diamond-structured porous titanium-alloy implants manufactured using the electron beam-melting technique.

The present study examined the bone bonding strength of diamond-structured porous titanium-alloy (Porous-Ti-alloy) manufactured using the electron beam-melting technique in comparison with fiber mesh-coated or rough-surfaced implants. Cylindrical implants with four different pore sizes (500, 640, 800, and 1000µm) of Porous-Ti-alloy, titanium fiber mesh (FM), and surfaces roughened by titanium arc spray (Ti-spray) were implanted into the distal femur of rabbits. Bone bonding strength and histological bone ingrowth were evaluated at 4 and 12weeks after implantation. The bone bonding strength of Porous-Ti-alloy implants (640µm pore size) increased over time from 541.4N at 4weeks to 704.6N at 12weeks and was comparable to that of FM and Ti-spray implants at both weeks. No breakage of the porous structure after mechanical testing was found with Porous-Ti-alloy implants. Histological bone ingrowth that increased with implantation time occurred along the inner structure of Porous-Ti-alloy implants. There was no difference in bone ingrowth in Porous-Ti-alloy implants with pore sizes among 500, 640, and 800µm; however, less bone ingrowth was observed with the 1000µm pore size. These results indicated Porous-Ti-alloy implants with pore size under 800µm provided biologically active and mechanically stable surface for implant fixation to bone, and had potential advantages for weight bearing orthopedic implants such as acetabular cups.

Bone formation using ß-tricalcium phosphate/carboxymethyl-chitin composite scaffold in rat calvarial defects.

OBJECTIVE: The aim of the present study was to evaluate the effects of ß-tricalcium phosphate/carboxymethyl-chitin material (ß-TCP/CM-chitin) on bone formation in rat calvarial defects. STUDY DESIGN: Eighteen animals surgically received 2 calvarial defects (5 mm) bilaterally in each parietal bone. ß-TCP/CM-chitin was implanted in one side of each defect, and the contralateral side of the defect was left empty as a control. The animals were humanely killed at 4, 8, and 12 weeks after surgery for histologic evaluation. RESULTS: New bone formation in the ß-TCP/CM-chitin group was significantly greater than that in the control group throughout the healing periods (P < .05). ß-TCP/CM-chitin was remarkably resorbed 12 weeks after surgery. CONCLUSIONS: These results indicate that ß-TCP/CM-chitin is useful as a scaffold for bone formation.

Carboxymethyl-chitin promotes chondrogenesis by inducing the production of growth factors from immune cells.

Many techniques have been tested for their ability to restore cartilage defects, but several problems still remain in the complete healing of injured cartilage. In our previous study, we found that a carboxymethyl-chitin/beta-tricalcium phosphate (CM-chitin/beta-TCP) composite induced cartilage regeneration in the osteochondral defects of rabbits in vivo. We also found that CM-chitin stimulated peritoneal exudate cells (PEC) in mice and induced several kinds of inflammatory cytokines and transforming growth factor beta-1 (TGF-beta1). In this study, we examined whether CM-chitin is responsible for the induction of chondrogenesis via the production of TGF-beta1 in vitro. The murine pluripotent cell line C3H10T1/2 was maintained as a micromass culture in conditioned medium prepared from PEC stimulated with and without CM-chitin. CM-chitin-conditioned medium induced RNA expression of the chondrogenic-factor Sox9 and the matrix proteins aggrecan, Col2a1, and Comp. Their expression levels were decreased in the presence of anti-TGF-beta1 antibody. The micromass tissues cultured in CM-chitin conditioned medium at day 21 were clearly stained by Toluidine blue or Alcian blue (histological staining) and collagen II antibody (immunohistological staining), showing the expression of acidic glycosaminoglycan and type II collagen. Similar results were observed in micromass tissue stimulated with TGF-beta1 as a positive control. However, no chondrogenesis occurred when CM-chitin was added directly to a C3H10T1/2 cell culture. These results indicated that CM-chitin is a potent inducer of chondrogenesis via the induction of TGF-beta1 in immune cells.

Subacute systemic toxicity assessment of beta-tricalcium phosphate/carboxymethyl-chitin composite implanted in rat femur.

The efficacy of a composite of beta-tricalcium phosphate particles and carboxymethyl-chitin (beta-TCP/CM-chitin) for bone repair has already been established in animal experiments. In the present study, subacute systemic toxicity was evaluated to further assess the biological safety of the implanted composite. beta-TCP/CM-chitin (approximately 4 mg/kg and 7 mg/kg in male and female rats, respectively) was implanted for 28 days into penetrating defects (2 mm diameter) made artificially in the shaft of the right femur of rats. Sham operation groups with the defect only were prepared as controls. Haematology, blood chemistry, urinalysis, and the histopathology of 44 organs and tissues were investigated. Body weight measurements and clinical observations were performed daily throughout the study. No subacute systemic toxicity possibly caused by the implantation of beta-TCP/CM-chitin was detected. These findings indicate that beta-TCP/CM-chitin composite is a highly biocompatible bone substitute, at least with an implantation dosage of < 4-7 mg/kg.

Biological roles of carboxymethyl-chitin associated for the growth factor production.

Many techniques to restore cartilage defection have been tried. However, the development is still under way because of problems, including loosening of artificial joint, degenerative change of compensated tissue, risk of viral transmission via allograft/autograft, and cost of therapeutic materials for repair. In the previous research, we found that complementing cartilage defective part with carboxymethyl-chitin (CM-chitin)/beta-tricalcium phosphate composite induced regeneration of cartilage in rabbits in vivo, and it is presumable that CM-chitin plays a key role in chondrogenesis causing the regeneration of cartilage. However, the induction mechanism of chondrogenesis with CM-chitin is still unclear. In this study, we investigated the cell responses to CM-chitin by using peritoneal exudate cell (PEC) in mice and found that CM-chitin induced the expression of inflammatory cytokines and growth factors, both of which are both considered to correlate with chondrogenesis. After intraperitoneal injection CM-chitin showed enhanced expressions of mRNA of interleukin-1beta (IL-1beta), interleukin-6 (IL-6), keratinocyte-derived chemokine, tumor necrosis factor-alpha, and transforming growth factor-beta1 (TGF-beta1) in PEC as observed by reverse transcriptase polymerase chain reaction. Productions of TGF-beta1 protein were confirmed by enzyme linked immunosorbant assay. It was also shown that mononuclear cells in PEC were responsible for the TGF-beta1 production. These results suggest that CM-chitin is an inductor of inflammatory cytokines and growth factors and may contribute to regeneration of cartilage.

Repair of 20-mm long rabbit radial bone defects using BMP-derived peptide combined with an alpha-tricalcium phosphate scaffold.

In previous studies, we have reported that the BMP-2-derived peptide KIPKASSVPTELSAISTLYL, corresponding to BMP-2 residues 73-92, binds to a BMP-2-specific receptor, and elevates both alkaline phosphatase activity and osteocalcin mRNA in the murine mesenchymal cell line, C3H10T1/2. This 73-92 peptide conjugated to a covalently crosslinked alginate gel induced ectopic bone formation in rat calf muscle, and activated osteoblasts to promote the repair of rat tibial bone defects. Here, we report repair of 20-mm long rabbit radial bone defects using the 73-92 peptide combined with a porous alpha-tricalcium phosphate (TCP) scaffold. In vitro, the 73-92 peptide was released from the porous alpha-TCP scaffold over more than one week. In vivo, radiomorphometric analysis showed that the 73-92 peptide combined with the porous alpha-TCP scaffold promoted calcification in the implanted area in a dose-dependent manner, and that 5 mg of the 73-92 peptide induced connection of 20-mm long defects, defects of critical size, 12 weeks after implantation. Histological examination revealed newly formed bone and a marrow cavity in the implanted area. The area of bone denser than 690 mg/cm(3) induced by the 73-92 peptide was nearly equal to that of the contralateral radius.

Mechanical and histologic examination of titanium alloy material treated by sandblasting and anodic oxidization.

To evaluate the biocompatibility and the bone-bonding strength of new titanium alloy materials treated by sandblasting and anodic oxidization, 3 cylindric test pieces having different surface roughnesses were manufactured and implanted into the diaphyses of the femurs of New Zealand white rabbits. Six weeks later, shear loading tests and histologic examination were carried out. Strong interfacial bonding strength and active new bone formation were confirmed in the peripheral area of the test pieces having a surface roughness (Ra = 2.7 microm and Ra = 4.7 microm). Judging from stable fixation to shear loading in bone tissue, it was concluded that group C (Ra = 2.7 microm) had the best surface condition of the 2 groups. Further detailed examination is required to demonstrate that the surface treatment used for group C (a micro rough surface on a macro rough surface structure) can enhance active bone formation and stable fixation in bone tissue.

In vitro cytocompatibility assessment of beta-tricalcium phosphate/carboxymethyl-chitin composite.

A novel bioabsorbable bone substitute composed of a beta-tricalcium phosphate (beta-TCP) and a carboxymethyl-chitin (CM-chitin) sodium has been developed. Rabbit tibia defects (4 mm in diameter) were repaired after 4 weeks more effectively by the composite compared with a sham-operation group. To further investigate the biological safety of the components, genotoxicity and carcinogenicity of an extract prepared from the composite were determined using four different in vitro assays. The main extract component was identified as CM-chitin sodium [average molecular weight (Mw) approximately 230 kDa] as determined by Fourier transform infrared spectroscopy and gel permeation chromatography analysis. The concentrations of P and Ca possibly derived from beta-TCP were 17.7 and 37.1 microg/g, respectively, as determined by inductively coupled plasma mass spectroscopy. Both the metabolic activation and nonactivation (-S9) systems of the rat microsome S9 fraction were used to perform a genotoxicity evaluation using the Ames test and chromosome aberration assay on Chinese hamster lung fibroblast cells treated with the extract. In these assays, no genotoxicity was detected with doses < or =5 mg/mL (maximum concentration). The cell transformation assay using BALB/c 3T3 cells and the metabolic cooperation assay with V79 cells both showed negative results for any tumor-promoting activity caused by the extract (approximately 5 mg/mL). These results indicate that the bioabsorbable beta-TCP/CM-chitin composite is a highly biocompatible bone substitute.