Shunsuke Fujibayashi

Osteoinduction of porous bioactive titanium metal

This is the first report of bone induction in a non-osseous site by titanium metal, which has long been recognized as a non-bioactive material. After undergoing specific chemical and thermal treatments, porous bioactive titanium induced bone formation without the need of additional osteogenic cells or osteoinductive agents. Four types of titanium implants were implanted in the dorsal muscles of mature beagle dogs, and were examined histologically after periods of 3 and 12 months. Chemically and thermally treated titanium, as well as pure titanium, was implanted either as porous blocks or as fibre mesh cylinders. Bone formation was found only in the chemically and thermally treated porous block implants removed after 12 months. The present study shows that even a non-soluble metal that contains no calcium or phosphorus can be an osteoinductive material when treated to form an appropriate macrostructure and microstructure. This finding may elucidate the nature of osteoinduction, and lead to the advent of epochal osteoinductive biomaterials for tissue regeneration.

A comparative study between in vivo bone ingrowth and in vitro apatite formation on Na2O-CaO-SiO2 glasses.

This study compared in vivo bioactivity with the in vitro apatite-forming ability of biomaterials. Granules of five kinds of P(2)O(5)-free Na(2)O-CaO-SiO(2) glasses, showing different apatite-forming ability in simulated body fluid (SBF), were implanted into a defect on the femoral condyle of rabbits. Bone ingrowth was evaluated using scanning electron microscopy among five kinds of glasses at 1, 2, 3, 6, and 12 weeks. Quantitative analysis was performed measuring the depth of new bone ingrowth from the periphery. In addition, the total areas of newly formed bone among glass particles were examined at 3 and 6 weeks using confocal laser scanning microscopy (CLSM) after weekly administration of fluorescent calcein. The depth of bone ingrowth among glass particles increased in proportion to their apatite-forming ability in vitro. The CLSM study showed a correlation between the quantities of labeled newly formed bone and in vitro apatite-forming ability. In the P(2)O(5)-free Na(2)O-CaO-SiO(2) glasses, the periods within 3-6 days for inducing apatite in SBF considered a necessary condition to convey bioactivity in vivo, and in vivo evaluations at 2-3 weeks is important to determine this. The in vivo bioactivity was precisely reproduced by apatite-forming ability in SBF. Therefore, evaluating apatite formation in SBF is a good screening test for the in vivo bioactivity of materials, resulting in reduction of the need for animal sacrifices and savings in experimental time.

The clinical risk of vertebral artery injury from cervical pedicle screws inserted in degenerative vertebrae.

Study Design. Postoperative outcomes of cervical pedicle screw (CPS) placement were evaluated. The screws were inserted in degenerative vertebrae using anatomic landmarks. Objective. To evaluate the risks of this procedure caused by misplacements and subsequent complications. Summary of Background Data. The CPS gives superior vertebral fixation but risks causing vertebral artery and spinal cord injury. However, few clinical reports have been published concerning CPS misplacement and subsequent complications. Therefore, the risk of this procedure is still unclear. Methods. There were 18 consecutive patients, who submitted to CPS fixation of degenerative vertebrae C2–C6, evaluated using computerized tomography (CT). In 5 of 9 patients in whom the screw critically violated the transverse foramen, viability of the vertebral artery was evaluated using multi-detector row CT angiography. Results. There were no intraoperative vertebral artery or spinal cord injuries and no serious postoperative complications. A total of 86 screws were inserted, and 25 (29%) breached the pedicle cortex. Of them, 84% deviated laterally and violated the transverse foramen. There were 13 screws (15%) that deviated more than 2 mm. However, multi-detector row CT angiography showed the continuity of the vertebral artery in all cases. Conclusions. The deviation rate of CPS placement using anatomic landmarks was higher than anticipated, although no serious complications were encountered. Refinement of the technique (e.g., using a computer-assisted navigation system) is strongly recommended.

Bioactive Ti metal analogous to human cancellous bone: Fabrication by selective laser melting and chemical treatments

Selective laser melting (SLM) is a useful technique for preparing three-dimensional porous bodies with complicated internal structures directly from titanium (Ti) powders without any intermediate processing steps, with the products being expected to be useful as a bone substitute. In this study the necessary SLM processing conditions to obtain a dense product, such as the laser power, scanning speed, and hatching pattern, were investigated using a Ti powder of less than 45 μm particle size. The results show that a fully dense plate thinner than 1.8 mm was obtained when the laser power to scanning speed ratio was greater than 0.5 and the hatch spacing was less than the laser diameter, with a 30 μm thick powder layer. Porous Ti metals with structures analogous to human cancellous bone were fabricated and the compressive strength measured. The compressive strength was in the range 35-120 MPa when the porosity was in the range 75-55%. Porous Ti metals fabricated by SLM were heat-treated at 1300 °C for 1h in an argon gas atmosphere to smooth the surface. Such prepared specimens were subjected to NaOH, HCl, and heat treatment to provide bioactivity. Field emission scanning electron micrographs showed that fine networks of titanium oxide were formed over the whole surface of the porous body. These treated porous bodies formed bone-like apatite on their surfaces in a simulated body fluid within 3 days. In vivo studies showed that new bone penetrated into the pores and directly bonded to the walls within 12 weeks after implantation into the femur of Japanese white rabbits. The percentage bone affinity indices of the chemical- and heat-treated porous bodies were significantly higher than that of untreated implants.

Expression of the p16INK4A Gene Is Associated Closely with Senescence of Human Mesenchymal Stem Cells and Is Potentially Silenced by DNA Methylation During In Vitro Expansion

The precise biological characteristics of human mesenchymal stem cells (hMSCs), including growth regulatory mechanisms, have not yet been defined. Using 29 strains of hMSCs isolated from bone marrow, we have performed extensive analyses of the growth profiles of hMSCs in vitro. All 29 strains stopped proliferating with a mean population doubling (PD) of 28, although there was a considerable difference among strains. The mean telomere restriction fragment length of the cells passaged twice correlated well with the final number of PDs in each strain, suggesting the value of this measurement to be predictive of the growth potential of hMSCs. The expression level of the p16INK4A gene was associated closely with the PD number of each strain (p = .00000001). Most of the p16INK4A‐positive cells were Ki67‐negative and senescence associated β‐galactosidase‐positive, and the suppression of p16INK4A gene expression by small interfering RNA in senescent hMSCs reduced the number of senescent cells and endowed them with the ability to proliferate. Twenty‐five of the 29 strains showed a steady gradual increase in the expression of p16INK4A. The remaining four strains (13.8%) showed different profiles, in which DNA methylation in the promoter region occurred in vitro. One of the four strains continued to proliferate for much longer than the others and showed chromosomal aberrations in the later stages. These results indicated p16INK4A to be a key factor in the regulation of hMSC growth, and, most importantly, careful monitoring of DNA methylation should be considered during the culture of hMSCs, particularly when a prolonged and extended propagation is required.

Bioactive macroporous titanium surface layer on titanium substrate

A macroporous titanium surface layer is often formed on titanium and titanium alloy implants for morphological fixation of the implants to bone via bony ingrowth into the porous structure. The surface of titanium metal was recently shown to become highly bioactive by being subjected to 5.0 M-NaOH treatment at 60 degrees C for 24 h and subsequent heat treatment at 600 degrees C for 1 h. In the present study, the NaOH and heat treatments were applied to a macroporous titanium surface layer formed on titanium substrate by a plasma spraying method. The NaOH and heat treatments produced an uniform amorphous sodium titanate layer on the surface of the porous titanium. The sodium titanate induced a bonelike apatite formation in simulated body fluid at an early soaking period, whereby the apatite layer grew uniformly along the surface and cross-sectional macrotextures of the porous titanium. This indicates that the NaOH and heat treatments lead to a bioactive macroporous titanium surface layer on titanium substrate. Such a bioactive macroporous layer on an implant is expected not only to enhance bony ingrowth into the porous structure, but also to provide a chemical integration with bone via apatite formation on its surface in the body.

Osteoinduction of porous Ti implants with a channel structure fabricated by selective laser melting.

Many studies have shown that certain biomaterials with specific porous structures can induce bone formation in non-osseous sites without the need for osteoinductive biomolecules, however, the mechanisms responsible for this phenomenon (intrinsic osteoinduction of biomaterials) remain unclear. In particular, to our knowledge the type of pore structure suitable for osteoinduction has not been reported in detail. In the present study we investigated the effects of interconnective pore size on osteoinductivity and the bone formation processes during osteoinduction. Selective laser melting was employed to fabricate porous Ti implants (diameter 3.3mm, length 15 mm) with a channel structure comprising four longitudinal square channels, representing pores, of different diagonal widths, 500, 600, 900, and 1200 μm (termed p500, p600, p900, and p1200, respectively). These were then subjected to chemical and heat treatments to induce bioactivity. Significant osteoinduction was observed in p500 and p600, with the highest observed osteoinduction occurring at 5mm from the end of the implants. A distance of 5mm probably provides a favorable balance between blood circulation and fluid movement. Thus, the simple architecture of the implants allowed effective investigation of the influence of the interconnective pore size on osteoinduction, as well as the relationship between bone quantity and its location for different pore sizes.

Vertebral artery injury during cervical spine surgery: a survey of more than 5600 operations.

Study Design. Retrospective survey. Objective. To clarify the present incidence and management of iatrogenic vertebral artery injury (VAI) during cervical spine surgery. Summary of Background Data. VAI is a rare complication of cervical spine surgery, but it may be catastrophic. Anterior cervical decompression (ACD) and posterior atlantoaxial transarticular screw fixation (Magerl fixation) have been the main causes, with reported incidences of 0.3% to 0.5% and 0% to 8.2%, respectively. Popular new surgical techniques, such as cervical pedicle screw or C1 lateral mass screw fixation, also entail the potential risk of VAI. Methods. A questionnaire was sent to our affiliated hospitals requesting information regarding iatrogenic VAI during cervical spine surgery. Results. Seven spine surgeon groups and 25 general orthopedist groups responded to the questionnaire, with a response rate of 89%. The overall incidence of VAI was 0.14% (8 cases among 5641 cervical spine surgeries). The incidence in anterior cervical decompression procedures was 0.18% and that in Magerl fixation was 1.3%. Inexperienced surgeons tended to commit VAI more frequently. One case of VAI during C1 lateral mass screw fixation was included, whereas there was no case of VAI caused by cervical pedicle screw fixation. In the case of “VAI in the screw hole,” hemostasis was obtained by tamponade or screw insertion, whereas “VAI in the open space” sometimes caused uncontrollable bleeding, in which embolization eventually stopped the bleeding. There were no deaths or apparent neurologic sequelae. Conclusion. The incidence of VAI during cervical spine surgery from this survey was similar to or slightly less than that in the literature. Tamponade was effective in many cases, but prompt consultation with an endovascular team is recommended if the bleeding is uncontrollable. Preoperative careful evaluation of the vertebral artery seems to be most important to prevent iatrogenic VAI and to avoid postoperative neurologic sequelae.

Comparison of the anatomical risk for vertebral artery injury associated with the C2-pedicle screw and atlantoaxial transarticular screw.

Study Design. We evaluated the trajectories of atlantoaxial transarticular and C2-pedicle screws in 3 dimensions using computerized tomography. Objective. To compare the anatomic risk for vertebral artery injury associated with C2-pedicle and atlantoaxial transarticular screws. Summary of Background Data. The atlantoaxial fixation technique using C1-lateral mass screws combined with C2-pedicle screws is considered a safer technique for preventing vertebral artery injury than atlantoaxial transarticular fixation. However, few reports have compared the anatomic risk of vertebral artery injury associated with C2-pedicle screws with that of transarticular screws. Methods. A total of 62 consecutive patients with cervical lesions were evaluated using 3-dimensional images reconstructed by a computer-assisted navigation system. We compared the maximum possible diameters of the atlantoaxial transarticular screw and C2-pedicle screw trajectories, and examined whether the maximum possible diameters were limited by the height or width of the bony structure in screw trajectories ≤4 mm in diameter. Results. Mean maximum possible diameters did not differ significantly between the trajectories of 124 atlantoaxial transarticular and 124 C2-pedicle screws. In screw trajectories ≤4 mm in diameter, 57.1% of transarticular screw trajectories were limited by the height of the bony structure, and all pedicle screw trajectories were limited by the width. Conclusions. C2-pedicle screw placement has nearly the same anatomic risk of vertebral artery injury as transarticular screw placement. Preoperative 3-dimensional evaluation may be useful for choosing the best surgical technique.

Bioactive titanium: effect of sodium removal on the bone-bonding ability of bioactive titanium prepared by alkali and heat treatment.

As reported previously, bioactive titanium is prepared by simple alkali and heat treatment, and can bond to living bone directly. The purpose of this study was to accelerate the bioactivity of bioactive titanium in vivo. In in vitro study, sodium removal by hot water immersion enhanced the apatite-forming ability of bioactive titanium in simulated body fluid dramatically. The specific anatase structure of titania gel was effective for apatite formation in vitro. In the current study, we investigated the in vivo effect of sodium removal on the bone-bonding strength of bioactive titanium. Sodium-free bioactive titanium plates were prepared by immersion in an aqueous solution of 5 M NaOH at 60 degrees C for 24 h, followed by immersion in distilled water at 40 degrees C for 48 h before heating them at 600 degrees C for 1 h. Three kinds of titanium plates were inserted into rabbit tibiae, including untreated cp-Ti, conventional alkali- and heat-treated Ti, and sodium-free alkali- and heat-treated Ti. In vivo bioactive performance was examined mechanically and histologically after 4, 8, 16, and 24 weeks. Sodium removal enhanced the bone-bonding strength of bioactive titanium at 4 and 8 weeks postoperatively; however, its bone-bonding strength was inferior to that of conventional alkali- and heat-treated titanium at 16 and 24 weeks. Histological examinations after the detaching test revealed breakage of the treated layer in the sodium-free alkali- and heat-treated titanium group. In conclusion, sodium removal accelerated the in vivo bioactivity of bioactive titanium and achieved faster bone-bonding because of its anatase surface structure, but the loss of the surfaces graded structure due to the complete removal of sodium decreased the adhesive strength of the treated layer to the titanium substrate. Further investigations are required to determine the optimum conditions for preparation of bioactive titanium.