Takayasu Kubo

Continuous Administration of Basic Fibroblast Growth Factor (FGF-2) Accelerates Bone Induction on Rat Calvaria— An Application of a New Drug Delivery System:

Some studies have shown that locally applied basic fibroblast growth factor (FGF-2) enhances bone regeneration at a fracture site, while others have not been in agreement. We developed a new continuous FGF-2 delivery system designed to accelerate cytokine-induced new bone formation. A subperiosteal pocket was surgically formed in 36 eight-week-old male Wistar rats. The rats were administered 0, 1, 10, or 100 ng of FGF-2 contained in a collagen minipellet, mixed with allogeneic demineralized bone matrix in a dome-shaped Millipore® filter and then placed into the pocket. New bone formation in the dome was evaluated at 2, 4, and 8 wks after placement. Soft x-ray radiographs disclosed an apparently larger radiopaque region in the 1-ng group at 4 wks compared with those in the other groups. Morphometrical analysis revealed that the new bone area in the 1-g group was significantly larger than that in the 0-g group (p < 0.01). In the 100-ng FGF-2 group, new bone formation seemed suppressed. We concluded that continuous slow administration of a small amount of FGF-2 accelerates bone-derived osteogenic cytokine-induced new bone formation.

Initial bone regeneration around fenestrated implants in Beagle dogs using basic fibroblast growth factor-gelatin hydrogel complex with varying biodegradation rates

PURPOSE The purpose of this study was to compare the effectiveness of fast and slow biodegradation of basic fibroblast growth factor (bFGF)-gelatin hydrogel complex on bone regeneration around fenestrated implants as a new augmentation drug delivery system. METHODS Nine titanium implants (3.3mm diameter and 10mm length) were placed into the edentulous areas of the mandibles of three adult beagle dogs with four screws exposed at the upper buccal side. The effectiveness of bFGF-gelatin hydrogel complexes of varying degradation types used to cover implant screws without membrane were compared with 1 microg and 10 microg bFGF-98 wt% gelatin as the fast degradation type and 10 microg bFGF-95 wt% gelatin as the slow degradation type. After 4 weeks, bone regeneration around the screws was evaluated histologically and histomorphometrically. RESULTS With use of 10 microg bFGF, regenerated bone around exposed screws was clearly seen in both the fast and slow degradation type groups. In contrast, little bone formation was seen in the fast degradation-type group with 1 microg bFGF. Height of regenerated bone for the slow degradation-type complex group was significantly greater than for the fast degradation-type group with 1 microg bFGF (P<0.05). CONCLUSION These results suggest that use of slow degradation-type bFGF-gelatin hydrogel complex may accelerate bone regeneration around fenestrated implants at an early stage of bone regeneration.

Effect of combined application of bFGF and inorganic polyphosphate on bioactivities of osteoblasts and initial bone regeneration

Basic fibroblast growth factor (bFGF) and inorganic polyphosphate (poly(P)) have been recognized as therapeutic agents that enhance bone regeneration. It has also been shown that poly(P) may enhance the mitogenic activity of bFGF. The purpose of this study is to evaluate the combined effect of bFGF and poly(P) on bioactivities of osteoblasts and initial bone regeneration in vitro and in vivo. MC3T3-E1 cells were treated with bFGF, poly(P) or bFGF+poly(P), then subjected to cell proliferation assay, alkaline phosphatase (ALP) activity measurement, quantitative real-time reverse transcription-polymerase chain reaction and Alizarin S Red staining. In an in vivo study, bFGF-, poly(P)- and bFGF+poly(P)-modified interconnected porous hydroxyapatite (IPHA) complexes were fabricated, and placed into the femurs of rabbits to evaluate new bone formation histologically and histomorphometrically. The highest enhancement of cell proliferation were observed in those treated with bFGF+poly(P) on days 5 and 7. Cells treated with bFGF+poly(P) also exhibited increased ALP activity on days 5 and 10, up-regulated mRNA levels of osteocalcin and osteopontin, and enhanced calcification when compared to the non-treated cells. In vivo, the highest bone formation ratio was observed in bFGF+poly(P)-modified IPHA complexes. This study indicated that co-application of bFGF and poly(P) may provide enhanced bone formation by modulating cell proliferation and the mineralization process. It is anticipated that a combined application of bFGF and poly(P) can provide a novel method for bone regeneration in clinical use.

Enhanced initial bone regeneration with inorganic polyphosphate-adsorbed hydroxyapatite.

Inorganic polyphosphate (poly(P)) can promote binding between fibroblast growth factors and their receptors and enhance osteoblastic cell differentiation and calcification. This study evaluated the possibilities for poly(P) adsorbed onto interconnected porous calcium hydroxyapatite (IP-CHA) as a new bone regeneration material. Prepared 1%, 5%, 25% and 50% poly(P)/IP-CHA composites showed the elution peak of poly(P) between 15 and 20 min, respectively, with the highest value from 50% poly(P)/IP-CHA in vitro. Histologically, at 1 week of placement into the femur of rabbits, granulation tissue had penetrated into the pores in all composites and IP-CHA as a control. In contrast, at 2 weeks of placement, newly formed lamellar bone was found in all groups, although a higher amount of bone regeneration was obviously formed in the 25% and 50% poly(P)/IP-CHA with a significantly higher value of bone regeneration ratio of 50% poly(P)/IP-CHA. These results indicate that 25% and 50% poly(P)/IP-CHA composites may enhance initial bone regeneration.

The three-dimensional bone interface of an osseointegrated implant. I: A morphometric evaluation in initial healing

Direct bone-implant interface as an indicator of endosseous implant success appears to have been overinterpreted because 100% bone apposition is not necessarily obtained at the surface of the endosseous dental implant. The purpose of this study was to obtain quantitative information about the three-dimensional bone structure around three hydroxyapatite-coated titanium alloy dental implants. Implants were placed in the mandible in three monkeys, and the surface bone contact ratio in the buccal, lingual, mesial, and distal directions was computed. Computer graphics were generated by the integration of data for serial ground surfaces obtained at 75 microm intervals of the tissue block involved with the implant. The bone contact ratio of the whole surface of each of the three implants was 80.8%, 68.1%, and 68.8%, and the bone contact ratio for each direction and portion varied with the conditions of implant placement. The bone volume ratios around the implant at the 0 to 300 microm zone were also calculated, and total ratios ranged from 58% to 81%. These results may provide useful quantitative information about the bone structure around the hydroxyapatite-coated implants and contribute to the development of realistic finite element analysis models based on the biologic bone structure around the implants.

Comparative evaluation of bone regeneration using spherical and irregularly shaped granules of interconnected porous hydroxylapatite. A beagle dog study

PURPOSE Bone regeneration stimulated by two different shapes of interconnected porous calcium hydroxyapatite (IP-CHA) granules was evaluated in the mandibles of 3 beagle dogs. METHODS Deferent shapes of IP-CHA were used, spherical (spherical shaped IP-CHA granules) and irregular shapes (irregularly shaped IP-CHA granules). Two bone sockets (3mm in diameter and 5mm in depth) were prepared in the right edentulous mandible of each animal where right premolars had been extracted and sites healed for 3 months. The two types of IP-CHA were filled into the sockets to stimulate bone regeneration. New bone formation was evaluated histologically at 4, 8 and 12 weeks after filling. RESULTS At 4 weeks, little bone formation was apparent in any of the bony sockets. At 8 weeks, newly formed bone was detected between the granules but not in the pores. In contrast, at 12 weeks, bone formation was clearly observed not only between the granules but also inside the granule pores. Comparing the two sites at 8 and 12 weeks, more bone formation was detected in sites receiving irregularly shaped IP-CHA than in sites receiving spherical IP-CHA. CONCLUSION These results indicate that use of irregularly shaped IP-CHA may enhance bone regeneration. The results of this preliminary study suggest that irregularly shaped IP-CHA granules may have more possible usefulness than spherically shaped granules as a scaffold for bone regeneration.

Polyphosphate-Mediated Inhibition of Tartrate-Resistant Acid Phosphatase and Suppression of Bone Resorption of Osteoclasts

Inorganic polyphosphate (poly(P)) has recently been found to play an important role in bone formation. In this study, we found that tartrate-resistant acid phosphatase (TRAP), which is abundantly expressed in osteoclasts, has polyphosphatase activity that degrades poly(P) and yields Pi as well as shorter poly(P) chains. Since the TRAP protein that coprecipitated with anti-TRAP monoclonal antibodies exhibited both polyphosphatase and the original phosphatase activity, poly(P) degradation activity is dependent on TRAP and not on other contaminating enzymes. The ferrous chelator α, α’-bipyridyl, which inhibits the TRAP-mediated production of reactive oxygen species (ROS), had no effect on such poly(P) degradation, suggesting that the degradation is not dependent on ROS. In addition, shorter chain length poly(P) molecules were better substrates than longer chains for TRAP, and poly(P) inhibited the phosphatase activity of TRAP depending on its chain length. The IC50 of poly(P) against the original phosphatase activity of TRAP was 9.8 µM with an average chain length more than 300 phosphate residues, whereas the IC50 of poly(P) with a shorter average chain length of 15 phosphate residues was 8.3 mM. Finally, the pit formation activity of cultured rat osteoclasts differentiated by RANKL and M-CSF were markedly inhibited by poly(P), while no obvious decrease in cell number or differentiation efficiency was observed for poly(P). In particular, the inhibition of pit formation by long chain poly(P) with 300 phosphate residues was stronger than that of shorter chain poly(P). Thus, poly(P) may play an important regulatory role in osteoclastic bone resorption by inhibiting TRAP activity, which is dependent on its chain length.

Development of implant/interconnected porous hydroxyapatite complex as new concept graft material.

Background Dental implant has been successfully used to replace missing teeth. However, in some clinical situations, implant placement may be difficult because of a large bone defect. We designed novel complex biomaterial to simultaneously restore bone and place implant. This complex was incorporated implant into interconnected porous calcium hydroxyapatite (IP-CHA). We then tested this Implant/IP-CHA complex and evaluated its effect on subsequent bone regeneration and implant stability in vivo. Methodology/Principal Findings A cylinder-type IP-CHA was used in this study. After forming inside of the cylinder, an implant was placed inside to fabricate the Implant/IP-CHA complex. This complex was then placed into the prepared bone socket in the femur of four beagle-Labrador hybrid dogs. As a control, implants were placed directly into the femur without any bone substrate. Bone sockets were allowed to heal for 2, 3 and 6 months and implant stability quotients (ISQ) were measured. Finally, tissue blocks containing the Implant/IP-CHA complexes were harvested. Specimens were processed for histology and stained with toluidine blue and bone implant contact (BIC) was measured. The ISQs of complex groups was 77.8±2.9 in the 6-month, 72.0±5.7 in the 3-month and 47.4±11.0 in the 2-month. There was no significant difference between the 3- or 6-month complex groups and implant control groups. In the 2-month group, connective tissue, including capillary angiogenesis, was predominant around the implants, although newly formed bone could also be observed. While, in the 3 and 6-month groups, newly formed bone could be seen in contact to most of the implant surface. The BICs of complex groups was 2.18±3.77 in the 2-month, 44.03±29.58 in the 3-month, and 51.23±8.25 in the 6-month. Significant difference was detected between the 2 and 6-month. Conclusions/Significance Within the results of this study, the IP-CHA/implant complex might be able to achieve both bone reconstruction and implant stability.

DIRECT BONE INDUCTION IN THE SUBPERIOSTEAL SPACE OF RAT CALVARIA WITH DEMINERALIZED BONE ALLOGRAFTS

In order to clarify the understanding of bone induction with crude bone morphogenetic protein (BMP)-containing allografts in subperiosteal conditions, chondrogenesis and osteogenesis were histologically evaluated following the implantation of the demineralized bone (DB) in the subperiosteal space of calvaria of 30 Wistar rats. On the forehead of the rat, DB particles were placed onto the denuded calvarial bone and covered by the skin-periosteum flap without any perforations of the marrow space of the calvaria. Sintered hydroxylapatite particles (HA) were also placed as a control. In the DB group, new bone formation on the surface of calvaria was achieved between 2 and 8 weeks after the operation. However, no chondrogenesis was seen throughout the experimental period. In the HA implantation group, fibrous tissue encapsulation of HA particles was generally seen. These results suggest that DB containing crude BMP might have the capacity for direct osteoblast induction from undifferentiated mesenchymal progenitor cells in vivo in specific situations, that is, in a subperiosteal space of uninjured rat calvaria.

Influence of Implant Surface Topography on Primary Stability in a Standardized Osteoporosis Rabbit Model Study

Evaluating primary stability is important to predict the prognosis of dental implant treatment. Primary stability is decreased in a low bone density site such as osteoporosis. However, it is difficult to apply in small animal and the effect of the different implant surface topography for the primary stability at low bone density site has not yet fully been investigated. The purpose of the present study was to evaluate the influence of implant surface topography on primary stability in a standardized osteoporosis animal model. Six rabbits underwent ovariectomy and administrated glucocorticoid to induce an osteoporosis model. Sham-operations were performed in additional six rabbits. Implants with machined or oxidized-surfaces were inserted into the femur epiphyses and insertion torque (IT) and implant stability quotient (ISQ) were measured. In sham model, the IT and ISQ did not differ significantly between the both implant. However, the IT value of oxidized-surface implant was significantly higher than that of the machined implant in the osteoporosis model. Meanwhile, ISQ did not significantly differ between the machined and oxidized-surfaced implants. In conclusion, the IT of implants is higher with rough than with smooth surfaces but that there are no differences in ISQ value between different surfaces in a standardized osteoporosis bone reduced rabbit model.