Enhanced bone bonding of the hydroxyapatite/beta-tricalcium phosphate composite by electrical polarization in rabbit long bone.

A review of the osteogenic cell activity and new bone growth in the regions bordering negatively charged surfaces of polarized Hydroxyapatite/beta-tricalcium phosphate (HA/TCP) composites implanted in the long bone in rabbits was conducted. Polarized and non-polarized HA/TCP specimens were implanted into the right and left femoral condyle, respectively (each n = 10). After 3 and 6 weeks, five rabbits were sacrificed in each group, and histological analysis was administered. Large cuboidal-shaped osteoblastic cells were predominantly observed lining the newly formed bone on the negatively charged surface (N-surface) in the polarized HA/TCP implants. The TRAP-positive multinucleated cells were observed extensively in the newly formed bone on the N-surfaces compared with the 0-surface and adhered directly to the HA/TCP composite. The bone area (B.Ar) value, newly formed bone area contacting the implant, and contact length (C.Le) value, percentage length of newly formed bone directly attaching to the implant, on both the 0- and N-surface increased significantly with time in each group. Both the B.Ar and C.Le value on the N-surface were significantly greater than those on the 0-surface after 3 and 6 weeks. The number of TRAP-positive cells/total length value on the N-surface was significantly greater than that on the 0-surface after 3 and 6 weeks postoperatively. It is hypothesized that electrical charge acquired by electrical polarization treatment may modify the biochemical and biophysical processes of the osteogenic cells, resulting in enhanced new bone formation and direct bonding between the recipient bone and implants.

Enhanced new bone formation in canine maxilla by a graft of electrically polarized ß-tricalcium phosphate.

We succeeded in the electrical polarization of ß-tricalcium phosphate (ß-TCP) granules and performed an unprecedented attempt to implant them into maxillary bone defects in canines to confirm their ability to facilitate new bone formation. Two holes were drilled into each maxilla half of a canine and filled with electrically polarized and nonpolarized ß-TCP granules (grouping assignment was decided randomly). The implanted specimens were dissected en bloc and used for microcomputed tomography (µCT) observations and histological analyses 4 and 8 weeks after the operation. New bone ingrowth in the bone hole progressed over time from the superficial layer of the cortex toward the inner cancellous bone. The percentage area of new bone in the bone hole, as measured by µCT in the sagittal plane, was significantly larger after 4 and 8 weeks, and that measured by H&E-stained specimens in the transverse plane after 4 weeks was significantly larger in the polarized group than in the nonpolarized group. In addition to the structural stability and chemical characteristics of the ß-TCP granules, electrical stimulation bears influence not indirectly but directly on osteogenic and vessel cells, which might work cooperatively for the early initiation of the bone formation process.

The axonal regeneration across a honeycomb collagen sponge applied to the transected spinal cord.

We developed a honeycomb-shaped lyophilized Type I atelocollagen (Honeycomb Collagen: HC) with different pore sizes, and the effectiveness of the honeycomb shape on nerve regeneration was examined. We analyzed neurite outgrowth of dorsal root ganglion (DRG) explants on HC, both in vitro and, with direct implantation of HC into the defects of adult rat spinal cords, in vivo. The neurites of DRGs on HC extended linearly through the pores. HC with a 400 microm-pore size enhanced neurite extension, and YIGSR laminin peptide coating to the HC extended more neurites than fibronectin coating. The HC scaffolds coated with YIGSR were implanted into 2 mm-defects of spinal cords at the level of T8-9. Four weeks after implantation, the implants had degraded and been replaced with self-tissues, repairing the injured site. Neurofilament-positive fibers were observed in the implantation area and passed the borders between the HC and spinal cord stumps. Functionally, a motor-evoked potential was observed in the quadriceps femoris muscle 10 weeks after implantation. The electrophysiological examination showed reconstruction of axon tracts over the implant. This result indicates that our developed honeycomb shape is advantageous for host spinal cord compared to the random pored sponge shape, and that it promotes axonal regeneration after spinal cord injury.

Enhanced bone bonding of hydroxyapatite-coated titanium implants by electrical polarization.

Hydroxyapatite (HAp) coatings are applied to orthopedic and dental implants made of titanium (Ti) and its alloys in order to increase their bioactivities and to offset the mechanical weakness of HAp. We examined the in vivo effects of electrical polarization on the bone bonding of HAp-coated Ti. Polarized samples with a negatively or positively charged HAp-coated surface (N- or P-surface, respectively) were randomly implanted in the femora and tibiae of canines. As controls, nonpolarized HAp-coated Ti substrates with 0-surfaces were implanted. Direct bonding between the newly formed bone and HAp-coated Ti was observed with the O-, N-, and P-surfaces. The results of a pullout test were consistent with the amount of newly formed bone bonded directly to the surface of HAp-coated Ti. Electrically polarized HAp-coated Ti substrates, especially those with N-surfaces, exposed to recipient bone enhance bone bonding and could enable earlier weight-bearing loads after operations.

Regeneration of peripheral nerves by bioabsorbable polymer tubes with fibrin gel.

We developed two types of nerve conduits, straight tubes, and bellows tubes, for peripheral nerve regeneration with bioabsorbable polymer membranes. Mechanical properties of these straight and bellows tubes were analyzed. 30 straight tubes and 30 bellows tubes were implanted to a nerve defect made in a rat sciatic nerve and the nerve regeneration in the tube was investigated. A half of these tubes were utilized alone and the others were filled with fibrin gel made from coagulated plasma and implanted. Bellows tubes were superior in mechanical characteristics to straight tubes and the inner cavities of the bellows tubes were suitably maintained after implantation. At 4 weeks after operations, remyelinations were observed in the regenerated tissue at the location of the middle parts of the tubes filled with fibrin gel whereas no remyelinations in the tubes without fibrin gel. The use of fibrin gel as filling materials within the tubes significantly enhanced the nerve regeneration. The fibrin gel might be soft nanomaterials significantly enhance the regeneration of the peripheral nerves. We concluded that the developed bioabsorbable bellows tubes filled with fibrin gel were effective for peripheral nerve regeneration.

Enhanced bone ingrowth into hydroxyapatite with interconnected pores by Electrical Polarization.

Hydroxyapatite (HA) ceramics are used as implants to repair damaged/removed bone, and negative or positive electrical polarization enhances osteoblast and decreases osteoclast activity, respectively, in vivo. We compared the ability of electrically polarized and non-polarized HA with interconnected pores (IPHA) implants to promote bone growth. Polarized or non-treated IPHAs were implanted into the right or left femoral condyle of rabbits (N = 10 in each group), and we performed histological examination, including enzymatic staining for osteoblasts and osteoclasts, 3 and 6 weeks after implantation. We observed improved bone ingrowth and increased osteoblast activity in polarized implants with complete bone penetration into polarized implants occurring as early as 3 weeks after surgery. In contrast, non-polarized implants were not fully ossified at 6 weeks after surgery. Furthermore, positively charged implant regions had decreased osteoclast activity compared to negatively charged or uncharged regions. We propose two different models to explain these observations.

Comparison of nerve regenerative efficacy between decellularized nerve graft and nonwoven chitosan conduit.

BACKGROUND: Recently decellularized nerves with various methods are reported as highly functional nerve grafts for the treatment of nerve defects. OBJECTIVE: To evaluate the efficacy of decellularized allogeneic nerve, compared with oriented chitosan mesh tube, and an autologous nerve. METHODS: Sciatic nerves harvested from Sprague-Dawley (SD) rats were decellularized in combination with Sodium dodecyl sulfate and Triton X-100. A graft into the sciatic nerve in Wistar rats was performed with the decellularized SD rat sciatic nerves or oriented chitosan nonwoven nanofiber mesh tubes (15 mm in length, N=5 in each group). A portion of sciatic nerve of Wistar rat was cut, reversed and re-sutured in-situ as a control. Nerve functional and histological evaluations were performed 25 weeks postoperatively. RESULTS: It was revealed that functional, electrophysiological and histological recoveries in the decellularized nerve group match those in the autograft group. Recovery of sensory function and nerve maturation in the decellularized nerve group were superior to those in the chitosan mesh tube group. CONCLUSIONS: Nerve regeneration in the decellularized nerves could match that in the autografts and is somehow superior to artificial chitosan mesh tube. Detergents wash of SDS and Triton X-100 could obtain highly functional nerve grafts from allografts.

Immobilization of laminin peptide in molecularly aligned chitosan by covalent bonding.

We developed a new biomaterial effective for nerve regeneration consisting of molecularly aligned chitosan with laminin peptides bonded covalently. Molecularly aligned chitosan was prepared from crab (Macrocheira kaempferi) tendons by ethanol treatment and 4 wt%-NaOH aqueous solutions to remove proteins and calcium phosphate, followed by deacetyl treatment using a 50 wt%-NaOH aqueous solution at 100 degrees C. Molecularly aligned tendon chitosan was chemically thiolated by reacting 4-thiobutyrolactone with the chitosan amino group. The introduction of thiol groups and their distribution to tendon chitosan and chitosan cast film were confirmed using ATR FT-IR, (1)H-NMR, and EDS. The 1.24 micromol/g of thiol groups introduced on the surface of tendon chitosan and the chitosan cast film was confirmed using ultraviolet (UV) spectra. Thiol groups of cysteine located at the end of synthetic laminin peptides were then reacted chemically with thiolated chitosan to form chitosan-S-S-laminin peptide. YIGSR estimated at 0.92 micromol/g and IKVAV estimated at 0.28 micromol/g on thiolated tendon chitosan were confirmed using UV spectra. YIGSR was estimated at 0.85 micromol/g and IKVAV was estimated at 0.34 micromol/g on the thiolated chitosan cast film.

Effects of a laminin peptide (YIGSR) immobilized on crab-tendon chitosan tubes on nerve regeneration.

Thiolated and nonthiolated hydroxyapatite-coated crab-tendon chitosan (t-chitosan/HAp-SH and t-chitosan/HAp, respectively) tubes, both alone and conjugated with CDPGYIGSR (YIGSR) peptide, were compared, in order to determine their biocompatibility and efficacy as nerve conduits. YIGSR peptide was adsorbed on the t-chitosan/HAp (HAp) tubes, and covalently bound on the t-chitosan/HAp-SH (HAp-SH) tubes (Y/HAp and Y/HAp-SH tubes, respectively). HAp, HAp-SH, Y/HAp, or Y/HAp-SH tubes measuring 15 mm were bridge grafted into the sciatic nerve of SD rats. Grafting of 15-mm-long Type I atelocollagen tubes and isografting of sciatic nerves were also carried out (N = 6 in each group). After 12 weeks, evoked muscle action potentials were recorded to calculate the terminal latency quotient. Histological observation and analysis of myelinated axons were also carried out. Nerve-tissue regeneration did not occur directly on the tubes' surfaces in the YIGSR peptide-unconjugated groups. Transplantation of YIGSR-conjugated tubes, however, gave rise to regenerated nerve tissue attached to thin layers of epineurium-like structure formed on the inner-tube surface. Histological and electrophysiological analyses suggested that although thiolation retards nerve-tissue regeneration, adsorbed YIGSR, and, to a lesser extent, peptide that had been covalently bound onto the tube surfaces, enhance nerve regeneration, promoting sprouting from the proximal nerve stump and bridging of regenerated axons throughout the tube.

Development of a novel biomaterial, hydroxyapatite/collagen (HAp/Col) composite for medical use.

A hydroxyapatite/type I collagen (HAp/Col) composite, aligning hydroxyapatite nano-crystals along collagen molecules, has been synthesized. The biocompatibility, osteoconductivity and efficacy as an rhBMP-2 carrier of this novel biomaterial implanted in the weight-bearing site have been examined. The HAp/Col implants adsorbing 0 or 400 microg/ml of rhBMP-2 were implanted into bone defects of tibiae in 3 beagle dogs and fixed according to the Ilizarov method. As a control, bone defects of 20 mm remaining in 2 beagle dogs and the dogs were allowed to walk using a Ilizarov external skeletal fixator. The radiological and histological findings suggest that the implants induce bone remodeling units and are a superior carrier of rhBMP-2 due to the stimulation of early callus and new bone formation. As a next step, anterior fusion was carried out on 6 beagle dogs with the implants adsorbing 400 microg/ml of rhBMP-2, and 9 dogs with the implants without rhBMP-2. In 3 dogs of the rhBMP-treated group, as well as 6 dogs of the non-rhBMP-treated group, the implant was fixed with a poly-L-lactide plate. Histological and radiographical analysis suggest that enhancement of callus formation and bone bridging by rhBMP-treatment is effective to prevent collapse of the implant.

In vivo evaluation of a Ti-based bulk metallic glass alloy bar.

Ti-based bulk metallic glasses are reported with high strength, low Young modulus and high corrosion resistance, suggesting their potentials in biomedical applications. However a thorough in vivo evaluation of its biocompatibilities has not been conducted yet. In this study, we implanted bars of Ti-based bulk metallic glass in the femoral bone of rats, followed up local tissue reaction as well as its component ions' diffusion in local area and whole body. The Ti-based BMG (Ti40Zr10Cu34Pd14Sn2) alloy exhibited favorable features of both high strength and high elasticity. In vivo implant evaluation showed that it has a good tissue compatibility, equivalent bone integration and bonding ability with Ti sample. No component ion diffusion was detected up to 3 months post implantation. The possibility and efficacy of its use for bone implant is confirmed. Thus further long term implant study is recommended.

The chitosan prepared from crab tendons: II. The chitosan/apatite composites and their application to nerve regeneration.

The chitosan tubes derived from crab tendons form a hollow tube structure, which is useful for nerve regeneration. However, in order to use the chitosan tubes effectively for nerve regeneration, there remain two problems to be solved. First, the mechanical strength of the tubes is quite high along the longitudinal axis, but is somewhat low for a pressure from side. Second, the chitosan tube walls swell to reduce the inner space of the tubes in vivo. These two problems limit the clinical use of the chitosan tubes. In this study, to solve the problems, apatite was made to react with the chitosan tubes to enhance the mechanical strength of the tube walls. Transmission electron microscopy showed that apatite crystals were formed in the walls of the chitosan tubes. The c-axis of the crystals aligned well in parallel with chitosan molecules. These results indicate that the apatite crystals grow in the tubes starting from the nucleation sites of the chitosan molecules, probably by forming complexes with amino groups of chitosan and calcium ions. Further, the tubes were thermally annealed at 120 degrees C to prevent from swelling, and simultaneously formed into a triangular shape to enhance the stabilization of the tube structure. By these treatments, the hollow tubes could keep their shape even in vivo after implantation. Animal tests using SD rats further showed that the chitosan tubes effectively induced the regeneration of nerve tissue, and were gradually degraded and absorbed in vivo.

The chitosan prepared from crab tendon I: the characterization and the mechanical properties.

Crystalline chitosan was prepared from crab tendon consisting mainly of chitin, including various proteins and calcium phosphates. The crab tendon has high mechanical properties due to its aligned molecular structure. Crab tendon components, i.e. proteins and calcium phosphates, were removed by deacetyl treatment using 50wt% NaOH aqueous solution at 100 degrees C, and a subsequent ethanol treatment. As judged from microscopic observations using an optical polarizer, the treated chitosan remained intact regarding its aligned molecular structure, and had a high tensile strength of 67.9+/-11.4MPa. The tensile strength was further enhanced to 235+/-30MPa by a thermal treatment at 120 degrees C, corresponding to the formation of the intermolecular hydrogen bonds.

Evaluation of cross-linking procedures of collagen tubes used in peripheral nerve repair.

Three different cross-linking methods were compared to prepare collagen tubes, namely irradiation by ultraviolet (UV), heating and immersing in glutaraldehyde (GA). Bridge grafting of 15 mm was carried out with these tubes, as well as with non-cross-linked collagen tubes for comparison, in a defect of rat sciatic nerves (N = 21 in each group). As a control, isografting was carried out (N = 6). The specimens were taken from the grafted site in each experimental group for histological observation after, respectively 1, 2, 4, 6 and 8 weeks (N = 3 each). Evoked muscle action potentials were recorded on the calf muscle in the experimental and control groups after 12 weeks, and the grafted material and tibial nerve were harvested for histological analysis (N = 6). The inner space of UV-irradiated tubes was preserved with almost no cell infiltration and nerve regeneration matching for isograft was obtained. Rapid degradation of the heat-treated tubes occurred and many macrophages were mobilized to remove the collagen debris. The non-treated tube swelled and the regenerated nerve tissue in the tube was constricted with time. The GA-immersed tubes showed less cellular activity and poor regenerated nerve tissue compared with the other cross-linking methods. Therefore, UV irradiation to collagen tubes is recommended as a cross-linking method for nerve conduit.

Development of an artificial vertebral body using a novel biomaterial, hydroxyapatite/collagen composite.

Hydroxyapatite/collagen (HAp/Col) composites having a bone-like nanostructure were synthesized and shaped into implants. This study was designed to develop an artificial vertebra system using this novel implant for anterior fusion of the cervical spine. Anterior fusion was carried out on 6 beagle dogs with the implants adsorbing rhBMP-2 (400 microg/ml). and 9 dogs with the implants without rhBMP-2. In 3 dogs of the rhBMP-treated group, as well as 6 dogs of the non-rhBMP-treated group, the implant was fixed with a poly-L-lactide plate and 2 titanium screws. Implants were taken out after 13 weeks from each 3 dogs in the rhBMP(-):plate(-). rhBMP(-):plate(+) and rhBMP(+):plate(+) groups. Also, the implants were removed from each 3 dogs in the rhBMP(-):plate(+) and rhBMP(+):plate(+) groups after 24 weeks. Histological and radiographical analysis suggested that since the larger part of the composite material was absorbed within 13 weeks, reduction of the intervertebral distance was caused, and that enhancement of callus formation and bone bridging by rhBMP-treatment was effective to prevent collapse of the implant, even though an effect of anterior plate-fixation was not obvious. The HAp/Col implant adsorbing rhBMP-2 may be a suitable replacement for the existing ceramics in anterior interbody fusion of the cervical spine.

Development of a hydroxyapatite/collagen nanocomposite as a medical device.

The effect of cross-linking of a hydroxyapatite/collagen (HA/Col) nanocomposite, in which HA nanocrystals and collagen fibers are aligned like natural bone by a self-organization mechanism between HA and collagen in vitro, on mechanical properties was examined. The influence of degree of cross-linking, as well as rhBMP-2 preadsorption to the composite on the substitution pattern and rate with bone, was examined. In Experiment 1, anterior fusion was carried out at the C3-C4 vertebrae on 10 dogs and they were implanted as follows: without cross-linking and without adsorbed rhBMP-2 (three dogs), with cross-linking and without adsorbed rhBMP-2 (three dogs), without cross-linking and with adsorbed rhBMP-2 (two dogs), and with cross-linking and adsorbed rhBMP-2 (two dogs). Implants were removed from each dog for histology determinations after 12, 16, and 24 weeks in the non-rhBMP-treated groups, and after 16 and 24 weeks in the rhBMP-treated groups. In Experiment 2, the HA/Col composites with cross-linking and both with and without rhBMP-2 pretreatment were implanted into a bone defect of 20 mm made in the central part of tibiae in dogs (N = 3 in each group). As a control, bone defects of 20 mm remained without implantation (N = 3). The dogs were allowed to walk using an Ilizarov extra skeletal fixator. The implants were removed after 12, 16, and 24 weeks from one dog in each group. The cross-linking of the HA/Col composite was effective in controlling both the mechanical strength and bioresorbability. A "self-organization process" on the HA/Col implant surface resulted in the formation of bone remodeling units in and around the implant. Radiographic and histological findings suggest that a combined treatment of cross-linking of the HA/Col composite with preadsorption of rhBMP-2 molecules may be a very suitable replacement of existing ceramic systems in the anterior fusion of the cervical spine, as well as inlay grafting of bone defects in weight-bearing sites.

Hydroxyapatite-coated tendon chitosan tubes with adsorbed laminin peptides facilitate nerve regeneration in vivo.

On the inner surface of tendon chitosan tubes having a triangular shape and a hydroxyapatite coating (t-chitosan/HAp tube), laminin-1 and laminin peptides (YIGSR, IKVAV) have been adsorbed in order to develop nerve growth conduits. The mechanical property, biocompatibility and efficacy of these tubes for nerve regeneration were examined. Step-1: bridge grafting (15 mm) into the sciatic nerve of Sprague-Dawley (SD) rats was carried out using either t-chitosan or t-chitosan/HAp tubes having either a circular or triangular cross section (N=12 in each group). Specimens were taken after 2-, 4-, 6- and 8-week post-implantation (N=3 in each group) for histology determinations. Step-2: t-chitosan/HAp tubes having a triangular cross section with adsorbed laminin-1, CDPGYIGSR or CSRARKQAASIKVAVSAD, as well as control tubes without pre-adsorption were used for implantation (N=18 in each group). Isografting was also carried out (N=6). Histological evaluation was carried out similarly as in Step-1. Furthermore, evoked muscle and sensory nerve action potentials were recorded, and the percentage of myelinated axon area measured at 10 mm distance of the distal anastomosed site in the experimental, control and isograft groups after 12 weeks (N=6 in each group). The results of histological findings, as well as mechanical properties, suggest that a triangular tube shape with a HAp coating benefits nerve regeneration. The effect of laminin peptides (YIGSR, followed by IKVAV) to enhance the growth of regenerating axons has been found comparable with intact laminin-1. Although histological regeneration in both the YIGSR- and laminin-1-treated t-chitosan/HAp tubes matches the isografts, the functional recovery is however delayed.

Muscle-specific protein MDP77 specifically promotes motor nerve regeneration in rats.

This study has examined the effects of recombinant human MDP77 (rhMDP77) on sciatic motor nerve regeneration in vivo. We carried out bridge grafting (14 mm) into the sciatic nerve of Sprague-Dawley rats using silicone tubes containing a mixture of type-I collagen and 0, 5, 10, or 20 microg/ml of rhMDP77, or containing phosphate-buffered solution (N = 6 in each group). Electrophysiological and histological evaluations carried out 12 weeks after implantation suggest that rhMDP77 has a positive effect on terminal and collateral sprouting of regenerating nerves and thereby promotes motor nerve regeneration in a dose-dependent manner.

Dental pulp can be a good candidate for nerve grafting in a xeno-graft model.

Dental pulp is discarded after extirpation of dental pulp and after tooth extraction. However, it contains nerve tissue abundantly and could be used more effectively. This study was designed to examine whether a dental pulp could be a candidate of donor for nerve grafting in xenografting model. The dental pulp was obtained from a human vital extracted tooth for orthodontic treatment, and treated with freezing and thawing method for reducing antigenicity. The treated sample was inserted into chitosan mesh tube for easy suturing, and then the complex was implanted into transected sciatic nerve in Sprague-Dawley (SD) rats (dental pulp group). As controls, chitosan tubes with and without sciatic nerve harvested from another SD rats were implanted (isograft group and tube group, respectively). As early as 4 weeks after grafting, regenerating axons accompanied by host Schwann cells were found to grout out through basal laminae by electron microscopy. The intact structure of basal laminae at this period suggested that they were derived from the original structure of donor graft. Twelve weeks after grafting, sporadic axonal regeneration was confirmed by light microscopy in the dental pulp group. Thirty-two weeks after implantation, aggregation of axons was observed in this group and matched that in isograft group. The average diameter of axons in dental pulp group was comparable to that in isograft group, whereas number of minifascicles and axon proportion were smaller. It was suggested that some delay occurred in dental pulp group because of the phagocytosis and absorption of tissue debris components remained after the freezing and thawing treatment. These findings clearly demonstrate that even dental pulp can act as conduits for regenerating axons.

Efficacy of platelet-rich plasma gel and hyaluronan hydrogel as carriers of electrically polarized hydroxyapatite microgranules for accelerating bone formation.

The technology for electrical polarization and characterization of hydroxyapatite (HA) microgranules has been developed. This study aimed to examine and compare the efficacy of composites comprising electrically polarized HA (pHA) microgranules and platelet-rich plasma (PRP) or hyaluronan (HAN) in osteoconductivity. Composites of HA microgranules with or without electrical polarization and either PRP or HAN (PRP+pHA, PRP+HA, HAN+pHA, and HAN+HA, respectively), as well as pHA and HA microgranules were implanted randomly into holes created in the medial femoral condyle or tibial tuberosity of rabbits. As a control, PRP or HAN gel alone was implanted, or the bone holes were left empty. Each group included six animals. After 6 weeks, histological examination was performed, and osteoclastic and osteoblastic cell activities were assessed by cell counting. Although PRP alone could not induce bone formation, PRP+pHA and PRP+HA composites, especially the former, activated osteogenic cells and enhanced bone formation. This effect was not prominent in the HAN+pHA and HAN+HA composites. PRP+HA composites formed a gel in which the ceramic particles were dispersed and entrapped in the fibrin network of PRP. It is assumed that these particles provide scaffolds for osteogenic cells, and when electrically polarized, can activate the cells in co-operation with the positive effects of the PRP, resulting in enhanced bone formation. Conversely, it is conceivable that this composite gel cannot act as an accelerator for woven bone formation, because HAN with low viscoelasticity is absorbed rapidly after implantation, the hydrated network containing HA microgranules is destroyed, and the HA microgranules effuse with HAN from the bone hole.