PMC:4137170 / 6739-15685 JSONTXT

{"target":"https://pubannotation.org/docs/sourcedb/PMC/sourceid/4137170","sourcedb":"PMC","sourceid":"4137170","source_url":"https://www.ncbi.nlm.nih.gov/pmc/4137170","text":"Materials and methods\n\nInjectable rhBMP-2 carrier fabrication and an rhBMP-2 release test\n\nPorous β-tricalcium phosphate microsphere\nThe porous β-TCP (Cerectron Co., Kimpo, South Korea) microspheres were prepared by the spray-dry method. The resulting spherical particles were subsequently sintered at 1,250 °C for 2 h, resulting in diameters ranging from 45 to 75 μm (Fig. 1). The β-TCP that was sintered at high temperature had a porosity of 59.3 %, as measured with a mercury porosimeter.\nFig. 1 SEM images of β-TCP microspheres generated by the spray-dry method after sintering at 1,250 °C\n\nHyaluronic acid (HA)-based powder gel\nThe powder gel was prepared to facilitate the loading of rhBMP-2 and to improve the fixation force of the carrier at the site of a bone defect by absorbing the blood and body fluids generated at the defect site. The powder gel was a cross-linked hyaluronic acid-based hydrogel consisting of HA (Bioland Co., Ochang, South Korea) with a high molecular weight of 3 million Daltons. This HA-based hydrogel was prepared through cross-linking by adding butanediol diglycidyl ether (Sigma-Aldrich, St. Louis, USA), a cross-linking agent, to 2.7 wt% of HA solution [17]. The remaining reagents were removed without resuspending by dialysis against 1X PBS (Sigma-Aldrich, USA) for 5 days. The resulting HA-based hydrogel was lyophilized for 4 days and then ground and sieved to yield a gel powder with particles less than 100 µm in diameter.\n\nCarrier preparation\nConformational change of the rhBMP-2 due to sterilization was avoided by mixing the rhBMP-2 solution with a powder gel composite to a final concentration of 1 mg/ml rhBMP-2. The in situ mixing process is a method used to generate an rhBMP-2 loaded injectable carrier by mixing the powder gel and the porous spherical particles for loading rhBMP-2.\nIn other words, one syringe contained porous β-TCP microspheres and the other syringe held the HA-based powder-gel containing 1 mg/ml rhBMP-2, and the relative weight ratio of the materials was 1:9. These two syringes were interconnected through a 2-way connector and 30 cycles of piston movement of each syringe were performed to ensure that the mixture was homogeneous. Collectively, β-TCP microspheres and HA were used as an rhBMP-2 carrier in this study.\n\nAnalysis of the physical properties of the HA-based powder gel\nThe reticular microstructure of the HA-based powder gel was induced by cross-linking and analyzed using a scanning electron microscope (SEM, S-4700, Hitachi, Tokyo, Japan). The swelling properties of the HA-based powder gel were measured by incubating it in PBS at room temperature. The swelling ratio was measured by comparing the change in the wet weight of the hydrogel before and after 3 days of incubation. The percentage of water absorbed (Wa) was calculated by the following formula: Swelling ratio (%) = ((Ww-Wi)/Wi) X 100 % (Ww: wet weight of hydrogel; Wi: initial weight of hydrogel).\n\nrhBMP-2 release rate study\nThe release rate for rhBMP-2 was evaluated by impregnating rhBMP-2 into the hyaluronic acid-based powder gel composite. The experiment was carried out by impregnating rhBMP-2 into the composite and then analyzing the rhBMP-2 release, as detected by an antigen-antibody reaction and ELISA analysis. A collagen type I sponge (Bioland, Ochang, South Korea), which is currently used as a periodontal tissue regeneration-inducing agent in dentistry, was impregnated with 100 µg rhBMP-2 and used for comparison. The release patterns from the sponge and the composite were analyzed following treatment with hyaluronidase (100 units/ml) and collagenase (20 CDU/ml) (Sigma, USA) for 7 days, and incubation at 37 °C in PBS [18, 19].\n\nIn vivo study\n\nAnimals and implantation\nA total of 17 New Zealand white male rabbits (3–3.5 kg) underwent general anesthesia with Zoletil® and xylazine. This study was approved by the Institutional Animal Care and Use Committee at the Clinical Research Institute of Seoul National University Hospital Biomedical Research Institute (IACUC No. 13-0038). The hind limb was disinfected with betadine after hair removal. The anterior aspect of the tibia was exposed and two holes were generated by pre-drilling. The two holes were spaced no less than 15 mm apart in order to minimize the interaction between them. After the two holes for the implant fixture in the tibia were placed using a lance drill, they were enlarged to a diameter of 2 mm using a twist drill. Next, a straight drill was used to further enlarge each of the holes first to 2.8 mm, then to 3.3 mm, and finally to 3.8 mm; and then the holes were countersinked. The depth of each of the holes was 8.5 mm and dental implant fixtures, 4 mm in diameter, (Ø4, 8.5 mm length, MegaGen, Seoul, South Korea) were inserted. The insertion torque of the implant was 45 Ncm. Three groups were allocated as follows: the implant only group (implant group), the injected β-TCP-hydrogel power gel composite implant group (the hydrogel group), and the rhBMP-2 (200 μg) loaded powder gel β-TCP-hydrogel composite implant group (BMP-2 group). After two tibiae of a rabbit were exposed, two implants of the same group were inserted per each tibia. The block randomization was used to decide which implant to place into each tibia. After inserting a cover screw, and fascia layer, the wound was then sutured (Fig. 2). Immediately after the surgery, 300 mg cefazolin was injected intramuscularly and antibiotics were administered for 2 days. The experimental animals were euthanized 4 weeks later.\nFig. 2 Surgical procedures. a Tibial diaphysis in the tibial medial aspect was exposed and two, 4 mm diameter holes were generated; b A composite of rhBMP-2 loaded porous β-TCP microspheres and hyaluronic acid powder-gel was injected; c A dental implant fixture was inserted\n\nEvaluation methods\n\nPlane radiographs\nThe experimental animals were sacrificed and plane radiographs of the full length of the tibia were obtained at 45 kV for 12 ms. The implants were evaluated for loosening, pullout, or differences in the surrounding osseous tissue.\n\nMicro-CT evaluation\nThe experimental animals were sacrificed and micro-CT was performed on all specimens by harvesting samples that included the implanted part of the tibia. Micro-CT (SkyScan 1173, Zwijnaarde, Belgium) was performed using an aluminum filter in a mid-resolution of 20 μm at 30 kV, 60 μA. The newly formed bone area ratio (the ratio of the area of new bone formation at the space between two threads in the pitch) and the bone-to-implant contact ratio (the ratio of bone directly attached to the thread of the screw) were evaluated. The quality of new bone was evaluated based on a total of 42 pictures analyzed by CTAn, a micro-CT analysis program. The bony tissue of the new bone was analyzed with an ROI (region of interest) at a width of 0.99 mm and a height of 2.48 mm. The bone fraction directly attached to the screw thread was analyzed with an ROI at a width of 0.97 mm and a height of 2.48 mm. For quantification of the quantity and quality of the new bone, the following items were evaluated: percent bone volume (bone volume/trabecular volume), specific surface (bone surface/volume ratio), trabecular bone pattern factor (the parameter of the connectedness of these bone patterns), trabecular thickness (the thickness of the trabeculae), trabecular number (the number of trabeculae), and trabecular separation (the distance between the trabeculae).\n\nHistologic evaluation\nThe full length of the tibia specimen was fixed in formalin for 5 days and the tibiae containing each specimen were divided into two parts. Gross section tissue was put into a cassette and washed for 6 h and then dehydrated in 100 % alcohol. The tissue was put into methacrylate-based chemical curing resin and stirred for 2 days, and then it was stirred and embedded by dissolving in benzoyl peroxide. The block was trimmed and sectioned along the longitudinal axis of the dental implant fixture using an EXAKT cutting instrument (BS-3000 N). Moreover, a 4 μm section was made at the right center of the implant along the sagittal plane and included the surrounding tibia. Grinding was carried out using an EXAKT grinding machine (4,110), and an acrylic slide attachment was performed. The slide was stained with hematoxylin and eosin (H\u0026E) staining and viewed with a light microscope to determine the bond between the bone and the implant and the new bone formation around the implant. Histomorphometric evaluations of the dental implant fixtures were carried out after a scaled calibration using a morphometry program (LEICA IM50 Image Manager, version 4.0). The bone-to-implant contact ratio was measured in the marrow space of the tibia. All measurements were performed using a (X12.5) magnification objective.\n\nStatistical analysis\nThe three groups were compared using a nonparametric Kruskal–Wallis test. 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