Abstract
Bone scrapers are commonly used to harvest autologous bone in oral and implant surgery. The angle of the cutting blade is a variable that distinguishes bone scrapers. In the present study, the impact of the angle of the cutting blade on the in vitro characteristics of harvested bone was determined. Bone scrapers with blade angles of 15°, 25°, 35°, 45°, and 55° were used to harvest porcine cortical mandibular bone. The number and characteristics of the cells that grew out from the bone chips were examined. The data showed that, independent of the angle of the cutting blade, viable cells were barely detectable in fresh bone grafts. However, cells with a fibroblastic morphology appeared within 1 week in the culture dishes. After 21 days, the number of cells did not differ significantly between the five preparations. Moreover, cells responded to incubation with bone morphogenetic protein 7 (BMP-7) with an increased alkaline phosphatase activity, irrespective of the preparation. The data suggest that bone scrapers with different cutting angles produce bone chips with comparable in vitro characteristics.
Introduction
The bone scraper is an established tool for harvesting autologous bone grafts in oral and implant surgery. Autografts obtained using bone scrapers can be used for surgical procedures such as sinus floor elevation, guided bone regeneration, and filling of extraction sockets. Bone can be harvested at the time of surgery from various intraoral anatomical regions, including the ramus, symphysis, and tuberosity. Thus, bone scrapers are an alternative to the use of bone harvesting procedures such as trephine burs, saws, and chisels. Autografts obtained by bone scrapers can be used alone or in combination with bone substitutes and membranes. Since bone scrapers have become standard instruments for harvesting small amounts of bone, the manufacturers now provide reusable instruments with fixed and replaceable blades as well as disposable instruments. All instruments basically harvest autologous bone grafts.
Only a few studies reporting the cellular aspects of the harvested bone are available in the literature. The investigation of the cellular aspects of autografts is inspired by the hypothesis that the transplanted cells can contribute to bone regeneration. Even though the exact mechanism by which autografts enhance bone formation remains unclear, convincing evidence from preclinical research has shown that bone regeneration occurs faster at sites with autografts compared to sites with bone substitutes. These and other studies have led to in vitro studies with autologous bone chips, basically focusing on the number of outgrowing cells and their phenotypic characterization, for example by determining the marker enzyme for the bone-forming osteoblasts, alkaline phosphatase.
With the development of bone scrapers in oral surgery routine, also the respective bone particles underwent in vitro examination. However, the impact of the shape of bone scrapers has not been considered so far. Among the potential key variables is the angle of the cutting blade. The manufacturers of bone scrapers dictate the angle of the cutting blade. However, the individual anatomy and the anatomical region from which the bone is harvested further affect the actual angle of the cutting blade. There are clearly large variations between bone scrapers and their individual uses – it would thus be of value to assess whether the angle of the cutting blade generates autologous bone of similar biological activity. We therefore examined bone grafts obtained from porcine cortical mandibular bone using bone scrapers with cutting blade angles of 15°, 25°, 35°, 45°, and 55° in vitro .
Materials and methods
Preparation of bone chips and explant culture
The bone scrapers, ‘Buser’ model with cutting blades of 15°, 25°, 35°, 45°, and 55°, were kindly provided by Hu-Friedy Mfg. Co., LLC (Chicago, IL, USA). Bone scrapers were tested on the buccal surface of pig mandibles within 12 h post mortem ( Sus scrofa domestica ). The animals were in the growing phase, at 8 months of age. Bone chips were harvested in two separate sessions by two independent investigators (UK and CS). The bone chips were immediately placed into sterile Petri dishes containing Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% foetal calf serum, antibiotics, and antimycotics (all Life Technologies Corp., Grand Island, NY, USA). Bone chips were washed twice with medium. One gram wet-weight of the bone chips was placed onto a culture plate with an area of 10 cm 2 (Greiner Bio-One GmbH, Frickenhausen, Germany) in culture medium and then placed in a humidified atmosphere at 37 °C in 5% CO 2 . The culture medium was replaced twice a week. After 3 weeks, cells that had grown out from the bone explants were released using trypsin (Life Technologies Corp.) and counted with a Neubauer chamber. The number of cells that had grown out was calculated per gram of wet bone tissue. Bone cells were further expanded to reach the first or second passage.
Viability of cells on the surface of freshly prepared bone chips
Random samples of bone chips were selected from the pool of bone chips harvested with a contact angle of 15°, 25°, 35°, 45°, and 55°. For staining of viable cells, freshly prepared bone chips were exposed to a solution containing 0.5 mg/ml MTT, which is 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (Sigma–Aldrich, St. Louis, MO, USA). MTT is converted to insoluble formazan crystals, allowing viable cells to become visible under a light microscope.
Alkaline phosphatase activity of cells growing out from the bone grafts
Bone cells harvested after 3 weeks were further expanded and seeded at an initial density of 30,000 cm 2 on microtitre plates. The next day, the medium was changed to growth medium supplemented with recombinant bone morphogenetic protein 7 (BMP-7; ProSpec-Tany TechnoGene Ltd., Rehovot, Israel) at 100 ng/ml for 72 h. For histochemical staining of alkaline phosphatase, cells were fixed with neutral buffered formalin and incubated with a substrate solution containing 4 mg of naphthol AS-TR phosphate in 0.15 ml of N , N -dimethylformamide and 12 mg of fast blue BB salt (all from Sigma–Aldrich, St. Louis, MO, USA) in 15 ml of 100 mmol/l Tris–HCl (pH 9.6). After rinsing with distilled water, the cultures were photographed.
Statistical analysis
The cell counts were compared with the non-parametric Kruskal–Wallis test; P < 0.05 was considered significant. All other observations are descriptive in nature as this was a pilot study. Experiments were performed twice by two of the authors (UK and CS).
Materials and methods
Preparation of bone chips and explant culture
The bone scrapers, ‘Buser’ model with cutting blades of 15°, 25°, 35°, 45°, and 55°, were kindly provided by Hu-Friedy Mfg. Co., LLC (Chicago, IL, USA). Bone scrapers were tested on the buccal surface of pig mandibles within 12 h post mortem ( Sus scrofa domestica ). The animals were in the growing phase, at 8 months of age. Bone chips were harvested in two separate sessions by two independent investigators (UK and CS). The bone chips were immediately placed into sterile Petri dishes containing Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% foetal calf serum, antibiotics, and antimycotics (all Life Technologies Corp., Grand Island, NY, USA). Bone chips were washed twice with medium. One gram wet-weight of the bone chips was placed onto a culture plate with an area of 10 cm 2 (Greiner Bio-One GmbH, Frickenhausen, Germany) in culture medium and then placed in a humidified atmosphere at 37 °C in 5% CO 2 . The culture medium was replaced twice a week. After 3 weeks, cells that had grown out from the bone explants were released using trypsin (Life Technologies Corp.) and counted with a Neubauer chamber. The number of cells that had grown out was calculated per gram of wet bone tissue. Bone cells were further expanded to reach the first or second passage.
Viability of cells on the surface of freshly prepared bone chips
Random samples of bone chips were selected from the pool of bone chips harvested with a contact angle of 15°, 25°, 35°, 45°, and 55°. For staining of viable cells, freshly prepared bone chips were exposed to a solution containing 0.5 mg/ml MTT, which is 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (Sigma–Aldrich, St. Louis, MO, USA). MTT is converted to insoluble formazan crystals, allowing viable cells to become visible under a light microscope.
Alkaline phosphatase activity of cells growing out from the bone grafts
Bone cells harvested after 3 weeks were further expanded and seeded at an initial density of 30,000 cm 2 on microtitre plates. The next day, the medium was changed to growth medium supplemented with recombinant bone morphogenetic protein 7 (BMP-7; ProSpec-Tany TechnoGene Ltd., Rehovot, Israel) at 100 ng/ml for 72 h. For histochemical staining of alkaline phosphatase, cells were fixed with neutral buffered formalin and incubated with a substrate solution containing 4 mg of naphthol AS-TR phosphate in 0.15 ml of N , N -dimethylformamide and 12 mg of fast blue BB salt (all from Sigma–Aldrich, St. Louis, MO, USA) in 15 ml of 100 mmol/l Tris–HCl (pH 9.6). After rinsing with distilled water, the cultures were photographed.
Statistical analysis
The cell counts were compared with the non-parametric Kruskal–Wallis test; P < 0.05 was considered significant. All other observations are descriptive in nature as this was a pilot study. Experiments were performed twice by two of the authors (UK and CS).
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