| BackgroundBone defect repair has never been an outdated topic in medical science. In the field of oral implant rehabilitation,insufficient bone mass is often the challenge to bone defect repair. The most important prerequisite of obtaining and maintaining long-term implant osseointegration is to have adequate healthy bone mass available near implant sites, as well as appropriate bone height and thickness allowing implants of suitable length and diameter. However, since more than half of the planting site lacks enough bone mass,bone augmentation is a necessary practice corresponding the time of implant or in stages. Autologous bone is considered the "gold standard" in bone reconstruction, but because of its limited sources, as well as potential to cause postoperative bleeding, pain and complications such as infections in donor site, artificial osteoinductive bone graft material becomes the main resource for bone defect repair in clinical practice. Artificial bone graft material helps maintaining osteogenic space, stabilize blood clot and promotes healing. It has limited effect on the growth of new bone. To achieve a better osteogenic effect and improve the overall quality of bone formation through compounding different bone growth factors has been a hotspot in the field of bone repair research.In recent years, scholars took note of a bone growth factor,-1,25 (OH) 2D3, that has never been topically applied before. It is found, in many vitro experiments, to have the ability to promote osteoblasts and bone maturation. The growth factor also has a synergistic effect with BMP-2, hence promotes mineralization and osteogenesis. At the same time, it suppresses the expression of c-Fms and RANK, and therefore inhibits osteoclast generation.1,25 (OH) 2D3 is the most biologically active form of vitamin D. It is the active ingredient produced by a series of vitamin D’s metabolism involving liver and kidney. It is ultimately released into the blood circulation to impact on the regulation of bone formation and bone development. Currently, there are many animal models (chicken, rats, rabbits) that support the positive correlation between the consumption of vitamin D and the effect of bone healing. In such experiments, metabolite of vitamin D promoted the healing of fractures and increased callus intensity. However, very few of 1,25 (OH) 2D3 can reach the target organ through systemic circulation. The amount is often only enough to maintain basic body bone metabolism. For postmenopausal women and patients with abnormalities in osteoporosis bone metabolism, the amount of vitamin D inside their body is a lot less than needed to effectively facilitate the repair of bone defects. However, overconsumption of vitamin D3 to increase the amount of 1,25 (OH) 2D3that reaches the target organ would result in toxic reaction, hypercalcemia, loss of appetite, vomiting, diarrhea, and even soft tissue heterotopic ossification. Thus, scholars began to study the effect of topical application of vitamin D. When Salomo-Coll put implants in the mandible of beagle dogs together with topical application of vitamin D2, he found that it reduces loss of parietal bone and increases the area of ossenointegration. The substance he used is precursor of 1,25 (OH) 2D3, which is not the highest form of vitamin D physiological activity.A research led by Hsiang-Hsi Hong has recorded 1,25 (OH) 2D3’s role in promoting osteogenesis and repairing the circular bone defect of beagles. However, the choice of administration adopted is injection. They injected a finished drug that is low in 1,25 (OH) 2D3, called Luogaiquan around the bone defect area. Such choice of administration still makes the amount of the drug that reaches the target organ suffer a great loss. Moreover, such weekly practice increases the times of surgery, repeatedly causing irritation in defect areas, hence adding additional interfering factors to osteogenesis. Moreover, the bone defect prepared for the experiment is not a standard bone defect and therefore, is not conducive to testing the drug’s effectiveness on osteogenesis. With all that being said, this paper focuses on the study of using topical application as a way to get vitamin D to reach target organ, in order to avoid loss of dosage and prevent toxic reaction. By using the composite of the most physiologically active form of vitamin D,1,25 (OH) 2D3 and inorganic calf bone substitude to repair the standard defects in rabbit skull bones, the paper studies the effect of topical application of 1,25 (OH) 2D3 on osteogenesis in its early and middle stages.PurposeResearch on the effect of topical application of 1,25 (OH) 2D3in the early and mid stages of osteogenesisMethod1. Animals and the Key Materials18 female New Zealand white rabbits,15-16 weeks old, weighted 2.9-3.2kg, are provided by the Guangdong Medical Experimental Animal Center. Treatment of the animals during and after the experimental is in line with animal ethical standards. After one week’s adaptive feeding, the experiment is carried out at Guangdong Animal Lab. The experiment is designed to have three groups, including a blank control group (A), a simple bone substitude group (group B), and a 1,25 (OH) 2D3 composite bone substitude group (group C). Each group has six rabbits and the rabbits are killed at three specific time points, in 2 weeks,4 weeks and 6 weeks respectively. To prepare for the procedure, on each rabbit’s parietal, four 8 mm circular bone defects are created. At each killing time, a total of 24 circular bone defects are ready for observation. A, B, C three types of bone repairing materials are randomly assigned to all 24 bone defects, with each type to 8 bone defects.The inorganic calf bone substitude is consisted of Bio-Oss, which is 0.25mm-lmm small bone particles (Switzerland Geiger Pharmaceutical Co., Ltd., Registration No:State Food and Drug Administration armed (import) word 2014 No. 3460922, standard number of imported products:YZB/SWI8428-2013). To make 1,25 (OH) 2D3, Sigma Chemical, Co. la 25-Dihydroxyvitamin D3 D1530, is dissolved in anhydrous ethanol, and then mixed with Bio-Oss bone mass at a ratio of 6μg:5 mg. After that, it is lyophilized and frozen using low-temperature vacuum freeze dryer, and sterilized using cobalt 60 radiation.2. Surgical Procedures and the Establishment of Animal Experiment ModelTo prevent intraoperative and postoperative infection, the rabbits are performed intramuscular injection of gentamicin (1 mg/kg) 30 minutes before the surgery. After surgical skin preparation, the rabbits are performed intramuscular induction of anesthesia by intramuscular injection of Sumianxin (0.2 ml/kg) at hind legs. When the induction of anesthesia takes effect,3% pentobarbital sodium (30 mg/kg) is injected through ear vein to administer anesthesia. After general anesthesia, the rabbits are set at a prone position. Their surgical areas are disinfected with iodine and laid on with disposable holely towels. The area of operation is performed local subcutaneous anesthesia using 2% lidocaine hydrochloride. Vertical incision is performed on palpable skull along the midline where the seam and bony protrusion is. The cut is deep to the bone surface and the full thickness flap along the seam is flipped. The front end of the cut reaches to the junction of parietal bone and frontal bone. The rear end of the cut reaches to the bottom of parietal bone. On both sides, the edges of parietal bone are fully exposed. So is the operation area. An annular shaped bone drill with an outer diameter of 8mm is used to drill 4 ring parietal bone defects, with the distance between each hole larger than 2mm. During the procedure, the operational area is adequately cooled to 4℃ with saline. The procedure is carefully conducted to not hurt the dura of the rabbits. Simple bone substitude or 1,25 (OH) 2D3 composite bone substitude is put to Group A, Group B, Group C accordingly and the layers are surgically sutured.Post operation, antibiotics is used to prevent infection.13 days and 14 days before the rabbits are killed, subcutaneous injection of tetracycline (30 mg/kg) are performed in the neck area of the rabbits.3 days and 4 days before the rabbits are killed, subcutaneous injection of calcein (6 mg/kg) are performed in the neck area of the rabbits. All animals in accordance with the observation time points at 2 weeks, 4 weeks,6 weeks, are killed respectively by air embolism in the ear vein. Samples are taken and placed in a 10% formaldehyde solution for 48 h. After that, they are placed in running water for another 24h, and then in 10% EDTA solution for decalcification for a period of approximately 3 month. After that, paraffin section and hard tissue sections are made.3. Main Outcome MeasuresGross observation, histological observation, bone metrology testing, observation of staining calcium deposition.Results1. Intraoperative Observation and General Postoperative ObservationA total of 72 circular bone defects are prepared for the surgical experiment. None has touched or damaged the dura of the rabbits. After the surgical procedure, all of the animals are in good spirit. Their food intake and excretion seem to be within the normal range. The wounded surgical areas are slightly swollen.1 week after the surgery, the swelling and wounded areas are mostly subsided. There is no loose stitches or fall-off. No sign of infection is observed in the wounded areas, which qualifies a primary healing.2 weeks after the surgery, the surgical areas are completely healed and the surgical sutures natural shed. In group A, the areas of bone defects are covered with fibrous tissue. The boundary is clearly visible and has a soft touch to it. In group B and group C, the areas of bone defects also have a soft touch to it. However, in the blank control group, the areas of bone defects feel hard and firm. In between the particles on the bone substitude is fibrous tissue. The edge of the bone defect area is clear and visible.4 weeks after the procedure, at the edge of the bone defects in Group A, a small amount of new bone formation can be observed. The edge of the defective areas, however, is not as visible. The center of the bone defect is dominated by soft tissue, which has a soft touch to it. In group B and group C, touch to the bone defect areas is hard. The edges of bone substitude particles are less visible compared to they are at 2 weeks after the procedure.6 weeks after the procedure, the new bone tissue at the edge of the bone defects in group A continues to grow toward the center. The entire bone defect area is not yet fully covered. In Group B and Group C, in between the particles of the bone substitude, bone tissue can be visibly observed. The touch of the bone defect area is extremely hard, the edge of the bone defect area is considerably blurred and difficult to recognize.2. Histological Observation2 weeks after the procedure, a small amount osteoid is formed at the edge of the bone defect area in Group A. There is fibrous tissue observed in the central area, with extensive infiltration of inflammatory cells. There is also a small amount of collagen found in the connective tissue mass; In group B, the formation of a small amount of new bone and osteoid is observed near the bone particles in the center or at the edge of the bone defect area. As it is the case in group B, group C has similar new bone and osteoid formation, but the area of the new formation is larger than it is found in group B.4 weeks after the procedure, the newly formed bone at the edge of the bone defect area has extended toward the center, covering larger the larger area. The central area is still filled with fibrous tissue. Among the visible coarse collagen fibers scattered a small amount of inflammatory cells. The newly form bone surrounding the bone substitude observed in group B is deeply stained. It seem to be more matured and the area of the newly form bone has increased. Area of newly formed bone in group C is found to be larger compared to that in group B. The trabecular bones are sparse and intertwined like a web, and surrounded by a flat monolayer of osteoblasts. A large quantity of immature bone cells transformed from osteoblast can be observed. The gap of the bone lacuna surrounding the bone cells is narrow, appeared in a fence-like arrangement near where the new bone is found.6 weeks after the procedure, the newly formed bone at the edge of the bone defect area in group A group continue to extend toward the center, but not yet docking each other; in group B, the newly formed bone can be widely seen to have surrounded the particles of the bone substitude. The newly formed bone seem to have matured; area of the newly formed bone found in group C is considered larger than that of the newly formed bone found in group B. The newly formed bone found in group C also seem more mature. A large amount of trabecular bone is observed to have fused into the shape of tablet and formed lamellar bone. Among the tablets of trabecular bone scattered a small amount of irregular gaps. Surrounding the gaps are uniformly and continuously arranged osteoblasts.3. Bone Histomorphometry and Observation2 weeks after the procedure, the ratio of area of newly formed bone in group A over the original total area is 9.58±1.21%. The number of osteoblast cells is 15.2± 1.68; the ratio of area of newly formed bone in group B over the original total area is 12.52±1.09%. The number of osteoblast cells is 23.8±1.25; the ratio of area of newly formed bone in group C over the original total area is 15.12±0.80%. The number of osteoblast cells is 24.8±2.12.4 weeks after the procedure, the ratio of area of newly formed bone in group A over the original total area is 12.75±1.13%. The number of osteoblast cells is 20.3±2.15; the ratio of area of newly formed bone in group B over the original total area is 15.88±1.04%, osteoblast cells was 24.1±2.23; group C newborn bone area ratio of 20.95±1.34%. The number of osteoblast cells is 23.8±1.79.6 weeks after the procedure, the ratio of area of newly formed bone in group A over the total original area is 18.94±1.20%. The number of osteoblast cells is 25.0±2.26; the ratio of area of newly formed bone in group B over the original total area is 21.37±1.32%. The number of osteoblast cells is 25.8±1.69; the ratio of area of newly formed bone in group C over the original total area is 25.10±1.27%. The number of osteoblast cells is 26.6±3.50. Through an one-way analysis of the variance and repeated measures analysis of the result, we find that results of the three group’s area of the newly formed bone observed at three different time points are significantly different from each other. (P<0.05) The result being group C> group B> A group. However, as time extends, the difference of results between the three groups shows a diminishing trend. As for the number of osteoblast cells from each of three groups is not significantly different from each other.4. Bone HistomorPhometry2 weeks after the procedure, the amount of calcium deposits observed in group A is relatively small. Only partial bright green calcium deposition is observed at the edge of the bone defect area; in B group, compared to the blank control group, calcium deposits is more extended in its area. In the central part of the bone defect area also shown partial calcium deposits. The area of calcium deposits in group C is relatively larger compared to the area and range of calcium deposits in the previous two groups. Its level of brightness is also higher, indicating that the amount of calcium deposits of group C is, to some extent, higher than those in both group A and group B.4 weeks after the procedure, area and range of calcium deposits in all three groups expanded in area compared to what they were before. The amount of calcium deposition still ranks as group O group B> A group.6 weeks after the procedure, in the center of the bone defect area in group A formed a bone island. At the edge of the bone defect area, traces of calcium deposits are visible, but some area near the center of the bone defect area still shows no trace of calcium deposits. In both group B and group C, calcium deposits are widely seen in the bone defect areas. The area of calcium deposits in group C is larger than it is in group C. In group C, it also has a higher level of brightness than in group B.Conclusion1.15 to 16 weeks, weight 2.93.2 kg of male New Zealand white rabbit cranial parietal bone can make 4 circular bone defects with 8 mm diameter.2. The rabbit skull 8 mm diameter of circular bone defect in six weeks time can be used as a standard of bone defect on drug osteogenesis performance for related research.3.Topical application of 1,25 (OH) 2D3 does not promote osteoblast differentiation and proliferation.4. Topical application of 1,25 (OH) 2D3 stimulates the maturation of osteoblasts, increases calcium deposition, promotes mineralization, and thereby enhances osteogenesis. |
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