• Korean Journal of Clinical Laboratory Science
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• v.48 no.2
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• pp.114-117
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• 2016

In vivo labeling of bone with fluorochromes is a widely used method for assessment of bone formation and remodeling processes. In particular, calcein is used as a marker for identification of bone growth, which is indicated by a green color. Calcein green is a calcium chelator that adheres to regions of mineralizing bone thereby allowing localization of new bone. Bone formation and remodeling in vivo can be assessed by calcium-binding calcein labeling. In this study, changes in the femoral bone of a normal mouse model at both 4 and 8 weeks were evaluated using calcein labeling. Intense deposition of calcium in the bone was observed after application for 8 weeks. A mouse model is suitable for application in in vivo experiments using genetically modified mice, such as knock-out mice, however data regarding femoral cross sectional bone in young mice are limited. The current study confirmed calcein as a useful marker for identification of bone growth, which was indicated by a green color on photomicrographs. This methodological process may provide basic information for interpreting bone formation and regeneration to pharmacologic or genetic manipulation in mice.

• Nutritional Sciences
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• v.8 no.4
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• pp.242-249
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• 2005

It is well established that zinc plays an important role in bone metabolism and mineralization. The role of zinc in bone formation is well documented in animal models, but not much reported in cell models. In the present study, we evaluated zinc deficiency effects on osteoblastic cell proliferation, alkaline phosphatase activity and expression, and extracellular matrix bone nodule formation and bone-related gene expression in osteoblastic MC3T3-E1 cells. To deplete cellular zinc, chelexed-FBS and interpermeable zinc chelator TPEN were used. MC3T3-E1 cells were cultured in zinc concentration-dependent (0-15 ${\mu}M\;ZnCl_2$) and time-dependent (0-20 days) manners. MC3T3-E1 cell proliferation by MTT assay was increased as medium zinc level increased (p<0.05). Cellular Ca level and alkaline phosphatase activity were increased as medium zinc level increased (p<0.05). Alkaline phosphatase expression, a marker of commitment to the osteoblast lineage, measured by alkaline phosphatase staining was increased as medium zinc level increased. Extracellular calcium deposits measured by von Kossa staining for nodule formation also appeared higher in Zn+(15 ${\mu}M\;ZnCl_2$) than in Zn-(0 ${\mu}M\;ZnCl_2$). Bone formation marker genes, alkaline phosphatase and osteocalcin, were also expressed higher in Zn+ than in Zn-. The current work supports the beneficial effect of zinc on bone mineralization and bone-related gene expression. The results also promote further study as to the molecular mechanism of zinc deficiency for bone formation and thus facilitate to design preventive strategies for zinc-deficient bone diseases.

• Journal of Nutrition and Health
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• v.39 no.2
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• pp.109-114
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• 2006

The effects of the erect bipedal stance exercise on bone mass and the biomarkers of bone formation and resorption were investigated in rats. Five-week old rats were assigned into control and exercise groups. The rats of exercise group were weight-bearing-trained for 13 weeks in the cage designed to adjust progressively the height from 26.5 cm to 31.5 cm to force the rats rising an erect bipedal stance for feeding and drinking. There was no significant difference in food intakes between two groups. But body weight gain was significantly increased in control group. The lengths of femur, tibia, humerus and radius were significantly longer in control group than exercise group, but the femur and tibia weights per body weight were significantly higher in exercise group than control group. Also the breaking force of femur and tibia in exercise group were higher than control group significantly. The calcium contents of femur and tibia were significantly increased in exercise group than control group. The activity of bone specific alkaline phosphatase (B-ALP) and the osteocalcin contents of serum (the biomarkers of bone formation) in exercise group were higher than control group, but the carboxyterminal propeptide of type I procollagen (P1CP) contents of serum did not show any difference between two groups. However the urinary deoxypridinolin (DPD) excretion, biomarker of bone resorption, was significantly lower in exercise group than control group. From these results, it has been indicated that the erect bipedal stance exercise enhanced the density and the strength of femur and tibia by increasing biomarkers of bone formation and suppressing a biomarker of bone resorption in rats.

• Journal of Periodontal and Implant Science
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• v.38 no.1
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• pp.75-82
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• 2008

Purpose: The purpose of this study was to evaluate exophytically vertical bone formation in the mandibular premolar area of beagle dogs by the concept of guided bone regeneration with a titanium reinforced e-PTFE membrane combined with human demineralized freeze-dried bone. Materials and Methods: Four one-year old beagle dogs were divided into control and experimental group. All mandibular premolars were extracted and surgical vertical defects of 5 mm in height were created in the extracted sockets. At 8 weeks after the extraction, TR e-PTFE membrane sized with 8 mm in length, 5 mm in width, and 4 mm in height was placed on the decorticated mandible, fixed with metal pins and covered with full-thickness flap and assigned as control group. In experimental group, decorticated mandibule was treated with TR e-PTFE membrane and human demineralized freeze-dried bone. The animals were sacrificed at 16 weeks after the regenerative surgery, and new bone formation was assessed by histomorphometric as well as statistical analysis. Results: Average of new bone formation was 38% in the control group, whereas was 25% in the experimental group (p<0.05). Average of connective tissue formation was 42% in the experimental group, whereas was 30% in the control group (p<0.05). The lamellar bone formation with haversian canals was observed in the both groups. In the experimental group, the particles of human demineralized freeze-dried bone were observed after 16 weeks and complete resorption of graft was not observed. Conclusion: On the basis of these findings, we conclude that titanium reinforced e-PTFE membrane may be used alone for vertical guided bone regeneration, but demineralized freeze-dried bone has no additional effect on vertical guided bone regeneration.

• Maxillofacial Plastic and Reconstructive Surgery
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• v.15 no.1
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• pp.63-79
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• 1993

To repair bony defects with tansplanted bone in the body, fresh autogenous bone is undoubtly, the most effective bone graft for clinical applications. But the demineralized bone has the matrix-induced bone formation which was suggested by Urist in 1965. Many authors assisted that demineralized bone powder induces phenotypic conversion of mesenchymal cells into osteoblasts, with high-density bone formation. The process of inducing differentiated cells becomes osteogenic properties. The purpose of this study was to evaluate the osteoinductive capacity of allogenic freeze-dried demineralized bone block (FDD, $7{\times}7mm$) and to compare FDD with the same sue of deep-frozen allogenic bone(DF), fresh autogenous bone (A) after implantation. The histological and ultrastructural features of tissue responses were examined after 1, 2, 4, 6, 8 weeks implantation of each experimental groups in the operative site of the New Zealand white rabbits. The results were as follows : 1. Inflammatory cell infiltration generally has appeared at 1 week, but reduced at 4 weeks in each group, but most severe in DF group. 2. Osteoblastic activity has increased for 4 weeks, but decreased at 6 weeks in each group and there was no significant difference among experimental groups. 3. New bone formation has begun at 1week, least activations in A groups, and showed the revesal line of bone formation among each group at 6 to 8 weeks. 4. Bone resorption has appeared at 1 week, but disappeared at 4 weeks in both A and DF groups, but more severe in DF than A groups. 5. In ultrastructural changs, the DF group have showed the most remarkable osteoclastic activities among experimental groups. 6. Osteoid or tangled collagen fibrils near the implanted sites were replaced by more mature, lamellated bony trabeculae during bone remodeling. There was little difference among each experimental groups. 7. During the convertion osteoblasts to osteocytes which embedded within the bone matrix, there was organ-less-poor cytoplasm, increased nuclear chromatin, abundant rough endothelial reticulum (RER) in each groups. From the above the findings, the DF group shored more bone resorption and foreign body reaction than FDD and A groups, and FDD group showed more new bone formation or osteoblastic activity than DF and A groups in early stage. There was no significant difference of cellular activities among the FDD DF, and A groups according to the time.

• Journal of the Korean Association of Oral and Maxillofacial Surgeons
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• v.29 no.6
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• pp.374-378
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• 2003

Aim : Several injectable materials have been used in the application of osteogenic bone substitute; however, nothing has won universal acceptance. This study was performed to investigate whether chitosan-alginate gel/MSCs/BMP-2 composites are potentially injectable materials for new bone formation. Material and Methods : The composites were injected into the subcutaneous space on the dorsum of the nude mouse to investigate whether new bone would be tissue engineered in the mouse. The composites were examined histologically over a 12-week period. Results : The composites implanted in the mouse were able to tissue engineer new bone, and the newly formed bone consisted of trabecular bone and calcified bone matrix. Conclusions : The present study shows that chitosan-alginate gel/MSCs/BMP-2 composites have the potential to become real injectable materials for new bone formation.

• Imaging Science in Dentistry
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• v.34 no.1
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• pp.35-48
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• 2004

Purpose: To investigate the effect of rhBMP-2 on the healing of bone defect in the low calcium diet rat. Materials and Methods: To prepare the experimental model, control group was fed a normal diet and experimental group was fed a low calcium diet for 3 weeks. And then, 4 mm bicortical perforated bone defect was made on mandibular body of each rats. Experimental group was subdivided into two groups; experimental group 1 (rats given a low calcium diet before and after bone defect) and experimental group 2 (rats given a low calcium diet before and after bone defect with rhBMP-2 application). At 1, 3, 5 and 7 weeks after bone defect formation, the rats were terminated. The healing of bone defect was assessed by three-dimensional computerized tomography, soft x-ray radiography, and histopathological examination. Results : The wound healing of the bone defect for control group, experimental group 1, and experimental group 2 showed a increase from 3 weeks after bone defect formation. The experimental group 2 showed a more increase in healing amount than control group and experimental group 1 from 5 weeks after bone defect formation and the experimental group 2 showed a complete recovery of bone defect at 7 weeks after bone defect formation. Conclusion: The healing process of bone defect is accelerated by rhBMP-2 application in the low calcium diet rats.

• Maxillofacial Plastic and Reconstructive Surgery
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• v.16 no.3
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• pp.290-302
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• 1994

This study was aimed to clarify the histopathological changes in the experimental animal model subjcted to rigid fixation performed across the frontonasal sutrue in growing rabbits. Sixteen rabbits aged 6 weeks used. In experimental group(n=12), rigid fixation with miniplates and screws was performed across the frontonasal suture. Control group(n=4) was those with periosteal elevation only. Experimental animals were sacrificed on the 2nd, 4th, 8th, and 12th week after operation, and frontonasal suture area was excised for light microscopic and scanning electron microscopic examination. The results obtained were as follows : 1. In control groups, collagen fiber bundles ran in the midportion of bone sutrue and cambial layers were seen at bone surface. Sutural surfaces are beveled and external and internal bony projected portions were observed. 2. In experimental groups, distance of bone suture was decreased by new bone formation on the 2nd week, while increased by bone resorption at the miniplate applied area and bone formation in the adjacent bone on the 4th week. 3. In experimental groups, the original bone surface was almost resorbed and new bone formation was found on the 8th week. Regulary-run collagen fibers, smooth and dense bone surfaces were similar to the bone patterns of control groups on the 12th week. Above results suggest that bone formation is restricted where the miniplate is applied, while compensatory growth is appeared in the adjacent bont. It is considered that rigid fixation with miniplates and acrews results in a little disturbance of sutural growth of the craniofacial bone in infancy and children when applied for short duration.

• Journal of Periodontal and Implant Science
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• v.35 no.2
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• pp.383-399
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• 2005

The successful implantation necessitate tissue regeneration m site of future implant placement, there being severe bone defect. Therapeutic approaches to tissue regeneration in the site have used bone grafts, root surface treatments, barrier membranes, and growth factors, the same way being applied to periodontal tissue regeneration. Great interest in periodontal tissue regeneration has lead to research in bone graft, guided-tissue regeneration, and the administration of growth factors as possible means of regenerating lost periodontal tissue. The blood component separated by centrifuging the blood is the platelet-rich plasma. There are growth factors, PDGF, $TGF{beta}1$, $TGF{beta}2$ and IGF in the platelet-rich plasma. The purpose of this study was to study the histopathological correlation between the use of platelet-rich plasma and the healing of bone defect around implant fixture site. Implant fixtures were inserted and graft materials were placed into the left femur of in the experimental group, while the control group received only implant fixtures. In the first experimental group, platelet-rich plasma and BBP xenograft were placed at the implant fixture site, and the second experimental group had platelet-rich plasma, BBP xenograft, and the e-PTFE membrane placed at the fixture site. The degree of bone regeneration adjacent to the implant fixture was observed and compared histopathologically at 2, 4, and 8 weeks after implant fixture insertion. The results of the experiment were as follows: 1. Bone remodeling in acid etched surface near the implant fixture of all experimental groups was found to be greater than new bone formation. 2. Bone remodeling in acid etched surface distant to the implant fixture of all experimental groups was decreased and new bone formation was not changed. 3. Significant new bone formation in machined surface near the implant fixture of bothl experimental groups was observed in 2 weeks. 4. New bone formation in machined surface distant to the implant fixture of both experimental groups was observed. Bone remodeling was significant in near the implant fixture and not in distant to the implant fixture. The results of the experiment suggested that the change of bone formation around implant. Remodeling in machined surface distant to the implant fixture of both experimental groups, and new bone formation and remodeling near the implant fixture were significant.

• Journal of Periodontal and Implant Science
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• v.25 no.2
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• pp.438-457
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• 1995

The origin of fibroblasts, their proliferative activity and roles in the early stages of periodontal regeneration were investigated in order to better understand the periodontal healing process in furcation defects of the beagle dog after guided tissue regeneration. Newly divided cells were identified and quantitated by immunolocalization of bromodeoxyuridine (BrdU) injected 1 hour prior to sacrificing the animals. The results were as follows :1. During periodontal healing in horizontal furcation defect, three different stages, namely the granulation tissue, connective tissue, and bone formation stages, were identified on the basis of major types of cells and tissue. 2. In the early stages of periodontal regeneration, both the remaining periodontal ligament and alveolar bone compartment were the major sources. 3. The majority of BrdU-labeled fibroblasts were located at the following areas ; 1) the coronal zone of the defect in case of the connective tissue fanned on the root surface. 2) the area within an 400 ${\mu}m$ distance from the remaining bone level in case of the periodontal ligament. 3) the area within an 100 ${\mu}m$ distance from the bone surface in case of areas of active bone formation.4. The highly proliferative fibroblasts adjacent to bone surface played a major role in the formation of osteoblast precursor cells, whereas both paravascular and endosteal cells played a minor role in new bone formation, In conclusion, it was suggested that the fibroblasts in the remaining periodontal ligament and bone will play a major role in periodontal regeneration, whereas both paravascular and endosteal cells will play a minor role in new bone formation.