To investigate the efficiency of a fibrous collagen membrane(FCM) composed of bovine skin type I atelocollagen as a carrier for BMP, partially purified bovine BMP/FCM($0.3mg/10{\times}5{\times}1mm$) composites were implanted into the dorsal subcutaneous tissue of rats. FCM alone was also implanted as a control. The implants were harvested at 1, 2, 3, and 10 weeks after implantation, then prepared for routine light microscopic observation. The FCM alone did not induce osteogenesis and revealed no specific foreign body reaction nor was there any definite resorptive evidence for 10 weeks after implantation, while the BMP/FCM composites induced favorable bone formation in a process that resembled an endochondral and direct ossification mode. At 10 weeks, the well formed bone confined to embedded collagen fibers revealed hematopoietic marrow between bony trabeculae without evidence of resorptive or degenerative changes . These findings support the suggestion that BMP may induce undifferentiated mesenchymal cells into either chondroblasts or osteoblasts or both independantly according to the chemico- physical characteristics of the carrier, which develops the endochondral and/or direct bone formation process, and suggest that the FCM may be a favorable carrier for BMP.
Bone morphogenetic proteins(BMPs) are a group of transforming growth factor beta(TGF-${\beta}$)-related factors and multifunctional proteins, especially the only known biologic factors capable of inducing endochondral bone formation at an extraskeletal site. This study was performed to investigate the effect of the partially purified porcine BMP(pBMP) at an ectopic site. PBMP was partially purified from porcine bone matrix and its activity was monitored by an in vivo bioassay. The purification method utilized extraction of the bone-inducing activity with 4M guanidine, followed by chromatography on heparin-Sepharose. Active fractions were assayed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. And the fractions were reconstituted with inactive insoluble collagenous bone matrix from rats, acid soluble type I collagen from rat tail and chondroitin-6-sulfate sodium salt and implanted into the pectroralis muscle pouches of Sprague-Dawley rats. And the carrier complex was implanted on the opposite side as control. The rats were sacrificed at the day of 1st, 3rd, 5th, 7th, 11th, 14th and 21st after implantation and examined histologically, radiologically and biochemically. And alkaline phosphatase activity and calcium content were used as indices of bone formation. The results were as follows ; 1. Active fractions were localized in a zone between 31 and 40 KDa on SDS-PAGE. 2. The implanted 3.0mg of the partially purified pBMP induced cartilage and bone in the muscle tissue of rats through an endochondral ossification process. 3. Inactive insoluble bone matrix, type I collagen and chondroitin-6-sulfate have functioned as carriers for pBMP, but revealed some foreign body reactions. 4. Soft X-ray didn't reveal significant change between the experimental and the control group. 5. The alkaline phosphatase activities in the experimental group of 5th, 7th, 11th, 14th and 21st were increased significantly compared with control (p<0.01) with the peak in the group of 11th day. 6. With time, the calcium content of the experimental group increased. And the calcium contents in the experimental group of 11th, 14th and 21st were increased significantly compared with control (p<0.01).
Objectives : The process through which mesenchymal cells condense and differentiate into chondrocytes to form new bone is known as endochondral bone formation. Chondrogenic differentiation and hypertrophy are essential steps in bone formation and are influenced by various factors. The stem bark and root bark of Eleutherococcus sessiliflorus (ES) have been widely used to treat growth retardation and arthritis in traditional Korean Medicine. In this study, we aimed to investigate the possible role of the stem bark of ES in the stimulation of chondrogenic differentiation in clonal murine chondrogenic ATDC5 cells. Methods : In ATDC5 cells treated with ES extract, cell viability and extracellular matrix production were determined using CCK-8 assay and Alcian blue staining, respectively, and alkaline phosphatase activity was measured. We also examined mRNA and protein expression levels of genes related to chondrogenic expression in ATDC5 cells using reverse transcription-polymerase chain reaction and western blot analyses. Results : ES extract increased the accumulation of Alcian blue-stained cartilage nodules and alkaline phosphatase activity in ATDC5 cells. It increased the mRNA expressions of chondrogenic markers including bone sialoprotein (BSP), cartilage collagens, Runt-related transcription factor-2 (RUNX-2), osteocalcin (OCN), β-catenin, and bone morphogenetic protein-2 (BMP-2), as well as the protein expressions of β-catenin, RUNX-2, BMP-2, and alkaline phosphatase (ALP). Conclusion : Taken together, these results suggest that ES extract exhibits a chondromodulating activity and therefore may be a possible agent for the treatment of bone growth disorders.
Receptor activator of nuclear factor-${\kappa}B$ ligand (RANKL) is an osteoblast/stromal cell-derived essential factor for osteoclastogenesis. During endochondral bone formation, hypertrophic chondrocytes calcify cartilage matrix that is subsequently resorbed by osteoclasts in order to be replaced by new bone. Hypoxia-induced upregulation of RANKL expression has been previously demonstrated in an in vitro system using osteoblasts; however, the involved mechanism remains unclear in chondrocytes. In the present study, we investigated whether hypoxia regulates RANKL expression in ATDC5 cells, a murine chondrogenic cell line, and hypoxia-inducible factor-$1{\alpha}$ (HIF-$1{\alpha}$) mediates hypoxia-induced RANKL expression by transactivating the RANKL promoter. The expression levels of RANKL mRNA and protein, as well as HIF-$1{\alpha}$ protein, were significantly increased in ATDC5 cells under hypoxic condition. Constitutively active HIF-$1{\alpha}$ alone significantly increased the levels of RANKL expression under normoxic conditions, whereas dominant negative HIF-$1{\alpha}$ reduced hypoxia-induced RANKL expression. HIF-$1{\alpha}$ increased RANKL promoter reporter activity in a HIF-$1{\alpha}$ binding element-dependent manner in ATDC5 cells. Hypoxia-induced RANKL levels were much higher in differentiated ATDC5 cells, as compared to proliferating ATDC5 cells. These results suggested that under hypoxic conditions, HIF-$1{\alpha}$ mediates induction of RANKL expression in chondrocytes; in addition, hypoxia plays a role in osteoclastogenesis during endochondral bone formation, at least in part, through the induction of RANKL expression in hypertrophic chondrocytes.
Seo, Jae-Jin;Kim, Tak;Pi, Sung-Hee;Yun, Gi-Yun;Yu, Hyung-Keun;Shin, Hyung-Shik
Journal of Periodontal and Implant Science
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v.30
no.3
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pp.553-569
/
2000
Many natural medicines have been studied for their capacity and effects of antibacterial, anti-inflammatory and regenerative potential in periodontal tissues. Safflower seed has been traditionally used as a drug for treatment of bone fracture in oriental medicine. The purpose of the present study was to compare the effects of safflower seed extract and bone substitute on bone formation and regeneration in artificial defects in mongrel dogs. The bony defects were made with round bur at mandible and tibia. Extracts of safflower seed and bovine bone were placed directly at each defect for experimental group, and the defect of control group was sutured without any other treatment. Experimental animals were sacrificed at 8 weeks. And then histopathologic reading and histomorphometric study was done. There was not significant differences between control and experimental groups in osteoclastic activity and infiltration of inflammatory cells. However, new capillary proliferation, fibrosis and new bone formation were prominent in safflower seed extract group. The mandibular defects of safflower seed extract group were healed with dense connective and bony tissues, and endochondral bone formation was observed in tibial defect of safflower seed extract group only. New bone area of safflower seed extract group was more significantly increased than that of control and that of bone substitute group. These results indicate that direct local application of safflower seed extracts on bony defects seems to reduces the early inflammatory response and to promotes the bone regeneration.
The purpose of this study was to observe the healing process and the distribution of fibronectin in injured condylar cartilage and bone by using LM and SEM. In order to perform this study, 40 male rat, weighing about 250g were selected. Under general anesthesia with Pentobarbital sodium, condylar cartilage and neck bone were resected. Then, the wound was irrigated with saline and closed with 5-0 chromic catgut and 4-0 silk by layer-to-layer suturing. The experimental rats were sacrificed by perfusion with 3% paraformaldehyde at 1st and 4th week after operation. The condylar process and surrounding tissues were cut, demineralized, dehydrated and embedded in paraffin. The histological observation of the specimens in LM level was performed after H-E stain and Azan stain. For localization of fibronectin, immunostaining was achieved by the avidin-biotin complex method. To study the change on condylar surface, the specimens were dehydrated, dried, gold coated and were observed with a scanning electron microscope(Hitachi S-2300). The results were as follows ; 1. The cartilage group and the bone group were repaired with epiphyseal cartilage layer on the cut surface as the normal control group. 2. The cut surface was repaired more quickly in the cartilage group than in the bone group. 3. Chondrocytes, diferentiated during healing, were stained strongly to anti-fibronectin, and fibronectin was supposed to participatein chondrocyte differentiation and cartilagenous matrix formation. 4. Fibronectin was distributed more in the new bone than in the old bone, and the osteoblasts surrounding it were also stained strongly. Fibronectin was supposed to participate in new bone matrix formation. 5. Fibronectin is supposed to be associated with the differentiation, migration and adhesion of chondrocyte and osteoblast and to participate in endochondral bone formation.
Approximately 200 individual skeletal elements, which differ in shape and size, are the building blocks of the vertebrate skeleton. Various features of the individual skeletal elements, such as their location, shape, growth and differentiation rate, are being determined during embryonic development. A few skeletal elements, such as the lateral halves of the clavicle and parts of the skull are formed by a process called intramembranous ossification, whereby mesenchymal cells differentiate directly into osteoblasts, while the majority of skeletal elements are formed via endochondral ossification. The latter process starts with the formation of a cartilaginous template, which eventually is being replaced by bone. This requires co-regulation of differentiation of the cell-types specific for cartilage and bone, chondrocytes and osteoblasts, respectively. In recent years it has been demonstrated that Wnt family members and their respective intracellular pathways, such as non-canonical and the canonical $Wnt/{\beta}$-catenin pathway, play important and diverse roles during different steps of vertebrate skeletal development. Based on the recent discoveries modulation of the canonical Wnt-signaling pathway could be an interesting approach to direct stem cells into certain skeletal lineages.
Journal of the Korean Association of Oral and Maxillofacial Surgeons
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v.30
no.4
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pp.261-270
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2004
The purpose of this experiment was to examine the histological changes and the pattern of expression of type I, II collagen in the elongated area by distraction osteogenesis in the rabbit mandible. Sixteen rabbits weighing 2.5kg-3kg were used for this experiment. Experimental group was distracted at the rate of 0.7mm, twice/day for 7days, and control group was only osteotomized. After 5 days latency, osteotomic site is distracted for 7days. Consolidation period is 28days. The animal was sacrificed at the 3rd, 7th, 14th, 28th day after the operation. The distracted bone was examined by histological analysis and RT-PCR analysis. The results were summarized as follows: 1. Experimental group was observed that the gaps between the distracted bone edges were occupied by new bone. 2. Expression of Type I collagen were detected throughout the experiment in both groups and Expression of Type I collagen were markedly increased during distraction and consolidation period in experimental group than control group. 3. Expression of Type II collagen were detected throughout the experiment in both groups and expression of Type II collagen were maintained at high level during distraction and consolidation period in experimental group than control group. From these results, in contrast to type II collagen, type I collagen seemed to be more expressed by mechanical stimuli during distraction and consolidation period. The predominent mechanism of new bone formation in the distraction gap was intramembranous bone formation, but some of the regenerated bone was formed by endochondral ossification.
Endochondral bone formation is the process by which mesenchymal cells condense into chondrocytes, which are ultimately responsible for new bone formation. The processes of chondrogenic differentiation and hypertrophy are critical for bone formation and are therefore highly regulated. The present study was designed to investigate the effect of aloe-emodin on chondrogenic differentiation in clonal mouse chondrogenic ATDC5 cells. Aloe-emodin treatment stimulated the accumulation of cartilage nodules in a dose-dependent manner. ATDC5 cells were treated with aloe-emodin and stained with alcian blue. Compared with the control cells, the ATDC5 cells showed more intense alcian blue staining. This finding suggested that aloe-emodin induced the synthesis of matrix proteoglycans and increased the activity of alkaline phosphatase. Aloe-emodin also enhanced the expressions of chondrogenic marker genes such as collagen II, collagen X, BSP and RunX2 in a time-dependent manner. Furthermore, examination of the MAPK signaling pathway showed that aloe-emodin increased the activation of extracellular signal-regulated kinase (ERK), but had no effect on p38 and c-jun N-terminal kinase (JNK). Aloe-emodin also enhanced the protein expression of BMP-2 in a time-dependent manner. Thus, these results showed that aloe-emodin exhibited chodromodulating effects via the BMP-2 or ERK signaling pathway. Aloe-emodin may have potential future applications for the treatment of growth disorders.
Journal of the Korean Association of Oral and Maxillofacial Surgeons
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v.26
no.6
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pp.620-627
/
2000
The purpose of this study is to investigate the clinical and histologic changes in distraction osteogenesis according to different distraction rates in the rat's tibia. Eighteen adult rats underwent open osteotomy and attachment of an external unilateral distraction device in the middle of left tibia. Latency was allowed for 7 days before distracton began. The distraction device was activated with varying distraction rates of 0.5mm, 1mm, 2mm and same rhythm of twice a day until 5mm length gain was achieved. The animals were sacrificed at post-distraction 4, 8 weeks to observe the bony healing states. At each group, clinical, radiographic and histologic studies were done. The results obtained from this study were as follows: 1. The 0.5mm group showed excellent osteogenesis than other groups. The new bone was formed by intramembranous bone formation mostly and endochondral bone formation partly. 2. The 1mm group showed delayed osteogenesis and incomplete bony healing at 8 weeks. 3. The 2mm group showed weak osteogenesis and fibrous union or nonunion at 8 weeks. From these results, it could be stated that distraction rate of 0.5mm per day was most useful in rat's tibia. The rate of 1mm showed delayed bony healing and needed more consolidation period. Distraction osteogenesis is a excellent clinical method for regenerating local bone deficiencies in limbs and craniofacial area. The more studies needed for the higher animals and human about distraction rates and other biomechanical factors on the basis of this study.
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