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Ultrastructural Changes of Gastrocnemius Muscles after Shortening of Tibia and Neurectomy in the Rat.

Khun Sung WHANG ; Choong Kyu CHOI ; Jong Koo KIM ; Doo Jin BAEK

Journal of Korean Orthopaedic Research Society.1998;1(2):248-258.

This study was performed to compare the ultrastructural changes of muscles in fracture(group A), fracture with shortening(group B), neurectomy(group C) and fracture with neurectomy(group D) and to search for factors that lead to muscular atrophy. A total of 65 rats weighing about 350g were used as experimental animals. These animals were sacrificed in 3 weeks, 5 weeks and 7 weeks after the operation. The superficial portion (3 X 1 X 1mm3) of the gastrocnemius muscle was dissected and was prepared by routine methods. The results were as follows. 1. The contracted gastrocnemius muscle returned to its normal size at 7th week in group B. However in group C and D, the gastrocnemius muscles atrophied gradually. 2. Under light microscope, fibrotic change and atrophy in the muscles in group C and D progressed as time passed. 3. Myofibrillolysis and kinking of myofibrils in the muscles was observed in group B, and segmentation of Z-line was seen in group 4. At 7th week in group B, the muscles returned to its normal ultrastructures. Irregular shaped myofibrils, streaming of Z-line, dilation of cisternae of sarcoplasmic reticulum and mitochondria with dilation of cristae and electron lucent matrix in group C and the helical complex in the myofibrils of muscle fibers of the rats at 7th week in group D were observed. Consequently, it is suggested that 7 weeks after 20% shortening of tibia in length, contracted gastrocnemius muscles were reformed to its normal shape and the loss of nerve stimuli was a more significant factor that lead to atrophy of the muscle than the decrease of working tension by shortening of bone.
Animals ; Atrophy ; Mitochondria ; Muscle, Skeletal ; Muscles* ; Muscular Atrophy ; Myofibrils ; Rats* ; Rivers ; Sarcoplasmic Reticulum ; Tibia*

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Plasticity of Cloned Human Marrow Adipocytes between Osteogenesis and Adipogenesis.

So Ra PARK

Journal of Korean Orthopaedic Research Society.1998;1(2):239-247.

Information on the interconversion potential of adipocytes, the key in the understanding of bone turnover in metabolic diseases such as osteoporosis has been limited by the lack of pure adipocytic cell population. The object of the present study was i) to isolate pure populations of adipocytes from human bone marrow, ii) to clone single adipocytes from these populations, and iii) to examine in vitro the interconversion potential of the progeny of these single cloned adipocytes between the osteogenic and adipogenic phenotypes. Adipogenic colonies were isolated from the low-density floating fraction of normal bone marrow cultured in adipogenic media for 4 days. Single adipocytes were isolated and cloned by limiting dilution. The cloned adipocytes were found to dedifferentiate into fibroblastlike cells, and subsequently to differentiate into two morphologically distinct cell types: osteoblasts and adipocytes in appropriate culture systems. The adipocytic phenotype was confirmed by morphology, oil red O staining and immunocytochemistry using antiserum to aP2. The osteogenic phenotype was confirmed by alkaline phosphatase, osteocalcin immunostaing using an osteocalcin antiserum OS35 and mineralized nodule formation. These findings confirm that there is plasticity between the differentiation of adipocytic and osteogenic cells in human bone marrow stromal cell cultures. In conclusion, we have shown the ability of isolated clonal adipogenic cells to redifferentiate into cells of the osteogenic and adipogenic lineage and defined the conditions required for the in vitro modulation of adipogenic differentiation. These results provide further evidence that the osteogenic and adipogenic cells share a common multipotential precursor.
Adipocytes* ; Adipogenesis* ; Alkaline Phosphatase ; Bone Marrow* ; Clone Cells* ; Humans* ; Immunohistochemistry ; Mesenchymal Stromal Cells ; Metabolic Diseases ; Osteoblasts ; Osteocalcin ; Osteogenesis* ; Osteoporosis ; Phenotype ; Plastics*

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The Affinitty of Human Osteoblast to A-W Glass Ceramics.

Nam Yong CHOI ; Yong Sik KIM ; Hyun Seok SONG

Journal of Korean Orthopaedic Research Society.1998;1(2):230-238.

The purpose of this study was to evaluate the biocompatibility of polystyrene, titanium, and A-W ceramic focusing on the affinity of osteoblasts to the metals. Human osteoblasts were cultivated with each material (group I : polystyrene; group II :titanium; and group III:A-W ceramic) for seven days. Serial scanning electromicroscopic (SEM) examination and quantitative analysis of cellular protein synthesis were performed in each group of material. SEM examination showed that at the first day, the osteoblasts in group II and group III had longer and abundant cytoplasmic spindles than those in group 1. At the seventh day, the particulate networks (diameter 1-2 urn) were observed on the surfaces of osteoblasts in group I and group II but not in group III. In all groups, the amount of protein synthesis tended to increase with days of culture. Furthermore the actual amount was significantly larger in the orders of group III, group II, and group I (p<0.05, in all comparisons). This in vitro study suggests that when other factors are controlled, the superior affinity of human osteoblast to A-W ceramic might be attributable to the better biocompatibility than that of titanium and polystyrene.
Ceramics* ; Cytoplasm ; Glass* ; Humans* ; Metals ; Osteoblasts* ; Polystyrenes ; Titanium

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Transplantation of Culture-Expanded Bone Marrow Mesenchymal Cells and Type I Collagen Gel-Xenograft Complex.

Tae Joon CHO ; Jae Hoon AHN ; In Ho CHOI ; Kye Yong SONG

Journal of Korean Orthopaedic Research Society.1998;1(2):223-229.

Bone marrow contains mesenchymal stem cells capable of differentiating into osteoblastic lineage. To test the ability of purified bone marrow mesenchymal cell loaded onto xenograft with type I collagen gel to heal bone defect, mesenchymal stem cells were isolated from bone marrow of Lewis rat, and culture-expanded. Northern blot hybridization revealed that osteocalcin and osteopontin mRNA expression persisted for 8 weeks of in-vitro culture. When the bone marrow mesenchymal cells became 80-90% confluent in 2-3 weeks, they were loaded onto chips of xenograft (Lubboc(R) with type I collagen gel. The cell-gel-xenograft composite was transplanted subcutaneously into the dorsum of nude mice. It was also transplanted into tibial diaphyseal defect made in syngeneic Lewis rats, and compared with the control group where only the xenograft was planted. There was no ectopic bone formation at the dorsum of nude mice around the transplanted cell-gel-xenograft complex. In the long bone defect model, the cell-gel-xenograft group induced fibrosis around it and the bone healing was inferior to the control group. Type I collagen gel is not a suitable carrier of bone marrow mesenchymal cells to heal the long bone defect.
Animals ; Blotting, Northern ; Bone Marrow* ; Collagen Type I* ; Fibrosis ; Heterografts ; Mesenchymal Stromal Cells ; Mice ; Mice, Nude ; Osteoblasts ; Osteocalcin ; Osteogenesis ; Osteopontin ; Plants ; Rats ; RNA, Messenger

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Dose-Dependent Effects of Transforming Growth Factor-beta1 on Cell Proliferation and Matrix Synthesis in Relation to the Degree of Chondrocyte Dedifferentiation in Monolayer Culture.

Tae Gyun KIM ; Myung Chul LEE ; In Ho SEONG ; Sung Tae KIM ; Hee Joong YONG ; Hoon Park SANG ; Chul PARK ; Sang Cheol SEONG

Journal of Korean Orthopaedic Research Society.1998;1(2):204-211.

Transforming growth factor-beta1(TGF-beta1) has been suggested to be a useful growth facto. that could maintain the phenotypic characteristics of cultured chondrocytes. We performed this study to investigate the dose-dependent effects of TGF-beta1 on cell proliferation and matrix synthesis in relation to the degree of chondrocyte dedifferentiation in monolayer culture. Chondrocytes were isolated from the articular cartilage of distal femur of neonatal Sprague-Dawley rats and were cultured in monolayer. When the cell population reached 70-80% of confluence, the cells were subcultured successively for 6 weeks for the following studies. Every weak, the dose dependent effects of rhTGF-beta1 (0ng/ml ,1ng/ml, 5ng/ml, 10ng/ml, 20ng/m1) on DNA synthesis, proteoglycan synthesis and collagen synthesis were evaluated by 3H-thymidine incorporation, 35S-sulfate incorporation and 3H-proline incorporation respectively. The DNA synthesis decreased with time of culture. The DNA synthesis was significantly decreased by the addition of rhTGF-beta1 at all concentrations. The dose-dependent inhibitory action was most prominent at initial 3 weeks. Proteoglycan synthesis was in the highest level at the second week and markedly decreased after 2 weeks. At 2 weeks, TGF-beta1 treatment increased the proteoglycan synthesis at all concentrations and this stimulatory effect was prominent at the concentrations of 5ng/ml and 10ng/ml. There was no significant difference in the level of collagen synthesis during the whole experimental period. TGF- beta1 treatment increased collagen synthesis at all concentrations and this stimulatory effect was prominent at the concentrations at 5ng/ml and 10ng/ml. The results of this study suggest that if the cultured chondrocytes treated by TGF-beta 1 are used for transplantation, the time for TGF-beta1 treatment and transplantation to be recommended is before 3 weeks and the beneficial concentration of TGF-beta1 is 5ng/ml to 10 ng/ml for the maintenance of the phenotypic properties.
Cartilage, Articular ; Cell Proliferation* ; Chondrocytes* ; Collagen ; DNA ; Femur ; Proteoglycans ; Rats, Sprague-Dawley ; Transforming Growth Factor beta ; Transforming Growth Factor beta1

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Expression of Exon 2 of Type II Procollagen mRNA in Primary Culture of Rat Chondrocyte.

Tae Gyun KIM ; In Ho SEONG ; Sung Tae KIM ; Hee Joong KIM ; Sang Cheol SEONG

Journal of Korean Orthopaedic Research Society.1998;1(2):196-203.

Type II procollagen mRNA exists in two forms, type II A and II B, by alternative splicing of exon 2. Type II A procollagen mRNA has been known to be expressed in chondroprogenitor, periosteal and perichondrial cells, whereas type II B procollagen mRNA in chondrocytes. Previous reports indicate that both types of procollagen mRNA exist in primary culture of rat chondrocyte. In this study, the time-dependent change in the type II A and II B mRNA expression in the primarily cultured rat chondrocytes was analyzed using reverse transcription polymerase chain reaction (RT-PCR) and Northern hybridization. Most chondrocytes in the early stage of primary cell culture were oval or polygonal in their shape. However, since 2 to 3 weeks of primary cell culture, most cells changed their shapes into fibroblast-like or ameboid cells. RT-PCR revealed that until 1 week of culture, the expression of the type II B mRNA was greater than that of the type II A. After 2 weeks of culture, the expression of the type II A mRNA became greater. Northern blot analysis showed a gradual decrease in the expression of the total type II procollagen mRNA with time of culture. On the other hand, the expression of the type II A procollagen mRNA increased significantly at 2 weeks and the increased level was maintained thereafter. This study confirmed that in primary culture of chondrocyte, the cells changed their morphological and molecular characteristics at the relatively early stage. This study implicates that in order to delineate the changes of phenotypes of chondrocytes, quantitative analysis of both type II A and II B procollagen mRNA is necessary instead of performing analysis of total type II procollagen mRNA.
Alternative Splicing ; Animals ; Blotting, Northern ; Chondrocytes* ; Collagen Type II* ; Exons* ; Hand ; Phenotype ; Polymerase Chain Reaction ; Primary Cell Culture ; Procollagen ; Rats* ; Reverse Transcription ; RNA, Messenger*

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Immunohistochemical Study of the Expression of Transglutaminase Isoenzymes in the Healing Process of Rat's Articular Cartilage Defect.

In Ho SEONG ; Myung Chul LEE ; Sang Cheol SEONG

Journal of Korean Orthopaedic Research Society.1998;1(2):188-195.

Injury to the articular cartilage is clinically troublesome because its regeneration is limited, but until now no treatment method was proved to be satisfactory. Transglutaminases(TGase) are a group of enzymes which catalyze intermolecular crosslinking reaction to contribute to the structural stability of the tissue, and several authors suggested their use as potential biological glue. However there have been few studies on the expression of TGase and the roles of any specific subtype in the healing process of cartilge injury. So the authors observed the expression of tissue TGase(TGase C) and prostate TGase(TGase P) in the healing process of rat's articular cartilge defect. Into the knee articular cartilage of 5 month-old Sprague-Dawley rat, a defect was made with diameter 1.2mm drill bit. At 1, 3, 5, 7 days and 2, 4, 6, 8 weeks after the operation, the animals were sacrificed and their distal femurs were harvested for the histological studies. The sections were stained with HandE, alcian-blue and Safranin-O. Immunohistochemical study for the expression of TGase C and TGase P was done with the sections. Between 4 and 6 weeks after the operation, the articular cartilage defect was regenerated to the level of intact portion, but histologically it was regenerated not with hyaline catilage but with fibrocartilage. TGase C was expressed in fibroblasts, osteoblasts and fibrous matrix in the early healing phase, but after 4 weeks, it was expressed in chondrocytes and chondroid matrix. TGase P was not expressed in any of the normal articular cartilage, subchondral bone and reparative cells and tissue of the defect. As one of the new methods for the treatment of articular cartilage defect, chondrocytes were incorporated with biodegradable carriers and grafted into the injured site. To maximize the condition of the graft, addition of any specific material into these chondrocyte-carrier composite graft can be considered. In this study, TGase was found to be expressed in the healing process of articular cartilage defect. So, TGase might be supposed to play an important role in the healing process of cartilage injury, and could be tried as one of the additives for the chondrocyte graft.
Adhesives ; Animals ; Cartilage ; Cartilage, Articular* ; Chondrocytes ; Femur ; Fibroblasts ; Fibrocartilage ; Hand ; Humans ; Hyalin ; Infant ; Isoenzymes* ; Knee ; Osteoblasts ; Prostate ; Rats ; Rats, Sprague-Dawley ; Regeneration ; Transplants

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Experimental Study of the Histologic Findings of the Neoendothelialization according to Length of Polytetrafluoroethylene in Rabbit.

Kwang Suk LEE ; Ki Hoon KANG ; Kyu Ho KYON

Journal of Korean Orthopaedic Research Society.1998;1(2):174-187.

The purpose of this study was to investigate the patency rates and the histologic findings of neoendothelialization according to the length of implanted polytetrafluoroethylene (PTFE) with an internal diameter of 3mm. Under the operating microscope, grafts of 8 and 24mm in length were implanted in the right carotid arteries of thirty rabbits by interrupted end-to-end microanastomosis. They were divided into two groups according to the length of implanted PTFE. Each group had fifteen rabbits. All implanted grafts were 25micro meter in fibril length and 0.39mm in wall thickness. Three grafts per group were harvested at 1, 2, 4, 8, and 12 weeks after implantation and all grafts were observed for patency rates and the histologic findings with a light microscope and scanning electron microscope. In conclusion, there was no difference in patency rates according to the length of implanted PTFE. However, the formation of neointima and subintimal tissue was delayed and incomplete in the longer implanted PTFE group.
Carotid Arteries ; Neointima ; Polytetrafluoroethylene* ; Rabbits ; Transplants

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Wear Properties of Surface Modified Ultra-high Molecular Weight Polyethylene.

Kyung Tae KIM ; Jae Bong CHOI ; Kui Won CHOI

Journal of Korean Orthopaedic Research Society.1998;1(2):167-173.

The wear debris of ultra-high molecular weight polyethylene (UHMWPE) used as a bearing material in total joint arthroplasty evokes a serious adverse biological reaction (osteolysis). In this study, therefore, the wear resistance of UHMWPE is enhanced by attaching a hydrophilic graft on the UHMWPE surface and by improving surface hardness without deteriorating the mechanical properties, which may result in the formation of lubrication film and the increased resistance to penetration, scratching or deformation during articulation. This was done by ion-implantation technique. The results showed that the wettability and the surface hardness of the modified UHMWPE by the above treatments increased due to the increased cross-linking density and hydrophilic graft attachment on the surface. The wear test was performed by a wear testing machine of pin-on-disk type. The modified UHMWPE revealed about 30~50% less volumetric wear than the control specimen. It was also shown that the volumetric wear depended not only on the optimal treatment condition but on the applied load and the sliding speed.
Arthroplasty ; Hardness ; Joints ; Lubrication ; Molecular Weight* ; Osteolysis ; Polyethylene* ; Transplants ; Wettability

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Experimental Study of the Histologic Findings of the Neoendothelialization according to Diameter of Polytetrafluoroethylene in Dog.

Kwang Suk LEE ; Hae Il PARK ; Ki Hoon KANG ; Dong Hoon SUH

Journal of Korean Orthopaedic Research Society.1998;1(2):154-166.

The purpose of this study was to investigate the patency rates and the histologic findings of neoendothelialization according to the diameter of polytetrafluoroethylene (PTFE) which is the most widely used synthetic graft for blood vessel. Under the operating microscope, grafts of 3 and 5mm in diameter were implanted in both femoral arteries of ten mongrel dogs by interrupted end-to-end microanastomosis. They were divided into two groups according to the diameter of implanted PTFE (3 and 5mm). Each group had five dogs. The lengths of implanted PTFE were 20mm in both groups. All implanted grafts were 25 urn in fibril length and 0.39mm in wall thickness. Two grafts from one dog were harvested at 1, 2, 4, 8, and 12 weeks after implantation and all PTFE grafts were observed for patency rates. The histologic analysis was performed under a light microscope and scanning electron microscope. In conclusion, PTFE with larger diameter than that of recipient vessel is recommended as the grafts for implantation because PTFE is not as elastic as normal vessel.
Animals ; Blood Vessels ; Dogs* ; Femoral Artery ; Polytetrafluoroethylene* ; Transplants

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