PURPOSE: To confirm the adhesion and matrix formation of chondrocytes which were cultured on chitosan beads and to elucidate the difference between the porous chitosan beads and non-porous chitsan beads as scaffold for chondrocytes. MATERIALS AND METHODS: Chondrocytes isolated from rabbit articular cartilage were cultured in vitro on porous and non-porous chitosan bead for 2 weeks. Histochemical (H&E stain, Toluidin blue stain) and scanning electromicroscopic approaches were used to compare the differences between two groups. RESULTS: In both groups, adhesion and proliferation of chondrocytes were observed on scanning electron microscopy. which were more active in the porous chitosan bead group. On histochemical staining with toluidine blue, the porous chitosan bead group showed stronger metachromasia than that of the non-porous chitosan bead. CONCLUSION: It is concluded that both chitosan beads could work as an effective scaffold for culturing chondrocytes, and that porous chitosan bead may be a better scaffold than non-porous chitosan bead because of cavities in former bead.
Cartilage, Articular ; Chitosan* ; Chondrocytes* ; Microscopy, Electron, Scanning ; Tolonium Chloride

The periosteum provides a good cell source for bone formation because it possesses osteoprogenitors. In this study, the osteogenic activity of the rabbit periosteum and periosteal cells was investigated. The periosteum was harvested from the long bone surface of New Zealand White rabbits. The periosteal cells were isolated from the periosteum and expanded. In vitro examination, the morphological changes, mineralization, and osteogenicity of the periosteal cells were evaluated. The amount of osteocalcin released was measured to check the maximal activity of the cells. The periosteum and periosteal cells-scaffold composites were transplanted into athymic mice to observe the in vivo bone formation. Radiological and histological changes were checked every 2 weeks. The isolated periosteal cells were uniformly proliferated in a monolayer culture, and mineral deposition was confirmed after 10 weeks. The osteogenic markers such as osteopontin, osteonectin, type I collagen, and alkaline phosphatase were expressed from the periosteal cells after 2 weeks. Osteocalcin was expressed after 3 weeks and was maximally expressed at 4 weeks. In 3-dimensional culture, the periosteal cells were well adhered and proliferated on the poly L-lactic-co-glycolic acid scaffold. The periosteal cells-scaffold composite transplant produced of osteoid without calcification in vivo. Radiological examination showed minimal changes of bone formation during the experimental period. However, the periosteum transplant was converted to a cartilaginous structure at 2 weeks, bony calcification after 4 weeks, and complete bony trabecula and marrow space formation at 12 weeks. The radiological bone density also increased during the experimental period. The periosteum may be one of good cell sources for osseous tissue engineering. Engineered periosteal cells may be used for the treatment of nonunion, osteolysis, avascular necrosis and fusion of the spine.
Alkaline Phosphatase ; Animals ; Bone Density ; Bone Marrow ; Collagen Type I ; Mice ; Mice, Nude ; Necrosis ; Osteocalcin ; Osteogenesis ; Osteolysis ; Osteonectin ; Osteopontin ; Periosteum* ; Rabbits ; Spine ; Tissue Engineering

PURPOSE: We investigated the reference line closer to the true femoral anteversion on only the cutting surface of the proximal femoral neck during the femoral stem insertion in cementless total hip arthroplasty. MATERIALS AND METHODS: A postoperative CT of both hips was taken to observe the positioning of the stem and its correlation with the true anteversion of the contralateral side of 33 consecutive patients after unilateral primary cementless total hip arthroplasty with avascular necrosis of the femoral head. We defined several parameters and measured the true anteversion on the contralateral side and the stem anteversion on the lesion side. RESULTS: The average of the midcortical angle was 13.9 degrees +/-6.8 degrees on the lesion side and 0.1 degrees +/-1.3 degrees less than the true anteversion on the contralateral side. CONCLUSION: The study has shown that anteversion using a midline between the anterior cortical line and the posterior cortical line is compatible with the true femoral anteversion.
Arthroplasty, Replacement, Hip* ; Femur Neck ; Head ; Hip ; Humans ; Necrosis

PURPOSE: The objective of this study was to investigate the use of cultured rib perichondrial cells embedded in alginate bead on healing in a rabbit osteochondral defect model. The degree of articular cartilage repair was evaluated histologically, histomorphometrically, and biochemical characteristics of the neocartilage. MATERIALS AND METHODS: A single defect, 3.5 mm wide by 3 mm deep, was created in the weight bearing area of the medial femoral chondyle in thirty New Zealand rabbits. The right defect filled with two alginate beads embedded with rabbit rib perichondrial cells, the left defect was empty as the control. The animals were killed at 1, 3, and 12 months, and the repair tissues were examined histologically, and histomorphometric differences were evaluated by an image analysis system. The defects also were examined biochemically for the glycosaminoglycan (GAG) and type II collagen to compare the results with normal articular cartilage. RESULTS: The attachment of repair tissue with the surrounding host tissue was incomplete, many specimens exhibited degenerative changes in adjacent tissue over a post transplant time period. Histomorphometric results showed that the repair groups (0.24+/-0.11 mm, -26.97 (25.62 mm) was decreased in surface roughness, and depression than controls (0.32+/-0.06 mm, -48.73 (32.59 mm) at 12 months. Repair area, repair area ratio, and repair thickness of the repair groups (6.89+/-2.1 mm2, 39.5+/-19.5%, 0.11 (0.01 mm) were increased than controls (2.65+/-2.35 mm2, 2.85+/-2%, 0.09+/-0.04 mm) at 12 months. After 12 months, the content of GAGs of neocartilage (36.45 microgram/mg) was similar to those of normal artilcular cartilage (36.74 microgram/mg), the percentage of type II collagen of the neocartilage increased up to 95%. CONCLUSION: Transplanted rib perichondrial cells were seen to proliferate to fill the osteochodral defect with neocartilage. Histomorphometric analysis should allow a more quantitative described the degree of articular cartilage repair.
Animals ; Cartilage ; Cartilage, Articular ; Collagen Type II ; Depression ; Rabbits ; Ribs ; Weight-Bearing

PURPOSE: We explored the possibility of DNA methylation as a mechanism of loss of type II collagen expression in dedifferentiating chondrocytes by culturing in monolayer. MATERIALS AND METHODS: Dedifferentiation was induced by low density subculturing primary porcine chondrocytes in vitro. The mRNA expression of Type I collagen, Type II collagen and DNA methyltransferase (DNMT) was measured by RT-PCR. Induction of redifferentiation in dedifferentiated chondrocytes was performed in 3-dimensional alginate bead culture system. As stimulating factors for reexpression of genes in dedifferentiated chondrocytes, 10 ng/ml TGF-beta1 and 5 micrometer 5-azacytidine were used. RESULTS: Type II collagen mRNAs was expressed strongly in freshly isolated cells but had decreased in monolayer cultured cells after 3 weeks up to 40%. In contrast, type I collagen expression was increased from 21 days and kept increasing during the 86 days of study. After treatment of 5 micrometer 5-azacytidine, fibroblast like morphology was changed to round shape such as traditional chondrocyte morphology at day 4. At day 10, type II collagen expression was increased by 5-azacytidine and TGF-beta1 marginally and also integrin beta1 expression was increased in all groups. RT-PCR analysis demonstrated that DNMT3A expression increased in dedifferentiating chondrocytes when compared with control cells for 40 days. CONCLUSION: Loss of type II collagen mRNA expression and increase of DNMT 3A expression were showed similar patterns during dedifferentiation. These results suggest that type II collagen gene expression may be influenced by DNA methylation. As stimulating factors, TGF-beta1 and 5-azacytidine have potential activity to increase the type II collagen expression in alginate culture system.
Antigens, CD29 ; Azacitidine ; Cells, Cultured ; Chondrocytes* ; Collagen Type I ; Collagen Type II* ; DNA Methylation* ; DNA* ; Fibroblasts ; Gene Expression ; RNA, Messenger ; Transforming Growth Factor beta1

PURPOSE: The purpose of this study is to find out the possibilities of the new treatment via continuous stimulation by photo-immobilization of growth factor in the anterior cruciate ligament(ACL) injury. MATERIALS AND METHODS: Photo-reactive epidermal growth factor(EGF-Az) synthesized by conjugating EGF with N-(4-azidobenzoyloxy)succinimide was immobilized onto biodegradable tissue-regenerative mesh(Bio-Mesh(R)) using UV irradiation. We sutured the BioMesh containing photo-reactive EGF on the artificial defect area of ACL in the left knee of beagle dogs and sutured the BioMesh that was not treated with EGF-Az on the defect area of ACL in the right knee. Four weeks later, we obtained the tissues from the defect area of both ACLs and compared the results of HE stain, immunohistochemistry and RT-PCR in both tissues. RESULTS: In results of HE stain and immunohistochemistry, we observed better cellular proliferation and more definite spindle shape of cells in EGF-immobilized area than in non-EGF defect area. Also cellular density was higher in EGF-immobilized area. On the other hand, cells had shaped more round and cellular density was lower in non-EGF defect area. In results of RT-PCR of GAPDH(Glyceraldehyde-3-phosphate dehydrogenase), COL I(Collagen type I), COL III and EGF, except for TGF-beta1(Transforming growth factor-beta 1), we also observed more definite expressions in EGF-immobilized area than in non-EGF defect area. CONCLUSION: We were able to confirm rapid cellular proliferation via artificial stimulation effect induced by photo-immobilized EGF. If EGF is immobilized onto biodegradable materials for clinical application, it will contribute to the treatment of ACL injury.
Animals ; Anterior Cruciate Ligament* ; Cell Proliferation ; Dogs* ; Epidermal Growth Factor ; Hand ; Immunohistochemistry ; Knee*

PURPOSE: To investigate the effects of transglutaminase in the environment of extracellular matrix on perichondrocyte in alginate culture. MATERIALS AND METHODS: Perichondrocyte cells were isolated from articular cartilage of New Zealand white rabbits by enzymatic digestion and maintained in monolayer culture. After 7 days, the cells were trypsinized and cultured in an alginate bead system. Four groups of the alginate beads were prepared as follow: containing 1 mg/ml of transglutaminase, 10 microgram/ml of fibronectin, mixture of 1mg/ml of TGase and 10 microgram/ml of fibronectin and only perichondrocytes as a control group. Cell proliferation was measure by [Methyl-3H] Thymidine uptake, and proteoglycan synthesis was measure by [35S] Sulfate uptake. The gene expression of integrin-alpha5, integrin-beta1 and type II collagen was analyzed by reverse transcription-polymerase chain reaction. Safranin-O staining was utilized for histological assessment of proteoglycan in extracellular matrix. One-way ANOVA was used to analyze the results statistically. RESULTS: Mixture of transglutaminase and fibronectin exhibited high synthesis rates of proteoglycan and active cell proliferation compared with other groups. The gene expression of type II collagen did not show significant difference between groups. The gene expression of integrin-alpha5 was down-regulated in all groups with time. The gene expression of integrin-beta1 was not down-regulated with time only in mixture of transglutaminase and fibronectin. Histological staining of the secretions by Safranin-O staining was in agreement with the data of proteoglycan synthesis, and Safranin-O staining showed that more cell-to-cell aggregates is developed in the mixture of transglutaminase and fibronectin. CONCLUSION: Mixture of transglutaminase and fibronectin can stimulate chondrocyte proliferation and proteoglycan synthesis, and integrin seems to modulate such interactions.
Cartilage, Articular ; Cell Proliferation ; Chondrocytes ; Collagen Type II ; Digestion ; Extracellular Matrix ; Fibronectins ; Gene Expression ; Proteoglycans ; Rabbits ; Thymidine ; Trypsin

PURPOSE: This study validated the musculoskeletal model of the human lower extremity by comparative study between calculated muscle parameters through simulation using modified hill-type model and measured muscle parameters through isokinetic exercise. The relationship between muscle forces and moments participated in motion was quantified from the results of simulation. MATERIALS AND METHODS: For simulation of isokinetic motion, a three-dimensional anatomical knee model was constructed using gait analysis. The EMG-force model was used to determine muscle activation level exciting muscles. The modified Hill-type model was used to calculate individual muscle force and moment in dynamic analysis. This method was validated by comparing analytical data with experimental data. RESULTS: The results showed that there was a significant correlation between calculated torques from simulation and measured torque from isokinetic motion experiments (R=0.97). We also found that muscle forces and moments during knee flexion and extension have nonlinearly proportional or inversely proportional relationship, since lower extremity muscles were simultaneously involved in flexion/extension motion and inner/outer rotation. CONCLUSION: We concluded that the simulation by using musculoskeletal model may be a useful mean to predict and recover musculoskeletal-related diseases, and analyze complicated experiment such as clash condition.
Gait ; Humans ; Knee Joint* ; Knee* ; Lower Extremity ; Muscles ; Torque

PURPOSE: This study was performed to create a traumatic brain injury (TBI) animal model of up-regulated bone formation, and to show the possibility of comprehensive analysis of early gene expression from the hard tissue by cDNA microarray technique. MATERIALS AND METHODS: Thirty Sprague-Dawley rats underwent either a severe brain injury operation procedure using the water pump with femur fracture (Group I, N=15), or femur frecture only (group II, N=15). The femur was nailed with a 20-gauge needle and fractured. The rats were euthanized at the day 1, 3, 8, 14, and 28 day. The volume of callus was calculated using GE PACS(R). The total RNA was isolated from the 3rd day's callus and the gene expression was compared using microarray chips. RESULTS: The average time until union was 28 days for control group and 14 days for TBI group, which was significantly shorter. The volume of the callus (27.4+/-8.5 mm2) in the TBI animals was clearly greater than that of the control group (13.0+/-6.3 mm2). The mRNA was successfully extracted from the callus and analysed using the c-DNA microarray, and. 312 genes were significantly increased and 227 genes were decreased in the TBI group. CONCLUSION: This study successfully made a model for a accelerated fracture healing in traumatic brain injury. We showed that cDNA microarray technique can be used to compare the expression of the mRNA in fracture callus. This method will be helpful in analysing the mechanism of accelerated fracture healing after brain injury.
Animals* ; Bony Callus ; Brain Injuries* ; Femur ; Fracture Healing* ; Gene Expression* ; Models, Animal* ; Needles ; Oligonucleotide Array Sequence Analysis ; Osteogenesis ; Rats ; Rats, Sprague-Dawley ; RNA ; RNA, Messenger

PURPOSE: To investigate the expression of Vascular Endothelial Growth Factor (VEGF) in diabetic frozen shoulders. MATERIALS AND METHODS: We preformed arthroscopic adhesiolysis on 9 diabetic frozen shoulder patients, and observed the arthroscopic findings. Also, we examined the potential role of VEGF by using samples of synovial tissues from 5 patients, and 2 normal synovial tissues. Immunohistochemical staining and Western blotting were performed using polyclonal antibodies against VEGF. RESULTS: There was hyperemic synovitis in the 9 diabetic frozen shoulder patients. In the 5 patients' tissue samples, there was strong immunostaining and expression to VEGF, but there was little staining and expression in the control group. CONCLUSION: We postulate that VEGF is synthesized and secreted in the synovium of diabetic frozen shoulders and that secreted VEGF binds specific receptors on the endothelial cells of nearby small blood vessels, and leads to the subsequent development of frozen shoulders in diabetic patients.
Antibodies ; Blood Vessels ; Blotting, Western ; Bursitis* ; Endothelial Cells ; Humans ; Shoulder ; Synovial Membrane ; Synovitis ; Vascular Endothelial Growth Factor A*