BACKGROUND: The objective of this study was to explore whether individual variations in the concentration of growth factors (GFs) influence the biologic effects of platelet-rich plasma (PRP) on human mesenchymal stem cells (HMSCs). METHODS: The concentrations of 7 representative GFs in activated PRP (aPRP) were measured using ELISA. The effects of PRP on the proliferation and alkaline phosphatase (ALP) activity of HMSCs were examined using several concentrations of aPRP from 3 donors; the relationships between the GF levels and these biologic effects were then evaluated using 10% aPRP from 5 subgroups derived from 39 total donors. HMSCs were cultured in DMEM with the addition of aPRP for 4 or 12 days; then, DNA content and ALP activity were measured. RESULTS: The quantity of DNA increased significantly at a 10% concentration of aPRP, but the ALP activity was suppressed at this concentration of aPRP. The GF concentrations varied among donors, and 5 subgroups of characteristic GF release patterns were identified via cluster analysis. DNA levels differed significantly between groups and tended to be higher in groups with higher concentrations of transforming growth factor-beta1 (TGF-beta1) and platelet-derived growth factors (PDGFs). DNA quantity was positively correlated with TGF-beta1 concentration, and was negatively correlated with donor age. ALP activity was negatively correlated with PDGF-BB concentration. CONCLUSIONS: The varying GF concentrations may result in different biologic effects; thus, individual differences in GF levels should be considered for reliable interpretation of the biologic functions and standardized application of PRP.
Alkaline Phosphatase/metabolism ; Blood Donors ; Cell Differentiation ; Cells, Cultured ; Culture Media/chemistry ; DNA/analysis ; Humans ; Intercellular Signaling Peptides and Proteins/*pharmacology ; Mesenchymal Stem Cells/*cytology/drug effects ; Platelet-Derived Growth Factor/pharmacology ; Platelet-Rich Plasma/*metabolism ; Transforming Growth Factor beta1/pharmacology

PURPOSE: Bone morphogenetic proteins (BMPs) play an important role in the formation of cartilage and bone, as well as regulating the growth of chondroblasts and osteoblasts. In this study, we investigated whether recombinant human BMP adenoviruses are available for ex vivo gene therapy, using human fibroblasts and human bone marrow stromal cells in an animal spinal fusion model. MATERIALS AND METHODS: Human fibroblasts and human bone marrow stromal cells were transduced with recombinant BMP-2 adenovirus (AdBMP-2) or recombinant BMP-7 adenovirus (AdBMP-7), referred to as AdBMP-7/BMSC, AdBMP-2/BMSC, AdBMP-7/HuFb, and AdBMP-2/HuFb. We showed that each cell secreted active BMPs by alkaline phosphatase staining. Since AdBMP-2 or AdBMP-7 tranducing cells were injected into the paravertebral muscle of athymic nude mice, at 4 weeks and 7 weeks, we confirmed that new bone formation occurred by induction of spinal fusion on radiographs and histochemical staining. RESULTS: In the region where the AdBMP-7/BMSC was injected, new bone formation was observed in all cases and spinal fusion was induced in two of these. AdBMP-2/BMSC induced bone formation and spinal fusion occurred among one of five. However, in the region where AdBMP/HuFb was injected, neither bone formation nor spinal fusion was observed. CONCLUSION: The osteoinductivity of AdBMP-7 was superior to that of AdBMP-2. In addition, the human bone marrow stromal cells were more efficient than the human fibroblasts for bone formation and spinal fusion. Therefore, the results of this study suggest that AdBMP-7/ BMSC would be the most useful approach to ex vivo gene therapy for an animal spinal fusion model.
Adenoviridae* ; Alkaline Phosphatase ; Animals ; Bone Morphogenetic Protein 7 ; Bone Morphogenetic Proteins ; Cartilage ; Chondrocytes ; Fibroblasts ; Genetic Therapy* ; Humans* ; Mesenchymal Stromal Cells ; Mice ; Mice, Nude ; Osteoblasts ; Osteogenesis ; Spinal Fusion* ; Spine

To evaluate availability of the BMP-7 adenovirus (AdBMP-7) as a gene therapy for osteoinduction, we investigated in morphological aspect at 1, 2, 4, 6 weeks after cells injection. Primary cultured human dermal fibroblasts, transduced with AdBMP-7, were injected into gastrocnemius muscle of the nude mice. One week after fibroblasts transplantation new tissue was observed in the muscle. Majority of new tissue was evaluated as cartilage and calcification in the matrix was confirmed by Von Kossa stain as well as electron microscopy. Two weeks after transplantation, spongy bone was built up and adipocytes were observed in intertrabecular spaces. Osteoblasts and osteoclasts were observed in the bony tissue surface. In the result of Von Kossa-Van Gieson stain, osteolysis was dominant in bony trabeculae. Bone marrow was established in 4th weeks with intertrabecular space filled up by hematopoietic cells. At the 6th weeks, the number of trabeculae decreased and thickness of the cortical bone was increased. A great part of bone matrix has laminar structure which run paralleled to surface and which included osteocytes and canaliculi. These data demonstrate that cell mediated AdBMP-7 for gene therapy initiate development of cartilage and calcification of matrix within 1 week and complete bone and bone marrow formation within 4 weeks, so then, could be made practical application for promotion of osteoinduction.
Adenoviridae* ; Adipocytes ; Animals ; Bone Marrow ; Bone Matrix ; Bone Morphogenetic Protein 7 ; Cartilage ; Fibroblasts* ; Genes, vif ; Genetic Therapy ; Humans* ; Mice ; Mice, Nude ; Microscopy, Electron ; Muscle, Skeletal ; Osteoblasts ; Osteoclasts ; Osteocytes ; Osteolysis

BACKGROUND: This study was conducted to analyze the height and width of the pedicle of the upper and lower levels on the concave and the convex sides. In addition, we checked for the appropriate pedicle screw size which could be screwed in without complications. MATERIALS AND METHODS: Taking a simple AP radiography in a standing position, 99 vertebrae on the major curve with the possibility of 3-D reconstruction were analyzed after checking the CT in a supine position of 22 idiopathic scoliosis. We measured Cobb's angle from a simple radiograph, and measured the size of the isthmus by the Inner Space 3-D Editor after 3-D reconstruction with the Inner Space 3-D program in the DICOM file transformed from CT image. We then analyzed the size of pedicles of the upper and lower levels on the concave and the convex sides by measuring the height and width of the pedicle. RESULTS: All pedicles on the concave side were smaller than those on the convex side. Their size increased as the measurement moved from the upper to lower vertebra, except for the upper thoracic vertebra. When the width of the pedicle through 3-D reconstruction was compared with the narrowest width of the pedicle measured by using CT, the width of the pedicles through 3-D reconstruction was statistically smaller (P< 0.01). Most of the pedicles were tear-drop or kidney shaped rather than cylindrical. CONCLUSION: These results suggest that the use of the coronal plane through 3-D reconstruction would be necessary for an accurate measurement of the size of the pedicle. It is important to pay careful attention to the screw size and the screwing method considering the pedicle shape through 3-D reconstruction.
Kidney ; Radiography ; Scoliosis* ; Spine ; Supine Position

As a preceding study to apply recombinant BMP-7 gene to the human, we investigated bone formation in immunodeficient mice by using tissue engineering and gene transplatation. Human dermal fibroblasts were transduced with AdBMP-7 and cultured with type I collagen solution to form collagen sponge. The collagen sponge containing AdBMP-7 transduced fibroblasts was transplanted into hypodermis of the mice and osteogenesis in the spongy was investigated by histochemical, electronmicroscopic, and radiologic methods at 1, 2, 4, 6, and 8 weeks. At one week after transplantation, there were fluent cells infiltration around the collagen sponge and capsular structure was formed with fibers arranged in concentric circles. New vessel formation was observed in the capsule and subcapsular area of the sponge, but there were nucleus condensation and obscure cell boundary in the cells of the central region. Lacuna containing eosinophilic structures were observed in the capsular structure at two weeks. This structures were enlarged with time and were confirmed to be bone tissue by showing positive reaction for Von Kossa stain. Cartilaginous structure was not observed in light microscopic level, but a few chondroblasts were observed in pericapsular area in electron microscopic observation. After 6 weeks, radiopaque shadows were observed at the region of transplantation. Cortical bone was formed in periphery of the sponge while marrow like structure was observed in central region; some trabecula bone, adipocytes, and well developed vessels. The percentage of bone formation in transplanted sponge at 1, 2, 4, 6, and 8 weeks were 0, 63, 88, 100, and 100% (n = 8), respectively. From these results, bone formation by BMP-7 transduced human dermal fibroblasts using collagen sponge scaffolds in immunodeficient mouse shows another potential way of human gene transplantation using recombinant BMP-7 adenovirus.
Adenoviridae ; Adipocytes ; Animals ; Bone and Bones ; Bone Marrow ; Bone Morphogenetic Protein 7* ; Chondrocytes ; Collagen Type I ; Collagen* ; Eosinophils ; Fibroblasts* ; Humans* ; Mice ; Osteogenesis* ; Porifera* ; Subcutaneous Tissue ; Tissue Engineering

To induce bone formation at ectopic site by tissue engineering and gene therapy, we transplanted collagen sponges containing rhBMP-7 transduced HEK 293 cells in the hypodermis of nude mice. Bone formation was investigated by histological and electron microscopic method at 3, 6, and 9 weeks after transplantation. At 9 weeks after transplantation, eosinophilic bony tissue was observed in the implanted collagen sponge and was confirmed as bone tissue by Von Kossa stain. In the transmission electron microscopic observation, the cells in newly formed bone tissue had eccentrically located nucleus and well developed rough endoplasmic reticulum (rER). Therefore, the cells were evaluated as osteoblasts. Those results suggest that it is possible to form a bone tissue in the ectopic site by transplantation of rhBMP-7 transduced HEK 293 cells. This will be contributed to push more advanced gene therapy for bone formation. However, the HEK 293 cell is unable to apply to the clinical gene therapy. Therefore it is worth to find more compatible cells for clinical application. In addition, collagen sponge is considered as an excellent scaffold and/or carrier for gene therapy and a good biomaterial for tissue engineering.
Animals ; Bone and Bones ; Bone Morphogenetic Protein 7 ; Collagen ; Endoplasmic Reticulum, Rough ; Eosinophils ; Genetic Therapy ; HEK293 Cells* ; Mice ; Mice, Nude* ; Osteoblasts ; Osteogenesis* ; Porifera ; Subcutaneous Tissue ; Tissue Engineering

To induce bone formation at ectopic site by tissue engineering and gene therapy, we transplanted collagen sponges containing rhBMP-7 transduced HEK 293 cells in the hypodermis of nude mice. Bone formation was investigated by histological and electron microscopic method at 3, 6, and 9 weeks after transplantation. At 9 weeks after transplantation, eosinophilic bony tissue was observed in the implanted collagen sponge and was confirmed as bone tissue by Von Kossa stain. In the transmission electron microscopic observation, the cells in newly formed bone tissue had eccentrically located nucleus and well developed rough endoplasmic reticulum (rER). Therefore, the cells were evaluated as osteoblasts. Those results suggest that it is possible to form a bone tissue in the ectopic site by transplantation of rhBMP-7 transduced HEK 293 cells. This will be contributed to push more advanced gene therapy for bone formation. However, the HEK 293 cell is unable to apply to the clinical gene therapy. Therefore it is worth to find more compatible cells for clinical application. In addition, collagen sponge is considered as an excellent scaffold and/or carrier for gene therapy and a good biomaterial for tissue engineering.
Animals ; Bone and Bones ; Bone Morphogenetic Protein 7 ; Collagen ; Endoplasmic Reticulum, Rough ; Eosinophils ; Genetic Therapy ; HEK293 Cells* ; Mice ; Mice, Nude* ; Osteoblasts ; Osteogenesis* ; Porifera ; Subcutaneous Tissue ; Tissue Engineering

PURPOSE: To establish the basis for a non-invasive and non-destructive assessment of the mechanical properties during natural fracture healing by analyzing the vibrational property of the fracture healing and comparing the vibrational property, the bone healing status (as determined by X-ray) and the mechanical strength parameters. MATERIALS AND METHODS: The tibial shafts of rabbits were broken under general anesthesia. The rabbits were sacrificed at 2, 4, 6 weeks after the fracture, then X-rays of the fractured tibias were sequentially taken to detect the fracture healing. The vibration mode and the biomechanical strength were measured. RESULTS: According to the results of a multiple regression analysis, the standardized coefficients of callus, apposition, lateral angulation, DAMP1, FREQ1 in the fractured tibias, were -0.80, -0.23, -0.21, -0.25, -0.25. In normal contralateral tibias, the standardized coefficients of the area, FREQ1, DAMP1, FREQ2, DAMP3 were -0.73, 0.28, 0.41, 0.39, -0.25. CONCLUSION: A monitoring of the fracture healing process that utilizes the frequency response function is thought to be useful in detecting the early phase of healing within 4 weeks. Additional studies on the vibrational characteristics of the healing bones after a clinical union or after simillar pathologies should be pursued so that future diagnostic applications ca be made.
Anesthesia, General ; Bony Callus ; Fracture Healing ; Fractures, Bone* ; Pathology ; Rabbits ; Tibia ; Vibration

No abstract available.
Aging*

A clinical analysis was done on 23 patients (24 hips) with fracture of the femoral neck, who had been admitted and treated at our Orthopedic department during the period of 4 years, from Jan. 1984 to May 1988. The results were as follows 1. 23 patients were comprised of 4 males and 19 females, and 11 patients were over 65 years old. 2. 18 cases of 24 cases were due to minor traumas such as slipping down, and for over 65 years old, all cases were due to simple minor traumas, 8 cases were showed a severe osteoporosis, below grade 3 of the Singh's index. 3. 14 cases of 24 cases were displaced subcapital fractures, and 6 cases displaced transcervical fractures. Only 4 cases were the undisplaced transcervical fractures. 4. In treatment of fractures internal fixations after manipulation were performed in 14 cases and primary arthroplasties in 10 cases. Secondary arthroplasties were done in complicated 4 cases of 14 cases treated with internal fixations. 5. Complications after internal fixation were developed in 7 cases out of 14 cases, avascular necrosis in 6, nonunions in 2, pin migrations in 3, and metal failure in 1 case. 6. In 14 arthroplasty immediate surgical fitness of femoral stem were related to late loosening of femoral stem (correlation coefficient γ=–0.68, p<0.01).
Arthroplasty ; Clinical Study* ; Female ; Femoral Neck Fractures* ; Femur Neck* ; Femur* ; Humans ; Male ; Necrosis ; Orthopedics ; Osteoporosis