[The construction of recombinant adenovirus containing p38MAPK gene and its expression in mouse osteoblast-like cells (MC3T3-E1)].

AIM: To construct a recombinant adenovirus vector containing p38MAPK gene and to identify its expression in mouse osteoblast-like cells, MC3T3-E1, in vitro. METHODS: The p38MAPK gene was amplified by PCR from mouse liver cDNA library, and inserted into pMD18-T vector and sequenced. Double digested with Bgl II and Hind III, p38MAPK gene was inserted into pShuttle-CMV. This recombinant plasmid was linearized by Pme I and electronically transfected into BJ5183 to get the recombinant adenovirus vector Ad-p38MAPK. Then the Ad-p38MAPK was obtained by packaging Pac I linearized in AD293 cells. The recombinant adenovirus expressing p38MAPK was infected into osteoblast- like cells(MC3T3-E1).The expression of exogenous p38MAPK was observed by Western blot. RESULTS: The recombinant plasmid Ad-p38MAPK was successfully generated, which increased p38MAPK protein expression levels in MC3T3-E1. CONCLUSION: The bioactive recombinant adenovirus Ad-p38MAPK has been successfully constructed. The study laid a foundation for further research of the function of p38MAPK in osteoblast.

Low-intensity pulsed ultrasound enhances posterior spinal fusion implanted with mesenchymal stem cells-calcium phosphate composite without bone grafting.

STUDY DESIGN: Experimental study on the effect of low-intensity pulsed ultrasound (LIPUS) on rabbit spinal fusion with mesenchymal stem cell (MSC)-derived osteogenic cells and bioceramic composite. OBJECTIVE: To investigate the efficacy of LIPUS in enhancing fusion rate and bone formation with porous tricalcium phosphate (TCP) bioceramic scaffold impregnated with MSCs without any bone grafts. SUMMARY OF BACKGROUND DATA: The goal of spinal fusion in the corrective spinal surgery for spinal deformities is to achieve solid bony fusion between selected vertebral segments. Previous studies with bone morphogenetic proteins and genetically manipulated materials revealed significant difficulties in actual clinical application. Alternative such as LIPUS has been shown to be effective in enhancing healing of fracture and nonunion clinically. Its potential for enhancing spinal fusion warrants further in-depth study. METHODS: Posterolateral intertransverse processes spinal fusion at the L5 and L6 levels were evaluated in New Zealand white rabbit model. The animals were divided into three groups with (A) TCP alone, (B) TCP with differentiated MSCs, and (C) TCP with differentiated MSCs and LIPUS treatment. At week 7 postoperation, manual palpation, peripheral quantitative computed tomography, and histomorphometric assessments were performed. RESULTS: At week 7 postoperation, a statistically significant increase in clinical fusion by manual palpation was observed in group C animals treated with LIPUS (86%) in comparing with groups A (0%) and B (14%) without LIPUS. With peripheral quantitative computed tomographic analysis, the bone volume of group C fusion mass was significantly larger than the other two groups. Group C fusion also had better osteointegration length between host bone and implanted composite and more new bone formed in the TCP implants. Importantly, all the group C animals had osteochondral bridging--early stage of bony fusion histologically. Endochondral ossification was observed at the junction between the cartilaginous and osseous tissues at the intertransverse processes area. Quantitative analysis showed that the fusion mass in group C had significantly smaller gap and larger area of cartilaginous tissue between the transverse processes. CONCLUSION: The present study showed that the combination of synthetic biomaterials, autologous differentiated MSCs, and LIPUS could promote clinical fusion in rabbit posterior spinal fusion model. The mechanism was likely to be mediated through better osteointegration between the host bone and implanted materials and enhanced endochondral ossification at the fusion site.

One-stage treatment and reconstruction of Gustilo Type III open tibial shaft fractures with a vascularized fibular osteoseptocutaneous flap graft.

OBJECTIVES: This study evaluated the usefulness of a single-stage, free-fibular vascularized osteoseptocutaneous flap transfer for Type III open tibial shaft fractures with segmental bone loss for the reconstruction of combined bone and soft tissue defects. DESIGN: Nonrandomized retrospective study. SETTING: University Level I trauma center. PATIENTS/PARTICIPANTS: All Gustilo Type III open tibial shaft fractures with segmental bone loss that were treated at one institution between 2000 and 2007 were identified from a trauma registry. The study group consisted of 28 patients with Type III open tibial fractures: 27 were Gustilo-Anderson Type IIIB and one was Grade IIIC. The cause of tibial injury included eight industrial accidents, seven motor vehicle accidents, five crushing injuries caused by heavy objects, five falls from a height, and three motorcycle crashes. The lengths of the preoperative segmental tibial bone loss ranged from 9 to 17 cm and the size of the associated soft tissue defects ranged from 8 × 6 cm to 15 × 7 cm. INTERVENTION: The free fibular vascularized osteoseptocutaneous flap was used to graft and reconstruct combined bone and soft tissue defects. The radical wound débridement, soft tissue and bone revision, fracture stabilization, and early soft tissue coverage were achieved by this technique in a one-stage procedure. The average duration from injury to one-stage reconstruction was 15.8 hours (range, 5.3 hours to 6.5 days). MAIN OUTCOME MEASUREMENT: Radiographic and functional evaluation of the lower extremity. RESULTS: All free fibular osteoseptocutaneous flaps survived completely. The average time to overall union for the entire group was 32 weeks after surgery (range, 26-41 weeks). None of the patients in this series had a nonunion. Acceptable radiographic alignment, defined as 5° of angulation in any plane, was obtained in 22 patients (78.6%). Malunion affected six (21.4%) fractures. According to the lower extremity functional assessment, excellent and good results were achieved for 82.1% (23 of 28), fair results were seen in 14.3 % (four of 28), and a poor result occurred in one case (3.5%). CONCLUSION: The free fibular vascularized osteoseptocutaneous flap grafting is an effective alternative in management of Type III open tibial fractures using a one-stage procedure. The grafted fibula offers good fracture stabilization plus a vascularized bone graft, and the fibular flap can also provide a large piece of mobile skin to cover the soft tissue defect in Type III open tibial fractures. The free osteoseptocutaneous flap also serves as a visible monitor of the adequacy of the circulation of the grafted fibula.

Skeletal repair in rabbits using a novel biomimetic composite based on adipose-derived stem cells encapsulated in collagen I gel with PLGA-beta-TCP scaffold.

In bone tissue engineering, the cell distribution mode in the scaffold may affect in vivo osteogenesis. Therefore, we fabricated a novel biomimetic construct based on a combination of rabbit adipose-derived stem cells (rASCs) encapsulated in collagen I gel with a PLGA-beta-TCP scaffold (rASCs-COL/PLGA-beta-TCP, group A), the combination of rASCs and PLGA-beta-TCP (rASCs/PLGA-beta-TCP, group B), the combination of collagen I gel and PLGA-beta-TCP (COL/PLGA-beta-TCP, group C), and PLGA-beta-TCP scaffold (group D). The composites were implanted into a 15-mm length critical-sized segmental radial defect. The results were assessed by histology, radiographs, bone mineral density (BMD), and mechanical testing. After 24 weeks, the medullary cavity recanalized, bone was rebuilt, and molding finished, the bone contour remodeled smoothly and the scaffold degraded completely in group A. The BMDs and mechanical properties were similar to normal. However, the bone defect remained unrepaired in groups B, C, and D. Moreover, the scaffold degradation rate in group A was significantly higher than the other groups. Thus, enhanced in vivo osteogenesis of rASCs wrapped in collagen I gel combined with PLGA-beta-TCP was achieved, and the bone defect was repaired. We hope this study provides new insights into ASCs-based bone tissue engineering.

Collagen I gel can facilitate homogenous bone formation of adipose-derived stem cells in PLGA-beta-TCP scaffold.

Cell-based tissue engineering is thought to be a new therapy for treatment of bone defects and nonunions after trauma and tumor resection. In this study, we explore the in vitro and in vivo osteogenesis of a novel biomimetic construct fabricated by using collagen I gel to suspend rabbit adipose-derived stem cells (rASCs) into a porous poly(lactic-co-glycolic)acid-beta-tricalcium phosphate (PLGA-beta-TCP) scaffold (rASCs-COL/PLGA-beta-TCP). In vitro and in vivo studies of the rASCs-COL/PLGA-beta-TCP composite (group A) were carried out compared with the single combination of rASCs and PLGA-beta-TCP (rASCs/PLGA-beta-TCP; group B), the combination of acellular collagen I gel and PLGA-beta-TCP (COL/PLGA-beta-TCP; group C), and the PLGA-beta-TCP scaffold (group D). Composites of different groups were cultured in vitro for 2 weeks in osteogenic medium and then implanted into the autologous muscular intervals for 8 weeks. After 2 weeks of in vitro culture, alkaline phosphatase activity and extracellular matrix mineralization in group A were significantly higher than in group B (p < 0.01, n = 4). In vivo osteogenesis was evaluated by radiographic and histological analyses. The calcification level was radiographically evident in group A, whereas no apparent calcification was observed in groups B, C and D (n = 4). In group A, woven bone with a trabecular structure was formed, while in group B, only osteoid tissue was observed. Meanwhile, the bone-forming area in group A was significantly higher than in group B (p < 0.01, n = 4). No bone formation was observed in groups C or D (n = 4). In conclusion, by using collagen I gel to suspend rASCs into porous PLGA-beta-TCP scaffold, osteogenic differentiation of rASCs can be improved and homogeneous bone tissue can be successfully formed in vivo.

[Three dimensional induction of autologous mesenchymal stem cell and the effects on depressing long-term degeneration of tissue-engineering cartilage].

OBJECTIVES: To induce autologous bone marrow derived mesenchymal stem cell (aMSC) into chondrocyte, and to confirm the effects of 3 dimensional (3D) dynamic inducing in vitro and their long-term animal model repairing in vivo. METHODS: aMSC were separated from rabbits bone marrow aspirates, then respectively experienced 3D dynamic inducing in alginate drops in modified rotating wall bioreactor culture or in two dimensional (2D) inducing (culture flask) for 10 d. The induced cells were harvest and then mixed with fibrin sealant (FS) to repair rabbit knee femoral trochlea cartilage defects model. After 8, 12, 24, 48 weeks animals were euthanized. Gross appearance, histological appearances were examined. RESULTS: Flask culture groups showed a little chondrocyte differentiation, 3D inducing group showed obviously chondrocyte differentiation, improved collagen II and proteoglycan production. For 3D inducing ones in vivo, the cartilage defects were smoothly repaired by white translucent hard tissue with obvious hyaline-like cartilage histological appearance after 8, 12 weeks, and the defects boundary were hard to be identified with hyaline like cartilage with sustained histological appearance and score after 24, 48 weeks. For 2D ones in vivo, the cartilage defects were smoothly repaired after 8 weeks by hyaline like cartilage which showed accelerated degeneration after 24 weeks and lose cartilage performance completely after 48 weeks. CONCLUSIONS: 3D dynamic inducing may assist aMSC on differentiating into chondrocyte, improve its long-term in vivo repairing effects, and enlighten its further applications in tissue engineering cartilage.

A novel TAT fusion protein with osteoinductive activity.

Osteoblasts are thought to be differentiated from pluripotent mesenchymal stem cells. Several intracellular and extracellular osteoinductive proteins are involved in this process. Such proteins include the bone morphogenetic proteins (BMPs) and the LIM mineralization proteins (LMPs) etc. LMP-1 is a novel LIM domain protein promoting the differentiation of osteoblasts during bone formation. It contains three LIM domains/motifs, one PDZ domain and a unique sequence. Through analysis of the amino acid sequence and the function of the LMPs, it has been found that the PDZ domain (1-93 aa) and a unique region (94-133 aa) appear to be critical for bone formation. The TAT protein of human immunodeficiency virus can be fused with other macromolecules, peptides or proteins and transport them into cells successfully. Once being transduced into cells, the fusion protein can recover its biological activity through being rapidly refolded. We supposed that TAT could be fused with LMP-1 (1-133 aa) and LMP-1 (94-133 aa) and the fusion proteins could be easily transduced through biological membranes and generate biological activity. The clinical application of BMPs has been limited for their relatively high cost and the unstable osteoinductivity. If the hypothesis proved to be practical, we would have a more effective new way to promote bone repair and regeneration.

[Morphology research of the rat sciatic nerve bridged by collage-heparin sulfate scaffold].

OBJECTIVE: To observe the treating effect of collage-heparin sulfate after the 10 mm rat sciatic nerve defect was bridged by it. METHODS: A new kind of nervous tissue engineering scaffold was produced by freeze-drying technique from collagen-heparin sulfate. Thirty-two SD rats were randomly divided into A, B, C and D groups. Sciatic nerve defect in group A was bridged by collagen-heparin sulfate. In group B, sciatic nerve was bridged by auto-nerve transplantation. Group C was the blank control group. Animals in group D were normal. And 10 mm sciatic nerve defect was bridged in the experiment. Thirty-six weeks after the operation, the experimental animals were detected by HRP labeled retrograde trace, HE staining, toluidine staining, silvering staining, S100, GAP-43 and NF immunohistological staining, MBP immunofluorescence staining and transmission electron microscope to observe the nerve regeneration inducing effect of this new scaffold. RESULTS: Nine months after operation, the collage-heparin sulfate scaffold was replaced by newly regenerated nerve. The number of HRP labeled spinal cord anterior horn cells and the area of sensation nerve fiber at the posterior horn were similar with that was repaired by auto-nerve. GAP-43, NF and S100 labeled regenerated nerve fiber had passed the total scaffold and entered the distal terminal. The regenerated nerve fibers were paralleled, lineage arranged, coincide with the prearranged regenerating "channel" in the collagen-heparin sulfate scaffold. MBP immunofluorescence staining also proved that the newly regenerated nerve fiber could be ensheathed. In the experimental group, the area of myelinated nerve fiber and the thickness of the myelin sheath had no obvious difference with that of the group repaired by auto-nerve, except that the density of the regenerated myelinated sheath fiber was lower than that of the control group. CONCLUSION: Nervous tissue engineering scaffold produced by collagen-heparin sulfate can guide the regeneration of nerve fibers. The nerve function recovers fine. This kind of material has great application potential.

[Repair of the radial defect of rabbit by polyester/tricalcium phosphate scaffolds prepared by rapid prototyping technology].

OBJECTIVE: To evaluate the repairing effect of the rabbits radial defects of by polyester/tricalcium phosphate scaffolds prepared by rapid forming technology loaded with bovine BMP, and find a new carrier for growth factor. METHODS: Polyester/Tricalcium phosphate scaffolds prepared by rapid prototyping (RP) technology loaded with and without bovine BMP were used to repair the 15 mm radial defect of rabbit. Then results of radiography, histology, scaffolds degrade rates and bone density were appraised to examine the repairing effects of the scaffolds at 12 weeks. RESULTS: At 12 weeks, all defects treated with bBMP were radiographically repaired. No radii implanted polyester/tricalcium phosphate scaffolds alone showed radiographic and historical union. At experimental groups, longitudinal alignment of lamellar structure was observed histologically at 12 weeks, indicating that remodeling of regenerated bone almost completed, the scaffolds degradation rates were different by 12 weeks, and no abnormalities were observed in the surrounding soft tissue in all groups. CONCLUSION: Polyester/tricalcium phosphate scaffolds prepared by rapid prototyping technology loaded with bovine BMP can repair the rabbits radical defects. As for the effects, the poly (L-lactic-co-glycolide)/tricalcium phosphate (PLGA/TCP) scaffold are ideal and better than poly (L-lacide-co-D, L-lactide)/tricalcium phosphate (PDLLA/TCP) scaffold, but the poly (L-lactic acid)/tricalcium phosphate (PLLA/TCP) is not so good for its low degradation rates.

Effect of type I collagen on the adhesion, proliferation, and osteoblastic gene expression of bone marrow-derived mesenchymal stem cells.

OBJECTIVE: To investigate the effects of porous poly lactide-co-glycolide (PLGA) modified by type I collagen on the adhesion, proliferation, and differentiation of rabbit marrow-derived mesenchymal stem cells (MSCs). METHODS: The third generation MSCs isolated from mature rabbits by density gradient centrifugation were cultured at different initial concentrations on 0.3 cm x 1.2 cm x 2.0 cm 3-D porous PLGA coated by type I collagen in RPMI 1640 containing 10% fetal calf serum, while cultured on PLGA without type I collagen as control. The cells adhesive and proliferative behavior at 7, 14, and 21 days after inoculation was assessed by determining the incorporation rate of [(3)H]-TdR. In order to examine MSCs differentiation, the expression of osteoblasts marker genes, osteocalcin (OCN), alkaline phosphatase (ALP), osteopontin (OPN) mRNA, were evaluated by reverse transcription-polymerase chain reaction (RT-PCR), and further more, the cell morphology at 21 days was also observed by scanning electron microscope (SEM). RESULTS: Type I collagen promoted cell adhesion on PLGA. The valve was significantly higher than controls (6 h, 2144 cpm+/-141 cpm vs. 1797 cpm+/-118 cpm, P=0.017; 8 h, 2311 cpm+/-113 cpm vs. 1891 cpm+/-103 cpm, P=0.01). The cells which cultured on PLGA coated with type I collagen showed significantly higher cell proliferation than controls on the 7 th day (1021 cpm+/-159 cpm vs. 451 cpm+/-67 cpm, P=0.002), the 14th day (1472 cpm+/-82 cpm vs. 583 cpm+/-67 cpm, P<0.001) and 21 th day (1728 cpm+/-78 cpm vs. 632 cpm+/-55 cpm, P<0.001). Osteoblasts markers, OCN, ALP, OPN mRNA, were all detected on PLGA coated by type I collagen on the 21 th day, but OCN, OPN mRNA could not be found in controls. Spindle and polygonal cells well distributed on the polymer coated by type I collagen while cylindric or round cells in controls. CONCLUSIONS: Type I collagen is effective in promoting the adhesion, proliferation and differentiation of MSCs on PLGA.

[Effect of marrow stromal cells derived chondrocytes on repair of full-thickness defects of rabbit articular cartilage].

OBJECTIVE: To investigate the feasibility of cartilaginous implants containing bone marrow stromal cells (MSCs) derived from chondrocytes in biological resurfacing procedures for repairing articular cartilage defect. METHODS: MSCs derived from chondrocytes were obtained with high initial cell density subculture. An implant was constructed by dispersing the chondrocytes in a acid soluble type I collagen gel(5 x 10(6) cells/ml, final cell concentration). A full-thickness defect 3 mm x 5 mm was created in the trochlear groove of femur in 36 rabbits. A piece of cotton soaked in 0.5% trypsin was laid into the defect for 5 minutes, then the defect was filled with MSC/collagen gel implant on one side (n = 36), filled with a plain collagen gel on the other side (n = 18), and left empty as controls on the other side (n = 18). The animals were sacrificed at 4, 8, 12, 24, 32, and 48 weeks. The repaired tissue was examined and evaluated with Pineda grading scale. RESULTS: In MSCs group, the implanted cells resembled well differentiated chondrocytes and were surrounded by metachromatic matrix and the reparative tissue resembled hyaline cartilage after 4 weeks; bone was formed at the base of the defects, the thickness of new cartilage was larger than tht of normal one after 8 weeks; the thickness was reduced proximally, approximating to that of normal cartilage, and chondrocyte columns was formed and subchondral bone and tidemark reappeared after 12 weeks; the thickness of the new tissue was about 55% of the normal tissue, with smooth surface and there were hypertrophic chondrocytes near the tidemark after 24 weeks; no hypertrophic chondrocytes were observed, indicating cessation of endochondral ossification after 32 weeks; the tissue architecture was the same as that at 32 weeks, hyaline-like cartilage persisting, with subchondral bone and tidemark in continuity after 48 weeks. The four layer cell orientation was not as clear as that of normal cartilage. The defects were partially filled with fibrous tissue in controls. At 32 weeks, erosive cartilage, naked subchondral bone and proliferative synovial membrane indicated the presence of osteoarthrosis. There were no statistical difference according to Pineda tissue scales in the specimens from the MSCs group between 24, 32, and 48 weeks, but there was significant difference between 4 weeks and 24, 32 and 48 weeks (P < 0.05). The joint function recovered after 2 weeks in MSCs group, while it deteriorated progressively in controls. CONCLUSION: MSCs derived from chondrocytes improve repair of large full-thickness defect in articular cartilage. The reparative hyaline-like cartilage is stable differentiation after 24 weeks, maintains good joint function after 48 weeks.

[Expression of human osteoprotegerin gene in E. Coli and bioactivity analysis of expression product].

OBJECTIVE: To express human osteoprotegerin (OPG) in E. Coli and analyze its bioactivity in vitro. METHODS: Synthetic oligonucleotides were used to amplify human OPG gene by RT-PCR from total RNA of human osteosarcoma cell line MG63. The OPG cDNA coding for 380 amino acid residues was inserted into prokaryotic expression vector pRSET-A, transformed into competent E. Coli BL21, and induced by 0.1 mmol/l IPTG. SDS-PAGE and Western blot were performed to identify OPG-6His fusion protein. After purified by affinity chromatography, 1,000 microg/L or 1,500 microg/L of OPG-6His were added into the mouse bone marrow cells culture medium. The number of tartrate-resistant acid phophatase (TRAP)-positive multinucleated cells and resorption pits were counted to assess the bioactivity of expression products. RESULTS: The sequence of OPG mature peptide encoding cDNA obtained in this experiment was as same as reported. SDS-PAGE showed 24% of total bacterial protein was of OPG-6His fusion protein. Western blot assay demonstrated that the molecular weight of recombinant protein was about 46 KD and could react specifically with human anti-OPG antibody. The mouse bone marrow cells were induced by 1alpha, 25-dihydroxyvitaminD3 (10(-8) mol/L) and Dexamethasone (10(-7) mol/L) to form osteoclastic-like multinucleated cells. 1,500 microg/L of purified OPG-6His protein could decrease the number of resorption pits and TRAP-positive multinucleated cells in vitro (P < 0.05), but it didn't show the same effects when the concentration of OPG-6His fusion protein was of 1,000 microg/L. CONCLUSIONS: Human OPG-6His fusion protein is expressed and purified in E. Coli. The expression products have moderate inhibitory effects on osteoclast differentiation and bone resorption in vitro only when excessive amount of proteins are added into the culture medium, indicating that prokaryotic expression of fuctionalal OPG protein awaits further investigation.

[Co-expression of human bone morphogenetic protein-2 and osteoprotegerin in myoblast C2C12].

OBJECTIVE: To construct a co-expressing vector of human bone morphogenetic protein 2 (BMP-2) and osteoprotegerin (OPG) and to determine the expression of BMP-2 and OPG in myoblast C2C12. METHODS: Using the isolated total RNA from osteosacoma cell line MG63 as a template, the cDNA encoding region of human OPG was amplified by reverse transcription-polymerase chain reaction (RT-PCT) method and cloned into sites EcoR 1 and BamH I of mammalian expressing vector pIRES2-EGFP, and the cDNA encoding region of human BMP-2 was cloned into endonucleases site BstX I. Then the recombinant plasmid pIRES2-BMP-2-OPG was transformed into C2C12 cell line, the expression of OPG and BMP-2 were determined by Western blot assay. RESULTS: The sequence of OPG cDNA obtained was the same as that reported, recombinant plasmid pIRES2-BMP-2-OPG was constructed successfully. Human OPG and BMP-2 co-expression cell line C2C12 was selected and confirmed by Western blot analysis. CONCLUSION: The co-expressing vector of OPG and BMP-2 is constructed and can expressed stably in myoblast C2C12. The co-expression of human OPG and BMP-2 may be logical approach for treatment of osteoporosis and bone metastasis.

[Massive frozen allografts for skeletal reconstruction: styles and affecting factors of bone union].

OBJECTIVE: To investigate the styles and affecting factors of bone union after massive frozen allografting for skeletal reconstruction owing to excision of bone tumor. METHODS: From 1992 to 1999, 85 patients suffering from bone malignant tumor were given the excision of large bone segment and treated with allografting in different methods of operation: large bone allografts with condylar articular surface in 16 cases, osteoarticular allografts in 57 cases, bone allografts in combination with prosthetic replacement of hip in 9 cases, and prosthetic replacement of knee in 3 cases. The average follow-up was 2 years and 9 months. The union time and styles of host-donor junction were determined by X-ray characters, and the results of operations were assessed according to Enneking's functional evaluation system of reconstructive procedures after surgical treatment of tumors for the musculoskeletal system. RESULTS: There were 4 kinds of basic bone union styles by the X-ray characters, there were no significant difference in the time span of bone union after fixation with different methods. Of the 85 fresh-frozen allografting procedures, more than 80% of the patients were treated with interlocked intramedullary nail and allograft-prosthesis combination, and the overall result was excellent and good. Sufficient blood supply was important for host-donor junction healing, but the function of immune response was uncertain. CONCLUSION: There were different styles of bone union after massive allografting. The recommended operative methods for massive allografts are stable internal fixation, sufficient blood supply, soft tissue repair and periosteal flap coverage.

[In vivo endochondral bone formation by implanting human bone morphogenetic protein-2-producing fibroblasts into nude mouse muscle].

OBJECTIVE: To determine whether fibroblasts can be used to promote endochondral bone formation in vivo by transfer of human bone morphogenetic protein-2(hBMP-2) into fibroblasts. METHODS: pcDNA3-hBMP-2 was constructed by use of gene clone and recombined technique. NIH3T3 fibroblasts were transfected with pcDNA3-hBMP-2. The positive cell clones were selected with G418. In NIH3T3 fibroblasts transferred with pcDNA3-hBMP-2, the expression of hBMP-2 was determined by in situ hybridization and immunohistochemical analysis; alkaline phosphatase activity was measured. hBMP-2-producing fibroblasts were implanted into nude mouse muscle to observe endochondral bone formation in vivo. RESULTS: pcDNA3-hBMP-2 was successfully constructed. In NIH3T3 fibroblasts transfected with pcDNA3-hBMP-2, the BMP-2 expression was stable; alkaline phophatase activity was much higher than that in non-transfected NIH3T3 cells. Endochondral bone formation in vivo was observed at the site of implantation 4 weeks later. CONCLUSION: Fibroblasts transfected by hBMP-2 gene can be used to promote endochondral bone formation in vivo.

[Application of gene therapy mediated by adenovirus vectors for bone trauma and bone disease].

OBJECTIVE: To review the current concepts of gene therapy approaches mediated by adenovirus vectors for bone trauma and bone disease. METHODS: The recent literature concerned gene therapy mediated by adenovirus vectors was reviewed, which provides new insights into the treatments of bone trauma and bone disease. RESULTS: Adenovirus vectors was efficient, achieved high expression after transduction, and could transfer genes to both replicating and nonreplicating cells, such as osteoblasts, osteoclasts, fibroblasts, chondrocytes, bone marrow stromal cells, etc. Gene therapy mediated by adenovirus vectors achieved affirmative results in enhancing bone union and in curing bone diseases, such as osteoporosis and rheumatoid arthritis. CONCLUSION: Gene therapy mediated by adenovirus offers an exciting avenue for treatment of bone trauma and bone diseases.

[Construction and biological activity study of human osteoprotegerin expressing adenoviral system].

Using the isolated total RNA from osteosacoma cell line MG63, the cDNA encoding human OPG was amplified by RT-PCR. A recombinant adenoviral vector carrying cDNA of OPG was constructed and OPG expression in mouse myoblast C2C12 cells was confirmed by Western blot and ELISA. The secreted expression of OPG protein persisted more than 6 weeks in vitro, and the growth of C2C12 cells infected by recombinant adenoviral were in good state. Osteoclasts derived from mouse bone marrow cells infected with recombinant adenoviral made less number of TRAP positive cells and resorption pits formed on dentine slices.

[Effect of insulin-like growth factor-I on cultured articular chondrocytes of rabbits].

OBJECTIVE: To investigate the influence of insulin-like growth factor-I (IGF-I) on biological characteristics of articular chondrocytes cultured in vitro of rabbits. METHODS: Monolayer articular chondrocytes of 4-week old rabbits were cultured in medium with IGF-I, at the concentrations of 3, 10, 30, 100, and 300 ng/ml. The DNA content in cells and glucuronic acid content in matrix were detected on the 2nd, 4th, 6th days after culture. RESULTS: The DNA content in cells and the glucuronic acid content in matrix in articular chondrocytes cultured in medium with IGF-I at concentrations of 3-300 ng/ml were all significantly higher than those in control group (P < 0.01), which reached the peak at the concentrations of 30-100 mg/ml on the 4th day. CONCLUSION: IGF-I could obviously promote the proliferation of articular chondrocytes in vitro, and there exist time-dependent and dose-dependent effect.

[Preparation of rhBMP-2/BCB reconstituted bone xenograft and assay of its osteoinductivity].

OBJECTIVE: To investigate a new grafting material of bone xenograft with strong bone inductive and conductive capacity. METHODS: Based on successful clinical application of the reconstituted bone xenograft (RBX), a new xenograft was made by combining recombinant human bone morphogenetic protein-2 (rhBMP-2) with antigen-free bovine cancellous bone (BCB). Sixty male BALB/C mice aged 4 weeks were divided into study group of 30 and control group of 30 randomly. rhBMP-2/BCB was implanted in the left thigh muscle pouch in the study group and BCB in the control group. The mice were sacrificed at 7 d, 14 d and 21 d after implantation. Inductivity of rhBMP-2/BCB was detected by histological observation and biochemical determination of the samples. RESULTS: Histological examination showed that rhBMP-2/BCB induced chondrogenesis on the 7th day, with woven bone formed on the 14th day, and lamellar bone and marrow on the 21st day, while BCB failed to induce chondrogenesis or osteogenesis on the 7th, 14th and 21st days. The alkaline phosphatase activities and calcium content in study group were higher than those in control group with significant difference (P < 0.01). CONCLUSION: rhBMP-2/BCB is an ideal grafting material with strong bone inductive and conductive capacity without evoking immune reaction.