BACKGROUND: How to deal with bone defect is a big problem to surgeons. In recent years, the development in the technology of molecular biology and tissue engineering provides broad prospect for the clinical treatment of bone defect, which is one of the important study directions in department of orthopedics. The transforming growth factor-beta 1(TGF-β1),one of the important factors in bone formation, plays an important role in bone metabolism and recovery.OBJECTIVE: To observe the therapeutic effects of bone defects with osteoblasts transfected . By TGF-β1 combining with biomimetic biodegradable polymer scaffolds.DESIGN: Randomized controlled trial.SETTING: Department of Orthopedics, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology.MATERIALS: Siderophilin, trypsin, 3H-proline and sirius red, etc.MATERIALS: The experiment was completed in Union Hospital, Tongji Medical College, Huazhong University of Science and Technology from March to August in 2003. Twenty healthy adult Sprague-Dawley(SD) rats of SPF grade, weighing 200-250 g, were provided by the Experimental Animal Center of Tongji Medical College.METHODS: The osteoblasts transfected by TGF-β1 gene, combining with poly-DL-lactic acid scaffolds modified with poly-L-lysine, were transplanted into rat tibia defect. Radiographs and histological analysis were performed to evaluate the repair effects.MAIN OUTCOME MEASURES: The X-ray evaluation and histology observation were performed at the 4th and 8th weeks after the operation.RESULTS: Totally 20 SD rats were included in result analysis without one rat missing. ①In the experiment group, X-ray image indicated callus formation, while histology observation showed osteoid tissue and new bone formation, and osteoblasts attached to the surface of the materials after 4 weeks. Eight weeks later, the defect was essentially repaired, and the bone density of new bone was similar to that of the autogenous bone. ②In the control group, there was no formation of callus and osteoid tissue, and few osteoblasts attached to the surface of the materials, and a lot of lymphocytes infiltrated and blood capillary grew in the lacune of materials after 4 weeks. Eight weeks later, the imbedded materials were substituted mostly by fibrous tissue.CONCLUSION: The ideal repair effect of bone defect can be obtained through the combination of molecular biology with tissue engineering.

To discuss and evaluate the method and effect of using calcaneal anatomic plate in treatment of intra-articular fractures of the calcaneus with assistant of arthroscope, 86 intra-articular fractures of the calcaneus in 78 patients were reduced by open reduction, and rigid fixation was made with calcaneal anatomic plate under assistant of arthroscope. The average follow-up duration was 18 months (range 12-30 months). The effect of treatment was evaluated according to AOFAS and X-ray before and after operation. The results showed that 86 patients have obtained satisfactory reduction according to X-ray, and there was significant difference before and after operation (P < 0. 01), the total excellent and fine rate was 91.86%. Treating intra-articular fractures of the calcaneus with calcaneal anatomic plate under arthroscope may provide more chance to achieve anatomical reconstruction, which can lead to satisfied recovery of function and few complication.

The protective effect of niacinamide on interleukin-1beta (IL-1beta)-induced annulus fibrosus (AF) type II collagen degeneration in vitro and the mechanism were investigated. Chiba's intervertebral disc (IVD) culture models in rabbits were established and 48 IVDs from 12 adult Japanese white rabbits were randomly divided into 4 groups: normal control group, niacinamide-treated group, type II collagen degneration group (IL-1beta) and treatment group (niacinamide+IL-1beta). After culture for one week, AFs were collected for inducible nitric oxide synthase (iNOS), cysteine containing aspartate specific protease-3 (Caspase-3) and type II collagen immunohistochemical examination, and type II collagen reverse transcription polymerase chain reaction (RT-PCR). The results showed that rate of iNOS positive staining AF cells in the 4 groups was 17.6%, 10.9%, 73.9% and 19.3% respectively. The positive rate in treatment group was significantly lower than in the type II collagen degeneration group (P<0.01). Rate of Caspase-3 positive staining AF cells in the 4 groups was 3.4%, 4.2%, 17.6% and 10.3% respectively. The positive rate in treatment group was lower than in the type II collagen degeneration group (P<0.01). Type II collagen staining demonstrated that lamellar structure and continuity of collagen in treatment group was better reversed than in the degeneration group. RT-PCR revealed that the expression of type II collagen in treatment group was significantly stronger than that in type II collagen degeneration group (P<0.01). It was concluded that niacinamide could effectively inhibit IL-1beta stimulated increase of iNOS and Caspase-3 in AF, and alleviate IL-1beta-caused destruction and synthesis inhibition of type II collagen. Niacinamide is of potential for clinical treatment of IVD degeneration.

BACKGROUND: Presently, the biomaterial used in ligament tissue engineering such as collagen protein, polylactic acid, polyglycolic acid, small intestinal submucosa, glycan and nanomaterial are characterized by rapid degradation, resulting in inflammatory reaction after applying in host.OBJECTIVE: To investigate mechanical strength and in vitro degradation of silk scaffold and explore the reaction to macrophages.DESIGN: Controlled experiment.SETTING: Experiments were performed at the Department of Orthopaedics, Union Hospital, Huazhong University of Science and Technology from September 2004 to January 2005.MATERIALS: White raw Bombyx mori silkworm fibers of size 20/22 (according to the manufacturer) were obtained from the market. Bundles of 30 parallel fibers were prepared for a bundle of scaffold, which was put into fervens 5g/L Na2CO3for degumming. Ratio of Na2CO3 solution (Ml) to raw silk (g) was 1000.METHODS: In vitro degradation: 8cm long silk scaffold was weighed after drying. Subsequently, the silk scaffold was separately dipped into phosphate buffer saline (PBS) and 1.0g/L collagenase prepared with PBS. Twelve weeks later, silk scaffold was weighed to calculate weight loss rate. Simultaneously, tensile test was performed to detect the ultimate tensile strength (UTS) of samples. Culture of monocyte strain RAW264.7:2×108L-1 macrophage suspension (1mL) were separately added in a silk scaffold group, a control group and a lipopolysaccharide (LPS) group. At days 1 and 7, cell supernatant was collected from each group. Tumor necrosis factor-α(TNF-α) levels were measured by enzyme linked immunosorbent assay (ELISA).MAIN OUTCOME MEASURES: ① Changes in weight loss rate and UTS of the silk matrices after incubated with collagenase and the PBS. ②TNF-αlevels in the supernatant of each groups at days 1 and 7.RESULTS: Mass of silk matrices reduced by over 50% after incubated with collagenase for 8 weeks, but no change was found in PBS. UTS decreased by over 50% 8 weeks after incubated with collagenase, but no change was detected in PBS. At days 1 and 7, TNF-α levels in the supernatant was less in the silk scaffold group; TNF-α levels in the supernatant was significantly higher in the LPS group than in the silk scaffold group (P<0.01), but no significant difference in TNF-α levels was measured between the silk scaffold group and the control group (P>0.05).CONCLUSION: After 12-weeks degradation, silk scaffold still has good mechanical properties. Macrophages possess immunological inertia at days 1 and 7 after inoculated with macrophages.

Objective To construct the recombinant rAAV2-hGAD65 vector and detect its function both in vitro and in vivo. Methods The cDNA of human glutamic acid decarboxylase 2 (hGAD65) gene, which was one of gamma-aminobutyric acid (GABA) synthetase, cloned by the method of RT-PCR, was subcloned into the adeno-associated virus (AAV) vector and formed the recombinant vector of AAV-hGAD65 (rAAV2-hGAD65). The recombinant vector was packaged by the AAV Helper-Free System and its titer was detected. The primary fibroblast, cultured from the rat lung, was transfected by the rAAV2-hGAD65. The expression of the hGAD65 in fibroblast was detected by immunohistochemical method and the level of GABA in the media was assayed by high performance liquid chromatograph (HPLC). In vivo, the hGAD65 was detected by immunohistochemical method in STN and the concentration of the GABA in the reticular part of substantia nigra (SNr) was assayed by HPLC after the rAAV2-hGAD65 was injected into the subthalamic nucleus (STN) by the stereotaxic method. Results The sequence of hGAD65 cDNA was in accordance with that in the Genebank. The amino acid sequence of hGAD65 had no mutation and the titer of rAAV2-hGAD65 was reached 4.5 ×10~(11) per milliliter. The efficiency of infection to the rat primary firoblasts was 80%, and the concentration of GABA in the media of fibroblasts was (45.66±6.07)nmol/L per liter. In vivo, hGAD65 could be detected in STN, and the concentrateion of the GABA in the SNr was increased from (5.66±1.07)nmol/g to (12.66±2.59)nmol/g.Conclusion The cDNA of hGAD65 was cloned by RT-PCR and the recombinant vector of rAAV2-hGAD65 was constructed. The AAV can infect the primary fibroblast in vitro and the hGAD65 can catalyse the glutamic acic to GABA. In vivo, the concentration of GABA in the SNr was heighten after the rAAV2-hGAD65 was injected into the STN.

BACKGROUND:There are several reports about the application of fresh bone marrow aspirate being injected directly to repair partial ligament injury, but the application about fresh bone marrow aspirate directly being planted on scaffolds to build tissue-engineered ligament is rarely mentioned.
OBJECTIVE:To evaluate the feasibility of applying fresh bone marrow aspirate planted directly on scaffolds to construct tissue-engineered ligament
METHODS:We constructed fibroin fiber/smal intestinal submucosa composite scaffold, then planting fresh bone marrow directly to built bone marrow seeding group and planting seed cel s (bone marrow mesenchymal stem cel s) on the scaffold to built cel seeding group. The control group had no treatment. After that, we detected the density of cel adhesion, cel proliferation ability and extracel ular matrix secretion. Then, the composite in the bone marrow seeding group was implanted into the broken anterior cruciate ligament in rabbits, and material biocompatibility in vivo was evaluated after 12 weeks.
RESULTS AND CONCLUSION:After 4 hours of incubation, bone marrow seeding group was significantly higher than the cel seeding group in cel adhesion density and proliferation rate (P<0.05). Bone marrow seeding group and cel seeding group showed higher type I, III col agen secretion compared with the control group (P<0.05), but the col agen secretion of bone marrow seeding group and cel seeding group showed no significant difference. Composite cel scaffold implantation in vivo did not cause fatal immune rejection and severe inflammatory reaction, and no significant ligament regeneration and vascularization occurred. These findings indicate that fresh bone marrow aspirate can be seeded directly on scaffolds to construct tissue-engineered ligament, and the short-term biocompatibility in vivo is good.

OBJECTIVE: To explore the possible mechanism of Huoxue Qianyang Formula (HXQYF), a compound traditional Chinese herbal medicine, in reversing the left ventricular hypertrophy (LVH) of spontaneous hypertensive rats (SHRs) by analyzing the expressions of mRNAs and proteins of proto-oncogenes c-fos and c-myc in left ventricular muscle. METHODS: The experimental study was carried out in SHRs, the sex- and age-matched Wistar-Kyoto (WKY) rats were served as normal control (n=5, normal saline 10 ml/kg daily). Twenty-five SHRs were randomly divided into five groups: untreated group (n=5, normal saline 10 ml/kg daily), high-dose HXQYF-treated group (n=5, 0.84 g/ml HXQYF, 10 ml/kg daily), medium-dose HXQYF-treated group (n=5, 0.42 g/ml HXQYF, 10 ml/kg daily), low-dose HXQYF-treated group (n=5, 0.21 g/ml HXQYF, 10 ml/kg daily) and cilazapril-treated group (n=5, 1 mg/ml cilazapril, 10 ml/kg daily). The drugs were intragastrically administered once daily for 14 weeks. The expressions of mRNAs and proteins of proto-oncogenes c-fos and c-myc in left ventricular muscle were detected separately by in situ hybridization histochemical method and immunohistochemical method. RESULTS: Compared with the normal control group, the expressions of mRNAs and proteins of proto-oncogenes c-fos and c-myc in left ventricular muscle were significantly increased in untreated group (P<0.01). After treatment, the expressions of c-fos and c-myc mRNAs in left ventricular muscle in HXQYF-treated groups were significantly down-regulated as compared with those of the untreated group (P<0.05). The expressions of c-myc protein were also significantly decreased in high- and medium-dose HXQYF-treated groups as compared with the untreated group (P<0.05), but it had no significant effects in protein expression of c-fos in the three HXQYF-treated groups. CONCLUSION: HXQYF can inhibit the expression of c-myc in ventricular hypertrophy tissue, which may be the mechanism in treating LVH of hypertension.

BACKGROUND: The cells of biomaterial compound cytokine gene or compound transfected cytokine gene that are transplanted into the area of bone defect can promote bone repair.OBJECTIVE: To investigate the feasibility of gene therapy for bone defect after rat transforming growth factor (TGF)β1 gene is transfected into osteoblasts.DESIGN: A controlled and observational experiment.SETTING: Department of Orthopedics, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology.MATERIALS: Five newborn Sprague-Dawley rats of either gender were included.METHODS: The experiment was conducted in the laboratory of Orthopedic Department, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, from February to September 2000.Rat osteoblasts were transfected with pcDNA3-TGF-β1 plasmid by lipofectamine mediated gene transfer, and the plasmid pcDNA transfected cells were set as control group. The transient expression of TGF-β1 was detected by strept avidin-biotin-peroxidase complex (SABC) method and in situ hybridization detection 24 hours later. The cells transfected by G418 for 2 weeks were detected with SABC to investigate the stable expression of TGF-β1 gene.MAIN OUTCOME MEASURES: SABC method and in situ hybridization detection were applied to detect gene expression.of pcDNA3-TGF-β1 transfected osteoblasts and in situ hybridization detection: After the osteoblasts were transfected for 24 hours, cytoplast was full of brown granules and there were no brown granules in the cytoplast of blank carrier transfected cells, indicating that TGF-β1 mRNA was signifiG418: transfected cells still had high expression of TGF-β1 after 2-week G418 screening.CONCLUSION: Osteoblasts can express cytokine immediately with high effect using gene transfection technique. The stable and high expression is presented after TGF-β1 gene is transfected into osteoblasts at the moment of transfection and after 2-week screening, proving the feasibility of gene therapy for bone defect when cytokine gene is transfected into osteoblasts.

The mobilization efficiency of granulocyte colony-stimulating factor (G-CSF) and stem cell factor (SCF) to bone marrow mononuclear cells (MNCs) in mice was observed, and the changes of CXCL12/CXCR4 signal were detected in order to find out the mobilization mechanism of stem cells. Kunming mice were randomly divided into two groups. The mice in treatment group were subjected to subcutaneous injection of G-CSF at a dose of 100 mug/kg and SCF at a dose of 25 mug/kg every day for 5 days, and those in control group were given isodose physiological saline. The MNCs were separated, counted and cultured, and the colony-forming unit-fibroblast (CFU-F) was evaluated. CD34(+)CXCR4(+) MNCs were sorted by flow cytometry. The expression of CXCL12 protein in bone marrow extracellular fluid was detected by ELISA, and that of CXCL12 mRNA in bone marrow was measured by RT-PCR. The results showed that the counts of MNCs in peripheral blood and bone marrow were increased after administration of G-CSF/SCF (P<0.01). The factors had a dramatic effect on the expansion capability of CFU-F (P<0.05). Flow cytometric of bone marrow MNCs surface markers revealed that CD34(+)CXCR4(+) cells accounted for 44.6%+/-8.7% of the total CD34(+) MNCs. Moreover, G-CSF/SCF treatment induced a decrease in bone marrow CXCL12 mRNA that closely mirrored the fall in CXCL12 protein. In this study, it is evidenced that G-CSF/SCF can effectively induce MNCs mobilization by disrupting the balance of CXCL12/CXCR4 signaling pathway in the bone marrow and down-regulating the interaction of CXCL12/CXCR4.

In order to investigate the effect of Arg-Gly-Asp (RGD) peptide-modified silk biomaterial on the adhesion and proliferation of bone marrow-derived mesenchymal stem cells (MSCs), MSCs of third generation were seeded onto the surface of RGD-decorated silk (silk-RGD group), silk alone (silk group) or tissue culture plate (TCP group). After incubation for 4 or 12 h, MSCs were examined quantitatively by using precipitation method for cell attachment. The cell proliferation, which was defined as cell density, was compared among the three groups after culture for 1, 2, 3, and 4 days. Cell skeleton, which was labeled fluorescently, was observed under laser confocal microscope after 24 h of culture. The results showed that cell adhesion rate in silk-RGD group was higher than in silk group (P<0.05), but similar to that in TCP group after incubation for 4 or 12 h (P>0.05). There were no significant differences in the cell proliferation among the three groups at different time points (P>0.05 for all). Laser confocal microscopy revealed that in silk-RGD group, MSCs, strongly fluorescently stained, spread fully, with stress fibers clearly seen, while in silk group, actin filaments were sparsely aligned and less stress fibers were found. It was concluded that RGD peptide could improve the adhesion of MSCs to the silk scaffold, but had no impact on the proliferation of the cells.
Biocompatible Materials/*chemistry ; Bone Marrow Cells/cytology ; Cell Adhesion/drug effects ; Cell Proliferation/drug effects ; Mesenchymal Stem Cells/*cytology ; Oligopeptides/*chemistry ; *Silk/chemistry ; Tissue Scaffolds