Discovery of core genes and intercellular communication role in osteosarcoma.

Osteosarcoma is a primary malignant bone tumor that affects children and young adults. Understanding the molecular mechanisms underlying osteosarcoma is critical to develop effective treatments. This study aimed to identify core genes and explore the role of intercellular communication in osteosarcoma. We used GSE87437 and GSE152048 dataset to conduct a weighted correlation network analysis (WGCNA) and identify co-expression modules. The enriched biological processes and cellular components of the genes in the steelblue module were analyzed. Next, we explored the expression, diagnostic value, correlation, and association with immune infiltrate of CCSER1 and LOC101929154. Finally, we utilized CIBERSORT algorithm to predict the infiltrated immune cells in osteosarcoma tissues. Our results identified 44 co-expression modules, and the steelblue module was mainly associated with axon development, axonogenesis, and innervation. CCSER1 and LOC101929154 were significantly upregulated in osteosarcoma tissues with poor response to preoperative chemotherapy. Moreover, the expressions of CCSER1 and LOC101929154 were positively correlated. The area under the receiver operating characteristic curve of CCSER1 and LOC101929154 was 0.800 and 0.773, respectively. The expression of CCSER1 was negatively correlated with follicular helper T cells and positively correlated with M0 macrophages, while LOC101929154 was negatively correlated with activated mast cells. Besides, CD4 memory-activated T cells were observed at lower levels in patients who responded well to chemotherapy. Our study identified core genes CCSER1 and LOC101929154 and provided insight into the intercellular communication profile in osteosarcoma. Our results suggested that targeting CCSER1, LOC101929154, and CD4 memory-activated T cells may be a promising strategy for the treatment of osteosarcoma.

Efficacy of Diosmin in Reducing Lower-Extremity Swelling and Pain After Total Knee Arthroplasty: A Randomized, Controlled Multicenter Trial.

BACKGROUND: Many patients experience lower-extremity swelling following total knee arthroplasty (TKA), which impedes recovery. Diosmin is a semisynthetic flavonoid that is often utilized to treat swelling and pain caused by chronic venous insufficiency. We aimed to evaluate the efficacy and safety of diosmin in reducing lower-extremity swelling and pain as well as in improving functional outcomes following TKA. METHODS: This study was designed as a randomized, controlled multicenter trial and conducted in 13 university-affiliated tertiary hospitals. A total of 330 patients undergoing TKA were randomized to either receive or not receive diosmin postoperatively. The diosmin group received 0.9 g of diosmin twice per day for 14 consecutive days starting on the day after surgery, whereas the control group received neither diosmin nor a placebo postoperatively. The primary outcome was lower-extremity swelling 1, 2, 3, and 14 days postoperatively. The secondary outcomes were postoperative pain assessed with use of a visual analogue scale, Hospital for Special Surgery score, range of knee motion, levels of the inflammatory biomarkers C-reactive protein and interleukin-6, and complications. RESULTS: At all postoperative time points, diosmin was associated with significantly less swelling of the calf, thigh, and upper pole of the patella as well as with significantly lower pain scores during motion. However, no significant differences in postoperative pain scores at rest, Hospital for Special Surgery scores, range of motion, levels of inflammatory biomarkers, or complication rates were found between the diosmin and control groups. CONCLUSIONS: The use of diosmin after TKA reduced lower-extremity swelling and pain during motion and was not associated with an increased incidence of short-term complications involving the outcomes studied. However, further studies are needed to continue exploring the efficacy and safety of diosmin use in TKA. LEVEL OF EVIDENCE: Therapeutic Level I . See Instructions for Authors for a complete description of levels of evidence.

[Comparison of early clinical outcomes between SuperCap and direct anterior approaches for total hip arthroplasty].

OBJECTIVE: To compare the short-term clinical efficacy of SuperCap approach and direct anterior approach in total hip arthroplasty. METHODS: Clinical data of 70 patients who underwent minimally invasive SuperCap approach and DAA THA in January 2016 to June 2017 were retrospective analyzed. These patients were divided into two groups:SuperCap approach group(SuperCap group) and direct anterior approach group(DAA group). There were 15 males and 15 females in SuperCap group, aged from 45 to 71 years old, and the follow-up time ranged from 24 to 30 months. There were 24 males and 16 females in Group B, aged from 51 to 76 years and the follow-up time ranged from 24 to 36 months. Hemoglobin level of the 3rd day after operation, transfusion rate, acetabular abduction angle, anteversion angle and creatine kinase level of the 3rd day after operation, Harris score of 3 months and the last time, VAS score of 1 week and the last time were recorded and compared. Complications were recorded at the final follow-up. RESULTS: All patients were followed up, the follow-up time of SuperCap group ranged from 24 to 30 months, that of DAA group ranged from 24 to 36 months. No significant differences were found in hemoglobin level on the 3rd day after operation, transfusion rate, Harris score or VAS score between two group (P>0.05). There was no significant difference in Harris score between 3 months after operation and the final follow-up in both groups (P>0.05). There were no significant difference in VAS scores of 6 weeks after operation and on the final follow-up neither(P>0.05). The level of creatine kinase in SuperCap group was significant lower than that in DAA group(P<0.05). Until the final follow-up, there was no significant difference in the incidence of complications between the two groups(P>0.05). CONCLUSION: The clinical effect of minimally invasive SuperCap approach after total hip arthroplasty is comparable to that of DAA approach with less soft tissue injury. Patients can recover rapidly after operation and it is a safe and effective surgical approach for surgeons with short learning curve.

[Short-term effectiveness of structural bone graft and total hip arthroplasty through direct anterior approach in lateral decubitus position for Crowe type â ¢ and â £ developmental dysplasia of the hip].

Objective: To explore the feasibility and effectiveness of total hip arthroplasty (THA) with acetabulum structural bone grafting using autogenous femoral head through direct anterior approach (DAA) in lateral decubitus position in the treatment of Crowe type Ⅲ and Ⅳ developmental dysplasia of the hip (DDH). Methods: Between June 2016 and July 2020, 12 patients with Crowe type Ⅲ and Ⅳ DDH were treated with THA with acetabulum structural bone grafting using autogenous femoral head through DAA in lateral decubitus position. There were 2 males and 10 females with an average age of 60.2 years (range, 50-79 years). Crowe classification was type Ⅲ in 10 hips and type Ⅳ in 2 hips. The preoperative Harris score of hip joint was 48.8±7.5, the difference in length of both lower extremities was (3.0±0.7) cm, and the visual analogue scale (VAS) score during activity was 7.2±0.9. The surgical incision length, operation time, intraoperative blood loss, and complications were recorded; the position and press-fitting of acetabulum and femoral prosthesis were observed after operation, and the difference in length of both lower extremities was measured; the horizontal coverage of acetabular cup and bone graft were measured, the healing with the host bone and the loosening of the prosthesis were evaluated; Harris score was used to evaluate hip joint function, and VAS score was used to evaluate patients' pain during activity. Results: The average surgical incision length was 9.3 cm, the average operation time was 117 minutes, and the average intraoperative blood loss was 283 mL. Two patients (16.7%) received blood transfusion during operation. There was no acetabular and femoral fractures during operation. All incisions healed by first intention, without dislocation, periprosthetic infection, sciatic nerve injury, deep venous thrombosis, and other complications. One patient had lateral femoral cutaneous nerve injury after operation. X-ray films at discharge showed a total acetabular cup level coverage of 93%-100%, with an average of 97.8%, and a bone graft level coverage of 25%-45%, with an average of 31.1%. All the 12 patients were followed up 22-71 months, with an average of 42.2 months. At last follow-up, the Harris score of hip joint was 89.7±3.9, the difference in length of both lower extremities was (0.9±0.4) cm, and the VAS score during activity was 1.1±0.6, which were significantly different from those before operation (P<0.05). During follow-up, there was no patient who needed hip revision surgery because of prosthesis loosening. At last follow-up, there was no translucent line between the graft and the host bone, the graft was fused, the position was good, and there was no obvious movement. One patient had one screw fracture and bone resorption at the outer edge of the graft, but the bone graft did not displace and healed well. Conclusion: THA with acetabulum structural bone grafting using autogenous femoral head through DAA in lateral decubitus position in the treatment of Crowe type Ⅲ and Ⅳ DDH is safe and reliable, and has satisfactory short-term effectiveness.

Long non-coding ribonucleic acid AFAP1-AS1 promotes chondrocyte proliferation via the miR-512-3p/matrix metallopeptidase 13 (MMP-13) axis.

Long-chain non-coding RNAs are reported to be involved in cartilage damage. However, less research on the role of actin filament-associated protein 1 antisense RNA 1 (AFAP1-AS1) in osteoarthritis. To investigate AFAP1-AS1 function in osteoarthritis development, AFAP1-AS1 and miR-512-3p expression levels in osteoarthritis cartilage and cells were evaluated using RT-qPCR. The downstream target genes of AFAP1-AS1 and miR-512-3p were predicted and validated using luciferase reporter assays. Moreover, a knee osteoarthritis model was established by injecting monoiodoacetate into the knee joints of mice. The effects of AFAP1-AS1 and miR-512-3p on osteoarthritis chondrocyte proliferation and MMP-13, collagen II, and collagen IV expressions were detected in vivo using CCK-8 assay and Western blotting and RT-qPCR, respectively. AFAP1-AS1 expression was upregulated in osteoarthritis cartilage and cells. MiR-512-3p expression was downregulated in osteoarthritis cartilage. AFAP1-AS1 overexpression inhibited miR-512-3p expression in chondrocytes. Furthermore, AFAP1-AS1 over-expression promoted chondrocyte proliferation, and miR-512-3p mimic inhibited chondrocyte proliferation in vivo. AFAP1-AS1 overexpression reduced type II and type IV collagen expression, while miR-512-3p overexpression promoted type II and type IV collagen in vivo. AFAP1-AS1 overexpression enhanced MMP-13 expression in vivo. AFAP1-AS1 overexpression regulated chondrocyte proliferation by inhibiting miR-512-3p expression in vivo. AFAP1-AS1 could be a potential target to treat osteoarthritis by inhibiting miR-512-3p and subsequently inducing chondrocyte proliferation and regulating matrix synthesis.

Euxanthone Impairs the Metastatic Potential of Osteosarcoma by Reducing COX-2 Expression.

Osteosarcoma (OS) is one of the most common malignancies of bone. This study was aimed to explore the anti-metastatic effect of euxanthone on OS. Adhesion assay and Transwell assay were used to examine the effect of euxanthone on adhesion, migration and invasion of OS cells. COX-2-over-expressing plasmid was applied to transfect OS cells to assess whether COX-2 affects the anti-metastatic function of euxanthone. PDCD4 knockdown and miR-21 mimic were applied to assess whether euxanthone suppresses the transactivation of c-jun via modulating miR-21-PDCD4 signaling. The effect of euxanthone in vivo was also examined by lung metastasis assay. Euxanthone, a xanthone derivative extracted from Polygala caudata, has been found to exhibit anti-neoplastic activities. In present study, our results showed that euxanthone suppressed cell adhesion, migration, and invasion in OS cells. Our experimental data also showed that repression of COX-2 by euxanthone mediated its anti-metastatic activities. Moreover, our findings revealed that euxanthone modulated the COX-2 expression through the miR-21/PDCD4/c-jun signaling pathway. The anti-metastatic activities of euxanthone were also validated in a pulmonary metastasis model. Taken together, our results highlighted the potential of euxanthone to be used in the treatment of OS. Anat Rec, 302:1399-1408, 2019. © 2018 Wiley Periodicals, Inc.

Co-implantation of bone marrow mesenchymal stem cells and chondrocytes increase the viability of chondrocytes in rat osteo-chondral defects.

Replacement of chondrocytes by adult stem cells was believed to improve the performance of autologous chondrocytes transplantation, since less chondrocytes were needed. Previous studies have demonstrated that the increased cartilage production in pellet co-cultures of chondrocytes and bone marrow mesenchymal stem cells (BMSCs) is due to the trophic effects of the MSC by stimulating chondrocyte proliferation and matrix production. However, the destination of MSCs or chondrocytes after implanted in osteo-chondral defects is not clear. The aim of the present study is to investigate the viability of MSCs and chondrocytes after co-implantation into a rat osteo-chondral defect model. MSCs were isolated from bone marrow and chondrocytes were extracted from knee joints of neonatal rats. Results of sulfated glycosaminoglycans (GAG) and collagen quantification demonstrated that co-culture pellets of BMSCs and chondrocytes have more GAG deposition than that of BMSCs or chondrocytes alone. Tracking cells with fluorescence protein demonstrated that MSCs disappeared following co-culture. In a rat knee injury model, co-implantation of BMSCs and chondrocytes contained more viable chondrocytes than chondrocytes implanted alone. To conclude, BMSCs were replaced by chondrocytes in pellet co-culture and BMSCs increased the viability of chondrocytes following co-implantation in a osteo-chondral defects model. Co-implantation of BMSCs and chondrocytes may be a promising approach to repairing osteo-chondral defects in the clinical setting.

Inhibitory effects of forkhead box L1 gene on osteosarcoma growth through the induction of cell cycle arrest and apoptosis.

Forkhead box L1 (FOXL1), which is considered a novel candidate tumor suppressor, inhibits proliferation and invasion in certain types of cancer. However, the regulation and function of FOXL1 in osteosarcoma remains unclear. The expression of FOXL1 gene in osteosarcoma tissues and cell lines was examined using RT-PCR, immunohistochemistry and western blot analysis. pcDNA-FOXL1 carrying full-length FOXL1 cDNA was constructed to upregulate the level of FOXL1 expression in osteosarcoma cell lines. The proliferation, cell cycle and apoptosis of osteosarcoma cells in vitro or in vivo were examined following transfection with pcDNA-FOXL1. In addition, the expression of p21, p27, cytochrome c and caspase-3, which may be involved in the regulation of the cell cycle and apoptosis was examined using western blot analysis. The results showed that FOXL1 expression was downregulated in osteosarcoma tissues and cell lines. Loss or downregulation of FOXL1 was associated with poor prognosis. Ectopic FOXL1 expression inhibited cell proliferation in vitro and in vivo. The ectopic FOXL1 expression increased the expression of p21 and p27, which induced G1 arrest in the U-2 OS cells. In addition, the ectopic FOXL1 expression induced cytochrome c release between mitochondria and cytoplasm, which disrupted the mitochondrial transmembrane potential and triggered intrinsic pathway apoptosis. In conclusion, the downregulation of FOXL1 expression was associated with osteosarcoma cell growth. Restoration of FOXL1 gene expression by gene therapy may have a therapeutic potential for patients with osteosarcoma.

Clinical Analysis of Internal Fixation Treatment of Intra-articular Calcaneal Fractures with Titanium Plate.

To explore the clinical effect of internal fixation treatment of intra-articular calcaneal fractures with titanium plate, we used open reduction and internal fixation with titanium plate to 48 treated feet from 42 patients with intra-articular calcaneal fractures. The efficacy of surgical treatment was evaluated based on assessment of pain, function, and line of force aspects according to the American Orthopedic Foot and Ankle Society scoring system. Our data show that internal fixation with titanium plate is an effective treatment for calcaneal fractures. It provides satisfactory reduction, reliable fixation, and early rehabilitation.

[ALLOTRANSPLANTATION OF CRYOPRESERVATED VASCULARIZED BONE IN LIMB SALVAGE SURGERY FOR CHILDREN AND ADOLESCENTS WITH OSTEOSARCOMA].

OBJECTIVE: To investigate the effectiveness and technical key points of limb salvage surgery by allotransplantation of cryopreservated vascularized bone in children and adolescents with osteosarcoma. METHODS: A retrospective analysis was made on the clinical data of 21 children and adolescents with osteosarcoma receiving limb salvage surgery by allotransplantation of cryopreservated vascularized bone from their relatives between February 2004 and April 2012. There were 13 males and 8 females, aged from 7 to 16 years (mean, 12.6 years). According to Enneking stage system, 15 cases were rated as stage IIA and 6 cases as stage IIB. The tumors located at the distal femur in 10 cases, at the proximal femur in 1 case, at the proximal tibia in 8 cases, at the proximal humerus in 1 case, and at the distal radius in 1 case. Imaging examination showed that epiphyseal extension of malignant bone tumors in 7 cases. The iliac bone allograft with deep iliac vessels was obtained from their lineal consanguinity. After preservation by a two-step freezing schedule, the iliac bone allograft with deep iliac vessels was implanted into the bone defect area after tumor resection. The size of iliac bone flap was 8.0 cm x 3.0 cm x 2.0 cm-14.0 cm x 5.0 cm x 2.5 cm. Reserved joint surgery was performed on 16 cases and joint fusion surgery on 5 cases, and external fixation was used in all cases. The chemotherapy was given according to sequential high-dose methotraxate, adriamycin, and cisplatine before and after operation. RESULTS: All 21 cases were followed up from 5 months to 11 years (mean, 6.4 years). At 2 weeks after operation, the erythrocyte rosette forming cells accounted for 56.7% ± 3.9%, showing no significant difference when compared with that of normal control (58.3% ± 4.3%) (t = 1.56, P = 0.13), which suggested no acute rejection. At 4 weeks after operation, single photon emission computerized tomography bone scan indicated that the blood supply of bone graft was rich, and the metabolism was active. At 12 weeks after operation, the digital subtraction angiography showed the artery of iliac bone flap kept patency. X-ray films showed that malunion and non-union occurred at 5 and 6 months after operation in 1 case, respectively. The bone graft healed in the other patients, and the healing time was 3.2-6.0 months (mean, 4.4 months). At last follow-up, American Musculoskeletal Tumor Society (MSTS) score was significantly improved to 26.80 ± 2.14 from preoperative value (17.15 ± 1.86) (t = -4.15, P = 0.00). The survival rate was 85.7% (18/21) and the recurrence rate was 9.5% (2/21). CONCLUSION: Allotransplantation of cryopreservated vascularized bone from the relatives provides a new method for the treatment of osteosarcoma in children and adolescents. A combination of allotransplantation and chemotherapy can achieve the ideal treatment effect. The correct cutting, preservation, and transplantation of the donor bone, and indication are the key to improve the effectiveness.

Effects of adenoviral vector expressing hIGF-1 on apoptosis in nucleus pulposus cells in vitro.

The excessive apoptosis of cells of the nucleus pulposus may plays an important role in intervertebral disc (IVD) degeneration. It has been shown that the pro-inflammatory cytokine tumour necrosis factor (TNF)-α can induce disc cell apoptosis. Insulin-like growth factor (IGF)-1 can promote nucleus pulposus cell proliferation; however, whether or not IGF-1 inhibits TNF-α-induced apoptosis in the nucleus pulposus has not yet been elucidated. In this study, our objective was to create a potentially therapeutic viral vector, which could be used to achieve the enforced expression of IGF-1 in rabbit nucleus pulposus cells. Furthermore, we investigated the ability of IGF-1 to reverse TNF-α-induced apoptosis in cells of the nucleus pulposus. Isolated nucleus pulposus cells were cultured to a confluent monolayer, digested with collagenase Ⅱ and purified using trypsin and differential adhesion methods. Nucleus pulposus cells were positively identified using type Ⅱ collagen immunohistochemistry. Following transfection with adenoviral vectors engineered to overexpress recombinant human IGF-1 (Ad-hIGF-1) or TNF-α, the cells were observed under a light microscope. Terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick end-labeling (TUNEL) and flow cytometry (FCM) were used to assess the rate of apoptosis. The Ad-hIGF-1 viral vector was effectively transduced into the nucleus pulposus cells and increased IGF-1 expression as confirmed by RT-PCR and western blot analysis. In the TNF-α-treated group, a large number of apoptotic cells was observed that exhibited morphological changes associated with this form of cell death. Minimal apoptosis was observed in the Ad-hIGF-1-treated group and the control group showed no obvious signs of apoptosis. TUNEL assay revealed that the rate of apoptosis in the Ad-hIGF-1 group was significantly reduced compared with the TNF-α-treated group (P<0.01). This result was confirmed using FCM. The rate of apoptosis was also significantly increased in the TNF-α-exposed cells compared with the control group (P<0.01). Our findings strongly suggest that the adenoviral vector expressing hIGF-1 can successfully infect nucleus pulposus cells in vitro and effectively enhance the expression of IGF-1. In addition, IGF-1 reversed the TNF-α -induced apoptosis of nucleus pulposus cells. Thus, Ad-hIGF-1 may be useful in the development of clinical interventions for disc degeneration.

[Influence on adjacent lumbar bone density after strengthening of T12, L1 segment vertebral osteoporotic compression fracture by percutaneous vertebroplasty and percutaneous kyphoplasty].

OBJECTIVE: To observe the influence on adjacent lumbar bone density after strengthening of T12, L1 segment vertebral osteoporotic compression fracture by percutaneous vertebroplasty (PVP) and percutaneous kyphoplasty (PKP) in postmenopausal female. METHODS: Between January 2008 and June 2011, 59 patients with T12, L1 segment thoracolumbar osteoporotic compression fracture were treated with PVP in 29 cases (PVP group) and PKP in 30 cases (PKP group), who were in accordance with the inclusion and exclusion criteria. No significant difference was found in gender, duration of menopause, disease druation, causes of injury, fractured vertebral body, and vertebral fracture classification between 2 groups (P > 0.05). The kyphosis Cobb angle of surgical area was measured at preoperation, 1 week after operation, and last follow-up; the lower three lumbar spine bone mineral density (BMD) of the surgical area, the femoral neck BMD, and body mass index (BMI) of patients were measured at perioperative period and last follow-up to find out the statement of anti-osteoporosis; FRAX online tools were used to evaluate the probability of major osteoporotic fracture and hip fracture of the next 10 years. RESULTS: The average follow-up was 25.5 months (range, 12-48 months) in 2 groups. There was significant difference in kyphosis Cobb angle of T12, L1 between preoperation and last follow-up in 2 groups (P < 0.05); the Cobb angle of PKP group was significantly less than that of PVP group at 1 week after operation and last follow-up (P < 0.05). No significant difference was found in BMI between 2 groups, and between perioperative period and last follow-up in the same group (P > 0.05). The lower three lumbar spine BMD of the surgical area and its T value at last follow-up was improved significantly when compared with BMD at perioperative period (P < 0.05); there was no significant difference in the lower three lumbar spine BMD and its T value between 2 groups at perioperative period (P > 0.05), but significant difference was found between two groups at last follow-up (P < 0.05). Difference was not significant in the femoral neck BMD and its T value between 2 groups, and between perioperative period and last follow-up in the same group (P > 0.05). The probability of major osteoporotic fracture and hip fracture of the next 10 years was not significantly different between 2 groups and between perioperative period and last follow-up in the same group (P > 0.05). CONCLUSION: The increased BMD of adjacent lumbar spine can improve the strength of the vertebral body and reduce the incidence of adjacent vertebral fracture in patients with T12, L1 segment vertebral osteoporotic compression fracture after PVP/PKP, and PKP is superior to PVP increasing BMD of adjacent lumbar spine.

Histologic and biomechanical studies of tendon-to-bone healing after autologous and allogeneic bone transplants.

OBJECTIVES: Compare histologic and biomechanical differences of tendon-to-bone healing between autologous and allogeneic bone transplants. MATERIALS AND METHODS: Adult, healthy, New Zealand white rabbits were used to establish the extra-articular tendon-to-bone healing model with the left hind limb transplanted with allogeneic bone and the right hind limb transplanted with autologous bone. After 3, 6, and 12 weeks after the transplant, the rabbits were killed to collect tendon-to-bone specimens, and then the healing processes in tendon-to-bone interfaces were examined. RESULTS: All rabbits grew well after incision without infection and can freely move. Histologic observations 3 and 6 weeks after surgery and biomechanical test results 6 weeks after surgery were statistically different between the autologous and the allogeneic transplants (P < .05). After 12 weeks, histologic observations and biomechanical test results showed no difference between the 2 transplants (P > .05). CONCLUSIONS: Allogeneic bone transplant has a relatively slower tendon-to-bone healing than does autologous bone transplant, but finally allogeneic and autologous bone transplants have the same extent of tendon-to-bone healing.

Effects of IGF-1 on IL-1ß-induced apoptosis in rabbit nucleus pulposus cells in vitro.

Excessive apoptosis in intervertebral disc (IVD) cells is important in IVD degeneration. Interleukin (IL)-1ß has been shown to induce apoptosis in these cells. However, whether insulin-like growth factor-1 (IGF-1) inhibits IL-1ß-induced apoptosis in the nucleus pulposus remains unclear. The purpose of this study was to investigate the effects of IGF-1 on IL-1ß-induced apoptosis in the nucleus pulposus. Cells isolated from the nucleus pulposus were grown in culture to a monolayer. These cells were identified using immuno-histochemistry for type II collagen and toluidine blue staining for glycosaminoglycans. Following exposure to IGF-1 or IL-1ß, the cells were observed using light microscopy. Giemsa staining, TdT-mediated dUTP-biotin nick end-labeling (TUNEL) and flow cytometry (FCM) were used to detect the rate of early cell death, which served as an indicator of apoptosis. In the IL-1ß group, a large number of these cells underwent apoptosis and demonstrated morphological changes associated with apoptosis. A small proportion of cells exposed to IGF-1 alone underwent apoptosis. No obvious signs of apoptosis were observed in the control group. TUNEL results revealed that the rate of apoptosis in the IGF-1 group was significantly reduced compared with that in the IL-1ß group (P<0.01), confirmed using FCM. Compared with the control group, the apoptotic rate was also significantly increased in IL-1ß-exposed cells (P<0.01). These findings strongly suggested that IGF-1 inhibits IL-1ß-induced apoptosis in the nucleus pulposus.

[Effects of recombinant adenovirus vector carrying human insulin-like growth factor 1 gene on the apoptosis of nucleus pulposus cells in vitro].

OBJECTIVE: To investigate the effects of human insulin-like growth factor 1 (hIGF-1) gene transfected by recombinant adenovirus vector (Ad-hIGF-1) on the apoptosis of rabbit nucleus pulposus cells induced by tumor necrosis factor a (TNF-alpha). METHODS: The intervertebral disc nucleus pulposus were harvested from 8 healthy adult domestic rabbits (male or female, weighing 2.0-2.5 kg). The nucleus pulposus cells were isolated with collagenase II digestion and the passage 2 cells were cultured to logarithm growing period, and then they were divided into 3 groups according to culture condition: DMEM/ F12 medium containing 10% PBS, DMEM/F12 medium containing 10% PBS and 100 ng/mL TNF-alpha, and DMEM/F12 medium containing 10% PBS, 100 ng/mL TNF-alpha, and Ad-hIGF-1 (multiplicity of infection of 50) were used in control group, TNF-alpha group, and Ad-hIGF-1 group, respectively. The results of transfection by adenovirus vector carrying hIGF-1 gene were observed by fluorescent microscopy; the expression of hIGF-1 protein was detected by Western blot, hIGF-1 mRNA expression by RT-PCR, and the cell apoptosis rate by TUNEL and flow cytometry. RESULTS: Green fluorescence was observed by fluorescent microscopy in Ad-hIGF-1 group, indicating that successful cell transfection. The expressions of hIGF-1 protein and mRNA were detected in Ad-hIGF-1 group by Western blot and RT-PCR, while the control group and TNF-alpha group had no expression. The cell apoptosis rates of TNF-alpha group, Ad-hIGF-1 group, and control group were 34.24% +/- 4.60%, 6.59% +/- 1.03%, and 0.40% +/- 0.15%, respectively. The early apoptosis rates of TNF-alpha group, Ad-hIGF-1 group, and control group were 22.16% +/- 2.69%, 5.03% +/- 0.96%, and 0.49% +/- 0.05%, respectively; the late cell apoptosis rates were 13.96% +/- 4.86%, 10.68% +/- 3.42%, and 0.29% +/- 0.06%, respectively. Compared with TNF-alpha group, the cell apoptosis rates of Ad-hIGF-1 group and control group were significantly reduced (P < 0.05); the cell apoptosis rate of Ad-hIGF-1 group was significantly higher than that of control group (P < 0.05). CONCLUSION: Ad-hIGF-1 could ingibit the apoptosis of nucleus pulposus cells induced by TNF-alpha.

[Effects of impaction on tissue engineered bone modified by BMP-2 gene].

OBJECTIVE: To observe effects of the direct impaction on the cell survival and the bone formation of the tissue engineered bone modified by the adenovirus mediated human bone morphogenetic protein 2 (Adv-hBMP2) gene and to verify the feasibility of the impacted grafting with it. METHODS: The marrow stromal cells (MSCs) were separated from the canine bone marrow and were cultured. MSCs were transfected with the Adv-hBMP2 gene and combined with the freeze-dried cancellous bone (FDB) to form the tissue engineered bone. Four days after the combination, the tissue engineered bone was impacted in a simulated impactor in vitro and implanted in the mouse. The cell survivals were evaluated with SEM 1 and 4 days after the combination, immediately after the impaction, and 1 and 4 days after the impaction, respectively. The bone formation and the allograft absorption were histologically evaluated respectively. RESULTS: There were multiple layers of the cells and much collagen on FDB before the impaction. Immediately after the impaction, most of the cells on the direct contact area disappeared and there was much debris on the section. Some of the cells died and separated from the surface of FDB at 1 day, the number of the cells decreased but the collagen increased on the surface at 4 days. Histologically, only the fibrous tissue was found in FDB without the cells, the bone formation on FDB was even in distribution and mass in appearance before the impaction, but declined and was mainly on the periphery after the impaction in the Adv-hBMP-2 modified tissue-engineered bone. CONCLUSION: The simulated impaction can decrease the cells survival and the bone formation of the Adv-hBMP-2 modified tissue-engineered bone. The survival cells still function well. It is feasible to use the tissue engineered bone in the impaction graft.

[Preliminary observation of biological characters of chondrocytes in articular loose body].

OBJECTIVE: To observe the biological characters of chondrocytes in articular loose body and to find out seeding cells for cartilage tissue engineering. METHODS: Samples from 5 loose body cartilages, 2 normal articular cartilages and 6 osteoarthritis articular cartilages were collected. Part of each sample's cartilage was histologically studied to observe the chondrocytes distribution the morphologic changes by toluidine-blue staining, chondrocytes' apoptosis by terminal deoxynucleotidyl transferase mediated deoxyuridine triphosphate-biotin nick end-labeling (TUNEL). The rest of each cartilage was digested and isolated by 0.25% trypsin and 0. 2% collagenase II, and then were cultivated in 10%DMEM. Their morphologic changes were observed 24h later. Comparison was made between three cartilages. RESULTS: Compared with normal cartilage and osteoarthritis articular cartilage, the cells density was higher, their lacunary were larger, cells distribution was irregular, and apoptosis was more apparent in loose body cartilage. CONCLUSION: The characters of chondrocytes from loose body is more like fibroblasts so they can not serve as seeding cells directly for cartilage tissue engineering.

[Effects of bone morphogenetic protein-2 gene therapy on the bone-implant interface: an experimental study with dogs].

OBJECTIVE: To investigate the effects of bone morphogenetic protein-2 (BMP-2) gene therapy on the bone-implant interface in the reconstruction of periprosthetic bone defect. METHODS: Transverse defects were caused in the external condylae of both femurs of 14 adult Beagle dogs. Titanium alloy implants were inserted and a bone defect 3 mm wide around the titanium alloy implant was preserved. Then the total 28 defects were divided into 4 groups: 8 bone defects remained untreated (blank control group); 8 bone defects were implanted with heterogeneous freeze-dried bone by impaction grafting technique (non-cell group); 8 bone defects were implanted with heterogeneous freeze-dried bone loaded with autogenous bone marrow stromal cells (BMSCs) from the greater trochanter of the same dog (cell group); and 10 bone defects were implanted with freeze-dried allograft loaded with autogenous BMSCs from the greater trochanter of the same dog which were transfected by Adv-BMP-2 gene (gene group). Three, 6, and 12 weeks after implantation X-ray examination was carried out to observe the place of the implant and the absorption of the implants. Six and 12 weeks after the dogs were killed and their bone defects were taken out to undergo histological, histomorphometric and biomechanical examination to observe the healing and oseeointegration of the bone-implant interface. RESULTS: Histological examination showed that 6 weeks after implantation new bone formation was found on the implant surface and there was point contact between the bone and implant in the gene group with the bone-to-impact contact (BIC) of about 10%; and continuous soft tissue was found at bone-implant interface in all other groups. Twelve weeks after, there was thick soft tissue membrane between the new bone and implant in the blank control group; most of the interface was connective fibrous tissue in the non-cell group and cell group with point contact between the bone and implant and a BIC lower than 10%; and in the gene group the interface consisted mainly of bone tissue and continuous bone-implant contact was found with the BIC of 50%, significantly higher than those of the other 2 groups (both P < 0.01). The mechanical strength of interface increased time-dependently in all groups, that of the gene group being significantly higher than those of the other 2 groups at any time-points (both P < 0.01). CONCLUSION: BMP-2 gene therapy can improve the osseointegration of bone-implant interface.