Therapeutic Applications of Genes and Gene-Engineered Mesenchymal Stem Cells for Femoral Head Necrosis.

Osteonecrosis of the femoral head (ONFH) is a common and disabling joint disease. Although there is no clear consensus on the complex pathogenic mechanism of ONFH, trauma, abuse of glucocorticoids, and alcoholism are implicated in its etiology. The therapeutic strategies are still limited, and the clinical outcomes are not satisfactory. Mesenchymal stem cells (MSCs) have been shown to exert a positive impact on ONFH in preclinical experiments and clinical trials. The beneficial properties of MSCs are due, at least in part, to their ability to home to the injured tissue, secretion of paracrine signaling molecules, and multipotentiality. Nevertheless, the regenerative capacity of transplanted cells is impaired by the hostile environment of necrotic tissue in vivo, limiting their clinical efficacy. Recently, genetic engineering has been introduced as an attractive strategy to improve the regenerative properties of MSCs in the treatment of early-stage ONFH. This review summarizes the function of several genes used in the engineering of MSCs for the treatment of ONFH. Further, current challenges and future perspectives of genetic manipulation of MSCs are discussed. The notion of genetically engineered MSCs functioning as a "factory" that can produce a significant amount of multipotent and patient-specific therapeutic product is emphasized.

Mid-term outcomes of primary constrained condylar knee arthroplasty for severe knee deformity.

This study aimed to examine the clinical and radiographic outcomes of primary total knee arthroplasy (TKA) with use of NexGen® Legacy® Constrained Condylar Knee (CCK) prosthesis for severe knee deformity. Clinical data of 46 patients (48 knees in total, aged 61 years on average) with severe knee deformity who underwent TKA with NexGen® Legacy® CCK prosthesis between December 2007 and February 2012 were retrospectively analyzed. There were 34 knees with severe valgus with incompetent medial collateral ligament, 11 knees with severe flexion contracture with inability to achieve knee balancing in flexion and extension by posterior soft tissue release, 2 knees with Charcot arthritis with severe varus and bone loss, and 1 with traumatic osteoarthritis with severe varus and ligamentous instability. The mean duration of follow-up was 71 months (range 40-90 months). The New Knee Society scoring (NKSS) system and the Hospital for Special Surgery (HSS) score were used to evaluate the functional and clinical outcomes. Visual Analogue Scale (VAS) was used for pain measurement and Knee Society criteria for evaluation of radiological images. The results showed that, in the total 48 knees, 1 case of loosening due to short-stem tibial component at 3 months post-operatively underwent revision. The 6-year prosthesis survival rate in this cohort was 97.9%. There was no component infection occurring within 6 years. Significant post-operative improvements were found in NKSS and HSS scores. Patient satisfaction was significantly increased. Pain score was decreased significantly. Total functional score was improved from 31.46±11.43 to 86.42±8.87, range of motion (ROM) from 42.42°±23.57° to 95.31°±23.45° and the flexion contracture from 5.31°±7.87° to 0.92°±1.80°. Preoperative radiographic study showed excessive valgus (≥7°) in 37 knees, and varus deformity in 3 knees. Post-operative femorotibial alignment was valgus 3.88°±1.76° in 48 knees. Antero/posterior (A/P) view of X-ray films showed 4 radiolucent lines (RLL) in 48 tibial components. It was concluded that TKA with CCK is effective for the treatment of the severe unstable knee that cannot be balanced by soft tissue.

Low-intensity treadmill exercise promotes rat dorsal wound healing.

In order to investigate the promoting effect of low-intensity treadmill exercise on rat dorsal wound healing and the mechanism, 20 Sprague-Dawley rats were randomly divided into two groups: exercise group (Ex) and non-exercise group (non-ex). The rats in Ex group were given treadmill exercise for one month, and those in non-ex group raised on the same conditions without treadmill exercise. Both groups received dorsal wound operation with free access to food and water. By two-week continuous observation and recording of the wound area, the healing rate was analyzed. The blood sample was collected at day 14 post-operation via cardiac puncture for determination of the number of endothelial progenitor cells (EPCs) by flow cytometry, and the concentrations of relevant cytokines such as basic fibroblast growth factor (bFGF), endothelial nitric oxide synthase (eNOS) and vascular endothelial growth factor (VEGF) were measured by ELISA. The skin tissue around the wound was dissected to observe the vascular density under the microscope after HE staining, to detect the mRNA level of VEGFR2 and angiopoietin-1 (Ang-1) receptor using RT-qPCR, and protein expression of a-smooth muscle actin (αSMA) and type III collagen (ColIII) using Western blotting. It was found that the wound area in Ex group was smaller at the same time point than in non-ex group. The number of circulating EPCs was greater and the concentrations of vasoactive factors such as VEGF, eNOS and bFGF were higher in Ex group than in non-ex group. HE staining displayed a higher vessel density in Ex group than in non-ex group. Moreover, the mRNA expression of VEGFR2 and Ang-1 detected in the wound tissue in Ex group was higher than in non-ex group. Meanwhile, the protein expression of αSMA and ColIII was more abundant in Ex group than in non-ex group. Conclusively, the above results demonstrate Ex rats had a higher wound healing rate, suggesting low-intensity treadmill exercise accelerates wound healing. The present work may provide some hint for future study of treating refractory wound.

Total hip arthroplasty with subtrochanteric femoral shortening osteotomy for high hip dislocation.

OBJECTIVE: To evaluate the outcomes of total hip arthroplasty (THA) with subtrochanteric femoral shortening osteotomy for high hip dislocation. METHODS: In this retrospective study, the results of 24 primary THAs with acetabular reconstruction and subtrochanteric femoral shortening osteotomy in 21 patients with high hip dislocation were evaluated. The acetabula were reconstructed with cemented or uncemented cups and bone grafting. Transverse subtrochanteric femoral shortening osteotomies were applied and the osteotomy sites treated by bone grafting and cable fixation. Assessment was by Hip Harris scores and radiographic evaluation. RESULTS: The mean follow-up time was 42 months (18-108 months), three cases being lost to follow-up 18-27 months postoperatively. The HHS improved from 47.5 ± 8.7 to 88.5 ± 3.1. The mean length of femoral segments removed was 2.5 ± 0.8 cm (range, 1.0-4.5 cm) and mean acetabular inclination 43° ± 5° (range, 31°-54°). Caudalization of the femoral head center was 3.2 ± 3.0 mm (range, -3 to 12 mm) and lateralization 4.0 ± 4.0 mm (range, -9 to 11 mm). Mean greater trochanter height relative to theoretical hip center was 5.2 ± 1.0 cm (range, 3.5-7.1 cm) preoperatively and 0.2 ± 0.6 cm (range, -0.9 to 1.2 cm) postoperatively. Intraoperative trochanteric fractures occurred in three cases and sciatic nerve palsy in one. CONCLUSION: THA with subtrochanteric femoral shortening osteotomy is an effective technique for treating high hip dislocation. Its advantages include improvement in limb imbalance and decreased risk of sciatic nerve injury.

Decreased in the number and function of circulation endothelial progenitor cells in patients with avascular necrosis of the femoral head.

INTRODUCTION: Once non-traumatic avascular necrosis of the femoral head (ANFH) happened, vascular impairment and feeble collateral circulation are followed by poor outcomes. Circulating endothelial progenitor cells (EPCs) may substantially contribute to vascular homeostasis such as vascular repair and new blood vessel growth. We investigated whether abnormalities in EPCs levels and functions are present in ANFH patients. METHODS: 54 ANFH patients were enrolled, including steroid-induced (n=21), alcohol-induced (n=15) and idiopathic ANFH (n=18), and 30 healthy subjects as control (HC). The numbers of circulation EPCs were determined by fluorescence-activated cell-sorting (FACS) analysis. EPCs cultured from peripheral blood mononuclear cells on fibronectin to induce the expression of receptors for acetylated low-density lipoprotein and ulex-lectin. EPCs colony-forming units (CFUs) were observed from 54 patients and 30 healthy controls. Migratory capacity to chemo-attractants (vascular endothelial growth factor) cellular senescence levels and in vitro angiogenesis ability were assessed in age-matched subjects (n=10 per groups). RESULTS: Mean numbers of circulating EPC were 1460+/-265 cells/ml in HC, 545+/-177 in ANFH, (P<0.001). Mean numbers of CFUs were 26.2+/-6.2 in HC, 19.6+/-7.7 in ANFH,(P<0.001). Although there were not significant differences in circulating EPC and CFUs among the steroid-induced, alcohol-induced or idiopathic three groups, all these risk factors contributed to the decreased circulating EPCs numbers and CFUs. In addition, EPCs from ANFH patients showed reduced migratory capacity and increased cellular senescence compared with EPCs from normal subjects, furthermore the ability of angiogenesis in vitro was also impaired. CONCLUSION: Circulating endothelial progenitor cells (EPCs) numbers and functions are reduced in ANFH patients, suggesting that risk factors of ANFH may alter EPCs biology in angiogenesis and vascular repair.

Shoulder hemiarthroplasty for the treatment of complex proximal humeral fractures.

OBJECTIVE: To investigate the indication, perioperative announcements, selection of prosthesis and clinical results of shoulder hemiarthroplasty for the treatment of complex proximal humeral fractures. METHODS: A total of 55 patients who suffered from complex proximal humeral fractures were treated by shoulder hemiarthroplasty. The mean age was 55.6 years and mean follow-up period was 25.1 months. The scoring system modification for hemiarthroplasty (SSMH) had been adopted for evaluation at the latest follow-up. RESULTS: The pain was obviously relieved in all patients. Fifty patients were painless and 5 patients had slight pain. The mean range of motion was 100 degrees (90 degrees-110 degrees) in abduction, 95 degrees (80 degrees-100 degrees) in forward flexion, 35 degrees (30 degrees-40 degrees) in external rotation and internal rotation was confined at L2 level (L1-L3). The mean SSMH score was 27.9 (24-29). Fifty patients (90.1%) were satisfied with the clinical outcome. CONCLUSIONS: Shoulder hemiarthroplasty is an effective method to treat complex proximal humeral fractures. The proper selection of patients and prosthesis, good operation skill and enough functional exercise are the key points of successful treatment.

Clinical result of structural augmentation with cannulated bone screws for the treatment of osteonecrosis of the femoral head.

OBJECTIVE: To evaluate the results of treatment of osteonecrosis of the femoral head by structural augmentation through a routine core decompression procedure combined with insertion of cannulated bone screws incorporating autogenous bone graft and biomaterial containing decalcified bone matrix. METHODS: From February 2002 to February 2005, 31 patients (33 hips) with femoral head necrosis were treated in our hospital using insertion of cannulated bone screws incorporating autogenous bone graft. There were 18 men and 13 women with an average age of 37 years (range, 27-49). The Steinberg classification was stage I for 20 hips (61%) and stage II for 13 hips (39%). Clinical and radiographic evaluations were performed on all patients. The patient's satisfaction was also assessed. RESULTS: All 31 patients (33 hips) were retrospectively studied after a mean follow-up of 38 months (range, 18-48). The average Harris hip score was 76 before surgery and 91 at the final follow-up. All patients stated that they were satisfied and had significantly reduced pain. According to the Harris hip score system, 21 cases were excellent, 8 good and 2 fair. No complications, such as wound infection, subtrochanteric fracture, neuropathy and deep vein thrombosis, were found. CONCLUSION: Structural augmentation using the insertion of cannulated bone screws incorporating autogenous bone graft is an effective option for Steinberg I-II stages of femoral head necrosis. Further study is needed to confirm mid- and long-term results.

Total hip arthroplasty for treatment of elderly patients with comminuted intertrochanteric fracture accompanied by femoral head necrosis.

OBJECTIVE: To assess the curative effect and investigate the indications of total hip arthroplasty for treatment of comminuted intertrochanteric fractures. METHODS: Total hip arthroplasty was carried out in 9 cases of severe intertrochanteric fracture. The patients included two men and seven women. The average age of the patients was 68 years (48-75 years). The period from fracture to operation was 5 days (2-10 days). The mean follow-up period lasted for 11 months (3 months-2 years). There was one patient with comminuted intertrochanteric fracture accompanied by femoral head necrosis and 2 patients with intertrochanteric fracture and stroke. Other 6 patients had severe osteoporosis. The Harris score before operation was 63 points (45-71 points). RESULTS: At the last follow-up, the patients gained 86 points (70-100 points) according to the Harris score. The effects of the 8 cases were good. The Harris score of all patients improved after treatment. Only two hemiplegia patients needed sticks to walk. The others could walk without hip pain. No radiographic evidence of acetabular wear and prosthesis dislocation or other major complications happened during the follow-up. CONCLUSIONS: Prosthetic replacements can well treat unstable intertrochanteric fracture if operative indication is correctly selected. It is suitable for elderly patients and the operation should be performed by experienced surgeons.

[Assessment of the expression profile during the entochondrostosis of vascular endothelial growth factor in bone morphogenetic protein 2 induced osteogenesis].

OBJECTIVES: To examine the gene expression profile of bone morphogenetic protein 2 (BMP-2) and vascular endothelial growth factor (VEGF) during entochondrostosis of mice and explore the expression rules and effects between BMP-2 and VEGF, and to detect the expression of VEGF in BMP-2 induced entochondrostosis in vivo. METHODS: cDNA microarray technique with 34,000 genes was used to analyze the gene expression profiles during entochondrostosis in the limbs of mice embryo from E10 to E14. Pathway analysis of BMP-2 and VEGF was performed with GCOS1.2 software. An experimental model of femoral muscular pouch in 20 mice was adopted. The expression of VEGF was examined by in situ hybridization method and immunohistochemical method in BMP-2 induced entochondrostosis in vivo. RESULTS: The expression signals of VEGF mRNA and VEGF appeared in cytoplasm during condensation of mesenchymal cell. As the mesenchymal cells differentiated into precartilage, the expression signals decreased in mesenchymal cells, but increased in chondrocytes and kept getting denser in the process of cartilage maturity. The peak expression of VEGF mRNA and VEGF in the experimental group appeared on the 14th day, accompanied by numerous hypertrophic chondrocytes. When mature cartilage calcified and new bone trabecula formed, the expression of VEGF mRNA and VEGF decreased in chondrocytes, but still expressed moderately in the osteoblasts and osteocytes. CONCLUSIONS: The finding reveals a complex pattern of gene coexpression of BMP-2 and VEGF during the critical period of entochondrostosis. It's feasible for the clinical application of BMP-2 in orthopedics.

[Effect of silencing HIF-1alpha by RNA interference on expression of vascular endothelial growth factor in osteosarcoma cell line SaOS-2 under hypoxia].

BACKGROUND & OBJECTIVE: Hypoxia-inducible factor-1 alpha (HIF-1alpha) is a key regulator for hypoxia tolerance and angiogenesis of tumor. This study was to investigate the expression of HIF-1alpha and vascular endothelial growth factor (VEGF) in human osteosarcoma cell line SaOS-2 under hypoxia, to explore the effect of HIF-1alpha on hypoxia-activated angiogenesis regulation pathway in osteosarcoma. METHODS: CoCl2 was used as chemical hypoxia-inducing reagent to mimic tumor hypoxic microenvironment. mRNA and protein levels of HIF-1alpha and VEGF at different hypoxic culture phases were detected by semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemistry. Small hairpin RNAs (shRNAs) eukaryotic expression vector targeting HIF-1alpha was constructed, and transfected into SaOS-2 cells. Western blot was used to detect gene silencing effect on HIF-1alpha. RT-PCR and enzyme-linked immunosorbent assay (ELISA) were used to observe the change of VEGF gene expression after HIF-1alpha gene silence. RESULTS: Under hypoxia, mRNA level of HIF-1alpha kept stable, while its protein level increased obviouslyu both mRNA and protein levels of VEGF were up-regulated. The shRNAs plasmid targeting HIF-1alpha gene was constructed successfully, and down-regulated HIF-1alpha gene in SaOS-2 cells efficiently followed by VEGF gene down-regulation. CONCLUSIONS: Hypoxia can increase protein level of HIF-1alpha in osteosarcoma. HIF-1alpha up-regulates the gene expression of VEGF via transcription activation which promotes angiogenesis in osteosarcoma under hypoxic microenvironment.

[Therapeutic effects of RNA interference targeting HIF-1 alpha gene on human osteosarcoma].

OBJECTIVE: To investigate the inhibition effect of the small hairpin RNA (shRNA) targeting HIF-1alpha gene on the growth of osteosarcoma in vitro and in vivo. METHODS: The small hairpin RNA (shRNA) eukaryotic expression vector targeting HIF-1alpha gene, named pSilencer-HIF, was constructed and transfected into cultured human osteosarcoma cell of line SaOS-2 via liposome reagent. Then the osteosarcoma cells were cultured under chemical hypoxia conditions. The inhibition effects on HIF-1alpha gene were determined by semi-quantitative reverse transcription PCR and Western blot analysis. The in vitro cellular growth activities were assayed by MTT colorimetry. The cell apoptosis was studied by electron microscopy, TUNEL assay, and annexin V/PI double staining. Eighteen Balb/C mice were randomly divided into 3 equal groups to be inoculated with SaOS-2/shRNA, SaOS-2/neo (blank vector), or SaOS-2 subcutaneously respectively and then the appearance and size of tumors were observed. Four weeks later the mice were killed and the volumes of tumor were calculated so as to evaluate the therapeutic effects of shRNA. RESULTS: The successful construction of pSilencer-HIF plasmid was identified with sequencing. After the shRNA expression vector was transfected into the SaOS-2 cells, the expression of HIF-1alpha gene was inhibited significantly (by 90%). The cellular growth activities in the SaOS-2 cells transfected with pSilencer-HIF plasmid decreased obviously in hypoxia culture. After 72 hours of exposure to hypoxia, electron microscopy and TUNEL assay showed classic apoptosis characters in the SaOS-2 cells transfected with pSilencer-HIF plasmid with an apoptosis rate of 18.71% +/- 0.98%, significantly higher than those in the negative control group transfected with pSilencer-neo and in the nontransfected group (both P < 0.01). The growth speed and formation rate of xenograft tumor in pSilencer-HIF transfected mice slowed down significantly. A lot of necrotic tissues could be observed in the pSilencer-HIF transfected group by HE staining, however, there was no similar inhibitive effect in the control groups. CONCLUSION: shRNA targeting HIF-1alpha gene blocks the hypoxia transduction pathway efficiently and inhibits the growth of osteosarcoma cells.