Objective To determine the feasibility of using Tet-on regulatory system to transfect primary rabbit bone marrow stem cells, and to control the activities of the target gene. Methods The Tet-on regulatory system were used to transfect bone marrow stem cells by lipofectamine with the increasing concentration of doxycycline (Dox), the activity of the fluorescence caused by the expression of the target gene (the luciferase gene) which transfected to bone marrow stem cells with Tet-on regulatory system were analyzed. Results Plasmids DNA after being digested by enzymes and agarose electrophoreses were analyzed. The DNA segments were cored with the plasmid map. The two DNA segments with the size of 6.02 kb and 1.38 kb could be detected after the plasmid of pTet-on, 5.2 kb DNA segment could be seen after the plasmid of pTRE-luciferase was digested by Hind Ⅲ. With the increasing concentration of Dox, the activity of the fluorescence caused by the expression of the target gene increased gradually. Although the uninfected bone marrow stem cells were induced, the relative-luciferase-unit(RLU) value was almost the same as that of the blanket control. It showed that uninfected bone marrow stem cells were not induced by Dox. The RLU value in infected bone marrow stem cells before induction was slightly high, but similar with the blanket control. This experiment shed light on that infected bone marrow stem cells showed good response to the induction by Dox. Conclusion With the help of Lipofectamine, the cultured bone marrow stem cells can be transfected by Tet-on regulatory system successfully. The expression of the target gene transfected to bone marrow stem cells with Tet-on regulatory system could be regulated by Dox.

Objective To construct vectors based on adeno-associated virus type 2 (rAAV2) carrying bone morphogenetic protein-7 (BMP-7) and observe their expression in adipose-derived adult stem cells, which can be served as a new gene therapy method and cell source for bone tissue engineering. Methods The coding sequence (1. 3 kb) of BMP-7 was amplified by PCR from the pcDNAl. 1 ( + ) plasmid containing the human BMP-7 cDNA. After purified, the gene fragments were cloned into a plasmid pUC18 and termed plasmid pUC18-hBMP7. The recombinant pUC18-hBMP7 was digested and further ligated to the pSNAV by T4DNA ligase and termed plasmid pSNAV-hBMP7. BHK-21 cells were transfected with the purified pSNAV-BMP7 plasmid according to a standard calcium phosphate precipitation method. The cells were isolated and the integrity of hBMP7 gene was determined by polymerase chain reaction (PCR) using the above PCR primers. To package the virus, stably transfected BHK-21 cells were subsequently infected with recombinant herpes simplex virus type 1 (rHSV-1). The collected cells were processed by chloroform treatment, PEG8000/NaCl precipitation and chloroform extraction for purification. The tiler was determined using quantitative DNA dot blots and the purity was examined by sodium dodecyl sulphatepolyacrylamide gel electrophoresis. Following infection with rAAV2-BMP7 at multiplicities of infection of 1?105 vector genomes per cell and subsequent culture, adipose-derived adult stem cells were assessed qualitatively for BMP7 production. Results The recombinant plasmid pSNAV-hBMP7 was identified by PCR and digested with restriction enzyme. Transfection showed an efficiency of 90 % in ADAS cells. BMP-7 expression in ADAS cells was identified by Western blot. Conclusions The hBMP-7 recombinant adeno-associated virus vectors can be successfully constructed in vitro and rAAV2-hBMP7 can infect ADAS cells.

BACKGROUND: At present, bone morphogenetic protein (BMP) is the only cytokine characterized as the ability of osteoblast, and it can promote marrow mesenchymal stem cells (MSCs) differentiating into chondroblast and osteoblast.OBJECTIVE: To investigate the differentiation into osteoblast from MSCs of rabbits transfected by human bone morphogenetic protein-7 (hBMP-7) gene with genetic engineering technique. DESIGN: Single sample observation. SETTING: Department of Orthopaedics, the First Hospital Affiliated to Sun Yat-sen University. MATERIALS: pcDNA1.1/AMP-hBMP7 plasmid.METHODS: The experiment was completed at the Surgery Laboratory of the First Hospital Affiliated to Sun Yat-sen University from May 2004 to March 2005. MSCs of rabbits were cultured with whole bone marrow technique, transfected with pcDNA1.1/AMP-hBMP7 and pcDNA1.1/AMP plasmids respectively in vitro, and left the blank controls. Transcription and expression of transfected genes were detected so as to observe form and growth of cells. Component of calcium content in cytoplasm was measured and osteogenic phenotype was identified with alkaline phosphatase (ALP) staining, calcium salts staining and transmission electron microscopy (TEM).MAIN OUTCOME MEASURES: ① Form and growth of MSCs of rabbits; ② evaluation of expressive product; ③ results of ALP staining (Ca-Co technique); ④ results of chinalizarin staining; ⑤ results of TEM; ⑥ results of scanning electron m icroscopy (SEM); ⑦ assay of osteocalcin.RESULTS: hBMP-7 gene was contained in MSCs after transfection and expressed the relevant mRNA. Cellular form changed a little after expression of amid genes, but growth curve did not changed as compared with that in non-transfected group. Expression of osteocalcin was increased as compared with that in non-transfected group, and ALP staining and calcium salts staining of transfected cells were positive.CONCLUSION: Transfection of hBMP-7 gene can promote MSCs of rabbits differentiating into osteoblast.

BACKGROUND:Periprosthetic infection after artificial joint replacement is a disastrous complication for a patient. Currently, during treating for the periprosthetic infection, most of patients need two-stage revision, which bring about enormous physical and psychological pain for patients, and a heavy burden for the families and society. To make matters worse, the effect is not very perfect, and some of these cases require multi-stage revision, even amputation. OBJECTIVE:To investigate the therapeutic effect of vacuum sealing drainage combined with iodophor douche for the infection after artificial joint replacement. METHODS:Nine cases (six knees and three hips) of infection after artificial joint replacement were col ected, with an average age of 63.4 years. Infection occurred at 7 days-14 months, and a median time of 1 month after replacement. Al patients suffered from purulent or purulent blood secretion. Fistula formed in two cases and incision and drainage sites were not healed in one case. According to the bacterial culture results, above symptoms were accorded with clinical diagnosis of prosthesis infection. The prosthesis was remained for debridement. Vacuum sealing drainage was performed. Iodophor douche (30-50 mL) was conducted every day. The drainage tube at proximal end was occluded for 30 minutes, and then vacuum sealing drainage was performed. Al patients were regularly fol owed up to assess the therapeutic effects of vacuum sealing drainage combined with prosthesis infection after replacement. RESULTS AND CONCLUSION:(1) Except that one case was stil in treatment, one case was dead, and one case of tumor prosthesis was failure and final y amputated, the remaining six patients were healed. (2) The time of vacuum sealing drainage and iodophor douche was 10 to 84 days, with the median time of 57 days. No adverse reactions or complications occurred. Al healed patients were fol owed up for 12-60 months, without recurrence. (3) These results indicated that vacuum sealing drainage combined with iodophor douche retained the prosthesis to the greatest degree, is simple, safe, and effective for the infection after artificial joint replacement, needs a low cost, and is a kind of therapy for prosthesis infection after artificial joint replacement.

Objective To investigate the effect of selectively upward placement of acetabular implants on limb length and post?operative function of developmental dysplasia of the hip patients with shortened legs during total hip arthroplasty (THA). Methods Twenty?six cases of developmental dysplasia of the hip received THA between January 2008 and December 2013, in?cluding 12 cases of Crowe typeⅠ, 8 of Crowe typeⅡ, 6 of Crowe typeⅢ. There were 5 males and 21 females with an average age of 62.7 years (range, 36-80 years). The left hip was involved in 9 cases and the right hip in 17 cases. The preoperative mean Har?ris score was 42.30±12.84, and the preoperative mean WOMAC score was 59.08±13.84 at the last follow?up. The anteroposterior X?ray films and CT scan of the pelvis, anteroposterior and lateral X?ray films of the femur, and TraumaCad analysis were conducted routinely preoperation. More than 70%of the bone?implant interface was covered by appropriate upward distance of acetabular im?plant. Results The follow?up time ranged from 6 to 73 months (mean, 36 months). The Harris score improved to 91.18±7.09, and WOMAC score reduced to 9.85±3.75. According to postoperative measurement, affected limb had been lengthened by 0-5 mm in 8 cases, 6-10 mm in 5 cases, 11-15 mm in 5 cases,>15 mm in 7 cases, and shortening increased 1 mm in 1 case, but the average lengthening was 9.23±7.54 mm. The upward distance of acetabular implant was 0-5 mm in 10 cases, 6-10 mm in 7 cases and>10 mm in 9 cases. The average lengthening was 6.60±6.72 mm in patients having 0-5 mm upward distance, 11.90±5.64 mm in patients having 6-10 mm upward distance and 10.11 ± 9.35 mm in patients having>15 mm upward distance, showing no significant differ?ence. The leg length discrepancy was-3.70±6.43 mm in patients having 0-5 mm upward distance, 1.71±6.24 mm in patients having 6-10 mm upward distance and 0.56 ± 7.70 mm in patients having>15 mm upward distance, showing no significant difference. Con?clusion The limb length could be improved by selectively upward placement of acetabular implants in developmental dysplasia of the hip patients with anatomically abnormal acetabulum during THA, with reasonable preoperative design and corrective operation.

BACKGROUND:The effect of advanced glycation end products (AGEs) on bone resorption is controversial. Our previous study has shown that bone resorption is significantly inhibited when AGEs present with pre-osteoclast cells RAW 264.7, while the effect of AGEs on osteoclastic acidification remains unknown. OBJECTIVE:To investigate the effect of AGEs on osteoclastic acidification and the underlying mechanism. METHODS:RAW 264.7 cells were induced by RANKL (15μg/L;normal group) to generate osteoclasts, and AGEs (50-400 mg/L;experimental group) or bovine serum albumin (100 mg/L;control group) were added at the beginning of the induction. The effect of AGEs on bone resorption was evaIuated by anaIyzing the area of bone resorption on the Osteo Assay Surface plates, and the effect of AGEs on osteoclastic acidification was evaluated by acridine orange staining. Furthermore, the expression levels of V-ATPase a3 and CIC-7 were detected to investigate the underlying mechanism. RESULTS AND CONCLUSION:The bone resorption area in the AGEs group was significantly decreased compared with the normal group (P<0.05). Acridine orange staining reveaIed that the red fluorescence (620 nm) intensity in the AGEs group was significantly decreased compared with the normal group (P<0.05), and this inhibitory effect became obvious with the increase of AGEs concentration. Immunocytochemistry, western blot assay and PCR findings showed that the expression levels of V-ATPase a3 and CIC-7 in the AGEs group were decreased significantly compared with the normal group (P<0.05). To conclude, AGEs exert inhibitory effect on osteoclastic acidification, probably by inhibiting the expression of V-ATPase a3 and CIC-7.

BACKGROUND:The effects of advanced glycation end products (AGEs) on osteoclast-induced bone resorption is controversial and the underlying mechanisms remain unclear. Most of the studies indicate that AGEs can enhance bone resorption, while some othersshowthe opposite effects.
OBJECTIVE:To investigate the effects of AGEs on osteoclast-induced inorganicmatrixdissolution and organic componentdegradation and the underlying mechanisms.
METHODS:RAW 264.7 cels were induced to generate osteoclasts,and AGEs (50-400 μg/mL) or control-bovine serum albumin (100 μg/mL) was added since the beginning of the induction. The effect of AGEs on bone resorption was evaluated by analyzing the area of resorption pits on the Osteo Assay Surface plates and the expression of cathepsin K. Furthermore, the number of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cels, nuclei per osteoclasts and the expression of integrinανβ3were detected.
RESULTS AND CONCLUSION:The area of resorption pits and expression of cathepsin K in AGEs groups were significantly decreased compared withthecontrol group, and this inhibiting effect became more obvious with the increase of AGEs concentration. TRAP staining also showed that number of TRAP-positivemultinucleated celsand nuclei per osteoclast were significantly reduced in an AGE dose-dependent manner. Quantitative PCR revealed that the expression of integrin ανβ3decreased significantly with the extension of AGEs incubation time. These data indicate that AGEs can exert inhibitory effects on organic and inorganicmatrixdegradation induced by osteoclasts. The underlying mechanism may be involved in the inhibitory effects of AGEs on directed differentiation and cel fusion of osteoclast precursor cels, and migration and adhension of osteoclasts.

Objective To introduce a self-developed computer-assisted design/rapid prototyping and guidance system used for precise placement of the acetabular component in total hip arthroplasty.Methods We collected the preoperative pelvic CT scanning data of 10 hips with aeetabular dysplasia that had undergone primary total hip arthroplasty from January 2016 to January 2017.The total time for import of radiographic images,model reconstruction,model segmentation,acetabular component position design and STL model export was calculated and compared between our self-designed software and Mimics vl 7.0.Three kinds of STL model from each case were imported into our self-developed 3D printing device,Stratasys Objet30 and Stratasys Demension SST1200es respectively for rapid prototyping.The printing efficiency and accuracy were compared among the 3 printers.The accuracy of placing acetabular component with guidance system was evaluated.Results The average time forpreoperative planningwas7.7±1.3 minbyourself-designedsoftware and 52.5 ± 15.9 min by Mimics v17.0,showing a significant difference (P ＜ 0.001).In morphological point-based comparison for each case,the 3D models exported by the 2 different kinds of software showed an average difference of 0.072 1 ± 0.069 1 mm.The average durations for rapid prototyping by the 3 different printers were 5.3 ± 0.6 h,10.8 ± 0.5 h,and 9.3 ± 0.6 h,respectively,showing significant differences (P ＜ 0.001).The guidance system resulted in precise placement.The locations of the acetabular component achieved by guide-assisted placement were not significantly different from the target ones (P ＞ 0.05).Conelusion Our self-developed preoperative planning software,rapid prototyping device and guidance apparatus for acetabular component placement may lead to good accuracy and high efficiency.

BACKGROUND:Previous studies have indicated that resistin stimulates a large set of chemokines in chondrocytes that are known to be important in inflammatory joint lesions.
OBJECTIVE:To further investigate the mechanism of co-regulation roles of transcription and post-transcription in the up-regulation of two chemokine genes CCL3 and CCL4 in chondrocytes in response to resistin.
METHODS:Human chondrocytes, T/C-28a2 and ATDC5 cels were cultured. The function of resistin on the chemokine genes, and the expression of C/EBPβ, nuclear factor-κB isoforms and chondrogenic specific miRNAs were tested by qPCR. The co-regulation of C/EBPβ and nuclear factor-κB was investigated by nuclear factor-κB inhibitor (IKK-NBD) and C/EBPβ inhibitor (SB303580) treatments, and subcelular localization was detected with or without resistin stimulation.
RESULTS AND CONCLUSION:Resistin could increase the expression of chemokine genes independently. Chondrocytes reacted in a non-restrictedly cel-specific manner to resistin; C/EBPβ inhibitor, nuclear factor-κB and some chondrogenic specific miRNAs in a combinatorial manner regulated chemokine gene expression. The activity of C/EBPβ was augmented by a transient increase in activity of nuclear factor-κB, and both transcription factors acted independently on the chemokine genes, CCL3 and CCL4.