BACKGROUND:Staphylococcal infections and its biofilm formation can occur when orthopedic implants or wound is healing, and are regulated by bacterial population sensing mechanism. RNAIII inhibiting peptide intervenes the quorum-sensing system of staphylococcal and blocks the signal transduction among staphylococcal cells, and inhibits staphylococcal biofilm formation, and then prevents staphylococcal infections.
OBJECTIVE:To investigate the influence of RNAIII inhibiting peptide on the adhesion of staphylococcus epidermis to the Hela cells.
METHODS:The Hela cells were cultured in vitro. There were four groups in this study. In the blank group, saline with dimethyl sulfoxide was added in each wel . In the RNAIII inhibiting peptide group, dimethyl sulfoxide solution containing RNAIII inhibiting peptide was added. In the levofloxacin group, levofloxacin was added. In the combination group, the dose was in accordance with above methods. Using intergroup control method, the adhesion of staphylococcus epidermis to the Hela cells was compared under the effects of saline, RNAIII inhibiting peptide and levofloxacin and their combination.
RESULTS AND CONCLUSION:In the blank group, abundant bacterial adhered to Hela cells. The number of adhered bacteria was significantly lower in each medicine group than in the blank group (P<0.001). The spot count was significantly lower in the levofloxacin group than in the RNAIII inhibiting peptide group (P<0.05). In the combination group, the number of bacteria adhered to Hela cells was decreased (P<0.01). Results verified that RNAIII inhibiting peptide effectively suppressed the adhesion of staphylococcus epidermis to the host cells, and showed synergistic effects on antibiotics.

OBJECTIVE: To evaluate the histocompatibility of poly (lactic-co-glycolic acid)/RNA Ⅲ inhibiting peptide (PLGNRIP) sustained release microspheres.METHODS: The crude peptide comprising N to C-terminals was synthesized using Fmoc method. The crude synthetic RNAⅢ peptide was purified by reverse phase high performance liquid chromatography, followed by component harvesting according to ultraviolet absorption peak, and freeze-drying. PLGNRIP sustained release microspheres with a diameter of 50-70 pm were prepared using liquid-phase multiple emulsion method. The histocompatibility of PLGNRIP sustained release microscopes were preliminarily evaluated through the use of acute general toxicity test, MTT cytotoxicity test, intramuscular implantation test, sensitivity test, and pyrogen test.RESULTS: Acute general toxicity test results showed that all included animals survived and presented with no toxicosis reaction and obviously changed body mass. MTT cytotoxicity test results revealed that the average relative growth rate of cells from two eluents was over 85%, with cytotoxicity grade 1, which indicated no cytotoxicity. Intramuscular implantation tests showed that at 4 weeks after implantation of RiP powder or PLGNRIP microscopes, no obviously congested, degenerated, or necrotic tissue was observed. All RIP powder and a part PLGNRIP microscopes were degraded. Fibroblasts accounted for a large proportion in all cells. NO inflammatory cell infiltration, involving neutrophits and multinucleated giant celts, was observed. Sensitivity test rasults displayed that the average primary irritation index was 0.38, 0.33, arid 0.31 in the eluent stock solution, 2% dinitoflruorobenzene, and physiological saline-administerd groups, respectively. Pyrogen test results showed that fervescence of each rabbit in the experiment was under 0.5 ℃ and the sum of fervescence was under 1.3 ℃ .This is in coincidence with evaluation criteria of pyrogen test.CONCLUSION: PLGNRIP sustained release microspheras exhibit good histocompatibility.

OBJECTIVE: To evaluate blood compatibility of polyaiticglycolic acid/RNA Ⅲ inhibiting peptide (PLGA/RIP) delayed release microspheres.METHODS: ① Preparation of PLGA/RIP microspheres: The solid-phase synthesis (Fmoc) method was used to synthesize RIP crude sample from C end to N end; the synthesized crude peptide was purified by the reverse phase high performance liquid chromatography. According to UV absorption peak, the components were collected and freeze-dried, to obtain RIP purifications. Then liquid-phase multiple emulsion method was used to prepare PLGA/RIP microspheres at the diameter of 50-70 μm. ② Preparation of eluent: The PLGA/RIP microsphere powders were eluted with sterile physiological saline at 37 ℃, to prepare 1 g/L eluent; then 0.5 g/L eluent was obtained adding equal volume of sterile physiological saline. The hemolysis test, blood clotting test, and platelet aggregation test were conducted to measure prothrombin time and activated partial thromboplastin time, to observe the influence on rabbit leucocytes, erythrocytes and thrombocytes, and to preliminarily evaluate the blood compatibility of PLGA/RIP microspheres. RESULTS: ①The haemolysis rates of eluent stock solution and 0.5 g/L eluents were 3.24% and 2.67% respectively, which were in coincidence with the criteria of medical biomaterials, less than 5%. ② The eluent stock solution and 0.5 g/L eluents of PLGA/RIP microspheres had no significant effect on rabbit clotting time, prothrombin time and activated partial thromboplastin time, the number of rabbit leucocytes, erythrocytes and thrombocytes, as well as platelet aggregation.CONCLUSION: PLGA/RIP delayed release microspheres have a good blood compatibility.

AIM: RNAⅢ inhibiting peptide (RIP) has been previously proved to possess good histocompatibility and safety for preventing and curing staphylococcal infection, and this study evaluated histocompatibility of polyaiticglycolic acid/RIP (PLGA/RIP) sustained release microsphere. METHODS: The experiment was performed in the Orthopedic Institute, Pharmacologic Research Institute and Animal Experimental Center of General Hospital of Chinese PLA from October 2005 to October 2007.①Preparation of PLGA/RIP: The solid-phase synthesis (Fmoc) method was used to synthesize RIP from C end to N end, then the synthesized peptide was purified by the reverse phase high performance liquid chromatography, and composition was collected by means of ultraviolet absorption peak. The purified RIP was obtained after freezing and drying. Liquid-phase multiple emulsion method was used to synthesize PLGA/RIP microsphere of 50-70 ?m diameter.②Acute general toxicity test was studied in PLGA/RIP. Effect of PLGA/RIP on the cell proliferation was detected with cytotoxicity test by MTT method. Intramuscular implanting test was used to observe the irritation reaction of muscles by implantation materials. Sensitivity test was used to observe the sensitization of PLGA/RIP. Changes of animal's body temperature were determined with pyrogen test. RESULTS: ①Acute general toxicity test: Neither toxicosis reaction nor animal death was found after animals were injected with 100% and 50% eluents of PLGA/RIP peritoneally. Animal's body weight was not changed significantly.②Cytotoxiciity test by MTT method: The average proliferation rate of cell in two kinds of eluents exceeded 85% and cytotoxicity was graded in 1 rank, indicating no cytotoxicity.③Intramuscular implanting test: At 4 weeks after RIP and PLGA/RIP were implanted into the animals, there was not obvious synathresis, denaturation or necrosis in tissues. No inflammatory cell infiltration occurred around the materials. There had been the fibrous capsules around the materials.④Sensitivity test: Average primary irritation index of three groups were 0.38, 0.33 and 0.31 respectively. There was no significant difference among three groups.⑤Pyrogen test: Fervescence of each animal in the experiment was under 0.5 ℃, confirming that the materials had no pyrogenic characteristics. This was in coincidence with evaluation criterion of pyrogen test. CONCLUSION: PLGA/RIP has good histocompatibility and safety, without general toxic reaction, cytotoxicity, immunological rejection, hypersensitive response or pyrogenic characteristics.

OBJECTIVE:To evaluate the histocompatibility of poly(lactic-co-glycolic acid)/RNA Ⅲ inhibiting peptide(PLGA/RIP)sustained release microspheres.METHODS:The crude peptide comprising N to C-terminals was synthesized using Fmoc method.The crude synthetic RNAIII peptide was purified by reverse phase high performance liquid chromatography,followed by component harvesting according to ultraviolet absorption peak,and freeze-drying.PLGA/RIP sustained release microspheres with a diameter of 50-70?m were prepared using liquid-phase multiple emulsion method.The histocompatibility of PLGA/RIP sustained release microscopes were preliminarily evaluated through the use of acute general toxicity test,MTT cytotoxicity test,intramuscular implantation test,sensitivity test,and pyrogen test.RESULTS:Acute general toxicity test results showed that all included animals survived and presented with no toxicosis reaction and obviously changed body mass.MTT cytotoxicity test results revealed that the average relative growth rate of cells from two eluents was over 85%,with cytotoxicity grade 1,which indicates no cytotoxicity.Intramuscular implantation tests showed that at 4 weeks after implantation of RIP powder or PLGA/RIP microscopes,no obviously congested,degenerated,or necrotic tissue was observed.All RIP powder and a part PLGA/RIP microscopes were degraded.Fibroblasts accounted for a large proportion in all cells.No inflammatory cell infiltration,involving neutrophils and multinucleated giant cells,was observed.Sensitivity test results displayed that the average primary irritation index was 0.38,0.33,and 0.31 in the eluent stock solution,2% dinitoflruorobenzene,and physiological saline-administerd groups,respectively.Pyrogen test results showed that fervescence of each rabbit in the experiment was under 0.5℃ and the sum of fervescence was under 1.3℃.This is in coincidence with evaluation criteria of pyrogen test.CONCLUSION:PLGA/RIP sustained release microspheres exhibit good histocompatibility.

BACKGROUND: A new material of porous carbonated hydroxyapatite cement (PCHC) is discovered using foaming technique.The new material characterizes original solidification and forms porous structure.OBJECTIVE: To investigate the biomechanical effect of PCHC on repairing cancellous bone defect.METHODS: Among 30 New Land rabbits, 25 ones were considered as surgery group, whose bilateral condyles of femur was used to establish bone defect model (5.5 mm diameter and 12 mm depth). PCHC was implanted into the left side, which was considered as the experimental group, and carbonated hydroxyapatite cement (CHC) was implanted into the right side, which was considered as the control group. Another 5 rabbits were used as normal mechanical control group. Both PCHC and CHC were dip in simulated body fluid (SBF) to test mechanical intension. PCHC and CHC were then implanted into muscles of back in the surgery group. Rabbits Were-sacrificed after 2, 4, 8, 12, and 16 weeks postoperatively. Mechanical analysis was tested following intra-bone and intramuscular implantation, and compressive strength was then tested following dipping into SBF.RESULTS AND CONCLUSION: PCHC: Intra-bone mechanical strength was lower at 2 weeks, the lowest at 4 weeks, but then closed to intension of normal cancellated bone at 8 weeks, higher than normal cancellated bone at 12 weeks, and recovered to the level of normal cancellated bone at 16 weeks. CHC: Intra-bone strength was higher than that of PCHC at 2 weeks, decreased at 4 weeks, gradually increased at 8, 12, and 16 weeks, but still lower than intension of normal cancallated bone. Compressive strength of both PCHC and CHC was not changed following dipping in SBF; however, compressive strength was changed remarkably following intramuscular implantation. The results demonstrated that PCHC characterized by immobilization in situ and mechanical supporting. Thus it could be used for one kind of bone substitute material to repair the bone defect.

BACKGROUND: Carbonated hydroxyapatite cement is a new type material for skeletal repair and hydroxyapatites have been applied in the clinical treatment of skeletal defect.OBJECTIVE: To observe the effective characteristics of carbonated hydroxyapatite cement on repair of skeletal defect by animal experiment.DESIGN: Paired design, self-controlled and verified experiment was applied in the research.SETTING: Orthopedic Institute and Animal Experimental Center of Chinese PLA.MATERIALS: The experiment was performed in Orthopedic Institute and Animal Experimental Center of Chinese PLA from May 2002 to January 2003, in which, 10 healthy adult male mongrel dogs were applied, body mass weighted varied from 20 to 22 kg.METHODS: Animal model of skeletal defect was prepared on proximal ends of humeri of 10 mongrel dogs thydroxyapatitet were randomized into experimental side and control side. Ceramics repair of skeletal defect was done by carbonated hydroxyapatite cement and high-temperature sintered hydroxyapatite respectively. The animals were sacrificed on the 5th day, 4th, 8th, 12th and 16th weeks successively after operation. The repair effects were performed with X-ray and histological observation.staining.Results of stereomicroscopic and X-ray observations on bilateral skeletal defect: Osseointegration with carbonated hydroxyapatite cement was tight on the experimental side and the interface became unclear gradually with time lasting. The interface between hydroxyapatite and bone was still clear on the and eosin staining and thydroxyapatitet of ground bone with Gimsa staining:On the 8th week on the experimental side, the new bone grew into carbonated hydroxyapatite cement, on the 16th week, the two parts were intermixed and integrated and the bone island was formed around newly generated vessels in carbonated hydroxyapatite cement. On the control side, hydroxyapatite still maintained integrated and the bone interface was clear between hydroxyapatite and bone. On the 16th week, the aggradation of newly generated bone presented on hydroxyapatite surface.CONCLUSION: Carbonated hydroxyapatite cement possesses solidification property in situ, biocompatibility and osseous conductive activity. It is the satisfactory new type material for repair of skeletal defect.

OBJECTIVE To investigate the in vitro effect of 4 kinds of implant materials on the biofilm formation of Staphylococcus epidermidis.METHODS S.epidermidis was cultured,purified and identified.Susceptibility test was done for S.epidermidis and the ability to produce biofilm was proven.The test samples were made into wafer shape for titanium alloy,Co-Cr-Mo alloy,ultrahigh molecular weight polyethylene(UHMWPE)and home-made polymethyl methacrylate(PMMA).S.epidermidis was cultured with 4 kinds of test samples for 5 days respectively.Bacteria adhering on surfaces of 4 kinds of test samples were dissolved with trypsin,and then diluted into bacterial suspensions.Each bacterial suspension was inoculated quantitatively and CFU were counted.Biofilm on surfaces of 4 kinds of test samples prepared by vacuum drying method was observed with SEM.RESULTS The strain proven to be S.epidermidis,was resistant to semisynthetic penicillins,and could produce biofilm.CFU count showed that CFU were the most on the UHMWPE surface and the number of CFU were(24.96?1.459)?105.CFU were(17.44?1.883)?105 on the PMMP surface.(0.424?0.065)?105 CFU were discovered on the surfaces of titanium alloy and(0.382?0.075)?105 CFU on Co-Cr-Mo alloy.When each group compared with UHMWPE and domestic PMMA respectively,all P value was

OBJECTIVE To determine the feasibility of clindamycin-loaded calcium phosphate cement(CLCPC) as a local antibiotic delivery system.METHODS The initial setting time(tI) and the final setting time(tF) were measured for 0%,2% and 5% CLCPC according to ASTM C266-89 method.Clindamycin concentrations eluting from the samples of 2% and 5% CLCPC in PBS were analyzed by HPLC at different times.The bacteriostasis tests were done by plate diffusion method for 2% and 5% CLCPC and 2% Palacos R-40 bone cement(PMMP) samples,and the diameters of the bacteriostasis ring and bacteriostasis duration were observed.The setting product and crystal size of 0%,2% and 5% CLCPC were analyzed and observed by X-ray diffraction(XRD) and scanning electron microscopy(SEM).RESULTS The setting time could be shortened by adding clindamycin(tI,tF) of 2% and 5% CLCPC.Clindamycin was with burst-release from CLCPC within the intial 6-hour period and the release rate slowed down on 4th day.Clindamycin could still release until to the 42th day.The ring of 2% Palacos R-40 bone cement(PMMP) bacteriostasis was smaller than that of 2% CLCPC,and the ring of 2% CLCPC bacteriostasis was smaller than that of 5%CLCPC.The bacteriostasis still existed to the 42th day of test for 2% and 5%CLCPC and 2% Clindamycin-loaded Palacos R-40 bone cement(CLPMMP).From the 30th day,many bacterial colonies were seen in the culture media laying 2%CLPMMP sample.On the contrary,bacterial colony was not found in the media putting 2% and 5%CLCPC.XRD and SEM showed that clindamycin did′t have an influence on setting product,crystal size and structure of CPC.CONCLUSIONS Clindamycin-loaded calcium phosphate cement can be used as a local antibiotic delivery system.

Objective To investigate the influences of survivin down-regulation on cell G2/M phase arrest,apeptosis and sensitivity to carbon ion irradiation. Methods Small interfering RNA (siRNA) targeting survivin mRNA was designed, in vitro chemo-synthesized and transfected into SMMC-7721 cells. Survivin mRNA expression in SMMC-7721 cells was measured by real-time PCR, and the apeptotic rates by Annexin-FTTC at 24 and 48 h after transfection. Cell G2/M phase arrest after transfection was assessed with flow eytometry as well. Cellular sensitivity to high-LET carbon ions was determined by means of colony-forming assay. Results The expressions of survivin at mRNA level were down-regulated to be 59% and 39% in relation to the non-treated cells at 24 and 48 h after siRNA transfeetion, respectively. G2/M phase arrest in SMMC-7721 cells at 24 h after transfection was observed while much more obvious at 48 h. The apeptotic rate of SMMC-7721 cells was 21.41 % at48 h after survivin siRNA transfection, which was significantly higher than that of the cells transfected with negative siRNA. Moreover, a decreased clonogenic survival in siRNA treated group was shown. Conclusion Down-regulation of survivin gene expression in SMMC-7721 cells by siRNA could effectively induce cell apeptosis and G2/M phase arrest, and enhance the cellular radiosensitivity to high-LET heavy ions.