Objective To choose the best procedure on preparation of acellularbovine pericardium (ABP) guided bone regeneration (GBR) material. Methods The BP was decellularized with 0.25% Trypsin+0.5% Triton X-100. The acellular bovine pericardiums (ABPs) were treated with phosphatebuffered saline(PBS) (group A), 95% glycerol (group B), EDAC (group C), and EDAC and 95% glycerol (group D) respectively. The treated ABPs were implanted subcutaneously in the back of SD rats respectively at random and no material was implanted as control. Seven rats were sacrificed at 2 weeks, twelve at 4 weeks, twelve at 8 weeks, seven at 16 weeks. Local reaction was studied grossly. The amount of antigen presenting cell (APC) and the percentage of ABP degeneration were reckoned by images analysis system. Results The ABPs were replaced by fibroblasts completely in group A at 8 weeks， in group C at 16 weeks, but only less than 50% till 16 weeks in groups B and D. In all groups, the depth of surrounding fibres attenuated timedependingly. The APC amount of the groups B and D was higher than that of the control group, and the ABP of the groups B and D degraded partly at 16 weeks. Conclusion The ABP treated with EDAC can be replaced by the surrounding tissues and has good biocompatibility.
Objective To evaluate the biocompatibility and safety of a novel orthopedics materials-graded zirconia(ZrO2)hydroxyapatite(HA) composite biomaterials. Methods First, ultrafine powers of ZrO2 and HA powder were prepared by chemical precipitation method, then graded ZrO2-HA composite was synthesized by dry-laying and sintering method. After the physiological saline and culture medium extracts of the composite were prepared, four experiments were conducted as follows:① The mouse acute toxic test consists of 2 groups(n=10). The extracts were intravenously injected to mice in the first group, and physiological saline to mice in the second group. The dose was 50 g/kg. Their toxicity manifestation, morality and the change of weight were recorded.② The standard curve of proliferation and metabolism of L929 cells was established. ③ The cytotoxinic test consists of 3 groups: materials group (extracts of the materials), positive control group (culture fluid with 0.64% phenol), and negative control group (RPMI-1640 culture fluid). Each of three was cultured with cell suspension, and then the morphology of the cells was observed, the relative proliferation rate (RGR) was calculated, and the toxicity was classified. ④ In vitrohemolytic test was divided into 3 groups: extracts, sterile distilled water (positive control) and 0.9% physiological saline. In each of three, 0.2 ml anticoagulant diluted fresh rabbit blood was added. The percentage of hemolysis was tested. ⑤ The muscle and implantation test were divided into 4 groups(n=3). The composite biomaterials were implanted into pygal muscleson either side and lateral condyles of femurs. After surgery, the rats of four groups were sacrificed at 12 and 24 weeks respectively.Tissue slice and scanning electronic microscopy were performed. Results General acute toxic test: no mouse died within 3 weeks; no toxicity symptom or adverse effects were shown within 3 days. The weight of materials group increased by 3.57±0.49 g, and the control group by 3.62±0.61 g, showing no statistically significant difference（Ρgt;0.05）.The standard curve of L929 cell perliferation and metabolism showed that their existed a positive correlation between the number of L929 cells and the perliferation. ③ Cytotoxinic test: cytosomes in the positive control group diminished and appeared round, there were pyknotic nucleus, the attached cells agglomerated; the toxicity was level Ⅳ. The morphology of cells in materials groupand negative control group was normal, and the number of them increased; the toxicity was level Ⅰand level 0, respectively. The MTT color experiments showed that positive control group was significantly lower than materials group and negative control group, showing statistically significant difference (Plt;0.01); there was no statistically significant difference between materials group and negative group.④ Hemolytic test: in vitrohemolytic rate of negative control group was0, of positive control group was 100%, and of materials group was 1.66%, which accords with the standard that hemolytic rate should be lower than 5% specified in ISO. ⑤ Implant test:No apparent rejection reaction took place after the composite was implanted; the composite bonded with the bones of the receptors firmly, which had good bonedinduced effect. Conclusion Graded ZrO2-HA composite bioceramic has good biocompatibility and is suitable for orthopedic biomaterials.
OBJECTIVE To study the biocompatibility of skin reproductive membrane. METHODS According to ISO’s standards, the extractions of the skin reproductive membrane were prepared, and the acute systematic toxicity test, primary skin irritant test, cytotoxicity test, gene expression of type I collagen and fibronectin were detected to evaluate the biocompatibility of skin reproductive membrane. RESULTS All of those tests showed negative results. CONCLUSION The skin reproductive membrane has excellent biocompatibility in the level of the systematic, cellular and molecular biology.
Objective To investigate the biocompatibility of acellular urinary bladder submucosa (AUBS). Methods The acellular collagen matrix of human urinary bladder submucosa was developed using freeze-thawed enzymatic treatment and freeze-drying technique. Human oral keratinocytes were cultured and seeded on AUBS at a density of 2×106/ml in vitro.The proliferation of the cells were observed. Pockets were created in the abdominal muscle wall of 18 SD rats. AUBS in size 1 cm×1 cm was implanted into the pocket. The grafts were observed by light microscope 3, 6, 10, 14, 21 and 28 days after operation. Results AUBSmainly consisted of collagen fibers with a three-dimensional network structure. After the oral keratinocytes were seeded, continous oral epithelium layer was formed on the surface of AUBS after 10 days in vitro. Histological observation of the grafted AUBS showed progressive cell infiltration at 6 days. New capillaries formed at 14 days. The collagen fibers arranged regularly at 28 days after implantation. Conclusion Freeze-dried AUBS may be used as a suitable scaffold for tissue regeneration, which can induce cell proliferation both in vivo and in vitro and has good biocompatibilty.
Objective To evaluate the biocompatibility and in vivo degradation of novel chest wall prosthesis materials and provide some data for their clinical application. MethodsAccording to the standard for the biological evaluation of the medical devices, several tests were performed to evaluate the tissue toxic effects induced by polydioxanone (Group A), chitosan (Group B), and hydroxyapitite/collagen (Group C),which were tested as component materials of the chest wall prosthesis. In the hemolysis test, 0.2 ml of the anticoagulant rabbit blood was added to the component materials and the normal saline (negative control) and to the distilled water(positive control). Five samples were made in each group. Absorbency was measured and the hemolysis rate was determined. In the acute systemic toxicity test, 20 mice were randomly divided into 4 groups (Groups A, B and C, and the normal saline group, n=5). The leaching liquid (50 ml/kg) was injected through the caudal vein, which was observed at 24, 48 and 72 hours. In the pyrogen test, 12 rabbits were randomly divided into 4 groups (Groups A, B, C and the normal saline group, n=3) the leaching liquid(10 ml/kg) was injected through the ear vein,and the body temperature was recorded within 3 hours. In the in vivo degradable test, the component materials (10 mm×10 mm) were implanted in 12 rabbits at 2, 4, 8, 12, 16 and 24 weeks, respectively, after operation. Two rabbitswere sacrificed for the macroscopic and the microscopic examinations. Results The chest wall component materials had no hemolytic reaction, no acute systemic toxicity, and no pyrogen reaction. The results demonstrated that the implanted materials had only a mild inflammatory reaction during the early days of the grafting, which subsided gradually. There was no tissue denaturation, necrosis or pathological hyperplasia when the prosthesis materials were degraded. Conclusion The degradable materials of the chest wall prosthesis have a good biocompatibility and agreat biological safety though their surgical application still requires a further clinical research.
Objective To investigate the biocompatibility of diamond-like carbon(DLC) coated NickelTitanium shape memory alloy with osteoblasts cultured invitro. Methods Rabbit’s osteoblasts were incubated with DLCcoated NickelTitanium shape memory alloy disks and uncoated ones of equal size for 5 days. The control group（without shape memory alloy in culture media） was performed simultaneously. The cultured cells were counted and graphed. The samples from culture media were collected and the concentrations of alkaline phosphatase (ALP) and nickel（Ni2+） were measured from the 1st to 5th day respectively. Results The proliferation of osteoblasts and the concentration of ALP in both DLC-coated group and control gruop was higher than uncoated group. The proliferation of osteoblasts on the 3rd, 4th, and 5th day in both DLC-coatedgroup and control group was significantly higher than that in the uncoated group(P＜0.05). The concentration of ALP in DLC-coated group on the 2nd, 3rd, and 5th day and in the control group on the 3rd, 4th, and 5th day was significantly higher than that in the uncoated group(P＜0.05). The concentration of Ni2+ on the 3rd, 4th, and 5th day was significantly lower than that in the uncoated group(P＜0.05). Conclusion DLC- coated NickelTitanium shape memory alloys appears to have better biocompatibility with osteoblast cultured in vitro compared to uncoated ones.
Objective To evaluate the biocompatibil ity of a new nano TCP/ gelatin / velvet antler polypeptide material. Methods The nano TCP/ gelatin / velvet antler polypeptide material was prepared, and the morphous was observed by scanning electron microscope. L929 and NIH/3T3 cell l ines were cultured conventionally. Acute toxicity test, hemolysis test, cell prol iferation and cytotoxicity test were used to evaluate the biocompatibil ity of the material. Results The compositemicrosphere material was about 10 μm in diamerter and had good spherical geometry, high monodispersity with nanometer size holes on the surface. Toxic symptoms such as hyperspasmia, palsy and death did not appear during the observing stage in acute toxicity test. Maximum hemolysis rate of the material was less than 5% which met the requirement of hemolysis test standard as a medical material. Different concentrations of the materials leaching l iquor could enhance the prol iferation of NIH/3T3 cells, which showed the good biologic activity. Toxicity grade was 0, and the material was no cytotoxic. Conclusion Nano TCP/ gelatin / velvet antler polypeptide material has good biocompatibil ity.
To explore the histological and the hematological change of rabbits after implanting novel injectable artificial nucleus prostheses, and to evaluate the biological safety. Methods In accordance with Biological Evaluation of Medical Devices, materials of polyurethane, sil icone rubber and macromolecular polyethylene for medical use were made into short column 1 cm in length and 0.3 cm in diameter. Forty-eight SPF New Zealand white rabbits weighing 2.5-3.0 kg were used, and cavity 1 cm in depth was made in the area 2 cm away from the spinal midl ine by separating muscle.Then according to different material being implanted, the rabbits were divided into 3 groups (n=16): Group A, polyurethane; group B, sil icone rubber; group C, macromolecular polyethylene for medical use as negative control. General condition of the rabbits was observed after operation. Gross and histology observation were conducted 1, 4, 12 and 26 weeks after operation. Blood routine, biochemical function and electrolyte assays were performed 26 weeks after operation to observe pathological changes of organs. Meanwhile, physicochemical properties of the materials were detected, and the material in the same batch was used as negative control. Results All rabbits survived until the end of experiment, and all wounds healed by first intention. In each group, red swollen muscles were observed 1 week after operation and disappeared 4 weeks after operation, connective tissue around the implanted materials occurred 12 and 26 weeks after operation. At 26 weeks after operation, there were no significant differences among three groups in blood routine, biochemical function and electrolyte assays (P gt; 0.05). Organs had smooth surface without ulceration, ecchymosis, obvious swell ing, hyperemia or bleeding, and nodules. There were no significant differences among three groups in percentage weight of each organ (P gt; 0.05). Histology observation: granulation tissue prol iferation and inflammatory cell infiltration were observed in each group 1 week after operation, fibrous capsule formation around the materials and the disappearance of inflammatory cell infiltration were evident 4 weeks after operation, cyst wall grewover time and achieved stabil ity 12 weeks after operation. The inflammatory response and the fiber cyst cavity of groups A and B met the standard of GB/T 16175 and were in l ine with group C. No specific pathological changes were discovered in the organs 26 weeks after operation. For group A, no significant difference was evident between before and after material implantation in terms of weight average molecular weight, number average molecular weight, tensile strength at break and elongation at break (P gt; 0.05). For group B, no significant difference was evident between before and after material implantation in shore hardness (P gt; 0.05). Conclusion Novel injectable nucleus pulposus prostheses do not damage local tissue and function of organs, but provide good biocompatibil ity and biological safety.
ObjectiveTo explore an optimized protocol of decellularization to fabricate an ideal scaffold derived from porcine skeletal muscle acellular matrix. MethodsSerial-step protocol of homogenating-milling-detergent method was used to fabricate decellularized porcine muscle tissue (DPMT) derived from native porcine skeletal muscle tissue from adult pig waist. Histological method was used to assess the effects of decellularization and degreasing. Sirius red staining was used to analyze collagen components. Scanning electron microscopy, BCA assay, and PicoGreen assay were used to evaluate the ultrastructure, total protein content, and DNA content in DPMT. The adipose derived stem cells (ADSCs), NIH3T3 cells, and human umbilical vein endothelial cells (HUVECs) were cultured in extraction liquor of DPMT in different concentrations for 1, 3, and 5 days, then the relative growth rate was calculated with cell counting kit 8 to assess the toxicity in vitro. Live/dead cell staining was used to evaluate the cytocompatibility by seeding HUVECs on the surface of DPMT and co-cultured in vitro for 3 days. For in vivo test, DPMT was subcutaneously implanted at dorsal site of male specific-pathogen free Sprague Dawley rats and harvested after 3, 7, 14, and 28 days. Gross obersvation was done and transverse diameter of remained DPMT in vivo was determined. HE staining and immunohistochemical staining of CD31 were used to assess inflammatory response and new capillary rings formation. ResultsDecellularization of the porcine skeletal muscle tissue by homogenating-milling-detergent serial steps protocol was effective, time-saving, and simple, which could be finished within only 1 day. The decellularizarion and degreasing effect of DPMT was complete. The main component of DPMT was collagen type I and type IV. The DNA content in DPMT was (15.902±1.392) ng/mg dry weight, the total protein content was 68.94% of DPMT dry weight, which was significantly less than those of fresh skeletal muscle tissue[(140.727±10.422) ng/mg and 93.14%] (P<0.05). The microstructure of DPMT was homogeneous and porous. The result of cytocompatibility revealed that the cytotoxicity of DPMT was 0-1 grade, and HUVECs could stably grow on DPMT. In vivo study revealed DPMT could almost maintain its structural integrity at 14 days and it degraded completely at 28 days after implantation. The inflammatory response peaked at 3 days after implantation, and reduced obviously at 7 days. Difference was significant in the number of inflammatory cells between 2 time points (P<0.05). Neovascularization was observed at 7 days after implantation and the number of new vessels increased at 14 days, showing significant difference between at 7 and 14 days (P<0.05). ConclusionThe homogenating-milling-detergent serial-steps protocol is effective, time-saving, and reproducible. The DPMT reveals to be cell and lipid free, with highly preserved protein component. DPMT has good biocompatibility both in vitro and in vivo and may also have potential in promoting neovascularization.
Objective To study the biocompatibility of tendon mixedextraction of bovine collagen(tMEBC) and to explore the feasibility of using the threedimensional framework as periodontal tissue engineering scaffold. Methods After being prepared, the tMEBC were cultured with the P4P6 of human periodontal ligament fibroblasts (HPDLFs) in vitro. Threedimensional framework was prepared from bovine tendon. The P4-P6 of HPDLFs (with an initial density of 5×106 cells／ml) were cultured in vitro. Cell attachment andproliferation were measured by cell counting 1 day, 3,5, and 10 days after cell seeding. Histological examination was performed with light microscope and scanning electron microscope 5 and 10 days after cell seeding. Results Porous structure, which supported the proliferation and attachment of HPDLFs, was found in tMEBC. The density of cell increased from 0.556×104 cells／ml 24 hours after cell seeding to 3.944×104 cells／ml 10 days after seeding. Light and scanning electron microscope examinationindicated that HPDLFs were attached and extended on the three-dimensional scaffolds and were well embedded in the newly formed tissue matrix. ConclusiontMEBC has good biocompatibility with the HPDLFs, and can be used as scaffold for cell transplantation in periodontal tissue engineering.