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 influence of different dose levels of hydroxyapatite/tricalcium phosphate (HA/TCP) on the proliferation and alkalinephosphatase (ALP) activity of rabbit osteoblasts. Methods Three different doselevels of HA/TCP (10%, 40%, 70%) were co-cultivated with rabbit osteoblasts respectively. The proliferation and ALP expression capacity of osteoblasts were examined with MTT method and enzyme histochemistry once every 24 hours until 5 days. Three control groups of other materials were treated and examined in the sameway: rabbit osteoblasts as normal control; polyvinylchloride as positive control; titanium alloy as negative control. Results There was remarkable timeeffect relationship in the proliferation of osteoblasts. Ten percent HA/TCP did not affect osteoblasts growth while 40% HA/TCP could slow the cell growth rate down though time-effect relationship still existed. The proliferation of osteoblasts stagnated when co-cultivated with 70% HA/TCP. On the other hand, 10% HA/TCP could cause reversible damage on ALP activity of osteoblasts, whereas when the dose was40%, and the cultivation lasted 6 days the damage was irreversible. Three different dose levels of titanium alloy (10%, 40%, 70%) had no effect on the proliferation or ALP activity of osteoblasts. Conclusion Dosage is an important factor affecting the biocompatibility evaluation of biomaterial. It suggests that dose choosing should be more specified upon each individual biomaterial. It also indicates that ALP may be a good supplementary index of the cell compatibility of material.
ObjectiveTo prepare of a novel functional self-assembling peptide nanofiber hydrogel scaffold RADKPS designed with linking the short functional motif of bone morphogenetic protein 7 (BMP-7) and to evaluate its biocompatibility so as to provide the experimental basis for in vivo studies on regeneration of degenerated nucleus pulposus tissue.
MethodA functional self-assembling peptide RADA-KPSS was designed by linking the short functional motif of BMP-7 to the self-assembling peptide RADA16-I. And the novel functional self-assembling peptide RADKPS was finally prepared by isometric mixing RADA16-I with RADA-KPSS. The structure characteristic of the functional self-assembling peptide nanofiber hydrogel scaffold RADKPS was evaluated by general observation and atomic force microscopy. Bone marrow mesenchymal stem cells (BMSCs) were isolated from 3-month-old New Zealand white rabbits and cultured. After the 3rd generation BMSCs were seeded on the peptide nanofiber hydrogel scaffold RADKPS for 7 days, the cellular compatibility of RADKPS was evaluated through scanning electron microscopy assay, cellular fluorescein diacetate/propidium iodide staining, and MTT assay. 1%RADKPS was injected into isolated intervertebral disc organs from 6-month-old New Zealand white rabbits, then the organs were cultured and the cellular activity of the intervertebral disc organs was observed. The blood compatibility of RADKPS was evaluated with hemolytic assay. After RADKPS was implanted into subcutaneous part of Kunming mice (aged 6-8 weeks) for 28 days, general observation and HE staining were carried out to evaluate the tissue compatibility.
ResultsThe functional self-assembling peptide solution RADKPS presented a homogeneous transparent hydrogel-like. Atomic force microscopy revealed that the RADKPS could self-assemble into three-dimensional nanofiber hydrogel scaffolds; the fibre diameter was (25.68±4.62) nm, and the fibre length was (512.42±32.22) nm. After BMSCs cultured on RADKPS for 7 days, scanning electron microscopy showed that BMSCs adhered to the scaffolds. And cell viability was maintained over 90%. MTT assay revealed that RADKPS of 0.1%, 0.05%, and 0.025% could increase the proliferation of BMSCs. The result of hemolytic assay revealed that the hemolysis rates of the RADKPS solutions with different concentrations were less than 5%, indicating that it met the requirement of hemolytic assay standard for medical biomaterials. After subcutaneous implantation, no vesicle, erythema, and eschar formation around injection site were observed. Meanwhile, HE staining showed inflammatory cells infiltration (lymphocytes), substitution of hydrogel scaffold by fibrous tissue, and good tissue compatibility.
ConclusionsThe novel functional self-assembling peptide nanofiber hydrogel scaffold RADKPS has good biocompatibility and biological reliability, which would be suitable for tissue engineering repair and regeneration of nucleus pulposus tissue.
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 the biocompatibility of the poly-lactide-co-glycolide (PLGA)/collagen type I scaffold with rat vaginal epithelial cells, and the feasibility of using PLGA/collagen type I as scaffold to reconstruct vagina by the tissue engineering.
MethodsPLGA/collagen type I scaffold was prepared with PLGA covered polylysine and collagen type I. The vaginal epithelial cells of Sprague Dawley rat of 10-12 weeks old were cultured by enzyme digestion method. The vaginal epithelial cells of passage 2 were cultured in the leaching liquor of scaffold for 48 hours to detect its cytotoxicity by MTT. The vaginal epithelial cells were inoculated on the PLGA/collagen type I scaffold (experimental group) and PLGA scaffold (control group) to calculate the cell adhesion rate. Epithelial cells-scaffold complexes were implanted subcutaneously on the rat back. At 2, 4, and 8 weeks after implantation, the epithelial cells-scaffold complexes were harvested to observe the cell growth by HE staining and immunohistochemical analysis. The epithelial cells-scaffold complexes were transplanted to reconstruct vagina in 6 rats with vaginal defect. After 3 and 6 months, the vaginal length was measured and the appearance was observed. The neovagina tissues were harvested for histological evaluation after 6 months.
ResultsThe epithelial cells grew and proliferated well in the leaching liquor of PLGA/collagen type I scaffold, and the cytotoxicity was at grade 1. The cell adhesion rate on the PLGA/collagen type I scaffold was 71.8%±9.2%, which significantly higher than that on the PLGA scaffold (63.4%±5.7%) (t=2.195, P=0.005). The epithelial cells could grow and adhere to the PLGA/collagen type I scaffolds. At 2 weeks after implanted subcutaneously, the epithelial cells grew and proliferated in the pores of scaffolds, and the fibroblasts were observed. At 4 weeks, 1-3 layers epithelium formed on the surface of scaffold. At 8 weeks, the epithelial cells increased and arranged regularly, which formed the membrane-like layer on the scaffold. The keratin expression of the epithelium was positive. At 3 months after transplantation in situ, the vaginal mucosa showed pink and lustrous epithelialization, and the majority of scaffold degraded. After 6 months, the neovagina length was 1.2 cm, without obvious stenosis; the vaginal mucosa had similar appearance and epithelial layer to normal vagina, but it had less duplicature; there were nail-like processes in the basal layer, but the number was less than that of normal vagina. The immunohistochemistry staining for keratin was positive.
ConclusionThe PLGA/collagen type I scaffolds have good cytocompatibility with the epithelial cells, and can be used as the biodegradable polymer scaffold of the vaginal tissue engineering.
Objective To evaluate the tissue response induced by three kinds of bone transplantation materials implanted in rat so as to provide proper evidence for their cl inical appl ication. Methods Thirty-six healthy mature Sprague- Dawly mice, weighing from 229 g to 358 g, were randomly assigned to groups A and B (n=18). Three kinds of materials wereimplanted into muscles of rats. Calcium sulfate (CS) granular preparations and allogeneic demineral ized bone matrix (DBM) were transplanted into the left (group A1) and right (group A2) thigh muscle pouches of group A. Respectively, whereas xenogenic DBM were transplanted into the left (group B1) thigh muscle pouches of group B and the right (group B2) sites were taken as control without implant. The samples (n=6) were collected to make the observation of gross and histology and to analyze histological score after 2, 4, and 6 weeks. Results The gross observation: implanted materials were gradually absorbed at late stage in group A1. No obvious degradation and absorption, but fibrosis of tissues were observed in group A2 and B1. The inflammatory reactions were more severe in groups A2 and B1. In group B2, only the changes of scar were seen at operative site. The histological observation: no obvious inflammatory reactions were seen in group A1, CS were gradually absorbed and completely absorbed at 6 weeks, while fibrosis of tissues increased at late stage. Inflammatory reactions in group A2 and group B1 were alleviated gradually, no obvious absorption and degradation were observed. The different two DBM could induce granulation tissues and bone formation at different sites and secondary fibrosis with no obvious immune response was observed. In group B2, there was an increase in collagen fiber density and angiogenesis at late stage. The scores of inflammatory infiltration were significantly higher in groups A2, B1 than in groups A1, B2 (P lt; 0.05), and the scores of fibrosis was larger in groups A1, A2 and B1 than in group B2 (P lt; 0.05). Conclusion CS has rapid dissolution and good biocompatibil ity. It is a good replaceable packing materials of bone defects in some upper l imb’s or acute bone fracture. Both of two DBM have biocompatibil ity and osteoinductive potential, which dissolution are very slow. Due to these capacity, they can be served as an ideal materials in treatment of lower l imb’s bone defect and nonunion.
Objective
To research in vitro biocompatibility of silicon containing micro-arc oxidation (MAO) coated magnesium alloy ZK60 with osteoblasts.
Methods
The surface microstructure of silicon containing MAO coated magnesium alloy ZK60 was observed by a scanning electron microscopy (SEM), and chemical composition of the coating surface was determined by energy dispersive spectrum analysis. The experiments were divided into 4 groups: silicon containing MAO coated magnesium alloy ZK60 group (group A), uncoated magnesium alloy ZK60 group (group B), titanium alloy group (group C), and negative control group (group D). Extracts were prepared respectively with the surface area to extraction medium ratio (1.25 cm2/ mL) according to ISO 10993-12 standard in groups A, B, and C, and were used to culture osteoblasts MC3T3-E1. The α-MEM medium supplemented with 10% fetal bovine serum was used as negative control in group D. The cell morphology was observed by inverted phase contrast microscopy. MTT assay was used to determine the cell viability. The activity of alkaline phosphatase (ALP) was detected. Cell attachment morphology on the surface of different samples was observed by SEM. The capability of protein adsorption of the coating surface was assayed, then DAPI and calcein-AM/ethidium homodimer 1 (calcein-AM/EthD-1) staining were carried out to observe cell adhesion and growth status.
Results
The surface characterization showed a rough and porous layer with major composition of Mg, O, and Si on the surface of silicon containing MAO coated magnesium alloy ZK60 by SEM. After cultured with the extract, cells grew well and presented good shape in all groups by inverted phase contrast microscopy, group A was even better than the other groups. At 5 days, MTT assay showed that group A presented a higher cell proliferation than the other groups (P lt; 0.05). Osteoblasts in groups A and C presented a better cell extension than group B under SEM, and group A exhibited better cell adhesion and affinity. Protein adsorption in group A [ (152.7 ± 6.3) μg/mL] was significantly higher than that of group B [(96.3 ± 3.9) μg/mL] and group C [ (96.1 ± 8.7) μg/mL] (P lt; 0.05). At each time point, the adherent cells on the sample surface of group A were significantly more than those of groups B and C (P lt; 0.05). The calcein-AM/EthD-1 staining showed that groups A and C presented better cell adhesion and growth status than group B. The ALP activities in groups A and B were 15.55 ± 0.29 and 13.75 ± 0.44 respectively, which were significantly higher than those in group C (10.43 ± 0.79) and group D (10.73 ± 0.47) (P lt; 0.05), and group A was significantly higher than group B (P lt; 0.05).
Conclusion
The silicon containing MAO coated magnesium alloy ZK60 has obvious promoting effects on the proliferation, adhesion, and differentiation of osteoblasts, showing a good biocompatibility, so it might be an ideal surface modification method on magnesium alloys.
OBJECTIVE To study the biocompatibility on bioactive glass ceramics (BGC) and polylactic acid (PLA) combined with cultured bone marrow stromal cells (BMSCs) in bone tissue engineering. METHODS BMSCs were cultured combined with BGC and PLA in vitro, and the morphological characters, cell proliferation, protein content, and alkaline phosphatase activity were detected. RESULTS: BMSCs could be attached to and extended on both BGC and PLA, and normally grown, proliferated, had active function. BGC could promote cell proliferation. CONCLUSION The results show that both BGC and PLA have good biocompatibility with BMSCs, they can be used as biomaterials for cell transplantation in tissue engineering.
Objective To explore the method of preparing the electrospinning of synthesized triblock copolymers of ε-caprolactone and L-lactide (PCLA) for the biodegradable vascular tissue engineering scaffold and to investigateits biocompatibil ity in vitro. Methods The biodegradable vascular tissue engineering scaffold was made by the electrospinning process of PCLA. A series of biocompatibil ity tests were performed. Cytotoxicity test: the L929 cells were cultured in 96-wellflat-bottomed plates with extraction media of PCLA in the experimental group and with the complete DMEM in control group, and MTT method was used to detect absorbance (A) value (570 nm) every day after culture. Acute general toxicity test: the extraction media and sal ine were injected into the mice’s abdominal cavity of experimental and control groups, respectively, and the toxicity effects on the mice were observed within 72 hours. Hemolysis test: anticoagulated blood of rabbit was added into the extracting solution, sal ine, and distilled water in 3 groups, and MTT method was used to detect A value in 3 groups. Cell attachment test: the L929 cells were seeded on the PCLA material and scanning electron microscope (SEM) observation was performed 4 hours and 3 days after culture. Subcutaneous implantation test: the PCLA material was implanted subcutaneously in rats and the histology observation was performed at 1 and 8 weeks. Results Scaffolds had the characteristics of white color, uniform texture, good elasticity, and tenacity. The SEM showed that the PCLA ultrafine fibers had a smooth surface and proper porosity; the fiber diameter was 1-5 μm and the pore diameter was in the range of 10-30 μm. MTT detection suggested that there was no significant difference in A value among 3 groups every day after culturing (P gt; 0.05). The mice in 2 groups were in good physical condition and had no respiratory depression, paralysis, convulsion, and death. The hemolysis rate was 1.18% and was lower than the normal level (5%). The SEM showed a large number of attached L929 cells were visible on the surface of the PCLA material at 4 hours after implantation and the cells grew well after 3 days. The PCLA material was infiltrated by the inflammatory cells after 1 week. The inflammatory cells reduced significantly and the fiber began abruption after 8 weeks. Conclusion The biodegradable vascular tissue engineering scaffold material made by the electrospinning process of PCLA has good microstructure without cytotoxicity and has good biocompatibil ity. It can be used as an ideal scaffold for vascular tissue engineering.
ObjectiveTo explore the influence of three central venous catheter biomedical materials (polyurethane, silicone, and polyvinyl chloride) on the proliferation, apoptosis, and cell cycle of Xuanwei Lung Cancer-05 (XWLC-05) cells so as to provide the basis for clinical choice of central venous catheter. MethodsXWLC-05 cells were cultured and subcultured, and the cells at passage 3 were cultured with polyurethane, silicone, and polyvinyl chloride (1.0 cm × 1.0 cm in size), and only cells served as a control. At 24, 48, and 72 hours after cultured, MTT assay was used to detect the cellular proliferation and flow cytometry to detect the cell cycle and apoptosis. At 72 hours after cultured, inverted microscope was used to observe the cell growth. ResultsInverted microscope showed the cells grew well in control group, polyurethane group, and silicone group. In polyvinyl chloride group, the cells decreased, necrosed, and dissolved; residual adherent cells had morphologic deformity and decreased transmittance. At 24 and 48 hours, no significant difference in proliferation, apoptosis, and cell cycle was found among 4 groups (P gt; 0.05). At 72 hours, the proliferations of XWLC-05 cells in three material groups were significantly inhibited when compared with control group (P lt; 0.05), and the cells in polyvinyl chloride group had more significant proliferation inhibition than polyurethane group and silicone group (P lt; 0.05), but there was no signifcant difference in proliferation inhibition between polyurethane group and silicone group (P gt; 0.05). Compared with the control group, three material groups had significant impact on the rate of apoptosis and cell cycle: polyvinyl chloride group was the most remarkable, followed by silicone group, polyurethane group was minimum (P lt; 0.05). ConclusionPolyvinyl chloride can significantly impact the proliferation, apoptosis, and cell cycle of XWLC-05 cells; polyurethane has better biocompatibility than polyvinyl chloride and silicone
