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 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 biocompatibil ity of silk fibroin nanofibers scaffold with olfactory ensheathing cells (OECs) and to provide an ideal tissue engineered scaffold for the repair of spinal cord injury (SCI). Methods Silk fibroin nanofibers were prepared using electrospinning techniques and were observed by scanning electron microscope (SEM). Freshly isolated OECs from SD rats purified by the modified differential adherent velocity method were cultured. The cells at passage 1 (1 × 104 cells/cm2) were seeded on the poly-l-lysine (control group) and the silk fibroin nanofibers (experimental group) coated coversl ips in Petri dish. At desired time points, the morphological features, growth,and adhesion of the cells were observed using phase contrast inverted microscopy. The OECs were identified by the nerve growth factor receptor p75 (NGFR p75) immunofluorescence staining. The viabil ity of OECs was examined by l ive/dead assay. The prol iferation of OECs was examined by MTT assay. The cytotoxicity of the nanofibers was evaluated. Results The SEM micrographs showed that the nanofibers had a smooth surface with sol id voids among the fibers, interconnecting a porous network, constituted a fibriform three dimensional structure and the average diameter of the fibers was about (260 ± 84) nm. The morphology of OECs on the experimental group was similar to the cell morphology on the control group, the cells distributed along the fibers, and the directions of the cell protrusions were in the same as that of the fibers. Fluorescence microscopy showed that the purity of OECs was 74.21% ± 2.48% in the experimental group and 79.05% ± 2.52% in the control group 5 days after culture. There was no significant difference on cell purity between two groups (P gt; 0.05). The OECs in the experimental group stained positive for NGFR p75 compared to the control group, indicating that the cells in the experimental group still maintained the OECs characteristic phenotype. Live/dead staining showed that high viabil ity was observed in both groups 3 days after culture. There was no significant difference on cell viabil ity between two groups. The prol iferation activity at 1, 3, 5, 7, and 10 days was examined by MTT assay. The absorbency values of the control group and the experimental group had significant differences 3 and 5 days after culture (P lt; 0.05). The relative growth rates were 95.11%, 90.35%, 92.63%, 94.12%, and 94.81%. The cytotoxicity of the material was grade 1 and nonvenomous according to GB/T 16886 standard. Conclusion Silk fibroin nanofibers scaffold has good compatibility with OECs and is a promising tissue engineered scaffold for the repair of SCI.
Objective To prepare silver-containing hydroxyapatite coating (hydroxyapatite/Ag, HA/Ag) and investigate its antibacterial property and biocompatibil ity in vitro. Methods Vacuum plasma spraying technique was adopted to prepare HA/Ag coating on titanium alloy substrate (3% Ag). After incubating the HA/Ag and the HA coating under staphylococcus aureus and pseudomonas aeruginosa suspensions of 2% tryptic soy broth (TBS) medium for 2, 4 and 7 days, respectively, the biofilm on the coatings was examined by confocal laser scanning microscope, and the bacterial density and viable bacterial percentage of bacterial biofilm were calculated. Meanwhile, the micro-morphology of bacterial biofilm was observed by SEM, the cytotoxicity was detected via MTT and the biocompatibil ity of biofilm was evaluated by acute aemolysis test. Results Compared with HA coating, the bacterial biofilm’s thickness on the surface of HA/Ag coating witnessed no significant difference at 2 days after culture (Pgt; 0.05), but decreased obviously at 4 and 7 days after culture (P lt; 0.01). The bacterial density of the biofilm increased with time, but there was no significant difference between two coatings (P gt; 0.05) at 2, 4 and 7 days after culture. The viable bacterial percentage of the biofilms on the surface of HA/Ag coating decreased obviously compared with that of HA coating at 2, 4 and 7 days after cultureP lt; 0.01). The MTT notified the cytotoxic grade of both coatings was zero. The acute haemolysis assay showed that the hemolytic rate of HA/Ag and HA coating was 0.19% and 0.12%, respectively. Conclusion With good biocompatibil ity, significant antibacterial property against staphylococcus aureus and pseudomonas aeruginosa, no obvious cytotoxicity and no erythrocyte destruction, the vacuum plasma sprayed HA/Ag coating is a promising candidate for the surface of orthopedic metal implants to improve their osseointegration and antibacterial property.
This paper is to evaluate the biocompatibility and cytotoxicity of a new Ni-free Zr-based bulk metallic glass (BMG), Zr60.14Cu22.31Fe4.85Al9.7Ag3, by comparing it with conventional Ti6Al4V alloy. According to ISO 10993-5:1999 and GB/T 16886.5-1997 standards, Zr60.14Cu22.31Fe4.85Al9.7Ag3, pure Zr and Ti6Al4V materials were extracted with surface area of sample/volume of medium ratio being 1 cm2/mL and 0.5 cm2/mL, respectively. The viabilities of MG-63 cells (Human osteosarcoma cell line) cultured in the BMG medium extracts for 1, 3 and 5 days were determined by CCK-8 assay. The cellular morphology of MG-63 cells cultured on the surface of samples for 3 days was tested through laser scanning confocal microscopy (LSCM) and scanning electron microscopy (SEM). The relative growth rate (RGR) of MG-63 cells cultured in Zr60.14Cu22.31 Fe4.85 Al9.7Ag3 and pure Zr were both more than 85%, indicating that the cytotoxicity of BMG was relatively low and met the national biomedical material eligibility standard. There was insignificant difference in the morphology of MG-63 cells cultured in the BMG medium extracts and the control group through LSCM and SEM, which showed the BMG had excellent biological compatibility. The Zr-based bulk metallic glass Zr60.14Cu22.31Fe4.85Al9.7Ag3 and the conventional Ti6Al4V alloy both had no obvious cytotoxicity to MG-63 cells. These results provided evidence that the new Zr-based bulk metallic glass could be potential replacement material for the orthopedic surgical implant.
ObjectiveThe tissue engineered osteochondral integration of multi-layered scaffold was prepared and the related mechanical properties and biological properties were evaluated to provide a new technique and method for the repair and regeneration of osteochondral defect.MethodsAccording to blend of different components and proportion of acellular cartilage extracellular matrix of pig, nano-hydroxyapatite, and alginate, the osteochondral integration of multi-layered scaffold was prepared by using freeze-drying and physical and chemical cross-linking technology. The cartilage layer was consisted of acellular cartilage extracellular matrix; the middle layer was consisted of acellular cartilage extracellular matrix and alginate; and the bone layer was consisted of nano-hydroxyapatite, alginate, and acellular cartilage extracellular matrix. The biological and mechanics characteristic of the osteochondral integration of multi-layered scaffold were evaluated by morphology observation, scanning electron microscope observation, Micro-CT observation, porosity and pore size determination, water absorption capacity determination, mechanical testing (compression modulus and layer adhesive strength), biocompatibility testing [L929 cell proliferation on scaffold assessed by MTT assay, and growth of green fluorescent protein (GFP)-labeled Sprague Dawley rats’ bone marrow mesenchumal stem cells (BMSCs) on scaffolds].ResultsGross observation and Micro-CT observation showed that the scaffolds were closely integrated with each other without obvious discontinuities and separation. Scanning electron microscope showed that the structure of the bone layer was relatively dense, while the structure of the middle layer and the cartilage layer was relatively loose. The pore structures in the layers were connected to each other and all had the multi-dimensional characteristics. The porosity of cartilage layer, middle layer, and bone layer of the scaffolds were 93.55%±2.90%, 93.55%±4.10%, and 50.28%±3.20%, respectively; the porosity of the bone layer was significantly lower than that of cartilage layer and middle layer (P<0.05), but no significant difference was found between cartilage layer and middle layer (P>0.05). The pore size of the three layers were (239.66±35.28), (153.24±19.78), and (82.72±16.94) μm, respectively, showing significant differences between layers (P<0.05). The hydrophilic of the three layers were (15.14±3.15), (13.65±2.98), and (5.32±1.87) mL/g, respectively; the hydrophilic of the bone layer was significantly lower than that of cartilage layer and middle layer (P<0.05), but no significant difference was found between cartilage layer and middle layer (P>0.05). The compression modulus of the three layers were (51.36±13.25), (47.93±12.74), and (155.18±19.62) kPa, respectively; and compression modulus of the bone layer was significantly higher than that of cartilage layer and middle layer (P<0.05), but no significant difference was found between cartilage layer and middle layer (P>0.05). The osteochondral integration of multi-layered scaffold was tightly bonded with each layer. The layer adhesive strength between the cartilage layer and the middle layer was (18.21±5.16) kPa, and the layer adhesive strength between the middle layer and the bone layer was (16.73±6.38) kPa, showing no significant difference (t=0.637, P=0.537). MTT assay showed that L929 cells grew well on the scaffolds, indicating no scaffold cytotoxicity. GFP-labeled rat BMSCs grew evenly on the scaffolds, indicating scaffold has excellent biocompatibility.ConclusionThe advantages of three layers which have different performance of the tissue engineered osteochondral integration of multi-layered scaffold is achieved double biomimetics of structure and composition, lays a foundation for further research of animal in vivo experiment, meanwhile, as an advanced and potential strategy for osteochondral defect repair.
Objective To evaluate the biocompatibil ity of manufactured heterogeneous demineral ized bone matrix(DBM) particles and to provide basis for further experimental study and cl inical application. Methods Heterogeneous DBMparticles A (degreased and demineralized) and B (degreased, demineralized and acellular), particle size from 250 to 810 μm, and leaching l iquor were made with a series of physical and chemical methods from pig l imbs cortical bone. The residual calcium and phosphorus contents of bone particles were measured after degreased and demineral ized. The acute toxicity test, skin stimulating test, pyrogeneous test, hemolysis test, cellular toxicity test and muscular embedded test were carried out according standard toxicological method. Results The contents of calcium and phosphorus in cortical bone were (189.09 ± 3.12) mg/g and (124.73 ± 2.87) mg/g, and in demineral ized bone matrix particles were (3.48 ± 0.09) mg/g and (3.46 ± 0.07) mg/ g. The residual calcium content was 1.87%, of phosphorus was 2.69%. The activity of mice was normal in the acute toxicity test. No animal died and no toxicity symptom or adverse effects were shown within 7 days. The mean weight daily increased showed no statistically significant difference (P gt; 0.05) between two groups after 7 days. Skin stimulating reactions were not found in the two experimental groups and negative control group by intradermal stimulation test. The maximal increase of body temperature in two experimental groups were 0.4℃ , which meet the national standard (lt; 0.6 ). The rate of haemolysis to the leaching liquor was 1.14% (A) and 0.93% (B), which was lower than the national standard (lt; 5%). The cell prol iferation rates of two experimental groups when compared with control group showed no statistically significant difference (P gt; 0.05). The toxicity of DBM particlesleaching liquor was graded from 0 to 1, which means the material has no cytotoxicity. All the animals survived well. There was no tissue necrosis, effusion or inflammation at all implantation sites. For the index of HE and Masson staining, there were no effusion around the material and inflammatory cell infiltrate obviously in two experimental groups. Inflammatory cell infiltrate is sl ight in control group 2 weeks postoperatively. The inflammatory cell infiltration was mitigate gradually over time in two experimental groups after 4, 8 and 12 weeks. New bone and collagen fibers formation were observed when the material was degraded and absorpted. Score evaluation of local cellular immune response at different time after operation of two experimental groups showed no statistically significant difference (P gt; 0.05). Conclusion Heterogeneous DBM has no obvious toxicity, skin irritation, pyrogenicity, and no cytotoxicity with a rate of haemolysis lt; 5%, so it has good biocompatibility and partial osteoinductive.
Decellularized tissue engineering scaffolds appear to have the properties of similar structure and mechanical characteristics to native tissues，good biocompatibility，suitability for cell adhesion，growth and angiogenesis induction，and non-immunogenicity. Genipin has anti-inflammatory，antithrombotic and antioxidative features which can considerably suppress vascular and endothelial inflammatory activation，increase mechanical strength of biological scaffolds，inhibit inflammatory response and decrease degradation rate of biological scaffolds. By cross-linking with decellularized matrices，Genipin can further improve corresponding performance of tissue engineering matrices，which is very helpful to promote the application of tissue engineering into clinical practice of cardiothoracic surgery. This review focuses on recent research process and possible prospects of Genipin cross-linking in tissue engineering in the field of cardiothoracic surgery.
Objective To prepare carboxymethyl-chitosan/hyaluronic acid/poly(vinyl alcohol) (CHP) blend membrane, evaluate its physicochemical properties and intraocular biocompatibil ity and to investigate its feasibil ity to be appl ied to glaucoma filtering surgery. Methods CHP blend membrane was prepared using solution casting method after blending carboxymethyl-chitosan, HA and poly(vinyl alcohol) in a proportion of 5 ∶ 4 ∶ 1 (M/M). Its water absorption rate, swell ing rate, permeabil ity, and mechanical properties were detected. Subconjunctival fibroblasts separated from subconjuncitival tissue of New Zealand white rabbits were cultured, and the cells at passage 4 were cultured on cell culture plate with or without the CHP blend membrane, serving as the experimental group and the control group, respectively. Effectof the CHP blend membrane on the subconjunctival fibroblasts was tested by MTT method 24, 48, and 72 hours after culture. Six New Zealand white rabbits were randomly divided into two groups (n=3 rabbits per group), and the CHP blend membrane and SK gel were implanted into the rabbits’ subconjunctival space and anterior chamber in the experimental group and the control group, respectively. Sl it lamp observation and binocular reaction record were conducted 1, 3, 5, 9, 11, 20, 30, 45, and 60 days after operation. Corneal tissue harvested from the experimental group was observed using scanning electron microscope 15 days after operation to study ophthalmic biocompatibil ity and biodegradabil ity. Results The water absorption rate and the swell ing rate of the CHP blend membrane was 83.8% ± 1.3% and 3.59 ± 0.50, respectively. The tensile strength of the dry and the wet CHP blend membrane was (20.59 ± 1.73) and (0.51 ± 0.13) MPa, respectively. The breaking elongation rate of the dry and the wet CHP blend membcane was 10.69% ± 1.16% and 53.15% ± 2.46%, respectively. The CHP blend membrane had good permeabil ity to NaCl and L-tyrosine. Absorbance (A) value of the experimental group 24, 48, and 72 hours after breeding was 0.207 ± 0.083, 0.174 ± 0.080, and 0.181 ± 0.048, respectively, while the A value of the control group was 0.284 ± 0.011, 0.272 ± 0.083, and 0.307 ± 0.056, respectively. Significant difference was evident between two groups (P lt; 0.05). In the experimental group, a small amount of floccus was exuded around the implanted membrane 1 day after operation; the floccus was absorbed on the third day, and there was no obvious inflammatory reaction occurring on the eleventh day. Most of the membrane degraded on the sixtieth day. Scanning electron microscope observation showed that the hexagonal morphology of the corneal endothel ial cells was intact, and no degradation particles adhered to the surface. In the control group, the implantation of SK gel into anterior chamber was unsuccessful because the SK gel was quite soft and easily broken. In the experimental group, mild hyperemia emerged around the implanted membrane 1 day after the subconjunctival implantation of the membrane, and it became normal on the ninth day. No corneal edema and inflammatory reaction of anterior chamber occurred till the sixtieth day. The results in the control group and the experiment group were similar. Conclusion Due to its good physicochemical properties and biocompatibil ity, the CHP blend membrane has potential appl ications in glaucomafiltering surgery.
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.