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 of a new bone matrix material (NBM) composed of both organic and inorganic materials for bone tissue engineering. Methods Osteoblasts combined with NBM in vitro were cultured. The morphological characteristics was observed; cell proliferation, protein content and basic alkaline phosphatase(ALP) activity were measured. NBM combined with osteoblasts were implanted into the skeletal muscles of rabbits and the osteogenic potential of NBM was evaluated through contraat microscope, scanning electromicroscope and histological examination. In vitro osteoblasts could attach and proliferate well in the NBM, secreting lots of extracellular matrix; NBM did not cause the inhibition of proliferation and ALP activity of osteoblasts. While in vivo experiment of the NBM with osteoblasts showed that a large number of lymphacytes and phagocytes invading into the inner of the material in the rabbit skeletalmuscle were seen after 4 weeks of implantation and that no new bone formation was observed after 8 weeks. Conclusion This biocompat ibility difference between in vitro and in vivo may be due to the immunogenity of NBM which causes cellular immuno reaction so as to destroy the osteogenic environment. The immunoreaction between the host and the organic-inorganic composite materials in tissue engineering should be paid more attention to.
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.
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.
In recent years, bone implant materials such as titanium and titanium alloys have been widely used in the biomedical field due to their excellent mechanical properties and good biocompatibility. However, in clinical practice, bacterial adhesion to the material surface and postoperative infection issues may lead to implantation failure. Based on the antibacterial mechanism, this review elaborated on the antibacterial surface design of titanium implants from the aspects of anti-bacterial adhesion, contact sterilization and photocontrol sterilization. Surface modification of titanium or titanium-based alloy implants with different techniques can inhibit bacteria and promote osseointegration. Thus, the application range of multifunctional titanium-based implants in the field of orthopedics will be expanded.
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 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 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.
ObjectiveTo study the hydrophilicity and the cell biocompatibility of the poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) and poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx) coated with a fusion protein polyhydroxyalkanoates granule binding protein (PhaP) fused with Arg-Gly-Asp (RGD) peptide (PhaP-RGD).
MethodsPHBV and PHBHHx films were fabricated by solvent evaporation.Scanning electronic microscope (SEM) was used to study the morphology of the films.PhaP-RGD fusion proteins were expressed and purified by the technology of protein engineering; PHBV and PHBHHx films were immersed in the PhaP-RGD with an amount of 3.5 mg/mL protein/per sample respectively.The hydrophilicity of the surface were detected by the contact angle measurements.Septal cartilage cells obtained from human septal cartilage were cultured in vitro.The 2nd passage chondrocytes were incubated on PHBV unmodified with PhaP-RGD in group A1,PHBV modified with PhaP-RGD in group A2,PHBHHx unmodified with PhaP-RGD in group B1,PHBHHx modified with PhaP-RGD in group B2,and on the cell culture plates in group C.After cultured for 3 days,the proliferation of cells was detected by the DAPI staining; the proliferation viability of cells was detected by the MTT assay after cultured for 3 and 7 days; after cultured for 7 days,the adhesion and morphology of the cells on the surface of the biomaterial films were observed by SEM and the matrix of the cells was detected through the toluidine blue staining.
ResultsSEM observation showed that PHBV and PHBHHx films had porous structures.The contact angle of the surface of the PHBV and PHBHHx films modified with PhaP-RGD fusion proteins were significantly reduced when compared with the films unmodified with PhaP-RGD fusion proteins (P<0.05).Chondrocytes of human nasal septal cartilage incubated on the films could grow in all groups.After 3 days of cultivation in vitro,the cell proliferation and viability of group B2 were the strongest among all groups (P<0.05); the cell proliferation after cultured for 7 days was significantly stronger than that after cultured for 3 days in groups A1,A2,B1,and B2 (P<0.05); and the cell proliferation was significantly stronger in groups B1 and B2 than groups A1,A2 and C,in group B2 than group B1,and in group A1 than group A2 (P<0.05).The results of toluidine blue staining showed that blue metachromasia matrixes were observed in groups A1,A2,B1,and B2; group A1 and group A2 had similar staining degree,and the staining of group B2 was deeper than that of group B1.The adhesion of cells in all groups was good through SEM observation; and the connection of cells formed and stretched into the pores of the materials.
ConclusionThe biomaterial films of PHBHHx modified with PhaP-RGD fusion protein can promote its biocompatibility with chondrocytes.
