Objective To prepare and study the biocompatibil ity of selectively decellular xenoskin which has the character of the lower antigen, continuous epidermis, and the dermal matrix without any cellular components. Methods The porcine skin was treated with glutaraldehyde solution, trypsin, and detergent solution TritonX-100 to prepare the selectivelydecellular xenoskin. The cytotoxicity was tested according to GB/T16886.5-2003 biological evaluation of medical devices for in vitro cytotoxicity, and the levels of cytotoxicity were evaluated with the United States Pharmacopeia. Subdermal implantation was tested according to GB/T16886.6-1997 biological evaluation of medical devices for local effects after implantation. Seventytwo mature Wistar rats were randomly assigned to groups A, B, and C (n=24). Three kinds of materials were implanted into subcutaneous of rats back. Selectively decellular xenoskin was transplanted into group A, fresh porcine skin was transplanted into group B, and allogeneic skin was transplanted into group C. The samples were collected to make the observation of gross and histology after 1, 2, 4, 8, 12, and 16 weeks. Results The cytotoxicity was proved to be first grade by biocompatibil ity test. The gross and histological observation of subdermal implantation: after implantation, the most severe inflammatory reactions were seen in group B which dispersion was very slow. Inflammatory reactions in groups A and C alleviated gradually. In groups A and C, there was an increased collagen fiber density and angiogenesis at late stage; the transplanted skin was gradually degraded and absorbed. In group B, no obvious degradation and absorption were observed. Conclusion Selectively decellular xenoskin, prepared with glutaraldehyde solution, trypsin, and detergent solution, possesses characteristics of integral skin structure andexcellent biocompatibil ity, so it can be used as a new type substitute to repair the burn wound.
The study aimed to evaluate the safety and function of poly(lactic-acid-co-ε-caprolactone) (PLCL)/fibrinogen nanofibers (P/F-Ns), and provide theoretical basis for the clinical application. The surface morphology, mechanical properties, the hydrophilicity and the fibrinogen content of P/F-Ns were tested by scanning electron microscope, the material testing machine, the contact angle meter and the microplate reader, respectively. The cell adhesion, proliferation and ligament remodeling genes expression of Hig-82 cells on P/F-Ns were conducted through cell counting kit-8 (CCK-8) and real-time quantitative PCR analyses, respectively. The results showed that with the increase of the fibrinogen content, the pore sizes and hydrophilicity of three P/F-Ns increased, but the mechanical properties decreased. Cell adhesion and proliferation tests showed that P/F-N-2 held the best ability to promote cell adhesion and proliferation. The ligament remodeling genes expressions of Hig-82 cells on P/F-N-1, P/F-N-2 and P/F-N-3 were all up-regulated compared to P/F-N-0 on days 3 and 7. All the three P/F-Ns containing fibrinogen (P/F-N-1, P/F-N-2 and P/F-N-3) had better biocompatibility compared to P/F-N-0, and could be efficiently applied to the reconstruction of anterior cruciate ligament.
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 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 fabricate in situ crosslinking hyaluronic acid hydrogel and evaluate its biocompatibility in vitro. Methods The acrylic acid chloride and polyethylene glycol were added to prepare crosslinking agent polyethylene glycol acrylate (PEGDA), and the molecular structure of PEGDA was analyzed by Flourier transformation infrared spectroscopy and 1H nuclear magnetic resonance spectroscopy. Hyaluronic acid hydrogel was chemically modified to prepare hyaluronic acid thiolation (HA-SH). And the degree of HA-SH was analyzed qualitatively and quantitatively by Ellman method. HA-SH solution in concentrations (W/V) of 0.5%, 1.0%, and 1.5% and PEGDA solution in concentrations (W/V) of 2%, 4%, and 6% were prepared with PBS. The two solutions were mixed in different ratios, and in situ crosslinking hyaluronic acid hydrogel was obtained; the crosslinking time was recorded. The cellular toxicity of in situ crosslinking hyaluronic acid hydrogel (1.5% HA-SH and 4% PEGDA mixed) was tested by L929 cells. Meanwhile, the biocompatibility of hydrogel was tested by co-cultured with human bone mesenchymal stem cells (hBMSCs). Results Flourier transformation infrared spectroscopy showed that most hydroxyl groups were replaced by acrylate groups; 1H nuclear magnetic resonance spectroscopy showed 3 characteristic peaks of hydrogen representing acrylate and olefinic bond at 5-7 ppm. The thiolation yield of HA-SH was 65.4%. In situ crosslinking time of hyaluronic acid hydrogel was 2 to 70 minutes in the PEGDA concentrations of 2%-6% and HA-SH concentrations of 0.5%-1.5%. The hyaluronic acid hydrogel appeared to be transparent. The toxicity grade of leaching solution of hydrogel was grade 1. hBMSCs grew well and distributed evenly in hydrogel with a very high viability. Conclusion In situ crosslinking hyaluronic acid hydrogel has low cytotoxicity, good biocompatibility, and controllable crosslinking time, so it could be used as a potential tissue engineered scaffold or repairing material for tissue regeneration.
Objective To study the preparation method of acellular vascular matrix and to evaluate its biocompatibil ity and safety so as to afford an ideal scaffold for tissue engineered blood vessel. Methods Fresh caprine carotids (length, 50 mm) were harvested and treated with repeated frozen (—80 )/thawing (37℃), cold isostatic pressing (506 MPa, 4 ), and 0.125% sodium dodecyl sulfate separately for preparation of acellular vascular matrix. Fluorescence staining and DNA remain test were used to assess the cell extracting results. Biological characteristics were compared with the raw caprine carotids using HE staining, Masson staining, scanning electron microscope (SEM), and mechanical test. Biocompatibil ity wasdetected using cell adhesion test, MTT assay, and subcutaneously embedding test. Ten SD rats were divided into 2 groups (n=5). In experimental group, acellular vascular matrix preserved by the combination of repeated frozen/thawing, ultrahigh pressure treatment and chemical detergent was subcutaneously embedded; and in control group, acellular vascular matrix preserved only by repeated frozen/thawing and ultrahigh pressure treatment was subcutaneously embedded. Results HE staining and Masson staining revealed that no nucleus was detected in the acellular vascular matrix. SEM demonstrated that a lot of collagen fibers were preserved which were beneficial for cell adhesion. Fluorescence staining and DNA remain test showed that the cells were removed completely. There was no significant difference in stress and strain under the maximum load between before and after treatment. Mechanical test revealed that the acellular vascular matrix reserved mechanical properties of the raw caprine carotids. Cell adhesion test and MTT assay confirmed that cytotoxicity was grade 0-1, and the acellular vascular matrix had good compatibil ity to endothel ial cells. After subcutaneously embedding for 8 weeks, negl igible lymphocyte infiltration was observed in experimental group but obvious lymphocyte infiltration in control group. Conclusion The acellular vascular matrix, which is well-preserved by the combination of repeated frozen/thawing, ultrahigh pressure treatment, and chemical detergent, is an ideal scaffold for tissue engineered blood vessel.
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.
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.
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 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.