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.
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.
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.
【Abstract】 Objective To investigate the manufacture and biocompatibil ity of a bioabsorbable poly-D,L-lactic acid(PDLLA) plate containing rhBMP-2. Methods rhBMP-2 was composited with PDLLA (0.05 mg/plate) through vacuum to repare PDLLA plate containing rhBMP-2. Thirty-two New Zealand rabbits (male or female) weighted (3.0 ± 0.5) kg were used in he study. A 2.5 mm middle ulna osteotomy was made bilaterally. The bones as well as periosteum were removed. The right side of all the animals was experimental side (n=32), was fixed internally by PDLLA plate containing rhBMP-2.The left side of all the animals was control side (n=32), was fixed by common PDLLA plate. After a follow-up of 2, 4, 8 and 12 weeks, the ulnas were examined visually, radiographically, histologically, and by computer graph analysis to compare the biocompatibil ity. Re sults Po rosity of PDLLA plate containing rhBMP-2 was 90%, aperture was 150 μm, tensile strength was higher than 50 MPa, three point flexural strength was higher than 90 MPa and intrinsic viscosity was 1.6 dL/g (chloroform solvent). All animals had a good heal ing 1 or 2 weeks after operation. All the animal’s diet and movement were normal. All the fractures were stable. The plates in the experimental group degraded faster than those in the control group. Relative values of callus density evaluated by X-ray at 2, 4, 8 and 12 weeks after operation in the experimental group were 39.22 ± 2.48, 48.79 ± 1.26, 63.78 ± 1.78 and 78.60 ± 1.25 respectively. Those in the control defects were 33.83 ± 1.13, 41.28 ± 1.25, 55.23 ± 0.68 and 66.54 ± 1.33. At each time point, the experimental side produced better and more trabeculae than the control side did (P ＜ 0.01). Histological examination showed that the newbone formation in the experimental side at 2, 4, 8 and 12 weeks after operation was 0.106% ± 0.015%, 0.292% ± 0.019%, 0.457% ± 0.048% and 0.601% ± 0.037%, while those in the control side was 0, 0.193% ± 0.019%, 0.339% ± 0.029% and 0.574% ± 0.047%respectively. At each time point, the experimental side produced better new bone formation than the control side did (P lt; 0.05). The experimental side showed better compatibil ity between plates and surrounding tissue, faster bone formation, more bone regeneration mass and better medullary canal structure than the control side. Conclusion PDLLA plate containing rhBMP-2 has good biocompatibil ity and osteoinducibil ity to enhance fracture heal ing.
Objective To investigate the biocompatibil ity of sil ica gel embedded permanent magnets of themicturition alert device dedicated to neurogenic bladder. Methods According to the national standards of biologicalevaluation of medical equipment (GB/T 16886), Shanghai Biomaterial Research and Test Center was confided to evaluate the biocompatibil ity of sil ica gel embedded permanent magnets both in vitro and in vivo, including cytotoxicity test, sensitization test, primary skin irritant test and acute general toxicity test. The cytotoxicity test was performed according to the agar diffusion method. The L929 cell discoloration index and cell lysis index were counted at 24 hours after the action of the specimen. The sensitization test was performed according to the maximal dose method. The skin response was evaluated in 30 male albino guinea-pigs at 24 and 48 hours after the routine induction and provocation of leaching l iquors of the specimen. The primary skin irritant test was evaluated in 2 male healthy New Zealand rabbits according to the local tissue response at 24, 48 and 72 hours after intradermal injection of leaching l iquors of the specimen. The acute general toxicity test was evaluated in 10 male Kumming mice musculus albus according to animal condition at 4, 24, 48 and 72 hours after injection of leaching l iquors of the specimen through the caudal vein. Both the general reaction of canines and the pathology of the local bladder walls were observed at 2, 4 and 8 weeks after a permanent magnet was fixed on the anterior wall of urinary bladder in three canines. Results No sensitization, no stimulation and no acute general toxicity were observed except sl ight cytotoxicity to sil ica gel embeddedpermanent magnets. After implantation of a permanent magnet, the canines showed excellent tolerace, which manifested as no abnormal ity in spirit, appetite, urine and stool, healed wounds and no infection. Adhesions occurred between the epiploon and the bladder wall around the permanent magnet in two canines at 2 and 4 weeks, and between the lower abdominal wall and the bladder wall around the permanent magnet in the other canine at 8 weeks. The local bladder wall below permanent magnet was thickened, the fibrous capsule around the permanent magnet was thin, but the bladder mucosa was normal. Inflammatory reaction such as congestion, edema and inflammatory cells lessened from the serosa layer to the mucosa layer microscopically. Conclusion Sil ica gel embedded permanent magnets used in the micturition alert device dedicated to neurogenic bladde has excellent biocompatibil ity and meet the criteria for cl inical appl ication．
Objective To produce a decellularized cartilage matrix (DCM) and investigate its possibil ity to be used as a scaffold for tissue engineering. Methods Fresh bovine articular cartilage from knee joints was sl iced, freeze-dried and freeze-ground into fine powder, and then was treated sequentially with Trypsin, Triton X-100 and hypotonic solution for decellularization. The decellularized matrix was freeze-dried for shaping and cross-l inked by UV radiation. Histological, immunohistological, SEM, porosity assays and biomechanical assays were used to characterize the DCM. BMSCs were isolated from rabbit bone marrow aspirate and cultured in DCM extraction medium of different concentration (100%, 10% and 1%) for 0, 24, 48 and 72 hours, respectively, to detect the release rate of the lactate dehydrogenase (LDH). The DMEM medium (5% FBS) served as the control. Biocompatibil ity was evaluated using BMSCs (1 × 107/mL) cultured with DCM. Results The DCM showed white spongy appearances, and histological analysis showed that the material was constructed by cartilage particles without any cells or cell fragments left in the matrix. Immunohistology staining and alcian blue staining showed that DCM retained the collagen and glycosaminoglycan components of cartilaginous matrix. SEM scanning showed that DCM had a porous spongy-l ike structure with the aperture ranging 30-150 μm .The porosity assay showed that the average porosity was 89.37% and the average aperture was 90.8 μm. The mechanical assay showed that there was no difference for the compress module before and after the decellularization process, which was (17.91 ± 0.98) MPa and (15.12 ± 0.77) MPa, respectively (P gt; 0.05), but were both statistical different from normal articular cartilage [(26.30 ± 1.98) MPa, P lt; 0.05]. The LDH release rate in the DCM extraction medium of different concentration was not significantly different from that in the normal DMEM medium (P gt; 0.05). The cell adhesion test showed BMSCs grew well on DCM without any signs of growth inhibition. Conclusion Articular cartilage can be decellularized and fabricated into a scaffold which retains the major components of cartilaginous matrix. DCM has goodbiochemical, biophysical characteristics and good biocompatibil ity with cultured BMSCs and may be used as a novel scaffold for tissue engineering studies.