The change of Ig-forming cells in the gallbladder mucoderm were studied in the rabbit models.One hundred rabbits were randomly divided into the control group(Con,n=10),simple biliary obstruction group(BO,n=45)and biliary obstruction and infection group(BOI,n=45).The results showed that only a few Ig-forming cells presented in the gallbladder mucoderm of normal rabbit.At the 3rd,7th and 14th day,the quantities of IgG and IgA-forming cells in the mucoderm in BO group remained unchange,but increased much higher in BOI group(Plt;0.001),especially in IgG formation.This study suggests that the gallbladder of rabbit may be the important place of Ig-formation.The quantities of Ig-forming cells in bilitary tract may have a close relationship with the gallstone formation.
Objective To study the biological characteristic of rabbit bone marrow mesenchymal stem cells (BMSCs) double-labeled by PKH26 and BrdU in vitro, and to construct tissue engineered cardiac patch in vitro. Methods The BMSCs were harvested from 6-month-old New Zealand rabbits and labeled with PKH26 and BrdU. The growth and fluorescent intensitywere observed by inverted phase contrast microscope, fluorescent microscope, flow cytometry, and MTT detection. Thecharacteristics of double-labeled BMSCs differentiating into osteoblasts and adipocytes, respectively, in vitro were identified by alkal ine phosphatase (ALP) staining, Al izarin red staining, Oil red O staining, immunocytochemical technique of collagen type I, and osteocalcin expression. The labeled BMSCs were seeded on the small intestinal submucosa (SIS) and co-cultured for 5-7 days to construct tissue engineered cardiac patch. The patches were tested by inverted phase contrast microscope, fluorescent microscope, scanning electron microscope, and HE staining to observe the cell prol iferation. Results The double-labeled cells grew well and showed red fluorescence. There was no significant difference in the growth characteristic between the labeled and unlabeled cells. There was no significant difference in the expression of stem cell specific surface antigen between before lebel ing and after lebel ing. After osteogenic induction of labeled BMSCs, ALP staining and Al izarin red staining were positive, and the cells expressed collagen type I and osteocalcin. After adipocytes induction, l ipid droplets could be observed in cytoplasm by Oil red O staining. After the co-culture in vitro for 5-7 days, the double-labeled cells grew well, showing a multi-layer cellular structure on the surface of SIS. Conclusion Rabbit BMSCs can be double-labeled with PKH26 and BrdU stably. The labeled cells still have the potential of self-renewal abil ity and multipotent differentiation abil ity; tissue engineered cardiac patch can be constructed by co-culturing labeled BMSCs and SIS in vitro.
Objective To compare the growth and extracellular matrix biosynthesis of nucleus pulposus cells (NPCs)and bone marrow mesenchymal stem cells (BMSCs) in thermo-sensitive chitosan hydrogel and to choose seed cells for injectable tissue engineered nucleus pulposus. Methods NPCs were isolated and cultured from 3-week-old New Zealand rabbits (male or female, weighing 150-200 g). BMSCs were isolated and cultured from bone marrow of 1-month-old New Zealand rabbits (male or female, weighing 1.0-1.5 kg). The thermo-sensitive chitosan hydrogel scaffold was made of chitosan, disodium β glycerophosphate, and hydroxyethyl cellulose. Then, NPCs at the 2nd passage or BMSCs at the 3rd passage were mixed with chitosan hydrogel to prepare NPCs or BMSCs-chitosan hydrogel complex as injectable tissue engineered nucleus pulposus. The viabil ities of NPCs and BMSCs in the chitosan hydrogel were observed 2 days after compound culture. The shapes and distributions of NPCs and BMSCs on the scaffold were observed by scanning electron microscope (SEM) 1 week after compound culture. The histology and immunohistochemistry examination were performed. The expressions of aggrecan and collagen type II mRNA were analyzed by RT-PCR 3 weeks after compound culture. Results The thermo-sensitive chitosan hydrogel was l iquid at room temperature and sol idified into gel at37 (after 15 minutes) due to crossl inking reaction. Acridine orange/propidium iodide staining showed that the viabil ity rates of NPCs and BMSCs in chitosan hydrogel were above 90%. The SEM observation demonstrated that the NPCs and BMSCs distributed in the reticulate scaffold, with extracellular matrix on their surfaces. The results of HE, safranin O histology and immunohistochemistry staining confirmed that the NPCs and BMSCs in chitosan hydrogel were capable of producing extracellular matrix. RT-PCR results showed that the expressions of collagen type II and aggrecan mRNA were 0.564 ± 0.071 and 0.725 ± 0.046 in NPCs culture with chitosan hydrogel, and 0.713 ± 0.058 and 0.852 ± 0.076 in BMSCs culture with chitosan hydrogel; showing significant difference (P lt; 0.05). Conclusion The thermo-sensitive chitosan hydrogel has good cellular compatibil ity. BMSCs culture with chitosan hydrogel maintains better cell shape, prol iferation, and extracellular matrix biosynthesis than NPCs.
Objective Platelet-rich plasma (PRP) contains high concentrations of platelets and leucocytes, which play a key role in antimicrobial host defense system. To evaluate the antimicrobial efficacy of autologous PRP in vitro and in vivo and to explore the mechanism of action so as to provide the experimental basis for the prevention and treatment of bone infection. Methods PRP was prepared with the method of two centrifugation from 15 health volunteers. Platelet-leukocytegel (PLG) was obtained after activation of PRP with bovine thrombin. Next, PLG was incubated with Staphylococcus aureus (1 × 106 cfu/mL) in vitro compared with PRP, platelet-poor plasma (PPP) and PBS. Samples were taken out after 2, 4, 6, 8, 12, and 24 hours for bacterial culture and colony count. Thirty-six New Zealand adult rabbits, weighing (2.85 ± 0.11) kg, were divided into 4 groups: PLG (n=10), antibiotic (n=10), infection (n=10), and PBS (n=6) groups. The osteomyel itis models were made by injecting 0.1 mL Staphylococcus aureus suspension (1 × 106 cfu/mL) into the tibial canal in PLG group, antibiotic group, and infection group; equal volumes of PBS was injected in PBS group as a control. Autologous PLG was injected immediately after operation in PLG group. Cefazol in (30 mg/kg) was injected through the auricular vein from 1 hour before operation to 72 hours after operation in antibiotic group, once per 8 hours. No treatment was given in infection and PBS groups. The efficacy of PLG for osteomyel itis prophylaxis was evaluated by microbiological, X-ray and histological observation within 28 days. Results The contents of leucocyte and platelet of PRP were 6.2 times and 5.5 times of whole blood, showing signficant differences ((P lt; 0.05); the contents of leucocyte and platelet of PPP were significantly lower than those of whole blood and PRP ((P lt; 0.05). In vitro test showed that PLG had the most obvious bacteriostasis effect. The bacterial count reached a minimum value at 4 hours after incubation in PLG and at 6 hours after incubation in PRP. PPP had slow and no obvious bacteriostasis effect and PBS had no bacteriostasis effect. At 2, 4, 6, 8, 12, and 24 hours of incubation, the bacterial count reduced significantly when compared PLG with PRP and PPP (P lt; 0.05), when compared PRP with PPP (P lt; 0.05). In PLG group and antibiotic group, 1 rabbit died, respectively; 34 rabbits survived to the end of the experiment. There was no significant difference (P gt; 0.05) in temperature, body weight, erythrocyte sedimentation rate and content of leucocyte between 28 days after operation andbefore operation in 4 groups. After 28 days, the X-ray scores were 2.78 ± 1.39, 1.55 ± 1.48, 4.17 ± 1.25, and 0 in PLG, antibiotic,infection, and PBS groups, respectively, which was significantly higher in infection group than in other 3 groups ((P lt; 0.05). Also, the histological scores were 5.89 ± 3.92, 3.00 ± 2.31, 10.33 ± 4.03, and 0, respectively, which was significantly higher in infection group than in other 3 groups (P lt; 0.05), and was significantly lower in antibiotic group than in PLG group ((P lt; 0.05). The results of bacterial culture showed that the infection rates of PLG group (44.4%) and antibiotic group (20.0%) were significantly lower ((P lt; 0.05) than that of infection group (88.9%). The quantitative analysis of bacteria showed that the number of bacteria was signifcantly lower ((P lt; 0.05) in PLG and antibiotic groups than in infection group. Conclusion PRP forms into PLG after activating, it can inhibit Staphylococcus aureus reproduction in vitro and can effectively prevent bone infection in vivo.
To investigate the preventive effect of TGF-β1 neutral izing antibody on collagen production and adhesion formation of flexor tendon. Methods Tendon fibroblasts, epitenon tenocytes, and endotenon tenocytes were obtained from 6 New Zealand rabbit flexor tendons. Each cell culture was supplemented with 1 ng/mL of TGF-β along with increasing dose of TGF-β1 neutral izing antibody. Col I production was measured by enzyme-l inked immunoabsorbent assay after 3 days. Eighty-four adult New Zealand White rabbits forepaws underwent sharp transection of middle digit flexor digitorumprofundus and immediate repair. Then the rabbits were divided into three groups: the normal saline (NS group, n=36), 1.0 µg/ mL TGF-β1neutral izing antibody (1.0 µg/mL TGF-β1group, n=36) and 2.0 µg/mL TGF-β1 neutral izing antibody (2.0 µg/mL TGF-β1 group, n=12) were injected in tendon sheath respectively. Tendons were harvested at 4 and 8 weeks for biomechanics testing, histological evaluation and scanning electron microscope observation. Tendons were harvested at 1, 2, 4 and 8 weeks to determine the mRNA expression of TGF-β1 and Col I by in situ hybridization. Results ELISA exhibed that TGF-β1 enhanced Col I production and the neutral izing antibody significantly inhibited TGF-β1-induced Col I production in all 3 cell culture with a dose-dependent. At 4 and 8 weeks after operation the gl iding excursion of the tendon and the simulated active flexion in NS group were less than that of 1.0 µg/mL TGF-β1 group and 2.0 µ g/mL TGF-β1 group. There was significant difference between NS group and 1.0 µ g/mL TGF-β1 group, 2.0 µ g/mL TGF-β1 group (P lt; 0.05). The tendon anastomosis breaking strength showed no significant differences among three groups (P gt; 0.05). Scanning electron microscope and histological observation showed that collagen fibers arranged irregularly in NS group, but arranged regularly in 1.0 µ g/mL TGF-β1 group and 2.0 µ g/mL TGF-β1group at 4 and 8 weeks after operation. The in situ hybridization results revealed that TGF-β1 and Col I mRNA expression in 1.0 µ g/mL TGF-β1 group was lower than that in NS group at each time. There was significant difference between two groups (P lt; 0.05). Conclusion TGF-β1neutral izing antibody can inhibit the function of the TGF-β1 effectively and prevent adhesion formation after the flexor tendon injured and repaired.
【Abstract】 Objective To establ ish a stable animal model for glucocorticoid-induced avascular necrosis of femoral head in rabbits. Methods Thirty-six adult New Zealand rabbits were randomly divided into four groups:ten were injected twice with l i popolysaccharide (group A), ten were treated with a combination of l i popolysaccharideand methylprednisolone (group B), ten were injected three times with methylprednisolone (group C), and six wereinjected normal sal ine as a control (group D). MR imaging was performed in the rabbits before the first injection ofl i popolysaccharide or methylprednisolone, and at 2, 4, and 6 weeks after the last injection of l i popolysaccharide ormethylprednisolone. Histopathological changes in the femoral heads were observed by l ight microscope and transmission electron microscope at the end of six weeks after the injection. Vascular infusion with Chinese ink was made to evaluate the morphological changes of blood vessels in the femoral head. The percentage of trabecular bone area and empty lacunae and microvascular density were measured. According to the histological and MR imaging appearance of the femoral heads in all groups, the incidence of osteonecrosis of every group was calculated. Results Listlessness, blepharal hyperemia,less activity and reduced diet were found in the rabbits of groups A and B after injected with l ipopolysaccharide. At 3 weeks after the final injection, the body weight of groups B and C was decreased. At 4 weeks after the final injection, the body weight of groups A and D was increased. No abnormal signal could be detected on MR images in rabbits of all groupsbefore injection and at 2 weeks after the injection. At 4 weeks and 6 weeks after the last injection, irregular low signal on T1-weighted images and irregular low or high signal on T2-weighted images could be detected on MR images in rabbits of groups B and C, no abnormal signal could be detected on MR images in rabbits of groups A and D. At 6 weeks after the last injection,the trabecular bone of group B became thin and sparse, some were broken. The percentages of empty lacunae were 11.8% ± 4.7%, 34.4% ± 6.2%, 20.0% ± 4.7% and 9.3% ± 4.6%; the percentages of trabecular bone area were 59.2% ± 6.8%, 40.1% ± 6.0%, 51.5% ± 5.6% and 63.2% ± 8.3%; and the microvascular densities were 14.3% ± 2.7%, 4.5% ± 2.1%, 10.2% ± 3.1% and 15.4% ± 4.1% in groups A, B, C and D respectively. There were statistically significant differences between group B and groups A, C, D (P lt;0.01). The fatty tamponade accumulated in the medullary cavity and intramedullary vascular sinusoids were pressed by the l ipocytes and became narrow. Limposomes were found in osteocytes and vascular endothel ia of group B and group C. Osteocytes of group B crimpled and pyknosis or karyolysis of chromatin were observed in these osteocytes, nuclearmembrane of the osteocytes was discontinous. Vascular endothel ia became swollen and the cell junctions widened or were destroyed in groups A and B. The incidence of osteonecrosis in group B (88.9%) was higher than that in group C (22.2%, P lt; 0.05). There was no osteonecrosis occurred in groups A and D . Conclusion Methylprednisolone combined with l ipopolysaccharide can induce typical rabbit model for early avascular necrosis of femoral head.
【Abstract】 Objective To investigate the in vivo osteogenic feasibil ity of tissue engineered periosteum constructedby porcine SIS and BMSCs in allogenic New Zealand rabbit. Methods The tissue engineered periosteum constructed by SIS scaffold and BMSCs was prepared in vitro .Twelve 2-month-old New Zealand rabbits were used in the experiments. The 1.5-2.0 cm critical bone defects were made in the both sides of radius of the animals. The tissue engineered periosteum was grafted into one side defect randomly, while the other side defect was only grafted SIS. Four weeks after operation, the forearms of all animals were checked by X-ray. Then, animals were sacrificed to harvest the specimen which were treated promptly for HE and Masson staining.The X-ray film and the morphological tissue staining outcome were evaluated qual itatively. Results After operation,all animals had a normal behavior and diet; the incision healed normally; the forearm could move normally for bearing weight.The tissue engineered periosteum constructed by allogenic BMSCs and heterogeneic SIS scaffold could form new bone tissue, andbridged the bone defect which could be confirmed either in X-ray film or histological staining. The newly formed bone tissue had similar bone density to normal bone. A lot of irregular newly formed vessels and medullary cavity inserted in the newly borned tissue. No lymphocytes infiltrated in histological examination. While the control side had no any osteogenesis neithter in X-ray, nor in HE and Masson staining inspecting; the defect space only occupied with some connective tissue. Conc lu sion Tissue engineered periosteum can form new bone in allogenic rabbit and has the feasibil ity to repair the segmental diaphysis defect.
Objective To explore the biocompatibility of poly(lacticacid/glycolic acid/asparagic acid-co-polyethylene glycol) biomaterials (PLGA-ASP-PEG) and biological behaviors of cultured marrow stroml stem cells (MSCs) combined with this new type of scaffold in tissue engineering. Methods The PLGA-ASP-PEG tri-block copolymers were obtained through bulk ringopening copolymerization method.MSCs were isolated from the bone marrow of 4 week old New Zealand rabbits. The 3rdgeneration MSCs were cultured combining with PLGA-ASP-PEG in vitro, while cells cultured in PLGA as control group. The cell adhesion rate and the adhesivepower were examined by conventional precipitation method and micropipette aspiration technique respectively. The morphological features were studied by scanning electron microscope. The proliferation behavior of the cells was analyzed by MTT assay. The cell cycle, proliferation index, DNA index and apoptosis of the cells were detected by flow cytometry. The synthesis of protein and collagen were examined by Coomassie Brilliant Blue dyes and 3H-Proline incorporation test. Results The MSCs adhered and grew well on the surface of the biomaterial PLGA-ASP-PEG. The powers of cell adhesion, proliferation and protein and collagen synthesis of the cells were all significantly higher than those of PLGA group (P＜0.05), but the apoptosis rate was significantly lower than that of PLGA group (P＜0.05). The DNA indexes showed the cells of both PLGA-ASP-PEG group and PLGAgroup were normal diploid cells. Conclusion PLGA-ASP-PEG showedgood biocompatibilityand the biological properties improved greatly compared with the PLGA scaffold materials. These results demonstrated that the promise of PLGAASPPEG canbe used as an ideal scaffold material for construction of tissue engineered bone to restore bone defects in bone tissue engineering．
Objective To investigate the effect of the injectable osteoinductive material with fibrin sealant(FS) as a carrier compounded with bone morphogenetic protein (BMP) on the proliferation and differentiation of marrow stromal cells (MSCs) towards osteoblasts and to provide the experimental foundation for the clinical application. Methods MSCs were extracted and cultured from bone marrow of the 3-day-old rabbit, and the third generation culturedMSCs were studied. The experiment included the experimental group(FS,including 1 μg/ml rhBMP-2), FS control group（FS）and blank control group (no material).The proliferation rate, the adhesive rate, the expression of the collagen Ⅰ and alkaline phosphatase, cell growth condition in the material and the ultrastructure of MSCs were investigated by electron microscopy, histochemistry and cell culture. Results The proliferation rate and the adhesive rate of MSCs in experimental group was significantly higher than those in blank control group ,but lower than those in FS control group （P＜0.05）. The expression level of thecollagen Ⅰ and alkaline phosphatase in the experimental group was significantlyhigher than those in all control groups（Marrow stromal cells Fibrin sealant Bone morphogenetic protein Cell culture Rabbits0.05）. Scanning electron microscope showed that the surface of material was rough and had many pores and that celland material mixed. Transmission electron microscope showed that MSCs of the experimental group were mostly of the phenotype of osteoblasts with relatively lowproliferation activity and high differentiation degree toward osteoblasts and with plenty of extracellular matrix and collagen fibers. MSCs of FS control group had low differentiation degree toward osteoblasts with few extracellular matrix and collagen fibers and high proliferation activity. MSCs of blank control group had low differentiation degree toward osteoblasts with few extracellularmatrix and collagen fibers, and low proliferation activity. Conclusion The injectable osteoinductive material with fibrin sealant as a carrier compounded with BMP could significantly accelerate the differentiation of MSCs towards osteoblasts. But it could not significantly accelerate the proliferation activity of MSCs.
Objective To compare the effect of guiding boneregeneration between l-ethyl-3(3-diaminopropyol)-carbodiimide(EDAC)crosslinked acellular bovine pericardium (ABP) and medical collagen membrane (CM). Methods Defects of 7 mm×7 mm×5 mm were created in both mandibles of 24 rabbits, which weighted 2.6～3.5 kg. One side defect was covered with EDAC-crosslinked ABP(EDAC-crosslinked ABP group), the other side defect with medical CM as control(CM group). The ability of bone defect repair and change ofboth membrane materials were evaluated by gross observation, histological study and computer graphic analysis in the 4th, 8th, 16th and 24th weeks after operation. Results The surface of bone defects was even, consistent with adjacent normal bonein EDACcrosslinked ABP group, while that of bone defects was of no evenness in CM group in the 16th and the 24th weeks. The histological observation showed that bone trabecula formed in the EDAC-crosslinked ABP group and fibrous connective tissue was seen in CM group in the 16th and the 24th weeks. There were no significant differences in new bone percentage of bone defects between 2 groups inthe 4th and the 8th weeks(P＞0.05). In the 16th week new bone percentage of bone defects was 81.99%±3.92% in EDAC-crosslinked ABP group and 76.35%±4.29% in CM group, showing significant difference (Plt;0.05). The average percentage of absorption in EDAC-crosslinked ABP group was 16.57%, 27.94%, 65.61% and85.72% in the 4th, 8th, 16th and 24th weeks respectively, while that in CM group was more than 50% in the 4th week and completely degraded at the end of 8 weeks. Conclusion EDAC-crosslinked ABP has a better effect on guiding bone regeneration than CM in the repair of bone defects.