Objective To evaluate the effect on microvessel density (MVD) and vascular endothelial growth factor (VEGF) expression of combining radiofrequency ablation (RFA) with arsenious acid (AA) locally treating liver VX2 tumor in rabbits. Methods Twenty-eight New Zealand White rabbits with implanted liver VX2 tumors were randomly divided into four groups, control group (n=7), AA group (n=7), RFA group (n=7) and combination (RFA+AA) group (n=7). All rabbits were killed 14 days after treatment. MVD and VEGF expression were examined by immunohistochemistry. Results The MVD degraded one by one in control group,AA group,RFA group and RAF+AA group, which were (38.50±0.44), (23.07±0.47), (18.65±0.39) and (11.36±0.36)/HP respectively, compared while each two groups, P＜0.05. The VEGF expression also degraded one by one, the ratio of positive cases were 7/7, 5/7, 4/7 and 2/7 respectively, compared while each two groups, P＜0.05. There was positive correlation between VEGF expression and MVD (Person conefficient of product-moment correlation r=0.47, P＜0.01). Conclusion Combining RAF with AA therapy can greatly decrease MVD and VEGF expression of tumor tissue.
ObjectiveTo observe the change of stromal cell-derived factor 1α/cysteine X cysteine receptor 4 (SDF-1α/CXCR4) signaling pathway during the process of axial stress stimulation promoting bone regeneration, and to further explore its mechanism.MethodsA total of 72 male New Zealand white rabbits were selected to prepare the single cortical bone defect in diameter of 8 mm at the proximal end of the right tibia that repaired with deproteinized cancellous bone. All models were randomly divided into 3 groups (n=24). Group A was treated with intraperitoneally injection of PBS; Group B was treated with stress stimulation and intraperitoneally injection of PBS; Group C was treated with stress stimulation and intraperitoneally injection of AMD3100 solution. The X-ray films were taken and Lane-Sandhu scores of bone healing were scored at 2, 4, 8, and 12 weeks after operation, while specimens were harvested for HE staining, immunohistochemical staining of vascular endothelial growth factor (VEGF) and CXCR4, and Western blot (SDF-1α and CXCR4). The bone healing area was scanned by Micro-CT at 12 weeks after operation, and the volume and density of new bone were calculated.ResultsX-ray film showed that the Lane-Sandhu scores of bone healing in group B were significantly higher than those in groups A and C at 4, 8, and 12 weeks after operation (P<0.05). Micro-CT scan showed that the bone defect was repaired in group B and the pulp cavity was re-passed at 12 weeks after operation. The volume and density of new bone were higher in group B than in groups A and C (P<0.05). HE staining showed that the new bone growth in bone defect area and the degradation of scaffolds were faster in group B than in groups A and C after 4 weeks. The immunohistochemical staining showed that the expressions of VEGF and CXCR4 in 3 groups reached the peak at 4 weeks, and group B was higher than groups A and C (P<0.05). Western blot analysis showed that the expressions of SDF-1α and CXCR4 in group B were significantly higher than those in groups A and C at 4 and 8 weeks after operation (P<0.05).ConclusionAxial stress stimulation can promote the expression of SDF-1α in bone defect tissue, activate and regulate the CXCR4 signal collected by marrow mesenchymal stem cells, and accelerate bone regeneration in bone defect area.
The aim of this article is to study how andrographolide-releasing collagen scaffolds influence rabbit articular chondrocytes in maintaining their specific phenotype under inflammatory environment. Physical blending combined with vacuum freeze-drying method was utilized to prepare the andrographolide-releasing collagen scaffold. The characteristics of scaffold including its surface morphology and porosity were detected with environmental scanning electron microscope (ESEM) and a density instrument. Then, the release of andrographolide from prepared scaffolds was measured by UV-visible spectroscopy. Rabbit chondrocytes were isolated and cultured in vitro and seeded on andrographolide-releasing collagen scaffolds. Following culture with normal medium for 3 d, seeded chondrocytes were cultured with medium containing interleukin-1 beta (IL-1β) to stimulate inflammation in vitro for 7 d. The proliferation, morphology and gene transcription of tested chondrocytes were detected with Alamar Blue assay, fluorescein diacetate (FDA) staining and reverse-transcriptase quantitative polymerase chain reaction (RT-qPCR) test respectively. The results showed that the collagen scaffolds prepared by vacuum freeze-dry possess a high porosity close to 96%, and well-interconnected chambers around (120.7±17.8) μm. The andrographolide-releasing collagen scaffold continuously released andrographolide to the PBS solution within 15 d, and collagen scaffolds containing 2.22% andrographolide significantly inhibit the proliferation of chondrocytes. Compared with collagen scaffolds, 0.44% andrographolide-containing collagen scaffolds facilitate chondrocytes to keep specific normal morphologies following 7 d IL-1β induction. The results obtained by RT-qPCR confirmed this effect by enhancing the transcription of tissue inhibitor of metalloproteinase-1 (TIMP-1), collagen II (COL II), aggrecan (Aggrecan) and the ratio of COL II/ collagen I(COL I), meanwhile, reversing the promoted transcription of matrix metalloproteinase-1 (MMP-1) and matrix metalloproteinase-13 (MMP-13). In conclusion, our research reveals that andrographolide-releasing (0.44%) collagen scaffolds enhance the ability of chondrocytes to maintain their specific morphologies by up-regulating the transcription of genes like COL II, Aggrecan and TIMP-1, while down-regulating the transcription of genes like MMP-1 and MMP-13 which are bad for phenotypic maintenance under IL-1β simulated inflammatory environment. These results implied the potential use of andrographolide-releasing collagen scaffold in osteoarthritic cartilage repair.
ObjectiveTo investigate the effect of nicotinamide mononucleotide adenosyl transferase 3 (NMNAT3) on the mitochondrial function and anti-oxidative stress of rabbit bone marrow mesenchymal stem cells (BMSCs) under oxidative stress in vitro by regulating nicotinamide adenine dinucleotide (NAD+) levels.MethodsThe bone marrow of femur and tibia of New Zealand white rabbits were extracted. BMSCs were isolated and cultured in vitro by density gradient centrifugation combined with adherent culture. The third generation cells were identified by flow cytometry and multi-directional induction. Overexpression of NMNAT3 gene was transfected into rabbit BMSCs by enhanced green fluorescent protein (EGFP) labeled lentivirus (BMSCs/Lv-NMNAT3-EGFP), and then the expression of NMNAT3 was detected by real-time fluorescence quantitative PCR (qRT-PCR) and Western blot and cell proliferation by cell counting kit 8 (CCK-8) method. BMSCs transfected with negative lentivirus (BMSCs/Lv-EGFP) and untransfected BMSCs were used as controls. The oxidative stress injury cell model was established by using H2O2 to treat rabbit BMSCs. According to the experimental treatment conditions, they were divided into 4 groups: Group A was normal BMSCs without H2O2 treatment; untransfected BMSCs, BMSCs/Lv-EGFP, and BMSCs/Lv-NMNAT3-EGFP in groups B, C, and D were treated with H2O2 simulated oxidative stress, respectively. The effects of NMNAT3 on the mitochondrial function of BMSCs under oxidative stress [changes of mitochondrial membrane potential, NAD+ and adenosine triphosphate (ATP) levels], the changes of anti-oxidative stress ability of BMSCs [reactive oxygen species (ROS) and malondialdehyde (MDA) levels, manganese superoxide dismutase (Mn-SOD) and catalase (CAT) activities], and the effects of BMSCs on senescence and apoptosis [senescence associated-β-galactosidase (SA-β-gal) staining and TUNEL staining] were detected after 24 hours of treatment.ResultsThe rabbit BMSCs were successfully isolated and cultured in vitro. The stable strain of rabbit BMSCs with high expression of NMNAT3 gene was successfully obtained by lentiviral transfection, and the expressions of NMNAT3 gene and protein significantly increased (P<0.05). There was no significant difference in the trend of cell proliferation compared with normal BMSCs. After treatment with H2O2, the function of mitochondria was damaged and apoptosis increased in all groups. However, compared with groups B and C, the group D showed that the mitochondrial function of BMSCs improved, the membrane potential increased, the level of NAD+ and ATP synthesis of mitochondria increased; the anti-oxidative stress ability of BMSCs enhanced, the levels of ROS and MDA decreased, and the activities of antioxidant enzymes (Mn-SOD, CAT) increased; and the proportion of SA-β-gal positive cells and the rate of apoptosis decreased. The differences in all indicators between group D and groups B and C were significant (P<0.05).ConclusionNMNAT3 can effectively improve the mitochondrial function of rabbit BMSCs via increasing the NAD+ levels, and enhance its anti-oxidative stress and improve the survival of BMSCs under oxidative stress conditions.
An experimental model of proliferative vitretinopathy(PVR) induced by macrophages was used for the evaluation of drug efficacy of daunomycin encapsulated in liposomes in the treatment of PVR.Five mu;g daunomycin(n=40),10mu;g daunomycin-liposome(DL,n=30)and 0.1 ml saline or empty liposomes(n=40,as controls)were injected into the rabbit vitreous after macrophage injection.Retinal detachment developed in 77.5% of the control eyes on day 28,compared to 33.3% of the eyes treated with DL(P＜0.01)and 50% of the daunomycin-treated eyes(P＜0.05).The results suggest that encapsulation in liposomes of cytotoxic agents can enhance drug efficacy.The phasic course of development of PVR is important in the selection of particular drugs. （Chin J Ocul Fundus Dis,1993,9:77-80）
Objective To investigate the effects of long time different negative pressures on osteogenic diffe-rentiation of rabbit bone mesenchymal stem cells (BMSCs). Methods The rabbit BMSCs were isolated and cultured by density gradient centrifugation. Flow cytometry was used to analyze expression of surface markers. The third passage cells cultured under condition of osteogenic induction and under different negative pressure of 0 mm Hg (control group), 75 mm Hg (low negative pressure group), and 150 mm Hg (high negative pressure group) (1 mm Hg=0.133 kPa), and the negative pressure time was 30 min/h. Cell growth was observed under phase contrast microscopy, and the growth curve was drawn; alkaline phosphatase (ALP) activity was detected by ELISA after induced for 3, 7, and 14 days. The mRNA and protein expressions of collagen type I (COL-I) and osteocalcin (OC) in BMSCs were analyzed by real-time fluorescence quantitative PCR and Western blot. Results The cultured cells were identified as BMSCs by flow cytometry. The third passage BMSCs exhibited typical long shuttle and irregular shape. Cell proliferation was inhibited with the increase of negative pressure. After induced for 4 days, the cell number of high negative pressure group was significantly less than that in control group and low negative pressure group (P<0.05), but there was no significant difference between the low negative pressure group and the control group (P>0.05); at 5-7 days, the cell number showed significant difference between 3 groups (P<0.05). The greater the negative pressure was, the greater the inhibition of cell proliferation was. There was no significant difference in ALP activity between groups at 3 days after induction (P>0.05); the ALP activity showed significant difference (P<0.05) between the high negative pressure group and the control group at 7 days after induction; and significant difference was found in the ALP activity between 3 groups at 14 days after induction (P<0.05). The greater the negative pressure was, the higher the ALP activity was. Real-time fluorescence quantitative PCR and Western blot detection showed that the mRNA and protein expressions of COL-I and OC protein were significantly higher in low negative pressure group and high negative pressure group than control group (P<0.05), and in the high negative pressure group than the low negative pressure group (P<0.05). Conclusion With the increase of the negative pressure, the osteogenic differentiation ability of BMSCs increases gradually, but the cell proliferation is inhibited.
In order to repair cartilage defect in joint with transplantation of cryopreserved homologous embryonic periosteum, 30 rabbits were used and divided into two groups. A 4 mm x 7 mm whole thickness cartilage defect was made in the patellar groove of femur of each rabbit. The homologous embryonic rabbit skull periosteum (ERSP), preserved in two-step freezing schedule, was transplanted onto the cartilage defect of joints of one group and autogenous periosteal graft was done in the joint defect of the other group. The knees were not immobilized, following operation and 16 weeks later, the newly formed tissue in the defects were assessed by gross observation, histochemical examination and biochemical analysis. The results showed that new hyaline-like cartilage was formed in the cryopreserved ERSP grafted knee, and had no significant difference from that of the knee receiving autogenous periosteal graft, but had significant difference from that of the fresh ERSP grafted knee and the non-grafted knee. Furthermore, the new hyaline-like cartilage had the biochemical characteristics of a fibrous cartilage. The conclusion was that this method might be feasible to repair articular cartilage defects.
ObjectiveTo investigate the effect of microencapsulated transgenic bone marrow mesenchymal stem cells (BMSCs) transplantation on early steroid induced osteonecrosis of femoral head (SONFH) in rabbits.MethodsAlginate poly-L-lysine-sodium alginate (APA) microencapsulated transgenic BMSCs with high expression of Foxc2 were prepared by high-voltage electrostatic method. Part of the cells were cultured in osteoblasts and observed by alizarin red staining at 2 and 3 weeks. Forty New Zealand white rabbits were used to prepare SONFH models by using hormone and endotoxin. Thirty two rabbits who were successful modeling were screened out by MRI and randomly divided into 4 groups (groups A, B, C and D, n=8); another 6 normal rabbits were taken as normal control (group E). The rabbits in group A did not receive any treatment; and in groups B, C, and D were injected with normal saline, allogeneic BMSCs, and APA microencapsulated transgenic BMSCs respectively after core decompression. At 6 and 12 weeks after operation, specimens of femoral head were taken for HE staining to observe bone ingrowth; the expressions of osteocalcin (OCN), peroxisome proliferative activated receptor γ 2 (PPARγ-2), and vascular endothelial growth factor (VEGF) proteins were observed by immunohistochemistry staining. At 12 weeks after operation, the bone microstructure was observed by transmission electron microscope, and the maximum compressive strength and average elastic modulus of cancellous bone and subchondral bone were measured by biomechanics.ResultsAfter 2 and 3 weeks of induction culture, alizarin red staining showed the formation of calcium nodules, and the number of calcium nodules increased at 3 weeks when compared with 2 weeks. The rabbits in each group survived until the experiment was completed. Compared with groups A, B, and C, the trabeculae of group D were more orderly, the empty bone lacunae were less, there were abundant functional organelles, and obvious osteogenesis was observed, and the necrotic area was completely repaired at 12 weeks. Immunohistochemical staining showed that, at 6 and 12 weeks after operation, the expressions of OCN and VEGF in groups A, B, and C were significantly lower than those in groups D and E, while those in groups B and C were significantly higher than those in group A, and in group E than in group D (P<0.05). The expression of PPARγ-2 was significantly higher in groups A, B, and C than in groups D and E, and in group A than in groups B and C, and in group D than in group E (P<0.05). At 12 weeks after operation, biomechanical test showed that the average elastic modulus and maximum compressive strength of cancellous bone and subchondral bone in groups D and E were significantly higher than those in groups A, B, and C (P<0.05); there was no significant difference between groups A, B, and C and between groups D and E (P>0.05).ConclusionIn vivo transplantation of microencapsulated transgenic BMSCs can repair early SONFH in rabbits.
ObjectiveTo evaluate the effect of bone morphogenetic protein 7 (BMP-7)/poly (lactide-co-glycolide) (PLGA) microspheres on in vitro proliferation and chondrogenic differentiation of rabbit bone marrow mesenchymal stem cells (BMSCs).MethodsBMP-7/PLGA microspheres were fabricated by double emulsion-drying in liquid method. After mixing BMP-7/PLGA microspheres with the chondrogenic differentiation medium, the supernatant was collected on the 1st, 3rd, 7th, 14th, and 21st day as the releasing solution. The BMSCs were isolated from the bilateral femurs and tibias of 3-5 days old New Zealand rabbits, and the 3rd generation BMSCs were divided into 2 groups: microspheres group and control group. The BMSCs in microspheres group were cultured by 200 μL BMP-7/PLGA microspheres releasing solution in the process of changing liquid every 2-3 days, while in control group were cultured by chondrogenic medium. The cell proliferation (by MTT assay) and the glycosaminoglycan (GAG) contents (by Alician blue staining) were detected after chondrogenic cultured for 1, 3, 7, 14, and 21 days. The chondrogenic differentiation of BMSCs was observed by safranine O staining, toluidine blue staining, and collagen type Ⅱ immunohistochemistry staining at 21 days.ResultsMTT test showed that BMSCs proliferated rapidly in 2 groups at 1, 3, and 7 days; after 7 days, the proliferation of BMSCs in the control group was slow and the BMSCs in microspheres group continued to proliferate rapidly. There was no significant difference of the absorbance (A) value at 1, 3, and 7 days between 2 groups (P>0.05), but theA value at 14 and 21 days in microspheres group was significantly higher than that in control group (P<0.05). Compared with control group at 21 days, in microsphere group, almost all nuclei were dyed bright red by safranine O staining, almost all the nuclei appeared metachromatic purple red by toluidine blue staining, and the most nuclei were yellow or brown by immunohistochemical staining of collagen type Ⅱ. Alcian blue staining showed that the content of GAG in 2 groups increased continuously at different time points; after 7 days, the increasing trend of the control group was slow and the microspheres group continued hypersecretion. There was no significant difference of the GAG content at 1, 3, and 7 days between 2 groups (P>0.05), but the GAG content at 14 and 21 days in microspheres group was significantly higher than that in control group (P<0.05).ConclusionBMP-7/PLGA microspheres prepared by double emulsion-drying in liquid method in vitro can promote proliferation and chondrogenic differentiation of rabbit BMSCs.
Objective To realize the visualization of three-dimensional microstructure of rabbit sciatic nerve bundles by micro-CT and three-dimensional visualization software Mimics17.0. Methods The sciatic nerve tissues from 6 New Zealand rabbits were divided into 2 groups (n=3), and the sciatic nerve tissues were stained by 1% (group A) and 5% (group B) Lugol solution respectively. After staining for 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, and 3.5 hours, the imaging changes of specimens were observed by light microscope and micro-CT. The clear micro-CT images were exported to the Mimics software to complete the visualization of three-dimensional microstructure of rabbit sciatic nerve according to three-dimensional reconstruction tool. Results The clear three-dimensional microstructure images could be observed in group A at 2.5 hours after staining and in group B at 1.5 hours after staining by light microscope and micro-CT. The sciatic nerve of New Zealand rabbits were divides into 3 bundles and each of them was relatively fixed. There was no obvious crossing or mergers between each bundle. The cross-sectional area of each bundle was (0.425±0.013), (0.038±0.007), and (0.242±0.026) mm2 respectively. The digital model could clearly reflect the microstructure of the sciatic nerve at all cross sections. Conclusion The internal structure of New Zealand rabbits sciatic nerve can be clearly reflected by micro-CT scanning. It provides a reliable method for establishing a nerve microstructure database with large amount specimens.