【Abstract】 Objective To build nano-biomimetic tissue engineered blood vessel (NBTEBV) with nanotopology by using electrospinning (ELSP) technology. Methods Cony vascular endothel ial cell(VEC) on tubiform tooting in vitro was cultured. NBTEBV was built by use of multi-row nozzle with the suspension of cony vascular smooth muscle cell (VSMC) and mimic ECM (MECM) solution. NBTEBV was cultured with bioreactor in vitro . VEC and VSMC viabil ity and prol iferation were observed with MTT; and HE staining, scanning electron microscopy(SEM) observation and biomechanical test were carried out after 24 hours of static culture and 7 days of dynamic culture. Results After 7 days of culture, the length of NBTEBV was 57 mm, the external diameter was 4 mm and the thickness of wall was 0.4 mm. The NBTEBV’s color was white and the texture was even and flexible. MTT results indicated the viabil ity of cells cultured on NBTEBV for 7 days was normal(8.9 × 106 /mg, 3.5 ×105/mg for 24 hours). SEM and HE staining indicated that the topologic character of NBTEBV was similar to that of the naturalblood vessel. The NBTEBV showed a network scaffolds structure with 100 nm thick fiber and 600 nm aperture. The HE stainingresult showed that the NBTEBV was composed of VEC and VSMC by layer. Vascular mechanical results showed that the NBTEBVultimate hydrostatic pressure was 950 mmHg, the compl iance of the NBTEBV under physio-pressure (110/70 mmHg) was 3.0%; the ultimate tensile strength of 20 mm × 5 mm tissue sl ice was 18.5 MPa. Conclusion The technology of ELSP can use VSMC and MECM scaffold simultaneously to build tissue engineered blood vessel with nanotopology mimic native blood vessel.
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 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.
ObjectiveTo investigate the effect of power-assisted intravascular shunt in replantation of amputated limbs of rabbits. MethodsEighty rabbits weighing 1.8-2.5 kg (male or female) were selected to establ ish the model of circular amputation at the hind groin, only femoral arteries and veins were completely preserved. After the femoral artery was clamped in 60 rabbits, the rabbits underwent power-assisted intravascular shunt with high-flow rate (group A, n=20), powerassisted intravascular shunt with low-flow rate (group B, n=20), and no power-assisted intravascular shunt (group C, n=20) to reconstruct blood supply; the femoral artery was not clamped in another 20 rabbits of sham group (group D). Before and after intravascular shunt (1, 3, 6, and 12 hours), the malondialdehyde (MDA), lactate dehydrogenase (LDH), and creatine kinase (CK) of the serum were determined. The myeloperoxidase (MPO), MDA, and wet to dry weight ratio (W/D ratio) of the gastrocnemius muscle were measured, and the thrombogenesis and survival rate of limb were observed. ResultsBefore intravascular shunt, MDA, LDH, and CK of the serum and MPO, MDA, and W/D ratio of the muscle showed no significant difference among 4 groups (P>0.05). At each time point after intravascular shunt, no significant difference was found in all indexes between groups A and D (P>0.05); the indexes of groups B and C were significantly higher than those of groups A and D (P<0.05); the values were the highest in group C (P<0.05), and reached the peak at 12 hours. All limbs of group A survived with low thrombosis rate, and less limbs could survive with high thrombosis rate in group C. ConclusionThe power-assisted intravascular shunt with high-flow rate can effective ensure the blood supply of the amputated limbs of rabbits with lower limb injury and higher survival rate of amputated limbs after replantation.
ObjectiveTo investigate the early effects of acellular xenogeneic nerve combined with adipose-derived stem cells (ADSCs) and platelet rich plasma (PRP) in repairing facial nerve injury in rabbits.MethodsThe bilateral sciatic nerves of 15 3-month-old male Sprague-Dawley rats were harvested and decellularized as xenografts. The allogeneic ADSCs were extracted from the neck and back fat pad of healthy adult New Zealand rabbits with a method of digestion by collagenase type Ⅰ and the autologous PRP was prepared by two step centrifugation. The 3rd generation ADSCs with good growth were labelled with CM-Dil living cell stain, and the labelling and fluorescence attenuation of the cells were observed by fluorescence microscope. Another 32 New Zealand rabbits were randomly divided into 4 groups and established the left facial nerve defect in length of 1 cm (n=8). The nerve defects of groups A, B, C, and D were repaired with CM-Dil-ADSCs composite xenogeneic nerve+autologous PRP, CM-Dil-ADSCs composite xenogeneic nerve, xenogeneic nerve, and autologous nerve, respectively. At 1 and 8 weeks after operation, the angle between the upper lip and the median line of the face (angle θ) was measured. At 4 and 8 weeks after operation, the nerve conduction velocity was recorded by electrophysiological examination. At 8 weeks after operation, the CM-Dil-ADSCs at the distal and proximal ends of regenerative nerve graft segment in groups A and B were observed by fluorescence microscopy; after toluidine blue staining, the number of myelinated nerve fibers in regenerated nerve was calculated; the structure of regenerated nerve fibers was observed by transmission electron microscope.ResultsADSCs labelled by CM-Dil showed that the labelling rate of cells was more than 90% under fluorescence microscope, and the labelled cells proliferated well, and the fluorescence attenuated slightly after passage. All the animals survived after operation, the incision healed well and no infection occurred. At 1 week after operation, all the animals in each group had different degrees of dysfunction. The angle θ of the left side in groups A, B, C, and D were (53.4±2.5), (54.0±2.6), (53.7±2.4), and (53.0±2.1)°, respectively; showing significant differences when compared with the healthy sides (P<0.05). At 8 weeks after operation, the angle θ of the left side in groups A, B, C, and D were (61.9±4.7), (56.8±4.2), (54.6±3.8), and (63.8±5.8)°, respectively; showing significant differences when compared with the healthy sides and with the values at 1 week (P<0.05). Gross observation showed that the integrity and continuity of regenerated nerve in 4 groups were good, and no neuroma and obvious enlargement was found. At 4 and 8 weeks after operation, the electrophysiological examination results showed that the nerve conduction velocity was significantly faster in groups A and D than in groups B and C (P<0.05), and in group B than in group C (P<0.05); no significant difference was found between groups A and D (P>0.05). At 8 weeks after operation, the fluorescence microscopy observation showed a large number of CM-Dil-ADSCs passing through the distal and proximal transplants in group A, and relatively few cells passing in group B. Toluidine blue staining showed that the density of myelinated nerve fibers in groups A and D were significantly higher than those in groups B and C (P<0.05), and in group B than in group C (P<0.05); no significant difference was found between groups A and D (P>0.05). Transmission electron microscope observation showed that the myelinated nerve sheath in group D was large in diameter and thickness in wall. The morphology of myelin sheath in group A was irregular and smaller than that in group D, and there was no significant difference between groups B and C.ConclusionADSCs can survive as a seed cell in vivo, and can be differentiated into Schwann-like cells under PRP induction. It can achieve better results when combined with acellular xenogeneic nerve to repair peripheral nerve injury in rabbits.
ObjectiveTo compare the osteogenic effect of bone marrow mesenchymal stem cells (BMSCs) transfected by adenovirus-bone morphogenetic protein 2-internal ribosome entry site-hypoxia inducible factor 1αmu (Ad-BMP-2-IRES-HIF-1αmu) and by Ad-cytomegalovirus (CMV)-BMP-2-IRES-human renilla reniformis green fluorescent protein 1 (hrGFP-1) single gene so as to optimize the source of osteoblasts. MethodsBMSCs were separated and cultured from 1-month-old New Zealand white rabbit. The BMSCs at passage 3 were transfected by virus. The experiment was divided into 4 groups (groups A, B, C, and D) according to different virus: BMSCs were transfected by Ad-BMP-2-IRES-HIF-1αmu in group A, by Ad-CMV-BMP-2-IRES-hrGFP-1 in group B, by Ad-CMV-IRES-hrGFP-1 in group C, and BMSCs were not transfected in group D. The optimum multiplicity of infection (MOI) (50, 100, 150, and 200) was calculated and then the cells were transfected by the optimum MOI, respectively. The expression of BMP-2 gene was detected by immunohistochemistry staining after transfected, the expressions of BMP-2 protein and HIF-1α protein were detected by Western blot method. The osteogenic differentiation potential was detected by alkaline phosphatase (ALP) activity and Alizarin red staining. ResultsThe optimum MOI of groups A, B, and C was 200, 150, and 100, respectively. The expression of BMP-2 was positive in groups A and B, and was negative in groups C and D by immunohistochemistry staining; the number of positive cells in group A was more than that in group B (P ﹤ 0.05). The expression of BMP-2 protein in groups A and B was significantly higher than that in groups C and D (P ﹤ 0.05), group A was higher than group B (P ﹤ 0.05). The expression of HIF-1α protein in group A was significantly higher than those in the other 3 groups (P ﹤ 0.05), no significant difference was found among the other 3 groups (P ﹥ 0.05). ALP activity in groups A and B was significantly higher than that in groups C and D (P ﹤ 0.05), group A was higher than group B (P ﹤ 0.05). Calcium nodules could be seen in groups A and B, but not in groups C and D; the number of calcium nodules in group A was higher than that in group B (P ﹤ 0.05). ConclusionThe expression of BMP-2 and osteogenic effect of BMSCs transfected by Ad-BMP-2-IRES-HIF-1αmu (double genes in single carrier) are higher than those of BMSCs transfected by Ad-CMV-BMP-2-IRES-hrGFP-1 (one gene in single carrier).
Objective To develop an experimental model of abdominal aorta transplantation with nano-biomimetictissue engineered blood vessel (NBTEBV) and to investige the change of histomorphology in evolutionary process of degradation and remodel ing. Methods Twenty 6-month-old New Zealand rabbits were included, weighing 2-3 kg, male or female. The autologous seed cells of rabbits were harvested to build NBTEBV in vitro. After the branch of abdominal aorta under kidney was l igated, about 10 mm abdominal aorta was cut and replaced by NBTEBV; the anastomotic stoma was marked by Ti cl ips. NBTEBV’s evolutionary processes of degradation and rebuilding were observed. Twelve weeks after operation, DSA and color Doppler examinations were made. At 1, 4 and 12 weeks after operation, the gross and histological observations were made and 14C binding in PLGA was detected with X-ray photon spectroscopy. Results Of 20 rabbits, 17 showed that the NBTEBV was patency; 3 died from NBTEBV occlusion 36 or 72 hours after operation. The results of DAS and color Doppler showed the blood flow was patency, the blood flow rate was normal and there was no angiectasis. The lumen of transplanted blood vessel was covered with monolayer endothel ial cells. At 1 week, smooth muscle cells (SMCs) arranged regularly and much PLGA distributed in the EMCs. At 4 weeks, SMCs arranged in a layer, ECM was forming, mimic ECM degraded partly; PLGA decreased obviously. At 12 weeks, the SMCs arranged regularly, ECM formed, mimic ECM degraded, no PLGA was seen in the wall, the shape of graft was similar to the natural vessel. The decreasing crest value of 14C in specimen showed the degradation of PLGA. Conclusion NBTEBV has a good surgical maneuverabil ity and histocompatibil ity, its remodel ing evolutionary process fits in with tissue engineering specification. Building NBTEBV with ELSP is feasible.
Objective To investigate the therapeutic effect of BMSCs- chitosan hydrogel complex transplantation on intervertebral disc degeneration and to provide experimental basis for its cl inical appl ication. Methods Two mill il iter of bone marrow from 6 healthy one-month-old New Zealand rabbits were selected to isolate and culture BMSCs. Then, BMSCs at passage 3 were labeled by 5-BrdU and mixed with chitosan hydrogel to prepare BMSCs- chitosan hydrogel complex. Six rabbitswere selected to establ ish the model of intervertebral disc degeneration and randomized into 3 groups (n=2 per group): control group in which intervertebral disc was separated and exposed but without further processing; transplantation group in which 30 μL of autogenous BMSCs- chitosan hydrogel complex was injected into the center of defected intervertebral disc; degeneration group in which only 30 μL of 0.01 mol/L PBS solution was injected. Animals were killed 4 weeks later and the repaired discs were obtained. Then cell 5-BrdU label ing detection, HE staining, aggrecan safranin O staining, Col II immunohistochemical staining and gray value detection were conducted. Results Cell label ing detection showed that autogenous BMSCs survived and prol iferated after transplantation, forming cell clone. HE staining showed that in the control and transplantation groups, the intervertebral disc had a clear structure, a distinct boundary between the central nucleus pulposus and the outer anulus fibrosus, and the obviously stained cell nuclear and cytochylema; while the intervertebral disc in the degeneration group had a deranged structure and an indistinct division between the nucleus pulposus and the outer anulus fibrosus. Aggrecan safarine O stainning notified that intervertebral disc in the control and transplantation groups were stained obviously, with a clear structure; while the intervertebral disc in the degeneration group demonstrated a deranged structure with an indistinct division between the nucleus pulposus and the anulus fibrosus. Col II immunohistochemical staining showed that the tawny-stained region in the control group was located primarily in the central nucleus pulposus with a clear structure of intervertebral disc, the central nucleus pulposus in the transplantation group was positive with obvious tawny-stained intercellular substances and a complete gross structure, while the stained color in the degeneration group was l ighter than that of other two groups, with a indistinct structure.Gray value assay of Col II immunohistochemical staining section showed that the gray value of the control, the ransplantation and the degeneration group was 223.84 ± 3.93, 221.03 ± 3.53 and 172.50 ± 3.13, respectively, indicating there was no significant difference between the control and the transplantation group (P gt; 0.05), but a significant difference between the control and transplantation groups and the degeneration group (P lt; 0.05). Conclusion The rabbit BMSCs-chitosan hydrogel complex can repair intervertebral disc degeneration, providing an experimental foundation for the cl inical appl ication of injectable tissue engineered nucleus pulposus complex to treat intervertebral disc degeneration.
Objective To explore the expression and effect of heme oxygenase-1 ( HO-1) in ventilator-induced lung injury. Methods Twenty-four New Zealand rabbits were randomly assigned to three groups, ie. a conventional ventilation + PEEP group( C group) , a ventilator-induced lung injury group( VILI group) , and a VILI + HO-1 inducer hemin group( Hm group) .Western blot and immunohistochemistry assay were used to investigate the expression of HO-1 protein. Blood gas analysis, lung wet /dry ratio, lunghistopathology and lung injury score were used to evaluate lung injury. Results HO-1 protein expression significantly increased in the VILI group compared with the C group. HO-1 was found mainly in alveolar epithelial cells and vascular endothelial cells, as well as in alveolar macrophages and neutrophils. Compared with the VILI group, HO-1 protein and PaO2 /FiO2 increased, while lung wet/dry ratio and lung injury score decreased in the Hmgroup significantly( P lt;0. 05) . Conclusion High HO-1 expression can alleviate lung injury from large tidal volume ventilation, implying its protective role in lung pathogenesis.
Objective To study the effect of two cytokines, basic fibroblast growth factor(bFGF) and insulin-like growth factor-I(IGF-I), on cell proliferation in chondrocytes of adult rabbits. Methods The primary chondrocytes of adult rabbits were harvested and cultured with bFGF and IGF-I at different concentrations,respectively, as well as with the mixture of the two cytokines; the quantity of cultured chondrocytes was detected by MTT assay at the 24th, 48th and 72th hours; and the final fold increase of different groups was measured by cell count for the 3rd passage; and the proliferation index of the groups was recorded by flowing cytometer on the 14th day. Results ① The cultured chondrocytes with either bFGF, IGF-I or their mixture were significantly more than that of control group at the 24th, 48th and 72th hours (P＜0.01). ② After the 3rd passage, the final folds of proliferation were significantly higher in the groups with cytokinesthan in the control group (P＜0.01); and the final fold with the mixture ofcytokines was significantly higher than that of both IGF-I and bFGF (P＜0.01). ③ Theproliferation index was significantly higher in the groups with cytokines than in the control group (P＜0.01); the proliferation index with the mixture of cytokines was significantly higher than that of both IGF-I and bFGF (P＜0.05); besides, proliferation index was higher when cytokine was applied twice than once (P＜0.05). Conclusion bFGF and IGF-I could promote chondrocytes proliferation of adult rabbits obviously and they are synergistic in cell proliferation.