Objective To compare the difference of preparing the acellular larynx scaffold between perfusion method and immersion method, and find better way to make acellular larynx scaffold for tissue engineering. Methods Twenty 6-month-old male New Zealand rabbits, weighing 2.0-2.5 kg, were divided into perfusion group (n=10) and immersion group (n=10) at random. All the larynxes were excised in a sterile fashion. The acellular larynx scaffold was obtained by perfusionmethod and immersion method respectively, and then comparative examinations were performed by the macroscopicview, histological view, scanning electron microscope (SEM), cartilage vital ity assay and toluidine blue staining. ResultsMacroscopic view showed that the larynxes perfused by sodium dodecyl sulphate (SDS) became transparent after 2 hoursof perfusion, but the larynxes immersed by SDS over 16 hours still appeared pink-white. Histology and SEM indicated thatcompared with immersion group, perfusion group showed better acellular effect, more ventages and collagen fibers wereretained, no intact cell or nuclei remained in acellular matrix and chondrocytes were still survival. The porosity was 85.39% ± 3.16% in perfusion group and 34.72% ± 4.51% in immersion group, showing significant difference (P lt; 0.01). The chondrocyte vital ity rate of perfusion group (86.93% ± 1.52%) was higher than that of immersion group (77.73% ± 1.66%), showing significant difference (P lt; 0.01). Toluidine blue staining showed that the chondrocyte heterochromaty was ber in perfusion group than that in immersion group. Conclusion Compared with immersion method, perfusion method is a better way to construct acellular larynx scaffold because it can achieve better acellular effect and retain chondrocyte vital ity at the greatest extent in the acellular larynx scaffold.
One eye each in 3 groups of 12 pigmented rabbits after bilateral vitrectomy received 0.5mg, 1mg or 2mg triamcinolone acetonide (TA), respectively. The fellow eye received only balance saline solution as control. Ophthalmoscopy and electroretinography were performed during 1 day to 38 days after vitrectomy and drug injection. Light and electronmicroscopic studies were done on the 28th day. The particles of drug were visible on day 28 in all TA-treated eyes. Administration of 0. 5rug and 1mg TA did not result in different changes in ERG b-wave amplitudes compared with those in control eyes(P＞0. 05). There were significant elevations of ERG b-wave in 2mg TA eyes compared to the control eyes(Plt;0.05), Both ligbt and electronmicroscopy of the retina in these groups were almost normal. The results showed no Toxielties in TA treated eye up to 2mg after vitrectomy. This offers the experimental evidence as a baseline for combining TA with vitrectomy to reduce recurrence of proliferative vitreoretinopathy. （Chin J Ocul Fundus Dis,1996,12: 105- 107）
Objective To detect the cell density, apoptotic rate, and the expressions of BNIP3 in nucleus pulposus of degenerative intervertebral disc of rabbits, so as to further understand the mechanism of intervertebral disc degeneration. Methods Thirty male New Zealand white rabbits, aging 3 months and weighing (2.3 ± 0.2) kg, were divided into sham operation group (control group, n=10) and intervertebral disc degeneration model group (experimental group, n=20). Interbertebral disc degeneration models were establ ished by puncture of L3,4, L4,5, and L5,6 intervertebral discs in the experimental group; intervertebral discs were exposed only and then sutured in the control group. The degree of intervertebral disc degeneration was evaluated according to Pfirrmann classification by MRI at 4 and 8 weeks after establ ishing models. Apototic cells were determined by TUNEL and histological methods, and the immunohistochemical staining was performed to detect the expressions of BNIP3 in nucleus pulposus of intervertebral disc. Results MRI examination showed that the signal intensity decreased gradually at 4 and 8 weeks in the experimental group. There wassignificant difference in the degree of intervertebral disc degeneration between at 4 weeks and at 8 weeks in the experimental group (P lt; 0.05). The histological observation and TUNEL test showed that high density of nucleus pulposus cells and only a few apoptotic cells were observed in the control group; at 4 and 8 weeks, the density of nucleus pulposus cells decreased gradually with more apoptotic cells in the experimental group. There were significant differences in the nucleus pulposus cell density and positive rate of TUNEL staining between 2 groups, and between at 4 weeks and at 8 weeks in the experimental group (P lt; 0.05). The expression of BNIP3 of nucleus pulposus was negative in the control group; however, in the experimental group, the positive expression rates of BNIP3 of nucleus pulposus (the gray values) were 13.45% ± 1.16% and 32.00% ± 1.82% (194.32 ± 4.65 and 117.54 ± 2.11) at 4 and 8 weeks respectively, showing significant differences (P lt; 0.05). Conclusion The decrease of cell density in nucleus pulposus is involved in the development of intervertebral disc degeneration. Cell apoptosis is one of reasons in the decrease of nucleus pulposus cell; BNIP3 is involved in nucleus pulposus cell apoptosis in the degenerative intervertebral disc.
Objective To explore the regulator factor of osteogenes is induced by the fibroblast in vitro so as to provide enough seeding cells for the bon e tissue engineering. Methods The fibroblasts were isolated and purified from granu lation of New Zealand rabbits, and they were incubated in the media offibronectin (FN) 10, 20, 40, 60 and 80 μg/ml, respectively, in the experimenta l grou ps 1- 5，but there was no FN in the control group. The markers for osteogenic features were investigated by fibroblast morphogenesis,calcium nodules formationratios,labeling of tetracycline fluorescence, labeling of 3H-TdR, determination of o steocaline, and labeling of 3H-proline within 2 weeks. Results The morphologic al changes of the fibroblasts were manifested as transference from a long spindle to a round or multiple form, shifted nucleus increased in number, confluenced and formed multilayered structure. There was a piling-up of calcium crystals that were gradually merged into foggy substances. The foggy substances increased and formed nodules. The calcium nodules formation ratios were as follows: 15.35％± 3.45％in the control group, and 53.73％± 9.49％, 75.21％± 9.80％, 98.34％± 15.2 0％, 61.83％± 10.04％, and 45.11％± 8.70％ in the experimental groups 1.5 ,respectively. There was a significant difference between the control group and the 5 experimental groups at 14 days (Plt;0.05), and a significant differenc e be tween the experimental group 3 and the other experimental groups at 14 days (Plt;0.05). The histochemical study on the nodules with the specific labeling of tet racycline fluorescence indicated that the nodules were composed of new bones. Conclusion Fibronectin can stimulate the fibroblast to prolifer ate, secrete osteocaline, and synthesize collagen fibrils. Fibronectin, in an optimal dose of 40 -60 μg/ml, is capable of inducing the fibroblast to form the bone.
Objective To establ ish the experimental animal model of perforator sural neurocutaneous flap for laying a foundation of further study on its physiology and haemodynamics. Methods Thirty-five New Zealand rabbits were divided into four groups, weighing 2.5-3.0 kg and being male or female. In group A (n=5), vivisection was performed to observe thestarting point and arrangement of sural nerve, its concomitant vessels, posterior tibial artery and perforating vessel. In groups B and C (n=5), red latex and gelatin-lead oxide were injected into the concomitant arteries of sural nerve and the posterior tibial arteries respectively to observe their arrangement, the diameter and anatomasis. In group D, forty neurocutaneous flaps based on single perforator were elevated in the twenty rabbits with a size of 7 cm × 1 cm and a pedicle of 0.5 cm. The colour and condition of flaps were observed. Results The sural nerve originated from posterior tibial nerve, passed through the lateral head of the gastrocnemius at site of the popl iteal fossa, descended obl iquely to exterior, entered in the deep fascia at about (5.42 ± 0.15) cm above lateral malleolus, and descended vertically to lateral malleolus. Its concomitant artery originated from deep femoral artery with an initial diameter of (0.73 ± 0.11) mm and extended to the lateral malleolus along the sural nerve. A perforating branch of posterior tibial artery at the position of the calcaneus originated from the midpoint of the l ine connecting between the medial malleolus and the calcaneus with an initial diameter of (0.45 ± 0.01) mm. The perforating branch traversed the calcaneus to the region of the lateral malleolus, and anastomosed to the concomitant artery of the sural nerve, forming a vascular plexus around the sural nerve. In group D, two cases were excluded due to infection. The survival rate was 78.0% ± 1.5% in other 38 flaps 10days after operation. Conclusion The perforator based sural neurocutaneous flap in rabbit is a good experimental model,which has stable anamatic features and rel iable blood distribution.
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.
The damage effects of the pure tumor necrosis factor (TNF) on the normal animals were observed. Eighteeen rabbits were divided into two groups, eight in tested group and ten in control group. 0.5mg per kg of the pure rabbit TNF was given to each animal of the tested group. Results:The symptoms similar to that induced by endotoxin appeared after the TNF injection. The functions of the main organs were markedly damaged. The arterial blood pressure of most animal was low. The weight ratio of the orgen to the body was raised. The pathologic changes were similar to those of the multiple organ failure (MOF) model. Most of the animal died before the end of the experiment. The results suggest that pure TNF could indece multiple organ damages similar to those of MOF.
ObjectiveTo investigate the effect of KLD-12 polypeptide complexed with recombinant human bone morphogenetic protein 2 (rhBMP-2) on osteogenic activity of rabbit bone marrow mesechymal stem cells (BMSCs). MethodsBone marrow was harvested from 3-month-old New Zealand white rabbit, and density gradient method was used to isolate and culture BMSCs. The third generation BMSCs were used for three-dimensional culture of KLD-12 polypetide/rhBMP-2 in vitro (experimental group) and KLD-12 polypeptide (control group). The morphology of the cells in the gel was observed by inverted phase contrast microscope at 7 days; alkaline phosphatase (ALP) and osteocalcin protein content were dectected at 3, 7, 10, 14, and 21 days; collagen type I immunofluorescence staining was done and real-time fluorescent quantitative PCR was performed to detect the relative expression of collagen type I and osteocalcin gene at 14 days. ResultsUnder the inverted phase contrast microscope, the BMSCs in the gel of the experimental group and the control group showed circular growth, and the distribution was uniform at 7 days. There was no significant difference in the expressions of ALP and osteocalcin protein content between 2 groups at 3 and 7 days (P > 0.05); the above indexes in experimental group were significantly higher than those in the control group at 10-21 days (P < 0.05). Laser scanning confocal microscope observation showed that immunofluorescence staining for collagen type I was positive in the experimental group, and the expression was higher than that in the control group at 14 days. Real-time fluorescence quantitative PCR detection showed that the collagen type I and osteocalcin gene expressions were significantly higher than those in the control group (t=15.902, P=0.000; t=12.998, P=0.000). ConclusionBMSCs can normally grow and proliferate in the KLD-12 polypeptide, and KLD-12 polypeptide/rhBMP-2 has good biological activity to induce BMSCs differentiation into osteoblasts.
Objective To observe the effect of recombinant human osteoprotegerin (rhOPG) on osteoclasts which were stimulated by polyethylene particles and to investigate the feasibil ity of applying rhOPG for the prosthetic aseptic looseness. Methods The osteoclasts were isolated from the long bones of 5 New Zealand rabbits born within 24 hours, weighing 80-100 g, male or female, and were plated into the 24-well coversl ips (10 mm × 10 mm) and bone sl ices (8 mm × 8 mm × 50 μm) at the density of 1 × 105/mL. Based on the different concentration and density of rhOPG and polyethylene particles, the plates of culture were divided into 3 groups: the group with polyethylene particles of 1 × 109/mL (polyethylene group), the group co-cultured with polyethylene particles of 1 × 109/mL and rhOPG of 100 ng/mL (polyethylene/rhOPG group) and the control group . The glass cover sl ips and bone sl ices were exposed to HE, toluidine blue and tartrate-resistant acid phosphatase (TRAP) staining at 1, 3, 5 and 7 days, and TRAP positive multinucleated cells and bone resorption tips were counted. Scanning electron microscope was used to observe the pits of bone resorption. Results The osteoclast was rose-red when exposed to TRAP staining. For the number of the TRAP-positive osteoclasts, the polyethylene group witnessed an obviouse increase compared with the control group and the polyethylene/rhOPG group after 5 and 7 days of culture (P lt; 0.05). And no significant difference between the control group and the polyethylene/rhOPG group was evident (P gt; 0.05). The pits of bone resorption was blue-purple when exposed to toluidine blue staining. For the number of bone resorption pits in the bone sl ice, significant difference was evident between the polyethylene group and the control group after 5 and 7 days of culture (P lt; 0.05), and there was significant different between the polyethylene/rhOPG group and the polyethylene group 1, 3, 5 and 7 days after culture (P lt; 0.05). Conclusion rhOPG could inhibit the stimulated effect of polyethylene particles on osteoclasts, and might be used to prevent the prosthetic aseptic looseness after artificial joint substitution.
Histological studies and morphometry quantitative analysis have been performed on trial rabbit’s dilated common bile duct(CBD),which does not dilate simultaneously.The results shows:①Epithelia of rabbit’s CBD have a ber reparable function,which is fairly significant to the prevention of bile duct’s further injure under the pathogenic situation.②The smooth muscle cell(SMC)of the CBD is the histological basis of contraction,some SMC can be seen in contracting state under light microscope.This indicates that the SMC in rabbit’s CBD possess contracting function.③The collagenous and elastic fibers have the normal histological morphometric characteristics and quantity in it’s dilatation process,and no breekdown and degeneration of the fibers can be detected.Because of the morphological structure of these sections is quite similiar with normal ones,theoretically,we suspect that when pathological change of bile duct’s distal portion is relieved and the bile pressure is normal again.It is possible for this dilating bile duct to return to its formal shape and size.