【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 explore the possibilityof constructing tissue engineering muscles by combining allogeneic myoblasts with small instestinal submucosa(SIS) in rabbits.Methods A large number of purified myoblasts were obtained with multiprocedure digestion and repeated attachment method from skeletal muscles taken from extremities of immature rabbits which were born 7 days ago. The myoblasts were labeled with BrdU, and then combined with SIS to construct tissue engineering muscles. This kind of tissue engineering muscles were grafted into the gastrocnemius muscle defect (1.5 cm in length, 1.0 cmin width) of fifteen rabbits as the experimental group. The SIS was grafted into the same position in the control group. The rabbits were sacrificed 4, 6, 8 weeks after operation. The tissue engineering muscles were evaluated by macroscopic、histological and immunohistochemical observations, and by quantitative analysis of local immunocyte in the grafting site. Results Allogeneic myoblasts with SIS were combined perfectly in vitro. The SIS was connected tightly to surrounding skeletal muscles and inflammation response was obvious 4 weeks after grafting.The SIS began to break down and inflammation response became slight 6 and 8 weeks after operation. Compared with that of 8th week, the quantitative analysis oflocal immunocyte in 4th and 6th week in both experimental and control group hassignificance(Plt;0.05). Newly formed muscle tissues were found around SIS in the experimental group in 4th, 6th, and 8th week. Expression of BrdU and myosin immunohistochemical staining were positive in the experimental group and negative inthe control group.Conclusion Tissue engineering muscles of rabbits which are constructed by combining allogeneic myoblasts with SIS can survive and proliferate.
ObjectiveTo investigate the immunogenicity of freezing periosteum and bone marrow during allogeneic joint transplantation, and to explore proper pretreatment of allogeneic joint.
MethodsThe allogeneic periosteum and bone marrow were harvested from knee joints of 5 New Zealand white rabbits (aged, 6 months; weighing, 2.6-3.0 kg). After gradient cooling, the tissue was cryopreserved for 1 month. The freezing periosteum and bone marrow were grinded to pieces after rewarming to prepare the suspension of periosteum and bone marrow. Eighteen Chinchilla rabbits (aged, 6 months; weighing, 2.1-2.8 kg) were divided into 3 groups randomly:normal saline injection group (group A, n=6), periosteum injection group (group B, n=6), and bone marrow injection group (group C, n=6). The normal saline, periosteum suspension, and bone marrow suspension were injected into the peritoneal cavity in groups A, B, and C, respectively. The concentrations of interleukin 2 (IL-2), IL-6, and tumor necrosis factor α (TNF-α) in serum and the ratio of CD4+ T cell/CD8+ T cell in venous blood were measured before injection, at 1 week and 2 weeks after injection.
ResultsThere was no significant difference in the concentration of IL-2 between before and after injection in the same group (P=0.241), and between groups (P=0.055). The concentration of IL-6 after injection was significantly lower than that before injection in the same group (P=0.040), but no significant difference was found between groups (P=0.357). The concentration of TNF-α showed no significant difference between before and after injection in the same group (P=0.925), but the concentration of TNF-α in group B was significantly higher than that in groups A and C (P<0.05). The ratio of CD4+T cell/CD8+T cell of venous blood had no significant difference between before and after operation in the same group (P=0.248), and between groups (P=0.646).
ConclusionThe freezing periosteum and bone marrow are lowly immunogenic. In order to decrease the immunogenicity of the joint, preserving the periosteum and removing the marrow cavity are recommended.
【摘要】 目的 探討LIM礦化蛋白（LIM mineralization protein，LMP）-1和LMP-3雙基因共轉染骨髓間充質干細胞（bone mesenchymal stem cells，BMSC）的表達情況。 方法 采用人工設計合成人LMP-1和LMP-3基因片段，分別與質粒pEGFP-N2連接，經酶切、測序鑒定后。分離培養新西蘭兔BMSC，用脂質體包裹轉染BMSC，按轉染情況分為5組:未轉染組（A組）、轉染空載體組（B組）、轉染LMP-1基因組（C組）、轉染LMP-3基因組（D組）、LMP-1與LMP-3雙基因共轉染組（E組）。采用實時聚合酶鏈反應（real-time polymerase chain reaction，RT-PCR）和蛋白質印跡法檢測LMP-1和LMP-3的表達。 結果 酶切及測序表明真核表達質粒pEGFP-N2-LMP-1和pEGFP-N2-LMP-3構建成功。E組可同時較高水平表達LMP-1和LMP-3分子。對RT-PCR及蛋白質印跡法檢測結果行灰度值測量并行統計學分析顯示:LMP-1 mRNA及蛋白水平的表達，5組間差異有統計學意義（Plt;0.05），但E組與C組的差異無統計學意義（Pgt;0.05）；LMP-3 mRNA及蛋白水平的表達，5組間差異有統計學意義（Plt;0.05），且E組與D組差異也有統計學意義（Plt;0.05）。 結論 雙基因共轉染的BMSC能在體外同時表達LMP-1與LMP-3，為基因修復骨缺損帶來新思路。【Abstract】 Objective To study the expression of LIM mineralization protein (LMP)-1 and LMP-3 genes after cotransfecting them into bone mesenchymal stem cells (BMSC) of rabbit in vitro. Methods Fragments of LMP-1 gene and LMP-3 gene were gained through artificial synthesis, and were constructed respectively into the plasmid vector pEGFP-N2. The inserted target genes in plasmid were verified by nucleotide sequencing and enzymes. The plasmids carrying LMP-1 and LMP-3 genes were cotransfected into chondrocytes by liposome method. According to the transfected situation, the BMSC were divided into 5 groups: the non-transfected group (Group A), the group transfected by empty vector (Group B), the group transfected by LMP-1 (Group C), the group transfected by LMP-3 (Group D) and the group transfected by both LMP-1 and LMP-3 (Group E). The expressions of LMP-1 and LMP-3 were detected by RT-PCR and western bloting technique. Results The plasmid pEGFP-N2-LMP-1 and pEGFP-N2-LMP-1 were obtained successfully by cloning technique and verified by nucleotide sequencing and enzymes. The LMP-1 and LMP-3 molecules were both expressed at a high level in Group E. The results of RT-PCR and western bloting were measured with the grey value. For the expression of LMP-1 mRNA and protein of LMP-1, the differences between groups A, B and groups C, D, E were significant (P＜0.05), while the difference between groups C and E was not significant (P＞0.05); For the expression of LMP-3 mRNA and protein of LMP-3, the differences between groups A, B and groups C, D, E were significant (P＜0.05), and the difference between groups D and E was also significant（P＜0.05). Conclusion LMP-1 and LMP-3 genes can be expressed effectively after being cotransfected into BMSC, which provides a basis for gene therapy for treating bone defects.
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.
Objective It is difficult to treat chronic osteomyel itis due to the formation of the Staphylococcus aureus biofilms. Liposomal gentamicin-impregnated allogeneic cortical bone can inhibit the formation of the Staphylococcus aureusbiofilms. To explore the treatment of chronic osteomyel itis of rabbit by l iposomal gentamicin-impregnated allogeneic cortical bone. Methods The l iposomal gentamicin, l iposomal gentamicin-impregnated allogeneic cortical bone and gentamicinimpregnated allogeneic cortical bone were produced. Then the chronic Staphylococcus aureus osteomyel itis models of rabbit were made in left lower l imbs of 40 6-month-old rabbits and the right lower l imbs were used as controls. After 2 weeks, the observations of gross and X-ray were done. Four rabbits died within 10 days after the models were made and other 36 rabbits were devided into 6 groups: group A (no antibiotics), group B (intravenous injection of gentamicin), group C (intravenous injection of l i posomal gentamicin), group D (implantation of gentamicin-impregnated allogeneic cortical bone), group E (implantation of l i posomal gentamicin-impregnated allogeneic cortical bone), and group F (implantation of allogeneic cortical bone). After 2 weeks of treatment, the bacterial culture, X-ray and HE staining were done. Results The chronic Staphylococcus aureus osteomyel itis model of rabbit was made successfully. The X-ray showed dissolution of bone and periosteal reaction in groups A, B, C, and F, and no obvious dissolution of bone and periosteal reaction in groups D and E. The Norden scores were (2.5 ± 0.3), (2.1 ± 0.2), (1.5 ± 0.3), (1.5 ± 0.2), (0.9 ± 0.3), and (2.7 ± 0.3) points in groups A-F, respectively; showing significant differences between group A and groups B-E (P lt; 0.05), between groups B, E, F and other groups (P lt; 0.05). The results of blood and marrow cultures for Staphylococcus aureus were positive in groups A and F, and negative in other 4 groups; the results of bone marrow culture for Staphylococcus aureus were positive in 6 rabbits of group B, 4 rabbits of group C and 3 rabitts of group D; and the results were negative in group E. HE staining showed: in groups A and F, abscess and dead bone formed, and no new bone formation were observed; in groups B and C, different degrees of neutrophil accumulation was seen; in group D, some neutrophil accumulation occurred, and osteoprogenitor cells and osteoclasts were seen around implanted bone; and in group E, no neutrophil accumulation was observed, a lot of granulation tissues formed, and osteoprogenitor cells and osteoclasts were seen around implanted bone. Conclusion Implantation of l iposomal gentamicin-impregnated allogeneic cortical bone has remarkly better effect in treating chronic osteomyel itis than intravenous injection of l iposomal gentamicin and implantation of gentamicin-impregnated allogeneic cortical bone.
ObjectiveTo observe the morphological characteristic by implanting domestic porous tantalum in rabbit patellar tendon and to evaluate biocompatibility features so as to provide experimental basis for porous tantalum used as interface fixation between tendon and bone.
MethodsA total of 48 adult New Zealand white rabbits, male or female, weighing 2.5-3.0 kg, were selected. Porous tantalum flake (5 mm×5 mm×2 mm) was implanted in the left patellar tendon (experimental group) and the same size porous titanium flake in the right patellar tendon (control group). The animals were sacrificed at 2, 4, 8, and 12 weeks after implantation, then the specimens were harvested for gross observation, HE staining, scanning electron microscope (SEM) observation, and hard slices observation.
ResultsNo animal died after operation. Porous tantalum was bonded closely with host tendon and no inflammatory reaction was found. Loose and thick fibrous capsule was observed at the beginning and became density and thinner in the end by microscope, showing significant difference between different time points in 2 groups (P<0.05), but no significant difference was found between 2 groups at different time points (P>0.05). The SEM observation showed that fibrous tissue attached to the surface and inner walls of porous tantalum at early stage, and extended on the material to reach confluence at late period, but the experimental group was more than the control group. Hard slices observation showed that the collagen fibrils were seen on porous tantalum interface with host tendon, and blood vessels grew into the pores. The control group and the experimental group showed no significant difference.
ConclusionThe domestic porous tantalum has good biocompatibility. Connection and integration can be established between tendon and porous tantalum, and therefore it could be used in reconstruction of tendon-bone fixation device.
