Objective To evaluate the cell biological features and the effect of transplantation of transforming growth factor β3 (TGF-β3) gene-modified nucleus pulposus (NP) cells on the degeneration of lumbar intervertebral discs in vitro. Methods NP cells at passage 2 were infected by recombinant adenovirus carrying TGF-β3 (Ad-TGF-β3) gene (Ad-TGF-β3 group), and then the cell biological features were observed by cell vital ity assay, the expression of the TGF-β3 protein was determined by Western blot, the expression of collagen type II in logarithmic growth phase was determined by immunocytochemistry. The cells with adenovirus-transfected (Adv group) and the un-transfected cells (blank group) were used as controls. The model of lumbar disc degeneration was establ ished by needl ing L3, 4, L4, 5, and L5, 6 in 30 New Zealand rabbits (weighing 3.2-3.5 kg, male or female). Then Ad-TGF-β3-transfected rabbit degenerative nucleus pulposus cells (100 μL, 1 × 105/ mL, group A, n=12), no gene-modified nucleus pulposus cells (100 μL, 1 × 105/mL, group B, n=12), and phosphatebuffered sal ine (PBS, 100 μL, group C, n=6) were injected into degenerative lumbar intervertebral discs, respectively. L3, 4, L4, 5, and L5, 6 disc were harvested from the rabbits (4 in groups A and B, 2 in group C) at 6, 10, and 14 weeks respectively to perform histological observation and detect the expression of collagen type II and proteoglycan by RT-PCR. Results The viabil ity of nucleus pulposus cells was obviously improved after transfected by recombinant Ad-TGF-β3 gene. At 3, 7, and 14 days after transfected, TGF-β3 expression gradually increased in nucleus pulposus cells. The positive staining of collagen type II was seen in Ad-TGF-β3 group, and the positive rate was significantly higher than that of Adv group and blank group (P lt; 0.05). The disc degeneration in group A was sl ighter than that in groups B and C. The expressions of collagen type II mRNA and proteoglycan mRNA in group A were significantly higher than those in groups B and C at 6, 10, and 14 weeks (P lt; 0.05). Conclusion TGF-β3 can improve the biological activity of NP cells and promote the biosynthesis of collagen type II and proteoglycan in intervertebral discs, alleviate the degeneration of intervertebral discs after transplantation.
Objective To examine the effects of alendronate (ALN) on IL-1β-stimulated chondrocyte of rabbit in vitro and on cartilage and subchondral bone in rabbit osteoarthritis (OA) induced by anterior cruciate l igament transection (ACLT). Methods The chondrocytes from articular surface of healthy 3-month-old Japanese White rabbits were obtained by the method of enzyme digestion and cultured in vitro. The third generation chondrocytes were assigned into three groups: thechondrocytes were cultured in DMEM medium with 10 ng/mL IL-1β for 2 days, subsequently with (ALN group, group A1) orwithout (IL-1β group, group B1) 1 × 10-6 mol/L ALN for 3 days; the chondrocytes in vacant group (group C1) were cultured in DMEM medium for 5 days. The expression of Col II and MMP-13 were analyzed by immunocytochemical staining observation and real time RT-PCR test. Another twenty-four 3-month-old male Japanese White rabbits were randomized into three groups (n=8 per group). The OA model was made by ACLT in ACLT+ALN group (group A2) and ACLT group (group B2); the joint cave was sutured after exposure of ACL in sham group (group C2). After 4 days, the rabbits of group A2 received the subcutaneous injection of ALN at a dosage of 10 μg/(kg·d) for 8 weeks. Rabbits of group B2 and C2 received equal normal sal ine treatment. After 8 weeks, the rabbits were executed. The macro-pathologic changes of right knee joints were observed, so were the histological changes of femoral condyles. Expression levels of Col II and MMP-13 were detected by immunohistochemical staining. The bone histomorphometry analysis was appl ied to subchondral bone of proximal tibia. Results In vitro, the Col II immunocytochemical staining showed intensely positive staining in group C1, and the intensity of staining was sl ightly decreased in group A1, but the intensity of Col II immunocytochemical staining was extremely lower in the group B1. The integrated absorbance (IA) value for Col II in group A1 was significantly higher than that of group B1 (P lt; 0.05), but there was no significant difference between group A1 and group C1 (P gt; 0.05). Immunocytochemical detection of MMP-13 showed intense staining in group B1, and the intensity of staining was sl ightly decreased in group A1, but no MMP-13 expression was detected in the group C1. The IA value for MMP-13 in group A1 was significantly lower than that of group B1 (P lt; 0.05), but significantly higher than that of group C1 (P lt; 0.05). The real time RT-PCR analysis showed significantly higher mRNA levels of Col II in group A1 than in group B1 (P lt; 0.05), but there was no significant difference between group A1 and group C1 (P gt; 0.05). The MMP-13 mRNA level of the chondrocytes in group A1 was significantly lower than that of group B1 (P lt; 0.05), but significantly higher than that of group C1 (P lt; 0.05). In vivo, the gross appearance of surface of knee joint showed that there was no ulcer in group C2, and there was some ulcers in group A2, but many and all layers ulcers in group B2. Mankin score of group A2 was significantly lowerthan that of group B2 (P lt; 0.05), but significantly higher than that of group C2 (P lt; 0.05). Immunohistochemical staining showed that Col II in articular cartilage was intensely staining in group C2, the intensity of staining was sl ightly decreased in group A2, and the intensity of Col II immunohistochemical staining was extremely low in group B2, but there was no significant difference between group A2 and group C2 (P gt; 0.05..........
To observe the histology change of the insertion using different diamertrical bone tunnel in anterior cruciate l igament (ACL) reconstruction. Methods Ninety Japanese rabbits were selected, wihout female and male l imit, weighing 2.5-3.0 kg, and were randomly divided into 3 groups, 30 in each group. The ratio of transplantation l igament diameter and bone tunnel diameter was 1/1 (group A), the ratio was 1/1.5 (group B), and the ratio was 1/2 (group C). Bone tunnel observation and histology observation were carried out in the 4th, 8th and 16th weeks postoperat ively. Results Wound healed well in 3 groups. The mean time of walking functional recovery was 1.5, 2.0 and 3.5 days in groups A, B and C respectively. After 4 weeks of operation, more soft tissues at tunnel entry were observed in group A and group B than in group C; after 8 weeks of operation, there was no crevice at bone-tunnel entry of the groups A and B, there was no improvement in group C; after 16 weeks of operation, groups A and B showed the normal insertion, group C had no normal insertion. Histology observation: in groups A, B and C, bone-tunnel was filled with loose connective tissue after 4 weeks of operation; group A and group B emerged the discontinuation ACL insertion tidal l ine after 8 weeks of operation, group C had no insertion; groups A and B emerged the similarity normal ACL insertion tidal l ine structure after 16 weeks of operation, but group C had no this structure. The results of ultimate tensile strength in groups A, B and C were (75.44 ± 7.06), (91.37 ± 6.14) and (126.91 ± 4.61) N respectively at 4 weeks; the results were (74.31 ± 4.81), (88.30 ± 7.46) and (124.34±8.44) N respectively at 8 weeks; and the results were (62.20 ± 5.32), (71.53 ± 5.99) and (83.62 ± 5.69) N respectively at 16 weeks. There was no significant difference between group A and group B (P gt; 0.05), and there were significant differences between groups A, B and group C (P lt; 0.05). Conclusion In the ACL reconstruction, the ratioof transplantation l igament diameter and bone tunnel diameter being 1/1.5 will not affect the insertion outcome, but if theratio less than the l imit it will affect the insertion outcome.
Objective To study the effect of various doses of estrogen on tissue injury, blood supply and survival area of skin flap and to investigate its mechanism. Methods Thirty New Zealand white rabbits aged 3-4 months old and weighing 1.5-2.2 kg (male or female) were used. Random pattern skin flap (12 cm × 3 cm in size) taking the central l ine of the rabbit dorsum as axis and with the pedicle attached at the proximal end was prepared, and the flap pedicle division was performed 7 days after operation. The rabbits were divided randomly into three groups (n=10 rabbits per group). At 2, 4, and 6 days after operation, the proximal edge of flap in group A and B received 100 ?g/kg and 50 ?g/kg subcutaneous injection ofestradiol benzoate, respectively, while group C received no further treatment serving as control group. General condition ofthe rabbits was observed after injection, gross observation was performed 3 and 7 days after injection, survival area of the skin flap was measured 7 days after injection, contents of malondialdehyde (MDA) and nitric oxide (NO) were tested 5 days after injection, and the flaps were harvested 4 and 7 days after injection to receive histology and no significant difference was noted between group A and group B (P gt; 0.05). The NEU counts 4 days after injection were (18.20 ±6.24) cells/HP in group A, (21.27 ± 5.34) cells/HP in group B, and (28.78 ± 7.92) cells/HP in group C, and at 7 days after injection, there were (15.16 ± 7.02) cells/HP in group A, (18.12 ± 6.44) cells/HP in group B, and (29.67 ± 9.12) cells/HP in group C. The VEGF score 4 days after injection was (4.02 ± 0.48) points in group A, (4.19 ± 0.66) points in group B and (3.67 ± 0.49) points in group C, and at 7 day after injection, it was (4.96 ± 0.69) points in group A, (5.12 ± 0.77) points in group B, and (3.81 ± 0.54) points in group C. Significant difference was evident between 4 days and 7 days after injection in group A or B in terms of NEU counts and VEGF score (P lt; 0.05), and difference between 4 days and 7 days after injection in group C was not significant (P gt; 0.05), and the differences among 3 groups were significant (P lt; 0.05). Conclusion Estrogen injection can increase VEGF expression and NO content of flap, decrease MDA content and NEU infiltration of flat, and improve survival area of flap.
OBJECTIVE To assess the treatment effect of sodium hyaluronate (HA) on experimental temporomandibular joint (TMJ) osteoarthritis of rabbits in comparison with prednisolone (PS). METHODS The upper compartments of both TMJs of 12 Japanese White Ear Rabbits were injected with 0.2 ml of 1.6% papain, 3 days after the right TMJs were injected again with same amount of papain to induce osteoarthritis with different severity levels. Except 1 rabbit was died accidentally. After one week from final injection of papain, the upper compartments of both TMJs of 6 rabbits were injected with HA 1.3 mg, 5 rabbits with PS 1.6 mg weekly for 4 times. At 3, 5 and 7 weeks after the final injection, the rabbits were sacrificed and the TMJs were pathologically examined. RESULTS The TMJs receiving PS showed predominant structural disorganization, and the right TMJs had much severe pathology. The manifestations were fibrillation, thinner or flaking of the articular cartilage of the temporal part of the joint, and the articular surface was covered with fibrous tissue. Whereas the TMJs receiving HA injections demonstrated limited changes of cartilage, less fibrillation, only local loss of cartilage on outside layer of the surface. In vicinity of the defect area, cluster of the chondrocytes appeared. Pathological scores of the TMJs receiving HA were significantly less than those of the TMJs revieving PS. CONCLUSION The results suggest that hyaluronate have effect of cartilaginous reparation and protection for the osteoarthritis of rabbit. While prednisolone has no help or worsened for articular cartilage reparation.
Objective To study the effect of decorin in the suppression of postoperative flexor tendon adhesion. Methods Eighteen Japanese large ear white rabbits underwent complete transection of the Ⅱ digit flexor digitorum profundus tendon in zone Ⅱ and defects immediately were repaired using the modified Kessler technique with -0 nonabsorbable monofilament suture. The site of the right repaired tendon was then injected with 100 μl of decorin(0.25mg/ml) as test toe, the site of the left repaired tendon with 100 μl of PBS as control toe. Inevery group, rabbits were killed and the feet were prepared for biomechanical testing, macroscopic examination and histological inspection. Results In every group, biomechanical testing demonstrates that the sliding distances and the rangs of motion significantly increased in the test toe compared with the control toe(Plt;0.05); macroscopic examination demonstrated that the tendon adhesions of the test toe were significantly reduced when compared with the control toe. In the tese toe, hematoxylin and eosin staining revealed that the hyperplasia of fibroblast was significantly delayed and the collagen fibrils arranged regularly and hadthe normal diameters. Conclusion Decorin can significantly reduce the flexor tendon adhesion formation, adjust collagen fibrillogenesis and promote the tendon healing.
Objective To explore a method to isolate, culture and multiplicate the placentaderived mesenchymal stem cells (PMSCs) and the bone marrow-derived mesenchymal stem cells (BMSCs) of rabbit，and to compare their biological characteristics. Methods PMSCs were isolated from placenta of 1fetation rabbitby Percoll density gradient centrifuge and cultured in vitro. BMSCs were isolated from hindlimb bone marrow blood of 1 new born rabbit by direct plates culturemethod. The 3rd passage PMSCs and BMSCs were observed by inverted phase contrast microscope. The stem cell marker (CD44, CD105, CD34 and CD40L) were examined by immunohistochemistry. The 2nd passage PMSCs and BMSCs were co-cultured with biomaterials，(1.0-1.5)×106 cells in one biomaterial， and then observed by aematoxylinstaining after 5 days,and by SEM after 3 days and 8 days. Results PMSCs and BMSCs were both uniformly spondle-shaped in appearance and showed active proliferative capacity. The proliferative ability of PMSCs were quite b and declined with passages. After cultured 10 passages in vitro, its growthslowed. Both PMSCs and BMSCs expressed CD44 and CD105,but did not express CD34 and CD40L immunoreactivity. PMSCs and BMSCs poliferated and adhered to the surface of biomaterials, and cell formed clumps and network; the cells proliferation and the matrix were seen in the pore after 5 days of culture. The observation ofSEM showed that many cells adhered to the biomaterials with spindle-shape and polygon after 3 days; and that PMSCs and BMSCs grew,arranged in layers andsecreted many matrices; the reticular collagen formed arround cells after 8 days. Conclusion PMSCs and BMSCs have similar biological characteristics and PMSCs can be served as excellent seedingcells for tissue engineering.
We established acute cholangitis and endotoxiemia in 18 rabbits by ligating the common bile duct and injecting E coli(O111B4 strain)into the common bile duct. After perfusion through activated charcoal via femoral artery-vein pathway, the average blood levels of endotoxin decreased sighificantly from 2.24Eu/ml to 0.17Eu/ml(Plt;0.001). This result suggested that blood perfusion through activated charcoal may be a promising therapy for acute endotoxemia.
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.
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）