Objective To explore a new method for the pre-degeneration of peripheral nerve in vitro for obtaining many effective Schwann cells so as to provide a large number of seed cells for the research and application of tissue engineered nerves. Methods The bone marrow derived cells (BMDCs) from transgenic green fluorescent protein C57BL/6 mouse and the sciatic nerve segments from the C57BL/6 mouse were co-cultured to prepare the pre-degeneration of sciatic nerve in vitro (experimental group, group A), and only sciatic nerve was cultured (control group, group B). At 7 days after culture, whether BMDCs can permeate into the sciatic nerve in vitro for pre-degeneration was observed by gross and immunohistofluorescence staining. And then Schwann cells were obtained from the sciatic nerves by enzymic digestion and cultured. The cell number was counted, and then the purity of primary Schwann cells was determined using immunohistofluorescence staining and flow cytometer analysis. Results At 7 days after pre-degeneration, gross observation showed that enlargement was observed at nerve stumps, and neuroma-like structure formed; the group A was more obvious than group B. Immunohistofluorescence staining showed many BMDCs permeated into the nerve segments, with positive F4/80 staining in group A. After culture, the yield of Schwann cells was (5.59 ± 0.19) × 104 /mg in group A and (3.20 ± 0.21) × 104/mg in group B, showing significant difference (t=2.14, P=0.03). At 48 hours after inoculation, the cells had blue bipolar or tripolar cell nuclei with small size and red soma by immunohistofluorescence staining; fibroblasts were flat polygonal with clear nucleus and nucleolus, showing negative p75NTR staining; and there were few of fibroblasts in group A. The purity of Schwann cells was 88.4% ± 5.8% in group A and 76.1% ± 3.7% in group B, showing significant difference (t=2.38, P=0.04). And the flow cytometer analysis showed that the purity was 89.6% in group A and 74.9% in group B. Conclusion BMDCs can promote the pre-degeneration of peripheral nerve in vitro, and it is a new method to effectively obtain Schwann cells for tissue engineered nerve.
OBJECTIVE: To study the characteristics of, morphology histology and ultrastructure of anterior cruciate ligament(ACL) autograft and two-step cryopreserved ACL allograft after transplantation. METHODS: Sixty New Zealand rabbits and sixty Japanese rabbits were randomly divided into two groups: ACL autograft group and two-step cryopreserved ACL allograft group. Immunosuppressant were not used after transplantation. The histology and ultrastructure of the ACL of transplantation and normal knee were observed after 4 weeks and 12 weeks, respectively. RESULTS: The rate of remodeling process was faster in ACL autograft than in two-step cryopreserved ACL allograft, but there was similar remodeling process between two groups 12 weeks after transplantation. The proportions of large-diameter fibers(gt; or = 80 nm) of ACL autograft and cryopreserved ACL allograft were 6% and 24% in the 4th week, and were 0 and 2% in the 12th week, respectively. The proportions of small-diameter of fibers(lt; 80 nm) of ACL autogrft and cryopreserved ACL allograft were 94% and 76% in the 4th week, and 100% and 98% in the 12th week, respectively. Histologic incorporation in ACL autograft was similar to that in cryopreserved ACL allograft. CONCLUSION: Two-step cryopreserved bone-ACL-bone allograft were similar to bone-ACL-bone autograft cryopreserved in remodeling process and histology. The rate of remodeling process was faster in ACL autograft than in cryopreserved ACL allograft.
OBJECTIVE To search an optimal method for improving viability of cryopreserved articular cartilage. METHODS Articular cartilage which was sampled from the rabbits were randomly divided into 5 groups. Fresh cartilage was group I, other groups were frozen. Before frozen, other cartilage was exposured in 10% DMSO at 4 degrees C for 30 minutes(group II), 1 hour(group III), 2 hours (group IV), 4 hours(group V), then were stored in liquid nitrogen for 1 week. Viabilities of the chondrocytes were detected by Typan-blue staining, electron transmission microscope, and determination of incorporation 3H-TdR after the temperature returned to normal. RESULTS 1. The cells were injuried at different extent after the cartilage was frozen. In group I, survival rate of cells was 96% and incorporation of 3H-TdR was (4,953.13 +/- 583.27)%, statistic difference was significant between group I and other groups(P lt; 0.01). The microstructure of group I was normal while other groups all had damage of the organella, 2. Structures and functions of cells in group IV were best among frozen groups. Organella were less damaged than group II, III, V, survival rate of cells was 56% and incorporation of 3H-TdR was (1,139.88 +/- 146.39)%, statistic difference was significant between group IV and group II, III, V(P lt; 0.01). CONCLUSION If cartilage are exposured in 10% DMSO at 4 degrees C for 2 hours before frozen, optimal cryopreservation can be achieved.
Objective To review the osteogenic mechanism and osteogenic effects of bone morphogenetic protein 6 (BMP-6) so as to provide the basis for further research of BMP-6. Methods The related articles about the osteogenic mechanism and the osteogenic effects of BMP-6 in experimental animals were extensively summarized. Results BMP-6 from bone matrix can transduct the osteogenic signal to bone marrow mesenchymal stem cells (BMSCs) by means of Smad protein signal transduction pathway. And the BMSCs which received the signals will differentiate into osteoblasts and chondroblasts. Therefore, BMP-6 plays an important role in the development and maturation of bone and cartilage. In addition, BMP-6 has a close relation with bone diseases, such as fracture, osteoporosis, and bone tumor. Conclusion The deep research of BMP-6 is expected to provide a new therapeutic approach for treating bone diseases of nonunion, osteoarthritis, and osteoporosis.
Objective To investigate the effects of human insulin-like growth factor 1 (hIGF-1) gene transfected by recombinant adenovirus vector (Ad-hIGF-1) on the apoptosis of rabbit nucleus pulposus cells induced by tumor necrosis factor α (TNF-α). Methods The intervertebral disc nucleus pulposus were harvested from 8 healthy adult domestic rabbits (male or female, weighing 2.0-2.5 kg). The nucleus pulposus cells were isolated with collagenase II digestion and the passage 2 cells were cultured to logarithm growing period, and then they were divided into 3 groups according to culture condition: DMEM/F12 medium containing 10% PBS, DMEM/F12 medium containing 10% PBS and 100 ng/mL TNF-α, and DMEM/ F12 medium containing 10% PBS, 100 ng/ mL TNF-α, and Ad-hIGF-1 (multiplicity of infection of 50) were used in control group, TNF-α group, and Ad-hIGF-1 group, respectively. The results of transfection by adenovirus vector carrying hIGF-1 gene were observed by fluorescent microscopy; the expression of hIGF-1 protein was detected by Western blot, hIGF-1 mRNA expression by RT-PCR, and the cell apoptosis rate by TUNEL and flow cytometry. Results Green fluorescence was observed by fluorescent microscopy in Ad-hIGF-1 group, indicating that successful cell transfection. The expressions of hIGF-1 protein and mRNA were detected in Ad-hIGF-1 group by Western blot and RT-PCR, while the control group and TNF-α group had no expression. The cell apoptosis rates of TNF-α group, Ad-hIGF-1 group, and control group were 34.24% ± 4.60%, 6.59% ± 1.03%, and 0.40% ± 0.15%, respectively. The early apoptosis rates of TNF-α group, Ad-hIGF-1 group, and control group were 22.16% ± 2.69%, 5.03% ± 0.96%, and 0.49% ± 0.05%, respectively; the late cell apoptosis rates were 13.96% ± 4.86%, 10.68% ± 3.42%, and 0.29% ± 0.06%, respectively. Compared with TNF-α group, the cell apoptosis rates of Ad-hIGF-1 group and control group were significantly reduced (P lt; 0.05); the cell apoptosis rate of Ad-hIGF-1 group was significantly higher than that of control group (P lt; 0.05). Conclusion Ad-hIGF-1 could inhibit the apoptosis of nucleus pulposus cells induced by TNF-α.
Objective To observe the influence on adjacent lumbar bone density after strengthening of T12, L1 segment vertebral osteoporotic compression fracture by percutaneous vertebroplasty (PVP) and percutaneous kyphoplasty (PKP) in postmenopausal female. Methods Between January 2008 and June 2011, 59 patients with T12, L1 segment thoracolumbar osteoporotic compression fracture were treated with PVP in 29 cases (PVP group) and PKP in 30 cases (PKP group), who were in accordance with the inclusion and exclusion criteria. No significant difference was found in gender, duration of menopause, disease druation, causes of injury, fractured vertebral body, and vertebral fracture classification between 2 groups (P gt; 0.05). The kyphosis Cobb angle of surgical area was measured at preoperation, 1 week after operation, and last follow-up; the lower three lumbar spine bone mineral density (BMD) of the surgical area, the femoral neck BMD, and body mass index (BMI) of patients were measured at perioperative period and last follow-up to find out the statement of anti-osteoporosis; FRAX online tools were used to evaluate the probability of major osteoporotic fracture and hip fracture of the next 10 years. Results The average follow-up was 25.5 months (range, 12-48 months) in 2 groups. There was significant difference in kyphosis Cobb angle of T12, L1 between preoperation and last follow-up in 2 groups (P lt; 0.05); the Cobb angle of PKP group was significantly less than that of PVP group at 1 week after operation and last follow-up (P lt; 0.05). No significant difference was found in BMI between 2 groups, and between perioperative period and last follow-up in the same group (P gt; 0.05). The lower three lumbar spine BMD of the surgical area and its T value at last follow-up was improved significantly when compared with BMD at perioperative period (P lt; 0.05); there was no significant difference in the lower three lumbar spine BMD and its T value between 2 groups at perioperative period (P gt; 0.05), but significant difference was found between two groups at last follow-up (P lt; 0.05). Difference was not significant in the femoral neck BMD and its T value between 2 groups, and between perioperative period and last follow-up in the same group (P gt; 0.05). The probability of major osteoporotic fracture and hip fracture of the next 10 years was not significantly different between 2 groups and between perioperative period and last follow-up in the same group (P gt; 0.05). Conclusion The increased BMD of adjacent lumbar spine can improve the strength of the vertebral body and reduce the incidence of adjacent vertebral fracture in patients with T12, L1 segment vertebral osteoporotic compression fracture after PVP/PKP, and PKP is superior to PVP increasing BMD of adjacent lumbar spine.
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.
ObjectiveTo understand the location characteristics of the lumbosacral autonomic nerve plexus and the morphological changes so as to provide the anatomic theoretical basis for the protection of autonomic nerve during the lower lumbar anterior approach operation. MethodsA random anatomic investigation was carried out on 19 formalin-treated adult cadavers (15 males and 4 females; aged 44-78 years, mean 64 years). The anterior median line (connection of suprasternal fossa point and the midpoint of the symphysis pubis) was determined, and the characteristics of abdominal aortic plexus (AAP), inferior mesenteric plexus (IMP), and superior hypogastric plexus (SHP) were observed. The relationship between the autonomic nerve and the anterior median line was measured and recorded. ResultsAPP and IMP were found to be located chiefly in front of the abdominal aorta in a reticular pattern, and the nerve fibers of the two nerve plexuses were more densely at the left side of abdominal aorta than at the right side. Superior hypogastric plexus showed more distinct main vessel variations, including 4 types. The main vessel length of the SHP was (59.38±12.86) mm, and the width was (11.25±2.92) mm. The main vessels of SHP were mainly located at the left side of the ventral median line (10, 52.6%) and anterior lumbar vertebra (13, 68.4%). The main vessels extended down to form the left and right hypogastric nerves. ConclusionIt is applicable to expose the nerve from the right side of centrum and move the autonomic nerve and blood vessel as a whole during anterior lower lumbar operation. In this way, the dissection to separate nerve plexus is not needed, thus nerve injury can be avoided to the largest extent.
ObjectiveTo assess the long-term effectiveness of the transplantation of iliac bone flap pedicled with deep iliac circumflex vessels for treating avascular necrosis of femoral head (ANFH) at Ficat stage Ⅱ and Ⅲ. MethodsThirty-two cases (43 hips) of ANFH underwent iliac bone flap transplantation pedicled with deep iliac circumflex vessels between October 2000 and February 2006, and the clinical data were retrospectively reviewed. Of 32 cases, 27 were male (38 hips), and 5 were female (5 hips), aged 21-52 years (mean, 36.6 years); there were 8 cases (11 hips) of hormone ANFH, 18 cases (23 hips) of alcoholic ANFH, and 6 cases (9 hips) of idiopathic ANFH. The disease duration ranged from 2-52 months (mean, 8.2 months). According to Ficat staging criteria, 26 hips were classified as stage Ⅱ and 17 hips as stage Ⅲ. The preoperative Harris hip score (HSS) was 68.2±8.4. The HHS scores and X-ray photograph were compared between at pre-and post-operation to assess the outcomes clinically and radiologically. ResultsAll incisions healed by first intention. Two cases had numbness of the lateral femoral skin. Four patients (6 hips) failed to be followed up, and the other 28 cases were followed up 98-187 months (mean, 129.3 months). Five patients (6 hips) showed aggravation or no relief with progression to stage Ⅳ at 8-69 months, who received total hip arthroplasty. The 10-year survival rate was 83.78% (31/37). The HHS score was significantly increased to 86.7±9.0 at last follow-up (t=-48.313, P=0.000). The hip function was excellent in 9 hips, good in 13 hips, and fair in 9 hips, and the success rate was 75.68%. Radiographic examination showed signs of bone remodeling at 6-8 weeks. After bone healing, the femoral head density gradually became uniform. Until last follow-up, ANFH progressed from Ficat stage Ⅱ to Ⅲ in 5 hips, from Ficat stage Ⅱ to Ⅳ in 3 hips, and from Ficat stage Ⅲ to Ⅳ in 3 hips; complete hip shape, continuous Shenton line, and normal joint space were observed in the other 26 hips, and the radiographic success rate was 70.27% (26/37). ConclusionThe pedicled iliac bone flap transplantation for ANFH at Ficat stage Ⅱ and Ⅲ can provide good osteogenesis and vascular reconstruction of the femoral head. The long-term follow-up effectiveness is satisfactory.
ObjectiveTo investigate the effect of vascularized peroneus longus tendon graft reconstruction on anterior cruciate ligament (ACL) insertion in rabbits.MethodsEighty healthy New Zealand white rabbits were harvested to prepare ACL injury models and randomly divided into two groups (n=40). The ACL was reconstructed with vascularized peroneus longus tendon graft in group A and peroneus longus tendon graft without blood supply in group B. The survival of animals and the healing of incision were observed after operation; the grafts were taken for gross and histological observations at 4, 8, and 16 weeks; the biomechanical test of the grafts was carried out to record the maximum tensile strength and incidence of ACL insertion rupture at 8 and 16 weeks.ResultsAll animals survived until the experiment completed. General observation showed that the tunnel was combined with grafts, the vascular infiltration was abundant, and no obvious boundary between the tunnel and grafts existed at 16 weeks in group A; there was still an obvious boundary between the tunnel and graft in group B. Histological observation showed that the collagen fibers between tendon and bone in group A increased gradually, the dense fiber connection was formed, and the “tidal-line” like structure similar to the normal ACL insertion was formed at 16 weeks; but the“tidal-line” like structure was not obvious in group B. Biomechanical test showed that there was no significant difference in the incidence of ACL insertion rupture at 8 and 16 weeks between group A and group B (P=0.680; P=0.590), but the maximum tensile strength at 8 and 16 weeks were higher in group A than in group B (t=18.503, P=0.001; t=25.391, P=0.001).ConclusionThe vascularized peroneus longus tendon graft for ACL reconstruction can obviously promote the outcome of the ACL insertion.