[A PRELIMINARY STUDY ON SMALL INTESTINAL SUBMUCOSA-SILK COMPOSITE SCAFFOLD TO RECONSTRUCT ANTERIOR CRUCIATE LIGAMENT].

OBJECTIVE: To prepare the small intestinal submucosa (SIS)-silk composite scaffold for anterior cruciate ligament (ACL) reconstruction, and to evaluate its properties of biomechanics, biocompatibility, and the influence on synovial fluid leaking into tibia tunnel so as to provide a better choice in the clinical application of ACL reconstruction. METHODS: The silk was used to remove sericin and then weaved as silk scaffold, which was surrounded cylindrically by SIS to prepare a composite scaffold. The property of biomechanics was evaluated by biomechanical testing system. The cell biocompatibility of scaffolds was evaluated by live/dead staining and the cell counting kit 8 (CCK- 8). Thirty 6-week-old Sprague Dawley rats were randomly assigned to 2 groups (n = 15). The silk scaffold (S group) and composite scaffold (SS group) were subcutaneously implanted. At 2, 4, and 8 weeks after implanted, the specimen were harvested for HE staining to observe the biocompatibility. Another 20 28-week-old New Zealand white rabbits were randomly assigned to the S group and SS group (n = 20), and the silk scaffold and composite scaffold were used for ACL reconstruction respectively in 2 groups. Furthermore, a bone window was made on the tibia tunnel. At last, the electric resistance of tendon graft in the bone window was measured and recorded at different time points after 5 mL of 10% NaCl or 5 mL of ink solution was irrigated into the joint cavity recspectively. RESULTS: The gross observation showed that the composite scaffold consisted of the helical silk bundle inside which was surrounded by SIS. The maximal load of silk scaffold and composite scaffold was respectively (138.62 ± 11.41) N and (137.05± 16.95) N, showing no significant difference (P > 0.05); the stiffness was respectively (24.65 ± 2.62) N/mm and (24.21 ± 2.39) N/mm, showing no significant difference (P > 0.05). The live/dead staining showed that the cells had good activity on both scaffolds. However, the cells on the composite scaffold had better extensibility. In addition, the cell proliferation curve indicated that no significant difference in the absorbance (A) values was founded between groups at various time points (P > 0.05). HE staining showed less inflammatory cells and much more angiogenesis in SS group than in S group at 2, 4, and 8 weeks after subcutaneously implanted (P < 0.05), indicating good biocompatibility. Additionally, the starting time points of electric resistance decrease and the ink leakage were both significantly later in SS group than in S group (P < 0.05). The duration of ink leakage was significantly longer in SS group than in S group (P < 0.05). CONCLUSION: The SIS-silk composite scaffold has excellent biomechanical properties and biocompatibility and early vacularization after in viva implantation. Moreover, it can reducing the leakage of synovial fluid into tibia tunnel at the early stage of ACE reconstruction. So it is promising to be an ideal ACL scaffold.

[Free fibula flap and computed tomographic angiography in the functional reconstruction of oral and maxillofacial hard and soft tissue defects].

OBJECTIVE: Assessed the feasibility of application of free fibular flap and clinical significance of pre-operational contrast enhanced CT angiography in functional reconstruction of oral and maxillofacial hard and soft tissue defects. METHOD: Eight cases with mandibular and soft tissue defects received a free fibula flap using arteriovenous anastomosis anastomosis method. The relationship between fibula flap design, size, repair parts and survival were analyzed. Preoperative enhanced CT angiography (CTA) examination was conducted to detect any abnormal blood vessels in fibula flap valve area. RESULT: Peroneal artery and posterior tibial artery variation was identified in one case of gums cancer, who used other muscle flap. Free fibula flap in the other 7 cases survived, which carried a skin island with an area ranging from 6 cm x 2 cm to 10.0 cm x 3.5 cm. Postoperative facial appearance, functionality, dental occlusion and voice function was normal. Lower limb function returned to normal from 2 weeks to 4 months after surgery. CONCLUSION: CTA examination of the free vascularized fibula flap in the preoperative evaluation of the donor site is a valuable tool. Free fibula flap of bone, periosteum and bone marrow has a dual blood supply and are highly resistant to infection after transplantation. Personalized shaping osteotomy allowed for accurate recovery of mandibular alveolar patterns. Furthermore, the height and width of the fibula and the thickness of cortical bone is suitable for dental implants. Free fibula flap skin island can also be used to monitor the post-operative blood supply and is an ideal technique for mandible and soft tissue defects reconstruction as well as functional restoration.

Tissue engineered esophagus by mesenchymal stem cell seeding for esophageal repair in a canine model.

PURPOSE: Acellular porcine small intestinal submucosa (SIS) has been successfully used for esophagoplasty in dogs. However, this has not led to complete epithelialization and muscular regeneration. We undertook the present study to assess the effect of tissue-engineered esophagus generated by seeding bone marrow mesenchymal stem cells (BMSCs) onto an SIS scaffold (BMSCs-SIS) in a canine model. METHODS: We cultured, passaged, and measured autologous BMSCs and myoblasts with cell proliferation and immunohistochemical assays. We labeled the third passage of BMSCs with PKH-26, a fluorescent dye, before seeded it onto the SIS. We resected canine cervical esophagus to generate a defect 5 cm in length and 50% in circumference, which we repaired with BMSCs-SIS or SIS alone. RESULTS: Four weeks later, barium esophagram demonstrated that esophageal lumen surface of the patch graft was smoother in the BMSCs-SIS group compared with the SIS group. Histological examination suggested a strong similarity between BMSCs and esophageal myoblasts in terms of morphology and function. Although both BMSCs-SIS and SIS repaired the esophageal defects, we noted complete re-epithelialization with almost no inflammation only in the former group. By 12 wk after the surgery, we observed long bundles of skeletal muscles only in the BMSCs-SIS group, where the microvessel density was also much greater. CONCLUSIONS: Bone marrow mesenchymal stem cells on an SIS scaffold can promote re-epithelialization, revascularization, and muscular regeneration. This approach may provide an attractive option for esophageal regeneration.

[Clinical treatment of open pelvic fractures associated with perineal injury].

OBJECTIVE: To investigate the method and the effectiveness of open pelvic fractures associated with perineal injury. METHODS: Between August 2000 and July 2010, 16 cases of open pelvic fractures associated with perineal injury were treated. There were 12 males and 4 females with an average age of 41 years (range, 17-69 years). Injury was caused by traffic accidents in 9 cases, by falling from height in 6 cases, and by crushing in 1 case. The mean time between injury and admission was 8 minutes (range, 5-20 minutes). According to Tile classification, 2 cases were rated as type A, 6 as type B, and 8 as type C. The wound size ranged from 5 cm x 3 cm to 15 cm x 12 cm. The perineal injured location included intraperitoneal rectal injury in 2 cases and extraperitoneal anorectal injury in 14 cases. The average injury severity score (ISS) was 29 (range, 25-48). The main treatments included emergency resuscitation, colostomy, external fixation of fractures, repeated debridement with pulsatile irrigation followed by intravenous antibiotics, and vacuum sealing drainage (VSD). RESULTS: In 5 deaths, 3 cases died of hemorrhagic shock and 2 cases died of multiple system organ failure within 4 days of admission. The other 11 cases were followed up 6-46 months (mean, 14 months). The X-ray films showed that bone union was achieved after 2-4 months of operation. Infection in varying degree occurred at perineal wounds; second stage healing of wounds was achieved in 10 cases after debridement and VSD treatment, and wound healed in 1 case after gracilis muscle flap repair. No anal incontinence occurred in the patients having anorectal injury during follow-up. CONCLUSION: For patients with perineal injury and open pelvic fractures, the following treatments should be carried out so as to obtain good effectiveness: early anti-shock, protection of important organ function, treatment of complications, late resistance to infection and stability restoration of the pelvic ring, functional repair and reconstruction of rectum and anal canal and urinary tract.

[Experiment of oral mucosa epithelial cells cultured on small intestinal submucosa in vitro].

OBJECTIVE: To explore an effective method to culture oral mucosa epithelial cells (OMECs) of canine in vitro, and to observe the biological characteristics of OMECs growing on small intestinal submucosa (SIS) in order to provide the experimental basis for epithelium tissue engineering. METHODS: The primary OMECs were cultivated with DKSFM (defined keratinocyte serum free medium) containing 6% fetal bovine serum (FBS). The morphological characteristics and the growth curve of OMECs were observed. The expressions of OMECs marker (CK19) were examined by immunocytochemistry. The 2nd passage of OMECs were seeded on SIS, OMECs co-cultured with SIS were observed by hematoxylin-eosin staining, immunohistochemical staining, and scanning electron microscope (SEM). RESULTS: OMECs were grown well in DKSFM. Immunohistochemical staining of the 2nd passage cultured canine OMECs with broadly reacting anti-cytokeratin anyibodies (CK19) was positive. OMECs formed a single layer on the surface of SIS, and eight days later the cells were polygong and arranged like slabstone. CONCLUSION: Culture of canine OMECs in DKSFM containing 6% FBS is a simple and feasible method. SIS has good biocompatibility, it is a kind of good bioscafold in the tissue-engineered epithelium.

[Effects of epithelial cell conditioned medium on differentiation of BMSCs].

OBJECTIVE: To investigate the feasibility of inducing canine BMSCs to differentiate into epithelial cells in vitro with epithelial cell conditioned medium (ECCM). METHODS: Five mL BMSCs were obtained from iliac spine of a healthy adult male canine with weighing 10 kg, and then isolated and cultured. The oral mucosa was harvested and cut into 4 mm x 4 mm after the submucosa tissue was eliminated; ECCM was prepared. BMSCs of the 2nd passage were cultured and divided into two groups, cultured in ECCM as experimental group and in L-DMEM as control group. The cell morphological characteristics were observed and the cell growth curves of two groups were drawn by the continual cell counting. The cells were identified by immunohistochemical staining through detecting cytokeratin 19 (CK-19) and anti-cytokeratin AE1/AE3 on the 21st day of induction. The ultra-structure characteristics were observed under transmission electron microscope. RESULTS: The cells of two groups showed long-fusiform in shape and distributed uniformly under inverted phase contrast microscope. The cell growth curves of two groups presented S type. The cell growth curve of the experimental group was right shifted, showing cell proliferation inhibition in ECCM. The result of immunohistochemical staining for CK-19 and anti-cytokeratin AE1/AE3 was positive in the experimental group, confirming the epithelial phenotype of the cells; while the result was negative in the control group. The cells were characterized by tight junction under transmission electron microscope. CONCLUSION: The canine ECCM can induce allogenic BMSCs to differentiate into epithelial cells in vitro.

Grafts of porcine small intestinal submucosa with cultured autologous oral mucosal epithelial cells for esophageal repair in a canine model.

The acellular porcine small intestinal submucosa (SIS) has been successfully used for esophagoplasty. However, it does not lead to a complete epithelialization in a canine model. A cellular component may be required for better reconstruction. The present study was undertaken to investigate the feasibility and effectiveness of the combination of SIS and autologous oral mucosal epithelial cells (OMECs) for esophageal reconstruction. The OMECs harvested from beagle dogs were cultured and propagated, and the 3rd passage cells were seeded on a single-layer SIS. Male beagle dogs were subjected to surgical resection to produce cervical esophageal defects (5 cm in length, 180 degrees in range). SIS with or without OMECs was patched on the esophageal defects. Barium esophagram, immunohistochemistry, and histology were performed to evaluate the therapeutic effectiveness. Four weeks after surgery, the histological examination showed a complete re-epithelialization and almost no inflammation in the SIS with OMECs group. But in the SIS group, only a partial epithelialization was observed along with inflammation. Eight weeks after surgery, the squamous epithelium was found to cover the entire graft surface in both groups; however, the muscular regeneration was observed in the SIS with OMECs, but not in the SIS group. The graft of SIS combined with autologous OMECs promotes re-epithelialization and muscular regeneration. It is an effective alternative method for esophageal repair.

Regulation of cell proliferation by fast Myosin light chain 1 in myoblasts derived from extraocular muscle, diaphragm and gastrocnemius.

The extraocular muscle (EOM) suffers much less injury from Duchenne muscular dystrophy (DMD) than other skeletal muscles such as diaphragm and gastrocnemius. The present study was undertaken to test the hypothesis that differential expression of regulatory proteins between the EOM and other skeletal muscles is responsible for the observed difference in the sensitivity to DMD-associated damage. Protein expression in the tissue samples obtained from EOM, diaphragm or gastrocnemius of C57BL/6 mice was analyzed by two-dimensional gel electrophoresis and mass spectrometry. There were 35 proteins that were identified to be differentially expressed among different skeletal muscle tissues. Among the 35 proteins, a fast skeletal muscle isoform myosin light chain 1 (MLC1f) protein was further studied in relation to muscle cell proliferation. The EOM-derived myoblasts had much lower levels of MLC1f and higher rate of cell proliferation in contrast to the myoblasts derived from diaphragm or gastrocnemius, which displayed a higher expression of MLC1f along with a slow proliferation. Deletion of MLC1f using siRNA targeting MLC1f resulted in an increased rate of cell proliferation in the myoblasts. Cell cycle analysis revealed that MLC1f inhibited the transition of the cell cycle from the G1 to the S phase. Therefore, the present study demonstrates that MLC1f may negatively regulate proliferation of myoblasts through inhibition of the transition from the G1 to the S phase of the cell cycle. Low levels of MLC1f in myoblasts of EOM may ensure cell proliferation and enhance the repair process for EOM under the DMD disease condition, thus making EOM suffer less injury from DMD.

[An experimental study of coculture of esophageal mucosa epithelial cells with SIS and their biological characteristics].

OBJECTIVE: To explore an effective method to cultivate esophageal mucosa epithelial cells (EMECs) of canine in vitro, and to observe the biological characteristics of EMECs growing on SIS in order to provide an experimental basis for esophagus tissue engineering. METHODS: Esophageal tissues were obtained from five healthy dogs aged 2 to 5 weeks under sterile conditions. The primary EMECs were cultivated with defined keratinocyte serum free medium (DKSFM) containing 6% FBS. The morphological characteristics and the growth curve of EMECs of the 2nd generation were observed for 1 to 5 days. The expressions of the EMECs marker (cytokeratin 19, CK-19) were examined by immunocytochemistry. The 2nd generation of EMECs was seeded on SIS and observed by HE staining, immunohistochemical staining, and SEM for 4 and 8 days. RESULTS: The primary culture of canine EMECs arranged like slabstone. Immunohistochemical staining of CK-19 of the 2nd generation EMECs showed positive broadly. The cells growth reached the peak level at 2 days by MTT method. EMECs were polygon in shape and arranged like slabstone, and formed a single layer on the surface of SIS. The cells were contacted closely with each other for 4 days. Eight days later, 2 to 3 layers stratified structure was formed. Lots of EMECs were grown on SIS, and showed laminate arrangement. CONCLUSION: With mixed enzymatic digestion, the culture of EMECs in DKSFM containing 6% FBS is a simple and feasible method. SIS shows good biocompatibility and can be used as a good scaffold material in the tissue engineered esophagus.

[Repair of anterior tibial, dorsal pedal and calcaneal soft-tissue defects with lateral crural flaps].

OBJECTIVE: To report the methods and clinical effect of the lateral crural flaps in repairing anterior tibial, dorsal and calcaneal soft-tissue defects. METHODS: From August 1999 to December 2004, 18 cases of defects were repaired with lateral crural flap, including 15 cases of anterior tibial, dorsal and calcaneal soft-tissue defects with vascular pedicled island lateral crural flaps and 3 cases of dorsal pedal soft-tissue defects with free vascular lateral crural flaps. RESULTS: All flaps survived after operation. Insufficient arterial supply of the flap occurred in 2 cases after operation, the pedicled incision sewing thread was removed and lidocaine was injected around vascular pedicle, then the flap ischemia was released. Inadequate venous return and venous hyperemia occurred in 1 case because peroneal vein was injured during operation. The flap edge skin was cut and heparin was locally dripped for one week, the flap vascular cycle was resumed. All patients were followed up two months to one year, the flaps were not fat, and the elasticity was good. CONCLUSION: It is safe and reliable to use lateral crural flap to repair anterior tibial, dorsal pedal and calcaneal soft-tissue defects.