Objective To investigate the distribution of the perforating branches artery of distally-based flap of sural nerve nutrient vessels and its clinical application. Methods The origins and distribution of perforating branchesartery of distally-based flap were observed on specimens of 30 adult cadavericlow limbs by perfusing red gelatin to dissect the artery.Among the 36 cases, there were 21 males, 15 females. Their ages ranged from 6 to 66, 35.2 in average. The defect area was 3.5 cm×2.5 cm to 17.0 cm×11.0 cm. The flap taken ranged from 4 cm×3 cm to 18 cm×12 cm. Results The perforating branches artery of distally-based flap had 2 to 5 branches and originated from the heel lateral artery, the terminal perforating branches of peroneal artery(diameters were 0.6±0.2 mm and 0.8±0.2 mm, 1.0±1.3 cm and 2.8±1.0 cm to the level of cusp lateral malleolus cusp).The intermuscular septum perforating branches of peroneal artery had 0 to 3 branches. Their rate of presence was 96.7%,66.7% and 20.0% respectively(the diameters were 0.9±0.3, 1.0±0.2 and 0.8±0.4 mm, andtheir distances to the level of cusp of lateral malleolus were 5.3±2.1, 6.8±2.8 and 7.0±4.0 cm). Those perforating branches included fascia branches, cutaneous branches, nerve and vein nutrient branches. Those nutrient vessels formed longitudinal vessel chain of sural nerve shaft, vessel chain of vein side and vessel network of deep superficial fascia. The distally-based superficial sural artery island flap was used in 18 cases, all flaps survived. Conclusion Distally-based sural nerve, small saphenous vein, and nutrient vessels of fascia skin have the same origin. Rotation point of flap is 3.0 cm to the cusp of lateral malleolus, when the distally-based flap is pedicled with the terminal branch of peroneal artery.Rotation point of flap is close to the cusp of lateral malleolus, when the distally-based flap is pedicled with the heel lateral artery.
Objective To investigate the possibility of theadipose tissue-derived stromal cells(ADSCs) to differentiate into the neuron-like cells and to explore a new cell source for the transplantation related to the central nervous system. Methods Adipose was digested by collagenase, cultured in the fetal bovine serum containing a medium. Trypse was used to digest the cells and the cell passage was performed. The 3rd to the 9th passage ADSCs were used to make an induction. Isobutylmethylxanthine, indomethacin, insulin, and dexamethasone were used to induce the ADSCs to differentiate into the neuron-like cells and adipocytes. Sudan black B and immunocytochemistry were used to identify the cells. Results A population of the ADSCs could be isolated from the adult human adipose tissue, they were processed to obtain a fibroblast-like population of the cells and could be maintained in vitro for an extendedperiod with the stable population doubling, and they were expanded as the undifferentiated cells in culture for more than 20 passages, which indicated their proliferative capacity. They expressed vimentin and nestin, and characteristics of the neuron precursor stem cells at an early stage of differentiation. And the majority of the ADSCs also expressed the neuron-specific enolase and βⅢ-tubulin, characteristics of the neurons. Isobutyl-methyxanthine, indomethacin, insulin, and dexamethasone induced 40%-50% of ADSCs to differentiate into adipocytes and 0.1%0.2% of ADSCs into neuron-like cells. The neuron-like cells had a complicated morphology of the neurons, and they exhibited a neuron phenotype, expressed nestin, vimentin, neuron-specific enolase and βⅢ-tubulin, but some neuron-like cells also expressed thesmooth muscle actin (SMA), and the characteristics of the smooth muscle cells; however, the neurons from the central nervous system were never reported to express this kind of protein. Therefore, the neuron-like cells from the ADSCs could be regarded as functional neurons. Conclusion Ourresults support the hypothesis that the adult adipose tissue contains the stem cells capable of differentiating into the neuron-like cells, and they can overcome their mesenchymal commitment, which represents an alternative autologous stemcell source for transplantation related to the central nervous system.
Objective To explore the expression characteristics of chaperone interacting protein (CHIP) in normal, scar and chronic ulcer tissues and its relationship with wound healing. Methods Twenty biopsies including scar tissues(n=8), chronic ulcer tissues(n=4) and normal tissues(n=8)were used in this study. The immunohistochemical staining (power visionTMtwo-step histostaining reagent) was used to explore the amount and expression characteristics of such protein.Results The positive expression of CHIP was observed in fibroblasts, endothelial cells and epidermal cells in dermis and epidermis. It was not seen ininflammatory cells. The expression amount of CHIP in scar tissues, chronic ulcer tissues and normal tissues was 89%, 83% and 17% respectively. Conclusion Although the function of CHIP is not fully understood at present, the fact that this protein is expressed only at the mitogenic cells indicates that it may be involved in mitogenic regulation during wound healing.
Objective To assess the possibility of placing the posterior pedicle screw on atlas. Methods Twenty human cadaver specimens were used to insert pedicle screws in atlas, through the posterior arch or the pedicle of C1 into the lateral mass. The screw entry point was on the posterior surface of C1 posterior arch and at the intersection of the vertical line through the center of C2 inferior articular process and the horizontal line at least 3 mm below the superior rim of the C1 lamina. The screw of 3.5 mm in diameter was placed in a direction of 10° medial angle and 5° upwardangle. After placement of C1 pedicle screw, the distance from C1 screw entry point to the mediallateral midpoint of C1 pedicle, the maximum length of screw trajectory and the actual screw trajectory angles were measured. The direction of screw penetrating through the cortical of C1 pedicle or lateral mass and the injuries to the vertebral artery and spinal cord were observed.Results Forty pedicle screws were placed on atlas, the mean distance from C1 screw entry point to the medial-lateral midpoint of C1 pedicle was (2.20±0.42)mm, the maximum length of screw trajectory averaged (30.51±1.59)mm, and the actual screw trajectory angle measured (9.7±0.67)° in a medial direction and (4.6±0.59) ° in a upward direction. Only 1 screw penetrated the upper cortical bone of the atlas pedicle because the upward angle was too large, and 8 screws were inserted so deep that the inferior cortical bone of the C1 lateral mass was penetrated. But no injuries to the vertebral artery and spinal cord wereobserved. Conclusion C1 posterior pedicle screw fixation is quite accessible and safe, but the su
Objective To investigate the effects of asiaticoside onthe proliferation and the Smad signal pathway of the hypertrophic scar fibroblasts.Methods The hypertrophic scar fibroblasts were cultured with tissue culture method. The expressions of Smad2 and Smad7 mRNA after asiaticoside treatment were determined by reverse transcriptionpolymerase chain reaction 48 hours later. Thecell cycle, the cell proliferation, the cell apoptosis and the expression of phosphorylated Smad2 and Smad7 with(experimental group) or without(control group) asiaticoside were detected with flow cytometry, immunocytochemistry and Western blot. Results Asiaticoside inhibited the hypertrophic scar fibroblasts from phase S to phase M. The Smad7 content and the expression of Smad7 mRNA were (1.33±1.26)% and (50.80±22.40)% in experimental group, and (9.15±3.36)% and (32.18±17.84)% in control group; there were significant differences between two groups (P＜0.05). While the content and the mRNA expression of Smad2 had no significant difference between two groups. Conclusion Asiaticoside inhibits the scar formation through Smad signal pathway.
Objective To review research progress of adipose tissuederived stromal cells (ADSCs).Methods The recent articles on ADSCs were extensively reviewed, and the culture and differentiation ability of ADSCs were investigated.Results A population of stem cells could be isolated from adult adipose tissue, they were processed to obtain a fibroblast-like population of cells and could be maintained in vitro for extended periods with stable population doubling. The majority of the isolated cells were mesenchymal origin, with a few pericytes,endothelial cells and smooth muscle cells. ADSCs could be induced to differentiate intomultiple mesenchymal cell types, including osteogenic, chondrogenic, myogenic and adipogenic cells, they could also differentiate into nerve cells.Conclusion ADSCs can substitute mesenchymal stem cells and become an alternative stem cells source for tissue engineering.
Objective To explore an effective method of culturing the canine bladder smooth muscle cells, observe the morphological characteristics of the bladder smooth muscle cells growing on acellular small intestinal submucosa（SIS） and offer an experimental basis for reconstruction of the bladder smooth muscle structure by the tissue engineering techniques. Methods The enzymetreatment method and the explant method were respectively used to isolate and harvest the canine bladder smooth muscle cells, and then a primary culture of these cells was performed. The canine bladder smooth musclecells were seeded on the SIS scaffold, and the composite of the bladder smooth muscle cells and the SIS scaffold were co cultured for a further observation. At 5,7 and 9 days of the co culture, the specimens were taken; the bladder smooth muscle cells growing on the SIS scaffold were observed by the hematoxylin staining, the HE staining, and the scanning electron microscopy. The composite of the bladder smooth muscle cells on the SIS scaffold was used as the experimental group, and the bladder smooth muscle cells with no SIS were used as the control group. In each group, 9 holes were chosen for the seeded bladder smooth muscle cells, and then the cells were collected at 3, 5 and 7 days for the cell counting after the enzyme treatment. Morphological characteristics of the cells were observed under the phase contrast microscope and the transmission electron microscope. Expression of the cell specific marker protein was assessed by the immunohistochemical examinaiton. The proliferation of the cells was assessed by the cell counting after the seeding on the SIS scaffold. Results The primary bladder smooth muscle cells that had been harvested by the enzyme treatment method were rapidly proliferated, and the cells had good morphological characteristics. After the primary culture in vitrofor 5 days, the bladder smooth muscle cells grew in confluence. When the bladder smooth muscle cells were seeded by the explant method, a small amount of the spindleshaped bladder smooth muscle cells emigrated from the explant at 3 days. The cells were characterized by the welldeveloped actin filaments inthe cytoplasm and the dense patches in the cell membrane under the transmissionelectron microscope. The immunohistochemical staining showed the canine bladdersmooth muscle cells with positive reacting α actin antibodies. The bladder smooth muscle cells adhered to the surface of the SIS scaffold, growing and proliferating there. After the culture in vitro for 5 days, the smooth muscle cells covered all the surface of the scaffold, showing a singlelayer cellular structure. The cell counts at 3, 5 and 7 days in the experimental group were（16.85±0.79）×105，（39.74±2.16）×105 and （37.15±2.02）×105, respectively. Thecell counts in the control group were（19.43±0.54）×105，（34.50±1.85）×105 and （33.07±1.31）×105, respectively. There was a significant difference between the two groups at 5 days （P＜0.05）. ConclusionWith the enzyme treatment method, the primarily cultured canine bladder smooth muscle cells can produce a great amount of good and active cells in vitro. The acellular SIS can offer an excellent bio scaffold to support the bladder smooth muscle cells to adhere and grow, which has provided the technical foundation for a further experiment on the tissue engineered bladder reconstruction.
Objective To resolve the tough problem of how to observe the growing cells in an opaque vector. Methods The urethral epithelial cells from a young male New Zealand rabbit were inoculated, and were primarily cultured in vitro and subcultured for 3 passages. Then, the urethralepithelial cells were cultured in the collagen chitosan complex for 3, 7, 14 and 21 days. The cells were dyed with 6-carboxyfluorescein diacetateacetoxymethyl ester and propidium iodine, respectively. Then, Interactive Laser Cytometer was used to detect the growing cells. Results The urethral epithelial cells grew and proliferated very well in the collagen chitosan complex vector. After the urethral epithelial cells grew in the collagen-chitosan complex vector for 3 and 7 days, the fluorescent density amount of the surviving cells were(1.09±0.13)×10.8 and (2.04±0.13)×10.8, respectively. However, after 14and 21 days, the fluorescent density amount of the surviving cells was (0.55± 0.09)×10.8 and (0.47±0.03)×108, respectively. There was a significant difference when compared with the amount of the surviving cells at 3 and 7 days（P＜0.05）.Conclusion Using Interactive Laser Cytometer for measurement of the green and red fluorescent densities of different waves, the activity of the cultured urethral epithelial cells in vitro can be rapidlymeasured with the in situ quantitation method. This method solves a difficult problem of observing the growing cells in an opaque vector. The dynamic growing state of the engineering tissues can be observed.
Objective To study the differentiation of the human osteoblasts during the construction of the tissue engineered periosteum with the human acellular amniotic membrane(HAAM).Methods To construct the tissue engineered periosteum (n=60) with HAAM, the human fetal osteoblasts were used. The fetal osteoblasts were cultured for 2, 4, 6, 8, and10 days, and then their total RNA was extracted, which were reversely transcripted to cDNA. The realtime PCR analysis was used to reveal Cbfal and Osterix, and the cycle threshold (Ct) was also measured. The simplycultured osteoblasts were used as the control group (n=20).Results The expression of Cbfa1 was higher in the experimental group on the 2nd day when compared with that on the 4th, 6th, and 8th day(P＜0.05). The same result existed on the 10th day when compared with that on the 4th and 8th day. The expression of Osterix increased and was highest on the 8th day when compared with the other results(P＜0.05). Both of the 2 gene expressions were decreased in the control group when compared with those in the experimental group, but with no significant difference(P＞0.05). Conclusion Cbfa1 and Osterix can be normally expressed by the osteoblasts after their integration with HAAM. As a scaffold, HAAM can be used to keep the osteoblast phenotype and differentiation with an osteoconductive ability. Such a cell-scaffold complex may provide a basis for the osteogenesis.
Objective To introduce a new approach of neurotization to treatment of the shoulder syndrome after the radical neck dissection by using transpositional anastomosis of C7 posterior root and the spinal accessory nerve to reconstruct the function of trapezius muscle. Methods From March 1999 to February 2001, 10 patients underwent the neurotization during the radical neck dissection. In the operation, the apo-cranial part of spinal accessory nerve was preserved from the trapezius muscle (gt;3.0 cm in length) and anastomosed to C7 posterior root. Objective physical examinations and electromyography were conducted before and after operation.One, 6 and 12 months after operation the trapezius muscle function after the transpositional anastomosis was evaluated. Results One, 6 and 12 months after operation, the recovery rates of each part were as follows: 9.8%, 68.9% and 73.5% in upper part; 4.7%, 73.6% and 69.4% in middle part; and 6.2%, 70.5% and 70.3% in lower part. The range of abduction motion of upper arm in 7 cases (70%) exceeded 90°. The mean maximal abduction angle was more than 95°. Evaluation of the shoulder function showed that myoatrophy was mild and the disability of abduction was classified as grade Ⅱ in 7 cases and grade Ⅲ in 3 cases. Conclusion Transpositional anastomosis of the C7 posterior root to thespinal accessory nerve after radical neck dissection can well reconstruct the function of trapezius muscle. This approach provides a wide indication in comparison with the functional neck dissection without impairment of arm function afterthe cutting of C7.
