Objective To investigate the clinical feasibility of different types of the saphenous neuro-veno-fascial cutaneous flaps. Methods From June 1996 to October 2002, 18 cases of skin defects in the knee and the lower part of the limb were treated with proximally(4 cases) or distally(11 cases) based pedicles of saphenous neuro-venofascial cutaneous flap or crossleg flap (3 cases)according to the site of defects . The sizes of the flaps ranged from 4 cm×5 cm to 9 cm×20 cm. Results The flaps survived completely in 17 cases, distal 1/5 of the flap necrosed partially in 1 case because of vein drainage disturbance. The colour and texture of flaps were excellent, the appearance and function were satisfactory after a follow up of 6-24 months.Conclusion The saphenous neuro-veno-fascial cutaneous flap is an idea flap in repairing skin defects of the knee, the leg, the ankle and the foot because it is easy to be designed and dissected and it has reliable blood supply and preserved main artery. The relationship between the septal perforating branches of the tibial posteriorartery and survival size of flap need to be investigated further.
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 investigate the effect of the synthetic bone morphogenetic protein 2 (BMP-2)derived peptide on the osteogenic induction in the marrow mesenchymal stem cells (MSCs）and to evaluate the osteoinductivity and dosedependence of the BMP-2 derived peptide in vitro. Methods MSCs of 4-week old Wistar rats were separated and cultured. In the 3rd passage, the conditional culture medium was changed, in which the BMP-2-derived peptide in the following doses was added: 300,200, 100, 50, and 0 μg/ml, respectively (Groups A-E). The activity of alkaline phosphatase (ALP）and the amount of calciumdeposition were meassured at 5，10，15 and 20 days during the culture with the conditional culture medium. The real-time fluorescent quantitative polymerase chain reaction (FQ-PCR) was performed to measure the mRNA expressions of collagen type Ⅰ, osteopontin (OPN), and osteocalcin（OCN）and to measure the osteoinductivity of the BMP-2-derived peptide in the different concentrations.Results Under the inverted phase contrast microscope, MSCs cultured in the conditional culture medium for 3-4 days were changed in shape, from long fusiform to short fusiform or polygon. As the concentration of the BMP-2-derived peptide increased, the time for MSCs to change into the osteoblasts decreased. There was a significantly greater level of the ALP activity and amount of the calcium deposition in Groups A and B than in the other groups（Plt;0.05）. However,there was no significant difference between Group A and Group B （Pgt;0.05）. Theresult of FQPCR showed that after MSCs were cultured in the different doses of theconditional culture medium for 14 days, the mRNA expressions of collagen type Ⅰ, OPN andOCN were at higher levels. An increasing order in the level of the cycle threshold (Ct) was found in the following groups: Agt;Bgt;Cgt;D. Almost no expression was found in Group E. The Ct levels were significantly greater in Groups A and B thanin Groups C and D（Plt;0.05）. However, there was no significant difference between Group A and Group B （Pgt;0.05）.ConclusionThe BMP-2-derived peptide can greatly promote differentiation of MSCs into the osteoblasts, the promotion of osteogenesis has a dosedependent pattern, and the best inducing dosage is 200 μg/ml.
Objective To provide theoretical evidence for clinical application of the epidermal stem cells after an investigation on changes of the epidermal stem cells during the survival process after the fullthickness skin autograft. Methods On the backs of 42 Wistar rats, orthotopic transplantation models (1.5 cm×1.5 cm) of the fullthickness skin autograft were made. According to the time of the specimen taking, at 1, 3, 5, 7, 14, 21 and 30 days after operation, the rats were randomly divided in 7 groups (Groups 1-7). Specimens taken in each group before operation were used as controls. At each time point, the gross observation was made on the transplanted skin flaps, from which the skin tissues were harvested at each time point before and after operation. The routine pathological and the immunohistochemical examinations were performed on the specimens, which were stained by HE and were observed for immunohistochemical changes and the changes in the cells positive for integrinβ-1 and p63. Results All the fullthickness skin autografts survived 3 days after operation except the skin autograft in 1 rat in both Group 5 and Group 6, which was infected around the transplanted skin flap. In Groups 1-4, cell edema, inflammatory cell infiltration, and increased fibrocytes were observed. In Groups 5-7, the maturity degree of the epithelial cells became higher and higher, and the fibrocyte proportion was lowered. In each group the cell positivity rate for integrin β1 was lower than the cell positivity rate for p63. The positive cells were arranged in disorder, distributed into the layers of the epidermis and gradually concentrated in the basal layer of the epidermis and the bulge of the folliculus pili. The positive cells were also found in the other layers of the epidermis.The positive cells were gradually decreased in number, and reached the lowest level in Group 2. There was a significant difference in the above variables in Groups 1,2,3,5,6 and 7 between before and after operations (P＜0.05). Conclusion During the survival process of the fullthickness skin autograft, the proportion of theepidermal stem cells is gradually decreased at first; Then, the proportion isgradually increased, even beyond the normal level; finally, the proportion is decreased again. The distribution of the epidermal stem cells appear in disorder, almost distributed in the layers of the epidermis; finally, the almost normal distribution can be found.
Objective To study the mechanism of ectopic osteogenesis of nacre/Polylactic acid (N/P) artificial bone combined with allogenic osteoblasts, and to explore the possibility as a scaffold material of bone tissue engineering. Methods The allogenic- osteoblasts seeded onto N/P artificial bone were co-cultured in vivo 1 week.The N/P artificial bone with allogenic osteoblasts were implanted subcutaneously into the left back sites of the New Zealand white rabbits in the experimental group and the simple N/P artificial bone into the right ones in the control group. The complexes were harvested and examined by gross observation, histologic analysis and immunohistochemical investigation 2, 4 and 8 weeks after implantation respectively.Results In experimental group, the osteoid formed after 4 weeks, and the mature bone tissue withbone medullary cavities formed after 8 weeks; but in control group there was nonew bone formation instead of abundant fibrous tissue after 4 weeks, and more fibrous tissue after 8 weeks.Conclusion N/P artificial bone can be used as an optical scaffold material of bone tissue engineering.
Objective To introduce the treatment and clinical result of reconstructing femoral artery with external jugular vein graft. Methods From June 2002 to April 2006, 22 cases of femoral artery defects caused by pseudoaneurysm resection, were reconstructed withexternal jugular vein graft by microsurgical technique. There were 20 males and2 females, aging 25-46 years (mean 31.2 years). The length of femoral artery defects was 5-9 cm, with an average of 6.8 cm. The location was left in 14 cases and right in 8 cases.Results Ten cases achieved healing by the first intention, and 4 cases by the second intention. The other 8 cases need the regional flap repair because the wound splited open and became ulcer. Femoral artery defects were reconstructed successfully and the pulse of dorsal arteries of foot could be felt except 1 case of bleeding of anastomotic rupture. Eighteen patients were followed up 1-30 months, and no pseudoaneurysm recurred. Color ultrasound Doppler detection showed that the external jugular veins took place of the femoral artery defects in 12 cases. Conclusion External jugular vein is stable at anatomy and easy-to-obtain. Its calibre is close to that of the femoral artery. It can be used for reconstructing femoral artery defects as the vein material. The operation is easy and the clinical result is sure.
Objective To study the feasibility of using mice marrow stromal stem cells(MSCs) as seed cells for tissue engineering cartilage to embed the seed cells in acellular cartilage matrix of human auricle. Methods Acellular cartilage matrix was made from human auricle cartilage. The MSCs were isolated from the nucleated cells fraction of mice marrow by centrifuge.The MSCs were embedded in acellular cartilage matrix. After 10 day’s combined culture, the specimens were observed with optical and electrical microscope.Results The MSCs could well proliferate in the acellular cartilage matrix. The cells were not well-distributed in acellular cartilage matrix. There were more cells in the peripheral part of the matrix than in the central part of the matrix. Most of the cells were in cartilaginous lacunae. There were 1 or 2 cells in every cartilaginous lacunae.Conclusion The MSCs can be used as seed cells of tissue engineering and can well proliferate in the acellular cartilage matrix and become tissue engineering cartilage.
Objective To explore the effective autologous bone marrow stem cell dosage for treatment of severe lower limb ischemia. Methods From December 2003 to December 2004, 22 cases of bilateral lower limb ischemia were treated with autologous bone morrow cell transplantation. All the patients were randomly divided into two groups according to ischemia degree. In group A(severe ischemia side), the amount of transplanted autologous bone marrow cells was more than 1×108, and ingroup B(mild ischemia side), the amount was less than 1×105. A series of subjective indexes, such as improvement of pain, cold sensation and numbness, and objective indexes, such as increase of ankle/brachial index (ABI) and transcutaneous oxygen pressure (TcPO2), angiography, amputation rate, and improvement of foot wound healing were used to evaluate the effect of autologous bone marrow stem cells implantation. Results The rates of pain relief were 90.0% in group A and 16.7% in group B (Plt;0.01); the rates of cold sensation relief were 90.5% in group A and 5.3% in group B（Plt;0.01）;the improvement of numbness was 62.5% in group A and 9.1% in group B（Plt;0.01）. Increase of ABI was 31.8% and 0 in groups A and B respectively（Plt;0.01） at 4 weeks after implantation. Increase of TcPO2was 94.4% and 11.1% in groups A and B respectively（Plt;0.01） at 4 weeks after implantation. Twelve cases of angiography showed rich new collateral vessels in 100% of the limbs in group A while no remarkable new collateral vessel in group B. The amputation rates were 4.5% in group A and 27.3% in group B（Plt;0.05） at 4 weeks after implantation. The rate of improvement of foot wound healing was 75% in group A and there was no changein wound healing in group B after 4 weeks of implantation. Conclusion The effectiveness of autologous bone marrow stem cell implantation depends on the number of implanted stem cells. Effectiveness is expected in most patients if the implanted stem cell is more than 1×108, whereas there would be little effect if the cell number is less than 1×105.
Objective To introduce the latest advances of research on repair of the degenerative intervertebral disc with gene transduction.Methods The recentlypublished articles about the treatment of degenerative disc with gene transduction were reviewed, especially the articles published during the recent 5 years about the application of this therapy to regulating the synthesisand degradation of the extracellular matrix of the degenerative intervertebral disc.Results The shape and function of the normal intervertebral disc were reported to be closely related to the synthesis and degradation of the extracellular matrix of the intervertebral disc. The extracellular matrix of the intervertebral disc was a target for the gene transduction to repair the degenerative intervertebral disc. There was a great development of the treatment with gene transduction, especially in vector choice, target gene transduction, and transgene regulation and safety. Conclusion The advances of the research have indicated that repair of the degenerative intervertebral disc with gene transduction is a keyto curing the disease of the degenerative intervertebral disc.
Objective To investigate a change in the differentiation and biological function of the cultured rat fibroblast (FB) transfected by the myoblast determining gene (MyoD) and the connexin 43 (Cx43) gene and to explore the possible mechanism of the MyoD and Cx43 genes on treatment of ischemic heart disease (IHD). Methods The gene cloning technology was used to construct the eukaryotic expressed plasmid vector pLenti6/V5-DEST-MyoD and pLenti6/V5DEST-Cx43 in which MyoD cDNA or Cx43 cDNA was inserted. The RFL-6 FB cells were transfected with exogenetic MyoD cDNA or Cx43 cDNA via lipofectamine, followed by the Blasticidin (50 μg/ml) selection, according to the lentiviral expression system (ViraPower) protocol. The expression and the biological functions of MyoD and Cx43 in the transfectants were testified by RT-PCR, Western blot, and molecular and immunocytochemical methods. The mophological structure changes of the cells were observed under microscope before and after the transfection. Results The expression of MyoD and Cx43 was detected in the MyoD and Cx43 genes transfected FB with RT-PCR and Western blot. The immunocytochemical methods indicated the expressionsof the MyoD and Cx43 genes, while desmin and αactin were found in these cells. The myotubes were found from the cultures incubated a week in the differentiation medium, in which the transfected cells had a characteristic of the filamentsin their cytoplasm and showed a myoblast morphology. Conclusion MyoD cDNA can induce the cultured FB to differentiate into the myoblasts and Cx43 cDNA can enhance the gap junctional intercellular communication between the cell and the cell. Thus, a further experimental foundation for the therapy of IHD can be provided.