【Abstract】 Objective To investigate the biocharacteristics of c-kit+ human amniotic fluid-derived mesenchymal stemcells (HAFMSCs) and its capacity to differentiate into cardiomyocytes in vitro. Methods Fifty samples of human amnioticfluid were obtained from amniocenteses or voluntary termination of pregnancy and were expanded in vitro. c-kit+ HAFMSCs were sorted by flow cytometry and were recultured in the same media. c-kit+ HAFMSCs were amplified and identified, then exposed to osteogenic , adi pogenic, and myogenic media. The flow cytometry, and immunocytochemistry were used for identifying the cell phenotype, von Kossa staining for osteogenic differentiation, oil red O staining for adi pogenic differentiation, and real-time fluorescent quantitative PCR for the expressions of NKx2.5, Tbx5, GATA-4, and α-MHC genes. Results After the selection procedure, the percentage of c-kit+ HAFMSCs was 3.07% ± 1.03% of the total adherent cells. The cells expressed MSCs markers (CD29, CD44, CD73, CD90, CD105), and did not express hematopoietic stem cells markers (CD34, CD45). The cells were positive for human leukocyte antigen (HLA)-ABC, and negative for HLA-DR. They also expressed Oct-4, which characterized the undifferentiated stem cell state. The growth curves of c-kit+ HAMFSCs at passages 5, 10, and 15 were similar. Li pid droplet was observed by oil red O staining and calcium deposition by von Kossa staining in the cells at 21 days after adi pogenic and osteogenic induction. The myocardium special gene expressions of Tbx5, Nkx2.5, GATA-4, and α-MHC were significantly ber after myogenic induction than those before myogenic induction (P lt; 0.05). Conclusion Selected c-kit+ HAFMSCs by flow cytometry is a group of pure MSCs, which has potential to differentiate into cardiomyocytes and can be used as seeding cells for myocardium regeneration treatment.
【Abstract】 Objective To isolate and culture human amniotic fluid-derived mesenchymal stem cells (HAFMSCs),to investigate a better cryopreservation protocol of HAFMSCs and to observe the biocharacteristics and the multi-potential of HAFMSCs after cryopreservation for the further fundamental researches and cl inical appl ications. Methods HAFMSCswere isolated from the amniotic fluid of pregnant women during the second trimester by the improved two-step method.HAFMSCs were cryopreserved with different cryopreservation protocols (containing different contents of FBS and DMSO atcryoprotectant) in l iquid nitrogen for 12 weeks. The biocharacteristics of the HAFMSCs after cryopreservation were analyzed. The growth characteristics were observed by MTT method and the growth curves were drawn. The surface antigens of HAFMSCs were detected using flow cytometry, including CD29, CD34, CD44, CD45, CD73, and CD90. The adi pogenic and osteogenic differentiation abil ities of HAFMSCs were observed. The mRNA levels of Oct-4 and Nanog of the HAFMSCs were compared between before and after cryopreservations. Results At 12 weeks after cryopreservation, different protocols had different effects on the cell viabil ity; the better formula of cryoprotectant was 50% DMEM, 40% FBS, and 10% DMSO. After cryopreservation, the cells proliferated rapidly and the growth curves showed “S” shape, which was the same as the cells before cryopreservation. Phenotype showed that HAFMSCs were positive for the surface markers CD29, CD44, CD73, and CD90, and negative for CD34 and CD45. After 21 days of adi pogenic differentiation, the l ipid droplets were observed by oil red O staining. After 21 days of osteogenic differentiation, the calcium mineralizations were verified by von Kossa staining. There was no significant difference (P gt; 0.05) in the mRNA levels of Oct-4 and Nanog between before and after cryopreservations. Conclusion HAFMSCs have rapid proliferation and multi-potential in vitro. The cells have high viabil ities and no changes of the biocharacteristics and differentiation potential ities after cryopreservation for 12 weeks. Cryoprotectant containing 50% DMEM, 40% FBS, and 10% DMSO is a better cryopreservation protocol.
Objective To investigate the appl ication and cl inical result of flap in the repair of wounds with Achilles tendon exposure. Methods Between May 2006 and May 2010, 21 patients with Achilles tendon skin defects were treated with microsurgical reconstruction. There were 15 males and 6 females, aged 7-63 years with a median of 34 years. The defect causesincluded sport injury in 4 cases, wheel twist injury in 7 cases, crush injury in 5 cases, chronic ulcer in 3 cases, and Achilles tendon lengthening in 2 cases. The areas of wounds with Achilles tendon exposure ranged from 2 cm × 2 cm to 10 cm × 8 cm. After debridement, wounds were repaired with the medial malleolus fasciocutaneous flap (5 cases), sural neurocutaneous vascular flap (8 cases), foot lateral flap (2 cases), foot medial flap (2 cases), and peroneal artery perforator flap (4 cases). The size of the flaps ranged from 3 cm × 3 cm to 12 cm × 10 cm. The donor sites were either sutured directly or covered with intermediate spl it thickness skin grafts. The Achilles tendon rupture was sutured directly (2 cases) or reconstructed by the way of Abraham (2 cases). Results All flaps survived and wounds healed by first intention except 2 flaps with edge necrosis. Twenty-one patients were followed up 6-18 months (mean, 12 months). The flaps had good appearance and texture without abrasion or ulceration. The walking pattern was normal, and the two point discrimination was 10-20 mm with an average of 14 mm. The Ameritan Orthopedic Foot and Ankle Society (AOFAS) Ankle-Hindfoot Scale assessment revealed that 10 patients had an excellent result, 7 had a good result, 3 had a fair result, and 1 had a poor result with an excellent and good rate of 81.0%. Fourteen cases could l ift the heels with power; 5 cases could l ift the heels without power sl ightly; and 2 cases could not l ift the heels. Conclusion The wounds with Achilles tendon exposure should be repaired as soon as possible by appropriate flap according to the condition of wound.
Objective To investigate the operative procedures and cl inical outcomes of the modified superficial peroneal neuro-fasciocutaneous flap in repairing soft tissue defect of forefoot. Methods From May 2006 to May 2009, 5 male patients (aged 40-63 years) with soft tissue defect of forefoot were treated with the modified superficial peroneal eurofasciocutaneous flap. Tendons and bones were exposed in all cases. Defect was caused by object crash (4 cases) and traffic accident (1 case). The sizes of soft tissue defects of forefoot were 4 cm × 2 cm-8 cm × 4 cm. Rotating point of the modified superficial peroneal neuro-fasciocutaneous flap pedicled with the peripheral vessels network of ankle joint was at the level of tibiotalar joint. The flaps ranging from 5 cm × 4 cm to 10 cm × 6 cm were adopted to repair soft tissue defects of forefoot. The donor sites were either sutured directly or covered with intermediate spl it thickness skin grafts. Results All flaps survived and all wounds healed by first intention. Skin graft at donor site survived completely in all cases. All patients were followed up 6-18 months (mean 11 months). The appearance, texture, and function of the flap were satisfactory. There was a protective sensibil ity in all flaps without abrasion or ulceration, and the two-point discrimination of the flaps was 10-13 mm. The walking pattern was normal. No obvious discomfort was observed at the skin-graft donor sites. Conclusion With rel iable blood supply, no sacrifice of vascular trunks, favorable texture, and thickness, the modified superficial peroneal neuro-fasciocutaneous flap pedicled with the peripheral vessels network of ankle joint is useful to repair skin soft tissue defect of the forefoot.