Objective To explore the feasibility of applying poroushigh density polyethylene (Medpor) as framework for auricle reconstruction of congenital oracquired auricular defects. Methods From February 1999 to February 2004, 61 patients suffering from congenital or acquired auricular defects underwent auricle reconstruction with Medpor framework after expanding postauricular skin. Among them, there were 38 males and 23 females, aging from 5 to 61 years. In 40 cases of congenital microtia, two sides were involved in 1 case and one side in 39 cases. In21 cases of traumatic auricle damage, two sides were involved in 6 cases and one side in 15 cases. The operation was performed by two stages. First stage:the expander was implanted underneath postauricular skin or soft tissuesuch as notrophic scar tissue for the traumatic auricle defect. Second stage:the expander was removed and auricle reconstruction was performed by placing Medpor framework between the expanded skin/scar flap and the underlying fascial flap. Results Sixty-one patients obtained successfully reconstructed auricles. During a followup of 6 months to 5 years and 1 month (mean 2.8 years), the results were excellent and good in 49 cases (80.3%) , fair in 7 cases (11.5%) and poor in 3 cases (4.9%)，2 cases (3.3%) underwent replacement of Medpor framework with autogenous costal cartilage after 6 months of operation. Conclusion Medpor framework would be applied safely, simply and reliably in condition that auricular framework is unfit or reluctant to undergo auricle reconstruction by using autogenous costal cartilage.
To evaluate an improved treatment of an autologous fat injection for hemifacial atrophy to increase the survival rate of the fat graft and decrease complications including colliquation, necrosis, and absorption of the graft fat. Methods From March 1999 to October 2004, 31 patients with hemifacial atrophy underwent an improved treatment by an autologous fat injection for their diseases. There were 12 males and 19 females aged 1928 years (average, 23.5 years). The patients were divided into the following 3 groups according to the atrophy extent: the mild group (n=9), the moderate group (n=19), and the severe group (n=3). Based on the previous researches on the fat transplantation techniques, the improved treatment combined the following strategies that were simply called “3L3M”: low position for the fat donation, low pressure for the fat harvesting, and lowspeed centrifugation for purification of the fat; multipoint, multitunnel, and multiplane for injections of the fat graft. The preoperative and the postoperative photos were taken and the findings were compared to make clear whether the hard and firm masses and cysts existed; then, the decision was made about whether the patients needed another operation according to whether the patients had a natural facial expression and whether the patients had comfortable feelings as well as the ray findings. Results All the patients had a satisfactory symmetrical face after 1 injection of the fat in 15 patients, 2 injections in 13 patients, and 3 injections in 3 patients. The effect of the 3rd injection was better than that of the 2nd injection; the effect of the 2nd injection was better than that of the 1st injection; the fat volume for the injection could be gradually decreased. The fat volumes for injections were as follows: 814 ml (average, 11 ml) in the submaxillary region, 1525 ml (average, 20 ml) in the buccal region, 510 ml (average, 75 ml) in the zygomatic region, and 1820 ml (average, 19 ml) in the forehead region. The followup for 35 years revealed that there wasno infection, hard and firm mass, cyst or other complications. The pigmentationin the affected face was significantly improved. Conclusion Compared with the traditional treatments, the improved treatment of an autologousfat injection for hemifacial atrophy can achieve a satisfactory symmetry of theface with no injury to the donor site or complications in the recipient site. This improved method is an ideal treatment for hemifacial atrophy.
Objective To observe the characteristics and related gene expression of osteoblastic differentiation in porcine bone marrow mesenchymal stem cells (MSCs) during. Methods Bone marrow from 6 landrace pigs, 3-month-old about 50 kg, was aspirated from the medullary cavity of the proximal tibia. The MSCs were isolated, and purified by Ficoll density gradient centrifugation combined with adherent culture method. The MSCs from passage 1 were cultivated in DMEM with 1×10-8mmol/L dexamethasone (Dex), 10 mmol/L β-glycerophosphate (β-GP), 82 μg/ml ascorbic acid (Asc) and 10% inactivated fetal bovine serum (FBS) up to 21 days. The MSCs were cultivated in basic DMEM as a control. Cell morphology was observed by microscope. Cell proliferation was tested by using the fluorescent dye SYBR green I measurement. Osteoblastic differentiation was evaluated by alkaline phosphatase (ALP) histochemical staining, quantitative calcium deposit, and real-time PCR technology. Results Characterization of primary MSCs: At day 1, most cells depicted round and floating hematopoietic cells. Colonies consisting of fibroblastlike cells were observed at day 3 after removal of nonadherent cells, colonies grew to various sizes at day 7. Thirteen population doublings took place in primary culture. Osteoblastic differentiation: During osteogenic stimulation, cellular morphology of MSCs changed from a fibroblastic shape to a cubical form. Cell proliferation had no impact in osteogenic medium compared to basic medium (Pgt;0.05). At day 14, ALP staining presented b positive. Calcium deposit pronouncedly increased at day 21 (Plt;001). Furthermore, the mRNA levels of core binding factor α1 (Cbfα1), osterix, ALP, collagen Ⅰ(ColⅠ), osteonectin (ON) and osteocalcin (OC) increased gradually. Cbfα1, ON and ALP genes increased at early stage of osteoblastic differentiation. Osterix and OC at day 21 were significantly increased when compared with that at day 7 (Plt;0.05). ColⅠ was increased at day 14 (Plt;0.05). Conclusion Porcine MSCs harvested from bone marrow by density gradient centrifugation are capable of osteoblastic differentiation in vitro. The potential of osteoblastic differentiation relies upon upregulation of genes specific to this lineage under the osteogenic conditions.
Objective To investigate the relationship between the expression of hypoxia inducible factor 1α (HIF-1α) and the neuron apoptosis during a hypoxia ischemia brain damage and explore the role of HIF1α in regulating the neuron apoptosis and repairing the brain damaged by hypoxia and ischemia. Methods Forty SD rats aged 10 days were randomly divided into the experiment group and the control group, with 20 rats in each group. In the experimental group, the rats were anesthetized with ethylether. The right common carotid artery was exposed and ligated. Then, they were exposed to hypoxia ina normobaric chamber filled with 8% oxygen and 92% nitrogen for 2.5 hours. In the control group, the right common carotid artery was exposed but was not ligated or exposed to hypoxia. The brain tissues were harvested from the rats in the both groups at 4, 8, 24, 48 and 72 hours after the hypoxia and ischemia, and fromthe rats in the control group at the same time points. The HIF-1α protein expression and the cleaved caspase 3 (CC3) protein expression were detected with the immunohistochemistry method. The apoptosis cells were detected with the TUNEL staining method. Results In the experimental group, the HIF-1α expression was significantly increased at 4 hours after operation, at the peak level at 8 hours, and began to decrease at 24 hours. The CC3 protein was expressed at 4 hours after operation, and was slightly expressed at 8 hours, but was significantly increased at 24 hours; the higher levels were maintained at 48 and 72 hours. However, in the control group, both the expression levels of HIF-1α and the CC3 protein were extremely low. So, the expression levels of HIF-1α andthe CC3 protein were significantly higher in the experimental group than in the control group (P＜0.01). The TUNEL staining showed that in the experimentalgroup the positive cells were significantly increased after the hypoxia and ischemia, with a peak level at 72 hours after the hypoxia and ischemia; however, in the control group there were few positive cells.TUNEL positive cells in the experimental group were significantly more than that in the control group(P＜0.01).ConclusionThe expression tendency of HIF-1α is completely different from that of CC3.HIF-1α may have a protective role in regulating the neuron apoptosis in the neonatal hypoxia-ischemia brain damage and may promote the repairing and rebuilding process in the brain that was damaged by hypoxia and ischemia.
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 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 investigate the effects of the insulin-like growth factor 1 (IGF-1), the transforming growth factor β1(TGFβ1), and the basic fibroblast growth factor (bFGF) on proliferation and cell phenotype of the human fetal meniscal cells, and to find out the best combination and concentration of the growth factors for the meniscus tissue engineering. Methods The fetus came from the healthy woman accidental abortion and the procedure had got her approval.The human fetal meniscal fibrochondrocytes were cultured in vitro. The cell phenotype was identifiedby the collagen type Ⅱ immunohistochemistry and Aggrecan immunofluorescence. Inthe growth factor groups, the 3rd passage meniscal cells synchronized by the serum starvation method and were mixed with IGF-1 (1, 10, 50, 100 μg/L), TGF-β1 (0.1, 1.0, 5.0, 10.0, 50.0 μg/L), and bFGF (5, 10, 50, 100, 200 μg/L), respectively, and in the combination groups, the combinations of bFGF and TGF-β1, bFGF and IGF-1, TGF-β1 and IGF-1 were established at their optimal effect concentrations. The control group was also established for comparison. The dose-response relationship was studied at 48 h and 72 h bythe MTT colorimetric method. Results The 3rd passage meniscalcells could express collagen type Ⅱ and Aggrecan before and after the addition of the three growth factors. The proliferating effects of the growth factors (IGF-1 50 μg/L，TGF-β1 5 μg/L，bFGF 50 μg/L) on the 3rd passage cells at 48 h and 72 h were significantly better in the growth factor groups than in the control group (Plt;0.05)，and the combination groups of bFGF 50 μg/L and IGF-1 50 μg/L, IGF-1 50 μg/L and TGF-β1 5 μg/L showed a significantly higher proliferatingeffect than that in the single growth factor group (Plt;0.05). bFGF 50 μg/L and TGF-β1 5 μg/L had no synergetic effect (Pgt;0.05). Conclusion IGF-1, TGF-β1 and bFGF can promote the proliferation of the human fetal meniscal cells, respectively, and the combinations of bFGF and IGF-1, IGF-1 and TGF-β1 at their optimal concentrations can have better proliferating effects than the single growth factor. They can be used for the in vitro amplification of the meniscal seed cells.
Objective To investigate the feasibility of a new kind of porous β tricalcium phosphate (β-TCP) as a scaffold for the bone tissue engineering Methods The inverted phase contrast microscope was used to observe the growth of the marrow mesenchymal stem cells (MSCs) in the experimentalgroup and the control group at 10 days.In the experimental group, the MSCs were cultured with β-TCP(3 mm×3 mm×3 mm) in the 24-hole cultivation board, and in the control to control group, only MSCs were cultivated. The scanning electron microscope was used to observe growth of MSCs at 6 days. Cultivated with β-TCP at 3, 6, 9, 12 days, the MTT assay was used to judge the biocompatibility. The cytotoxicity was analyzed with the method that used the different density(100%， 50%， 10%， 1%，0%) leaching liquor gained from β-TCP to raise MSCs. MSCs were induced into the osteoblasts and were mixed with β-TCP, and the composite was used to repair a large radius bone defect in the rabbit. The specimens were made at 2,6,12 weeks. The histology imageology, and the radionuclide bone scan were used to analyze the bone formation. Results Some MSCs had a good adherence 4 hours after MSCs were inoculated and had a complete adherence at 12 hours. The cells were shaped like polyangle, spindle or converge monolayer after 8-10 days. The cells in the two groups had no difference. The cell adhesion was good, when observed by the inverted phase contrast microscope and the scanning electron microscope at 6 days. MTT showed that the absorbance (A)was not statistically different between the experimental group and the control group (P＞0.05); the different density leaching liquor had no cytotoxicity at the different time points. Histology, X-ray, and CT tomograph showed that itcould repair the large radius bone defect in the rabbit and its in vivo degradationrate was the same as the bone formation rate. Conclusion The new porous β-TCP has a unique three dimensional (3D) stereochemical structure and superordinary physicochemical property, and so it is a good scaffold for the bone tissue engineering.
Objective To establish a model of the human marrow mesenchymal stem cells (hMSCs) cultured under the hypoxic condition in adults and to investigate the biological features of MSCs under hypoxia.Methods The bone marrow was obtained by aspiration at the posterior superior iliac spine in 3 healthy adult subjects. hMSCs were isolated by the gradient centrifugation and were cultured in the DMEM-LG that contained 20％ fetal bovine serum. The serial subcultivation was performed 10-14 days later. The second passage of the hMSCs were taken, and they were divided into the following 4 groups according to the oxygen concentrations and the medium types: the normoxic group（20％O2, DMEM-LG, Group A）, the hypoxic group（1％O2, DMEM-LG,Group B）, the normoxic osteoblast induction group（20％O2, conditioned medium, Group C）, and the hypoxic osteoblast induction group（1％O2, conditioned medium, Group D）. The biological features of the cultured hMSCs under hypoxia were assessed bythe cell count, the MTT method, the colony forming unit-fibroblast, the real-time RT-PCR, and the alkaline phosphatase (ALP) activity, and the alizarinred staining. Results The hMSCs cultured in the Group B and Group D had a significantly higher proliferation rate than those in the Group A (Plt;0.01), and the culture effect was not influenced by the medium type. The hMSCs in the Group B had a significantly higher level of the colony-forming unit capability than the hMSCs cultured in the Group A(Plt;0.01). After the induction, hMSCs in the Group B had a decreasednumber of the osteoblasts than hMSCs in the Group C. The hMSCs in the Group D had a gradually-increasedactivity of ALP, which was significantly lower than that in the Group C(Plt;0.01). The RT-PCR examination revealed that ALP,osteocalcin, and mRNA expressions of collagen type Ⅰ and osteonectin in the Group Csignificantly increased （P＜0.01). By comparisonamong the 3 groups, after the 4-week culture the obvious calcium salt deposit and the red-stained calcium nodus could be observed.ConclusionHypoxia can promote the proliferation rate of hMSCs, enhance the colonyforming ability and inhibit the differentiation of the osteoblasts.
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.