ObjectiveTo study the feasibility of human adipose-derived stem cells (hADSCs) combined with small intestinal submucosa powder (SISP)/chitosan chloride (CSCl)-β-glycerol phosphate disodium (GP)-hydroxyethyl cellulose (HEC) for adipose tissue engineering.
MethodshADSCs were isolated from human breast fat with collagenase type I digestion, and the third passage hADSCs were mixed with SISP/CSCl-GP-HEC at a density of 1×106 cells/mL. Twenty-four healthy female nude mice of 5 weeks old were randomly divided into experimental group (n=12) and control group (n=12), and the mice were subcutaneously injected with 1 mL hADSCs+SISP/CSCl-GP-HEC or SISP/CSCl-GP-HEC respectively at the neck. The degradation rate was evaluated by implant volume measurement at 0, 1, 2, 4, and 8 weeks. Three mice were euthanized at 1, 2, 4, and 8 weeks respectively for general, histological, and immunohistochemical observations. The ability of adipogenesis (Oil O staining), angiopoiesis (CD31), and localized the hADSCs (immunostaining for human Vimentin) were identified.
ResultsThe volume of implants of both groups decreased with time, but it was greater in experimental group than the control group, showing significant difference at 8 weeks (t=3.348, P=0.029). The general observation showed that the border of implants was clear with no adhesion at each time point;fat-liked new tissues were observed with capillaries on the surface at 8 weeks in 2 groups. The histological examinations showed that the structure of implants got compact gradually after injection, and SISP gradually degraded with slower degradation speed in experimental group;adipose tissue began to form, and some mature adipose tissue was observed at 8 weeks in the experimental group. The Oil O staining positive area of experimental group was greater than that of the control group at each time point, showing significant difference at 8 weeks (t=3.411, P=0.027). Immunohistochemical staining for Vemintin showed that hADSCs could survive at each time point in the experimental group;angiogenesis was most remarkable at 2 weeks, showing no significant differences in CD31 possitive area between 2 groups (P>0.05), but angiogenesis was more homogeneous in experimental group.
ConclusionSISP/CSCl-GP-HEC can use as scaffolds for hADSCs to reconstruct tissue engineered adipose.
ObjectiveTo investigate the effectiveness of human placental decidua basalis derived mesenchymal stem cells (PDB-MSCs) in repairing full-thickness skin defect of nude mice.
MethodsHuman placenta samples were obtained from healthy donor mothers with written informed consent. PDB-MSCs were isolated through enzymic digestion and density gradient centrifugation; the 4th passage cells were identified by cellular morphology, cell adipogenic and osteogenic differentiation, and phenotype evaluation. Forty-two 4-5-week-old BALB/c female nude mice were randomly divided into experimental group (n=21) and control group (n=21). The 4th passage PDB-MSCs solution (200 μL, 5×106/mL) was injected into the mice of experimental group via caudal vein; the mice of control group were given equal volume of PBS. The full-thickness skin defect model of 1.5 cm×1.5 cm in size was made after 3 days. The wound healing was observed generally at 1, 2, 4, 7, 14, 18, 21, 25, and 30 days after operation, and the wound healing rate was calculated after wound decrustation. HE staining was used to observe the wound repair at 1, 7, 14, 21, and 31 days; immunofluorescent staining was used for cellular localization at 7, 14, and 31 days after operation.
ResultsCells isolated from human placenta were MSCs which had multipotential differentiation ability and expressed MSCs phenotype. Animals survived to the end of the experiment. The general observation showed that the experimental group had a faster skin repairing speed than the control group; the time for decrustation was 12-14 days in experimental group and was 14-17 days after operation in the control group. The wound healing rate of experimental group was significantly higher than that of control group at 14, 18, and 21 days (t=4.001, P=0.016; t=3.380, P=0.028; t=3.888, P=0.018), but no significance was found at 25 and 30 days (t=1.565, P=0.193; t=1.000, P=0.423). HE staining showed lower inflammatory reaction, and better regeneration of the whole skin and glands with time in the experimental group. The immunofluorescent staining was positive in skin defect area of experimental group at different time points which displayed that human PDB-MSCs existed.
ConclusionThrough enzymic digestion and density gradient centrifugation, PDB-MSCs can be obtained. Pre-stored PDB-MSCs can mobilize to the defect area and participate in repair of nude mice skin.
Objective To investigate the feasibility and characteristic of tissue engineered testicular prosthesis with highdensity polyethylene(HDPE，trade name: Medpor) and polyglycolic acid(PGA). Methods The chondrocytes were isolated from the swine articular.The PGA scaffold was incorporated with medpor which semidiameters were 6mmand 4mm respectively.Then, the chondrocytes (5×10 7/ml) were seeded onto Medpor-PGA scaffold and cultured for 2 weeks. The ten BALB／C mice were divided into two groups randomly(n=5). In the experimental group, the cell-scaffold construct was implanted into subcutaneous pockets on the back of nude mice. In the control group, the Medpor-PGA scaffold was implanted. The mice of two groups were sacrificed to harvest the newly formed cartilage prosthesis after 8 weeks. Macroscopy, histology and immunohistochemistry observations were made. Results The gross observation showed that on changes were in shape and at size, the color and elasticity were similar to that of normal cartilage and that the cartilage integrated with Medpor in the experimental group; no cartilage formed and fiberlike tissue was found in the control group. HE staining showed that many mature cartilage lacuna formed without blood vessel and some PGA did not degradated completely. Toluidine blue staining showed extracellular matrix had metachromia. Safranin O-fast green staining showed that many proteoglycan deposited and collagen type Ⅱ expression was bly positive. In the control group, Medpor was encapsulated by fiber tissue with rich blood vessel. Conclusion The newly formed complex of Medpor-PGA and cells was very similar to testicle in gross view and to normal cartilage in histology. This pilot technique of creating testicular prosthesis by incorporating tissue-engineered cartilage with Medpor demonstrated success.
The model of transplanted colonic SW480 cell line carcinoma in gymnomouse body was set up to observe the effect of octapeptide somatostatin (SMS 201-995,SMS) on the transplanted carcinoma and elucidate its mechanism. Results: the volume, weight, DNA and protein content in carcinoma cell, cell amount and proliferation index of S and G2M phase in SMS group and SMS+PG (pentagastrin) group were markedly lower than those in PG group and control group, those of PG group were markedly higher than those in control group.The cell amount of G0/G1 phase in SMS group and SMS+PG group was markedly higher than that in PG group and control group, and that of PG group was markedly lower than that in control group.All these suggested that somatostatin could not only inhibit the growth of transplanted human colonic SW480 cell line carcinoma directly but also inhibit the growthpromoting effect of gastrin on the transplanted carcinoma.The mechanism might be that somatostatin inhibit the synthesis of cAMP, DNA and protein in carcinoma cells, then inhibit the cell growing from G0/G1 phase to S and G2M phases.Our study might provide experimental basis for the homonotherapy with analogue of somatostatin in patients with large intestine carcinoma.
Objective
To investigate the mechanism of vascular stromal fraction (SVF) at the early stage after aspirated fat transplantation.
Methods
Fat was harvested from 5 cases of women undergoing abdominal liposuction operation, and SVF was isolated. Aspirated fat with (group B) or without (group A) SVF was injected subcutaneously into the back of nude mice, and the grafts were harvested at 1, 3, 5, and 7 days. Graft wet weight was measured; and immunohistochemical method (CD31) was performed and the secretion of vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF) were qnantified by Western blot assay.
Results
The wet weight of transplanted adipose tissue showed an increasing tendency in groups A and B with time, and no significant difference was found between groups A and B (P gt; 0.05). At 1 and 3 days after transplantation, no CD31 positive cells was seen in 2 groups; the CD31 positive cells of group B were significantly more than those of group A at 5 and 7 days (P lt; 0.05), and the CD31 positive cells at 7 days were significantly more than those at 5 days in 2 groups (P lt; 0.05). Western blot test showed that VEGF expression reached peak at 3 days , then decreased gradually; the expression of VEGF protein in group B was significantly higher than that in group A at 1, 3, and 5 days (P lt; 0.05). The expression of HGF protein in groups A and B remained at a high level within 5 days, but it tended to decrease at 7 days, which was significantly higher in group B than that in group A (P lt; 0.05).
Conclusion
SVF can enhance angiogenesis by secretion of growth factors at the early stage after aspirated fat transplantation.
【Abstract】 Objective To investigate the impact of dermal papillary cells on vascularization of tissue engineered skinsubstitutes consisting of epidermal stem cells and allogeneic acellular dermal matrix. Methods Human foreskins from routinecircumcisions were collected to separate epidermal cells by using dispase with trypsogen. Collagen type IV was used to isolateepidermal stem cells from the 2nd and 3rd passage keratinocytes. Dermal papilla was isolated by the digestion method of collagenaseI from fetus scalp and cultured in routine fibroblast medium. Tissue engineered skin substitutes were reconstructed by seedingepidermal stem cells on the papillary side of allogeneic acellular dermis with (the experimental group) or without (the controlgroup) seeding dermal papillary cells on the reticular side. The two kinds of composite skin substitutes were employed to cover skindefects (1 cm × 1 cm in size) on the back of the BALB/C-nu nude mice (n=30). The grafting survival rate was recorded 2 weeks aftergrafting. HE staining and immunohistochemistry method were employed to determine the expression of CD31 and calculate themicrovessel density at 2 and 4 weeks after grafting. Results Those adhesion cells by collagen type IV coexpressed Keratin 19 andβ1 integrin, indicating that the cells were epidermal stem cells. The cultivated dermal papillary cells were identified by expressinghigh levels of α-smooth muscle actin. The grafting survival rate was significantly higher in experimental group (28/30, 93.3%), thanthat in control group (24/30, 80.0%). HE staining showed that the epithelial layer in experimental group was 12-layered with largeepithelial cells in the grafted composite skin, and that the epithelial layer in control group was 4-6-layered with small epithelial cells.At 2 and 4 weeks after grafting, the microvessel density was (38.56 ± 2.49)/mm2 and (49.12 ± 2.39)/mm2 in experimental group andwas (25.16 ± 3.73)/mm2 and (36.26 ± 3.24)/mm2 in control group respectively, showing significant differences between 2 groups(P lt; 0.01). Conclusion Addition of dermal papillary cells to the tissue engineered skin substitutes can enhance vascularization,which promotes epidermis formation and improves the grafting survival rate.
ObjectiveTo explore the effect of Poria cocos on xenograft tumors of gastric cancer SGC-7901 cell line in mude mice.
Method①After establishment of xenograft tumor of gastric cancer SGC-7901 cell line, 10 nude mice were equally divided into normal control group and Poria cocos group. The nude mice of each group were gavaged with normal saline (NS) and Poria cocos (0.5 mL) for 32 days, respectively. Tumor volume were measured to draw tumor growth curves and the tumor weight inhibitory rate was calculated with tumor weight (on the 32-day, nude mice were sacrificed to get the xenograft tumors). The expressions of B cell lymphoma 2 (Bcl-2), Bcl-2 associated X protein (Bax), Caspase-3, Caspase-9, and vascular endothelial growth factor (VEGF) were detected by immunohistochemical staining. ②Preparation of drug serum containing Poria cocos. Gastric cancer SGC-7901 cell line were be divided into 2 groups: normal control group and Poria cocos group. Cells of normal control group were treated with serum containing NS, and cells of Poria cocos group were treated with drug serum containing 10% Poria cocos. After 24 hours and 48 hours, Western-blot was used to detect the expressions of Bcl-2 and Bax.
ResultsOn 32-day, the volume and weight of xenograft tumors in normal control group〔(2 652.17±225.01) mm3 and (2.48±0.21) g〕were both higher than those of Poria cocos group〔(1 247.56±277.23) mm3 and (1.28±0.28) g〕, P<0.050. The tumor inhibitory rate in Poria cocos group was 48.39%. The results of immunohistochemical staining showed that, compared with normal control group, Poria cocos could down-regulate the expressions of Bcl-2〔(4.20±1.10）score vs. (8.00±1.20) score〕and VEGF〔(3.80±0.45) score vs. (7.80±1.10) score〕, while up-regulate the expressions of Bax〔(7.40±1.34) score vs. (3.00±0.71) score〕, Caspase-3〔(6.60±1.34) score vs. (2.60±0.55) score〕, and Caspase-9〔(7.20±1.79) score vs. (4.00±1.22) score〕, P<0.050. Compared with normal control group (1.72±0.03), the expression value of Bcl-2 was all higher in 24 h-Poria cocos group (0.96±0.04) and 48 h-Poria cocos group (0.77±0.04), P<0.050, and the expression value was higher in 48 h-Poria cocos group than that of 24 h-Poria cocos group (P<0.050). Compared with normal control group (0.15±0.01), the expression value of Bax was higher in 48 h-Poria cocos group (0.55±0.01), P<0.050, but there was no significant difference between the normal control group and 24 h-Poria cocos group（0.19±0）, P>0.050.
ConclusionsPoria cocos can restrain the growth of xenograft tumors for gastric cancer SGC-7901 cell line in mude mice, and the mechanism may be related to mitochondrial apoptosis pathway and the inhibition of expression of VEGF.
ObjectiveTo investigate the survival rate of core fat tissue with different diameters by advanced fat harvesting instrument.
MethodsBased on core fat transfer by 1 mL syringe, the fat harvesting instrument was modified with different diameters, including 4, 6, 8, and 10 mm respectively. Between May 2014 and April 2015, the fat harvesting instrument with diameters of 4, 6, 8, and 10 mm was respectively used to harvest abdominal fat in 3 of 12 patients undergoing autologous fat transplantation. The glucose transportation quantities and the fat cell viability were measured. Then 64 nude mice at the age of 3-4 weeks were randomly divided into 4 groups (groups A, B, C, and D, n=16). And 0.5 mL fat harvested with diameters of 4, 6, 8, and 10 mm was implanted into the dorsal subcutaneous space. After fat transplantation, the mice survival and the appearance at the recipient site were observed. At 1, 2, 4, and 8 weeks after fat transplantation, the grafted fat was harvested for gross, histological and immunohistochemical observations; the intact adipocytes and capillary were counted.
ResultsThe glucose transportation quantities gradually increased with increased diameter, showing significant difference among groups (P<0.05). And the fat cell viability had a rising tendency, showing significant differences when comparing groups A and B with group D (P<0.05). With the time passing by, the protuberant appearance became flat at the recipient site, but the appearance of groups C and D was better than groups A and B. Normal shape of the fat and capillary were found in groups C and D. At immediate and 1 week after fat transplantation, there was no significant difference in fat weight among 4 groups (P>0.05); the fat weight of group A was significantly less than that of groups B, C, and D (P<0.05) at 2, 4, and 8 weeks after fat transplantation, and it was significantly less in group B than groups C and D (P<0.05), but no significant difference was found between groups C and D (P>0.05). Histological and immunohistochemical observations showed better integrity of the cells, less necrosis, and higher vascular density in group D than groups A, B, and C as time extension. The adipocyte integrity of group A was significantly worse than that of other 3 groups at other time points (P<0.05) except at 1 week (P>0.05). At each time point, the capillary counting had an increasing trend with increased diameter in all groups, showing significant difference among groups (P<0.05).
ConclusionWith diameters within 10 mm, the thicker the core fat is transferred, the better integrlity, higher vessel density, and quicker revascularization time can be predicted. So the postoperative appearance could be maintained longer.
Objective
To study the effect of platelet-rich plasma (PRP) on the survival and quality of fat grafts in the nude mice so as to provide a method and the experimental basis for clinical practice.
Methods
Fat tissue was harvested from the lateral thigh of a 25-year-old healthy woman and the fat was purified by using saline. The venous blood was taken from the same donor. PRP was prepared by centrifugation (200 × g for 10 minutes twice) and activated by 10% calcium chloride (10 : 1). Then 24 female nude mice [weighing (20 ± 3) g, 5-week-old] were allocated randomly to the experimental group and the control group (12 mice per group). Each subcutaneous layer of two sides of the back (experimental group) was infiltrated with 0.8 mL fat tissue-activated PRP mixtures (10 : 2); the control group was infiltrated with 0.8 mL fat tissue-saline mixtures (10 : 2); 0.14 mL activated PRP and 0.14 mL saline were injected into the experimental group and the control group respectively at 5 and 10 days after the first operation. At 15, 30, 90, and 180 days after the first operation, the samples were harvested for gross and histological observations.
Results
All nude mice survived to the end of the experiment. No inflammation and abscess formation of the graft were observed. Experimental group was better than control group in angiogenesis, liquefaction, and necrosis. The grafted fat weight and volume in the experimental group were significantly larger than those in the control group at 15, 30, and 90 days (P lt; 0.05); but there was no significant difference between the 2 groups at 180 days (P gt; 0.05). Histological observation showed good morphological and well-distributed adipocytes, increasing vacuoles, few necrosis and calcification in the experimental group; but disordered distribution, obvious necrosis, and calcification in the control group. The necrosis area ratio of the experimental group was significantly lower than that of the control group (P lt; 0.05), and the number of micro-vessels was significantly higher in the experimental group than in the control group at 15 and 180 days (P lt; 0.05).
Conclusion
The method of repeatedly using the PRP within 180 days in assisting fat grafts can obviously improve the survival and quality.
【摘要】 目的 觀察低頻超聲（20 kHz）輻照聯合靜脈注射微泡造影劑SonoVue對裸鼠前列腺癌（Du145）移植瘤的抑瘤效應，并探討其可能的抑瘤機制。 方法 通過細胞移植和瘤塊移植方法建立24只裸鼠前列腺癌Du145移植瘤模型，隨機分為超聲微泡組、單純超聲組、單純微泡組和對照組，每組各6只。超聲微泡組：尾靜脈注射0.2 mL SonoVue的同時對瘤體行20 kHz超聲輻照，輻照強度200 mW/cm2；單純超聲組：尾靜脈注射生理鹽水0.2 mL，同時超聲輻照2 min；單純微泡組：尾靜脈注射SonoVue 0.2 mL同時行假照，各組均隔天1次，共3次，對照組不做任何處理。治療后測量瘤體大小，繪制瘤體生長曲線，計算抑瘤率。首次治療后14 d剝離瘤體，通過光學顯微鏡、電子顯微鏡觀察腫瘤組織病理改變。免疫組織化學方法觀察CD34陽性染色血管，計算腫瘤微血管密度（micro vessel density，MVD），比較各組間MVD的差異。 結果 24只裸鼠均成功植瘤。治療后超聲微泡組瘤體體積均數明顯小于其他3組（Plt;0.05），抑瘤率為62.7%。光學顯微鏡下超聲微泡組瘤體組織大部分損傷壞死，電子顯微鏡下超聲微泡組腫瘤內微血管的內皮細胞損傷，線粒體腫大，基底膜斷裂。超聲微泡組瘤體內CD34陽性染色微血管數減少，其MVD值顯著低于其他各組。 結論 20 kHz低頻超聲輻照聯合微泡造影劑SonoVue可有效抑制裸鼠人前列腺癌移植瘤的生長，其抑瘤機制可能是通過超聲空化效應破壞腫瘤的微血管實現的。【Abstract】 Objective To investigate the anti-tumor effect induced by low-frequency ultrasound (20 kHz) radiation combined with intravenous injection of microbubbles on human prostate carcinoma xenograft in nude mice, and to discuss its probable mechanism. Methods Human prostate carcinoma xenograft model in 24 nude mice were established with human prostate carcinoma Du145 cells inoculation and sub-graft through mice, which were randomly divided into ultrasound+microbubble, ultrasound, microbubble, and control group, with 6 mice in each group. In the ultrasound+microbubble group, 0.2 mL SonoVue was injected intravenously, followed by 20 kHz ultrasound exposure of 200 mW/cm2 at every other day for 3 times totally. Mice in the ultrasound group and the microbubbles group were only treated with ultrasound radiation and microbubbles injection, respectively. The volume of gross tumors was measured, and tumor growth curve was drawn. The ratio of anti-tumor growth was calculated. The mice were sacrificed 14 days after the last ultrasound exposure. Specimens of the exposed tumor tissues were obtained and observed pathologically under light microscope and transmission electron microscope. CD34 positive vessels were counted in all the tumor slices by immunohistochemistry, and the micro-vessels density（MVD）of the tumor was also calculated. Results Du145 prostate tumor model was successfully established in all the mice. The average gross tumor volume of the ultrasound+microbubble group was significant lower compared with the other two groups after treatment (Plt;0.05), and the ratio of anti-tumor growth was 62.7%. Histological examination showed signs cell injury in the ultrasound+microbubble group. Electron microscopic examination revealed that the endothelium of vessels in the tumor was injured. The amount of CD34 positive vessels and MVD of the ultrasound+microbubble group was less than that of the other two groups. Conclusion The low-frequency ultrasound of 20 kHz exposure combined with microbubbles can be used to ablate human prostate carcinoma xenograft in nude mice, which is probably realized through micro-vessels destroyed by cavitation effect of ultrasound.
