Involvement of acid-sensing ion channel 1a in functions of cultured human retinal pigment epithelial cells / 华中科技大学学报（医学）（英德文版）

In the retina, pH fluctuations may play an important role in adapting retinal responses to different light intensities and are involved in the fine tuning of visual perception. Acidosis occurs in the subretinal space (SRS) under pathological conditions such as age-related macular degeneration (AMD). Although it is well known that many transporters in the retinal pigment epithelium (RPE) cells can maintain pH homeostasis efficiently, other receptors in RPE may also be involved in sensing acidosis, such as acid-sensing ion channels (ASICs). In this study, we investigated whether ASIC1a was expressed in the RPE cells and whether it was involved in the function of these cells. Real-time RT-PCR and Western blotting were used to analyze the ASIC1a expression in ARPE-19 cells during oxidative stress induced by hydrogen peroxide (H(2)O(2)). Furthermore, inhibition or over-expression of ASIC1a in RPE cells was obtained using inhibitors (amiloride and PCTx1) or by the transfection of cDNA encoding hASIC1a. Cell viability was determined by using the MTT assay. The real-time RT-PCR and Western blotting results showed that both the mRNA and protein of ASIC1a were expressed in RPE cells. Inhibition of ASICs by amiloride in normal RPE cells resulted in cell death, indicating that ASICs play an important physiological role in RPE cells. Furthermore, over-expression of ASIC1a in RPE cells prolonged cell survival under oxidative stress induced by H(2)O(2). In conclusion, ASIC1a is functionally expressed in RPE cells and may play an important role in the physiological function of RPE cells by protecting them from oxidative stress.

Involvement of acid-sensing ion channel 1a in functions of cultured human retinal pigment epithelial cells / 华中科技大学学报（医学）（英德文版）

In the retina, pH fluctuations may play an important role in adapting retinal responses to different light intensities and are involved in the fine tuning of visual perception. Acidosis occurs in the subretinal space (SRS) under pathological conditions such as age-related macular degeneration (AMD). Although it is well known that many transporters in the retinal pigment epithelium (RPE) cells can maintain pH homeostasis efficiently, other receptors in RPE may also be involved in sensing acidosis, such as acid-sensing ion channels (ASICs). In this study, we investigated whether ASIC1a was expressed in the RPE cells and whether it was involved in the function of these cells. Real-time RT-PCR and Western blotting were used to analyze the ASIC1a expression in ARPE-19 cells during oxidative stress induced by hydrogen peroxide (H(2)O(2)). Furthermore, inhibition or over-expression of ASIC1a in RPE cells was obtained using inhibitors (amiloride and PCTx1) or by the transfection of cDNA encoding hASIC1a. Cell viability was determined by using the MTT assay. The real-time RT-PCR and Western blotting results showed that both the mRNA and protein of ASIC1a were expressed in RPE cells. Inhibition of ASICs by amiloride in normal RPE cells resulted in cell death, indicating that ASICs play an important physiological role in RPE cells. Furthermore, over-expression of ASIC1a in RPE cells prolonged cell survival under oxidative stress induced by H(2)O(2). In conclusion, ASIC1a is functionally expressed in RPE cells and may play an important role in the physiological function of RPE cells by protecting them from oxidative stress.

Repair of calvarial defects with human umbilical cord blood derived mesenchymal stem cells and demineralized bone matrix in athymic rats / 中华整形外科杂志

<p><b>OBJECTIVE</b>To investigate the feasibility of using human umbilical cord blood derived mesenchymal stem cells (UCB-MSCs) and demineralized bone matrix (DBM) scaffolds to repair critical-sized calvarial defects in athymic rats.</p><p><b>METHODS</b>Human UCB-MSCs were isolated, expanded and osteogenically induced in vitro. Osteogenic differentiation of UCB-MSCs was evaluated by Alizarin Red staining and measurement of calcium content respectively, and then the cells were seeded onto DBM scaffolds. Bilateral full-thickness defects (5 mm in diameter) of parietal bone were created in an athymic rat model. The defects were either repaired with UCB-MSC/DBM constructs (experimental group) or with DBM scaffolds alone (control group). Animals were harvested at 6 and 12 weeks post-implantation respectively, and defect repair was evaluated with gross observation, micro-CT measurement and histological analysis.</p><p><b>RESULTS</b>Micro-CT showed that new bone was formed in the experimental group at 6 weeks post-implantation, while no sign of new bone formation was observed in the control group. At 12 weeks post-transplantation, scaffolds had been degraded almost completely in both sides. It was shown that an average of (78.19 +/- 6.45)% of each defect volume had been repaired in experimental side; while in the control side, only limited bone formed at the periphery of the defect. Histological examination revealed that the defect was repaired by trabecular bone tissue in experimental side at 12 weeks, while only fibrous connection was observed in the control group.</p><p><b>CONCLUSIONS</b>Tissue-engineered bone composed of osteogenically-induced human UCB-MSCs on DBM scaffolds could successfully repair the critical-sized calvarial defects in athymic rat models.</p>

Tissue-engineering bone with ADSCs and coral scaffold for repairing of cranial bone defect in canine / 中华整形外科杂志

<p><b>OBJECTIVE</b>To investigate the application of tissue-engineering bone with ADSCs (adipose-derived stem cells) and coral scaffold for repairing of cranial bone defect in canine.</p><p><b>METHODS</b>Autologous ADSCs isolated from canine subcutaneous fat were expanded, osteogenically induced, and seeded on coral scaffolds. Bilateral full-thickness defects (20 mm x 20 mm) of parietal bone were created (n = 7). The defects were either repaired with ADSC-coral constructs (experimental group) or with coral alone (control group). Radiological, gross, biomechanical and histological observations were done to evaluate the bone regeneration.</p><p><b>RESULTS</b>Three-dimensional CT scan showed that new bones were formed in the experimental group at 12 weeks after implantation, while coral scaffolds were partially degraded in the control group. By radiographic analysis at 24 weeks post-transplantation, it showed that an average repair percentage of each defect was (84.19 +/- 6.45)% in experimental group, and (25.04 +/- 18.82)% in control group (P < 0.01). The maximum compression loading was (73.45 +/- 17.26) N in experimental group, and (104.27 +/- 22.71) N in control group (P <0.01). Histological examination revealed that the defect was repaired by typical bone tissue in experimental group, while only minimal bone formation with fibrous connection in the control group.</p><p><b>CONCLUSIONS</b>The tissue-engineering bone with autologous osteogenic ADSCs and scaffold could successfully repair the cranial defects in canine models.</p>

Pilot study of using autologous bone marrow stromal cells and coral to repair canine segmental mandibular defects / 中华整形外科杂志

<p><b>OBJECTIVE</b>To repair segmental mandibular defects with autologous bone marrow stromal cells (BMSCs) engineered bone.</p><p><b>METHODS</b>Isolated BMSCs were expanded in vitro and osteogenic induced. In 12 canines, a 3 cm segmental mandibular defect at right mandible was created. 6 canine's defects were repaired with cell-scaffold constructs made from induced BMSCs and coral; others were repaired with coral as control. The engineered bone was evaluated by X-ray, CT, Dual Energy X-ray Absorptiometry (DXA), gross and histological examination, and biomechanical test post-operatively.</p><p><b>RESULTS</b>Induced BMSCs grew well on coral scaffold. At 12 weeks, X-ray showed more callus formed in experimental group, while evident scaffold duration in control group. At 32 weeks, gross observation, X-ray and CT demonstrated well bony-union in experimental group, while bony-nonunion in control group. Also DXA revealed significantly higher bone mineral density of experimental group than control group. Histologically, mature bone were commonly observed and there were bony healing in experimental group, while fibrous healing occurred in control group. Biomechanical test revealed no significant difference between experimental group and normal group.</p><p><b>CONCLUSIONS</b>Canine segmental mandibular defects can be repaired with the tissue-engineered bone generated by coral scaffold with autologous osteogenic BMSCs.</p>

The isolation, subculture and identification of human adipose derived endothelial progenitor cells / 中华整形外科杂志

<p><b>OBJECTIVE</b>To investigate the methods of isolating and identifying human adipose derived EPCs.</p><p><b>METHODS</b>The cells obtained from human lipoaspirates were plated on culture dishes coated with human fibronectin and were cultured in DMEM containing 2% FBS. Cells of passage 2 cultured in EGM-2 (2% FBS) served as the induced cells (experimental group), with cells cultured in DMEM (2% FBS) as the non-induced cells (control group) . Immunofluorescence was used to detect the expression of cell markers, including CD34, vWF and PECAM-1. FACS (fluorescence activated cell sorter) was used to quantitatively analyze the expression rate of cell markers (CD34, CD45, CD133 and PECAM-1). Fluorescence microscope was used to observe the function of taking up DiI-ac-LDL by the induced cells. To determine the ability of forming capillary-like structure in three-dimensional matrices, the induced cells were also cultured in methylcellulose.</p><p><b>RESULTS</b>The induced cells of passage 2 exhibited cobblestone morphology, similar to that of the endothelial cells. In contrast, these morphological changes were not observed in non-induced cells. Immunofluorescence detected expression of vWF, PECAM-1 in induced cells and CD34 in non-induced cells. FACS analysis showed (67.41 +/- 13.35)% of the induced cells expressed PECAM-1 and (6.73 +/- 2.21)% of the non-induced cells expressed PECAM-1 (P < 0.01), while (72.39 +/- 13.45)% of the non-induced cells expressed CD34 and (16.06 +/- 3.86)% of the induced cells expressed CD34 (P < 0.01). Fluorescence microscopy observed the induced cells took up low-density lipoprotein (LDL). The formation of "branch-like" structure confirmed their functional activity.</p><p><b>CONCLUSION</b>EPCs derived from human adipose may serve as another source of seeding cells for vascular tissue engineering.</p>

The effects of cryopreservation on growth and osteogenesis of human bone marrow stromai cells cultured on demineralized bone matrix / 中华创伤骨科杂志

Objective To investigate the effects of cryopreservation on the growth and osteogenesis capa- bility of human bone marrow stromal cells(BMSCs)on demineralized bene matrix(DBM).Methods Bone marrow aspirates were obtained from the lilac crests of three donors.The BMSCs were isolated from the bone marrow by density gradient centrifugation.Cells of passage 3 were cryopreserved in liquid nitrogen for 24 hours,and then re- covered.The non-cryopreserved BMSCs were used as the control,The cryopreserved and control BMSCs were cul- tured in osteogenic media,collected and labeled with Dil to be seeded onto the DBM when cells were confluent.The percentage of BMSCs adhered to the DMB was detected.The cell morphology and matrices secreted by BMSCs on the DBM were observed by the inverted phase-contrasted microscope,fluorescence microscope and scanning electron microscope(SEM).The growth and viability of BMSCs on the DBM were determined using the modified MTT ashy. The osteogenesis ability of BMSCs on the DBM was determined by assessment of the alkaline phosphatase(ALP) activity and osteocalcin(OCN)content.Results The percentages of the cryopreserved and control cells adhered to DBM were(97.25?1.17)% and(97.00?1.09)% respectively.The cells adhered well to the DBM and grew rapidly.Large amounts of matrices on the DBM were observed by the light microscope and SEM.The cells embedded in the matrices could be observed by fluorescence microscope.There were no significant differences in the assay values of MTT,ALP and OCN between the cryopreserved and control BMSCs on the DBM.Conclusion Since cryopreservation does not affect the growth and osteogenesis capability of BMSCs on DBM,the cryopreserved BMSCs can be used as a cell source in bone tissue engineering.

Repair of canine segmental mandibular defects by using autogenous bone marrow stromal cells and?-tricalcium phosphate / 中华创伤杂志

Objective To repair segmental mandibular defects with autogenous bone marrow stromal cells(BMSCs)and?-triealcium phosphate.Methods Isolated BMSCs were in vitro expand- ed.A 3 cm-long segmental mandibular defect was created at right mandible in 12 canines,of which de- fects in six canines were repaired with BMSCs and?-tricalcium phosphate(?-TCP)and that in other six cases repaired with?-TCP,which was used as control.The engineered bone was evaluated by X-ray, CT,DXA,gross and histological examination,immunohistochemistry and biomechanical test 4,12,26,32 weeks after operation respectively.Results In induced BMSCs,histochemistry showed AKP activity. Oral X-ray showed obvious callus formation 4-26 weeks after operation in experimental group but minimal bone formation in control group.At 32 weeks after operation,gross observation,X-ray and CT demonstra- ted well bony-union in experimental group but bony-nonunion in control group.DXA indicated that the bone density of experimental group was significantly higher than that of control group.Biomechanical test revealed no statistical difference upon mechanical strength of mandibula between experimental group and normal group.Conclusions Canine segmental mandibular defects can be well repaired with the tissue- engineered bone generated by autogenous osteogenic BMSCs and?-TCP scaffold.