中国组织工程研究与临床康复
中國組織工程研究與臨床康複
중국조직공정연구여림상강복
JOURNAL OF CLINICAL REHABILITATIVE TISSUE ENGINEERING RESEARCH
2009年
38期
7433-7436
,共4页
任大伟%刘菲%余喜讯%邓晓薇%张小华%万昌秀
任大偉%劉菲%餘喜訊%鄧曉薇%張小華%萬昌秀
임대위%류비%여희신%산효미%장소화%만창수
掺锶聚磷酸钙%内皮细胞%血管化%基质金属蛋白酶2
摻鍶聚燐痠鈣%內皮細胞%血管化%基質金屬蛋白酶2
참송취린산개%내피세포%혈관화%기질금속단백매2
背景:课题组研究的掺锶聚磷酸钙作为一种新型骨修复材料,具有良好的生物相容性和生物可降解性,经过前期的研究已证明有促血管化作用,但机制尚不清楚.目的:内皮细胞与掺锶聚磷酸钙支架于体外复合培养,观察细胞的增殖和促血管化因子基质金属蛋白酶2的分泌情况.设计、时间及地点:体外细胞学对比观察实验,于2008-09/2009-04在四川大学组织工程研究室完成.材料:在制备聚磷酸钙过程中掺入锶制备掺锶量为1%,2%,5%,8%,10%的掺锶聚磷酸钙骨组织工程支架材料.方法:①材料置于24孔培养板中,将浓度为3×10~7 L~(-1)的内皮细胞悬液300 μL接种于24孔培养板上,补加200 μL RPMI1640全培养液.分别于1,3,5,7 d通过MTT法观察内皮细胞的增殖情况.②聚磷酸钙和8%掺锶聚磷酸钙多孔支架放于24孔板中,将浓度为1×10~8L~(-1)的内皮细胞悬液300 μL接种于支架材料上,补加600 μL RPMI1640全培养液.培养5 d后取出材料,扫描电镜观察内皮细胞的形貌.③内皮细胞与不同掺锶量的掺锶聚磷酸钙支架材料复合培养5 d,至细胞汇合后,离心取上清液,采用酶联免疫吸附试验检测内皮细胞来源的基质金属蛋白酶2蛋白的分泌量.主要观察指标:观察掺锶聚磷酸钙和聚磷酸钙材料上内皮细胞的增殖及形貌,内皮细胞来源的基质金属蛋白酶2蛋白的分泌量.结果:MTT法实验结果表明,与聚磷酸钙组及其他掺锶量的掺锶聚磷酸钙比较,8%掺锶聚磷酸钙拥有更高的内皮细胞增殖度;扫描电镜表明在8%掺锶聚磷酸钙材料表面生长的内皮细胞具有更好的生长状态和生物活性;酶联免疫吸附试验法结果表明,与聚磷酸钙组相比,掺锶聚磷酸钙能上调基质金属蛋白酶2的蛋白分泌量,其中以8%掺锶聚磷酸钙最为明显(P＜0.05).结论:掺锶聚磷酸钙与内皮细胞具有良好的生物相容性,明显上调促血管化因子基质金属蛋白酶2的分泌,具有促血管化的作用.
揹景:課題組研究的摻鍶聚燐痠鈣作為一種新型骨脩複材料,具有良好的生物相容性和生物可降解性,經過前期的研究已證明有促血管化作用,但機製尚不清楚.目的:內皮細胞與摻鍶聚燐痠鈣支架于體外複閤培養,觀察細胞的增殖和促血管化因子基質金屬蛋白酶2的分泌情況.設計、時間及地點:體外細胞學對比觀察實驗,于2008-09/2009-04在四川大學組織工程研究室完成.材料:在製備聚燐痠鈣過程中摻入鍶製備摻鍶量為1%,2%,5%,8%,10%的摻鍶聚燐痠鈣骨組織工程支架材料.方法:①材料置于24孔培養闆中,將濃度為3×10~7 L~(-1)的內皮細胞懸液300 μL接種于24孔培養闆上,補加200 μL RPMI1640全培養液.分彆于1,3,5,7 d通過MTT法觀察內皮細胞的增殖情況.②聚燐痠鈣和8%摻鍶聚燐痠鈣多孔支架放于24孔闆中,將濃度為1×10~8L~(-1)的內皮細胞懸液300 μL接種于支架材料上,補加600 μL RPMI1640全培養液.培養5 d後取齣材料,掃描電鏡觀察內皮細胞的形貌.③內皮細胞與不同摻鍶量的摻鍶聚燐痠鈣支架材料複閤培養5 d,至細胞彙閤後,離心取上清液,採用酶聯免疫吸附試驗檢測內皮細胞來源的基質金屬蛋白酶2蛋白的分泌量.主要觀察指標:觀察摻鍶聚燐痠鈣和聚燐痠鈣材料上內皮細胞的增殖及形貌,內皮細胞來源的基質金屬蛋白酶2蛋白的分泌量.結果:MTT法實驗結果錶明,與聚燐痠鈣組及其他摻鍶量的摻鍶聚燐痠鈣比較,8%摻鍶聚燐痠鈣擁有更高的內皮細胞增殖度;掃描電鏡錶明在8%摻鍶聚燐痠鈣材料錶麵生長的內皮細胞具有更好的生長狀態和生物活性;酶聯免疫吸附試驗法結果錶明,與聚燐痠鈣組相比,摻鍶聚燐痠鈣能上調基質金屬蛋白酶2的蛋白分泌量,其中以8%摻鍶聚燐痠鈣最為明顯(P＜0.05).結論:摻鍶聚燐痠鈣與內皮細胞具有良好的生物相容性,明顯上調促血管化因子基質金屬蛋白酶2的分泌,具有促血管化的作用.
배경:과제조연구적참송취린산개작위일충신형골수복재료,구유량호적생물상용성화생물가강해성,경과전기적연구이증명유촉혈관화작용,단궤제상불청초.목적:내피세포여참송취린산개지가우체외복합배양,관찰세포적증식화촉혈관화인자기질금속단백매2적분비정황.설계、시간급지점:체외세포학대비관찰실험,우2008-09/2009-04재사천대학조직공정연구실완성.재료:재제비취린산개과정중참입송제비참송량위1%,2%,5%,8%,10%적참송취린산개골조직공정지가재료.방법:①재료치우24공배양판중,장농도위3×10~7 L~(-1)적내피세포현액300 μL접충우24공배양판상,보가200 μL RPMI1640전배양액.분별우1,3,5,7 d통과MTT법관찰내피세포적증식정황.②취린산개화8%참송취린산개다공지가방우24공판중,장농도위1×10~8L~(-1)적내피세포현액300 μL접충우지가재료상,보가600 μL RPMI1640전배양액.배양5 d후취출재료,소묘전경관찰내피세포적형모.③내피세포여불동참송량적참송취린산개지가재료복합배양5 d,지세포회합후,리심취상청액,채용매련면역흡부시험검측내피세포래원적기질금속단백매2단백적분비량.주요관찰지표:관찰참송취린산개화취린산개재료상내피세포적증식급형모,내피세포래원적기질금속단백매2단백적분비량.결과:MTT법실험결과표명,여취린산개조급기타참송량적참송취린산개비교,8%참송취린산개옹유경고적내피세포증식도;소묘전경표명재8%참송취린산개재료표면생장적내피세포구유경호적생장상태화생물활성;매련면역흡부시험법결과표명,여취린산개조상비,참송취린산개능상조기질금속단백매2적단백분비량,기중이8%참송취린산개최위명현(P＜0.05).결론:참송취린산개여내피세포구유량호적생물상용성,명현상조촉혈관화인자기질금속단백매2적분비,구유촉혈관화적작용.
BACKGROUND: Strontium-doped calcium polyphosphate (SCPP), as a new repairing material, has been demonstrated to have favorable biocompatibility and biodegradability and some effects on promoting self-angiogenesis. However, the mechanism remains still unknown. OBJECTIVE: Endothelial cells were cultured with SCPP scaffolds in vitro, as well as the cell proliferation and angiogenic factor matrix metalloproteinase-2 (MMP-2) secretion were observed. DESIGN, TIME AND SETTING: A contrast study was performed at the Laboratory of Tissue Engineering of Sichuan University from September 2008 to April 2009. MATERIALS: A series of calcium polyphosphate (CPP) respectively containing 1 %, 2%, 5%, 8%, and 10% Sr~(2+) were prepared.METHODS: ① Materials were plated on 24-well culture plate, and endothelial cell suspension (300 μL) were seeded on 24-well culture plate at the concentration of 3×10~7/L and cultured with 200 uL RPMI1640 culture media. Endothelial cell proliferation was observed using MTT method at days 1,3,5, and 7 after culture. ② CPP and 8% SCPP were plated on 24-well culture plate, and endothelial cell suspension (300 uL) was then incubated in 24-well culture plate at the concentration of 1x10~8/L and cultured with 600 uL RPMI1640 culture media. The morphology of endothelial cells was observed by scanning electron microscopy (SEM) at day 5 after culture.③ Endothelial cells were co-cultured with SCPP of various Sr~(2+) contents for 5 days. After confluence, cells were centrifuged to obtain supernatant. Angiogenic factor MMP-2 secretion was evaluated by ELISA assay.MAIN OUTCOME MEASURES: The proliferation and morphology of endothelial cells on SCPP and CPP were observed. The amount of endothelial cells-derived MMP-2 protein secretion was detected. RESULTS: MTT method demonstrated that the proliferation of endothelial cells on the 8% SCPP scaffold showed a higher level compared to CPP, and other SCPP groups. Scanning electron microscope results suggested that endothelial cells on 8% SCPP had a better growth status and biological activity. ELISA results indicated that angiogenic factor MMP-2 expression on the SCPP was promoted compared with that of CPP, and 8% SCPP showed the highest expression (P < 0.05). CONCLUSION: SCPP has good compatibility with endothelial cells, promoting angiogenesis and enhancing the angiogenic factor MMP-2 expression.
