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[1]丁曉明,張凱三,劉婷婷,等.絲素蛋白-羥基磷灰石三維大孔支架復(fù)合脂肪干細(xì)胞體外構(gòu)建組織工程骨的可行性研究[J].中醫(yī)正骨,2021,33(12):1-6.
　DING Xiaoming,ZHANG Kaisan,LIU Tingting,et al.A feasibility study of three-dimensional silk fibroin-hydroxyapatite macroporous scaffold-adipose-derived stem cells complex in constructing tissue-engineered bone in vitro[J].The Journal of Traditional Chinese Orthopedics and Traumatology,2021,33(12):1-6.
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絲素蛋白-羥基磷灰石三維大孔支架復(fù)合脂肪干細(xì)胞體外構(gòu)建組織工程骨的可行性研究()
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《中醫(yī)正骨》[ISSN:1001-6015/CN:41-1162/R]

卷:
第33卷
期數(shù):
2021年12期
頁碼:
1-6
欄目:
基礎(chǔ)研究
出版日期:
2021-12-20

文章信息/Info

Title:
A feasibility study of three-dimensional silk fibroin-hydroxyapatite macroporous scaffold-adipose-derived stem cells complex in constructing tissue-engineered bone in vitro
作者:
丁曉明1; 張凱三1; 劉婷婷2; 朱明1; 王祥杰1; 潘月興1; 劉玉田1; 郭振光1; 徐開民1; 楊彬1
(1.日照市中醫(yī)醫(yī)院,山東 日照 276800; 2.日照市人民醫(yī)院,山東 日照 276827)
Author(s):
DING Xiaoming1; ZHANG Kaisan1; LIU Tingting2; ZHU Ming1; WANG Xiangjie1; PAN Yuexing1; LIU Yutian1; GUO Zhenguang1; XU Kaimin1; YANG Bin1
1.Rizhao Hospital of TCM, Rizhao 276800,Shandong,China2.People’s Hospital of Rizhao,Rizhao 276827,Shandong,China
關(guān)鍵詞:
組織工程;  絲心蛋白;  羥基磷灰石類;  脂肪干細(xì)胞;  骨誘導(dǎo)
Keywords:
tissue engineering;  fibroins;  hydroxyapatites;  adipose derived stem cell;  osteoinduction
摘要:
目的:探討絲素蛋白(silk fibroin,SF)-羥基磷灰石(hydroxyapatite,HA)三維大孔支架復(fù)合脂肪干細(xì)胞(adipose-derived stem cells,ADSCs)體外構(gòu)建組織工程骨的可行性。方法:以SF和HA為原料,采用石蠟微球瀝濾技術(shù)制備三維大孔支架,以micro-CT掃描重建觀察其結(jié)構(gòu),并測定支架的彈性模量。將兔ADSCs接種到支架上,給予成骨誘導(dǎo)液培養(yǎng),分別取樣本進(jìn)行掃描電子顯微鏡觀察、HE染色、細(xì)胞增殖情況測定(CCK8)、Von Kossa染色、Ⅰ型膠原免疫組化染色及Ⅰ型膠原含量測定(Elisa),評估SF-HA三維大孔支架的生物相容性和ADSCs在支架上的成骨分化情況。結(jié)果:①SF-HA三維大孔支架性能評估結(jié)果。支架內(nèi)部可見均勻排列的大孔結(jié)構(gòu),連通性好。支架孔徑(365.30±27.10)μm,孔隙率(85.30±1.80)%,彈性模量(54.93±5.44)kPa。②SF-HA三維大孔支架生物相容性評估結(jié)果。掃描電子顯微鏡圖像顯示細(xì)胞能夠很好地在支架孔壁上黏附和伸展,細(xì)胞通過孔隙之間的連通結(jié)構(gòu)長入孔隙內(nèi)或在孔隙邊緣呈拉網(wǎng)樣覆蓋,細(xì)胞周圍可見基質(zhì)分泌。SF-HA三維大孔支架ADSCs復(fù)合體HE染色結(jié)果顯示,細(xì)胞核深染、胞漿紅染,均勻黏附在支架孔隙內(nèi)壁上,隨成骨誘導(dǎo)培養(yǎng)時間延長,細(xì)胞和基質(zhì)明顯增多。CCK8檢測結(jié)果顯示,培養(yǎng)21 d時SF-HA三維大孔支架上的細(xì)胞數(shù)量較培養(yǎng)7 d時增加(1.14±0.08,1.98±0.11,t=13.810,P=0.000)。③ADSCs在SF-HA三維大孔支架上的成骨分化情況評估結(jié)果。Von Kossa染色和Ⅰ型膠原免疫組化染色可觀察到鈣沉積和Ⅰ型膠原積累,而且隨時間延長不斷增多。成骨誘導(dǎo)培養(yǎng)1 d、7 d、21 d時,SF-HA三維大孔支架ADSCs復(fù)合體上Ⅰ型膠原含量比較,差異有統(tǒng)計學(xué)意義(0.90±0.24,1.26±0.27,3.89±0.82,F=49.780,P=0.000); 成骨誘導(dǎo)培養(yǎng)21 d時的Ⅰ型膠原含量高于成骨誘導(dǎo)培養(yǎng)1 d、7 d時(P=0.000; P=0.000); 成骨誘導(dǎo)培養(yǎng)1 d、7 d時的Ⅰ型膠原含量比較,差異無統(tǒng)計學(xué)意義(P=0.059)。結(jié)論:SF-HA三維大孔支架復(fù)合成骨誘導(dǎo)培養(yǎng)的ADSCs具有在體外構(gòu)建組織工程骨的可能。
Abstract:
Objective:To explore the feasibility of constructing tissue-engineered bone in vitro with the three-dimensional(3D)silk fibroin(SF)-hydroxyapatite(HA)macroporous scaffold-adipose-derived stem cells(ADSCs)complex.Methods:With SF and HA as raw materials,a 3D macroporous scaffold was prepared using the paraffin microsphere-leaching method,and its structure was reconstructed by micro-CT scanning,followed by the determination of its modulus of elasticity.The rabbit ADSCs were incubated onto this scaffold and cultured with the osteogenic induction fluid.The samples were then observed under the scanning electron microscope(SEM),followed by HE staining,cell proliferation assay(CCK8),Von Kossa staining as well as immunohistochemical staining of type I collagen for its content determination(Elisa)for assessing the biocompatibility of 3D SF-HA composite macroporous scaffold and the osteogenic differentiation of ADSCs on the scaffold.Results:①The performance evaluation results of 3D SF-HA macroporous scaffold.The homogeneously arranged macroporous structure was present inside the scaffold with good connectivity.The aperture,porosity and modulus of elasticity of the scaffold were 365.30±27.10 μm,85.30±1.80% and 54.93±5.44 kPa respectively.②Biocompatibility evaluation results of 3D SF-HA macroporous scaffold.As demonstrated by the SEM images,the cells displayed good adhesion and extension on the pore wall of the scaffold,and grew into the pores through the interconnected structure between the pores or covered the edges of the pores like a stretched mesh,with matrix secretion seen around the cells.The HE staining results of the 3D SF-HA macroporous scaffold-ADSCs complex showed that the deeply-stained cell nuclei and red-stained cytoplasm were uniformly adhered to the pore wall of the scaffold,and the number of cells and matrix increased significantly with the prolongation of osteogenic culture time.The CCK8 assay proved that the number of cells on 3D SF-HA macroporous scaffold increased on day 21 compared with that on day 7(1.14±0.08 vs 1.98±0.11,t=13.810,P=0.000).③The osteogenic differentiation evaluation results of ADSCs on 3D SF-HA macroporous scaffold.The calcium deposition and type I collagen accumulation were observed after Von Kossa staining and immunohistochemical staining of type I collagen,which were further enhanced over time.The differences in content of type I collagen in 3D SF-HA macroporous scaffold-ADSCs complex were statistically significant on day 1,7 and 21 osteogenic induction(0.90±0.24,1.26±0.27,3.89±0.82,F=49.780,P=0.000).The content of type I collagen was higher on day 21 compared with that on day 1 and 7(P=0.000; P=0.000),and there was no statistical difference between day 1 and day 7(P=0.059).Conclusion:It is possible to construct tissue-engineered bone in vitro with 3D SF-HA macroporous scaffold-osteogenic-induced ADSCs complex.

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備注/Memo

備注/Memo:
基金項目:山東省醫(yī)藥衛(wèi)生科技發(fā)展計劃項目(2016WS0330)
通訊作者:楊彬 E-mail:[email protected]
更新日期/Last Update: 1900-01-01