【摘要】：目的:利用3D打印技術及高壓靜電紡織技術聯(lián)合構建組織工程支架-細胞復合物,并通過體內(nèi)外實驗驗證其在構建血管化組織工程骨中的優(yōu)勢。方法:利用醫(yī)學圖像三維重建軟件(Mimics)及3D打印機將斷層掃描數(shù)據(jù)(CT)進行處理、編輯制作成個性化三維立體模型,在模型打印的同時利用同軸高壓靜電紡織技術制備芯層為P3HB4HB、殼層為PVA與人骨髓間充質(zhì)干細胞的(PVA-細胞)/P3HB4HB纖維。將一次成型的支架-細胞復合物向成骨方向誘導14日,植入前復合內(nèi)皮細胞。體外實驗中通過力學測試觀察經(jīng)3D打印技術制作的P3HB4HB外部支架的力學性能;通過光鏡、透射電鏡、親水性實驗及掃描電鏡觀察經(jīng)靜電紡織技術制備的PVA/P3HB4HB支架的微觀結構及生物相容性;通過掃描電鏡、DAPI免疫熒光染色、吖啶橙免疫熒光染色以及CCK-8實驗觀察細胞在支架結構中的黏附、增殖情況;體內(nèi)實驗中將支架-細胞復合物作為實驗組,不含細胞的支架材料作為對照組,標本分別于12周與24周后取出,行HE、VonKossa、天狼星紅、馬松三色、CD31免疫組織化學及Ⅰ型膠原免疫組織化學染色,觀察支架-細胞復合物成骨及血管的能力。結果:利用3D打印技術能夠制造外觀個性化很強的模型,模型能夠為組織工程支架提供一定的力學支撐;通過掃描電鏡及親水性實驗可觀察到通過靜電紡織技術制備的PVA/P3HB4HB支架具有較高的孔隙率、良好的生物相容性及仿生細胞外基質(zhì)的三維立體結構;利用吖啶橙免疫熒光染色、DAPI免疫熒光染色、掃描電鏡及CCK-8可顯示細胞與支架復合后仍具有增值、分化的能力,實現(xiàn)了材料與細胞的一次成型與精確種植;在體內(nèi)實驗中,分別對實驗組12周和24周的標本行HE染色、Von Kossa染色、天狼星紅染色、馬松三色染色、CD31免疫組織化學染色、Ⅰ型膠原免疫組織化學染色,結果均呈陽性,并隨著時間的延長骨組織及血管結構的數(shù)量及質(zhì)量明顯增加,對照組陰性。結論:利用3D打印技術及高壓靜電紡織技術聯(lián)合構建的支架-細胞復合物具有一定的力學性能及仿生細胞外基質(zhì)結構的結構與功能,通過復合內(nèi)皮細胞后,能夠在體內(nèi)構建出含血管的組織工程骨。
[Abstract]:Aim: to construct tissue engineering scaffold-cell complex using 3D printing technique and high voltage electrostatic textile technology, and to verify its advantages in the construction of vascularized tissue engineering bone in vitro and in vivo. Methods: the medical image 3D reconstruction software (Mimics) and 3D printer were used to process the scanning data (CT), and the 3D 3D model was edited and made into personalized 3D model. The PVA- cells / P3HB4HB fibers of PVA and human bone marrow mesenchymal stem cells (PVA- cells) were prepared by co-axial high-voltage electrostatic spinning technique and the core layer was P3HB4HB.The core layer was P3HB4HB. the core layer was P3HB4HB. The scaffold-cell complex was induced to osteogenic direction for 14 days, and the endothelial cells were mixed before implantation. The mechanical properties of P3HB4HB external scaffolds fabricated by 3D printing technology were observed by mechanical test in vitro. The microstructure and biocompatibility of PVA/P3HB4HB scaffolds were observed by light microscopy, transmission electron microscopy, hydrophilicity test and scanning electron microscopy. Scanning electron microscopy, DAPI immunofluorescence staining, acridine orange immunofluorescence staining and CCK-8 test were used to observe the adhesion and proliferation of the cells in the scaffolds. In vivo, the scaffold-cell complex was used as the experimental group, and the cell-free scaffold material was used as the control group. After 12 weeks and 24 weeks respectively, the specimens were taken out, HE,VonKossa, Sirius Red and Ma Song trichromatic, respectively. CD31 immunohistochemical staining and type 鈪，

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