在骨组织工程研究中，由于难以模拟自然组织的复杂环境（如血管化），严重限制了组织工程临床转化及应用。细胞片（CS）技术已被应用于心脏、肝脏、膀胱、骨骼、角膜和食道中等多种类型组织的构建中。由于CS技术可以维持完整的细胞基质，该技术可为血管化提供极好的微环境，这对血管生成至关重要。但单层CS力学性能较差，在构建3D骨组织中具有局限性。虽然通过叠层CS可以提高力学性能，但CS的厚度会限制血管的氧气和营养的供应。针对这一问题，葡萄牙阿威罗大学的João F. Mano和Ana S. Silva教授研究团队采用预血管化磁性CS构建3D组织，加速CS植入后的血管生成与长入，以确保3D组织的存活及与宿主组织之间的联系。
为开发分层的预血管化3D细胞构建体，研究人员利用负载氧化铁纳米粒子（MNPs）的细胞来构建分层的磁响应异型CS，即磁标记的脂肪基质细胞（ASCs）和人脐静脉内皮细胞（HUVECs）组装成ASCs / HUVECs / ASCs（异型CS）的三层结构。对照组中，则利用ASCs组装成ASCs/ ASCs（同型CS）的二层结构（图1）。所有细胞在接种形成CS前均以异硫氰酸-罗丹明B进行标记。CD31免疫荧光染色结果证实，在异型CS中，HUVECs层位于两个ACSs片层中间（图2A）。扫描电镜结果进一步证实预血管化CS具有较高的完整性，同时HUVECs还可形成管状结构（图2C）。
研究人员将上述同型和异型CS分别在基础和成骨培养基中培养。在7天、14天和21天时分别检测细胞的活性、DNA含量、ALP活性。结果显示，同型或者异型CS，不论在基础还是成骨培养基中，到14天时，其代谢活性、DNA含量都有明显提高（图2D、E）。培养21天后，大多数细胞仍然保持活性（图2F），说明CSs中细胞具有长期存活的能力而ALP活性检测显示，成骨培养基培养的异型CS的细胞在7天前就已经开始分化。到21天时，无论是在基础培养基还是成骨培养基中培养，异型CS均显示更强的ALP活性，表明HUVECs与ASCs共培养可促进ASCs细胞的成骨分化。
Fig. 1. Cellular uptake and graphical illustration of the CS fabrication. (A) Internalization of MNPs-RodB in ASCs and HUVECs: actin filaments of HUVECs and ASCs (green phalloidin), MNPs-RodB (red) and cell nucleus-DAPI (blue). (B) Flow cytometry analysis of MNPs-RodB uptake after 4 hours. (C) Schematic representation of the fabricated 3D vascularized heterotypic CS.
Fig. 2. Cell sheet integrity, metabolic activity, cell survival and proliferation, and in vitro osteogenic potential. (A) Confocal microscopy of the magnetically labeled CS demonstrating CD31 staining HUVECs in between two ASCs sheets–CD31: HUVECs (red) and ASCs (green); (B) 3D reconstruction of B and side sections exhibiting CD31 (HUVECs) in the middle of two ASCs sheets (Phalloidin–green, DAPI–blue). (C) SEM micrographs of the triple layer conformation of the developed CS: HUVECs (represented in red) and ASCs (represented in green). (D) Cell metabolic activity determined by MTS colorimetric assay, (E) Cell proliferation by DNA quantification and (F) Alkaline phosphatase (ALP) activity normalized by DNA content for both homotypic and heterotypic CS. (G) Live-dead fluorescence assay at day 7, 14 and 21 of culture in basal and osteogenic medium. Living cells were stained by calcein (green) and dead cells by propidium iodide (red).
之后，研究人员通过EDS和Osteoimage TM分析、Von Kossa染色及Masson Trichrome染色，表征了异型CS的矿化（图3A-E）。结果显示，不论在哪种培养基中培养，异型 CS在第14天都出现了明显的羟基磷灰石。另外，同型和异型CSs在两种培养基培养21天后均有晚期成骨标记骨桥蛋白和骨钙素的分泌（图3F-G）。在普通培养基条件下，同型CS骨钙素分泌相对较少，而普通培养基培养的异型CS与成骨诱导培养基培养的同型CS的分泌量相当，表明异型CS比同型CS具有更高的成骨分化能力。
Fig. 3. Mineralization of the developed 3D heterotypic CS in basal and osteogenic media. (A) The hydroxyapatite portion of bone-like nodules deposited by cells was visualized through fluorescent OsteoImage TM Mineralization Assay: cell nucleus-DAPI (blue) and hydroxyapatite (green). SEM micrographs displaying calcium deposits are depicted in the right panel; (B) Energy dispersive X-ray spectroscopy (EDS) spectra of minerals formed within the CS; (C) mineralization of the heterotypic CS accessed by Von Kossa staining and collagenous connective tissue fibers identified by Trichrome Masson staining on histological sections CS cultured in basal or osteogenic media after 21 days. (D) Percentage of collagen-matrixin the representative histological cut; (E) Number of Ca deposits in the denovo cell matrix; (F) Quantification of osteopontin and osteocalcin expressionby (G) ELISA.
此外研究人员还考察了各个时间点，不同CS中BMP-2和VEGF的分泌（图4A，B）。结果显示BMP-2和VEGF之间存在协同行为。在异型CS中，BMP-2的表达量更高，尤其是在成骨诱导培养基培养条件下，BMP-2的表达量进一步提高。而同型CS的VEGF的表达量更高。实际上，ASCs可以通过分泌血管生成和抗凋亡生长因子（例如VEGF）来刺激血管生长，而VEGF对内皮细胞的作用可以诱导BMP-2的释放，之后随着BMP-2值的升高，VEGF的水平则降低，这表明ASCs向成骨细胞发生了分化。该结果再次表明，即使在没有成骨诱导因子的情况下，ASCs和HUVECs之间的协同相互作用也可以促进成骨，同时产生基质并形成血管（图4C、D）。
Fig. 4. Pre-vascularization of the heterotypic CS cultured during 7 days with basal media. (A) Quantification of BMP-2 and (B) VEGF release by Elisa on the homotypic and heterotypic CS cultured over 21 days in basal and osteogenic media; (C) H&E staining of paraffin-embedded heterotypic CSs cultured under basal conditions for 21 days; (D) Immunostaining of CS: VWF (green) and FN (red); collagen IV (green) and CD31 (red); and VWF (green) and CD31 (red) demonstrating the presence of capillary-like structures (white arrows). DAPI (in blue) stains all nuclei.
研究人员接下来进行鸡胚绒毛尿囊膜（CAM）分析评估了基础培养基中培养的磁性异型CS的体内血管生成潜力。如图5A所示，借助磁铁收获CSs，然后将其植入CAM顶部的硅环内并培养4天。拍摄显微照片对新形成的血管进行计数（图5B）。结果表明预血管化CS可以募集新生血管，数量显著高于对照组。该预血管化磁性多层细胞片的开发为制备更复杂的3D组织开辟了一条新的道路。
Fig. 5. In vivo angiogenic potential of the heterotypic CS cultured during 7 days with basal media. PBS was used as negative control and bFGF as a positive control. (A) Schematic representation of the implementation of the CS into the CAM; (B) Photomicrographs of the newly formed vessels (C) Quantification of the newly formed vessels after 14 days of maturation.
该研究由来自葡萄牙阿威罗大学的João F. Mano和Ana S. Silva教授研究团队完成，于2019年12月发表于Biomaterials。
论文信息：
Ana S. Silva*, Lúcia F. Santos, Maria C. Mendes, João F. Mano*. Multi-layer pre-vascularized magnetic cell sheets for bone regeneration. Biomaterials 2019, 231: 119664.
供稿：余丽
审校：过倩萍
编辑：魏强
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