Department of Otorhinolaryngology, Mayo Clinic, Rochester, Minnesota, USA.
Tissue Eng Part A. 2021 Feb;27(3-4):237-245. doi: 10.1089/ten.TEA.2020.0117. Epub 2020 Nov 5.
Craniofacial reconstruction of critical bone defects typically requires a bone graft. As graft availability may be restricted by disease or comorbidities, tissue engineering approaches are actively sought. The pericranium could provide new bone graft material. During development and repair, bone transitions through a chondrogenic phase. However, with tissue engineering, pluripotent cells can differentiate directly into bone cells. Does ability to recapitulate bone formation affect osteogenesis and vascularization of pericranium grafts? To answer this, we obtained tissue from nine patients with preplanned craniotomy surgery and studied three-dimensional osteogenesis and angiogenesis of pericranium-derived spheroids. First, we established growth and differentiation conditions on Matrigel. For each spheroid sample, we investigated (i) continuous osteogenic differentiation (COD) and (ii) osteogenic differentiation preceded by chondrogenesis (CD → OD). The effect of vascular endothelial growth factor (VEGF) was compared to VEGF supplemented with fibroblast growth factor, interleukin (IL)-1, IL-6, platelet-derived growth factor, and tumor necrosis factor-α, a growth factor mix (GFM) with possible synergistic effects. In this limited sample, we observed no age- or sex-related differences in cell expansion. Similarly, no statistically significant differences in osteogenic or angiogenic scores between COD or CD → OD spheroids were noted with regular media. In COD, however, VEGF statistically significantly increased angiogenesis compared to control media ( = 0.007). Also, in COD, both VEGF and VEGF + GFM increased osteogenesis ( = 0.047 and = 0.038, respectively). By contrast, in CD → OD, neither VEGF nor VEGF + GFM yielded statistically significant angiogenesis or osteogenesis scores compared to control media. To understand these results, we characterized spheroid protein expression by nanoliquid chromatography coupled to tandem mass spectrometry. Nine angiogenic proteins were either uniquely expressed or upregulated in COD compared to CD → OD: (i) endothelial markers JUP, PTGIS, PTGS2, and TYMP, (ii) tissue remodeling factors CHI3L1 and MMP14, and (iii) metabolic pathways modulators ANGPTL4, ITGA5, and WNT5A. ANGPTL4, ITGA5, PTGIS, PTGS2, and WNT5A define a conserved angiogenic network and were >2-fold increased in VEGF compared to VEGF + GFM. Finally, we examined bone formation on printable poly-(propylene-fumarate) (PPF) scaffolds for individualized grafting. Under COD + VEGF conditions, PPF scaffolds loaded with pericranium-derived cells displayed hallmarks of spongiform-like bone formation. Thus, the human pericranium may be a potential repository for bone-generating cells with applications in craniofacial bone repair using tissue printing.
颅面骨严重缺损的重建通常需要骨移植。由于疾病或合并症可能限制了移植物的可用性，因此正在积极寻求组织工程方法。颅骨可以提供新的骨移植材料。在发育和修复过程中，骨骼经历一个软骨形成阶段。然而，通过组织工程，多能细胞可以直接分化为成骨细胞。是否具有骨形成的能力会影响颅骨移植的成骨和血管生成？为了回答这个问题，我们从 9 名计划行颅骨切开术的患者中获得组织，并研究了颅骨衍生球体的三维成骨和血管生成。首先，我们在 Matrigel 上建立了生长和分化条件。对于每个球体样本，我们研究了（i）连续成骨分化（COD）和（ii）软骨形成前的成骨分化（CD→OD）。比较了血管内皮生长因子（VEGF）与补充成纤维细胞生长因子、白细胞介素（IL）-1、IL-6、血小板衍生生长因子和肿瘤坏死因子-α的 VEGF 的作用，生长因子混合物（GFM）可能具有协同作用。在这个有限的样本中，我们没有观察到细胞扩增与年龄或性别相关的差异。同样，在常规培养基中，COD 或 CD→OD 球体之间的成骨或血管生成评分之间没有统计学上的显著差异。然而，在 COD 中，VEGF 与对照培养基相比，统计学上显著增加了血管生成（=0.007）。此外，在 COD 中，VEGF 和 VEGF+GFM 均增加了成骨作用（=0.047 和=0.038）。相比之下，在 CD→OD 中，VEGF 或 VEGF+GFM 与对照培养基相比，均未产生统计学上显著的血管生成或成骨评分。为了理解这些结果，我们通过纳升液相色谱-串联质谱法对球体蛋白表达进行了特征分析。与 CD→OD 相比，在 COD 中，有 9 种血管生成蛋白要么是特异性表达的，要么是上调的：（i）内皮标志物 JUP、PTGIS、PTGS2 和 TYMP，（ii）组织重塑因子 CHI3L1 和 MMP14，以及（iii）代谢途径调节剂 ANGPTL4、ITGA5 和 WNT5A。ANGPTL4、ITGA5、PTGIS、PTGS2 和 WNT5A 定义了一个保守的血管生成网络，与 VEGF+GFM 相比，它们的表达增加了 2 倍以上。最后，我们研究了用于个体化移植的可打印聚（丙交酯-富马酸酯）（PPF）支架上的骨形成。在 COD+VEGF 条件下，负载颅骨衍生细胞的 PPF 支架显示出海绵状骨形成的特征。因此，人类颅骨可能是一种具有成骨细胞生成潜力的潜在储存库，可应用于组织打印的颅面骨修复。
