Biofabrication of a 3D bone tissue analogue with immunomodulatory factors to induce neovascularization and osteogenesis

在英国，每年有超过50,000例的骨移植手术涉及自体骨移植(1)。自体移植物的严重局限性，如组织供应有限、供体部位发病率、感染或疾病传播的可能性以及骨整合不良，导致了许多骨组织工程方法(2)。然而，关键尺寸的3D工程组织的主要挑战是缺乏足够的血管供应，导致细胞坏死和组织衰竭。我们已经开发了骨髓来源的间充质干细胞（MSC）球体，形成富钙结节(3)，并在胶原凝胶中开发了内皮细胞和MSC共培养方法，用于工程构建体的预血管化(4)，该方法与天然水凝胶结合可诱导血管生成和成骨。当植入生物材料时，宿主组织会产生反应，导致持续的炎症。这反过来导致愈合和修复受损。因此，生物材料的骨和血管免疫调节特性会影响骨再生的结果(5)。越来越多的证据表明，巨噬细胞分泌的炎症细胞因子是介导组织-生物材料整合所必需的。在这里，我们的目标是通过在天然水凝胶（胶原蛋白、明胶或海藻酸盐）中嵌入骨祖细胞的细胞球体/类器官（骨髓来源的间充质干细胞）来生物构建血管化骨结构。其中，胶原凝胶中的内皮细胞和间充质干细胞将使用悬浮层增材制造（SLAM）方法进行生物打印，以创建悬浮血管网络。这些细胞将在巨噬细胞释放活性骨免疫调节因子的情况下进行培养，以增加骨再生。

Over 50,000 bone procedures performed annually in the UK involve autografts (1). Serious limitations in autologous-grafts, such as limited tissue supply, donor site morbidity, potential for infection or disease transmission and poor osseointegration, has led to many bone tissue engineering approaches (2). However, a major challenge of critically-sized 3D engineered tissues is the lack of adequate vascular supply which results in cell necrosis and tissue failure. We have developed bone marrow derived mesenchymal-stem-cells (MSC) spheroids that form calcium rich nodules (3) and an endothelial and MSC coculture methodology in collagen gel for the prevascularisation of engineered constructs (4) that combined with natural hydrogels induce angiogenesis and osteogenesis. When a biomaterial is implanted a response is initiated by the host tissue which could result in persistent inflammation. This in turn results in impaired healing and repair. Therefore the osteo- and angio-immunomodulatory properties of biomaterials can affect the outcomes of bone regeneration (5). Increasing evidence suggest that the inflammatory cytokines secreted by macrophages are necessary for mediating tissue-biomaterial integration. Here we aim to biofabricate vascularised-bone-constructs by embedding cellular spheroids/organoids of osteoprogenitor-cells (bone marrow derived mesenchymal-stem-cells) in a natural hydrogel (collagen, gelatin or alginate). In that, ECs and MSCs in collagen gel will be bioprinted using the suspended layer additive manufacture (SLAM) method for the creation of a suspended vascular network. These will be cultured with or without active osteo-immunomodulatory factors released by macrophages to increase bone regeneration.