Cell and Gene Therapy, Department of Biomedicine, Basel University Hospital and University of Basel, Hebelstrasse 20, 4031, Basel, Switzerland; Department of Plastic, Reconstructive, Aesthetic and Hand Surgery, Basel University Hospital, Petersgraben 4, 4031, Basel, Switzerland.
Cell and Gene Therapy, Department of Biomedicine, Basel University Hospital and University of Basel, Hebelstrasse 20, 4031, Basel, Switzerland.
Acta Biomater. 2022 Sep 1;149:111-125. doi: 10.1016/j.actbio.2022.07.014. Epub 2022 Jul 12.
Rapid vascularization of clinical-size bone grafts is an unsolved challenge in regenerative medicine. Vascular endothelial growth factor-A (VEGF) is the master regulator of angiogenesis. Its over-expression by genetically modified human osteoprogenitors has been previously evaluated to drive vascularization in osteogenic grafts, but has been observed to cause paradoxical bone loss through excessive osteoclast recruitment. However, during bone development angiogenesis and osteogenesis are physiologically coupled by VEGF expression. Here we investigated whether the mode of VEGF delivery may be a key to recapitulate its physiological function. VEGF activity requires binding to the extracellular matrix, and heterogeneous levels of expression lead to localized microenvironments of excessive dose. Therefore we hypothesized that a homogeneous distribution of matrix-associated factor in the microenvironment may enable efficient coupling of angiogenesis and bone formation. This was achieved by decorating fibrin matrices with a cross-linkable engineered version of VEGF (TG-VEGF) that is released only by enzymatic cleavage by invading cells. In ectopic grafts, both TG-VEGF and VEGF-expressing progenitors similarly improved vascularization within the first week, but efficient bone formation was possible only in the factor-decorated matrices, whereas heterogenous, cell-based VEGF expression caused significant bone loss. In critical-size orthotopic calvaria defects, TG-VEGF effectively improved early vascular invasion, osteoprogenitor survival and differentiation, as well as bone repair compared to both controls and VEGF-expressing progenitors. In conclusion, homogenous distribution of matrix-associated VEGF protein preserves the physiological coupling of angiogenesis and osteogenesis, providing an attractive and clinically applicable strategy to engineer vascularized bone. STATEMENT OF SIGNIFICANCE: The therapeutic regeneration of vascularized bone is an unsolved challenge in regenerative medicine. Stimulation of blood vessel growth by over-expression of VEGF has been associated with paradoxical bone loss, whereas angiogenesis and osteogenesis are physiologically coupled by VEGF during development. Here we found that controlling the distribution of VEGF dose in an osteogenic graft is key to recapitulate its physiological function. In fact, homogeneous decoration of fibrin matrices with engineered VEGF could improve both vascularization and bone formation in orthotopic critical-size defects, dispensing with the need for combined osteogenic factor delivery. VEGF-decorated fibrin matrices provide a readily translatable platform for engineering a controlled microenvironment for bone regeneration.
临床大小的骨移植物的快速血管化是再生医学中未解决的挑战。血管内皮生长因子-A（VEGF）是血管生成的主要调节因子。先前已经评估过通过基因修饰的人成骨前体细胞过表达 VEGF 来驱动成骨移植物中的血管化，但已观察到通过过度破骨细胞募集引起矛盾的骨丢失。然而，在骨发育过程中，血管生成和成骨通过 VEGF 表达在生理上耦合。在这里，我们研究了 VEGF 传递方式是否是重现其生理功能的关键。VEGF 活性需要与细胞外基质结合，并且表达水平的异质性导致局部微环境中存在过量剂量。因此，我们假设在微环境中均匀分布基质相关因子可能能够有效地将血管生成和骨形成偶联起来。这是通过用可交联的工程化 VEGF（TG-VEGF）修饰纤维蛋白基质来实现的，该 VEGF 只能通过入侵细胞的酶切释放。在异位移植物中，TG-VEGF 和表达 VEGF 的祖细胞在第一周内同样改善了血管化，但只有在因子修饰的基质中才能实现有效的骨形成，而细胞基的 VEGF 表达则导致明显的骨丢失。在关键大小的原位颅骨缺损中，与对照和表达 VEGF 的祖细胞相比，TG-VEGF 有效地改善了早期血管侵入、成骨前体细胞的存活和分化以及骨修复。总之，基质相关 VEGF 蛋白的均匀分布保留了血管生成和骨生成的生理偶联，为工程化血管化骨提供了一种有吸引力且具有临床应用前景的策略。
