Implication of CXCR2-Src-Map4k4-p38 MAPK Axis in the Angiogenic and Osteogenic Effects of Functional Proteins-Based Tissue-Engineered Bones
Authors: Xing Junchao , He Sihao , Hou Tianyong , Zhou Jiangling , Yuan Bo , Cai Juan , Luo Fei , Xu Jianzhong 
Published: December 2023

Background The application of tissue-engineered bones (TEBs) is hindered by technical and logistical challenges associated with the incorporation of viable cells, despite their effectiveness in treating bone defects. Previously, we sought to address this predicament by employing freeze-drying techniques on TEBs to devitalize mesenchymal stem cells (MSCs) while preserving the majority of functional proteins, yielding functional proteins-based TEBs (FP-TEBs). In this study, we aimed to elaborate on the in-vivo angiogenic and osteogenic capabilities of FP-TEBs and elucidate the underlying mechanisms.
Methods Utilizing mouse models featuring critical femoral bone defects, the infiltration of endothelial cells (ECs), especially Type-H ECs (CD31hiEmcnhi), and the newly formed vasculature and bones were compared among FP-TEBs, TEBs and blank scaffolds. Then, quantitative PCR (qPCR) arrays were employed to evaluate the potentially implicated chemokines and receptors governing EC migration within implants. Identified C-X-C chemokine receptors (CXCRs) were further substantiated using short hairpin RNAs (shRNAs), and the pivotal role of CXCR2 was validated via conditional knockout mice. Finally, signaling molecules downstream of CXCR2 were screened and identified using signal inhibitors and shRNAs, concerning angiogenesis and osteogenesis.
Results FP-TEBs exhibited a considerable capacity to attract ECs, thereby accelerating vascularization and bone regeneration. This effect was analogous to that observed with TEBs but significantly greater than that seen with demineralized bone matrix (DBM). Furthermore, compared to TEBs, FP-TEBs exhibited enhanced recruitment of Type-H ECs, which played a pivotal role in orchestrating angio-osteogenesis coupling during the process of bone regeneration. qPCR arrays implicated CXCR1 and CXCR2 as candidates responsible for FP-TEBs-induced EC migration. In-vitro experiments revealed that the blockade of CXCR2, but not CXCR1, abolished EC migration and tube formation induced by FP-TEBs. Then, cxcr2tek mice, in which cxcr2 was conditionally knocked out in ECs, were generated. This manipulation led to a marked impairment in the presence of Type-H ECs within FP-TEBs, as well as diminished new vessel and bone formation. Additionally, steroid receptor coactivator (Src), Mitogen-activated protein kinase kinase kinase kinase-4 (MAP4K4) and p38 mitogen-activated protein kinase (p38 MAPK) were identified indispensable for CXCR2 function, both in vitro and in vivo. Further investigations revealed that the regulation of p38 MAPK phosphorylation by Src is mediated by MAP4K4.
Conclusion In conclusion, we demonstrated that FP-TEBs promoted EC migration, angiogenesis and osteogenesis via the CXCR2-Src-Map4k4-p38 MAPK axis. These findings unveiled the functional mechanism of FP-TEBs and provided valuable insights for the development of homing-based strategies in tissue regeneration.