In this study, we examined the association between FPR1 expression and bone regeneration through a series of in vitro and in vivo experiments. Using FPR1 WT and KO models, including primary BMSCs as well as animals, we characterized FPR1’s impact on osteogenic differentiation, bone mechanical strength, and regenerative capacity of fractured bone. When BMSCs were induced osteogenesis in vitro, FPR1’s expression is associated with improved expression of osteogenic markers and mineralization (Figs. 1 & 2). It was also found that, compared to FPR1 KO mice, bones from WT mice exhibit significantly better biomechanical properties, bone density, volume, and cortical thickness (Figs. 3 & 4). In the femur fracture model, WT was associated with more dynamic bone healing compared to FPR1 KO mice (Figs. 5–7).
As discussed previously, formyl peptide receptor is a large group of receptors that has received a lot of research interest. Receptors within this family has been implicated in a wide range of settings, most notably as part of the innate immune system and the inflammation response (Rot et al. 1987) (Raoof et al. 2010). FPR1 specifically have also been explored for its role in bone metabolism. FPR1 has been implicated within the pathogenesis of osteoarthritis through its role in immune cell migration (Yang et al. 2021) (Gjertsson et al. 2012) (Yang et al. 2016). Other orthopedic diseases such as degenerative disc disease and rheumatoid arthritis also involved the FPR family (Duvvuri et al. 2021) (Xiao et al. 2019). Viswanathan et al in 2007 demonstrated that human mesenchymal stem cells (hMSCs) exhibited FPR1 and FPR2 expression in both mRNA and protein, which were involved in the chemotactic migration of these stem cells (Viswanathan et al. 2007). This is further supported by Kim et al who showed that ANXA1, a FPR ligand, induced hMSCs migration (Kim et al. 2007). Similar conclusions were drawn by the Gao group, who used corticosteroid, an agent that can upregulate FPR1 expression, to mobilize hMSCs (Gao et al. 2021). Given that human stem cells have been shown to possess the capacity to differentiate to different bone cell lines (Dominici et al. 2006), the ability to direct migration of these cells to specific areas of interest confer significant therapeutic possibilities.
FPR1’s chemotactic properties work synergistically with its ability to induce osteogenic differentiation in human mesenchymal stem cells. Shin et al noted that FPR1 signaling was significantly associated with differentiation into osteoblasts of hMSCs (Shin et al. 2011). The authors also used zebrafish and rabbit models to demonstrate that stimulation of the FPR1 receptor led to grossly and histologically more notable osteogenesis in vivo. The proposed mechanism of osteogenesis is through the FPR1/phospholipase C/phospholipase D-Ca2+-calmodulin-dependent kinase II-ERK-CREB signaling pathway. A previous work in our lab (Xiao et al. 2021) further expanded on Shin’s work by showing that following osteogenic induction of human adipose-derived MSCs, at day 7, FPR1 expression was increased in tandem with a variety of osteogenic markers as well as mineralization. We also found that without FPR1 signaling, mouse adipose-derived MSCs demonstrated decreased osteogenic differentiation and increased adipogenic tendencies. Furthermore, a critical transcription factor, FoxO1, was proposed as an intermediary in the signaling mechanism of FPR1, linking it to the eventual commitment to either osteoblasts or adipocytes. Building upon existing literature, we further illustrate FPR1’s association with osteogenic differentiation of BMSCs and how lack of FPR1’s expression causes weakened BMSCs’ capacity to make bone. We provided quantitative analysis of the impact of FPR1’s expression on in vivo bone formation and biomechanical strength (Figs. 3 & 4). It is clear that the knockout of FPR1 led to decreased quality of bone in mice.
As noted earlier, bone healing is a complicated process requiring mobilization of bone marrow mesenchymal stem cells, which is the motivation for our development of the FPR1 KO mice fracture model. The different stages of bone healing requires involvement of different types of cells including osteoblasts, osteoclast, and chondrocytes (Bahney et al. 2019). Our FPR1 KO mice model clearly show that without expression of FPR1, the recruitment of these cell types, likely from bone marrow stem cells, is delayed. At day 7 following surgery, the knockout mice showed only fibrous tissue in the bone callus at the site of fracture (Fig. 5). At day 35 following surgery, Xray showed that bone healing in FPR1 KO mice was at least delayed, maybe even showing signs of nonunion (Fig. 6). MicroCT confirmed the inferior quality of the newly formed bone in knockout mice (Fig. 7). Based on our knowledge of the existing literature, we are the first group to attempt using FPR1 KO mice to study the role of FPR1 in fracture healing, which is likely to be pivotal.
While our research is centered around FPR1, it would be remiss to not discuss the potential roles of FPR2 and FPR3. Similar to FPR1, FPR2 and FPR3 are expressed in a variety of immune cells (Murphy et al. 1992) (Nagaya et al. 2017). Within the rheumatoid arthritis literature, FPR2 has been recognized for its likely anti-inflammatory and anti-osteolytic properties through decreased production of IL-6 and osteoclast differentiation (Kao et al. 2014). FPR2 has also been observed to have a significant role in angiogenesis and promotion of fracture healing through increased exosome secretion and activation of M2 macrophages (Zhao et al.2021) (Corliss et al. 2016) (Heo et al. 2017) (Stegen et al. 2015). FPR3 is notable for its chemotaxis of monocytes and monocyte-derived dendritic cells as well as its presence in multiple key innate immune processes (Migeotte et al.2005) (Devosse et al.2009).
Based on current understanding, FPR1 has been found to have the ability to influence the migration and enhance osteogenic differentiation of human mesenchymal stem cells. This is a powerful synergistic combination with great potentials to be translated therapeutic tools to support stem cells therapy specifically for orthopedic disorders. As such, further characterization of FPR1’s mechanisms should be explored, specifically the signaling pathways leading to osteogenesis and increased bone quality. Furthermore, according to our fracture model, FPR1 is likely to play a significant role in the support of fracture healing, which a new avenue to address fracture nonunion. As such, more research could be extended to bone healing in an animal model with critical size bone defects under different conditions of normal, enhanced or impaired FPR1 expression. Use of FPR1 agonists and antagonists in such studies would lead to discovery of new pharmaceutical candidates for large-volume tissue reconstruction in clinic (Shin et al. 2011). Additionally, FPR2 has been noted for its contribution to fracture healing, and therefore should be explored in tandem with FPR1. Since these two receptors are believed to influence difference aspects of healing, it is possible that they might have synergistic effects. ANXA1, for instance, is a ligand previously described as capable of activating both FPR1 and FPR2 (Kim et al. 2007), which should be looked into for this purpose. In all these possible research directions, we advocate for the use of FPR1 KO mice models, which provides an opportunity to definitively describe the downstream effects of FPR1.