The process of bone healing can be mainly divided into three stages. The first stage is the hematoma formation stage. After an acute bone injury, blood vessels from within the bone and surrounding soft tissues rupture, leading to local hematoma formation. At this time, the hematoma serves as a temporary scaffold, recruiting inflammatory cells such as macrophages and neutrophils. These inflammatory cells are activated and initiate a cascade reaction, secreting inflammatory cytokines such as IL-6 and CCL2, which play an early role in clearing damaged and inactive tissue. The second stage is the procallus formation stage. The release of various cytokines and immunogenic factors during the hematoma formation stage promotes the recruitment and migration of mesenchymal stem cells (MSCs) to the site, initiating the repair process. MSCs have the dual potential to differentiate into osteoblasts and chondrocytes. The procallus forms through intramembranous or endochondral ossification, with intramembranous ossification occurring earlier than endochondral ossification and overlapping with it, ultimately leading to bony bridging of the fracture gap. The third stage is the callus remodeling stage. Intramembranous ossification directly deposits mineralized bone through osteoblasts, while endochondral ossification involves cell differentiation, formation of a cartilaginous callus bridging the bone ends, mineralization and extension of immature callus, and coordinated activity between osteoblasts and osteoclasts to remodel the callus, replacing woven bone with mechanically stronger lamellar bone. Ultimately, the marrow cavity is reconnected, and the fracture site returns to its normal structure[2, 11, 12].
Our research has found that the transcriptional changes in local tissues at different time points of fracture have significant differences and correspond to the biological processes of different stages of fracture healing. Most of the top 10 DEGs (Fig. 3) at each time point show a significant increase in expression within 3 days of fracture, gradually reaching a peak and then slowly decreasing but still maintaining a high level of expression. This is consistent with the first type of genes identified by temporal dynamics, indicating that the molecular mechanisms involved in fracture healing begin early and run through the entire process. According to GO analysis (Fig. 4), only biological processes related to inflammation, such as lymphocyte activation and proliferation, adaptive immune response, leukocyte proliferation, leukocyte-mediated immunity, and monocyte proliferation, were significantly upregulated on the 3rd day, but not on the 7th day and later. According to KEGG analysis (Fig. 5), the AGE-RAGE signaling pathway was significantly enriched. There is evidence that advanced glycation end products (AGEs) are the products of excessive sugar and protein binding, which interact with the AGE receptor (RAGE) and activate downstream factor nuclear factor-kappaB (NF-κB) through the AGE-RAGE signaling pathway, thereby promoting the inflammatory response[13]. These findings suggest that the most prominent feature of early fracture healing is a highly inflammatory response.
On the 7th day, in most of the temporal dynamics analysis, the first type of genes reached peak expression, such as collagen synthesis-related genes col1a1, col3a1, col5a1. Collagen is the main component of the extracellular matrix, and type I collagen constitutes 90% of the total organic components of bone matrix. Its synthesis, deposition, and remodeling play an important role in the development, formation, and stability of bone tissue. Mutations in the human Col1a1 gene can lead to osteogenesis imperfecta[14]. This indicates that as bone fracture healing progresses, collagen synthesis continuously increases, ensuring the toughness and mechanical strength of the newly formed bone callus. Osteogenesis-related genes Mmp2, Timp1, and ptprv also reached peak expression. Matrix metalloproteinases (MMPs) are a group of structurally related secreted and membrane-bound proteins that participate in the degradation of extracellular matrix (ECM) and other structural components of extracellular and non-matrix proteins. MMP-2 deficiency can lead to impaired osteoblast differentiation. Evidence suggests that osteoblasts and bone cells express Mmp2 and Timp1 during osteogenesis. Osteoblasts and osteocytes degrade ECM molecules together with MMPs while producing them, and regulate degradation by inhibiting MMP activity through tissue inhibitors of metalloproteinases (TIMPs). They can also reorganize ECM components, allowing the bone matrix to mature during the process of intramembranous ossification[15]. The Ptprv gene belongs to the transmembrane protein tyrosine phosphatase gene family and is specifically expressed in the cartilage and periosteum of immature long bone necks, but is hardly expressed in mature osteoblasts[16]. It may be involved in the transformation process of skeletal components from cartilage templates to bone. Platelet activation was significantly enriched according to KEGG analysis. Platelet activation often occurs in low-oxygen-induced inflammatory reactions. The substances released from activated platelet particles, such as VEGF and PDGF, have the ability to promote angiogenesis and regulate vascular maturation and stability[17], which leads to an increase in the peripheral vascular bed and its growth into the healing tissue, helping to restore the decreased blood supply in the fracture area caused by vascular injury and accelerating fracture healing. According to GO analysis, biological processes related to bone development, connective tissue development, collagen fiber tissue, and ossification began to be significantly upregulated. At the same time, the content of cell components such as collagen trimer and extracellular matrix containing collagen protein significantly increased.
According to GO analysis on the 14th and 28th days, the process of cartilage generation was significantly upregulated. Cartilage cells differentiate from bone marrow mesenchymal stem cells and can produce a cartilage matrix composed of collagen and proteoglycans. At the same time, the process of bone formation was significantly accelerated, indicating that bone calluses were formed on the bone surface and in the gap of adjacent fractures through intramembranous ossification and endochondral ossification. Bone mineralization was only significantly upregulated on the 28th day. As cartilage cells differentiate, the extracellular matrix of cartilage undergoes mineralization, transforming immature primitive bone calluses into mature mineralized bone calluses. Evidence suggests that Pentraxin 3 (PTX3) plays a key role in the mineralization and deposition of bone matrix, and the genetic variation of the PTX3 gene is closely related to osteoporosis[18]. We found that the expression changes of PTX3 were not significant within 3 days but continued to increase after the 7th day, indicating that the process of bone mineralization mainly occurs in the middle and late stages of bone fracture healing. KEGG analysis showed that osteoclast differentiation was significantly enriched during this period. Osteoclasts originate from the recruitment of osteoclast precursors by bone cells expressing RANKL. The expression of RANKL and M-CSF in the bone marrow cavity initiates the differentiation of osteoclast precursors into osteoclasts[19]. As osteoclast activity gradually exceeds that of osteoblasts, completing the remolding of the bone callus.
In addition, we were amazed to discover that neural regulation plays an important role in the early stages of bone fracture healing. By analyzing the temporal dynamics of gene expression clusters and conducting functional enrichment on each cluster, we excluded clusters that did not yield significant results. The first class of enriched pathway mainly includes the Cytokine-cytokine receptor interaction pathway, EMC-report interaction pathway, Protein digestion and absorption, and the PI3K-Akt signaling pathway. The second class consists of Phagosome, Neutrophil extracellular trap formation, and the Renin-angiotensin system (RAS). Interestingly, the EMC-report interaction pathway and the PI3K-Akt signaling pathway in the first class are closely related to neural function. The interaction between extracellular matrix (ECM) and neurons is an important condition for axonal regeneration after injury, and it may play a regulatory role in the regeneration of peripheral or central sensory neurons after nerve injury[20, 21]. In addition, existing research indicates that the PI3K/Akt signaling pathway improves neuronal survival and regulates axonal growth, playing an important role in functional recovery after spinal cord injury[22, 23]. In the second class, the RAS can participate in and affect neural activity. The massive activation of the RAS system can increase the levels of renin, angiotensin (AT), angiotensin-converting enzyme (ACE), and aldosterone in circulation and neural tissue. The excessive increase of these components can lead to neuronal damage and degeneration[24, 25], and there is evidence that RAS inhibitors have a positive effect on spinal cord and nerve root functional activity in patients with degenerative lumbar diseases[26]. Furthermore, the KEGG results suggest that on 3rd day the Axon guidance pathway is significantly activated, and the IPA enrichment results related to neural function show that CDH1 and L1CAM exhibit the most significant expression changes. CDH1 plays a role in regulating axonal growth, and knocking down CDH1 can promote axonal growth and increase axon length, while L1CAM is of great significance for the growth, regeneration, development, and maintenance of the nervous system. It also has the ability to regulate axonal sprouting during neuronal regeneration and improve behavioral outcomes after CNS injury. In the GO-CC analysis results three days after the fracture, we found that glutamatergic synapses and gamma-aminobutyric acid (GABA) synapses are among the top components, with the former depolarizing the postsynaptic membrane and the latter hyperpolarizing it. These two are the most common excitatory and inhibitory neurotransmitters in the central nervous system[27, 28]. Therefore, we speculate that the local intraosseous nerve repair is mainly to regulate axon growth, and the central nervous system may play an important regulatory role in the early stages.
Through IPA (Fig. 6), we have found that most of the nerve-related pathways are in an inhibited state at the 3rd day time point after fracture, and gradually become activated over time, such as the CREB Signaling in Neurons pathway, which is closely related to various biological reactions including neuron excitation, neurogenesis, synaptic plasticity, etc. CREB must be phosphorylated to form pCREB before it can act as a transcriptional activator. Growth factor/receptor tyrosine kinase-induced pathways (Ras/Erk/RSK2) and stress or inflammatory cytokine (MAPK; PI3/Akt) pathways can all phosphorylate CREB. In our previous time kinetic results, the 3rd day after fracture was the key node for the changes in the PI3/Akt and Ras pathways. In addition, the significant IPA changes on the 7th day included FHL2 and ALP. The former can enhance CREB transcriptional activation activity, while the latter, in addition to being a sensitive indicator of osteoblast activity, can also serve as a marker of the inflammatory environment. Therefore, we speculate that the inflammatory stimulation during the bone healing process promotes CREB phosphorylation, activates CREB Signaling in Neurons, and exerts neuroregulatory effects. Furthermore, the Neuroinflammation Signaling Pathway gradually becomes activated during the bone healing process. As a key signaling pathway for maintaining central nervous system (CNS) homeostasis, its function is to destroy and clear damage factors and damaged nerve tissue. When this beneficial inflammatory response is not controlled, excessive damage to cells and tissues can lead to the destruction of normal tissue and chronic inflammation, ultimately leading to the death of glial cells and neurons. This process can be accelerated by multiple pro-inflammatory cytokines expressed by neurons in an inflammatory state, including neurotransmitters or modulators (glutamate, fractalkine, nitric oxide, GABA), and neurotoxic proteins. In summary, local inflammation during bone healing may have adverse effects on the central nervous system, and promote the regulatory role of the central nervous system during bone healing.
In previous studies, many transcriptional analyses of local tissue after bone fracture have been conducted[29, 30]. In these studies, the molecular mechanisms affecting fracture healing were explored through bioinformatics methods. In comparison, our study focuses more on summarizing the biological characteristics of each stage of fracture healing and concludes the characteristics of the changes in each stage of fracture healing, which is consistent with the biological process of fracture healing. Additionally, we found that the peripheral and central nervous systems may play an active regulatory role in the process of fracture healing, which has not been deeply explored in previous sequencing studies. This contributes to further elucidating the biological process of fracture healing and provides new ideas for clinical treatment.