Biomechanical Effects of Mechanical Stress on Cells Involved in Fracture Healing

Abstract

Fracture healing is a complex staged repair process in which the mechanical environment plays a key role. Bone tissue is very sensitive to mechanical stress stimuli, and the literature suggests that appropriate stress can promote fracture healing by altering cellular function. However, fracture healing is a coupled process involving multiple cell types that balance and limit each other to ensure proper fracture healing. The main cells that function during different stages of fracture healing are different, and the types and molecular mechanisms of stress required are also different. Most previous studies have used a single mechanical stimulus on individual mechanosensitive cells, and there is no relatively uniform standard for the size and frequency of the mechanical stress. Analyzing the mechanisms underlying the effects of mechanical stimulation on the metabolic regulation of signaling pathways in cells such as in bone marrow mesenchymal stem cells (BMSCs), osteoblasts, chondrocytes, and osteoclasts is currently a challenging research hotspot. Grasping how stress affects the function of different cells at the molecular biology level can contribute to the refined management of fracture healing. Therefore, in this review, we summarize the relevant literature and describe the effects of mechanical stress on cells associated with fracture healing, and their possible signaling pathways, for the treatment of fractures and the further development of regenerative medicine.

Fracture healing requires multiple cells to participate in the repair process, such as in bone marrow mesenchymal stem cells (BMSCs), chondrocytes, osteoblasts, and osteoclasts. Under the stimulation of mechanical stress, various cells coordinate with each other to jointly complete the reconstruction of bone tissue.

Introduction

Bone tissue, a dynamic skeletal tissue with the capability for adaptation and regeneration, initiates a sophisticated repair mechanism following injury. This process, known as fracture healing, involves the generation of new bone tissue at the site of the fracture and the reconstruction of the bone's structure. ¹ , ² Predominantly, long bone fractures undergo healing through a synthesis of intramembranous and endochondral osteogenesis. ³ This process necessitates the involvement of a diverse array of cells, including bone marrow mesenchymal stem cells (BMSCs), chondrocytes, osteoblasts, and osteoclasts. ⁴ , ⁵ Influenced by mechanical stresses, these cells perform distinct roles at varying stages of fracture healing, collaboratively working towards the reconstitution of bone tissue (Figure 1). Historically, as early as 1892, Wolff discovered the high sensitivity of bone to mechanical influences, asserting that mechanical forces are crucial in regulating the fracture healing process. ⁶ , ⁷ Throughout this process, the callus tissue adeptly perceives and reacts to the local mechanical environment. ⁸ These biomechanical cues are relayed to the cells, modulating gene expression and consequently altering the fracture microenvironment, which is pivotal in guiding the regeneration of bone tissue.

FIGURE 1.

Fracture healing requires multiple cells to participate in the repair process, such as BMSCs, chondrocytes, osteoblasts, and osteoclasts. Under the stimulation of mechanical stress, various cells coordinate with each other to jointly complete the reconstruction of bone tissue.

During various stages of fracture healing, the predominant cell types within the callus tissue differ. It is critically important to comprehend accurately the physiological requirements of these cells, especially in relation to the intensity, duration, and frequency of mechanical stress stimuli, to optimize the fracture microenvironment and enhance fracture healing. Investigating the impact of mechanical stimulation on cellular signaling pathways, which regulate metabolism in BMSCs, osteoblasts, chondrocytes, and osteoclasts, poses a significant research challenge. Historically, studies have predominantly employed a singular mechanical stimulus on individual mechanosensitive cells, without a standardized approach to the magnitude and frequency of applied mechanical stress. This article elucidates how diverse mechanical signals influence the functions of various cells during fracture healing, thereby modulating the healing trajectory. The objective of this review is to offer more precise and scientifically grounded treatment strategies for fracture therapy, and to establish a foundation for interdisciplinary collaboration in the fields of biology, tissue engineering, and regenerative medicine.

Methods

Description of searching method: (i) searching platforms: web of science, Pubmed, Google scholar;

Databases: Embase, Medline; (ii) key words: mechanical stress, fracture healing, bone marrow mesenchymal stem cells, osteoblasts, chondrocytes, osteoclasts; mechanosensors; (iii) retrieving time: 2000–2023; and (iv) excluded based on abstracts: repeat studies, unable to obtain full text, no specific stress loading method provided, papers in languages other than English (Figure 2).

FIGURE 2.

The criteria and process of literature screening flowchart.

Effect of Mechanical Strain on Cells

Recruitment of BMSCs

The recruitment of enough bone marrow mesenchymal stem cells (BMSCs) at the beginning of a fracture is crucial for healing. When a fracture happens, the immune system activates, leading to an inflammatory phase. During this phase, macrophages near the fracture release cytokines, encouraging BMSCs to move to the injury site, aiding in tissue healing and repair. The SDF‐1/CXCR4 axis is vital in guiding these stem cells to their target. ⁹ , ¹⁰ Studies by Kitaori et al. ¹¹ showed that mice lacking SDF‐1 or CXCR4 had less BMSC migration and slower bone regeneration. Further, in vivo research ¹² linked mechanical stress to BMSC movement, emphasizing the SDF‐1/CXCR4 axis's role. Wei et al. ¹³ , ¹⁴ found that mechanical stimulation enhances BMSC migration, which is usually weak in normal cultures. This stimulation works through the SDF‐1/CXCR4 signaling pathway. Zhang et al. ¹⁵ discovered that cyclic stretching (10%, 8 h) increases BMSC migration via the FAK‐ERK1/2 pathway. Additionally, Yuan et al. ¹⁶ showed that shear stress (0.2 Pa) increases SDF‐1 secretion, activating CXCR4 in human MSCs through JNK and p38 MAPK. BMSCs also express metalloproteinases (MMPs) and tissue inhibitors (TIMPs) vital for cell movement. Reis et al. ¹⁷ found that reducing MMP and TIMP genes in BMSCs hindered their movement across the extracellular matrix. Kasper et al. ¹⁸ observed increased MMP and TIMP levels in BMSCs after mechanical stimulation. Moreover, low‐intensity pulsed ultrasound (LIPUS) at an intensity of 50 mW/cm² can boost BMSC migration by activating autophagy, but autophagy inhibitors can reduce this effect. ¹⁹ In conclusion, mechanical forces help heal fractures by moving stem cells to the injury, with various pathways, including the SDF‐1/CXCR4 axis, playing a role.

Osteogenesis Differentiation of BMSCs

After the hematoma and inflammation subside, BMSCs around the periosteum near the fracture site proliferate and differentiate into osteoblasts. They secrete extracellular matrix, mineralize, and repair the fracture through intramembranous osteogenesis. The osteogenic differentiation of BMSCs is regulated by transcription factors Osterix (OSX) and runt related transcription factor 2 (Runx2). As BMSCs mature into osteoblasts, they express collagen type I (Col‐I) and alkaline phosphatase (ALP), followed by osteocalcin (OCN) and osteopontin (OPN) secretion and mineralization. Mechanical stress plays a crucial role in this process. Lu et al. ²⁰ reported that mechanical signaling enhances BMSC osteogenic differentiation, marked by increased OSX and Runx2 expression. Bone tissue exposed to mechanical forces generates fluid shear stress (FSS) on the cell surface. ²¹ Yourek et al. ²² found that FSS promotes the BMSC osteogenic phenotype and increases BMP2 and OPN mRNA expression. BMSCs in three‐dimensional structures respond more to mechanical stimuli than in two‐dimensional ones. Vetsch et al. ²³ showed that high flow rate (0.061 m/s) and high shear force (0.55–24 MPa) in 3D culture scaffolds significantly boost osteogenic differentiation. However, excessive shear stress may lead to BMSC apoptosis. ²⁴ Liu et al. ²⁵ observed that intermittent FSS more effectively induces osteogenic gene expression (ALP, Runx2, Col‐I) and activates ERK1/2 and FAK, leading to osteogenic differentiation. During fracture healing, cyclic tensile stress stimulates periosteal stem cells. ²⁶ Kearney et al. ²⁷ found that cyclic tensile strain of 2.5% and 0.17 Hz leads to high and time‐dependent expression of osteogenic markers (Runx2, Col‐I, OCN) in BMSCs. Similarly, Wu et al. ²⁸ noted that 10% cyclic tension stress enhances osteogenic gene expression in BMSCs via the ERK1/2‐Runx2 pathway. Also, directed mechanical stresses, non‐directed stresses, like low‐amplitude, high‐frequency vibration, also enhance stem cells' osteogenic differentiation. This was demonstrated by Lu et al., ²⁹ who showed that such vibration notably increases the expression of osteogenic genes (Col‐I, ALP, OPN) via the p38 MAPK signaling pathway. These findings provide insights into how mechanical stress influences osteoblast differentiation in BMSCs, offering a theoretical foundation for stress regulation in intramembranous osteogenesis. ³⁰

Chondrocyte Differentiation of BMSCs

Most long bone fractures heal through a combination of intramembranous and endochondral osteogenesis. ³ In this process, fibrous vascular tissue at the fracture site becomes cartilage, and BMSCs differentiate into chondrocytes on fibrin scaffolds, leading to bone formation via endochondral ossification. This transformation of BMSCs into chondrocytes is influenced by mechanical factors. Heiner et al.'s research ³¹ using intramedullary nailing to alter the mechanical environment of fractures revealed higher expression of chondrocyte‐related genes with intramedullary fixation than with plate fixation. This finding indicates that mechanical stimulation plays a role in the chondrocyte differentiation of BMSCs. The main components of cartilage's extracellular matrix, Glycosaminoglycans (GAG) and collagen type II (Col‐II), are key indicators of cartilage differentiation. Sex Determining Region Y Box Protein 9 (Sox9) is vital for maintaining the chondrocyte phenotype and essential for cartilage formation. ³² It has been found that compressive stress forces have been shown to induce the expression of cartilage‐specific transcription factor Sox9 and the Col‐II in BMSCs. ³³ In addition, it has been reported that the higher the magnitude of compressive loading, the higher the chondrogenic potential of BMSCs. For example, Miyanishi et al. ³⁴ observed although 0.1 MPa compressive stress can increase the expression of Sox9, the application of 10 MPa compressive stress in order to significantly up‐regulate the expression of Col‐II. Angele et al. ³⁵ demonstrated that cyclic hydrostatic stimulation (1 Hz, 0.55 MPa −5.03 MPa) in chondrocyte culture medium significantly enhances GAG and Col‐II content compared with controls. Lu et al. ³⁶ discovered that fluid shear stress (FSS) rate influences BMSC differentiation; a faster flow rate favors chondrogenesis, whereas a slower rate supports osteogenesis. Schätti et al. ³⁷ observed that shear stress combined with dynamic compression enhances cartilage gene expression more than simple stress, highlighting the effect of biaxial stress on BMSCs cartilage differentiation. In summary, bone marrow mesenchymal stem cells (BMSCs) are sensitive to mechanical stress, which significantly influences their differentiation into osteoblasts or chondrocytes. The type and intensity of the stress are crucial in determining the direction of this differentiation. ²⁶ Specifically, tension and high‐level shear stress tend to drive stem cell differentiation towards osteoblasts, whereas compressive and low‐level shear stress favor chondrocyte differentiation.

Effects of Stress on Osteoblasts

Osteoblasts play a crucial role in callus formation during fracture healing. As healing progresses, periosteal osteoblasts proliferate, synthesizing and secreting bone matrix. The callus extends towards the fracture end, eventually forming a spindle shape. Osteoblasts within the cartilaginous callus continue to differentiate and replace chondrocytes, leading to woven bone formation through mineralization, which strengthens the new bone. ³⁸ Markers such as alkaline phosphatase (ALP), osteocalcin (OCN), and collagen type I (Col‐I) are indicative of osteoblast maturation during differentiation. Mechanical stimulation in vivo has been shown to increase prostaglandin production, ALP, and Col‐I, fostering an environment conducive to osteoblastic proliferation and mineralization. ³⁹ Later, Woodcock et al. ⁴⁰ found that osteogenic capacity was positively correlated with the magnitude of the force, that is, higher levels of stress had a more significant effect on osteoblast maturation and differentiation. A current study ⁴¹ found that the modes of mechanical stimulation of isolated osteoblasts can be categorized into several types, and these different modes of mechanical stress stimulation affect the proliferation and differentiation of osteoblasts. A study by Guo et al. ⁴² demonstrated that under mechanical tensile strain (0.5 Hz, 2500 με), osteoblasts showed increased mRNA levels of ALP, Col‐I, and OCN. Additionally, in shear stress studies, osteoblasts in a 3D culture model exhibited higher proliferation rates and ALP activity under oscillatory fluid shear compared with static culture or unidirectional fluid shear. ⁴³ Wang et al. ⁴⁴ discovered that fluid shear stress (FSS) may down‐regulate miR‐5‐5p, promoting osteoblast proliferation through the VEGFA/ERK5 signaling pathway. However, only appropriate levels of mechanical stress promote osteoblast proliferation and differentiation, while excessive stress may lead to apoptosis. ⁴⁵ , ⁴⁶ Tripuwabhrut et al. ⁴⁷ noted enhanced osteoblast differentiation with compressive stress ranging from 2.0 to 4.0 g/cm². Conversely, Goga et al. reported that compressive forces exceeding 2.04 × 10⁻⁴ N/cm² could increase apoptotic cell numbers, indicating that such levels are excessively high in vitro. ⁴⁸ Li et al. ⁴⁹ combined mechanical and electrical stimulation, enhancing osteoblast proliferation and differentiation. It has been observed that mechanical stress stimulation can enhance BMP expression in osteoblasts, which regulate the activation of bone formation‐related genes through BMPs/Smad signaling pathways. ⁵⁰

Effects of Stress on Chondrocytes

The transformation of cartilage into hard bone scabs is a crucial step in fracture repair and healing, characterized mainly by the replacement of chondrocytes with woven bone. Initially, chondrocytes undergo hypertrophy and differentiation, followed by extensive, orderly apoptosis, and are eventually replaced by osteoblasts to complete fracture repair. MMPs (MMP9, MMP13), along with collagen type X (Col‐X) can be expressed as hallmarks of chondrocyte hypertrophy. Wong et al. ⁵¹ observed that cyclic tensile stress induced Col‐X in chondrocytes, accelerating hypertrophy via the RUNX2/MMP13 pathway and upregulating MMP13. Studies have indicated that cartilage phenotype is maintained under compressive loads of 5% to 10%, but hypertrophy markers are highly expressed when the load increases to 15%–20%. ⁵² , ⁵³ Carter and Wong ⁵⁴ showed that low fluid shear and high hydrostatic pressure inhibit cartilage hypertrophy, whereas high fluid shear stress (FSS) promotes it, potentially leading to apoptosis. High FSS is known to activate the Wnt signaling pathway, promoting cartilage hypertrophy in chondrocytes. ⁵⁵ , ⁵⁶ Ren et al. ⁵⁷ applied a shear stress of 16 dyn/cm² to chondrocytes, noting decreased mitochondrial membrane potential and changes in mRNA expression levels of apoptotic markers. The previous view was that the hypertrophy and death was the ultimate fate of terminally differentiated chondrocytes. However, some recent studies ⁵⁸ , ⁵⁹ have demonstrated through genetic genealogy tracing experiments that chondrocytes remain active and develop osteoblastic properties inside the cartilage callus during fracture repair and proposed that chondrocytes can change directly into osteoblasts to complete the fracture healing, a phenomenon known as transdifferentiation of chondrocytes. ⁵⁹ This involves downregulation of chondrocyte markers (e.g., COL‐II, SOX‐9) and upregulation of osteoblast markers (Runx2, OSX). ³² Wong et al. ⁶⁰ observed the expression of cartilage‐specific genes in osteoblasts and bone lining cells within new bone in an unstable fracture model. This finding indicates that these cells originated from chondrocytes, suggesting that the mechanical stimulation from an unstable fracture prompted their transformation into osteoblasts. It has been found that mechanotransduction signals can act through Piezo1/2 in chondrocytes, resulting in promoted endochondral ossification. ⁶¹ , ⁶²

Effects of Stress on Osteoclasts

Callus remodeling, often regarded as the final stage of fracture repair, involves the degradation of initially formed woven bone and its replacement with mature lamellar bone. Towards the end of the healing process, osteoclasts are activated; inhibiting them during the remodeling phase leads to increased callus density. ⁶³ Differentiated osteoclasts, attaching to bone surfaces, express biomarkers such as translocon‐associated protein (TRAP), MMP‐9, and Cathepsin K (CTSK), facilitating their polarization and maturation. The RANK/RANKL pathway is essential for the differentiation of precursors into mature osteoclasts. Recent findings indicate that osteoclasts are mechanosensitive. ⁶⁴ , ⁶⁵ For instance, Ma et al. ⁶⁶ demonstrated that fluid shear at 12 dyn/cm² inhibits osteoclast differentiation and reduces CTSK, MMP‐9, and TRAP levels. Inhibiting ERK5 with XMD5‐8 disrupts this process, showing that fluid shear stress (FSS) inhibits osteoclast‐related proteins by activating ERK5. Rubin et al. ⁶⁷ applied a 5% intensity cyclic distraction force to osteoclasts and observed a down‐regulation of RANKL gene expression, reducing osteoclast differentiation. During endochondral ossification, thickening chondrocytes attract osteoclasts to the cartilage callus; these cells then absorb mineralized cartilage, allowing osteoblasts to reshape it into immature woven bone. Complete inhibition of osteoclast activity can impair this resorption process, negatively impacting fracture healing. Xiao et al. ⁶⁸ showed that silencing the PDK1 gene in osteoclasts reduces their activity, hindering the resorption of cartilage and the remodeling of immature woven bone. Muschter et al. ⁶⁹ found that applying a 10%, 1Hz cyclic tensile stress to mouse RAW264.7 cells upregulated RANK mRNA expression, enhancing osteoclast differentiation. These results suggest that mechanical stress modulates osteoclast differentiation, aiding in bone scab formation. YAP and TAZ, effectors of the Hippo signaling pathway, mediate physical signals in the extracellular environment, regulating a range of biological signals in response to mechanical stress. ⁶⁵ In conclusion, osteoclast activity varies at different stages of fracture healing, necessitating flexible regulation for dynamic fracture management.

Mechanobiology: Mechanosensation and Mechanotransduction

Mechanotransduction is the process by which a physical force is exerted on a cell in its natural environment, which is subsequently converted into biochemical and electrical signals that produce a cellular response. ⁷⁰ Mechanical signaling stress can affect intracellular signaling in the nucleus to alter protein activity and gene expression. ⁷¹ Various mechanosensors have been proposed by which stem cells can sense the mechanical environment (Figure 3).

FIGURE 3.

Schematic representation of mechanosensation and mechanotransduction (by Figdraw). (A) Macro view of cellular mechanosensors. (B) Microscopic view of cellular mechanosensors.

Extracellular Matrix (ECM)

The ECM consists of fibers, proteoglycans (PGs), and glycoproteins. The morphology, viscosity, and mechanical properties of the ECM are largely dependent on the number, type, and arrangement of these macromolecules. By applying mechanical stress to the cell, the cell can secrete structural components of the ECM and matrix metalloproteinases and in these ways alter the ECM composition and remodel the structure to adapt to mechanical stimuli. ⁷² Mechanical signaling may change cell shape and ECM composition, thereby creating a favorable ecological environment to induce mechanotransduction signals and influence cellular functions. ⁷³ Previous studies have shown that the composition of the ECM alters the biochemical and physical properties of cells, thereby determining cell fate. ⁷⁴ Basement membranes and connective tissues influence the morphology, viscosity, and mechanical properties of the ECM through elastic fibers, protofibrillar collagen, GAG, and associated PG, while collagen and elastin provide tensile strength. ⁷⁵ When the composition of the ECM changes, it may assume a geometrical shape providing topography and mechanical stimulation, both of which are necessary to alter cellular phenotype. Mechanical stimulation and ECM composition interact with each other and together are important factors affecting cell function. ⁷⁶

Ion Channels

Appropriate mechanical stimulation activates calcium channels on the cell membrane, facilitating the transport of extracellular calcium into the cell, increasing intracellular calcium concentration and promoting increased bone mass. Calcium acts as a second messenger to initiate the activation of many mechanically regulated signaling cascade responses, including ATP and nitric oxide (NO). Increased intracellular calcium stimulates the release of prostaglandin 2 (PGE2), a key anabolic regulator of bone formation and one of the key paracrine signaling agents. PGE2 is released from connexins in the cell membrane and communicates with other osteoblasts as an intercellular messenger through gap junctions. PGE2 has been shown to not only increase bone sensitivity to external loads in a positive feedback manner after release, but also to recruit bone after release. It acts in a positive feedback manner after release, but also recruits and stimulates precursor cells to differentiate into bone tissue. ⁷⁷ Piezo1 and Piezo2 were found to be important mechanosensitive channels through which osteoblasts sense and respond to changes in mechanical loading, and their deficiency in osteoblasts promotes bone resorption and leads to osteoporosis in mice. ⁷⁸ , ⁷⁹

Primary Cilia

The primary cilium is a single, immobile, antenna‐like structure that extends from the cell into the extracellular space. ⁸⁰ Primary cilia of degenerate structures have emerged as important signaling centers and have been shown to be important for mechanosensation in a variety of cell types. In cells, primary cilia act as mechanosensors in response to the flow of extracellular fluid generated during walking and running. When primary cilia bend, the increased tension in the membrane opens mechanosensitive ion channels, which leads to intracellular Ca2⁺ influx, membrane depolarization, and activation of nerve fibers, which then transmit stresses into the cell. ⁸¹ It has been shown ⁸² that fluid flow‐induced paracrine signaling in bone tissue may also be primary cilia‐dependent. Osteocytes were found to release a certain growth factor upon stimulation by fluid stress, which upregulates OPN gene expression in MSC cells. However, when the formation of primary cilia in osteoblast‐like cells is inhibited, the growth factor will not be released. ⁸² However, the current study could not fully explain the specific mechanism of primary cilia action under mechanical stress and needs to be further elucidated in future studies.

Cytoskeleton

The cytoskeleton links the nucleus to the ECM and thus can transmit mechanical stimuli from the extracellular environment ⁸³ The cytoskeletal structure is highly nonlinear, allowing the cell to sense deformations and changes, and the intact cytoskeleton helps maintain tight adhesion between the cell and the extracellular matrix (ECM). ⁸⁴ The cytoskeleton is composed of protein fibers, mainly microtubules, actin fibers, and intermediate filaments. The cytoskeleton consists mainly of microtubules, actin fibers and intermediate filaments. ⁸⁴ Myosin interacts with actin to generate cytoskeletal tension, which plays an important role in maintaining cellular morphology, withstanding external forces and maintaining the internal structure of the cell. ⁸⁵ Cells alter their internal cytoskeleton in response to external mechanical stimulus stresses through actin polymerization or microtubule assembly, and this reorganization activates a variety of intracellular signaling pathways that control cell migration, proliferation, and gene expression on tissue growth and function.

Integrin

Integrins are transmembrane proteins that bind ECM proteins (ligands) to the cytoskeleton, but also facilitate interactions with other cells and act as signaling receptors. ⁸⁶ Signaling between the inside and outside of the cell occurs through a key bidirectional mechanotransduction pathway called the ligand‐integrin‐cytoskeleton junction. Integrins undergo conformational changes by binding to ligands (ECM proteins) in the extracellular region to transmit physical signals into the cell to induce adhesion, migration, proliferation and differentiation. Moreover, changes in intracellular signals also affect conformational changes of integrins, altering the affinity for binding to ECM proteins and influencing the biological behavior of cells. This integrin‐dependent bidirectional signaling mechanism plays an important role in bone remodeling. Recent studies have shown that osteoclasts express integrins α2 and αV and play an important role in bone resorption. ⁸⁷ , ⁸⁸ Follow‐up studies should focus on further elucidating the important role of integrins in mechanical stimulation and bone metabolism.

Focal Adhesion Kinase (FAK)

Focal adhesion kinase (FAK) is one of the first molecules recruited for the development of focal adhesion in response to external mechanical stimuli. In response to external mechanical stimuli, FAK undergoes autophosphorylation, which in turn transduces downstream mechanotransduction by activating mechanical transducers in the cytoplasm. ⁸⁹ Phosphorylation of FAK, therefore, can be enhanced by the perception of exogenous stress. ⁹⁰ The interaction between FAK and the contractile cytoskeletal network is tightly controlled in the cell to maintain tension at critical cellular sites and regulate force transmission to the nucleus. ⁹¹ For example, during processes that require cell polarization and nuclear deformation (e.g., directed migration), FAK activation occurs at specific sites, facilitating local reorganization of the cytoskeleton and nucleus extrusion. ⁹²

Discussion

In summary, the repair of fractures requires coordination of biological signal transmission and gene function between multiple cell types. This process is influenced by many local factors, including inflammatory factors at the fracture end, blood supply, degree of injury, condition of soft issue, and mechanical stimulation, which constitute the fracture microenvironment. Several cell types involved in fracture healing, such as BMSCs, osteoblasts, chondrocytes, and osteoclasts, are responsive to mechanical stimuli. The main responsible cells involved in fracture healing are different at different stages of fracture healing, and interestingly, the mechanical responsiveness of each cell shows different variations depending on the type of stress versus the magnitude of the stress (Table 1). Research over the past decade has greatly increased our understanding of bone cell biology. The activities of multiple signaling molecules within the cell are affected in the presence of mechanical factors. Mechanical signals, like other signals, are converted into biological signals in the cell that affect the cell's gene expression and thus play a role in cellular behavior. Stimulated by mechanical stresses, various cells work together to coordinate the entire fracture healing process.

TABLE 1.

Molecular biological effects of different types of mechanical stress stimuli on cells involved in fracture healing.

Type of stress	Stress conditions	Cell	Phenotype	Effect	Reference
Cyclic mechanical strain	1.0 Hz, 10% strain	Rat BMSCs.	Migration.	Phosphorylation of FAK and ERK1/2.	Zhang et al. 15
FSS	0.2 Pa	Human BMSCs.	Migration.	Phosphorylation of JNK and p38 MAPK.	Yuan et al 16
FSS	9 dyn/cm2	Human BMSCs.	Osteogenic differentiation.	Upregulating the expression of BMP‐2, BSP, and OPN.	Yourek et al. 22
FSS	4.2 dyn/cm2 twice a day	Human BMSCs.	Osteogenic differentiation.	Upregulating the expression of ALP, Runx2, and Col‐I and phosphorylation of ERK1/2 and FAK.	Liu et al. 25
Tensile mechanical strain	2.5% at 0.17 Hz for 1–14 day	Rat BMSCs.	Osteogenic differentiation.	Upregulating the expression of Runx‐2, col‐1, OCN, and BMP2.	Kearney et al. 27
Cyclic mechanical strain	10% at 0.5 Hz	Rat BMSCs.	Osteogenic differentiation.	Upregulating the expression of ALP, OCN, Col‐I Runx‐2 and phosphorylation of ERK1/2.	Wu et al. 28
Intermittent hydrostatic pressure	10 MPa, 1 Hz	Human BMSCs.	Chondrogenic differentiation.	Upregulating the expression of SOX9, Col‐II, and ACAN.	Miyanishi et al. 34
Intermittent hydrostatic pressure	1 Hz, 0.55 MPa −5.03 MPa	Human BMSCs.	Chondrogenic differentiation.	Upregulating the expression of GAG and Col‐II.	Angele et al. 35
FSS	12 dyn/cm2	MC 3T3‐E1.	Osteoblast proliferation.	Upregulating the expression of VEGFA and downregulation of miR‐140‐5p.	Wang et al. 44
Cyclic tension	0.004 MPa/9%	Bovine chondrocytes.	Differentiation hypertrophic of the chondrocyte.	Upregulating the expression of cbfa1, MMP‐13 and Col‐II.	Wong et al. 51
FSS	16 dyn/cm2	Rat chondrocytes.	Apoptosis of the chondrocyte.	Upregulating the expression of Bax, PARP1 while downregulation the expression of Bcl‐2.	Ren et al. 57
FSS	12 dyn/cm2, 30 min/day)	RAW264.7.	Inhibition of osteoclast differentiation.	Downregulation the expression of MMP‐7, CTSK, and TRAP and Phosphorylation with ERK5.	Ma et al. 66
Mechanical strain	1 Hz and an amplitude of 10% stretch	RAW264.7.	Promotion osteoclast differentiation.	Upregulating the expression of RANK and downregulation of SP.	Muschter et al. 69
Mechanical strain	2% at 10 cycles/min	Murine bone marrow‐derived macrophages.	Inhibition of osteoclast differentiation.	Downregulation the expression of ODF/TRANCE.	Rubin et al. 67

Although there is now a consensus on the role of stress and dynamization in promoting fracture healing, there is still controversy regarding the timing of fracture dynamization. This review of the literature summarizes the biological behaviors of cells associated with fracture healing during fracture healing in a mechanical environment. Mechanical stimuli are certainly required throughout the entire process of fracture healing, but the type of stress stimulation required may vary at different stages of fracture healing depending on the dominant cell. Further understanding of the effects of different types of stresses on various cells, in terms of magnitude, frequency, and mode of action, will allow us to take different measures to promote fracture healing at different times, to achieve more precise fracture management. It also provides new ideas for cellular repair research and the development of regenerative tissue engineering.

Author Contributions

All the authors have made appropriate contributions to this review. Weiyong Wu designed the study and wrote the manuscript. Lili Li and Yongqing Wang approved the article submission. Zhuihui Zhao revised the manuscript. Genbao Zhu, Kemeng Tan, and Meiyue Liu searched and collected the literature.

Conflict of Interest Statement

The authors declare that they do not have any commercial or associative interest that represents a conflict of interest in connection with the work submitted.

ETHICS statement

The study was approved by the Ethics Committee of the Anhui Wanbei Coal‐Electricity Group General Hospital (WBZY‐2023‐033).