Mitochondrial dysfunction under hyperglycemic conditions is the central source of reactive oxygen species (ROS) burst. In the diabetic milieu, excessive glucose influx into cells—particularly in insulin-independent tissues such as skin, nerves, and vascular endothelium—increases tricarboxylic acid (TCA) cycle flux, leading to an imbalance in the mitochondrial NADH/NAD⁺ ratio. This causes over-reduction of the electron transport chain and ultimately results in massive leakage of superoxide ( O 2 − ⋅ ). This mitochondria-derived ROS storm is regarded as the “unifying upstream event” that activates all downstream pathological pathways [6]. In DFU, the accumulation of intracellular ROS under hyperglycemia promotes the formation of advanced glycation end products (AGEs), activates the polyol pathway and protein kinase C (PKC) signaling, and simultaneously suppresses the activity of endogenous antioxidant enzymes and compounds [7]. Upon binding to their specific receptor RAGE (Receptor for AGEs), AGEs activate key pro-inflammatory signaling cascades such as nuclear factor-kappa B (NF-κB), leading to sustained release of inflammatory cytokines including tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6). This perpetuates a state of chronic inflammation at the wound site, preventing the orderly transition to the proliferative phase of healing [8].

The normal wound healing process requires a moderate inflammatory response to clear necrotic tissue and pathogens; however, wounds in diabetic patients often exhibit a chronic inflammatory state, leading to healing stagnation. This dysregulation of the inflammatory response is characterized by the overexpression of pro-inflammatory factors (e.g., IL-1β, TNF-α, IL-6) and insufficient anti-inflammatory factors [9]. During normal wound healing, macrophages transition from the M1 (pro-inflammatory) phenotype to the M2 (anti-inflammatory/repair) phenotype, promoting tissue repair [10]. However, in DFU, this balance between M1 and M2 macrophages is disrupted, with persistent M1 macrophages leading to chronic inflammation and impaired healing [10].

Prolonged hyperglycemia causes microvascular damage and neuropathy, resulting in foot sensory loss, reduced blood flow, and tissue ischemia. Sensory neuropathy is one of the core initiating factors of DFU, primarily due to long-term hyperglycemia-induced axonal degeneration and dysfunction, which reduces the ability to perceive foot pain and increases the risk of trauma [11]. During the development of DFU, the accumulation of AGEs occurs; AGEs activate the NF-κB pathway, leading to the release of pro-inflammatory factors that cause neuronal apoptosis [12]. Consequently, patients lose sensitivity to minor stimuli, and abnormal foot pressure distribution triggers skin breakdown [13]. Approximately 15% of diabetic patients develop DFU over their lifetime, with neuropathy being a critical precursor; as indicated by recent epidemiological data [12], reflecting the prevalence of diabetic neuropathy in ulcer development.

BMSCs are a class of adult stem cells characterized by self-renewal capacity, multipotent differentiation potential, and immunomodulatory functions. They have demonstrated tremendous potential in tissue regeneration and repair, particularly attracting significant attention in research on DFU treatment. BMSCs are primarily derived from the bone marrow stroma. Bone marrow constitutes a complex microenvironment containing hematopoietic stem cells, mesenchymal stem cells, adipocytes, osteoblasts, as well as various cytokines and extracellular matrix components [14]. BMSCs can be isolated from bone marrow using several methodologies. Common isolation techniques include: Whole bone marrow adherent culture method: This is one of the most widely used techniques. Bone marrow aspirates are diluted and centrifuged, and the mononuclear cell-containing layer is then plated onto culture dishes, where BMSCs selectively adhere to the plastic surface. Density gradient centrifugation method: A representative example is Ficoll density gradient centrifugation. Research has demonstrated that conventional culture under standard atmospheric oxygen tension (approximately 21% O₂) leads to rapid downregulation of CXCR4 expression in BMSCs [15]. This hyperoxic condition starkly contrasts with the physiological hypoxic microenvironment of the bone marrow niche, which typically maintains oxygen levels between 1% and 5% O₂. This native hypoxic milieu is critical for preserving BMSC stemness and homing capacity. Consequently, optimizing culture conditions to mimic the physiological hypoxia of the bone marrow has been shown to significantly upregulate CXCR4 expression and enhance the chemotactic migratory response of BMSCs toward stromal cell-derived factor-1α (SDF-1α) gradients. This enhanced migratory ability is essential for improving the therapeutic efficacy of BMSC-based interventions, as efficient homing to injury sites—mediated by the SDF-1α/CXCR4 axis—is a key determinant of successful tissue repair and regeneration.

One of the most prominent biological characteristics of BMSCs is their multipotent differentiation potential—under specific microenvironmental cues or inductive conditions, BMSCs can differentiate into multiple cell lineages, including osteoblasts, chondrocytes, and adipocytes. This property endows them with broad application prospects in tissue engineering and regenerative medicine [16]. This multipotency forms the foundation for BMSC-mediated repair of damaged tissues and organs. For instance, in DFU healing, BMSCs can promote neovascularization and tissue regeneration by differentiating into vascular endothelial cells and fibroblasts, thereby accelerating wound closure [17]. Beyond these classical trilineage differentiation capabilities, studies have also shown that under specific conditions, BMSCs possess the ability to differentiate into other cell types. For example, in a neuro-inductive environment, BMSCs can differentiate toward neural cells, suggesting their potential application in neural injury repair [16]. A deep understanding and precise control of BMSC differentiation fate are crucial for developing more effective and targeted cell-based therapeutic strategies. Especially in the context of complex chronic wounds such as DFU, precisely directing BMSCs to differentiate into desired cell types holds promise for achieving optimal therapeutic outcomes.

In the context of DFU—a chronic, non-healing wound—persistent low-grade inflammation and immune imbalance are key factors impeding healing. BMSCs, through their dynamic and plastic immunomodulatory capacity, can effectively reshape the local immune microenvironment, creating favorable conditions for the transition from the inflammatory phase to the proliferative phase of wound healing. The immunomodulatory function of BMSCs is not static or intrinsic; rather, it is a highly context-dependent response shaped by surrounding inflammatory signals. Upon stimulation by pro-inflammatory cytokines such as interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α), BMSCs become activated and upregulate the expression of multiple immunosuppressive molecules, thereby initiating their immunoregulatory program. This mechanism ensures that BMSCs exert suppressive effects only when needed, avoiding interference with normal immune surveillance. Their core mechanisms involve both direct cell–cell contact and paracrine signaling. At the level of direct contact, BMSCs can bind to T cells via surface molecules such as ICAM-1 and induce apoptosis of activated T cells through the Fas/FasL pathway, thereby precisely eliminating hyperactive immune cells [18]. More importantly, BMSCs exhibit potent paracrine activity. They secrete a range of soluble factors with immunomodulatory properties, forming a complex regulatory network. Notably, BMSCs can promote macrophage polarization from the pro-inflammatory M1 phenotype to the anti-inflammatory, pro-repair M2 phenotype—evidenced by downregulation of iNOS and IL-1β (pro-inflammatory markers) and upregulation of Arg1, CD206, and IL-10 (M2 markers). This phenotypic shift is critical for clearing necrotic tissue, initiating angiogenesis, and facilitating extracellular matrix deposition in the wound bed [19].

Chemokines are a class of small secreted proteins that mediate chemotactic cell movement by binding to their specific G protein-coupled receptors. In the homing process of BMSCs, the SDF-1/CXCR4 axis is one of the most extensively studied and functionally well-defined signaling pathways. Stromal cell-derived factor-1 (SDF-1), also known as CXCL12, is abundantly secreted by endothelial cells, fibroblasts, and inflammatory cells within injured tissues, establishing a concentration gradient. BMSCs highly express CXCR4—the sole receptor for SDF-1—on their surface, enabling them to initiate directional migration under the guidance of this gradient [20]. Experimental studies have demonstrated that SDF-1 significantly enhances the in vitro migratory capacity of BMSCs and promotes their accumulation at diabetic wound sites in animal models, thereby accelerating wound closure and tissue regeneration [21]. Further mechanistic investigations revealed that the binding of SDF-1 to CXCR4 activates downstream PI3K/Akt and MAPK/ERK signaling pathways, which regulate actin cytoskeleton reorganization, pseudopod formation, and the expression of cell adhesion molecules, ultimately driving cellular migration [22].

Integrins are a family of heterodimeric transmembrane receptors that primarily mediate adhesion between cells and the extracellular matrix (ECM), playing a central role in BMSC homing. Among them, β1-family integrins—particularly α4β1 and α5β1—are especially critical. The α4β1 integrin (also known as VLA-4) recognizes vascular cell adhesion molecule-1 (VCAM-1) expressed on the surface of vascular endothelial cells, facilitating the rolling, firm adhesion, and subsequent transendothelial migration of BMSCs at inflammatory sites. Research has shown that in areas of tissue injury, inflammatory cytokines such as TNF-α and IL-1β upregulate VCAM-1 expression on endothelial cells, creating “adhesion hotspots” that favor stem cell capture [23]. Additionally, α5β1 integrin mediates the anchoring and spreading of BMSCs within the wound matrix by binding to the RGD motif in fibronectin, providing structural support necessary for their proliferation and differentiation.

Matrix metalloproteinases (MMPs) are a class of zinc-dependent endopeptidases capable of degrading ECM components such as collagen, fibronectin, and laminin, thereby creating structural pathways for BMSCs to traverse the vascular basement membrane and surrounding tissue barriers. In particular, MMP-2, MMP-9, and MT1-MMP act synergistically to degrade collagen and gelatin, serving as core effectors enabling BMSCs to complete transendothelial and interstitial migration [24][25]. Moreover, the inflammatory microenvironment of DFU—rich in cytokines such as TNF-α and IL-1β—upregulates MMP expression in BMSCs, thereby enhancing their directional migration toward ulcer sites [26]. Following successful homing, MMPs secreted by BMSCs continue to participate in ECM remodeling and collagen metabolism in the wound area, which are critical steps in tissue regeneration and wound healing. MMPs thus serve as a key molecular bridge linking BMSCs to the repair process in DFU. They not only “pave the way” for BMSC homing through enzymatic degradation but are also activated and upregulated within the DFU inflammatory microenvironment, thereby increasing the efficiency of MSC recruitment.

BMSCs have been demonstrated to promote angiogenesis through multiple mechanisms, thereby improving local blood perfusion and accelerating wound healing [27]. BMSCs secrete various pro-angiogenic factors, such as vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF). These factors can stimulate the proliferation, migration, and tube formation of endothelial cells, facilitating the generation of new blood vessels. Moreover, BMSCs can also differentiate into endothelial cells and directly participate in vessel formation [27]. A systematic review and meta-analysis highlighted the role of extracellular vesicles derived from bone marrow-derived stem cells (BMSC-EVs) in promoting angiogenesis, suggesting their potential as a cell-free therapy for DFU treatment, although this approach remains in the preclinical stage [28]. SDF-1 is abundantly secreted by damaged tissues under hypoxic and inflammatory conditions, forming a concentration gradient that guides the directional migration of BMSCs expressing CXCR4. When the expression level of CXCR4 is downregulated due to in vitro high oxygen culture (21% O₂), the chemotactic response of BMSCs to SDF-1 is significantly weakened, leading to homing failure, which in turn reduces their ability to locally release factors such as VEGF, ultimately affecting the efficiency of angiogenesis.

BMSCs possess robust paracrine capabilities and can secrete a wide array of bioactive molecules, including growth factors, cytokines, and chemokines, which play pivotal roles in the repair process of DFU. In addition to VEGF and bFGF mentioned above, BMSCs also secrete transforming growth factor-β (TGF-β), platelet-derived growth factor (PDGF), and epidermal growth factor (EGF). These growth factors can enhance fibroblast activity, promote ECM remodeling and collagen synthesis, thereby accelerating wound contraction and tissue regeneration [29]. For instance, in a diabetic rat model, transplantation of bone marrow mesenchymal stem cells was shown to promote delayed wound healing, which may be attributed to the growth factors they secrete [30]. The paracrine effects of growth factors also depend on effective cell homing as a prerequisite. If cells become sequestered in the lungs or other non-target organs, they cannot establish a sufficient local concentration gradient of bioactive molecules at the wound site, thereby impairing the processes of ECM remodeling and re-epithelialization.

Chronic inflammation is a key reason why DFU is difficult to heal [31]. BMSCs exhibit significant immunomodulatory properties, capable of suppressing the release of pro-inflammatory cytokines (such as tumor necrosis factor-α and interleukin-1β) and promoting the secretion of anti-inflammatory cytokines (such as interleukin-10), thereby creating a microenvironment conducive to tissue repair [32]. This immunomodulatory effect helps alleviate local inflammatory responses, prevents further tissue damage, and sets the stage for subsequent tissue regeneration. For example, exosomes derived from M2 macrophages have been confirmed to possess anti-inflammatory properties, capable of modulating macrophage phenotype and promoting diabetic wound healing [31]. Janus Liposozyme promotes healing by regulating the transformation of M1 macrophages into M2 macrophages, thereby reducing inflammatory responses [33]. This regulatory effect can only be effectively exerted after BMSCs successfully engraft into the inflammatory microenvironment, and the intensity of the immunomodulatory effect directly depends on the number of BMSCs reaching the lesion site and their functional integrity.

As multipotent stem cells, BMSCs have the capacity to differentiate into various cell types, including keratinocytes, fibroblasts, and endothelial cells—key cellular components in skin repair and tissue regeneration [34]. Through either direct differentiation or paracrine actions, BMSCs can facilitate the regeneration and functional recovery of damaged tissues. During the healing process of DFU, BMSCs contribute to the formation of high-quality granulation tissue, promote re-epithelialization, and enhance the tensile strength of the wound, ultimately achieving effective wound repair [35]. The relationship between tissue regeneration and BMSC homing is not a simple linear one; rather, it constitutes a dynamic closed loop of “homing - engraftment - activation - regulation - regeneration.” The efficiency of homing determines the threshold for initiating regeneration, while the functional state of the engrafted cells dictates the quality and durability of the regenerative outcome.

Genetic modification is an effective approach to enhance the targeting ability and functionality of BMSCs. By overexpressing specific chemokine receptors or pro-angiogenic factors in BMSCs, their homing capacity and therapeutic potential in vivo can be significantly improved. For instance, BMSCs engineered to overexpress the chemokine receptor CXCR4 exhibit enhanced responsiveness to stromal cell-derived factor-1 (SDF-1), which is highly expressed at DFU lesion sites, thereby improving homing efficiency [36]. Additionally, genetic modification of BMSCs to overexpress vascular endothelial growth factor (VEGF) or heme oxygenase-1 (HO-1) can augment their pro-angiogenic and antioxidant capacities, playing a more critical role in promoting DFU healing [37].

Preconditioning BMSCs in vitro to mimic the pathophysiological environment in vivo can enhance cell survival, migratory capacity, and paracrine function. Common preconditioning strategies include:

Hypoxic preconditioning: DFU lesions are often hypoxic. Preconditioning BMSCs under hypoxic conditions in vitro upregulates the expression of hypoxia-inducible factor-1α (HIF-1α), thereby enhancing cellular tolerance to hypoxia and increasing the secretion of pro-angiogenic factors [38].

Cytokine preconditioning: Pre-treatment of BMSCs with inflammatory cytokines such as IL-1β or TNF-α can activate intracellular signaling pathways, upregulate chemokine receptor expression, and improve homing ability [39].

Pharmacological preconditioning: Preconditioning BMSCs with specific drugs (e.g., statins, melatonin) or small-molecule compounds can enhance antioxidant capacity, reduce apoptosis, and promote migration and proliferation [37].

Combining BMSCs with biomaterial scaffolds is an effective strategy to improve cell retention and local therapeutic outcomes [40]. Biomaterial scaffolds provide a three-dimensional microenvironment for transplanted BMSCs, protecting them from the harsh local milieu and supporting their survival, proliferation, and functional activity [41]. While ensuring cell retention, the scaffold can be endowed with the active capability to recruit endogenous stem cells. For instance, sustained-release chemokines such as SDF-1α can be incorporated into PEG or collagen scaffolds. This factor can establish a stable concentration gradient that attracts host circulating CXCR4⁺ BMSCs to migrate toward the implantation site, thereby achieving synergistic enhancement between “exogenous cell retention” and “endogenous cell homing.”

Injectable hydrogels: Injectable polyethylene glycol (PEG)-based hydrogels are widely used for cell delivery due to their excellent biocompatibility and controllable release properties [42]. For example, studies have shown that injectable reactive oxygen species (ROS)-degradable PEG hydrogels enhance stem cell retention and provide antioxidant protection [41].

Collagen scaffolds: As a natural component of the extracellular matrix, collagen exhibits excellent biocompatibility and biodegradability. Local application of BMSCs seeded on collagen scaffolds has been shown to promote ulcer healing and angiogenesis in diabetic rabbit models [30].