Introduction
Fracture repair is among the most common organ-scale regenerative processes in humans. Although bone possesses intrinsic reparative capacity, about 10% of patients experience delayed union or nonunion, which markedly impairs functional recovery and quality of life [ 1 ]. Fracture healing is a highly dynamic and spatiotemporally coordinated regenerative program that typically begins with a tissue-formation phase characterized by angiogenesis together with recruitment and differentiation of mesenchymal progenitors, and progresses to a later phase in which woven bone is replaced and remodeled to restore function [ 2 ]. Developing effective approaches to accelerate fracture repair remains a major research priority.
Clinically, fractures in the elderly are characterized by a markedly elevated risk driven by osteoporosis and by higher lifetime fracture incidence [ 3 ]. Healing times are generally prolonged, and the risks of delayed union or nonunion are substantially higher in older patients [ 4 , 5 ]. These observations indicate that fractures in aging populations represent a significant and growing concern. At the biological level, older individuals exhibit reduced angiogenic capacity, diminished recruitment and differentiation potential of mesenchymal progenitors, and impaired osteoblast function, all of which constitute barriers to fracture repair [ [6] , [7] , [8] ]. According to Global Burden of Disease data, since 1990 the prevalence of hip fracture in older adults has increased by 173.07% and continues to rise [ 9 ]. From 2015 to 2050, the proportion of people aged 60 years or older will increase from 12% to 22%, rising from 700 million to 1.5 billion [ 10 ]. Elucidating age-related mechanisms of fracture healing is therefore essential to guide targeted interventions that improve outcomes in older adults and to reduce the medical and economic burden associated with fracture-related complications in the context of population aging [ 11 ].
During fracture healing, aging has been reported to influence multiple stages through distinct mechanisms. In mice, advancing age is associated with rarefaction of skeletal and bone marrow microvascular networks, a decline in type H vessels, and delayed expression of HIF-1α and VEGF, which together postpone the angiogenic response during repair [ 12 , 13 ]. At fracture sites, p21 + senescent-like cells with an inflammatory SASP phenotype, primarily neutrophils and osteochondral progenitors, create a positive-feedback inflammatory milieu via the THBS1/CD47 pathway, resulting in reduced osteogenic capacity, inferior callus quality, and delayed repair [ 14 ]. In aged BMSCs, promoter hypermethylation at loci such as RUNX3, BMP2, and SOST diminishes migration and osteogenesis through Wnt and Smad signaling, and the accumulation of DNA damage and ROS further suppresses osteogenic activity [ 15 ]. Senescent cells also inhibit fracture healing by secreting SASP factors including IL-6, TNF-α, and high levels of TGF-β [ 16 ]. Osteoprogenitors are widely recognized as the principal precursors of osteoblasts and play a central role in fracture repair [ 17 ]. However, in the context of aging-related delays in healing, it remains insufficiently defined whether their responsiveness to osteogenic cues is altered, whether their functional competence is impaired, and whether specific signaling pathways govern their osteogenic differentiation in a systematic manner.
The management of post-fracture bone defects remains challenging due to limited donor availability, the risk of immune rejection, and substantial treatment costs [ 18 ]. Applying biomaterials in fracture repair offers a promising therapeutic strategy. Hydrogels exhibit biocompatibility and aqueous processability and are widely applied in tissue engineering [ [19] , [20] , [21] ]. A variety of hydrogel modifications have been developed to promote fracture repair, including DNA-based hydrogels, nanoparticle hydrogels, and exosome hydrogels [ [22] , [23] , [24] , [25] ]. Although these exogenous designs can respond to the fracture microenvironment, a gap remains between such approaches and true in situ endogenous induction of repair. EGFR signaling in bone progenitor stem cells is indispensable for bone regeneration, yet whether in situ induction via its ligand HBEGF can be achieved has not been fully explored [ 26 , 27 ]. Here, we found that EGFR activity in skeletal stem/progenitor cells decreased as mice aged and thus led to delayed fracture healing. We hypothesized that targeting EGFR signaling in skeletal stem/progenitor cells could be an effective therapy for aging-induced delayed fracture healing. To test this, we designed a two-pronged approach. First, we and others have identified Prx1 as an ideal maker to label mouse skeletal stem/progenitor cells, and are localized within articular cartilage, the height of the growth plate, the cortical bone, as well as the cancellous bone and especially in the periosteal surface, plays an essential role in bone development and repair [ 27 , 28 ]. We then generated a skeletal stem/progenitor cell-specific EGFR inactivation mouse model ( Prx1-Cre Egfr flox/flox ) using Prx1-Cre . Next, we genetically enhanced EGFR activity by adopting a Rosa-diphtheria toxin receptor ( DTR ) model that is normally used for cell ablation [ 29 ]. DTR was originally used as a receptor for bacterial diphtheria toxin, later found to be human full-length heparin-binding EGF like growth factor (HBEGF), a ligand for EGFR, which shares 81% sequence identity with the mouse ortholog [ 30 , 31 ]. We crossed Rosa-DTR mice with stem/progenitor cell-specific Prx1-Cre mice to generate a Prx1-HBEGF overexpressing mouse line ( HBEGF Over Prx1 ). The results revealed that overactivation of EGFR signaling markedly enhanced fracture healing in aged mice, which was accompanied by promotion of osteogenesis and vascular coupling and inhibition of cell senescence. Based on these above results, we constructed a hyaluronic acid-grafted-dopamine (HADA) hydrogel that provides a stable and controllable platform for protein delivery. We designed an injectable HADA hydrogel loaded with HBEGF protein that produced significant therapeutic benefit in an aged mouse fracture model, and we demonstrate that HBEGF@HADA hydrogel promotes fracture healing in an EGFR/ERK-dependent manner in vivo, offering a new rationale for treating fractures in older adults.
Results
EGFR activity declines in the fracture callus of aged mice
Micro-CT at different time-points during fracture healing revealed that the aged group exhibited significantly smaller calluses compared with the young group at 10 dpf ( Fig. 1 A). Quantitative analysis of μCT images indicated that BV and TV both decreased significantly in the aged group at 10 dpf ( Fig. 1 B). Remodeling was also delayed in aged mice. By 28 dpf, clear evidence of fracture remodeling was observed, and remodeling was almost complete by 42 dpf in young mice. However, in fractures of aged mice, the remodeling process was lagging, with notably greater callus volumes, resulting in significant decreases in BV/TV compared to the young group at 28 dpf and 42 dpf ( Fig. 1 A and B). Safranin O/Fast green staining of the fractures further confirmed a delay in fracture healing and remodeling in aged mice. Whereas callus formation had occurred in both young and aged mice by day 10, cartilage calluses reached a peak at 10 dpf and then matured and remodeled more rapidly in young mice. By 28 dpf, young mice showed significant remodeling, with most of the initial woven bone in the callus having already been replaced. At 42 dpf, fractures in young mice had almost completed remodeling, and the femur regained a morphology resembling nonfractured bone. The total callus area was notably larger in young mice at 10 dpf but decreased by days 28 and 42 after remodeling. In contrast, these events were markedly delayed in the fracture callus from aged mice, which exhibited reduced callus formation and delayed chondrogenesis at 10 dpf, with remodeling not evident until 42 dpf ( Fig. 1 C and D). Fig. 1 EGFR signaling is reduced during the fracture healing process in aged mice. (A) Representative 3D reconstructions and coronal cross-sectional micro-computed tomography (μCT) images of fractured femurs at 10, 28 and 42 days post fracture (dpf). (B) Quantitative analysis of tissue volume (TV), bone volume (BV) and bone volume fraction (BV/TV) of the calluses of fractured femurs at 10, 28 and 42 dpf. n = 8. (C) Representative images of Safranin O/Fast green-stained fracture calluses at 10, 28 and 42 dpf; scale bar = 500 μm. (D) Quantitative analysis of total calluses area at 10, 28 and 42 dpf. n = 6. (E) Representative co-immunostaining images of p16 (green) and γ-H2AX (red) in fracture callus at 10 dpf, counterstained with DAPI (blue). Scale bar = 200 μm. (F) Quantification of the percentage of p16 + , γ-H2AX + cells. n = 3. (G) Representative co-immunostaining images of osteocalcin (OCN; green) and endomucin (Emcn; red) in fracture callus at 10 dpf, counterstained with DAPI (blue). Scale bar = 200 μm. (H) Quantification of the percentage of OCN + cells and endomucin + vessels. n = 3. (I) Immunohistochemical staining of EGFR, p-EGFR and HBEGF within the calluses of young and aged mice at 10 dpf. Scale bar = 200 μm. (J) Quantification of the percentage of EGFR + , p-EGFR + and HBEGF + cells. n = 3. (K) Western blotting analysis of EGFR, p-EGFR and HBEGF expression in fracture calluses isolated from young and aged mice. Data are presented as means ± SD. Groups were compared by a two-tailed Student's t -test. ns = not significant, * P < 0.05, ** P < 0.01, *** P < 0.001.
Next, we measured the expression levels of two well-established senescence markers (p16 and γ-H2AX) by double-IF staining. Results showed a significantly greater p16/γ-H2AX double-positive area in the callus of aged mice than in that of young mice at 10 dpf ( Fig. 1 E and F). Previous studies have established that vascularization is a sensitive indicator of endochondral ossification, with angiogenesis being tightly coupled with osteogenesis to facilitate successful bone regeneration during fracture repair [ 32 ]. For this reason, we performed IF staining of osteocalcin (OCN) and endomucin (Emcn) in fracture callus at 10 dpf and found fewer OCN + cells and Emcn + blood vessels within fracture callus in aged mice, compared with young mice ( Fig. 1 G and H), indicating delayed bone formation and vascularization in fractures from aged mice.
EGFR signaling has been reported to play a vital role in skeletal stem/progenitor cells during bone formation and repair [ 27 ]. However, it was unclear whether aging-induced delayed fracture healing resulted from EGFR downregulation in skeletal stem/progenitor cells. Therefore, we analyzed the single-cell RNA sequencing (scRNA-seq) datasets (OMIX015927, GSE272155 ) of fracture callus from young and aged mice, after integrating the data, we applied the Harmony algorithm to correct for batch effects and found that EGFR activity was significantly downregulated in aged mice ( Fig. S1A–B ). To further verify the changes of EGFR expression in fracture callus, fractured femurs from young and aged mice were collected for immunohistochemical (IHC) staining. In line with scRNA-seq data, the amount of EGFR was not changed, but p-EGFR (an indicator of EGFR activity) as well as its ligand, HBEGF, were both significantly reduced in calluses at 10 dpf in aged mice ( Fig. 1 I and J). Furthermore, we isolated the fracture calluses from young and aged mice and performed western blotting analysis. The results were consistent with our IHC staining ( Fig. 1 K). Together, these results suggested that EGFR activity is negatively correlated with aging after injury, and reduced EGFR expression in stem/progenitor cells is one of the underlying mechanisms of aging-induced delayed fracture healing.
Stem cell/progenitor-specific deletion of EGFR results in delayed fracture healing
To delineate the role of EGFR activity during fracture healing, we constructed stem cell/progenitor-specific EGFR knockout mice, Prx1 Egfr flox / flox ( Egfr iCKO ) mice. The levels of EGFR activity indicators, p-EGFR, p-ERK and HBEGF, were significantly decreased in the fracture calluses of Egfr iCKO mice compared to WT controls ( Fig. S2A–C ). Interestingly, Egfr iCKO mice showed significant decreases in trabecular bone mass and cortical bone thickness at 3 months of age, with decreases in bone volume fraction (BV/TV), trabecular number (Tb. N), trabecular thickness (Tb. TH) and cortical thickness (Ct. TH), compared to WT mice ( Fig. S3A–E ). More importantly, we observed that Egfr iCKO mice had much smaller callus, with significant reductions in TV and BV at 10 dpf. By 28 dpf in WT mice, robust remodeling was evident, with nearly complete remodeling observed at 42 dpf. In contrast, Egfr iCKO mice displayed negligible evidence of remodeling at the same time-point ( Fig. 2 A). Quantitative analysis of μCT images indicated BV/TV was significantly reduced in the Egfr iCKO mice at both 28 dpf and 42 dpf ( Fig. 2 B). Safranin O/fast green staining further revealed a smaller cartilage callus size at 10 dpf and areas of cartilage persisting through 28 dpf ( Fig. 2 C and D). Even at 42 dpf, fracture calluses in Egfr iCKO mice remained enlarged and retained areas of woven bone, indicating the arrest of the healing process in the Egfr iCKO mice. IF staining revealed that compared to WT mice, Egfr iCKO mice exhibited a marked reduction in OCN + osteoblasts and Emcn + vessels within the fracture callus ( Fig. 2 E and Fig. S3F ). Overall, these data suggest that conditional EGFR knockout in stem/progenitor cells results in impairments in both angiogenesis and osteogenesis, ultimately leading to delayed fracture healing. Fig. 2 Specific deletion of EGFR signaling in stem/progenitor cells inhibits the fracture healing process. (A) Representative 3D reconstructions and coronal cross-sectional μCT images of fractured femurs at 10, 28 and 42 dpf. (B) Quantitative analysis of TV, BV and BV/TV of the calluses of fractured femurs at 10, 28 and 42 dpf. n = 8. (C) Representative images of Safranin O/Fast green-stained fracture calluses at 10, 28 and 42 dpf; scale bar = 500 μm. (D) Quantitative analysis of the total callus area at 10, 28 and 42 dpf. n = 6. (E) Representative co-immunostaining images of OCN (green) and endomucin (red) in fracture callus at 10 dpf, counterstained with DAPI (blue). Scale bar = 200 μm. Data are presented as means ± SD. Groups were compared by a two-tailed Student's t -test. ns = not significant, * P < 0.05, ** P < 0.01, *** P < 0.001.
EGFR overactivation in stem/progenitor cells accelerates fracture healing of aged mice
To further determine whether overactivation of EGFR signaling in stem/progenitor cells could promote fracture healing in aged mice, we bred Prx1-cre and Rosa-DTR mice to generate a Prx1-HBEGF overexpressing mouse line ( HBEGF Over Prx1 ), and maintained them under standard conditions until 16 months of age before performing femoral fracture modeling. Western blots and IHC staining confirmed increased HBEGF expression as well as elevated p-EGFR in 16-month-old (16M)- HBEGF Over Prx1 mouse callus compared to 16M- WT mice ( Fig. S4A–C ). Consistent with data shown in Fig. 1 , 16M- WT mice exhibited smaller callus area, and delayed chondrogenesis and bone formation compared with young mice at 10 dpf. In contrast, 16M- HBEGF Over Prx1 mice showed significantly increased cartilage and bone area at 10 dpf, similar to that observed in young mice ( Fig. 3 A–D). By 28 dpf, both young and 16M- HBEGF Over Prx1 mice experienced extensive remodeling, with most of the initial woven bone within the callus having been remodeled. In contrast these events were markedly delayed in the fracture callus from aged mice, in which the fracture callus remains enlarged and retained areas of woven bone at the same time-point ( Fig. 3 A–D). Bone indices, including BV, TV, and BV/TV, were consistent with the aforementioned results ( Fig. 3 B). Moreover, double IF staining of OCN and Emcn confirmed that EGFR overactivation displayed a robust osteogenesis coupled to the angiogenic effect in aged mice ( Fig. 3 E and Fig. S4D ). Because EGFR is essential for the development and homeostasis of multiple organs, there has been concern that sustained expression of HBEGF may have side effects. However, we did not observe any significant morphological changes in major organs, such as heart, liver, spleen, lung and kidney in 16M HBEGF Over Prx1 mice ( Fig. S5 ). Thus, overactivation of EGFR signaling in stem/progenitor cells accelerates the endochondral phase of bone repair in aged mice and results in a pattern of healing that mimics fracture healing in young mice, and this stem/progenitor-specific EGFR activation was harmless in vivo. Fig. 3 Activation of EGFR signaling in stem/progenitor cells accelerates fracture healing in aged mice. (A) Representative 3D reconstructions and coronal cross-sectional μCT images of fractured femurs at 10, 28 and 42 dpf. (B) Quantitative analysis of TV, BV and BV/TV of the calluses of fractured femurs at 10, 28 and 42 dpf. n = 8. (C) Representative images of Safranin O/Fast green-stained fracture calluses at 10, 28 and 42 dpf; scale bar = 500 μm. (D) Quantitative analysis of the total calluses area at 10, 28 and 42 dpf. n = 6. (E) Representative co-immunostaining images of OCN (green) and endomucin (red) in fracture callus at 10 dpf, counterstained with DAPI (blue). Scale bar = 200 μm. Data are presented as means ± SD. Groups were compared by one-way ANOVA with Bonferroni's post-hoc test. ns = not significant, * P < 0.05, ** P < 0.01, *** P < 0.001.
EGFR overactivation reduces the expression of SASP factors and stimulates migration and differentiation of aged periosteal progenitors
Next, to validate the above findings in vitro, we established a culture protocol, in which we generated senescent periosteal progenitors by treating periosteal progenitors isolated from WT , Egfr iCKO and HBEGF Over Prx1 mice with H 2 O 2 , a commonly used senescence inducer in vitro [ 33 ]. Western blotting confirmed that H 2 O 2 effectively reduced the levels of EGFR activation indicators (p-EGFR, p-ERK and HBEGF) in periosteal progenitors, and this was more obvious in Egfr iCKO periosteal progenitors ( Fig. S6A ). H 2 O 2 had no effect on the expression of EGFR activation indicators in HBEGF Over Prx1 periosteal progenitors ( Fig. S6A ). Meanwhile, we found that both H 2 O 2 -treated WT and Egfr iCKO periosteal progenitors expressed high levels of senescence markers (p16, p21, γ-H2AX) ( Fig. 4 A), and stained positively for SA–β-gal ( Fig. 4 D). However, overexpressing EGFR signaling in periosteal progenitors ( HBEGF Over Prx1 periosteal progenitors) prevented the increase in senescence markers and SA–β-gal staining induced by H 2 O 2 ( Fig. 4 A and D). Senescent cells present a senescence-associated secretory phenotype (SASP), which is the group of bioactive substances secreted by these cells, including cytokines, chemokines and proteases [ 34 ]. We further examined the expression of SASP components under H 2 O 2 -inducing conditions and the results show that HBEGF overexpression in periosteal progenitors significantly reduces the secretion of SASP factors in response to H 2 O 2 treatment, whereas this effect is abrogated upon EGFR knockdown ( Fig. 4 B). These findings suggest that EGFR signaling may suppress the senescence phenotype by modulating the SASP, thereby contributing to the reduced expression of senescence markers such as p16, p21, γ-H2AX, and decreased SA-β-gal activity. Fig. 4 H 2 O 2 inhabits the migration, differentiation and pro-angiogenic ability of periosteal progenitors, which is prevented by EGFR overactivation. (A) Western blot analysis of p16, p21, γ-H2AX, RUNX2, SOX9 and VEGFA in periosteal progenitors derived from WT , HBEGF Over Prx1 and Egfr iCKO mice. Periosteal progenitors were treated with or without H 2 O 2 (50 μM). (B) Western blot analysis of SASP factors in periosteal progenitors derived from WT , HBEGF Over Prx1 and Egfr iCKO mice. Periosteal progenitors were treated with or without H 2 O 2 (50 μM). (C) CFU-F assay using periosteal progenitors isolated from WT , HBEGF Over Prx1 and Egfr iCKO mice. Periosteal progenitors were treated with or without H 2 O 2 (50 μM). Percentages of CFU + cells were quantified; n = 3. (D) SA–β-gal staining of the indicated periosteal progenitors was performed, and the percentage of SA–β-gal + cells was measured using ImageJ. n = 3. (E) Periosteal progenitors were seeded into the upper chamber in 1% FBS medium. Migrated cells on the lower surface of the membrane were stained with crystal violet, and the number of migrated cells was quantified; n = 3. Scale bar = 100 μm. (F) Alkaline phosphatase (ALP) and Alizarin Red S (ARS) staining of the WT , HBEGF Over Prx1 and Egfr iCKO periosteal progenitors after culture in osteogenic medium for 3 weeks with or without H 2 O 2 . ALP- or ARS-positive areas were quantified using ImageJ; n = 3. (G) Alcian blue staining of the WT , HBEGF Over Prx1 and Egfr iCKO periosteal progenitors after culture in chondrogenic medium for 2 weeks with or without H 2 O 2 . Alcian blue-positive areas were measured using Image J; n = 3. (H) HUVEC migration assay and tube formation assay were performed with conditioned medium collected form WT periosteal progenitors (control-CM), HBEGF Over Prx1 periosteal progenitors (HBEGF Over Prx1 -CM) or Egfr iCKO periosteal progenitors (Egfr iCKO-CM) in the absence or presence of H 2 O 2 . Scale bar = 100 μm. Data are presented as means ± SD. Groups were compared by one-way ANOVA with Bonferroni's post-hoc test. ns = not significant, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.
We next investigated the effects of EGFR signaling on the proliferation, migration and differentiation of H 2 O 2 -induced periosteal progenitors. We found that H 2 O 2 inhibited the proliferation and migration of periosteal progenitors, and EGFR inactivation in periosteal progenitors (H 2 O 2 + Egfr iCKO ) further aggravated these effects. Of note, overexpressing EGFR signaling in periosteal progenitors significantly blocked the inhibitory effects of H 2 O 2 on periosteal progenitor proliferation and migration ( Fig. 4 C and E). Similar to the observation that EGFR knockout exacerbated the inhibitory effect of H 2 O 2 -induced periosteal progenitor proliferation and migration, EGFR knockout also exacerbated the inhibitory effect of H 2 O 2 on osteogenic differentiation of periosteal progenitors, as shown by reduced ALP and ARS staining and lower expression of osteoblastic marker genes ( ALP , RUNX2 and OSTERIX ). Not surprisingly, overexpression of EGFR markedly reversed these inhibitory effects ( Fig. 4 A–F and S6B ). Evaluation of chondrogenic differentiation by alcian blue staining as well as chondrogenic marker genes ( Col2a1 , Acan and SOX9 ) showed similar results ( Fig. 4 A–G and S6C ).
Osteogenesis and angiogenesis are intimately coupled during bone regeneration. We then investigated whether EGFR activation in periosteal progenitors affected angiogenesis. Notably, treatment with H 2 O 2 significantly inhibited vascular endothelial growth factor A (VEGFA) expression in WT and Egfr iCKO periosteal progenitors, but not in HBEGF Over Prx1 periosteal progenitors ( Fig. 4 A). Immunofluorescence staining of VEGFA in the fracture callus at 10 dpf further confirmed our in vitro data ( Fig. S6D ). To validate the possible angiogenic function of EGFR signaling in periosteal progenitors, conditioned medium (CM) from WT , HBEGF Over Prx1 and Egfr iCKO periosteal progenitors after H 2 O 2 treatment was collected for subsequent analysis in HUVECs. As shown in Fig. 4 H and S6E , migration and tube formation of HUVECs were both blocked by CM from H 2 O 2 -treated WT and Egfr iCKO periosteal progenitors compared to vehicle-treated cells, and this inhibition could be significantly rescued by EGFR overactivation, in line with our previous in vivo data (Figs. 2E and 3 E). Furthermore, these increases of HBEGF Over Prx1 -CM could be significantly blocked by VEGFA inhibition. Thus, these results suggest that EGFR signaling plays a critical role in proliferation, migration and differentiation of periosteal progenitors, and EGFR activation upregulates a variety of osteogenic, chondrogenic and angiogenic mediators (RUNX2, SOX9, VEGFA, etc.) in the fracture region, thereby facilitating callus formation and consequent fracture repair in aged mice.
Design and synthesis of HBEGF@HADA hydrogel
As the loss of EGFR activity is likely a contributor to aging-induced delayed fractures, we developed an injectable hyaluronic acid-graft-dopamine (HADA) hydrogel to treat aged fractures with the ability to provide sustained release of HBEGF (a ligand of EGFR) at the fracture site. First, HADA was synthesized through an amidated reaction between the carboxylic acid group of hyaluronic acid (HA) and the primary amine group of dopamine (DA) ( Fig. 5 A). The grafting of DA to HA was confirmed by 1 H and 13 C nuclear magnetic resonance (NMR), which showed that the spectrum of HADA exhibited a new chemical shift at approximately 2.76 ppm, attributed to a characteristic peak of the methylene protons of DA, as well as the presence of chemical shifts at 6.7 ppm, which are indicative of the characteristic proton peaks of the DA phenyl ring ( Fig. 5 B). Meanwhile, HADA and DA exhibited a strong ultraviolet (UV) absorption peak attributed to the benzene rings of DA at around 280 nm [ 35 ], and no absorption peak between 230 and 400 nm for HA, indicating the successful grafting of DA onto HA ( Fig. 5 C). In addition, Fourier-transform infrared spectroscopy (FTIR) revealed characteristic absorption bands for HA: a stretching vibration peak of O–H and N–H at 3400 cm −1 , a C–H stretching vibration peak at 2920 cm −1 , and a carbonyl (C=O) stretching vibration of HA at 1640 cm −1 . These absorption peaks were also observed in the HADA spectrum. Furthermore, HADA exhibited distinct vibrational absorption bands between 1500 and 1450 cm −1 , attributed to the phenyl ring of DA, which were absent in HA, further corroborating the successful synthesis of HADA ( Fig. S7A ). Next, the HBEGF protein was added to lyophilized HADA to form a transparent and fluid aqueous solution. Subsequently, the H 2 O 2 and peroxidase solution was added to this liquid, which induced a transition to an opaque solid-like HBEGF@HADA hydrogel as its fluidity decreased ( Fig. 5 D). This liquid-to-solid transition verifies that hydrogels consist of three-dimensional networks resulting from the cross-linking of hydrophilic polymer chains. Rheological measurements indicated the energy storage modulus (G′) of both hydrogels were always higher than the loss modulus (G″) at 1–100 Hz, and showed a gradual increase, confirming hydrogel formation ( Fig. 5 E). The morphological structures inside the HADA hydrogel and the HBEGF@HADA hydrogel were further examined by SEM. As shown in Fig. 5 F, both hydrogels possessed porous three-dimensional mesh structures with a pore diameter of about 10 μm, and there was little significant difference between HADA hydrogel and HBEGF@HADA hydrogel. Such structure enables the hydrogels to preserve their shape and elasticity during water uptake, thereby endowing them with significant capacity for water absorption and retention. Fig. 5 The fabrication of HBEGF@HADA hydrogel. (A) Synthetic route of HADA, involving grafting DA onto HA using EDC/NHS as activators. (B) 1 H and 13 C NMR spectra of HA and HADA. (C) UV spectra of HA, DA and HADA. (D) Photographs of HBEGF@HADA solution and HBEGF@HADA hydrogel after mixing with horseradish peroxidase and H 2 O 2 . (E) Storage modulus (G′) and loss modulus (G″) of HADA hydrogel and HBEGF@HADA hydrogel in the rheological test over a frequency range of 0–100 Hz. (F) Scanning electron microscopy (SEM) images of HADA hydrogel and HBEGF@HADA hydrogel.
Material characterization of HBEGF@HADA hydrogel
Suitable injection and adhesion are crucial, ensuring ease of use during the application, and guaranteeing that the hydrogels will not be easily detached due to daily activities. Therefore, we tested the injection and adhesion of the hydrogels, and the results showed that the hydrogels exhibited good injectability, enabling them to be readily injected through a 21 G needle ( Fig. 6 A and B). The self-healing property of HADA hydrogel was also evaluated. As depicted in Fig. 6 C, the incised hydrogels quickly fused into an integrated hydrogel within 20 min, mainly attributed to the reversible dynamic quinone/catechol chemistry together with the inter- and intramolecular reversible noncovalent interactions [ 36 , 37 ]. Furthermore, this injectable hydrogel showed robust interfacial adhesion between the hydrogel and the bone, and the adhesion to the bone surface was around the fracture line, providing resistance against inevitable mechanical forces in vivo. ( Fig. 6 D). In addition, the degradation property of HADA hydrogel showed that the hydrogels can be gradually degraded within 14 days and loaded HBEGF had little effect on their degradation behavior ( Fig. 6 E). Fig. 6 Material characterizations of HBEGF@HADA hydrogel. (A, B) Extrusion from a 21 G needle showing the injection performance of HBEGF@HADA hydrogel. (C) Optical images showing self-healing performance of HBEGF@HADA hydrogel. (D) Images of HBEGF@HADA hydrogel adhering to the surface of the fractured femur. (E) Degradation rate of HADA hydrogel and HBEGF@HADA hydrogel. n = 3. (F) Release profile of HBEGF from HBEGF@HADA hydrogel over a 150-h period. (G) Representative in vivo fluorescence images of the fracture site over 21 days after injection of Cy5.5-labeled HBEGF or HBEGF@HADA hydrogel. (H) Quantitative analysis of the fluorescence intensity at the fracture site after injection of Cy5.5-labeled HBEGF or HBEGF@HADA hydrogel. n = 3. (I) H&E staining of the heart, liver, spleen, lung and kidney after administration of PBS, HADA hydrogel or HBEGF@HADA hydrogel for 42 days; scale bar = 100 μm. Data are presented as means ± SD.
With the aim of enhancing the therapeutic efficacy of HBEGF, we encapsulated them within a HADA hydrogel to achieve sustained release. Next, to check whether HADA hydrogel improved the stability of HBEGF, 100 μL of HBEGF@HADA hydrogel (HBEGF at 50 μg/mL) was placed in an EP tube. After swelling, the filtrate was collected and subjected to Western blot analysis. The results indicated that HBEGF@HADA hydrogel exhibited a band intensity comparable to that of HBEGF protein at the same concentration, demonstrating that HBEGF was successfully incorporated into the hydrogel and stably distributed ( Fig. S8A ). Drug release curves further showed that HADA hydrogel exhibited a time-release of HBEGF, which reached 60% cumulative release percentage at day 7 ( Fig. 6 F). Next, we directly injected HBEGF or HBEGF@HADA hydrogel labeled with CY5.5 into the fracture site to assess retention in vivo. Results showed that directly injected HBEGF dissipated rapidly, with detectable signal lasting less than 7 days. In contrast, the HADA@HBEGF hydrogel remained unequivocally detectable until day 21, indicating the sustained retention of HBEGF in HADA hydrogel ( Fig. 6 G and H). Lastly, to investigate the biocompatibility of the HADA hydrogel, we tested its toxicity on the main organs. There were no notable changes in the heart, liver, spleen, lung, and kidneys, as shown in Fig. 6 I. Together, such injectable, adhesive, self-healing and harmless properties contribute to the adhesion of our HADA hydrogel carrier to the surrounding native bone tissue, and its ability to maintain integrity after filling bone defects to support HBEGF release and bone repair make it suitable for medical implants.
HBEGF@HADA hydrogel promotes aging-induced delayed fracture healing
Finally, we investigated the effect of HBEGF@HADA hydrogel on the repair of aging-induced delayed fracture healing in vivo. The results of μCT, Safranin-O/Fast green and H&E staining showed that fractures in aged mice treated with PBS or HADA hydrogel developed abnormal bone calluses and exhibited retardation in the cartilage-to-bone transition. Administration of free HBEGF produced a moderate improvement over PBS controls, as evidenced by increased callus formation at 10 dpf, yet healing remained suboptimal, with persistently incomplete bony bridging at 42 dpf and only modest gains in BV/TV at later time points. In contrast, fractures in aged mice treated with HBEGF@HADA hydrogel showed abundant callus formation with maximal remaining cartilage at 10 dpf, and formation of completely bridged bone callus at 42 dpf, similar to young mice ( Fig. 7 A–E). At 28 dpf, TV gradually decreased in young and HBEGF@HADA hydrogel-treated aged mice, indicating progressive callus remodeling. Moreover, BV/TV was significantly increased in both young and HBEGF@HADA hydrogel-treated aged mice at later (28, 42 dpf) time-points, suggesting increased bone mineralization in the fracture healing process ( Fig. 7 A–E). To further study whether EGFR mediates the action of HBEGF@HADA hydrogel on bone repair in vivo, we administered aged mice with the HBEGF@HADA hydrogel after fracture, subsequently treated with gefitinib, the EGFR-specific inhibitor. The levels of EGFR activation indicators, p-EGFR, p-ERK and HBEGF increased significantly in the aged mice after HBEGF@HADA hydrogel treatment, but were significantly decreased in the gefitinib-treated mice ( Fig. 8A–D–E and Fig. S9A–C ). More importantly, HBEGF@HADA hydrogel-induced accelerated fracture healing progress was completely abolished by gefitinib ( Fig. 7 A–E). Thus, HBEGF@HADA hydrogel accelerates the endochondral phase of bone repair in aged mice and results in a pattern of healing that mimics fracture healing in young mice, and this effect could be completely blocked by the EGFR-specific inhibitor. Fig. 7 HBEGF@HADA hydrogel promotes delayed fracture healing in aged mice. (A) Representative 3D reconstructions and coronal cross-sectional μCT images of fractured femurs at 10, 28 and 42 dpf. (B) Quantitative analysis of TV, BV and BV/TV of the calluses of fractured femurs at 10, 28 and 42 dpf. n = 8. (C) Representative images of Safranin O/Fast green-stained fracture calluses at 10, 28 and 42 dpf; scale bar = 500 μm. (D) Representative images of H&E-stained fracture calluses at 10, 28 and 42 dpf; scale bar = 500 μm. (E) Quantitative analysis of the total calluses area at 10, 28 and 42 dpf. n = 6. Data are presented as means ± SD. Groups were compared by one-way ANOVA with Bonferroni's post-hoc test. ns = not significant, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. Fig. 8 HBEGF@HADA hydrogel signals through EGFR to promote osteogenesis and vascular coupling in vivo. (A) Representative co-immunostaining images of EGFR (red), p-EGFR (green) and HBEGF (grey) within the calluses of the indicated mice at 10 dpf, counterstained with DAPI (blue). Scale bar = 100 μm. (B) Representative co-immunostaining images of OCN (green) and endomucin (red) in fracture callus at 10 dpf, counterstained with DAPI (blue). Scale bar = 200 μm. (C) Representative co-immunostaining images of p16 (green) and γ-H2AX (red) in fracture callus at 10 dpf, counterstained with DAPI (blue). Scale bar = 100 μm. (D, E) Quantification of the ratio of p-EGFR + cells to EGFR + cells and the percentage of HBEGF + cells. n = 3. (F) Quantification of the percentage of OCN + cells and endomucin + vessels. n = 3. (G) Quantification of the percentage of p16 + , γ-H2AX + cells. n = 3. Data are presented as means ± SD. Groups were compared by one-way ANOVA with Bonferroni's post-hoc test. ns = not significant, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.
We performed further investigation of the role of hydrogels in osteogenesis–angiogenesis coupling using IF staining. The results showed that HBEGF@HADA hydrogel exhibited an obviously greater number of highly positive OCN + and Emcn + cells in the calluses compared to the other aged groups ( Fig. 8 B–F). Furthermore, double immunostaining with senescence markers of p16 and γ-H2AX confirmed that p16/γ-H2AX + calluses areas in the PBS and HADA hydrogel-treated aged mice were abnormally increased, while HBEGF@HADA hydrogel rescued this trend in aged mice ( Fig. 8 C–G). Once again, this robust osteogenesis coupled to the angiogenic and anti-senescence effects of HBEGF@HADA hydrogel were abolished by gefitinib ( Fig. 8 B, C, F, G). In conclusion, the above results confirm that fracture healing is delayed in aged mice and that HBEGF-loaded hydrogel signals through EGFR in vivo to promote fracture healing of aged mice by promoting osteogenesis and vascular coupling ( Fig. 9 ). In our study, the incomplete efficacy of free HBEGF is most consistent with short half-life and rapid clearance from the fracture site, as soluble growth factors are typically eliminated within hours after local injection. Thus, the limited therapeutic benefit of free HBEGF is more likely attributable to pharmacokinetic constraints than to insufficient intrinsic activity, underscoring the advantage of the HADA hydrogel in sustaining local HBEGF delivery and prolonging EGFR activation to achieve robust, youthful-like bone repair. Fig. 9 Schematic illustration of the therapeutic process for fracture healing in aged mice using HBEGF@HADA hydrogel. The HBEGF@HADA hydrogel can be constructed in situ through a 21 G needle injection process. HBEGF is then synchronously and sustainably released from the HADA hydrogel and binds to EGFR on the periosteal progenitor membrane at the fracture site, thereby promoting osteogenesis and angiogenesis coupling and accelerating fracture healing.
Discussion
Population aging is gradually imposing a heavy economic burden worldwide. Senescence is generally regarded as a protective damage response program, including DNA damage, oncogene activation, and telomere shortening, during which cells irreversibly cease division and enter a state of permanent growth arrest [ 38 ]. In human stem cells, aging leads to a decline in both endogenous and exogenous response rates, accompanied by enhanced DNA damage and activation of the DNA damage response, thereby impairing their self-renewal and differentiation potential [ 39 ]. The hallmark of bone tissue aging is bone loss and the accumulation of bone marrow adipose tissue, which directly contributes to the increased incidence of fractures [ 40 ]. Bone progenitor stem cells, as the resident stem cells of bone tissue, have been demonstrated in vivo to exhibit a SASP with aging in mice [ 40 ]. Our study further augments the evidence in this field by showing that, in a murine fracture healing model, aging is accompanied by the loss of EGFR signaling. In vitro experiments further demonstrate that the indicators of EGFR activity (p-EGFR, p-ERK and HBEGF) are markedly reduced in senescent stem/progenitor cells. It should be noted, however, that in vitro senescence was induced by acute exposure to H 2 O 2 , a protocol that imposes robust oxidative stress over a short period. H 2 O 2 -induced senescence is widely employed as an in vitro model of cellular aging, but it may recapitulate features of acute oxidative damage rather than bona fide aging-related senescence, which develops gradually over time under cumulative low-grade stress. Unlike aging-related senescence, acute oxidative stress may bypass certain upstream signaling events—such as DNA damage response kinetics and telomere attrition, and engage distinct downstream pathways, including more pronounced inflammatory or apoptotic responses [ 41 , 42 ]. Consequently, some of the molecular and functional changes observed in this model may reflect damage-driven adaptation rather than the progressive decline in cellular homeostasis associated with chronological aging. Future studies should complement these findings using more physiologically relevant models (such as replicative exhaustion of primary cells or cells derived from aged animals) to verify whether the observed effects are generalizable to natural aging. Nonetheless, our data here suggest that EGFR may represent a novel therapeutic target for improving fracture healing in elderly patients.
EGFR, as the second growth factor to be identified, is frequently expressed at elevated levels or shows increased activity in rapidly proliferating cells, such as those in lung cancer and brain tumors [ 43 ]. HBEGF, an EGFR ligand, possesses a heparin-binding domain that mediates membrane-bound paracrine activity, thereby inducing more sustained receptor activation signals, and is regarded as an important endogenous tissue repair factor [ 44 , 45 ]. Although in vitro experiments have demonstrated that HBEGF, via EGFR, can inhibit the differentiation of osteoblastic cell lines (such as MC3T3-E1 and C2C12 cells), the role of HBEGF in bone tissue in vivo has rarely been investigated. EGFR is expressed in osteoblasts but is almost absent in osteoclasts [ 46 ]. In multiple myeloma, HBEGF–EGFR signaling promotes tumor establishment by stimulating angiogenesis in endothelial cells [ 47 ]. Mice with EGFR −/− in myeloid cells exhibit reduced secondary ossification centers in long bones and irregular vertebral endplate calcification during development [ 48 ]. Osteoblasts can utilize EGFR signaling to promote the mineralization process through the IGF-1R/mTOR pathway [ 48 ]. However, the function of EGFR signaling in bone progenitor stem cells remains unexplored. Collectively, these findings suggest that the EGFR signaling pathway is closely associated with bone development and bone mass regulation within the bone microenvironment. Although Reeves et al. reported that bone progenitor stem cells under senescent conditions impair calvarial healing due to mitochondrial dysfunction, the underlying molecular mechanisms remain unclear [ 49 ]. In our study, we found that EGFR signaling in bone progenitor stem cells is essential for fracture healing in aged mice. Specifically, both bone progenitor stem cell–specific EGFR knockout mice and aged bone progenitor stem cell–specific EGFR overexpressing mice displayed significantly altered fracture healing phenotypes. These results deepen our understanding of bone progenitor stem cells and provide new directions for improving fracture healing in the elderly.
Mechanistically, activation of the EGFR pathway confers a strong capacity to drive cell proliferation and resist apoptosis, thereby providing powerful support for tissue repair [ 50 ]. EGFR transmits intracellular PI3K/AKT signaling, which promotes protein synthesis and plays a pivotal role in tumor cell proliferation [ 51 ]. In addition, EGFR-mediated RAF/ERK1/ERK2 signaling drives G1/S phase progression, leading to enhanced cell proliferation [ 51 ]. During the osteogenic differentiation of bone progenitor stem cells, ERK signaling is indispensable, as it regulates transcription factors and directly enhances RUNX2 activity [ [52] , [53] , [54] ]. In our study, HBEGF treatment significantly activated EGFR signaling in senescent bone progenitor stem cells and increased ERK phosphorylation levels, which partially explains the enhanced osteogenic effects observed in vivo. Notably, EGFR has multiple ligands, including EGF, HBEGF, BTC, AREG, EREG, and EPGN [ 55 ]. Among these, HBEGF is considered a high-affinity ligand and has the longest retention time on the cell membrane [ 55 , 56 ]. Although HBEGF has unique advantages in promoting osteogenesis, its clinical application is still limited by its short half-life in vivo, poor stability at the target site, and the risk of off-target effects from systemic administration [ 57 , 58 ]. Besides, HBEGF is known to signal through ErbB4 in addition to EGFR, but our data here indicate that ErbB4 is neither expressed nor functionally activated during HBEGF-mediated bone regeneration ( Fig. S10 ). Therefore, the mechanisms of action for HBEGF in bone repair are primarily EGFR-dependent. Nevertheless, we acknowledge that a minor or transient contribution of ErbB4 cannot be formally excluded. Future genetic loss-of-function studies targeting ErbB4 specifically would be valuable to further refine the mechanistic model. Taken together, the development of novel materials capable of locally delivering and prolonging the activity of HBEGF represents a valuable endeavor.
Hyaluronic acid (HA) is a natural polysaccharide that has been widely applied in bone and cartilage regeneration due to its excellent biocompatibility and biodegradability [ 59 ]. Dopamine (DA) grafting endows HA hydrogels with mussel-inspired adhesiveness, antioxidant capacity, and protein-binding ability, thereby markedly enhancing their tissue adhesion and sustained release performance [ 60 ]. The catechol groups in HADA can effectively immobilize HBEGF through electrostatic interactions and metal-assisted binding, enabling controlled release and prolonging its local activity. The HBEGF@HADA hydrogel not only preserves the biological activity of HBEGF but also achieves precise delivery to fracture defects via local injection, thereby continuously activating the EGFR signaling pathway in bone progenitor stem cells in situ, significantly enhancing downstream ERK phosphorylation, and promoting osteogenic differentiation and bone repair. Compared with conventional free protein administration, this strategy avoids the rapid degradation and nonspecific distribution of HBEGF in vivo, ensuring efficient action at the target site. More importantly, the mussel-inspired adhesive property of the hydrogel enables firm attachment within the fracture microenvironment, maintaining a stable drug release profile even under dynamic mechanical stress. In vivo, our HBEGF@HADA hydrogel demonstrated robust bone regenerative efficacy, providing a novel therapeutic approach to address fracture healing impairments caused by bone progenitor stem cell senescence in elderly patients, with strong clinical translational potential. However, several questions remain for future investigation. First, hyperactive EGFR signaling resulting from missense mutations, deletions, and insertions is frequently observed in various tumors, including those of the breast, lung, liver, and brain [ 61 ]. In our study, we attempted to minimize this possibility by locally overactivating EGFR in stem/progenitor cells. No obvious morphological abnormalities were observed in vital internal organs in HBEGF Over Prx1 mice up to 16 months of age or in mice injected with HBEGF@HADA hydrogel at the fracture site. However, long-term pharmacological studies remain essential before translating our findings to the clinic in future. Second, a limitation of this study is the use of the constitutive Prx1-Cre line, which mediates recombination from embryonic day 9.5 onward in the early limb bud mesenchyme [ 62 ]. This constitutive activity means that any gene deletion occurs during limb skeletal development, potentially giving rise to structural abnormalities that persist into adulthood and confound the interpretation of adult fracture healing phenotypes. Indeed, our baseline skeletal analyses show reduced trabecular and cortical bone parameters in Egfr iCKO mice, indicating that the delayed fracture-healing phenotype observed in this model cannot be attributed exclusively to EGFR function in adult skeletal progenitors during the repair phase. Therefore, future studies using inducible lineage models, such as Prx1-CreER; EGFR conditional knockout mice, will be necessary to definitively determine the adult skeletal progenitor-specific role of EGFR during bone regeneration.
Ethics approval and consent to participate
This study does not involve sex-based analysis and sex was not considered in study design. Therefore, in order to control variable, we used only male mice in this study. All animal procedures were approved by the Medical Ethics Committee of Tongji Medical College, Huazhong University of Science and Technology and conducted in accordance with institutional guidelines (IACUC Number: 4111).
CRediT authorship contribution statement
Yuxiang Hu: Data curation, Writing – original draft. Yi Wang: Data curation, Methodology. Yangyang Chen: Formal analysis, Software. Xiaoyao Peng: Formal analysis, Software. Hang Chu: Formal analysis, Software. Guosilang Zuo: Resources. Fashuai Wu: Resources. Min Cui: Funding acquisition, Supervision, Writing – review & editing. Zengwu Shao: Supervision, Writing – review & editing. Yulong Wei: Funding acquisition, Supervision, Writing – review & editing.
Declaration of competing interests
The authors have declared that no conflict of interest exists.
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Supplementary data
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Data availability
All data reported in this paper and mouse lines generated in this study will be provided by the corresponding author upon request.
Acknowledgements
This work was supported by the National Key R&D Program of China ( 2021YFA1102600 to Y.W.), the National Natural Science Foundation of China (No. 82372407 to Y.W., No. 82002315 to Y.W., No. 82202766 to M.C.), the Natural Science Foundation of Hubei Province of China (No. 2022CFB686 to M.C.), the Fundamental Research Funds for the Central Universities ( 2024BRB021 to Y.W., YCJJ20252117 to Y.H.).
Peer review under the responsibility of editorial board of Bioactive Materials.
Supplementary data to this article can be found online at https://doi.org/10.1016/j.bioactmat.2026.07.050 .