CYR61 delivery promotes angiogenesis during bone fracture repair

Abstract

Compromised vascular supply and insufficient neovascularization impede bone repair, increasing risk of non-union. CYR61, Cysteine-rich angiogenic inducer of 61kD (also known as CCN1), is a matricellular growth factor that has been implicated in fracture repair. Here, we map the distribution of endogenous CYR61 during bone repair and evaluate the effects of recombinant CYR61 delivery on vascularized bone regeneration. In vitro, CYR61 treatment did not alter chondrogenesis or osteogenic gene expression, but significantly enhanced angiogenesis. In a mouse femoral fracture model, CYR61 delivery did not alter cartilage or bone formation, but accelerated neovascularization during fracture repair. Early initiation of ambulatory mechanical loading disrupted CYR61-induced neovascularization. Together, these data indicate that CYR61 delivery can enhance angiogenesis during bone repair, particularly for fractures with stable fixation, and may have therapeutic potential for fractures with limited blood vessel supply.

Fig. 1. Spatial expression of YAP, CCN1/CYR61, EMCN, and OSX in the fractured bone at 14 dpf.

a Exemplary Movat’s pentachrome staining 14 dpf; black box indicates ROI as magnified in b–d. b Magnification of fracture gap and adjacent bone marrow areas (ROI from (a); black box indicates ROI for (g). c, d Overview stainings. e, f Quantifications (n = 6). One-way repeated measures ANOVA was used to determine the statistical significance; p-values are indicated with *p < 0.05; ***p < 0.001. g Magnifications from endochondral part of fracture gap. Scale bars indicate 1 mm (a) and 500 µm (b–d) and 100 µm (g).

Fig. 2. CYR61 accelerates in vitro angiogenesis, but not chondrogenesis or osteogenesis of hMSCs.

a Experimental setup. Created in BioRender. Boerckel, J. (2025) https://BioRender.com/ c35l812. b Representative images of Safranin-O-staining and c quantitative measurement of chondrogenic pellet diameter at two weeks (n = 2–3). d Relative mRNA expression of RUNX2, SP7, COL1A1 and CYR61 after three weeks of osteogenic differentiation normalized to housekeeping gene and control. e 3D in vitro angiogenesis assay combing HUVECs and hMSCs (n = 5–9). Created in BioRender. Boerckel, J. (2025) https://BioRender.com/k29p512. f Exemplary images of tube formation at 3 days. g Quantification of relative GFP+ cell area, relative tube number and tube length. Mean ± SEM and individual data points. Two-way ANOVA was used to determine the statistical significance; p-values from multiple comparisons are indicated with *p < 0.05; **p < 0.01; ***p < 0.001. Scale bars indicate 500 µm (b) and 200 µm (f).

Fig. 3. CYR61 treatment did not enhance fracture callus bone or cartilage formation at 14 dpf.

a Experimental setup. Created in BioRender. Boerckel, J. (2025) https://BioRender.com/ l41q360. b 3D Representative reconstruction images of microCT analysis. c MicroCT – quantification of bone volume (BV) and bone volume fraction (bone volume/BV; total callus volume/TV; n = 4–8). d Exemplary images of Movat’s Pentachrome staining—yellow = mineralized bone; green = cartilage; magenta = bone marrow; red = muscle tissue. e Quantification of mineralized bone and cartilage area in gap (n = 4–8 distinct samples). Mean ± SEM and individual data points. Two-way ANOVA was used to determine the statistical significance; p-values from multiple comparisons are indicated with *p < 0.05; **p < 0.01. Scale bars indicate 500 µm (d).

Fig. 4. Treatment with exogenous CYR61 promotes revascularization in the fracture gap 14 dpf.

a Representative images of EMCN and OSX staining and b quantifications (n = 4‒7). c Quantifications and d representative images of F4/80 and SOX9 staining (n = 4‒7). Mean ± SEM and individual data points. Two-way ANOVA was used to determine the statistical significance; p-values from multiple comparisons are indicated with *p < 0.05; ***p < 0.001. In Two-way ANOVA, compliant fixation significantly decreased OSX staining, but multiple comparisons were not statistically significant (fix = fixation). Scale bars indicate 200 µm (a, d).

Fig. 5. CYR61 promotes vascular maturation with or without loading in vitro.

a Experimental setup highlighting the components of the 3D vascular network-on-a-chip system. The devices were loaded longitudinally (as indicated by the arrows), ensuring that all cells in the vascular compartment (green) experienced uniform, unidirectional compression with consistent strain. Created in BioRender. Boerckel, J. (2025) biorender.com/n15u686. b Representative images. Quantifications of (c) vessel length, (d) vascular area, (e) number of vessel junctions, and (f) number of vessel endpoints as measure for vascular connectivity (n = 3). Absolute values are given for the whole chip area. Mean ± SEM and individual data points. Two-way ANOVA was used to determine the statistical significance; p-values from multiple comparisons are indicated with *p < 0.05; **p< 0.01; ***p < 0.001. Scale bars indicate 500 µm.