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. 2023 Feb 1;31(2):420-434.
doi: 10.1016/j.ymthe.2022.10.007. Epub 2022 Oct 17.

IL-1Ra gene transfer potentiates BMP2-mediated bone healing by redirecting osteogenesis toward endochondral ossification

Joseph A Panos  1 , Michael J Coenen  2 , Christopher V Nagelli  2 , Erin B McGlinch  3 , Aysegul Atasoy-Zeybek  2 , Consuelo Lopez De Padilla  2 , Ryan F Coghlan  4 , Brian Johnstone  5 , Elisabeth Ferreira  6 , Ryan M Porter  6 , Rodolfo E De la Vega  7 , Christopher H Evans  8
Affiliations

IL-1Ra gene transfer potentiates BMP2-mediated bone healing by redirecting osteogenesis toward endochondral ossification

Joseph A Panos et al. Mol Ther. .

Abstract

An estimated 100,000 patients each year in the United States suffer severe disability from bone defects that fail to heal, a condition where bone-regenerative therapies could provide substantial clinical benefits. Although recombinant human bone morphogenetic protein-2 (rhBMP2) is an osteogenic growth factor that is clinically approved for this purpose, it is only effective when used at exceedingly high doses that incur substantial costs, induce severe inflammation, produce adverse side effects, and form morphologically abnormal bone. Using a validated rat femoral segmental defect model, we show that bone formed in response to clinically relevant doses of rhBMP2 is accompanied by elevated expression of interleukin-1 (IL-1). Local delivery of cDNA encoding the IL-1 receptor antagonist (IL-1Ra) achieved bridging of segmental, critical size defects in bone with a 90% lower dose of rhBMP2. Unlike use of high-dose rhBMP2, bone formation in the presence of IL-1Ra occurred via the native process of endochondral ossification, resulting in improved quality without sacrificing the mechanical properties of the regenerated bone. Our results demonstrate that local immunomodulation may permit effective use of growth factors at lower doses to recapitulate more precisely the native biology of healing, leading to higher-quality tissue regeneration.

Keywords: bone regeneration; endochondral ossification; gene transfer; interleukin-1 receptor antagonist.

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Conflict of interest statement

Declaration of interests The authors declare no competing interests.

Figures

None
Graphical abstract
Figure 1
Figure 1
Histology of CSDs in the presence or absence of rhBMP2 (A–D) Safranin O/fast green staining of rat femoral CSDs with (A and B) or without (C and D) rhBMP2. (E–L) Serial histology evaluation of CSDs using hematoxylin and eosin with and without rhBMP2 treatment between weeks 3 and 12 after surgery. Dashed lines (I−L) indicate the bone perimeter; arrows (F, G, and J, inset) indicate multinucleated giant cells. For (A)−(D), magnification 4×, scale bars represent 500 μm; inset 20×, scale bars represent 100 μm. For (E)−(L), magnification 1.25×, scale bars represent 1 mm; inset 40×, scale bars represent 50 μm.
Figure 2
Figure 2
Transient osteoclast activation in CSDs treated with rhBMP2 (A and B) TRAP staining (A) and quantification (B) of CSDs with and without rhBMP2 treatment. For all treatments and time points, n = 3. For (B), one-way ANOVA with correction for multiple comparisons by controlling the false discovery rate (FDR). ∗∗∗∗p < 0.0001. For (A), magnification 10×; scale bars represent 200 μm.
Figure 3
Figure 3
In vivo induction of IL-1 with rhBMP2 treatment (A and B) Acute induction of inflammatory genes with rhBMP2 treatment. IL-1α, interleukin-1 alpha; IL-1β, interleukin-1 beta; IL-6, interleukin-6; TNF-α, tumor necrosis factor alpha. n = 3 per treatment per time point. (C–F) Protein expression and bioactivity of IL-1. Quantification of total IL-1 and individual isoforms was performed on protein lysate samples isolated from femoral CSDs with and without rhBMP2 treatment using ELISA, normalized to total protein content. Activity of detected IL-1 in protein lysate samples was determined using HEK-Blue IL-1 reporter cells. n = 4–5 per treatment per time point. For (A)−(F), Student’s t test or non-parametric equivalent; ∗p < 0.05, ∗∗∗p < 0.001.
Figure 4
Figure 4
IL-1Ra promotes bone regeneration with rhBMP2 (A) CSDs were treated with high-dose rhBMP2 plus sham, delayed, or immediate dosing regimens of recombinant IL-1Ra, as indicated in the experimental timeline. Blue dashed lines indicate a sham injection of PBS, and green dashed lines indicate injection of recombinant IL-1Ra. n = 3 per treatment. (B–D) Safranin O/fast green staining and corresponding radiographs (top) at week 3 after surgery. (E–G) TRAP staining of high-dose rhBMP2 plus sham, delayed, or immediate recombinant IL-1Ra. (H–J) Low-dose rhBMP2 and delayed recombinant IL-1Ra safranin O/fast green histology and radiographs (top) at week 8 after surgery. For (B)−(D) and (H)−(J), magnification 4×, scale bars represent 500 μm; inset 20×, scale bars represent 100 μm. For (E)−(G), magnification 10×, scale bars represent 200 μm. Dashed lines (H and I) indicate the bone perimeter.
Figure 5
Figure 5
Analysis of adenoviral transgene activity and bone formation in animals treated with low-dose rhBMP2 and Ad.IL-1Ra (A) In vivo adenoviral luciferase transgene activity in femoral CSDs as measured by bioluminescence after administration of D-luciferin. n = 3. (B) Quantification of BV formed in CSDs at week 12 after surgery. Filled circles indicate samples that demonstrated radiographic bridging by week 12; unfilled circles indicate samples that did not bridge by week 12. (C) MicroCT reconstructions with central cross-sections at week 12 after surgery. For (B) and (C), n = 3–9 per treatment, as indicated in (B). For (A) and (B), one-way ANOVA with correction for multiple comparisons by controlling the FDR. For (A), differences between time points are denoted by a connecting letters report, where groups that share the same letter do not differ statistically. For (B), ∗∗p < 0.01, ∗∗∗∗p < 0.0001.
Figure 6
Figure 6
Microarchitectural, biomechanical, and histological analyses of bones treated with low-dose rhBMP2 and Ad.IL-1Ra (A−D) Bone microarchitectural analysis at week 12 in CSD treated with low-dose rhBMP2 and Ad.IL-1Ra or high-dose rhBMP2. n = 9 per treatment. (E−G) Mechanical properties at week 12 after surgery, as determined via biomechanical torsional testing in CSDs treated with low-dose rhBMP2 and Ad.IL-1Ra or high-dose rhBMP2. n = 6 per treatment. The dashed lines indicate the relevant values for normal rat femora. (H−K) Safranin O/fast green staining of CSD regeneration at week 12 after surgery. n = 3 per treatment. Magnification 1.25×, scale bars represent 1 mm; inset 20×, scale bars represent 100 μm. For (A)−(G), Student’s t test or non-parametric equivalent. ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001. Dotted lines in (A), (F), and (G) indicate values for native uninjured femora.
Figure 7
Figure 7
Low-dose rhBMP2 and Ad.IL-1Ra restore endochondral bone regeneration CSDs were created in Col2-fLuc transgenic rats and treated with high-dose rhBMP2 or low-dose rhBMP2 and Ad.IL-1Ra. In vivo bioluminescence imaging was performed weekly between 2 and 7 weeks after surgery. Dashed lines indicate the boundary of the femur. (A and B) With quantification of bioluminescence activity (A) and normalized to the signal from vertebrae in the tail (B). n = 3 per treatment. (C) WT rats were treated with high-dose rhBMP2 or low-dose rhBMP2 and Ad.IL-1Ra, and serial serum draws were performed 1 day before surgery and weekly between 2 and 7 weeks after surgery. Circulating collagen X fragments (Cxm) were measured and normalized to preoperative values. n = 3 per treatment. (D and E) Safranin O/fast green staining of animals treated with high-dose rhBMP2 or low-dose rhBMP2 and Ad.IL-1Ra at week 4 after surgery reveals woven bone formation with high-dose rhBMP2 treatment (D), with focal evidence of cartilage at the leading edge of bone formation in animals treated with low-dose rhBMP2 and Ad.IL-1Ra (E). n = 3 per treatment. For (B) and (C), two-way ANOVA with correction for multiple comparisons by controlling the FDR. ∗p < 0.05, ∗∗p < 0.01. For (D) and (E), magnification 1.25×, scale bars represent 1 mm; insets 20×, scale bars represent 100 μm.
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