Effect of Microbial Stimuli and Bone Morphogenetic Protein 2 on Ectopic Bone Formation

Abstract

Advancements in biomaterials design increasingly focus on material–host immune interactions as one of the strategies to promote new bone formation, referred to as osteoimmunomodulation. Recent studies indicate that inflammatory stimuli can synergize with growth factors such as bone morphogenetic protein 2 (BMP-2) to promote bone formation. Pathogen-associated molecular patterns (PAMPs) are motifs expressed by microbes that are recognized by immune cells and induce an immune-stimulatory response. In this study, we combined PAMPs with low-dose BMP-2 on a biphasic calcium phosphate (BCP) scaffold and evaluated its effect on ectopic bone formation in a subcutaneous implantation model. The PAMPs tested include gamma-irradiated whole microbes (γi-Staphylococcus aureus and γi-Candida albicans), a vaccine (Bacillus Calmette-Guérin containing Mycobacterium bovis), bacterial cell wall components (peptidoglycan [PGN], lipopolysaccharide [LPS], lipoteichoic acid, and Pam3CysSerLys4), an exopolysaccharide (Curdlan), and nucleic acid analogues (polyinosinic:polycytidylic acid [Poly(I:C)] and Cytidine–phosphate–guanosine [CpG]-containing oligonucleotides type C). Implants consisting of BCP, PAMPs, and BMP-2 were placed subcutaneously in rabbits and evaluated for ectopic bone formation after 5 weeks. Implants with only BMP-2 served as controls. Of the PAMPs tested, only PGN and BMP-2 showed a positive bone volume compared with the control, with borderline significance (+4.4%, p = 0.08). Decreased bone volume was seen for LPS (−7.4%, p = 0.03) and Poly(I:C) (−6.3%, p = 0.04). Fluorochrome labeling at weeks 2 and 3 assessed mineralization onset, revealing no mineralization in the first 2 weeks and some implants showing onset at week 3. We observed variability in ectopic bone formation across animals, associated with higher osteoclast numbers in those where ectopic bone occurred versus those that did not (p = 0.004). PAMPs can modulate bone formation, but their effects are variable, requiring further refinement to harness their osteoimmunomodulatory properties effectively. Additionally, we highlight osteoclasts’ important role in stimulating ectopic bone formation.

Impact Statement

Immune mediators have been shown to modulate both bone formation and bone loss. Pathogen-associated molecular patterns (PAMPs) are potent immunomodulators that elicit a wide range of immune-stimulatory responses. To determine whether PAMPs potentially function for osteoimmunomodulation to promote bone formation, we combined bone-substitute material with different PAMPs and low-dose bone morphogenetic protein 2 (BMP-2). Implants were tested in a challenging extraskeletal (ectopic) implantation model and evaluated for de novo bone formation. We saw a minimal effect of PAMPs on ectopic bone formation for most PAMPs tested in our model. Only peptidoglycan, a prominent bacterial cell wall component, in combination with BMP-2 resulted in a positive bone volume compared with BMP-2-only controls, with borderline significance. Future studies using more predictive in vivo models are needed to evaluate the potential of PAMPs as osteoimmunomodulatory agents. Additionally, our results suggest osteoclasts to be associated with differences in ectopic bone formation across animals and therefore a relevant parameter to be included in future studies using ectopic implantation models.