Recent evidence indicates the potential of gamma-irradiated (γi) Staphylococcus aureus to be used as an osteo-immunomodulator for bone regeneration. This study aims at characterizing the inflammatory milieu caused by the stimulation of γi S. aureus in immune cells and investigates its effects on MSC osteogenic differentiation. Furthermore, we aimed to recreate the immune-modulatory response exhibited by γi S. aureus by using a mixture of various synthetic pathogen recognition receptor (PRR) ligands consisting of TLR2, TLR8, TLR9, and NOD2 agonists. Human peripheral blood mononuclear cells (hPBMCs), isolated from healthy human donors, were exposed to γi S. aureus or seven different ligand mixtures. After 24 h, the conditioned medium (CM) from the hPBMCs was collected and its effects on hMSC osteogenic differentiation were investigated by assessing alkaline phosphatase (ALP) activity and matrix mineralization. The hPBMCs and their CM were also analyzed by bulk RNA sequencing and for cytokine secretion. CM from the γi S. aureus and the mixture consisting of Pam3CSK4, C-class CpG oligodeoxynucleotide (CpG ODN C), and murabutide targeting TLR2, TLR9, and NOD2 showed a fivefold increase in ALP and matrix mineralization in a donor-dependent manner. These effects were due to the upregulation of inflammatory signaling pathways, which led to an increase in cytokines and chemokines TNF, interleukin (IL)-6, IFN-γ, IL-1α, CXCL10, CCL18, CCL17, CXCL1, and CCL5. Upregulation of genes like BMP2R, BMP6R, BGLAP, and others contributed to the upregulation of osteogenic pathways in the hPBMCs stimulated with γi S. aureus and the aforementioned mix. Thus, formulations with mixtures of PRR ligands may serve as immune-modulatory osteogenesis-enhancing agents.

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.

Therapeutic pathogen recognition receptor (PRR) ligands are reaching clinical practice following their ability to skew the immune response in a specific direction. We investigated the effects of various therapeutic PRR ligands on bone cell differentiation and inflammation. Following stimu-lation, alkaline phosphatase (ALP) activity (Day 10), osteocalcin, osteonectin expression (Day 14), and calcium deposition (Day 21) were quantified in bone marrow-derived human mesenchymal stem cells (hMSCs). The osteoclastogenic response was determined by measuring tartrate-resistant acid phosphate (TRAP) activity in human monocytes. TNF-α, IL-6, IL-8, and IL-10 expressions were measured by enzyme-linked immunosorbent assay as an indicator of the ligands’ inflammatory properties. We found that nucleic acid-based ligands Poly(I:C) and CpG ODN C increased early ALP activity in hMSCs by 4-fold without affecting osteoclast formation. These ligands did not enhance expression of the other, late osteogenic markers. MPLA, Curdlan, and Pam3CSK4 did not affect osteogenic differentiation, but inhibited TRAP activity in monocytes, which was associated with increased expression of all measured cytokines. Nucleic acid-based ligands are identified as the most promising osteo-immunomodulators, as they favor early osteogenic differentiation without inducing an exaggerated immune-cell mediated response or interfering in osteoclastogenesis and thus can be potentially harnessed for multifunctional coatings for bone biomaterials.