Antimicrobial bone cement device and methods of making and using the same

Abstract

Provided herein is a novel bone cement composition with inherent and added antimicrobial activity. The bone cement comprises a viscous porogen comprising honey and other bioactive agents. Also, provided here are methods of making and using the bone cement.

Description

TITLEANTIMICROBIAL BONE CEMENT DEVICE AND METHODS OF MAKING AND USING THE SAME CROSS REFERENCE TO RELATED APPLICATION

[0001] This application claims priority to U.S. Provisional Patent Application Serial No.63 / 753,805 filed February 4, 2025, which is incorporated by reference herein in its entirety.BACKGROUND1. Field

[0002] The present disclosure relates to antimicrobial bone cement and method of making and using the bone cement for therapeutic purposes.2. Discussion of Related Art

[0003] Bone injuries, particularly fractures, are a significant global health concern. In 2019, there were approximately 178 million new fracture cases worldwide. Osteoporosis, a condition characterized by decreased bone density, contributes notably to these injuries. The International Osteoporosis Foundation reports that osteoporosis causes more than 8.9 million fractures annually, equating to an osteoporotic fracture every three seconds. As the global population ages, the incidence of hip fractures is projected to double by 2050, further emphasizing the growing burden of bone injuries.

[0004] Bone cements are essential in orthopedic surgeries for stabilizing fractures and securing implants. The global bone cement and glue market was valued at approximately USD 1.3 billion in 2022 and is projected to reach USD 2.2 billion by 2032, growing at a compound annual growth rate (CAGR) of 5.4%. This growth is driven by an increasing number of surgical procedures, rising prevalence of osteoporosis, and advancements in medical technology. Polymethyl methacrylate (PMMA) remains the most commonly used bone cement due to its effectiveness in providing mechanical support and stability in various orthopedic applications.SUMMARY

[0005] In some aspects, the current disclosure encompasses a bone cement comprising: a) a polymer scaffold; and b) a viscous porogen comprising one or more types of honey, or derivatives thereof. In some aspects, the polymer scaffold comprises one or more polymers. In some aspects, the one or more polymers comprise natural polymers, or synthetic polymers, or any combination thereof. In some aspects, the one or more polymers are biocompatible and / or biodegradable. Non-limiting examples of suitable polymers include poly(methyl methacrylate) (PMMA), polycaprolactone (PCL), polydioxanone (PDO), poly (glycolic acid)301541711 - 1 -(PGA), poly(L-lactic acid) (PLA), poly(lactide-co-glycolide) (PLGA), poly(L-lactide) (PLLA), poly(D.L-lactide) (P(DLLA)), poly(ethylene glycol) (PEG), poly(e-caprolactone) (PCL), montmorillonite (MMT), poly(L-lactide-co-E-caprolactone) (P(LLA-CL)), poly(e-caprolactone-co-ethyl ethylene phosphate) (P(CL-EEP)), poly[bis(p-methylphenoxy) phosphazene] (PNmPh), poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), poly(ester urethane) urea (PEUll), poly(p-dioxanone) (PPDO), polyurethane (Pll), polyethylene terephthalate (PET), poly(ethylene-co-vinylacetate) (PEVA), poly(ethylene oxide) (PEO), poly(phosphazene), poly(ethylene-co-vinyl alcohol), a polymer nanoclay nanocomposite; a halogenated polymer solution containing metal compounds (e.g., graphite), poly(ethylenimine), grafted cellulosics, poly(ethyleneoxide), poly vinylpyrrolidone, or polystyrene (PS), or any combination thereof.ln some aspects, the polymer scaffold comprises PMMA.In some aspects, the bone cement comprises about 10% to about 90% PMMA by weight.

[0006] In some aspects, the bone cement comprises about 15% to about 60% by weight of the one or more types of honey or derivatives thereof. In some aspects, the bone cement further comprises about 0.01% to about 20% by weight of a radiopacifier. In some aspects, the bone cement further comprises one or more bioactive agents. In some aspects, the viscous porogen further comprises one or more hydrophilic polymers. Non-limiting examples of hydrophilic polymers comprise polyethylene glycol (PEG), hyaluronic acid, chitosan, alginate, polyvinyl alcohol (PVA), polyvinylpyrrolidone (PVP), or collagen, or any combination thereof.

[0007] In some aspects, the bone cement comprises: about 10% to about 90% by weight of poly(methyl methacrylate) (PMMA); about 0.01% to about 20% by weight of a radiopacifier; about 15% to about 60% by weight of one or more types of honey, or derivatives thereof; and about 0.001% to about 5% by weight of one or more bioactive agent.

[0008] Non-limiting examples of types of honey that may be incorporated in the bone cement include manuka honey, raw honey, tualang honey, buckwheat honey, acacia honey, eucalyptus honey, lavender honey, thyme honey, Sidr honey, heather honey, wildflower honey, or jarrah honey, or any derivative thereof, or any combination thereof. In some aspects, the bone cement comprises about 15% to about 30% by weight of the one or more types of honey or derivatives thereof. In some aspects, the bone cement comprises about 30% by weight of the one or more types of honey or derivatives thereof. In some aspects, the bone cement comprises about 30% to about 60% by weight of the one or more types of honey or derivatives thereof. In some aspects, the bone cement comprises about 50% by weight of the one or more types of honey or derivatives thereof. In some aspects, the bone cement is a nonload bearing formulation. In some aspects, the one or more types of honey is medical grade honey.301541711 - 2 -

[0009] In some aspects, the bone cement has inherent antimicrobial property against one or more bacterial species, one or more fungal species, and / or one or more viral species. In some aspects, the bacterial species comprise a streptococcal species, staphylococcal species including Staphylococcus aureus, methicillin-susceptible S. aureus (MSSA), and methicillin-resistant S. aureus (MRSA), Enterococcus species, Escherichia coli, Enterobacter species, Klebsiella pneumoniae, Pseudomonas aeruginosa, Salmonella species, Helicobacter pylori, or Cutibacterium acnes, or any combination thereof. In some aspects, the one or more viral species comprise varicella-zoster virus, rubella virus, influenza virus, herpes simplex virus, respiratory syncytial virus, or human immunodeficiency virus, or any combination thereof. In some aspects, the one or more fungal species comprise Candida albicans, Candida auris, Rhodotorula species, Aspergillus species, Mucor species, or dermatophytes such as Trichophyton rubrum, or any combination thereof.

[0010] In some aspects, the one or more bioactive agents is an antimicrobial agent, an immunotherapeutic, and / or a vaccine. In some aspects, the antimicrobial agent comprises antibacterial, antiviral, anti-parasitic, or an antifungal agent, or any combination thereof. In some aspects, the antimicrobial agent is incorporated into the viscous porogen. In some aspects, the antimicrobial agent is incorporated into a delivery particle incorporated into the bone cement.

[0011] In some aspects, the antibacterial agent comprises ampicillin, amoxicillin, benzylpenicillin, dalbavancin, oritavancin, phenoxymethylpenicillin, bacampicillin, pivampicillin, carbenicillin, cioxacillin, cyclacillin, dicloxacillin, methicillin, oxacillin, piperacillin, ticarcillin, flucioxacillin, cefuroxime, cefetamet, cefetrame, cefixine, cefoxitin, ceftazidime, ceftizoxime, latamoxef, levofloxacin, cefoperazone, ceftriaxone, cefsulodin, cefotaxime, cephalexin, cefaclor, cefadroxil, cefalothin, cefazolin, cefpodoxime, ceftibuten, aztreonam, tigemonam, erythromycin, dirithromycin, roxithromycin, azithromycin, clarithromycin, clindamycin, paldimycin, lincomycin, vancomycin, moxifloxacin, metronidazole benzoate, spectinomycin, tobramycin, paromomycin, metronidazole, tinidazole, ornidazole, amifloxacin, cinoxacin, ciprofloxacin, difloxacin, enoxacin, fleroxacin, norfloxacin, ofloxacin, linezolid, temafloxacin, teromyocin, doxycycline, minocycline, tetracycline, daptomycin, chlortetracycline, oxytetracycline, methacycline, rolitetracyclin, nitrofurantoin, nalidixic acid, gentamicin, rifampicin, amikacin, netilmicin, imipenem, cilastatin, chloramphenicol, furazolidone, nifuroxazide, sulfadiazin, sulfametoxazol, trimethoprim-sulfamethoxazole, bismuth subsalicylate, colloidal bismuth subcitrate, gramicidin, mecillinam, cloxiquine, dichlorobenzylalcohol, povidone, sugars, mucopolysaccharides, chlorobutanol, quaternary ammonium compounds such as benzalkonium chloride, organic mercurials, parahydroxy benzoates, aromatic alcohols, halogenated phenols, sorbic acid, benzoic acid, dioxin, EDTA,301541711 - 3 -BHT, BHA, TBHQ, gallate esters, NDGA, tocopherols, gum guaiac, lecithin, boric acid, citric acid, p-Hydroxy benzoic acid esters, propionates, sulfur dioxide and sulfites, nitrates and nitrites of potassium and sodium, diethyl pyrocarbonate, sodium diacetate, diphenyl, hexamethylene tetramine o-phenyl phenol, or sodium o-phenylphenoxide, or any variant thereof, or any combination thereof.

[0012] In some aspects, the bone cement has antimicrobial properties against Staphylococcal species (including Staphylococcus aureus, methicillin-susceptible S. aureus (MSSA), and methicillin-resistant S. aureus (MRSA)), coagulase-negative Staphylococci (including Staphylococcus epidermidis), Streptococcal species, Enterococcus species, Cutibacterium acnes, Corynebacterium striatum, Escherichia coli, Klebsiella pneumoniae, Enterobacter complex, Candida albicans and other yeast, Aspergillus species, and / or Pseudomonas aeruginosa. In some aspects, the antifungal agent comprises fluconazole, itraconazole, ketoconazole, voriconazole, amphotericin B, nystatin, clotrimazole, terbinafine, posaconazole, griseofulvin, caspofungin, micafungin, anidulafungin, rezafungin, or flucytosine, or any combination thereof. In some aspects, the antiviral agent comprises acyclovir, valacyclovir, ganciclovir, valganciclovir, tenofovir, entecavir, remdesivir, ritonavir, lopinavir, glecaprevir, pibrentasvir, sofosbuvir, foscarnet, oseltamivir, zanamivir, dolutegravir, raltegravir, maraviroc, enfuvirtide, palivizumab, sotrovimab, bebtelovimab, or interferons, or any combination thereof. In some aspects, the antiparasitic agent comprises chloroquine, artemether-lumefantrine, primaquine, metronidazole, tinidazole, nitazoxanide, pentamidine, albendazole, mebendazole, ivermectin, praziquantel, sodium stibogluconate, amphotericin B, eflornithine, nifurtimox, benznidazole, or diethylcarbamazine (DEC), or any combination thereof.

[0013] In some aspects, the bone cement comprises a vaccine. In some aspects, the vaccine is incorporated into the viscous porogen. In some aspects, the vaccine is incorporated into a delivery particle incorporated into the bone cement. In some aspects, the vaccine is a live attenuated vaccine, a killed microorganism, a protein-based vaccine, a polysaccharide-protein conjugate vaccine, a capsular polysaccharide vaccine, a toxoid based vaccine, a multicomponent vaccine, killed whole cell vaccine, a DNA based vaccine, or a mRNA vaccine, or any combination thereof.

[0014] In some aspects, the vaccine is against Staphylococcus aureus, methicillin-susceptible S. aureus (MSSA), methicillin-resistant S. aureus (MRSA), coagulase-negative Staphylococci (including Staphylococcus epidermidis), Streptococcal species, Enterococcus species, Cutibacterium acnes, Corynebacterium striatum, Escherichia coli, Klebsiella pneumoniae, Enterobacter complex, Candida albicans and other yeast, Aspergillus species, and / or Pseudomonas aeruginosa. In some aspects, the vaccine comprises inactivated Staphylococcus aureus, recombinant proteins vaccines: ClfA, IsdB, and SaeR or other301541711 - 4 -surface / intracellular proteins of interest, conjugates vaccines thereof, and / or DNA or mRNA vaccines encoding a surface antigen or intracellular antigen of Staphylococcus aureus, or any combination thereof.

[0015] In some aspects, the delivery particle is a liposome, a solid-lipid nanoparticle (SLN), a nanostructured lipid carrier, poly(lactic-co-glycolic acid) (PLGA) nanoparticles, a chitosan nanoparticle, a polystyrene nanoparticle, a metallic nanoparticle, a virus like particle (VLP), a ferritin nanoparticle, an albumin nanoparticle, a silica based nanoparticle, a viral particle, a dextran nanoparticle, an alginate microparticle, or an exosome. In some aspects, the delivery particle exhibits delayed dissolution compared to the viscous porogen. In some aspects, the delivery particle is a sustained release delivery particle.

[0016] In some aspects, the bone cement has a viscosity of 1,000 to 10,000 cp. In some aspects, the bone cement has a stiffness of about 1 MPa to about 2000MPa. In some aspects, the radiopacifier is barium sulphate, or zirconium dioxide, or both.

[0017] In some aspects, the one or more bioactive agents comprises both an antimicrobial agent and a vaccine. In some aspects, the one or more bioactive agents comprises an immunotherapeutic. In some aspects, the immunotherapeutic is an immunostimulator, costimulatory molecules, dendritic cell therapeutic, modified cell therapeutic (e.g., CAR-T cells), cytokine, adoptive T-cell therapeutic, or checkpoint inhibitors, or any combination thereof.

[0018] In some aspects, the bone cement may further comprise additional components, nonlimiting examples of which include stem cells, osteocytes, bone tissue, connective tissue cells, bioactive glass, fibrin, collagen, bioactive peptides, growth factors, calcium phosphate, or one or more trace metals, or any combination thereof. In some aspects, the bone cement exhibits improved interactions with host tissues in comparison to a bone cement without a viscous porogen. In some aspects, the bone cement is for treating a bone defect.

[0019] In some aspects, the current disclosure also encompasses a bioactive agent depot system comprising a bone cement as described herein.

[0020] Disclosed herein is a method of treating a bone defect in a subject in need thereof, the method comprising contacting the bone with a bone cement as described herein. In some aspects, the bone defect is a simple fracture, compound fracture, comminuted fracture, transverse fracture, oblique fracture, spiral fracture, stress fracture, pathological fracture, avulsion fracture, impacted fracture, segmental fracture, compression fracture, hairline fracture, torus fracture, bone cysts, osteoporosis, osteomalacia, Paget's disease, osteogenesis imperfecta, fibrous dysplasia, osteomyelitis, bone tumors, bone necrosis, metabolic bone diseases, congenital bone defects, trauma-induced bone loss, or bone graft failure, or any combination thereof. In some aspects, the bone cement has a bead, pellet, film,301541711 - 5 -disc, sheet, or layered architecture, or can be molded in the operating room to a custom geometry.

[0021] In some aspects, the current disclosure encompasses a method of fixing an implant on a bone in a subject in need thereof, the method comprising contacting the bone with the implant via a bone cement as described herein. In some aspects, the current disclosure encompasses a method of preventing or treating a bone site or a bone prosthetic site infection, the method comprising contacting the bone site or the bone prosthetic site with a bone cement as described herein.

[0022] In some aspects, disclosed herein is a method of localized delivery of a bioactive agent at a site in a subject in need thereof, the method comprising contacting the site with a bone cement as described herein, or implanting a bioactive agent depot system as described herein.

[0023] In some aspects, the current disclosure encompasses a bone cement comprising: about 10% to about 90% by weight of poly(methyl methacrylate) (PMMA); about 0.01% to about 20% by weight of a radiopacifier comprising barium sulfate or zirconium dioxide or both; about 15% to about 50% by weight of one or more types of honey, or derivatives thereof; and about 0.001% to about 5% by weight of one or more immunotherapeutic agents comprising one or more of, an antigen, an adjuvant, a chemokine, or any combinations thereof. In some aspects, the bone cement comprises about 30% by weight of the one or more types of honey or derivatives thereof. In some aspects, the bone cement further comprises one or more antibiotics. In some aspects, the antibiotic comprises about 3% by weight of vancomycin. In some aspects, the antigen comprises a microbial antigen, tumor-associated antigens, neoantigens, peptide antigens, protein antigens, or any combination thereof. In some aspects, the antigen is a microbial antigen comprising a live attenuated microorganism, an inactivated microorganism, a heat-killed microorganism, a chemically inactivated microorganism, an antibiotic killed microorganism, a microbial lysate, a microbial subunit antigen, or a fragment, epitope, or combination thereof. In some aspects, the microbial antigen comprises killed S. aureus. In some aspects, the one or more immunotherapeutic comprises killed S. aureus, granulocyte-macrophage colony-stimulating factor or GM-CSF. In some aspects, the adjuvant comprises CpG.

[0024] In some aspects, the bone cement comprises: about 10% to about 90% by weight of PMMA; about 0.5% to about 20% by weight of radiopacifier comprising barium sulfate and / or zirconium dioxide; about 15% to about 50% by weight of the one or more types of honey, or derivatives thereof; about 1% to about 5% by weight of an antibiotic; and about 0.001% to about 5% by weight of one or more immunotherapeutic agents comprising killed or attenuated S. aureus, adjuvant comprising CpG, and a chemokine comprising GM-CSF.301541711 - 6 -

[0025] In some aspects, the current disclosure encompasses a method of making a bone cement, comprising: 1) preparing a precursor solution comprising about 10% to about 90% by weight of PMMA, about 10% to about 35% by weight of MMA, about 0.5% to about 20% by weight of a radiopacifier, and about 10% to about 60% by weight of one or more types of honey, or derivatives thereof; 2) homogenizing the precursor solution; and 3) providing conditions suitable to form a polymer construct.

[0026] In some aspects, the method further comprises one or more of the steps of shaping, molding, curing, gelling, or washing, or any combination thereof, of the polymer. In some aspects, the method further comprises adding a bioactive agent to the bone cement.BRIEF DESCRIPTION OF THE DRAWINGS

[0027] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure. Aspects of the present disclosure may be better understood by reference to one or more of these drawings in combination with the detailed description of specific aspects presented herein:

[0028] FIG. 1 is a bar graph showing the device density as a function of amount of honey incorporation.

[0029] FIG. 2 is a bar graph showing data from device leaching studies (n=3 per biomaterial and antibiotic loading condition). Comparisons were made via Student’s t-test.

[0030] FIG. 3 is a bar graph showing the amount of mass lost following leaching cylindrical lABCs as a function of honey in an aqueous environment for 48 hours (n=5-6). Comparisons were made via ANOVA with post-hoc Tukey’s Honestly Significant Difference.

[0031] FIG. 4A provides representative cross-sectional microcomputed tomography images of conventional bone cement (PMMA) and a formulation of IABC (IABC50).

[0032] FIG. 4B provides representative three-dimensional reconstruction microcomputed tomography images of conventional bone cement (PMMA) and a formulation of IABC (IABC50). Color has been digitally added to three-dimensional reconstructions to better illustrate topography.

[0033] FIG. 5A is a bar graph showing radiographic measurements of total porosity of PMMA and IABC using three-dimensional reconstructions (n=3 per group).

[0034] FIG. 5B is a bar graph showing percent open pores in PMMA and IABC using three-dimensional reconstructions (n=3 per group).

[0035] FIG. 6A is a representative photograph of disc diffusion testing using PMMA and IABC devices loaded with or without gentamicin.301541711 - 7 -

[0036] FIG. 6B is a bar graph showing zone of inhibition results for PMMA (n=12) and IABC (n=11) devices with and without gentamicin. Comparisons were made via Student’s t-test.

[0037] FIGs. 7A-7B provide disc diffusion results of conventional bone cement (PMMA) and inherently antimicrobial bone cement (IABC) with 30 and 50% honey (IABC30 and IABC50) evaluated against: FIG. 7A methicillin-resistant Staphylococcus aureus JE2 and FIG. 7B methicillin-susceptible Staphylococcus aureus UAMS-1. Devices were either unloaded (no incorporated antibiotic “none”) or loaded with vancomycin or gentamicin per formulations in Table 1. Comparisons were made via ANOVA with post-hoc Tukey’s Honestly Significant Difference.

[0038] FIG. 8A provides a representative photograph of disc diffusion testing against P. aeruginosa using PMMA and IABC devices loaded with gentamicin.

[0039] FIG. 8B provides zone of inhibition results for PMMA and IABC devices (n=4 per group) with and without gentamicin. Comparisons were made via ANOVA with post-hoc Tukey’s Honestly Significant Difference.

[0040] FIG. 9A provides a gross image of IABC50 prior and after compression to material failure.

[0041] FIG. 9B is a bar graph providing the compressive strength of IABC50. Compressive testing was performed with n=5-6 per group. Comparisons were made via ANOVA with post-hoc Tukey’s Honestly Significant Difference.

[0042] FIG. 9C is a bar graph providing the compressive modulus of IABC50. Compressive testing was performed with n=5-6 per group. Comparisons were made via ANOVA with post-hoc Tukey’s Honestly Significant Difference.

[0043] FIG. 10A provides a representative image of S. aureus pigmentation changes in response to IABC without any additional antibiotics on a tryptic soy agar plate.

[0044] FIG. 10B provides growth curves of wild type S. aureus (wt, closed circle) and S. aureus with functional crtN deletion (AcrtN, open circle) in media with no honey (black lines), 5% honey (light yellow lines), or 10% honey (dark yellow lines) over 10 hours of incubation at 37 °C. The red arrow depicts the delayed growth between wt and AcrtN at the two different honey conditions.

[0045] FIG. 11 provides a schematic of the IABC system with embedded microparticles for staged drug delivery.301541711 - 8 -

[0046] FIG. 12 provides formulations of bone cement with or without the addition of conventional antibiotics at varying concentrations of medical-grade honey, resulting in nine groups.

[0047] FIGs. 13A-13B show that honey functions as a porogen when combined with poly(methyl methacrylate). FIG. 13A provides photographs of the lABCs. Scale bar = 3 mm.FIG. 13B shows representative scanning electron micrographs of device surfaces. Scale bar = 250 pm.

[0048] FIGs. 14A-14C show radiographic analysis of the IABC. FIG. 14A provides representative microcomputed tomography reconstructions. Scale bar = 1 mm. FIG. 14B provides calculated total porosity (%). FIG. 14C provides percentage of pores which are open (communicate outside of the device) versus closed (no communication). Error bars = standard deviation and n = 4 per group.

[0049] FIGs. 15A-15D provide data for mechanical testing of PMMA-H compared to PMMA using compressive testing of cylinders (n=6 per group). FIG. 15A is a bar graph showing data for compressive strength. FIG. 15B is a bar graph showing data for compressive modulus: bending testing of bars (n=5 per group). FIG. 15C is a bar graph showing data for bending strength. FIG. 15D is a bar graph showing data for bending modulus. Error bars = standard deviation.

[0050] FIGs. 16A-16B provide data on cumulative antibiotic elution. FIG. 16A is a bar graph showing data for the antibiotic Vancomycin. FIG. 16B is a bar graph showing data for the antibiotic Gentamicin. Circles are groups with 3 wt% antibiotic (loaded) and triangles are groups with 0 wt% antibiotic (unloaded). All loaded groups had significantly greater elution than unloaded groups at all time points (p<0.01). Loaded PMMA-HH had significantly greater elution than loaded PMMA for both vancomycin and gentamicin at all time points. Loaded PMMA-LH had significantly greater gentamicin elution than loaded PMMA for all time points. Error bars = standard deviation and n=4 per group per timepoint.

[0051] FIGs. 17A-17C show bacterial inhibition zone as measured by the standard Kirby-Bauer disc diffusion method. FIG. 17A is a bar graph showing the zone of inhibition in mm for methicillin-susceptible Staphylococcal aureus (MSSA). FIG. 17B is a bar graph showing the zone of inhibition in mm for methicillin-resistant Staphylococcal aureus (MRSA). FIG. 17C is a bar graph showing the zone of inhibition in mm for Staphylococcus epidermidis. No bars are visible for groups that had no measurable zones of inhibition. Error bars = standard deviation and n=4 per group per timepoint.

[0052] FIGs. 18A-18B show S. aureus U1 luminescence normalized to sterile media (background) from the first 5 hours of a high inoculum challenge. FIG. 18A is a bar graph301541711 - 9 -showing data for discs without antibiotic loading. At 1 hr, all groups are significantly different from one another. By 2hr, PMMA-HH is no longer significantly different from sterile PMMA (p>0.05). PMMA has greater luminescence than sterile PMMA at all time points. PMMA-LH is not different than sterile PMMA at 3 hr but otherwise always has greater luminescence. FIG.18B is a bar graph showing data for antibiotic-loaded discs. By 1 hr, PMMA-HH has significantly less luminescence than PMMA. By 3hr, PMMA-HH is no longer significantly different than sterile PMMA (p>0.05). By 4hr and onward, both PMMA-LH and PMMA-HH are no longer significantly different than sterile PMMA (p>0.05) but PMMA continues to have higher luminescence (p<0.05). FIG. 18C is a bar graph showing data for comparison of unloaded PMMA-HH from (A) and vancomycin-loaded PMMA from (B). There was no significant difference in luminescence between the two groups at any timepoint (p>0.05). Error bars = standard deviation and n=4 per group per timepoint.

[0053] FIGs. 19A-19B show bacteria in the surrounding media (planktonic) or attached to biomaterial discs (biofilm) 24 hours after a high inoculum challenge with S. aureus 111. FIG.19A is a bar graph showing planktonic bacterial burden. FIG. 19B is a bar graph showing biofilm bacterial burden. NG= no growth. Error bars = standard deviation and n=4 per group.

[0054] FIG. 20 provides temperature and setting kinetics of IABC compared to conventional bone cement poly(methyl methacrylate) (PMMA) as performed per ISO 5833.

[0055] FIGs. 21A-21C show pilot mouse subcutaneous implantation studies of Inherently Antimicrobial Bone Cement (IABC) and Inherently Antimicrobial Bone Cement Immunotherapy (IABCX). FIG. 21A is a bar graph showing implant nodule size seven days after surgery. FIG.21 B is a bar graph showing serum cytokines 28 days after surgery. FIG. 21 C provides subcutaneous implant histology with H&E stain at 20X magnification 28 days after surgery.

[0056] The drawing figures do not limit the present disclosure to the specific aspects disclosed and described herein. The drawings are not necessarily to scale, emphasis instead being placed on clearly illustrating principles of certain aspects of the present disclosure.DETAILED DESCRIPTION

[0057] The following detailed description references the accompanying drawings that illustrate various aspects of the present disclosure. The drawings and description are intended to describe aspects and aspects of the present disclosure in sufficient detail to enable those skilled in the art to practice the present disclosure. Other components can be utilized and changes can be made without departing from the scope of the present disclosure. The following description is, therefore, not to be taken in a limiting sense.301541711 - 10 -

[0058] In some aspects, the current disclosure describes the development of a new clinical bone cement with inherent antimicrobial activity, for the prevention and treatment of orthopedic surgical site infections. This synthetic polymer / natural polysaccharide viscous porogen composite formulation, or Inherently Antimicrobial Bone Cement (IABC), is novel in that it is inherently antimicrobial; has a unique porous architecture; and is able to deliver bioactive agents including conventional antimicrobials, immunotherapeutics, drugs and / or vaccines with improved kinetics compared to traditional bone cements. Surprisingly, IABC was found to have higher porosity, open porous networks that allow efficient leaching of incorporated bioactive agents, and tunable material and porosity characteristic than traditional poly(methyl methacrylate) (PMMA) based bone cement. This new biomaterial has many exciting clinical applications. Non-limiting examples include use as a device to maintain space and sterilize infected bones and joints; application to fix new devices into bones, such as prostheses, and prevention of subsequent surgical-site infection; or implantation to locally treat infection. In addition to better delivery of one or more bioactive agents, the material allows for incorporation of delivery particles such that different therapeutics are released at different timepoints. For example, antibiotics can be released quickly to sterilize an infected site followed by delayed release of immune-stimulating components, such as adjuvants and specific antigens, that are incorporated into microparticles, to promote adaptive immunity for prevention of infection relapse. The improved porosity of the bone cement due to the inclusion of the viscous porogen not only improves bioactive agent release, but also improves device interactions with host tissues by improving the ability of patient's tissues to grow on / grow into the device and promote biocompatibility.I. Terminology

[0059] The phraseology and terminology employed herein are for the purpose of description and should not be regarded as limiting. For example, the use of a singular term, such as, “a” is not intended as limiting of the number of items. Also, the use of relational terms such as, but not limited to, “top,” “bottom,” “left,” “right,” “upper,” “lower,” “down,” “up,” and “side,” are used in the description for clarity in specific reference to the figures and are not intended to limit the scope of the present disclosure or the appended claims.

[0060] Any term of degree such as, but not limited to, “substantially” as used in the description and the appended claims, should be understood to include an exact, or a similar, but not exact configuration. For example, “a substantially planar surface” means having an exact planar surface or a similar, but not exact planar surface.301541711 - 11 -

[0061] The terms “comprising,” “including,” and “having” are used interchangeably in this disclosure. The terms “comprising,” “including,” and “having” mean to include, but not necessarily be limited to the things so described.

[0062] The terms “or” and “and / or,” as used herein, are to be interpreted as inclusive or meaning any one, or any combination. Therefore, “A, B, or C” or “A, B, and / or C” mean any of the following: “A,” “B,” or “C”; “A and B”; “A and C”; “B and C”; “A, B, and C.” An exception to this definition will occur only when a combination of elements, functions, steps, or acts are in some way inherently mutually exclusive.

[0063] Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art to which this disclosure belongs. The following references provide one of skill with a general definition of many of the terms used in this disclosure: Singleton et al., Dictionary of Microbiology and Molecular Biology (3rd ed. 2006); The Cambridge Dictionary of Science and Technology (Walker ed., 1990); The Glossary of Genetics, 5th Ed., R. Rieger et al. (2008), The HarperCollins Dictionary of Biology (1991), all of which are incorporated by reference herein. As used herein, the following terms have the meanings ascribed to them below, unless specified otherwise.

[0064] The phraseology and terminology employed herein are for the purpose of description and should not be regarded as limiting. When introducing elements of the present disclosure or the preferred aspects(s) thereof, the articles “a,” “an,” “the,” and “said” are intended to mean that there are one or more of the elements. The terms “comprising,” “including,” and “having” are intended to be inclusive and mean that there may be additional elements other than the listed elements. Wherever the terms “comprising” or “including” are used, it should be understood the disclosure also expressly contemplates and encompasses additional aspects “consisting of” the disclosed elements, in which additional elements other than the listed elements are not included.

[0065] The term “about” or “approximately,” as used herein, can mean within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, e.g., the limitations of the measurement system. For example, “about” can mean within 1 or more than 1 standard deviation, per the practice in the given value. Where particular values are described in the application and claims, unless otherwise stated the term “about” can mean an acceptable error range for the particular value, such as 10% of the value modified by the term “about.” As used herein, the term “about,” can mean relative to the recited value, e.g., amount, dose, temperature, time, percentage, etc., ±10%, ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%, ±2%, or ±1%.301541711 - 12 -

[0066] Further, as the present disclosure is susceptible to aspects of many different forms, it is intended that the present disclosure be considered as an example of the principles of the present disclosure and not intended to limit the present disclosure to the specific aspects shown and described. Any one of the features of the present disclosure may be used separately or in combination with any other feature. References to the terms “aspect,” “aspects,” and / or the like in the description mean that the feature and / or features being referred to are included in, at least, one aspect of the description. Separate references to the terms “aspect,” “aspects,” and / or the like in the description do not necessarily refer to the same aspect and are also not mutually exclusive unless so stated and / or except as will be readily apparent to those skilled in the art from the description. For example, a feature, structure, process, step, action, or the like described in one aspect may also be included in other aspects but is not necessarily included. Thus, the present disclosure may include a variety of combinations and / or integrations of the aspects described herein. Additionally, all aspects of the present disclosure, as described herein, are not essential for its practice. Likewise, other systems, methods, features, and advantages of the present disclosure will be, or become, apparent to one with skill in the art upon examination of the figures and the description. It is intended that all such additional systems, methods, features, and advantages be included within this description, be within the scope of the present disclosure, and be encompassed by the claims.

[0067] As used herein, the term “viscous porogen” refers to a high viscosity substance used as a temporary pore-forming agent in biomaterial fabrication, particularly in scaffold development, biomaterials, and porous polymer synthesis. The porogen is typically mixed into a base material and may later be removed through dissolution, degradation, or thermal treatment, leaving behind a porous structure. Without being bound by theory, inclusion of a viscous porogen may provide better control over pore size and interconnectivity due to its fluid nature, which allows for tunable viscosity and precise incorporation into a matrix. Examples include high-molecular-weight polymers, honey, high fructose corn syrup, hydrogels, or viscous organic solvents used in tissue engineering, drug delivery, and filtration applications.

[0068] As used herein, the term “hydrogel” is given its ordinary meaning in the art and refers to a material comprising a polymer network that is able to trap and contain water. The hydrogel may include polymer chains that are crosslinked, either directly or via a crosslinking agent. In certain aspects, the hydrogel can form a physically-crosslinked network. In certain aspects, the hydrogel can form a chemically-crosslinked network. The degree of crosslinking may be varied, in some cases, to tailor the extent to which the gel absorbs or retains fluids. In certain aspects, the hydrogel can be an elastic synthetic hydrogel.301541711 - 13 -

[0069] A term “bone cement” as used herein refers to a medical material for use in orthopedic and trauma surgeries, for example, to anchor implants, fill bone voids, or stabilize fractures. Typically composed of biocompatible polymers or ceramics, it serves as a grout rather than a glue, creating a mechanical interlock between the implant and bone. Bone cement may be prepared as a paste by mixing a powder and liquid, which then undergoes a polymerization process, hardening in place to provide immediate stability, may be provided as a paste, injectable, or may be fabricated in various shaped ex vivo, and injected or applied to the desired site. Its applications includes surgery, parenteral injection, joint replacement procedures, such as hip or knee arthroplasty, and vertebroplasty for treating spinal fractures.

[0070] The term “administration” and variants thereof (e.g., “administering” a composition) in reference to a composition of the disclosure means introducing the composition into the system of the subject in need of treatment. When a composition of the disclosure or prodrug thereof is provided in combination with one or more other active agents (e.g., a cytotoxic agent, etc.), “administration” and its variants are each understood to include concurrent and sequential introduction of the composition and other agents. Thus, in the methods of treatment of the present disclosure, the term “administering” shall encompass the treatment of the various conditions described with the composition specifically disclosed or with a composition which may not be specifically disclosed, but which converts to the specified composition in vivo after administration to the patient. The administration of the compositions disclosed herein may comprise surgical administration, application to site of injury or defect, or parenteral injection of composition, or other routes of administration known in the art, or any combination thereof.

[0071] The term “therapeutically effective amount” as used herein means that amount of active compound or pharmaceutical agent that elicits the biological or medicinal response in a tissue, system, animal, or human that is being sought by a researcher, veterinarian, medical doctor or other clinician.

[0072] As used herein, the term “treating” refers to the application or administration of a composition including one or more active agents to a subject, who is in need of the treatment, for example, having a target disease or disorder, a symptom of the disease / disorder, or a predisposition toward the disease / disorder, or injury, with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve, or affect the disorder, injury, the symptom of the disease, or the predisposition toward the disease or disorder. Alleviating a target disease / disorder includes delaying the development or progression of the disease or reducing disease severity. Alleviating the disease does not necessarily require curative results.301541711 - 14 -

[0073] “Development” or “progression” of a disease means initial manifestations and / or ensuing progression of the disease. Development of the disease can be detectable and assessed using standard clinical techniques as well known in the art. However, development also refers to progression that may be undetectable. For purpose of this disclosure, development or progression refers to the biological course of the symptoms. “Development” includes occurrence, recurrence, and onset. As used herein “onset” or “occurrence” of a target disease or disorder includes initial onset and / or recurrence. In some aspects, the disease is an injury associated infection, implant associated infection or both.II. Bone Cement

[0074] In some aspects, the current disclosure encompasses a bone cement comprising a polymer scaffold and a viscous porogen comprising one or more types of honey, or derivatives thereof. In some aspects, the bone cement may further comprise a bioactive agent.

[0075] In some aspects, the bone cement comprises a polymer scaffold. In some aspects, the polymer scaffold comprises one or more natural polymers, or synthetic polymers, or any combination thereof. In some aspects, the bone cement comprises a natural polymer. Nonlimiting examples of natural polymers that can be incorporated in the bone cement include alginate, gelatin, chitosan, and hyaluronic acid. These polymers are often biocompatible and biodegradable, making them suitable for use in biomedical applications such drug delivery, bone implants, and tissue engineering. In some aspects, the polymer comprises a synthetic polymer. Non-limiting examples of synthetic polymers that may be incorporated in the bone cement include poly(methyl methacrylate) (PM MA), methyl methacrylate, polycaprolactone (PCL), polydioxanone (PDO), poly (glycolic acid) (PGA), poly(L-lactic acid) (PI_A), poly(lactide-co-glycolide) (PLGA), poly(L-lactide) (PLI_A), poly(D,L-lactide) (P(DLLA)), poly(ethylene glycol) (PEG), poly(e-caprolactone) (PCL), montmorillonite (MMT), poly(L-lactide-co-e-caprolactone) (P(LLA-CL)), poly(£-caprolactone-co-ethyl ethylene phosphate) (P(CL-EEP)), poly[bis(p-methylphenoxy) phosphazene] (PNmPh), Polyhydroxybutyrate (PHB), poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), poly(ester urethane) urea (PEUll), poly(p-dioxanone) (PPDO), polyurethane (Pll), polyethylene terephthalate (PET), poly(ethylene-co-vinylacetate) (PEVA), poly(ethylene oxide) (PEG), poly(phosphazene), poly(ethylene-co-vinyl alcohol), a polymer nanoclay nanocomposite; a halogenated polymer solution containing metal compounds (e.g., graphite), poly(ethylenimine), grafted cellulosics, poly(ethyleneoxide), or poly vinylpyrrolidone (PVP), polystyrene (PS), or any combination thereof. In some aspects, the disclosed bone cement can also be formed by combining natural and synthetic polymers to leverage the advantages of both, such as the biocompatibility of natural polymers and the tunability of synthetic ones. In some aspects, the bone cement comprises PM MA.301541711 - 15 -

[0076] In some aspects, the polymer is a synthetic polymer derived from or mimicking a naturally sourced polymer. Non-limiting examples of such synthetic polymers include polymers made from cellulose, for example cellulose acetate, cellulose nitrate, cellulose xanthate, carboxymethylcellulose, methyl cellulose, ethyl cellulose, or hydroxyethyl cellulose.

[0077] In some aspects, the one or more polymers comprise biocompatible polymers. In some aspects, the or more polymers comprise a biodegradable polymer. In some aspects, the one or more polymers are biocompatible polymers. Non-limiting examples include methylcellulose, Poly-p-hydroxybutyrate-co-p-hydroxy valerate (PHBV), Polyglycolic Acid (PGA), Polylactic Acid (PI_A), Poly (e-caprolactone) (PCL), and Polyhydroxybutyrate (PHB).

[0078] In some aspects, the bone cement comprises about 10% to about 15%, about 15% to about 20%, about 20% to about 25%, about 25% to about 30%, about 30% to about 35%, about 35% to about 40%, about 40% to about 45%, about 45% to about 50%, about 50% to about 55%, about 55% to about 60%, about 60% to about 65%, about 65% to about 70%, about 70% to about 75%, about 75% to about 80%, about 80% to about 85%, or about 85% to about 90% by weight of the one or more polymers disclosed herein. In some aspects, the bone cement comprises 10% to 15%, 15% to 20%, 20% to 25%, 25% to 30%, 30% to 35%, 35% to 40%, 40% to 45%, 45% to 50%, 50% to 55%, 55% to 60%, 60% to 65%, 65% to 70%, 70% to 75%, 75% to 80%, 80% to 85%, or 85% to 90% by weight of one or more polymers disclosed herein. In some aspects, the bone cement comprises about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% by weight of one or more polymers disclosed herein. In some aspects, the bone cement comprises at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% by weight of one or more polymers disclosed herein. In some aspects, the bone cement comprises at most 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% by weight of one or more polymers disclosed herein. In some aspects, the bone cement comprises a polymer scaffold comprising PMMA. In some aspects, the bone cement comprises a polymer scaffold comprising about 10% to about 90% by weight PMMA. In some aspects, the bone cement comprises about 10%, 11%, 12%, 13%, 14%, 16%, 17%, 18%, 19%, 21%, 22%, 23%, 24%, 26%, 27%, 28%, 29%, 31%, 32%, 33%, 34%, 36%, 37%, 38%, 39%, 41%, 42%, 43%, 44%, 46%, 47%, 48%, 49%, 51%, 52%, 53%, 54%, 56%, 57%, 58%, 59%, 61%, 62%, 63%, 64%, 66%, 67%, 68%, 69%, 71%, 72%, 73%, 74%, 76%, 77%, 78%, 79%, 81%, 82%, 83%, 84%, 86%, 87%, 88%, 89%, or 90% by weight of PMMA. In some aspects, the bone cement comprises a polymer scaffold comprising about 80% by weight PMMA.

[0079] In some aspects, the bone cement comprises a viscous porogen. In some aspects, the viscous porogen may comprise one or more types of honey, or derivatives thereof. In some301541711 - 16 -aspects, the honey may be derived from a natural source, or may be synthetic. Non-limiting examples of natural honey include manuka honey, clover honey, wildflower honey, tualang honey, buckwheat honey, acacia honey, orange blossom honey, sage honey, eucalyptus honey, lavender honey, blueberry honey, thyme honey, Sidr honey, heather honey, and jarrah honey, or any derivative thereof, or any combination thereof. In some aspects, the honey is a synthetic honey, designed to mimic natural (e.g., non-synthetic honey). By way of non-limiting examples, the synthetic honey may mimic the composition, viscosity, texture, pH, sugar content, immunogenic, wound-healing, and / or antimicrobial properties of natural honey. It is typically made by combining various sugars, water, and other ingredients to replicate the characteristics of honey without involving bees.

[0080] In some aspects, the honey may be a derivative of a natural honey. Derivative of honey includes any product or substance that is derived from honey or its components, often through processing or refinement. These derivatives retain one or more of the natural properties of honey. Non-limiting examples of suitable derivatives of honey include honey extract with one or more components of honey, or medical grade honey. In some aspects, the bone cement comprises about 1% to about 5%, or about 5% to about 10%, about 10% to about 15%, about 15% to about 20%, about 20% to about 25%, about 25% to about 30%, about 30% to about 35%, about 35% to about 40%, about 40% to about 45%, about 45% to about 50%, about 50% to about 55%, about 55% to about 60% by weight of honey or a hydrogel comprising honey. In some aspects, the bone cement comprises at about 15% to about 50% by weight of honey or a hydrogel comprising honey. In some aspects, the bone cement comprises at about 15% to about 30% by weight of honey or a hydrogel comprising honey. In some aspects, the bone cement comprises about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, or 60% by weight of honey or a hydrogel comprising honey. In some aspects, the bone cement comprises about 30% by weight of honey or a hydrogel comprising honey. In some aspects, the bone cement comprises about 50% by weight of honey or a hydrogel comprising honey. In some aspects, the bone cement comprises at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, or 60% by weight of honey or a hydrogel comprising honey. In some aspects, the bone cement comprises at most 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, or 60% by weight of honey or a hydrogel comprising honey. In some aspects, the amount of honey or hydrogel comprising honey is at most 50% or at most 60% by weight, of the bone cement.

[0081] In some aspects, the bone cement has inherent antimicrobial activity due to the presence of honey. As used herein, the term “inherent antimicrobial activity” refers to antimicrobial activity of the bone cement that comes from honey that is incorporated into the bone cement, and not due to added bioactive agent. In some aspects, the bone cement has301541711 - 17 -inherent antimicrobial activity against one or more of streptococcal species, staphylococcal species including Staphylococcus aureus and drug resistant variants thereof including methicillin-susceptible S. aureus (MSSA) and methicillin-resistant S. aureus (MRSA), Escherichia coli, Salmonella species, Helicobacter pylori, and Cutibacterium acnes. Honey has also been shown to have antiviral activity against multiple viruses. Non-limiting examples include varicella-zoster virus, rubella virus, influenza virus, herpes simplex virus, respiratory syncytial virus, and human immunodeficiency virus. Thus, in some aspects, the bone cement has inherent antiviral activity. Additionally, honey has also shown to have antifungal activity. Non-limiting examples of pathogenic fungal species that have been shown to be sensitive to honey include Candida albicans, Candida auris, Rhodotorula species, Aspergillus species, Mucor species, and dermatophytes such as Trichophyton rubrum.

[0082] In some aspects, the disclosed viscous porogen has a water content of about 15% to about 20%, about 20% to about 25%, about 25% to about 30%, about 30% to about 35%, about 35% to about 40%, about 40% to about 45%, about 45% to about 50%, about 50% to about 55%, about 55% to about 60%, about 60% to about 65%, about 65% to about 70%, about 70% to about 75%, about 75% to about 80%, about 80% to about 85%, about 85% to about 90%, or about 90% to about 95% by weight. In some aspects, the disclosed viscous porogen has a water content of at least 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% by weight. In some aspects, the disclosed viscous porogen has a water content of at most 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% by weight.

[0083] In some aspects, the viscous porogen may further comprise one or more hydrophilic polymers. Non-limiting examples of hydrophilic polymers that may be incorporated into the hydrogel include polyethylene glycol (PEG), hyaluronic acid, chitosan, alginate, polyvinyl alcohol (PVA), polyvinylpyrrolidone (PVP), and collagen, or any combination thereof. In such aspects, the viscous porogen is a hydrogel comprising one or more types of honey and a polymer matrix.

[0084] In some aspects, varying the honey to polymer ratio can result in varying material viscosity, varying viscous porogen dissolution rates, and bone cements of varying pore sizes and mechanical properties. In some aspects, this is an important feature of the current disclosure.

[0085] In some aspects, the bone cement may further comprise or exclude one or more inorganic salts and / or metal additives. These are frequently incorporated into bone cement to enhance its properties, such as radiopacity, mechanical strength, bioactivity, and antimicrobial capabilities. Non-limiting examples of inorganic salts for addition to the bone cement include301541711 - 18 -but are not restricted to calcium phosphate compounds like hydroxyapatite (HA), tricalcium phosphate (TCP), brushite, and biphasic calcium phosphate (BCP). Calcium sulfate is another common additive, valued for its bioresorbability. In some aspects, radiopacifiers like barium sulfate, zirconium dioxide, calcium carbonate, calcium sulfate, or strontium, or any combination thereof may be added. Barium sulfate and / or zirconium dioxide (ZrO2) may be included to improve radiopacity, making the cement visible during imaging procedures, while zinc oxide contributes both antimicrobial effects and radiopacity. Magnesium phosphate is sometimes added to enhance bioactivity and support bone healing. In some aspects, the disclosed bone cement may comprise about 0.01% to about 0.1%, or about 0.1% to about 0.5%, or about 0.5% to about 1.0%, about 1.0% to about 1.5%, about 1.5% to about 2.0%, about 2.0% to about 2.5%, about 2.5% to about 3.0%, about 3.0% to about 3.5%, about 3.5% to about 4.0%, about 4.0% to about 4.5%, about 4.5% to about 5.0%, about 5.0% to about 5.5%, about 5.5% to about 6.0%, about 6.0.% to about 6.5%, about 6.5% to about 7.0%, about 7.0% to about 7.5%, about 7.5% to about 8.0%, about 8.0% to about 8.5%, about 8.5% to about 9.0%, about 9.0% to at >ut 9.5%, about 9.5% to about 10.0%, about 10.0% to about 10.5%, about 10.5% to about 11.0%, about 11.0% to about 11.5%, about 11.5% to about 12.0%, about 12.0% to about 12.5%, about 12.5% to about 13.0%, about 13.0% to about 13.5%, about 13.5% to about 14.0%, about 14.0% to about 14.5%, about 14.5% to about 15.0%, about 15.0% to about 15.5%, about 15.5% to about 16.0%, about 16.0% to about 16.5%, about 16.5% to about 17.0%, about 17.0% to about 17.5%, about 17.5% to about 18.0%, about 18.0% to about 18.5%, about 18.5% to about 19.0%, about 19.0% to about 19.5%, or about 19.5% to about 20.0% by weight of one or more inorganic compounds. In some aspects, the disclosed bone cement may comprise at least 0.01%, 0.1%, 0.5%, 1.0%, 1.5%, 2.0%, 2.5%, 3.0%, 3.5%, 4.0%, 4.5%, 5.0%, 5.5%, 6.0%, 6.5%, 7.0%, 7.5%, 8.0%, 8.5%, 9.0%, 9.5%, 10.0%, 10.5%, 11.0%, 11.5%, 12.0%, 12.5%, 13.0%, 13.5%, 14.0%, 14.5%, 15.0%, 15.5%, 16.0%, 16.5%, 17.0%, 17.5%, 18.0%, 18.5%, 19.0%, 19.5%, or20.0% by weight of one or more inorganic compounds. In some aspects, the disclosed bone cement may comprise at most 0.01%, 0.1%, 0.5%, 1.0%, 1.5%, 2.0%, 2.5%, 3.0%, 3.5%, 4.0%, 4.5%, 5.0%, 5.5%, 6.0%, 6.5%, 7.0%, 7.5%, 8.0%, 8.5%, 9.0%, 9.5%, 10.0%, 10.5%, 11.0%, 11.5%, 12.0%, 12.5%, 13.0%, 13.5%, 14.0%, 14.5%, 15.0%, 15.5%, 16.0%, 16.5%, 17.0%, 17.5%, 18.0%, 18.5%, 19.0%, 19.5%, or 20.0% by weight of one or more inorganic compounds. In some aspects, the bone cement may comprise about 0.01% to about 20% by weight of one or more inorganic compounds, for example barium sulfate. In some aspects, the bone cement may comprise about 0.5% to about 20% by weight of one or more inorganic compounds, for example barium sulfate. In some aspects, the bone cement may comprise about 0.5% to about 20% by weight of one or more inorganic compounds, for example301541711 - 19 -zirconium sulfate. In some aspects, one or more of the inorganic salts listed above may be expressly excluded from the formulation.

[0086] In some aspects, the bone cement may further comprise or exclude one or more metal additives. Metal additives also play a crucial role in optimizing the performance of bone cement comprising the disclosed bone cement. For example, silver nanoparticles are frequently used for their potent antimicrobial properties, helping to reduce the risk of infection; titanium dioxide (TiO2) improves mechanical strength and provides photocatalytic antimicrobial effects, while zirconium dioxide (ZrO2) enhances both radiopacity and structural stability; strontium-based compounds, such as strontium carbonate or strontium ranelate, are added to stimulate osteogenesis and improve bone healing; copper ions or nanoparticles contribute antimicrobial and angiogenic properties, supporting tissue regeneration; Iron oxide (Fe3O4) is sometimes included in magnetic bone cements for advanced therapeutic applications, such as targeted drug delivery. Together, these additives enable the customization of bone cement formulations to meet specific clinical requirements. In some aspects, one or more of the metals, or all of the metals disclosed above may be expressly excluded from the formulation.

[0087] Additionally, bioactive glass may be added to bone cement primarily to enhance its bioactivity, osteoconductivity, mechanical properties, and compatibility with natural bone tissue. Bioactive glass is a type of silica-based material that interacts with biological systems to promote bone growth and regeneration. Its inclusion in bone cement formulations is particularly beneficial for applications like joint replacements, spinal surgeries, and bone defect repair, where both structural support and biological integration are critical.

[0088] In some aspects, the bone cements can be engineered to exhibit a wide range of viscosity. In some aspect, the bone cement may be engineered to mimic the viscosity of the tissue into which it is implanted. The viscosity of bone cements is influenced by factors such as polymer concentration, molecular weight, and temperature, and methods for tailoring the viscosity of the bone cement are known in the art. In some aspects, the bone cement has a viscosity of about 1,000 to about 10,000 cP.

[0089] In some aspects, bone cements can be engineered to exhibit a wide range of stiffnesses. By modifying the polymer concentration and crosslinking density, the compressive modulus can vary from approximately 10 Pa to 2,000 MPa, allowing customization to match the mechanical properties of various tissues. Thus in some aspects, the bone cement has a stiffness (compressive modulus) of about 10 Pa to about 100 Pa, about 100 Pa to about 1 KPa, about 1 KPa to about 10 Kpa, 10 Kpa to about 100 Kpa, about 100 KPa to about 1 MPa, about 1 MPa to about 10 MPa, or about 1 MPa to about 100 MPa, about 100 MPa to about 200 MPa, about 200 MPa to about 300 MPa, about 300 MPa to about 400 MPa, about 400301541711 - 20 -MPa to about 500 MPa, about 500 MPa to about 600 MPa, about 600 MPa to about 700 MPa, about 700 MPa to about 800 MPa, about 800 MPa to about 900 MPa, about 900 MPa to about 1,000 MPa, about 1,000 MPa to about 1,100 MPa, about 1,100 MPa to about 1,200 MPa, about 1,200 MPa to about 1,300 MPa, about 1,300 MPa to about 1,400 MPa, about 1,400 MPa to about 1,500 MPa, about 1,500 MPa to about 1,600 MPa, about 1,600 MPa to about 1,700 MPa, about 1,700 MPa to about 1,800 MPa, about 1,800 MPa to about 1,900 MPa, or about 1 ,900 MPa to about 2,000 MPa as measured by methods known in the art, for example, using a universal testing machine (UTM), atomic force microscopy (AFM), dynamic mechanical analysis (DMA), ultrasound elastography, rheometry, or resonance methods, or any combination thereof. In some aspects, the bone cement has a stiffness (compressive modulus) of about 1,200 MPa.

[0090] In some aspects, the bone cement has a porous structure, with a pore size ranging from about 10 mm to about 100 pm. In some aspects, the pore size is about 1.0 x 10-9, 1.0 x 10-8, 1.0 x 1 o-7, 1.0 x 1 o-6, 1.0 x 1 o-5, 1.0 x 1 o-4, or 1.0 x io-3m. In some aspects, the pore size is at least 1.0 x 10-9, 1.0 x 10-8, 1.0 x 10-7, 1.0 x 10-6, 1.0 x 10-5, 1.0 x 10-4, or 1.0 x 10-3m. In some aspects, the pore size is at most 1.0 x 10-9, 1.0 x 10-8, 1.0 x 10-7, 1.0 x 10-6, 1.0 x 10-5, 1.0 x 10-4, or 1.0 x io-3m. In some aspects, the pore size of the bone cement may vary temporally based on the degradation rate. In some aspects, the viscous porogen component of the bone cement may degrade faster than the polymer scaffold. In some aspects, the viscous porogen may degrade within about a few minutes to a few days depending on the composition of the hydrogel. In some aspects, the polymer scaffold can be engineered to degrade over days to months, depending on the application. In some aspects, the bone cement may degrade within at least, about, or at most 2 weeks, 3 weeks, 1 months, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 1 years, 2 years, 3 years, 4 years, 5 years, or more. In some aspects, the polymer scaffold in the bone cement may not degrade and be intended to be permanent. In some aspects, the degradation rate of the viscous porogen determines the rate of initial release of the inherent or added antimicrobial agent, and the degradation rate of the bone cement and / or its ability to adsorb drugs to its surface determines the rate of release of the remaining bioactive agent incorporated into the bone cement. In some aspects, the bone cement of the current disclosure also improve device interactions with host tissues. Porosity / roughness on biomaterial surfaces can improve the ability of patient's tissues to grow on / grow into the device and promote biocompatibility. In some aspects, this is an additional improvement over existing bone cements. Additionally, one or more bioactive agents may be added to the bone cement, non-limiting examples of which include antimicrobial agents, immunotherapeutics, vaccines, stem cells, osteocytes, bone tissue, connective tissue cells,301541711 - 21 -fibrin, collagen, bioactive peptides, drugs, and growth factors. In some aspects, the bone cement may comprise one or more antimicrobial agents, immunotherapeutics, and / or vaccines. In some aspects, the one or more antimicrobial agents or vaccines may be incorporated directly into the bone cement or in suitable delivery particles incorporated into the bone cement, as disclosed herein and / or known in the art.III. Bioactive Agents

[0091] A bioactive agent as used herein is a substance that interacts with biological systems to elicit a specific physiological or therapeutic effect by influencing cellular, molecular, or systemic processes. These agents include antimicrobials, which target and inhibit harmful microorganisms; vaccines, which stimulate the immune system to recognize and combat pathogens; and immunotherapeutics, which harness or modulate the immune response to treat conditions like cancer, infections, and autoimmune diseases. Other examples include growth factors, hormones, peptides, stem cells, osteocytes, bone tissue, connective tissue cells, fibrin, collagen, bioactive peptides, and growth factors, and nucleic acid-based therapies. In some aspects, the one or more bioactive agents included in the bone cement are such that they do not comprise the structural and / or biological properties of the bone cement. In some aspects, the one or more bioactive agents are added, or the specific bone cement composition used depends on patient specific allergies, renal function, and the site of infection.

[0092] In some aspects, the bioactive agent is an antimicrobial agent, for example an antibacterial, an antiviral, antiparasitic, or an antifungal agent, or any combination thereof. Infections are often associated with bone grafts and implants. Among bacteria, Staphylococcus aureus, particularly methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-sensitive Staphylococcus aureus (MSSA), is the most frequently implicated pathogen in orthopedic infections. Staphylococcus epidermidis and other coagulase-negative staphylococci are also common, especially in prosthetic joint infections, due to their ability to form biofilms on implant surfaces. Streptococcal species, enterococcal species, Cutibacterium acnes, Corynebacterium striatum, Escherichia coli, Klebsiella pneumoniae, Enterobacter species, and / or Pseudomonas aeruginosa can also cause bone and implant infections. Mycobacterial infections, such as those caused by Mycobacterium tuberculosis or non-tuberculous mycobacteria (NTM), may also lead to osteoarticular infections, particularly in endemic regions or among patients with weakened immune systems. In some aspects, the bioactive agent may target one or more species that are commonly associated with infections at bone grafts and implants. In some aspects, the bioactive agent may be a broad spectrum antibiotic effective against a wide range of bacterial species. In some aspects, the bioactive agent may be part of a targeted therapeutic regimen against a single or a few bacterial species.301541711 - 22 -

[0093] Fungal infections, although less common, can occur, especially in immunocompromised individuals. The most frequently encountered fungal pathogens include Candida species and Aspergillus species. Polymicrobial infections, involving multiple organisms, are often seen in cases of trauma, open fractures, or contaminated wounds. These infections can include anaerobic bacteria such as Bacteroides and Clostridium species. The risk of infection is influenced by several factors, including the patient’s immune status, the surgical technique employed, and the presence of biofilm-forming pathogens on the implant surface. Biofilms are particularly challenging because they protect microorganisms from the immune system and antibiotics, making these infections more difficult to treat effectively. In some aspects, the antimicrobial agent that is incorporated into the bone cement may target one or more of the microorganism provided herein or known in the art to infect wounds and / or bone fractures and other bone defects may be targeted by the antimicrobial agent.

[0094] In some aspects, the antimicrobial agent is an antibacterial agent. Non-limiting examples of antibacterial agents that may be incorporated into the bone cement include tetracyclines, penicillin, cephalosporins, carbopenems, aminoglycosides, macrolide antibiotics, lincosamide antibiotics, 4-quinolones, rifamycins, and nitrofurantoin. Suitable specific compounds include, without limitation, ampicillin, amoxicillin, benzylpenicillin, dalbavancin, oritavancin, phenoxymethylpenicillin, bacampicillin, pivampicillin, carbenicillin, cioxacillin, cyclacillin, dicloxacillin, methicillin, oxacillin, piperacillin, ticarcillin, flucioxacillin, cefuroxime, cefetamet, cefetrame, cefixine, cefoxitin, ceftazidime, ceftizoxime, latamoxef, cefoperazone, ceftriaxone, cefsulodin, cefotaxime, cephalexin, cefaclor, cefadroxil, cefalothin, cefazolin, cefpodoxime, ceftibuten, aztreonam, tigemonam, erythromycin, dirithromycin, roxithromycin, azithromycin, clarithromycin, clindamycin, paldimycin, lincomycirl, vancomycin, moxifloxacin, metronidazole benzoate, spectinomycin, tobramycin, paromomycin, metronidazole, tinidazole, ornidazole, amifloxacin, cinoxacin, ciprofloxacin, difloxacin, enoxacin, fleroxacin, norfloxacin, ofloxacin, linezolid, temafloxacin, teromyocin, doxycycline, minocycline, tetracycline, daptomycin, chlortetracycline, oxytetracycline, methacycline, rolitetracyclin, nitrofurantoin, nalidixic acid, gentamicin, rifampicin, amikacin, netilmicin, imipenem, cilastatin, chloramphenicol, furazolidone, nifuroxazide, sulfadiazin, sulfametoxazol, trimethoprim-sulfamethoxazole, bismuth subsalicylate, colloidal bismuth subcitrate, gramicidin, mecillinam, cloxiquine, dichlorobenzylalcohol, povidone, sugars, mucopolysaccharides, chlorobutanol, quarternary ammonium compounds such as benzalkonium chloride, organic mercurials, parahydroxy benzoates, aromatic alcohols, halogenated phenols, sorbic acid, benzoic acid, dioxin, EDTA, BHT, BHA, TBHQ, gallate esters, NDGA, tocopherols, gum guaiac, lecithin, boric acid, citric acid, p-Hydroxy benzoic acid esters, propionates, sulfur dioxide and sulfites, nitrates and nitrites of potassium and301541711 - 23 -sodium, diethyl pyrocarbonate, sodium diacetate, diphenyl, hexamethylene tetramine o-phenyl phenol, sodium o-phenylphenoxide, and levofloxacin, or any variant thereof. In some aspects, the antibiotic is vancomycin.

[0095] In some aspects, the antimicrobial agent is an antifungal agent. Non-limiting examples of antifungal agent include fluconazole, itraconazole, ketoconazole, rezafungin, voriconazole, amphotericin B, nystatin, clotrimazole, terbinafine, posaconazole, griseofulvin, caspofungin, micafungin, anidulafungin, and flucytosine, or any variant thereof.

[0096] In some aspects, the antimicrobial agent is an antiviral agent. Non-limiting examples of antiviral that may be incorporated into the bone cement include acyclovir, valacyclovir, ganciclovir, valganciclovir, tenofovir, entecavir, and remdesivir; protease inhibitors like ritonavir, lopinavir, glecaprevir, and pibrentasvir; polymerase inhibitors such as sofosbuvir and foscarnet; neuraminidase inhibitors like oseltamivir and zanamivir; integrase strand transfer inhibitors (INSTIs) such as dolutegravir and raltegravir; entry inhibitors like maraviroc and enfuvirtide; monoclonal antibodies such as palivizumab, sotrovimab, and bebtelovimab; and interferons, including interferon-alpha, or any combination thereof.

[0097] In some aspects, the antimicrobial agent is an antiparasitic agent. Non-limiting examples of antiparasitic agents include chloroquine, artemether-lumefantrine, primaquine, metronidazole, tinidazole, nitazoxanide, pentamidine, albendazole, mebendazole, ivermectin, praziquantel, sodium stibogluconate, amphotericin B, eflornithine, nifurtimox, benznidazole, and diethylcarbamazine (DEC), or any combination thereof.

[0098] In some aspects, the bioactive agent is a vaccine. In some aspects, any type of vaccine, and against any target may be incorporated into the bone cement. Non-limiting examples include live attenuated vaccine, protein based vaccine, polysaccharide-protein conjugate vaccine, capsular polysaccharide vaccine, toxoid based vaccine, killed whole cell vaccine, multi-component vaccine, DNA based vaccine, and a mRNA vaccine, or any combination thereof. In some aspects, the vaccine targets microorganisms that usually infect wounds, fractures, or are associated with implants, as discussed above, and known in the art. In some aspects, the vaccine is against Staphylococcus aureus, methicillin-susceptible S. aureus (MSSA), and methicillin-resistant S. aureus (MRSA), coagulase-negative Staphylococci including Staphylococcus epidermidis, streptococcal species, enterococcus species, Cutibacterium acnes, Corynebacterium striatum, Escherichia coli, Klebsiella pneumoniae, Enterobacter complex, Candida albicans and other yeast, Aspergillus species, and / or Pseudomonas aeruginosa. In some aspects, the vaccine is against Staphylococcus aureus. In some aspects, the vaccine is a protein based vaccine targeting one or more of the key surface proteins of S. aureus such as clumping factor A (ClfA), Iron-regulated Surface301541711 - 24 -Determinant B (IsdB), Staphylococcal Protein A (SpA), fibronectin-binding protein (FnBPA), or protein A, as well as exotoxins like toxic shock syndrome toxin-1 (TSST-1), and enterotoxins. Some vaccines may incorporate capsular polysaccharides, which are important for bacterial evasion of the immune system, or lipoproteins that play a role in the bacterium’s adhesion to host tissues. CP5 and CP8 are the most common capsular types in S. aureus and may be conjugated to a carrier protein (e.g., CRM 197 or tetanus toxoid) to enhance immunogenicity. In some aspects, the vaccine is a multicomponent vaccine. In some aspects, the vaccine is a DNA vaccine encoding one or more of the surface proteins, or fragments thereof. In some aspects, the vaccine is a mRNA vaccine encoding one or more surface or intracellular antigens. In some aspects, the vaccine is a live attenuated vaccine. In some aspects, the vaccine is a killed whole cell vaccine. In some aspects, the vaccine is a recombinant vaccine developed to express multiple antigens simultaneously, enhancing the immune response. In some aspects, immunostimulators and / or adjuvants may be included with or without the vaccine formulations into the bone cement to boost the effectiveness of the immune response.

[0099] The term “immunostimulator” as used herein refers to a compound that can stimulate an immune response in a subject and may include an adjuvant. In some aspects, an immunostimulator is an agent that does not constitute a specific antigen but can boost the strength and longevity of an immune response to an antigen. Such immunostimulators may include, but are not limited to stimulators of pattern recognition receptors, such as Toll-like receptors, RIG-1 and NOD-like receptors (NLR), mineral salts, such as alum, alum combined with monphosphoryl lipid (MPL) A of Enterobacteria, such as Escherichia coli, Salmonella minnesota, Salmonella typhimurium, or Shigella flexneri or specifically with MPL (ASO4), MPL A of above-mentioned bacteria separately, saponins, such as QS-21, Quil-A, ISCOMs, ISCOMATRIX, emulsions such as MF59, Montanide, ISA 51, and ISA 720, AS02 (QS21+squalene+MPL), liposomes and liposomal formulations such as AS01, synthesized or specifically prepared microparticles, and microcarriers such as bacteria-derived outer membrane vesicles (OMV) of N. gonorrhoeae, Chlamydia trachomatis and others, or chitosan particles, depot-forming agents, such as Pluronic block co-polymers, specifically modified or prepared peptides, such as muramyl dipeptide, aminoalkyl glucosaminide 4-phosphates, such as RC529, or proteins, such as bacterial toxoids or toxin fragments.

[0100] In some aspects, the immunostimulators and / or adjuvants may be proinflammatory stimuli released from necrotic cells (e.g., urate crystals). In some aspects, additional immunostimulators and / or adjuvants may be activated components of the complement cascade (e.g., CD21, CD35, etc.). In some aspects, the immunostimulators and / or adjuvants may be activated components of immune complexes. The immunostimulators and / or301541711 - 25 -adjuvants also include complement receptor agonists, such as a molecule that binds to CD21 or CD35. In some aspects, the complement receptor agonist induces endogenous complement opsonization of the synthetic nanocarrier. In some aspects, immunostimulators are cytokines, which are small proteins or biological factors (in the range of 5 kD to 20 kD) that are released by cells and have specific effects on cell-cell interaction, communication and behavior of other cells. In some aspects, the cytokine receptor agonist is a small molecule, antibody, fusion protein, or aptamer.

[0101] In certain aspects, the immunostimulator or adjuvant comprises an adjuvant system such as AS04, AS03, AS01 , or MF59. In some aspects, the immunostimulator and / or adjuvant can comprise any one or more components from these systems, as described below. In some aspects, the immunostimulator comprises an aluminum containing adjuvant such as aluminum hydroxide, aluminum phosphate, or potassium aluminum sulfate (Alum). In some aspects, the immunostimulator and / or adjuvant comprises a nucleic acid based adjuvant such as a CpG oligodeoxynucleotide, cytosine phosphoguanine (CpG) or RNA.

[0102] As noted, the adjuvant AS04 (Adjuvant System 04) is an adjuvant system comprising aluminum hydroxide and monophosphoryl lipid A (MPL) which are used in hepatitis and human papillomavirus (HPV) vaccines, as would be appreciated by one skilled in the art. AS03 (Adjuvant System 03) is a squalene based adjuvant which can be used in certain influenza vaccines, as would be appreciated by one skilled in the art. AS01 (Adjuvant System 01) is an adjuvant system comprising monophosphoryl lipid A (MPL) and QS-21, a natural compound extracted from the Chilean soapbark tree, combined in a liposomal formulation.

[0103] In some aspects, the bioactive agent is an immunotherapeutic agent. As used herein, “immunotherapy” refers to any therapy that harnesses or modulates the immune system to treat a condition and may be active, passive, or hybrid (active and passive), exploiting immune recognition of antigens including proteins, carbohydrates, and other macromolecules associated with infectious agents or cancer cells. Non-limiting immunotherapies include inhibition of co-stimulatory molecules, dendritic cell therapies, modified cell therapies (e.g., CAR-T cells), cytokine therapies, adoptive T-cell therapy, checkpoint inhibitors, cell-based immunotherapies involving immune effector cells (e.g., lymphocytes, macrophages, natural killer (NK) cells, dendritic cells, cytotoxic T lymphocytes (CTL)), antigen based immunotherapies, antibodies and antibody derivatives (e.g., monoclonal antibodies, conjugated monoclonal antibodies, polyclonal antibodies, antibody fragments, radiolabeled antibodies, chemolabeled antibodies), immune checkpoint inhibitors, immunomodulators (e.g., interleukins, cytokines, chemokines), topical immunotherapies (e.g., imiquimod), injection immunotherapies, oncolytic virus therapies, immunosuppressive drugs, helminthic therapies, other non-specific immunotherapies, and combinations thereof. In some aspects,301541711 - 26 -the immunotherapeutic may comprise one or more of immunomodulatory cytokines including interleukins, interferons, colony-stimulating factors, and transforming growth factors; immune checkpoint modulators including PD-1 inhibitors, PD-L1 inhibitors, CTLA-4 inhibitors, and combinations thereof; toll-like receptor agonists; stimulator of interferon genes (STING) agonists; nucleotide-based immunostimulatory agents including CpG oligonucleotides and RNA-based agonists; microbial antigens, tumor-associated antigens, neoantigens, peptide antigens, and protein antigens; dendritic cell activators; macrophage-polarizing agents including M1 -polarizing cytokines and small molecules; adoptive immune therapy components including cell-derived lysates, extracellular vesicles, and exosomes; monoclonal antibodies and antibody fragments; bispecific and multispecific antibodies; immune-targeting fusion proteins; cancer vaccines including peptide-based, protein-based, and nucleic acid-based vaccines; oncolytic viral components; adjuvants including aluminum salts, emulsions, and saponin-based adjuvants; immunogenic cell death inducers; small-molecule immunomodulators; kinase inhibitors with immunomodulatory activity; pattern-recognition receptor ligands; inflammasome activators; immune-tolerizing agents including regulatory T-cell-inducing factors; and combinations thereof. Immune checkpoint inhibitor immunotherapies include those that target PD-1 , PD-L1 , or CTLA-4, such as ipilimumab (also referred to herein as ilipimumab), nivolumab, and pembrolizumab; and, in some aspects, an immunotherapeutic incorporated into the bone cement can include an interleukin and / or interferon (IFN) and / or one or more suitable antibody-based reagents, such as denileukin diftitox and / or administration of an antibody-based reagent selected from the group consisting of ado-trastuzumab emtansine, alemtuzumab, atezolizumab, bevacizumab, blinatumomab, brentuximab vedotin, cetuximab, catumaxomab, gemtuzumab, ibritumomab tiuxetan, natalizumab, nimotuzumab, nivolumab, ofatumumab, panitumumab, pembrolizumab, rituximab, tositumomab, trastuzumab, vivatuxin, and the like. In some embodiments, an immunotherapy assigned or administered to a subject can include an indoleamine 2,3-dioxygenase (IDO) inhibitor, adoptive T-cell therapy, virotherapy (T-VEC), and / or any other immunotherapy whose efficacy extensively depends on anti-tumor immunity. In some aspects, the immunotherapeutic may comprise a microbial antigen, adjuvants as disclosed herein, and / or suitable chemokines as disclosed herein. In some aspects, the microbial antigen comprising a live attenuated microorganism, an inactivated microorganism, a heat-killed microorganism, a chemically inactivated microorganism, a microbial lysate, a microbial subunit antigen, or a fragment, epitope, or combination thereof. In certain aspects, the antigen comprises killed S. aureus provided with an adjuvant CpG and further comprising a chemokine, for example GM-CSF, and incorporated into a bone cement composition. In some aspects, the killed S. aureus is an antibiotic killed S. aureus.301541711 - 27 -

[0104] Those skilled in the art can determine appropriate immunotherapy options, including treatments that have been approved and those that are in clinical trials or otherwise under development. Any relevant immunotherapy treatment strategies, alone or in combination with one or more additional therapy, can be utilized in the practice of the present disclosure.

[0105] In some aspects, the bioactive agent is a growth factor. Non-limiting examples of growth factors that may be incorporated bone morphogenetic proteins (BMPs), transforming growth factor-beta (TGF-P), platelet-derived growth factor (PDGF), and fibroblast growth factors (FGFs). In some aspects, the bioactive agent is hormone, for example calcitonin.

[0106] In some aspects, the bone cement may comprise between about 0.001% to about 5% by weight of the one or more bioactive agents. In some aspects, the bone cement comprises less than, more than, or about 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.09%, 0.08%, 0.07%, 0.06%, 0.05%, 0.04%, 0.03%, 0.02%, 0.01%, 0.009%, 0.008%, 0.007%, 0.006%, 0.005%, 0.004%, 0.003%, 0.002%, 0.001% by weight of the one or more bioactive agents disclosed herein.

[0107] In some aspects, the one or more bioactive agents are homogenously distributed in the bone cement. In some aspects, the one or more bioactive agents are non-homogenously distributed within the bone cement, for example, as encapsulated agents or in delivery particles for controlled release, surface loaded allowing rapid release, layer-by-layer assembled bone cements with one or more bioactive agent distributed in one or more layers, etc. In some aspects, the added in a layer configuration within the bone cement. In some aspects, the bioactive agents can be incorporated into delivery particles as disclosed below.IV. Delivery Particles

[0108] In some aspects, the one or more bioactive agents disclosed herein may be incorporated into a delivery particle, that are added to the bone cement. Non-limiting examples of suitable delivery particles include liposome, dendrimers, solid-lipid nanoparticle (SLN), nanostructured lipid carrier, poly(lactic-co-glycolic acid) (PLGA) micro- and / or nano-particles, chitosan nanoparticle, polystyrene nanoparticle, metallic nanoparticle, virus like particle (VLP), ferritin nanoparticle, albumin nanoparticle, silica based nanoparticle, viral particle, dextran nanoparticle, alginate microparticle, or an exosome. Microparticles, typically ranging in size from 1 to 1000 micrometers, include types such as microspheres, microcapsules, and coated pellets. These microparticles can be made from proteins or synthetic polymeric materials and are used for controlled release, and protection of biomolecules. Nanoparticles, which are smaller than 100 nanometers, include a variety of materials such as liposomes, dendrimers, polymeric nanoparticles, and metallic nanoparticles like gold colloids and quantum dots. These nanoparticles offer advantages like high surface-area-to-volume ratio, tunable301541711 - 28 -properties, and the ability to cross biological barriers, making them ideal for targeted and controlled drug delivery. Delivery particles and methods of making and using them are well known in the art of drug delivery.

[0109] In some aspects, a single type of delivery particle may be incorporated into the disclosed bone cement. In some aspects, a bone cement composition may comprise more than one form of delivery particle, each type comprising the same or different bioactive agent. In some aspects, the delivery particle has a dissolution rate which is different from the rate of dissolution of the bone cement. This platform can be used to release different therapeutics at different timepoints. For example, antibiotics incorporated into the bone cement can be released quickly to sterilize an infected site followed by delayed release of immune-stimulating components, such as adjuvants and specific antigens, to promote adaptive immunity for prevention of infection relapse.V. Methods of Making

[0110] In some aspects, the current disclosure also encompasses methods of making the disclosed bone cement. Methods of making bone cements are well known in the art. In some aspects, the method may comprise one or more steps as disclosed herein:

[0111] Selection of a suitable precursor: appropriate monomers, polymers, or precursors based on the desired properties of the bone cement, such as hydrophilicity, mechanical strength, and biocompatibility can be selected. In some aspects, a suitable precursor may comprise PMMA and MMA. In some aspects, the bone cement formulation comprises PMMA as the polymer component, which may be present in the polymerization mixture at about 10%, 11%, 12%, 13%, 14%, 16%, 17%, 18%, 19%, 21%, 22%, 23%, 24%, 26%, 27%, 28%, 29%, 31%, 32%, 33%, 34%, 36%, 37%, 38%, 39%, 41%, 42%, 43%, 44%, 46%, 47%, 48%, 49%, 51%, 52%, 53%, 54%, 56%, 57%, 58%, 59%, 61%, 62%, 63%, 64%, 66%, 67%, 68%, 69%, 71%, 72%, 73%, 74%, 76%, 77%, 78%, 79%, 81%, 82%, 83%, 84%, 86%, 87%, 88%, 89%, or 90% by weight, or any sub-range thereof. In some aspects, the bone cement formulation comprises MMA as a monomeric precursor, present in trace or residual amounts. In such aspects, MMA may comprise about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, or 35% by weight of the polymerization mixture, or any sub-range therein. In some aspects, the polymerization mixture comprises a PMMA:MMA ratio of 2:1. In an exemplary aspect, the polymerization mixture comprises about 67% by weight of PMMA and about 33% by weight of MMA. Table 1 provides exemplary formulations.

[0112] Preparation of the precursor solution: Selected precursors may be dissolved in a solvent, typically water or an aqueous buffer, to form a homogeneous solution. This solution301541711 - 29 -may also contain additives like crosslinkers, initiators, or catalysts, depending on the method of bone cement synthesis. In some aspects, the solution comprises one or more types of honey, or derivatives thereof.

[0113] Polymerization or crosslinking: This step involves the formation of the bone cement network through chemical or physical crosslinking. Chemical crosslinking involves covalent bond formation, often facilitated by crosslinking agents, LIV light, or free-radical polymerization. Physical crosslinking relies on non-covalent interactions such as ionic bonding, hydrogen bonding, or hydrophobic interactions. PMMA is typically polymerized by free-radical polymerization with subsequent strong mechanical properties based on molecular weight and hydrophobic interactions between chains.

[0114] Shaping or molding: The bone cement can be shaped into any desired form, for example, injectable gel, layers, discs, beads, films, or scaffolds by casting the precursor solution into molds or using techniques like extrusion or 3D printing.

[0115] Curing or gelation: The bone cement can then be solidified or gelled by providing the necessary conditions, such as time, temperature, or light exposure, for the crosslinking or polymerization process to complete.

[0116] Washing and purification: unreacted monomers, crosslinkers, or other impurities may be removed by washing the bone cement with water or a suitable solvent. This step ensures biocompatibility and safety for applications like drug delivery or tissue engineering although may not always be necessary.

[0117] Characterization and testing: Bone cement’s properties, such as swelling behavior, mechanical strength, porosity, and biocompatibility can be tested to ensure it meets the requirements for its intended application.

[0118] Bioactive agents and / or delivery particles may be added at any suitable step as determined by a person skilled in the art.

[0119] In some aspects, the disclosed bone cement comprises PMMA and is synthesized using free-radical polymerization of PMMA and MMA monomers in the presence of an initiator, honey, and bioactive agent etc. Some exemplary formulations are provided in Table 1. In some aspects, the process may comprise the preparation of an aqueous solution of honey, containing MMA, PMMA, and a free-radical initiator like ammonium persulfate (APS) or benzoyl peroxide. Polymerization is initiated by heating or exposure to LIV light, depending on the initiator used, leading to the formation of a three-dimensional polymer network. The water content and viscosity of the viscous fluid during synthesis plays a critical role, as it ensures the bone cement’s ability to form a porous network following viscous fluid leaching. The honey301541711 - 30 -content of the viscous fluid during synthesis plays a critical role, as it has inherent antimicrobial activity. To enhance biocompatibility or introduce specific functionalities, PMMA bone cements can be modified by copolymerizing MMA with hydrophilic monomers like 2-hydroxyethyl methacrylate (HEMA) or polyethylene glycol methacrylate (PEGMA).VI. Methods of Use

[0120] In some aspects, the disclosed bone cement is a versatile system that can be used in multiple applications. Non-limiting examples include use as a cement to maintain space and sterilize infected bones and joints; application to fix new devices into bones, such as prostheses, and prevention of subsequent surgical-site infection; and formulated as a collection of beads or discs devices for implantation to locally treat infection. In some aspects, the bone cement is inherently antimicrobial due to the presence of honey. In some aspects, additional bioactive agents can be added to the formulation, allowing the bone cement to function as a delivery platform for the incorporated agent. In addition to better delivery of one or more bioactive agents, the bone cement allows for incorporation of delivery particles with bioactive agents, such that different therapeutics are released at different timepoints.

[0121] Also provided is a method of treating a subject for a bone defect comprising administering to a site of bone damage, loss or deficiency, a bone cement according to the description. The method may further comprise implanting into said subject a medical device or implant. The term treating may comprise an orthopedic, periodontal, neurosurgical, oral, or maxillofacial procedure. The term treating may comprise repair of a simple fracture, compound fracture, comminuted fracture, transverse fracture, oblique fracture, spiral fracture, stress fracture, pathological fracture, avulsion fracture, impacted fracture, segmental fracture, compression fracture, hairline fracture, or non-union; external or internal fixation; joint reconstruction, arthrodesis, arthroplasty or cup arthroplasty of the hip; femoral or humeral head replacement; femoral head surface replacement or total joint replacement; repair of the vertebral column, spinal fusion or internal vertebral fixation; tumor surgery; deficit filling; discectomy; laminectomy; excision of spinal cord tumors; an anterior cervical or thoracic operation; repair of a spinal injury; treatment of scoliosis, treatment of lordosis; kyphosis treatment; intermaxillary fixation of a fracture; mentoplasty; temporomandibular joint replacement; alveolar ridge augmentation or reconstruction; as part of an inlay osteoimplant; implant placement and revision; sinus lift; a cosmetic procedure; or the repair or replacement of the ethmoid, frontal, nasal, occipital, parietal, temporal, mandible, maxilla, zygomatic, cervical vertebra, thoracic vertebra, lumbar vertebra, sacrum, rib, sternum, clavicle, scapula, humerus, radius, ulna, carpal bones, metacarpal bones, phalanges, ilium, ischium, pubis, femur, tibia, fibula, patella, calcaneus, tarsal bones and / or metatarsal bones. The added301541711 - 31 -bioactive agent is suitable to meet specific requirements of orthopedic applications, such as arthroplasty, fracture augmentation, and bone void fillers. For example, the addition of an agent that would lead to increased levels of bone reformation after surgery can be combined with addition of an antimicrobial within the cement to decrease the ability for pathogens to grow in the interface between the bone and new implant.

[0122] In some aspects, disclosed herein is a method of treating a bone defect in a subject in need thereof, the method comprising contacting the bone with a disclosed bone cement. In some aspects, the bone cement can be formulated as an injectable gel to be injected locally. In some aspects, the bone cement can be molded as a bead, disc, pellet, film, sheet, scaffold, or layered architecture and applied to the site of defect and / or treatment. In some aspects, the molding can be done at the time of a procedure or prior to a procedure. In some aspects, the bone cement can be used during a clinical procedure. Some non-limiting use cases for the disclosed bone cement include arthroplasty, vertebroplasty, kyphoplastyis, fracture augmentation, bone void filling, or similar.

[0123] Arthroplasty is the surgical replacement of arthritic or destructive or necrotic joint or joint surface with a prosthesis. For example a hip joint that is affected by osteoarthritis may be replaced entirely (total hip arthroplasty) with a prosthetic hip. This would involve replacing both the acetabulum (hip socket) and the head and neck of the femur. The purpose of this procedure is to relieve pain, to restore range of motion and to improve walking ability, thus leading to the improvement of muscle strength. The disclosed bone cement can be used to attach the bone to the prosthetic. Indications for arthroplasty may include osteoarthritis (OA), rheumatoid arthritis (RA), avascular necrosis (AVN) or osteonecrosis (ON), congenital dislocation of the hip joint (CDH), hip dysplasia (human), acetabular dysplasia (shallow hip socket), frozen shoulder, loose shoulder, traumatized and mal-aligned joint, joint stiffness. Vertebroplasty is typically performed by a spine surgeon or interventional radiologist. During the procedure, the bone cement is injected with a biopsy needle into the collapsed or fractured vertebra. The needle is placed with fluoroscopic x-ray guidance. The cement quickly hardens and forms a support structure within the vertebra that provides stabilization and strength. The needle makes a small puncture in the patient’s skin that is easily covered with a small bandage after the procedure. Kyphoplasty is a variation of a vertebroplasty which attempts to restore the height and angle of kyphosis of a fractured vertebra (of certain types), followed by its stabilization using injected bone cement. The procedure typically includes the use of a small balloon that is inflated in the vertebral body to create a void within the cancellous bone prior to cement delivery. Once the void is created, the procedure continues in a similar manner as a vertebroplasty, but the bone cement is typically delivered directly into the newly created void. Fracture augmentation is the process by which bone cement is introduced via injection to301541711 - 32 -stabilize bones that have undergone a fracture. Bone cement can be injected through a small hole in the skin (percutaneously) into a fractured area, or applied around the fractured bone. Bone void fillers are injectable or moldable compositions that can be flowed or molded into a bone defect, such as a crack, fissure, gap or the like, such as a gap between a synthetic implant (such as a metal prosthesis) and a bone and where bone regeneration is desirable. Examples include the repair of a simple fracture, compound fracture, comminuted fracture, transverse fracture, oblique fracture, spiral fracture, stress fracture, pathological fracture, avulsion fracture, impacted fracture, segmental fracture, compression fracture, hairline fracture, or non-union, external or internal fixation, joint reconstruction, a cosmetic procedure, and the repair or replacement of the bones such as ethmoid, sphenoid, frontal, nasal, occipital, parietal, temporal, mandible, maxilla, orbital, zygomatic, cervical vertebra, thoracic vertebra, lumbar vertebra, sacrum, coccyx, rib, sternum, clavicle, scapula, humerus, radius, ulna, carpal bones, metacarpal bones, phalanges, ilium, ischium, pubis, femur, tibia, fibula, patella, calcaneus, talus, tarsal bones and / or metatarsal bones.

[0124] In some aspects, a suitable subject, or a subject in need thereof is a subject diagnosed with, showing symptoms of, suspected of having, a bone defect. Suitable subjects may include, without limit, humans, as well as companion animals such as cats, dogs, rodents, and horses; research animals such as rabbits, sheep, pigs, dogs, primates, mice, rats, and other rodents; agricultural animals such as cattle, pigs, goats, sheep, horses, deer, chickens, other fowl; zoo animals; and primates such as chimpanzees, monkeys, and gorillas. The subject can be of any age without limitation. In some aspects, the subject is diagnosed with, showing symptoms of, is susceptible to, or will benefit from prevention and / or treatment of a bone defect related, injury related, surgery related, or implant related infection.

[0125] In some aspect, the method comprises contacting the site of defect, or injury with the bone cement disclosed herein. In some aspects, the bone cement comprises an effective amount of one or more bioactive agents. Determination of whether an amount of the bioactive agent, for example small molecules, the nucleic acids, the polypeptides / peptide, or the delivery particle disclosed herein can have a desired therapeutic effect would be evident to one of skill in the art. Effective amounts vary, as recognized by those skilled in the art, depending on the condition being treated, the severity of the condition, the individual patient parameters including age, physical condition, size, gender and weight, injury, the duration of the treatment, the nature of concurrent therapy (if any), the specific method of administration and like factors within the knowledge and expertise of the health practitioner. These factors are well known to those of ordinary skill in the art and can be addressed with no more than routine experimentation. In some aspects, a maximum dose of the individual components or combinations thereof may be used, that is, the highest safe dose according to sound medical301541711 - 33 -judgment. In some aspects, empirical considerations, such as the half-life, generally will contribute to the determination of the dosage. In some aspects, frequency of administration may be determined and adjusted over the course of therapy, and is generally, but not necessarily, based on treatment and / or suppression and / or amelioration and / or delay of a target disease / disorder. In some aspects, sustained continuous release formulations may be appropriate. Various formulations and devices for achieving sustained release are known in the art and provided herein.Use of bone cement for delivery of bioactive agents

[0126] In some aspects, the disclosed bone cement compositions are suitable for localized delivery of bioactive agents at skeletal and peri-skeletal sites, including long bones, vertebrae, pelvis, mandible, maxilla, and locations of orthopedic implantation. In certain aspects, a method of treatment comprises placing a device formed from the bone cement into an osseous defect or against a bone surface during a surgical procedure, thereby providing sustained local release of one or more bioactive agents. The bioactive agent can be selected from antimicrobial agents including antibacterial, antifungal, antiviral and antiparasitic agents. In some aspects, the bioactive agent comprises one or more of vaccine, immunotherapeutics, and / or analgesics. Combinations of these and additional bioactive agents are also envisaged. The bioactive agent can be incorporated into the honey-based porogen phase, the PMMA scaffold phase, and / or into delivery particles such as poly(lactide-co-glycolide) microparticles dispersed within the bone cement to tune the release profile. In certain aspects, the total loading of a bioactive agent is between about 5% and about 0.0001% by weight of the bone cement. In some aspects, the bone cement comprises less than, more than, or about 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.09%, 0.08%, 0.07%, 0.06%, 0.05%, 0.04%, 0.03%, 0.02%, 0.01%, 0.009%, 0.008%, 0.007%, 0.006%, 0.005%, 0.004%, 0.003%, 0.002%, 0.001%, 0.0009%, 0.0008%, 0.0007%, 0.0006%, 0.0005%, 0.0004%, 0.0003%, 0.0002%, 0.0001% by weight of the bioactive agent.

[0127] In certain aspects, the method comprises preventing or treating bone site infection or prosthetic joint infection by contacting the bone site or the prosthetic interface with a device fabricated from the bone cement that is loaded with one or more antibacterial agents as described herein, at the loading levels described herein. Upon placement, the honey-mediated porous network provides an initial release window in the first one to two weeks to rapidly reduce bacterial burden, followed by staged release from embedded delivery particles over two to six weeks to suppress residual organisms and biofilm. The method is applicable to infections as disclosed herein above, for example those caused by Staphylococcus aureus, including methicillin-susceptible and methicillin-resistant strains, coagulase-negative301541711 - 34 -staphylococci including Staphylococcus epidermidis, Streptococci, Enterococci, Gramnegative organisms including Escherichia coli, Klebsiella pneumoniae, Enterobacter complex, and Pseudomonas aeruginosa, fungi including Candida and Aspergillus species, and mixed infections. The method can include intraoperative molding of the device to match patient anatomy and to contact contaminated surfaces, and may further comprise radiographic confirmation of device placement via incorporated barium sulfate. The method can optionally include systemic antimicrobial therapy administered concomitantly or sequentially with the localized delivery.

[0128] In certain aspects, the method comprises using the bone cement as a fixation medium for orthopedic implants, such as joint prostheses, plates, screws, or intramedullary devices, while simultaneously delivering local antimicrobials to reduce the risk of surgical-site infection. The device can be formulated with honey loading tailored to the mechanical demands of the indication, for example in a non-limiting sense, compositions comprising about 10% honey by weight may be used for load-bearing indications and compositions comprising at least 50% honey by weight are used for non-weight-bearing indications. In certain aspects, the device is deployed as beads, pellets, discs, films, sheets, spacers, or layered architectures to cover exposed bone or to occupy dead space following debridement. The method can further comprise sterilizing the device by ethylene oxide prior to implantation, whereby inherent antimicrobial activity is retained.

[0129] In certain aspects, the method comprises treating osteomyelitis or infected nonunion using a two-stage approach in which the device is implanted as a temporary space maintainer to sterilize the site and maintain anatomy, followed by removal and definitive reconstruction. During the temporary implantation, the porous network enhances antimicrobial elution and promotes integration of vascularized soft tissue at the device-tissue interface to support subsequent healing. The device can be formulated to reduce maximum polymerization temperature and setting time relative to conventional PMMA, thereby permitting incorporation of fragile bioactive agents including vaccines and immunotherapeutics without thermal denaturation.

[0130] In certain aspects, the method comprises localized delivery of immunotherapy. In some aspects, any suitable immunotherapeutic may be incorporated as disclosed herein above. The device can be formulated with an immunotherapy payload comprising an antigen, an adjuvant, and a chemokine, alone or in combination with an antimicrobial agent. In a particular aspect, the device comprises killed Staphylococcus aureus lysate as antigen, CpG oligodeoxynucleotide as adjuvant, and granulocyte-macrophage colony-stimulating factor as chemokine. The method comprises implanting the device at or near the infected or debrided site to achieve early release of the antimicrobial agent over one to two weeks to sterilize the301541711 - 35 -site, followed by continued release of the antigen, adjuvant, and chemokine over two to six weeks to stimulate innate and adaptive immune responses against staphylococcal antigens and reduce relapse risk. The induced immune response can be monitored by serum cytokine levels such as interferon-gamma and interleukin-2, local nodule formation, or other biomarkers. In certain aspects, the antigen is a recombinant protein such as ClfA, IsdB, or SaeR, a conjugate antigen, or a nucleic acid encoding a staphylococcal antigen; the adjuvant is selected from toll-like receptor agonists including CpG, monophosphoryl lipid A, or poly(l:C); and the chemokine or cytokine is selected from granulocyte-macrophage colony-stimulating factor, interleukin-12, interferons, or combinations thereof. In certain aspects, immunotherapeutic agents are encapsulated within poly(lactide-co-glycolide) microparticles having a lactic acid:glycolic acid ratio selected to achieve sustained release over two to six weeks, while the honey-mediated phase provides an earlier release window. The method can further comprise administering systemic immunomodulators or vaccines to complement the localized response.

[0131] In certain aspects, the method comprises delivering pro-regenerative agents to support healing of infected or contaminated defects. The device can be loaded with growth factors, bioactive peptides, calcium phosphate, and / or bioactive glass, alone or in combination with antimicrobial agents, and can be deployed to provide early antimicrobial coverage and subsequent pro-regenerative signaling. The porous network is selected to increase surface area for elution and to permit vascularized soft tissue integration, thereby supporting clearance of infection and tissue repair.

[0132] In certain aspects, the method comprises treating contaminated mandibular or craniofacial defects by placing a device formed from the bone cement as a space maintainer within the defect, optionally without additional antimicrobial loading, to reduce infection risk through inherent antimicrobial activity while maintaining soft tissue contours for staged reconstruction. The method can include subsequent removal and replacement with definitive grafts or implants after infection control.

[0133] In certain aspects, the method comprises delivering bioactive agents to mixed soft tissue and osseous environments by positioning the device at the interface of bone and soft tissue, whereby the porous network communicates with the exterior environment and permits diffusion of agents into both compartments. The method can include selecting honey loading to tune porosity and mechanical properties for the intended clinical application, such that release kinetics and device stiffness are matched to the anatomical site and therapeutic objectives.301541711 - 36 -

[0134] In certain aspects, the method comprises enhancing antibiotic activity against organisms less sensitive to honey by leveraging increased elution. For example, a gentamicin-loaded device can be placed at a site at risk for Pseudomonas aeruginosa infection, whereby honey-mediated porosity enhances gentamicin release and yields improved inhibition relative to gentamicin-loaded PMMA under identical loading and test conditions.

[0135] Across the foregoing aspects, non-limiting examples of device geometry include beads, pellets, discs, films, sheets, layered architectures, or custom intraoperative molds to conform to patient-specific anatomy. The methods optionally comprise imaging to confirm radiopaque device placement, and may include removal or replacement of the device after completion of the intended release schedule. The disclosed methods permit simultaneous or sequential localized delivery of antimicrobial and immunotherapeutic agents, thereby providing immediate decontamination of infected sites and subsequent immune stimulation to reduce recurrence, while supporting tissue integration and healing.EXAMPLESExample 1 : Inherently Antimicrobial Bone Cement (IABC) can be synthesized with high honey content

[0136] IABC was synthesized by free-radical polymerization. Briefly, sterile medical-grade honey in different quantities was added to a polymer powder containing poly(methyl methacrylate) and initiator. The resulting mixture was stirred until homogenous. Liquid methyl methacrylate monomer was then added to the mixture in a ratio of 2:1 polymer powder: liquid. The mixture was stirred by hand as polymerization occurred. The resulting IABC was placed in molds to cure in a shape with pre-defined geometries. Disc devices (6 mm in diameter, 1 mm in height) were created with 30, 40, 50, and 60% by mass of honey and allowed to cure for 24 hours at room temperature. At 60%, there was interference with polymerization and devices had incomplete curing. However, IABC devices were successfully formed at the other three concentrations. Compared with PMMA (conventional bone cement), IABC discs had greater initial density (FIG. 1).

[0137] Cylindrical devices (6 mm in diameter, 12 mm in height) were also fabricated from IABC. Given the synthesis technique, custom geometries were easily achievable. Barium sulfate could be added to the formulations to allow the IABC to be visualized by radiographs (X-rays) for imaging. The devices could be successfully fabricated at different concentrations of poly(methyl methacrylate), methyl methacrylate, honey, and gentamicin (a representative antibiotic), with and without 10 wt / wt% barium sulfate. Table 1 shows some exemplary formulations of lABCs.301541711 - 37 -TABLE 1. EXAMPLES OF DIFFERENT FORMULATIONS. POLY(METHYL METHACRYLATE) = PM MA. MMA = METHYL METHACRYLATE. BaSO4= BARIUM SULFATE.Example 2: IABC is a porous biomaterial

[0138] To better understand the ability of the IABC to release its viscous porogen (in this case, honey) into an aqueous media and create a porous biomaterial, IABC30 and IABC30G disc devices were compared to PMMA devices with and without antibiotic loading. Gentamicin was chosen as a model antibiotic due to its use in current clinical bone cement products. Disc devices underwent leaching in aqueous media under gentle agitation for 48 hours (n=3 per group). Even with antibiotic loading, conventional bone cement lost less than 0.5% of its mass (FIG. 2). IABC30 lost 35% of its mass with bone cement leaching. Loading with antibiotic (IABC30G) resulted in significantly further leaching (p=0.002), suggesting that the unique porosity created in the IABC facilitates diffusion.Example 3: Porosity of IABC is tunable

[0139] The amount of porosity in a biomaterial can affect parameters such as drug release kinetics (dependent on available surface area) as well as interface with surrounding soft tissues. To determine if IABC porosity is tunable based on amount of incorporated honey, cylinders of IABC (12 mm in height and 6 mm in diameter) were created with 10% barium sulfate. These devices were fabricated without any honey (conventional PMMA), with 30% mass honey, and with 50% mass honey (n=5-6 / group). Following leaching, the mass loss was dependent based on the mass of honey used (FIG. 3). The incorporated honey was able to successfully leach out without a significant portion being trapped within the polymer matrix. For example, the 30% honey group lost 31.3% mass and the 50% honey group lost 50.8% mass (p<0.0001). As expected, the conventional PMMA device lost less than 1%. This implies that the formed porous network is interconnected which is desirable for increasing the amount of antibiotic elution.301541711 - 38 -Example 4: IABC has an interconnected porous network

[0140] After leaching, devices fabricated from an IABC formulation (IABC50, Table 1) and a representative conventional bone cement (PM MA) were scanned via microcomputed tomography to analyze inner architecture. The IABC device had an interconnected network of pores compared to the relatively dense PMMA (FIG. 4A). Three-dimensional reconstruction highlighted the increased surface roughness in the porous groups (FIG. 4B).

[0141] Quantitative analysis was performed using computed tomography scans (FIG. 5A and 5B). The total porosity of IABC by three-dimensional radiographic computation was 54.9%, compared to 0.4% of conventional bone cement (PMMA) (p=0.0007, FIG. 5A). Pores in a biomaterial matrix can either be open or closed. A closed pore has no communication with the outside environment; any material within a closed pore remains within a closed pore in a non-biodegradable matrix. An open pore is part of an interconnected network that includes communication with the device surface. With the synthesis of conventional bone cement, the few pores (0.4% of the material) that were in the matrix were closed to the environment in 43% of cases (FIG. 5B). For IABC50, 99.9% of pores were open - in other words, only 0.01% of pores were closed, confirming the sacrificial viscous porogen in IABC50 succeeds in forming an interconnected network within the matrix.Example 5: IABC has inherent antimicrobial properties

[0142] In a pilot study, the ability of IABC to inhibit the growth of S. aureus, the most common pathogen associated with bone and joint infections, was compared to current bone cement. PMMA and IABC devices were synthesized either unloaded (without additional therapeutic) or loaded with gentamicin. Disc devices were prepared with the same geometry used for standard Kirby Bauer antimicrobial testing assays at 6 mm in diameter and 1 mm in height. Disc devices were challenged against Staphylococcus aureus UAMS-1, a well-characterized virulent strain of S. aureus that was originally isolated from a patient with bone infection. After 18 hours of incubation at 37°C, the diameter of the zone of inhibition (area where bacterial growth was inhibited) was measured (FIG. 6A).

[0143] PMMA without antibiotic was ineffective at suppressing bacterial growth. IABC devices without additional antibiotic inherently inhibited the growth of S. aureus (FIG. 6B). IABC loaded with gentamicin significantly outperformed PMMA loaded with gentamicin, resulting in an average zone of inhibition diameter 19% larger than conventional bone cement (p=0.0044).301541711 - 39 -Example 6: IABC is effective against Methicillin-Resistant Staphylococcus aureus (MRSA) and can release multiple antibiotic agents

[0144] Both methicillin-susceptible S. aureus (MSSA) and methicillin-resistant S. aureus (MRSA) cause hardware-associated infections. MRSA is particularly challenging to treat due to its resistance against important classes of antibiotics. IABC was inherently inhibitory against a representative MRSA strain (JE2, FIG. 7A) and MSSA strain (UAMS-1, FIG. 7B), whereas conventional bone cement had no inherent activity against either type. IABC was also capable of releasing vancomycin, a glycopeptide, and gentamicin, an aminoglycoside, with greater inhibition of MSSA and MRSA than conventional bone cement with the equivalent amount of antibiotic (Table 1, FIGS. 7A and 7B).Example 7: IABC Increases Antibiotic Release Compared to Conventional Bone Cement

[0145] Pseudomonas aeruginosa is a gram-negative organism that is a rare cause of orthopedic hardware infection. While rare, P. aeruginosa infection is challenging due to biofilm formation and inherent resistance of P. aeruginosa to many classes of antibiotics. P. aeruginosa was used to evaluate whether antibiotic release from IABC is more effective than conventional bone cement without any major antimicrobial effect from the honey itself, given that Pseudomonas is less sensitive to honey. As expected, neither PMMA nor IABC had any direct effect on P. aeruginosa (FIG. 8B). However, when both devices are loaded with the same amount of gentamicin, IABC caused a 25.4-27.2% greater zone of inhibition (p<0.0001), demonstrating improved drug release compared to conventional bone cement (FIGS. 8A and 8B)Example 8: IABC Has Tunable Mechanical Properties

[0146] The compressive stress and compressive modulus of cylinders of PMMA, IABC30, and IABC50 (Table 1) after leaching were evaluated per the International Organization for Standardization ISO 5833 (“Implants for surgery — Acrylic resin cements”). There was a statistically significant inverse relationship between amount of incorporated honey and compressive strength and modulus (FIGS. 9A-9C). While IABC has lower mechanical properties than conventional PMMA, IABC30 has properties sufficient for bone loading. Formulations with lower yield strength, such as IABC50, could be used in non-weight bearing clinical scenarios which are not uncommon in two stage arthroplasty revision.Example 9: IABC Upregulates Antioxidant Pathways

[0147] IABC, even without additional antibiotics, inhibits S. aureus growth. The underlying mechanism behind honey’s antimicrobial effect is unclear although there are several different301541711 - 40 -hypotheses. During the evaluation of IABC, an unexpected discovery was made showing that the S. aureus that were closest to the device (but not fully inhibited) expressed a greater degree of a yellow pigment (FIG. 10A). S. aureus pigmentation is due to carotenoid biosynthesis and acts as an antioxidant to protect S. aureus against immune-mediated free radicals. Therefore, given that S. aureus upregulated carotenoid biosynthesis as a defense against IABC, it was hypothesized that one mechanism of IABC effectiveness is oxide-based bacterial killing. To test this, wild-type (wt) S. aureus (MRSA JE2) and a S. aureus mutant with a defective gene along the carotenoid biosynthesis pathway (MRSA JE2crtN;;tn) were cultured in a media containing increasing amounts of honey at 37°C and 200 RPM. Both wildtype and mutant had inhibition of growth in the presence of honey. However, the mutant had greater dose-dependent inhibition at both 5 and 10% of honey (FIG. 10B). This may be due to the inability to upregulate carotenoid biosynthesis in response to the oxidative stress caused by honey. To our knowledge, this is the first time this mechanism has been demonstrated. Example 10: Addition of biodegradable microparticles as carriers for therapeutics.

[0148] While therapeutics can be incorporated into either the backbone or bone cement for drug delivery, IABC can act as a platform for more advanced strategies. For instance, biodegradable microparticles synthesized from poly(lactide-co-glycolide) can be added to the formulations (FIG. 11). These microparticles biodegrade through hydrolysis and the rate of their degradation can be controlled by altering the ratio of glycolic acid to lactic acid in their backbone. By adding these to IABC, a biphasic release pattern can be created where agents are released in the first week or two in the bone cement layer and then more slowly released over two to six weeks. This platform can be used to release different therapeutics at different timepoints. For example, antibiotics can be released quickly to sterilize an infected site followed by delayed release of immune-stimulating components, such as adjuvants and specific antigens, to promote adaptive immunity for prevention of infection relapse.Example 11: Summary

[0149] IABC is a novel bone cement formulation with inherent antimicrobial properties and improved architecture which facilitates release of conventional antibiotics and other bioactive agents. The platform has tunable porosity and mechanical properties which allows for different formulations for different clinical applications. Future work will further tune control over antibiotic release kinetics and ability to release other compounds, such as growth factors and immunotherapy components, to assist in tissue healing and boost host immunity, respectively.Example 12: Effect of honey amounts on IABC properties

[0150] These studies were conducted to determine how different amounts of honey affected PMMA-H effectiveness at releasing vancomycin and gentamicin, two of the most common301541711 - 41 -antibiotics used in bone cement formulations. Lastly, the ability of PMMA-H to inhibit bacterial growth was compared to PMMA against the organisms most often responsible for bone and joint infection as shown above, including methicillin-resistant Staphylococcus aureus (MRSA), methicillin-susceptible Staphylococcus aureus (MSSA), and Staphylococcus epidermidis. Example 13: Materials and MethodsBiomaterial Synthesis and Device Fabrication

[0151] Bone cement devices (FIG. 12) were prepared with 0 (PMMA), 15 (PMMA-low honey AKA PMMA-LH), or 30 (PMMA-high honey AKA PMMA-HH) wt% of medical-grade manuka honey (Activon, Beaver Dam, Wl). Bone cement formulations were evaluated as unloaded or loaded with gentamicin or vancomycin (Fisher Scientific, Waltham, MA) at a 3 wt% concentration in this study. All constructs were prepared by established methods.

[0152] Syntheses were performed using a 2:1 powder phase: liquid phase bone cement (Stoelting Corporation, Wood Dale, IL) wt / v ratio. Barium sulphate (Thermo Fisher Scientific, Waltham, MA) was added at 7 wt%. All powder components for the desired formulation were added to honey and mixed thoroughly until uniform. Methyl methacrylate (liquid phase) was then added and the resulting mixture was stirred to initiate polymerization. As the mixture began doughing, it was added to polytetrafluoroethylene molds before hardening. Constructs were allotted 24 hours in molds before storing at4°C until use. PMMA, PMMA-LH, and PMMA-HH discs of 6 mm in diameter and 1 mm in height were fabricated for elution and antimicrobial activity assays.Scanning Electron Microscopy

[0153] To leach honey as a porogen prior to scanning, each disc construct was leached in deionized water at room temperature under mild agitation for a minimum of 48 hours to remove soluble honey porogen. Constructs were then frozen at -80 °C and dried by lyophilization for a minimum of 48 hours (FreeZone Freeze Dryer, Labconco, Kansas City, MO). Scanning electron microscopy (SEM) was used to examine the surface topography and pores of disc surfaces. Discs were sputter coated with 2 nm of gold / palladium target at 30 mA with a Leica EM ACE600 sputter coater (Wetzlar, Germany) and then imaged with a Zeiss Sigma VP field emission scanning electron microscope (Oberkochen, Germany) at an accelerating voltage of 3 kV.Microcomputed Tomography

[0154] Microcomputed tomography (MicroCT) was performed on discs after leaching and lyophilization as described above for each group in FIG. 12 (n=4 per group). Specimens were301541711 - 42 -scanned using a SkySkan 1272 CMOS (Bruker, Billerica, MA) at an isotropic voxel size of 17.1 m, a rotation step of 0.2°, and 3 frame averaging. The X-ray source was used at a voltage of 80 kV and a current of 124 pA. Raw scans were reconstructed using N Recon (Bruker) with a smoothing factor of 3, a ring artifact correction of 5 and a beam hardening correction of 15%. Sample porosity was calculated in CTAN (Bruker), using a threshold of 35-255 on a combined 75 slices of sample excluding the outer edge of the constructs to avoid partial volume effects.Mechanical Characterization

[0155] Mechanical testing was performed to evaluate compressive and bending strength and moduli of PMMA, PMMA-LH, and PMMA-HH. Using the formulations in FIG. 12, cylinders (6 mm diameter, 12 mm in height) and bars (75 mm in length, 10 mm in length, and 3.3 mm in height) were fabricated for compressive and bending testing, respectively, per standard ISO 5833 [2], Constructs were leached in 40 mL deionized water at room temperature with mild agitation for at least 48 hours and dried by lyophilization as described previously. For testing, a TA HD Mechanical Testing System (Texture Technologies Corporation, South Hamilton, MA) was used for compression and bending tests as specified by ISO 5833. For compressive testing, a compressive force at a crosshead speed of 20 mm / min was applied until the construct underwent fracture or surpassed the upper yield point (n=6). For bending tests, the distance between inner and outer loading points was 20 mm and between the two outermost points was 60 mm. Per ISO 5833, a force at crosshead speed 5 mm / min was applied until failure (n=5). The slope of force over displacement was measured between tare force to yield force to calculate modulus. Compressive and bending properties were measured using Exponent Connect (Texture Technologies Corporation, South Hamilton, MA).Antibiotic Elution

[0156] Samples for Liquid Chromatography-Mass Spectrometry (LC / MS) analysis were generated by placing individual discs (n=4 per formulation in Figure 1) into 1 mL of phosphate-buffered saline (PBS) and incubating under mechanical agitation (200 RPM) at 37°C. Supernatant was removed and replaced with fresh PBS at 2, 6, 24, 48, 72, 96, and 168 hours. Samples were stored upon collection at -20°C until sterile filtering for analysis. Quantification of drug release by LC / MS was performed with a Triple Quad 4500 System (SCIEX, Framingham, MA). For unloaded groups, the release supernatants of the first three time points were assayed and the remaining time points were also considered negative if the first three time points did not demonstrate antibiotic release.301541711 - 43 -Antimicrobial1. Kirby-Bauer Testing

[0157] PMMA, PMMA-LH, and PMMA-HH were evaluated by Kirby-Bauer disc diffusion assays against bacteria commonly associated with orthopedic infection. The strains used were UAMS-1 (MSSA), JE2 (MRSA), and ATCC 14990 (Staphylococcus epidermidis).

[0158] Prior to antibacterial testing, discs of each formulation underwent sterilization by ethylene oxide (Anprolene AN76j, Haw River, NC). All discs were utilized based on standards defined by the Clinical and Laboratory Standards Institute Standard M02-A12 with n=4 per formulation per group per species. Briefly, assays were conducted by diluting an overnight culture of desired bacteria in tryptic soy broth (TSB; BD Biosciences, Franklin Lakes, NJ) within a McFarland range of 0.5-1.0. This was then streaked onto the surface of tryptic soy agar plates with 5% sheep’s blood (Thermo Scientific, Waltham, MA) using a sterile cotton swab. Sterile forceps were used to transfer each disc onto the plate. Plates were incubated for 18-24 hours at 37°C. The diameter of the zones of inhibition were measured in millimeters following incubation. Zones of inhibition that were not consistent in diameter were averaged between the smallest and largest diameters to the nearest millimeter.2. High Inoculum Challenge

[0159] To better understand PMMA-H activity against planktonic versus biofilm-embedded bacteria and temporal dynamics of inhibition, a strain of UAMS-1 S. aureus modified to constitutively express lux (U1) was used. UAMS-1 rapidly produces biofilm with peak material coverage at ~5 hours prior to exodus back into the planktonic state. Sterile discs of PMMA, PMMA-LH, and PMMA-HH with and without vancomycin were press-fit into the bottom of wells in a black 96-well plate (n=4 per group) with 198 pLof TSB added per well. At t=0, wells were inoculated with 3x 105colony-forming units (CFU) of S. aureus U1 in 2 pL of TSB. Sterile TSB (n=4) and PMMA discs without any inoculum (“sterile PMMA,” n=4) were added as controls. The plate was placed in a CLARIOstar® Plus plate reader (BMG LabTech, Cary, NC) and incubated at 37°C with double orbital shaking (200 RPM) during idle time. Every 10 minutes, luminescence was measured in each well from the top optic and recorded using MARS software (BMG LabTech, Cary, NC) for five hours (peak biofilm coverage prior to exodus phase. Afterwards, the plate was removed and further incubated for a total of 24 hours at 37°C for CFU quantification during the period of biofilm maturation. The supernatant was removed from discs and plated for CFU counting to assess planktonic bacteria. Each disc was then removed and placed individually in 1 mL of TSB containing 0.3% v / v Tween-80 and kept at 4°C to arrest further growth. Organisms attached to the discs and / or embedded in biofilm on the301541711 - 44 -discs were displaced via sonication for 10 minutes (Cole-Parmer® UC-200, Vernon Hills, IL), vortexed for 5 minutes, and plated for CFU counting.Statistical Analysis

[0160] Total porosity, percentage of open porosity, mechanical properties, cumulative release of antibiotics, zones of inhibition, luminescence relative light units, and bacterial CFU were compared by a one-way analysis of variance with post hoc analysis by Tukey’s honestly significant difference (a = 0.05) unless stated otherwise using R (v4.4.1, R Foundation, Vienna, Austria).Example 14: Results related to Example 12.The Effect of Honey on Architecture and Mechanical Properties

[0161] When combined with medical-grade honey prior to free radical generation, PMMA-H successfully polymerized to form a gold-colored bone cement construct (FIGs. 13A-13B).Following porogen leaching, honey-containing groups underwent a qualitative change from a golden color to white (FIG. 13A). The surfaces of low- and high-honey containing groups had increased number of pores in a dose-dependent manner (FIG. 13B). Leached disc devices underwent microCT scanning to characterize device porosity (FIG. 14A). PMMA-H groups had significantly greater porosity in a dose-dependent manner for unloaded, vancomycin-loaded, and gentamicin-loaded groups (FIG. 14B). The percentage of open pores, i.e. interconnected pores that communicate with the environment surrounding the device, was significantly highest in the PMMA-HH groups (FIG. 14C).

[0162] The introduction of honey as porogen decreased the compressive and bending strength and modulus of devices (FIGs. 15A-D). For example, unloaded PMMA had a compressive yield strength of~1.6xand ~3.6x higher than unloaded PMMA-LH and PMMA-HH, respectively (FIG. 15A). This effect was slightly less pronounced for bending; unloaded PMMA had a bending yield strength ~1.5x and 3.3x higher than unloaded PMMA-LH and PMMA-HH, respectively (FIG. 15C). These trends were similar for moduli. The loading of antibiotics into PMMA decreased its mechanical properties. For example, PMMA lost 12.1 ± 2.4% and 31.5 ± 15.6% of its compressive strength with vancomycin and gentamicin, respectively. Honey appeared to shield bone cement from the additional loss of mechanical properties with antibiotic loading- for example, PMMA-HH gained 24.1 ± 11.1% and 10.2 ± 15.4% compressive strength with vancomycin and gentamicin loading, respectively (FIG. 15A).301541711 - 45 -The Effect of on Bone Cement Antibiotic Elution

[0163] For elution and antimicrobial studies, no pre-leaching was performed. All devices containing antibiotic demonstrated antibiotic elution under in vitro release conditions (37°C, PBS, 200 RPM) (FIG. 16A). When normalized to percent loaded antibiotic (3 wt%), 46.5%, 78.9%, and 100.7% of loaded vancomycin was eluted cumulatively at one week by PMMA, PMMA-LH, and PMMA-HH, respectively (FIG. 16B). Likewise, 25.1%, 89.4%, and 85.9% of gentamicin was eluted cumulatively at one week by PMMA, PMMA-LH, and PMMA-HH, respectively (FIG. 16B). For both antibiotics, PMMA-HH groups had significantly greater elution than PMMA for all time points (p<0.01). PMMA-LH also had significantly greater elution than PMMA (p<0.01) for gentamicin although this was not statistically significant for vancomycin.The Effect of Honey on Bone Cement Antimicrobial Activity

[0164] In standardized Kirby-Bauer assays without the addition of antibiotics, PMMA did not inhibit the growth of MSSA, MRSA, or S. epidermidis (FIGs. 17A-17C). Unloaded PMMA-HH significantly inhibited the growth of all staphylococcal isolates; at lower honey concentrations (PMMA-LH), there was less consistent inhibition. At 3 wt% antibiotic loading, vancomycin-loaded PMMA-HH generated zones of inhibition 35.9% (MSSA), 41.9% (MRSA), and 77.4% (S. epidermidis) greater than PMMA. Gentamicin-loaded PMMA-HH had zones of inhibition 13.0% (MSSA), 11.0% (MRSA), and 20.4% (S. epidermidis) greater than PMMA. In most cases, PMMA-LH had a smaller inhibitory effect compared to PMMA-HH but was still significantly greater than PMMA in all cases other than for PMMA with gentamicin and MSSA (FIG. 17A).

[0165] In a high inoculum challenge of biomaterials without any antibiotic loading, PMMA-HH significantly reduced S. aureus bioluminescence to sterile levels by two hours whereas PMMA-LH decreased the rate of lux production and the overall peak compared to PMMA (FIG. 18A).With vancomycin loading, PMMA-H and PMMA were able to reduce luminescence from peak value, although only PMMA-HH and PMMA-LH were able to reduce luminescence to the level of sterile discs by 3 hours and 4 hours of exposure, respectively (FIG. 18B). When comparing across experiments, PMMA-HH without antibiotic loading had comparable inhibitory effects to PMMA loaded with vancomycin in terms of temporal inhibition of S. aureus bioluminescence (FIG. 18C).

[0166] Following 24 hours of culture after a high level of inoculation with S. aureus, bacteria in the surrounding media (planktonic) and attached to the disc and / or embedded in biofilm on the disc (biofilm) were counted (FIGs. 19A-19B). Compared to unloaded PMMA, PMMA-LH301541711 - 46 -and PMMA-HH reduced the amount of S. aureus by over 150- and 20,000-fold, respectively (FIG. 19A). When loaded with vancomycin, all PMMA-H groups had no growth and three of the four PMMA discs had no growth. In this rigorous model of high inoculum challenge, all unloaded groups grew S. aureus on the discs themselves with no significant differences between groups (FIG. 19B). With antibiotic loading, all PMMA-H groups had no growth. Three of four PMMA groups still had growth of S. aureus.Discussion

[0167] Bone and joint infections are a growing burden on the healthcare system and are expected to increase with an aging population requiring more implanted devices. Current antibiotic-loaded bone cements have been used for decades despite poor elution kinetics and loss of mechanical properties. In this study, medical-grade honey and PMMA was combined to synthesize PMMA-H with the goal of generating a new orthopedic biomaterial better capable of inhibiting bacteria. Physical properties were evaluated through SEM, microCT, and mechanical testing, its antibiotic elution kinetics through LC / MS, and its antimicrobial activity by Kirby-Bauer testing and a high inoculum challenge.

[0168] Honey Creates a Porous Architecture and Shields Against Antibiotic-induced Mechanical Loss

[0169] This study was novel in the application of a porogen with inherent antimicrobial properties (medical-grade honey) rather than an inert porogen to create a porous PMMA-based device (FIGs. 12 and 13). Porosity increases surface area to promote efficient elution of therapeutics from bone cements. PMMA-H formulations are significantly more porous than conventional bone cement (FIG. 14B). This porosity is interconnected and communicates with the environment outside of the device (FIG. 14C), facilitating antibiotic elution (FIGs. 16A-16B).For bone tissue engineering, bulk scaffold porosity is a critical factor in promoting osteogenesis. The degree of porosity of PMMA-H can be tuned by the amount of honey incorporated and may be a useful tool for both drug delivery and host tissue integration.

[0170] Consistent with the effect of other porogens, the introduction of porosity in PMMA-H does decrease compressive and bending strength and moduli compared to PMMA alone (FIGs.15A-15D). PMMA-HH did not exhibit compressive weakening with additional antibiotic loading (FIG. 15C); this may be attributed to antibiotic preferentially solubilizing in the porogen during phase separation of the hydrophobic methyl methacrylate and relatively more hydrophilic honey and therefore not interfering with polymerization. This novel shielding of bone cement antibiotic-induced mechanical loss by porogen introduces a new tool in bone cement development for anti-infective applications.301541711 - 47 -

[0171] The Incorporation of Honey Increases Antibiotic Elution

[0172] PMMA-HH had significantly greater antibiotic elution than antibiotic-loaded PM MA over 7 days, releasing 100.7% and 86.0% versus 46.5% and 25.1% of loaded vancomycin and gentamicin, respectively (FIGs. 16A-16B). PMMA-LH trended towards increased vancomycin elution and had significantly greater gentamicin elution than PMMA. Vancomycin (1,449.3 g / mol) has a higher molecular weight than gentamicin (477.6 g / mol) which may explain why greater porosity more closely correlated with greater release. PMMA-H released antibiotics more efficiently than PMMA but its duration of release was similar. This may further suggest preferential separation of antibiotic into the porogen phase rather than sequestration into the polymer phase where it would be embedded into a non-biodegradable matrix following polymerization with limited elution.

[0173] Honey Generates Antimicrobial Cement with or without Antibiotics

[0174] Unlike typical porogens, honey has inherent antimicrobial properties during leaching from PMMA-H that result in staphylococcal inhibition (FIGs. 17A-17C). Over the period when S. aureus attaches and produces biofilm on a material surface

[0013] , PMMA-H without any antibiotic was able to decrease the amount bioluminescence to sterile levels at the same rate as PMMA loaded with vancomycin (FIG. 18C). When combined with vancomycin, PMMA-LH and PMMA-HH were able to more rapidly decrease bioluminescence compared to PMMA at the same vancomycin loading levels (FIG. 18B). When directly inoculated with 300,000 organisms, PMMA-LH and PMMA-HH loaded with vancomycin had no growth after 24 hours whereas PMMA loaded with vancomycin still had viable S. aureus in 75% of cases (FIG. 19B), suggesting great improvement of PMMA-H in bacterial inhibition even in a rigorous biofilm model compared to conventional bone cement.

[0175] Chitosan was previously explored as a potential antimicrobial porogen but was found to not have efficacy against S. aureus or S. epidermidis when used in combination with PMMA and also decreased the release of gentamicin with antibiotic loading. Without being bound to theory the success of PMMA-H compared to chitosan may be due to the high molecular weight of chitosan preventing effective leaching and the possibility of its cationic charge increasing interactions with aminoglycosides and / or incidental conjugation.

[0176] While surface roughness can correlate with increased risk for bacterial colonization, porous PMMA (using carboxymethyl cellulose as porogen) has not been shown to increase infection rates in animal studies or a human case series including those for mandibular repair at high risk for contamination due to proximity to the oral mucosa even when used without loaded antibiotic. In our challenge where PMMA-H without antibiotic was unable to overcome a very high level of inoculation, the increased porosity / surface roughness did not cause worse301541711 - 48 -bacterial burden compared to conventional non-porous PMMA (FIG. 19B). With this strategy, PMMA-H has enhanced initial antibiotic release (FIGs. 16A-16B) as well as synergy with the antimicrobial effects of honey (FIGs. 17-19), decontaminating an infected site and / or providing protection against pathogens that are present at the time of surgery or shortly thereafter. After the antibiotic and honey elutes out, host tissues can integrate into the pores and provide further vascularization near the biomaterial interface which also theoretically reduces infection risk due to immune surveillance. Future work will further evaluate the ability of PMMA-H compared to PMMA with and without antibiotics to treat orthopedic infection and stimulate bony regeneration in an infected preclinical in vivo model.Conclusion

[0177] The present disclosure describes a new formulation of bone cement combining naturally derived polysaccharide-based honey and synthetic poly(methyl methacrylate). This formulation has honey dose-dependent porosity with inherent activity against S. aureus and S. epidermidis. When combined with antibiotics, PMMA-H has greater and more rapid inhibition of bacteria than antibiotic-loaded PMMA. By adding an antimicrobial porogen to bone cement, we have increased its capacity to mitigate infection as a biomaterial.Example 15: IABC Has Decreased Synthesis Temperature

[0178] A major limitation of conventional bone cement is the exothermic reaction of poly(methyl methacrylate) (PMMA) which denatures or otherwise destroys loaded therapeutics. This has historically limited bone cement to delivering only a few classes of heat stabile antibiotics. By incorporating an inherently antimicrobial gel (medical-grade honey) into the structure of PMMA at the time of synthesis, IABC decreases the maximum temperature as well as setting temperature during synthesis compared to PMMA, conventional bone cement (FIG. 20). The setting time was also significantly decreased for IABC. These assays were performed per the industry standard ISO 5833. Because the maximum temperature of synthesis is decreased by more than 25% and because the time of setting (when temperatures are elevated) has also been decreased, IABC could also be used to deliver more fragile therapeutics such as antigens, adjuvants, and immunogenic chemokines.Example 16: Use of IABC as Immunotherapy

[0179] Infection of orthopedic devices, especially by Staphylococcus aureus, is the most dreaded complication facing patients and surgeons in today’s clinical practice. Given that IABC is more effective at delivering molecules than conventional bone cement and IABC has decreased synthetic temperature which may facilitate encapsulation and elution of fragile therapeutics without destruction, a pilot study was conducted to transform IABC into an301541711 - 49 -immunotherapy. Antigen (antibiotic-killed S. aureus lysate) at 1.2 wt%, adjuvant (CpG) at 1 wt%, and a chemokine (granulocyte-macrophage colony-stimulating factor or GM-CSF) at 0.03 wt% were added to IABC at 30 wt% honey with 3 wt% vancomycin to create a new type of biomaterial immunotherapy (IABCX) that de-contaminates an infected wound (local release of vancomycin) while also vaccinating via delivery of antigen, adjuvant, and chemokine.

[0180] IABCX was compared to inherently antimicrobial bone cement with 30 wt% honey plus 3 wt% vancomycin and without vaccine components (IABC) in a subcutaneous implantation murine model. The goal was to determine if there was any concerning toxicity and to measure any apparent immune differences between the two groups.

[0181] Mice tolerated implantation of both devices well. Mice did not significantly lose more weight with IABCX compared to IABC over a 28-day period following implantation. A nodule formed over the implantation site, likely due to recruitment of immune cells. This nodule was nearly twice as large in the IABCX group by Day 7 (FIG. 21A). The nodule began to recede and was not appreciated by Day 28. When serum cytokines were measured at Day 28, the IABCX group had persistently greater interferon-gamma (IFN) and interleukin-2 (IL-2) compared to naive mice and mice that received IABC, suggesting sustained immune boosting activity (FIG. 21 B). Histology of the subcutaneous tissues surrounding the implant revealed a large and more organized membrane around IABCX compared to IABC, suggesting more persistent immune stimulation (FIG. 21 C). This study suggests that inherently antimicrobial bone cement can be transformed into an immunotherapy in addition to its prior established activity as an antimicrobial biomaterial.301541711 - 50 -

Claims

CLAIMSWhat is claimed is:

1. A bone cement comprising:a) a polymer scaffold; andb) a viscous porogen comprising one or more types of honey, or derivatives thereof.

2. The bone cement of claim 1, wherein the polymer scaffold comprises one or more polymers.

3. The bone cement of claim 2, wherein the one or more polymers comprise natural polymers, or synthetic polymers, or any combination thereof.

4. The bone cement of claim 2 or claim 3, wherein the one or more polymers are biocompatible and / or biodegradable.

5. The bone cement of claim 4, wherein the one or more polymer comprises one or more of poly(methyl methacrylate) (PMMA), polycaprolactone (PCL), polydioxanone (PDO), poly (glycolic acid) (PGA), poly(L-lactic acid) (PLA), poly(lactide-co-glycolide) (PLGA), poly(L-lactide) (PLLA), poly(D,L-lactide) (P(DLLA)), poly(ethylene glycol) (PEG), poly(e-caprolactone) (PCL), montmorillonite (MMT), poly(L-lactide-co-e-caprolactone) (P(LLA-CL)), poly(£-caprolactone-co-ethyl ethylene phosphate) (P(CL-EEP)), poly[bis(p-methylphenoxy) phosphazene] (PNmPh), poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), poly(ester urethane) urea (PEUU), poly(p-dioxanone) (PPDO), polyurethane (PU), polyethylene terephthalate (PET), poly(ethylene-co-vinylacetate) (PEVA), poly(ethylene oxide) (PEG), poly(phosphazene), poly(ethylene-co-vinyl alcohol), a polymer nanoclay nanocomposite; a halogenated polymer solution containing metal compounds (e.g., graphite), poly(ethylenimine), grafted cellulosics, poly(ethyleneoxide), poly vinylpyrrolidone, or polystyrene (PS), or any combination thereof.

6. The bone cement of claim 5, wherein the polymer scaffold comprises PMMA.

7. The bone cement of claim 6, wherein the bone cement comprises about 10% to about 90% PMMA by weight.

8. The bone cement of any one of claims 1-7, wherein the bone cement comprises about 15% to about 60% by weight of the one or more types of honey or derivatives thereof.301541711 - 51 -9. The bone cement of any one of claims 1-8, further comprising about 0.01% to about 20% by weight of a radiopacifier.

10. The bone cement of any one of claims 1-9, further comprising one or more bioactive agents.

11. The bone cement of any one of claims 1-10, wherein the viscous porogen further comprises one or more hydrophilic polymers.

12. The bone cement of claim 11 , wherein the one or more hydrophilic polymers comprise polyethylene glycol (PEG), hyaluronic acid, chitosan, alginate, polyvinyl alcohol (PVA), polyvinylpyrrolidone (PVP), or collagen, or any combination thereof.

13. The bone cement of claim 1, comprising:about 10% to about 90% by weight of poly(methyl methacrylate) (PMMA);about 0.01 % to about 20% by weight of a radiopacifier;about 15% to about 60% by weight of one or more types of honey, or derivatives thereof; andabout 0.001% to about 5% by weight of one or more bioactive agent.

14. The bone cement of any one of claims 1-13, wherein the one or more types of honey comprise one or more of manuka honey, raw honey, tualang honey, buckwheat honey, acacia honey, eucalyptus honey, lavender honey, thyme honey, Sidr honey, heather honey, wildflower honey, or jarrah honey, or any derivative thereof, or any combination thereof.

15. The bone cement of any one of claims 1-14, wherein the bone cement comprises about 15% to about 30% by weight of the one or more types of honey or derivatives thereof.

16. The bone cement of any one of claims 1-14, wherein the bone cement comprises about 30% by weight of the one or more types of honey or derivatives thereof.

17. The bone cement of any one of claims 1-14, wherein the bone cement comprises about 30% to about 60% by weight of the one or more types of honey or derivatives thereof.

18. The bone cement of any one of claims 115, wherein the bone cement comprises about 50% by weight of the one or more types of honey or derivatives thereof.301541711 - 52 -19. The bone cement of claim 18, wherein the bone cement is a non-load bearing formulation.

20. The bone cement of any one of claims 9-19, wherein the one or more types of honey is medical grade honey.

21. The bone cement of any one of claims 1-20, wherein the bone cement has inherent antimicrobial property against one or more bacterial species, one of more fungal species, and / or one or more viral species.

22. The bone cement of claim 21 , wherein the bacterial species is a streptococcal species, staphylococcal species including Staphylococcus aureus, methicillin-susceptible S. aureus (MSSA), and methicillin-resistant S. aureus (MRSA), Enterococcus species, Escherichia coli, Enterobacter species, Klebsiella pneumoniae, Pseudomonas aeruginosa, Salmonella species, Helicobacter pylori, or Cutibacterium acnes, or any combination thereof.

23. The bone cement of claim 21 , wherein the one or more viral species comprise varicellazoster virus, rubella virus, influenza virus, herpes simplex virus, respiratory syncytial virus, or human immunodeficiency virus, or any combination thereof.

24. The bone cement of claim 21, the one or more fungal species comprise Candida albicans, Candida auris, Rhodotorula species, Aspergillus species, Mucor species, or dermatophytes such as Trichophyton rubrum, or any combination thereof.

25. The bone cement of any one of claims 10-24, wherein the one or more bioactive agents is an antimicrobial agent, an immunotherapeutic, and / or a vaccine.

26. The bone cement of claim 25, wherein the antimicrobial agent is an antibacterial, antiviral, anti-parasitic, or an antifungal agent, or any combination thereof.

27. The bone cement of claim 26, wherein the antimicrobial agent is incorporated into the viscous porogen.

28. The bone cement of claim 26, wherein the antimicrobial agent is incorporated into a delivery particle incorporated into the bone cement.

29. The bone cement of any one of claims 26-28, wherein the antibacterial agent comprises of ampicillin, amoxicillin, benzylpenicillin, dalbavancin, oritavancin, phenoxymethylpenicillin, bacampicillin, pivampicillin, carbenicillin, cioxacillin, cyclacillin, dicloxacillin, methicillin, oxacillin, piperacillin, ticarcillin, flucioxacillin, cefuroxime, cefetamet,301541711 - 53 -cefetrame, cefixine, cefoxitin, ceftazidime, ceftizoxime, latamoxef, levofloxacin, cefoperazone, ceftriaxone, cefsulodin, cefotaxime, cephalexin, cefaclor, cefadroxil, cefalothin, cefazolin, cefpodoxime, ceftibuten, aztreonam, tigemonam, erythromycin, dirithromycin, roxithromycin, azithromycin, clarithromycin, clindamycin, paldimycin, lincomycirl, vancomycin, moxifloxacin, metronidazole benzoate, spectinomycin, tobramycin, paromomycin, metronidazole, tinidazole, ornidazole, amifloxacin, cinoxacin, ciprofloxacin, difloxacin, enoxacin, fleroxacin, norfloxacin, ofloxacin, linezolid, temafloxacin, teromyocin, doxycycline, minocycline, tetracycline, daptomycin, chlortetracycline, oxytetracycline, methacycline, rolitetracyclin, nitrofurantoin, nalidixic acid, gentamicin, rifampicin, amikacin, netilmicin, imipenem, cilastatin, chloramphenicol, furazolidone, nifuroxazide, sulfadiazin, sulfametoxazol, trimethoprimsulfamethoxazole, bismuth subsalicylate, colloidal bismuth subcitrate, gramicidin, mecillinam, cloxiquine, dichlorobenzylalcohol, povidone, sugars, mucopolysaccharides, chlorobutanol, quarternary ammonium compounds such as benzalkonium chloride, organic mercurials, parahydroxy benzoates, aromatic alcohols, halogenated phenols, sorbic acid, benzoic acid, dioxin, EDTA, BHT, BHA, TBHQ, gallate esters, NDGA, tocopherols, gum guaiac, lecithin, boric acid, citric acid, p-Hydroxy benzoic acid esters, propionates, sulfur dioxide and sulfites, nitrates and nitrites of potassium and sodium, diethyl pyrocarbonate, sodium diacetate, diphenyl, hexamethylene tetramine o-phenyl phenol, or sodium o-phenylphenoxide, or any variant thereof, or any combination thereof.

30. The bone cement of any one of claims 26-29, wherein the bone cement has antimicrobial properties against Staphylococcal species (including Staphylococcus aureus, methicillin-susceptible S. aureus (MSSA), and methicillin-resistant S. aureus (MRSA)), coagulase-negative Staphylococci (including Staphylococcus epidermidis), Streptococcal species, Enterococcus species, Cutibacterium acnes, Corynebacterium striatum, Escherichia coli, Klebsiella pneumoniae, Enterobacter complex, Candida albicans and other yeast, Aspergillus species, and / or Pseudomonas aeruginosa.

31. The bone cement of any one of claims 26-28, wherein the antifungal agent comprises fluconazole, itraconazole, ketoconazole, voriconazole, amphotericin B, nystatin, clotrimazole, terbinafine, posaconazole, griseofulvin, caspofungin, micafungin, anidulafungin, rezafungin, or flucytosine, or any combination thereof.

32. The bone cement of any one of claims 26-28, wherein the antiviral agent comprises acyclovir, valacyclovir, ganciclovir, valganciclovir, tenofovir, entecavir, remdesivir, ritonavir, lopinavir, glecaprevir, pibrentasvir, sofosbuvir, foscarnet, oseltamivir, zanamivir, dolutegravir, raltegravir, maraviroc, enfuvirtide, palivizumab, sotrovimab, bebtelovimab, or interferons, or any combination thereof.301541711 - 54 -33. The bone cement of any one of claims 26-28, wherein the antiparasitic agent comprises chloroquine, artemether-lumefantrine, primaquine, metronidazole, tinidazole, nitazoxanide, pentamidine, albendazole, mebendazole, ivermectin, praziquantel, sodium stibogluconate, amphotericin B, eflornithine, nifurtimox, benznidazole, or diethylcarbamazine (DEC), or any combination thereof.

34. The bone cement of any one of claims 25-33, wherein the bone cement comprises a vaccine.

35. The bone cement of claim 34, wherein the vaccine is incorporated into the viscous porogen.

36. The bone cement of claim 34, wherein the vaccine is incorporated into a delivery particle incorporated into the bone cement.

37. The bone cement of any one of claims 34-36, wherein the vaccine is a live attenuated vaccine, a killed microorganism, a protein-based vaccine, a polysaccharide-protein conjugate vaccine, a capsular polysaccharide vaccine, a toxoid based vaccine, a multi-component vaccine, killed whole cell vaccine, a DNA based vaccine, or a mRNA vaccine, or any combination thereof.

38. The bone cement of any one of claims 34-36, wherein the vaccine is against Staphylococcus aureus, methicillin-susceptible S. aureus (MSSA), methicillin-resistant S. aureus (MRSA), coagulase-negative Staphylococci (including Staphylococcus epidermidis), Streptococcal species, Enterococcus species, Cutibacterium acnes, Corynebacterium striatum, Escherichia coli, Klebsiella pneumoniae, Enterobacter complex, Candida albicans and other yeast, Aspergillus species, and / or Pseudomonas aeruginosa.

39. The bone cement of any one of claims 34-36, wherein the vaccine comprises inactivated Staphylococcus aureus, recombinant proteins vaccines: ClfA, IsdB, and SaeR or other surface / intracellular proteins of interest, conjugates vaccines thereof, and / or DNA or mRNA vaccines encoding a surface antigen or intracellular antigen of Staphylococcus aureus, or any combination thereof.

40. The bone cement of claim 28, wherein the delivery particle is a liposome, a solid-lipid nanoparticle (SLN), a nanostructured lipid carrier, poly(lactic-co-glycolic acid) (PLGA) nanoparticles, a chitosan nanoparticle, a polystyrene nanoparticle, a metallic nanoparticle, a virus like particle (VLP), a ferritin nanoparticle, a albumin nanoparticle, a silica based nanoparticle, a viral particle, a dextran nanoparticle, a alginate microparticle, or an exosome.301541711 - 55 -41. The bone cement of claim 40, wherein the delivery particle exhibit delayed dissolution compared to the viscous porogen.

42. The bone cement of any one of claim 40 or claim 41 , wherein the delivery particle is a sustained release delivery particle.

43. The bone cement of any one of claims 1-42, wherein the bone cement has a viscosity of 1,000 to 10,000 cp.

44. The bone cement of any one of claims 1-43, wherein the bone cement has a stiffness of about 1 MPa to about 2000MPa.

45. The bone cement of any one of claims 9-44, wherein the radiopacifier is barium sulphate, or zirconium dioxide, or both.

46. The bone cement of any one of claims 10-45, wherein the one or more bioactive agents comprises both an antimicrobial agent and a vaccine.

47. The bone cement of any one of claims 26-46, wherein the one or more bioactive agents comprises an immunotherapeutic.

48. The bone cement of claim 47, wherein the immunotherapeutic is an immunostimulator, co-stimulatory molecules, dendritic cell therapeutic, modified cell therapeutic (e.g., CAR-T cells), cytokine, adoptive T-cell therapeutic, or checkpoint inhibitors, or any combination thereof49. The bone cement of any one of claims 1-48, further comprising stem cells, osteocytes, bone tissue, connective tissue cells, bioactive glass, fibrin, collagen, bioactive peptides, growth factors, calcium phosphate, or one or more trace metals, or any combination thereof.

50. The bone cement of any one of claims 1-49, wherein the bone cement exhibits improved interactions with host tissues in comparison to a bone cement without a viscous porogen.

51. The bone cement of any one of claims 1-50, wherein the bone cement is for treating a bone defect.

52. A bioactive agent depot system comprising a bone cement of any one of claims 1-50.

53. A method of treating a bone defect in a subject in need thereof, the method comprising contacting the bone with a bone cement of any one of claims 1-50.301541711 - 56 -54. The method of claim 53, wherein the bone defect is a simple fracture, compound fracture, comminuted fracture, transverse fracture, oblique fracture, spiral fracture, stress fracture, pathological fracture, avulsion fracture, impacted fracture, segmental fracture, compression fracture, hairline fracture, torus fracture, bone cysts, osteoporosis, osteomalacia, Paget’s disease, osteogenesis imperfecta, fibrous dysplasia, osteomyelitis, bone tumors, bone necrosis, metabolic bone diseases, congenital bone defects, trauma-induced bone loss, or bone graft failure, or any combination thereof.

55. The method of claim 53 or claim 54, wherein the bone cement has a bead, pellet, film, disc, sheet, or layered architecture, or can be molded in the operating room to a custom geometry.

56. A method of fixing an implant on a bone in a subject in need thereof, the method comprising contacting the bone with the implant via the bone cement of any one of claims 1-50.

57. A method of preventing or treating a bone site or a bone prosthetic site infection, the method comprising contacting the bone site or the bone prosthetic site with a bone cement of any one of claims 1-50.

58. A method of localized delivery of a bioactive agent at a site in a subject in need thereof, the method comprising contacting the site with a bone cement of any one of claims 1-50, or implanting the bioactive agent depot system of claim 52.

59. A bone cement comprising:about 10% to about 90% by weight of poly(methyl methacrylate) (PMMA);about 0.01% to about 20% by weight of a radiopacifier comprising barium sulfate, and / or zirconium sulfate;about 15% to about 50% by weight of one or more types of honey, or derivatives thereof; andabout 0.001 % to about 5% by weight of one or more immunotherapeutic agents comprising one or more of, an antigen, an adjuvant, a chemokine, or any combinations thereof.

60. The bone cement of claim 59, wherein the bone cement comprises about 30% by weight of the one or more types of honey or derivatives thereof.301541711 - 57 -61. The bone cement of any one of claims 59-60, wherein the bone cement further comprises one or more antibiotics.

62. The bone cement of claim 61 , wherein the antibiotic comprises about 3% by weight of vancomycin.

63. The bone cement of any one of claims 59-62, wherein the antigen comprises a microbial antigen, tumor-associated antigens, neoantigens, peptide antigens, or protein antigens, or any combination thereof.

64. The bone cement of any one of claims 59-63, wherein the antigen is a microbial antigen comprising a live attenuated microorganism, an inactivated microorganism, a heat-killed microorganism, a chemically inactivated microorganism, an antibiotic killed microorganism, a microbial lysate, a microbial subunit antigen, or a fragment, epitope, or combination thereof.

65. The bone cement of claim 64, wherein the microbial antigen comprises killed S. aureus.

66. The bone cement of any one of claim 59-65, wherein the one or more immunotherapeutic comprises killed S. aureus, granulocyte-macrophage colony-stimulating factor or GM-CSF.

67. The bone cement of any one of claims 59-66, wherein the adjuvant comprises CpG.

68. The bone cement of any one of claims 59-67, comprising:about 10% to about 90% by weight of PMMA;about 0.5% to about 20% by weight of radiopacifier comprising barium sulfate and / or zirconium dioxide;about 15% to about 60% by weight of the one or more types of honey, or derivatives thereof;about 1% to about 5% by weight of an antibiotic; andabout 0.001 % to about 5% by weight of one or more immunotherapeutic agents comprising killed or attenuated S. aureus, adjuvant comprising CpG, and a chemokine comprising GM-CSF.301541711 - 58 -69. A method of making a bone cement, comprising:1) preparing a precursor solution comprising about 10% to about 90% by weight of PM MA, about 10% to about 35% by weight of MMA, about 0.5% to about 20% by weight of a radiopacifier, and about 10% to about 60% by weight of one or more types of honey, or derivatives thereof;2) homogenizing the precursor solution; and3) providing conditions suitable to form a polymer construct.

70. The method of claim 69, further comprising one or more of the steps of shaping, molding, curing, gelling, or washing, or any combination thereof, of the polymer.

71. The method of claim 69 or claim 70, further comprising adding a bioactive agent to the bone cement.301541711 - 59 -

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