Triggered Release of Ampicillin from Metallic Implant Coatings for Combating Periprosthetic Infections.

Periprosthetic infections caused by Staphylococcus aureus (S. aureus) pose unique challenges in orthopedic surgeries, in part due to the bacterium's capacity to invade surrounding bone tissues besides forming recalcitrant biofilms on implant surfaces. We previously developed prophylactic implant coatings for the on-demand release of vancomycin, triggered by the cleavage of an oligonucleotide (Oligo) linker by micrococcal nuclease (MN) secreted by the Gram-positive bacterium, to eradicate S. aureus surrounding the implant in vitro and in vivo. Building upon this coating platform, here we explore the feasibility of extending the on-demand release to ampicillin, a broad-spectrum aminopenicillin ß-lactam antibiotic that is more effective than vancomycin in killing Gram-negative bacteria that may accompany S. aureus infections. The amino group of ampicillin was successfully conjugated to the carboxyl end of an MN-sensitive Oligo covalently integrated in a polymethacrylate hydrogel coating applied to titanium alloy pins. The resultant Oligo-Ampicillin hydrogel coating released the ß-lactam in the presence of S. aureus and successfully cleared nearby S. aureus in vitro. When the Oligo-Ampicillin-coated pin was delivered to a rat femoral canal inoculated with 1000 cfu S. aureus, it prevented periprosthetic infection with timely on-demand drug release. The clearance of the bacteria from the pin surface as well as surrounding tissue persisted over 3 months, with no local or systemic toxicity observed with the coating. The negatively charged Oligo fragment attached to ampicillin upon cleavage from the coating did diminish the antibiotic's potency against S. aureus and Escherichia coli (E. coli) to varying degrees, likely due to electrostatic repulsion by the anionic surfaces of the bacteria. Although the on-demand release of the ß-lactam led to adequate killing of S. aureus but not E. coli in the presence of a mixture of the bacteria, strong inhibition of the colonization of the remaining E. coli on hydrogel coating was observed. These findings will inspire considerations of alternative broad-spectrum antibiotics, optimized drug conjugation, and Oligo linker engineering for more effective protection against polymicrobial periprosthetic infections.

Modulating On-Demand Release of Vancomycin from Implant Coatings via Chemical Modification of a Micrococcal Nuclease-Sensitive Oligonucleotide Linker.

Periprosthetic infections are one of the most serious complications in orthopedic surgeries, and those caused by Staphylococcus aureus (S. aureus) are particularly hard to treat due to their tendency to form biofilms on implants and their notorious ability to invade the surrounding bones. The existing prophylactic local antibiotic deliveries involve excessive drug loading doses that could risk the development of drug resistance strains. Utilizing an oligonucleotide linker sensitive to micrococcal nuclease (MN) cleavage, we previously developed an implant coating capable of releasing covalently tethered vancomycin, triggered by S. aureus-secreted MN, to prevent periprosthetic infections in the mouse intramedullary (IM) canal. To further engineer this exciting platform to meet broader clinical needs, here, we chemically modified the oligonucleotide linker by a combination of 2'-O-methylation and phosphorothioate modification to achieve additional modulation of its stability/sensitivity to MN and the kinetics of MN-triggered on-demand release. We found that when all phosphodiester bonds within the oligonucleotide linker 5'-carboxy-mCmGTTmCmG-3-acrydite, except for the one between TT, were replaced by phosphorothioate, the oligonucleotide (6PS) stability significantly increased and enabled the most sustained release of tethered vancomycin from the coating. By contrast, when only the peripheral phosphodiester bonds at the 5'- and 3'-ends were replaced by phosphorothioate, the resulting oligonucleotide (2PS) linker was cleaved by MN more rapidly than that without any PS modifications (0PS). Using a rat femoral canal periprosthetic infection model where 1000 CFU S. aureus was inoculated at the time of IM pin insertion, we showed that the prophylactic implant coating containing either 0PS- or 2PS-modified oligonucleotide linker effectively eradicated the bacteria by enabling the rapid on-demand release of vancomycin. No bacteria were detected from the explanted pins, and no signs of cortical bone changes were detected in these treatment groups throughout the 3 month follow-ups. With an antibiotic tethering dose significantly lower than conventional antibiotic-bearing bone cements, these coatings also exhibited excellent biocompatibility. These chemically modified oligonucleotides could help tailor prophylactic anti-infective coating strategies to meet a range of clinical challenges where the risks for S. aureus prosthetic infections range from transient to long-lasting.

Chemically Crosslinked Amphiphilic Degradable Shape Memory Polymer Nanocomposites with Readily Tuned Physical, Mechanical, and Biological Properties.

Facile surgical delivery and stable fixation of synthetic scaffolds play roles just as critically as degradability and bioactivity in ensuring successful scaffold-guided tissue regeneration. Properly engineered shape memory polymers (SMPs) may meet these challenges. Polyhedral oligomeric silsesquioxanes (POSSs) can be covalently integrated with urethane-crosslinked polylactide (PLA) to give high-strength, degradable SMPs around physiological temperatures. To explore their potential for guided bone regeneration, here we tune their hydrophilicity, degradability, cytocompatibility, and osteoconductivity/osteoinductivity by crosslinking star-branched POSS-PLA with hydrophilic polyethylene glycol diisocyanates of different lengths and up to 60 wt % hydroxyapatite (HA). The composites exhibit high compliance, toughness, up to gigapascal storage moduli, and excellent shape recovery (>95%) at safe triggering temperatures. Water swelling ratios and hydrolytic degradation rates positively correlated with the hydrophilic crosslinker lengths, while the negative impact of degradation on the proliferation and osteogenesis of bone marrow stromal cells was mitigated with HA incorporation. Macroporous composites tailored for a rat femoral segmental defect were fabricated, and their ability to stably retain and sustainedly release recombinant osteogenic bone morphogenetic protein-2 and support cell attachment and osteogenesis was demonstrated. These properties combined make these amphiphilic osteoconductive degradable SMPs promising candidates as next-generation synthetic bone grafts.

Surface-grafted zwitterionic polymers improve the efficacy of a single antibiotic injection in suppressing S. aureus periprosthetic infections.

Implant-associated bacterial infections are difficult to treat due to the tendency of biofilm formation on implant surfaces, which protects embedded pathogens from host defense and impedes antibiotic penetration, rendering systemic antibiotic injections ineffective. Here, we test the hypothesis that implant coatings that reduce bacterial colonization would make planktonic bacteria within the periprosthetic environment more susceptible to conventional systemic antibiotic treatment. We covalently grafted zwitterionic polymer brushes poly(sulfobetaine methacryate) from Ti6Al4V surface to increase the substrate surface hydrophilicity and reduce staphylococcus aureus (S. aureus) adhesion. Using a mouse femoral intramedullary (IM) canal infection model, we showed that the anti-fouling coating applied to Ti6Al4V IM implants, when combined with a single vancomycin systemic injection, significantly suppressed both bacterial colonization on implant surfaces and the periprosthetic infections, outperforming either treatment alone. This work supports the hypothesis that grafting anti-fouling polymers to implant surfaces improves the efficacy of systemic antibiotic injections to combat periprosthetic infections.

Significant Suppression of Staphylococcus aureus Colonization on Intramedullary Ti6Al4V Implants Surface-Grafted with Vancomycin-Bearing Polymer Brushes.

Orthopedic implant-associated bacterial infection presents a major health threat due to tendency for periprosthetic bacterial colonization/biofilm formation that protects bacteria from host immune response and conventional antibiotic treatment. Using surface-initiated atom transfer radical polymerization and copper-catalyzed azide-alkyne cycloaddition (CuAAC), alkynylated vancomycin is conjugated to azido-functionalized side chains of polymethacrylates grafted from Ti6Al4V. High-efficiency CuAAC across the substrate is confirmed by complete surface conversion of azides by X-ray photoelectron spectroscopy (XPS) and elemental mapping of changing characteristic elements. The vancomycin-modified surface (Ti-pVAN) significantly reduces in vitro adhesion and colonization of Staphylococcus aureus (S. aureus), a main bacterial pathogen responsible for periprosthetic infection and osteomyelitis, compared to untreated Ti6Al4V, supporting retained antibacterial properties of the covalently conjugated antibiotics. When the surface-modified intramedullary Ti-pVAN pins are inserted into mouse femoral canals infected by bioluminescent Xen29 S. aureus, significantly reduced local bioluminescence along with mitigated blood markers for infection are detected compared to untreated Ti6Al4V pins over 21 days. Ti-pVAN pins retrieved after 21 days are confirmed with ∼20-fold reduction in adherent bacteria counts compared to untreated control, supporting the ability of surface-conjugated vancomycin in inhibiting periprosthetic S. aureus adhesion and colonization.

Multifunctional scaffolds for facile implantation, spontaneous fixation, and accelerated long bone regeneration in rodents.

Graft-guided regenerative repair of critical long bone defects achieving facile surgical delivery, stable graft fixation, and timely restoration of biomechanical integrity without excessive biotherapeutics remains challenging. Here, we engineered hydration-induced swelling/stiffening and thermal-responsive shape-memory properties into scalable, three-dimensional-printed amphiphilic degradable polymer-osteoconductive mineral composites as macroporous, non-load-bearing, resorbable synthetic grafts. The distinct physical properties of the grafts enabled straightforward surgical insertion into critical-size rat femoral segmental defects. Grafts rapidly recovered their precompressed shape, stiffening and swelling upon warm saline rinse to result in 100% stable graft fixation. The osteoconductive macroporous grafts guided bone formation throughout the defect as early as 4 weeks after implantation; new bone remodeling correlated with rates of scaffold composition-dependent degradation. A single dose of 400-ng recombinant human bone morphogenetic protein-2/7 heterodimer delivered via the graft accelerated bone regeneration bridging throughout the entire defect by 4 weeks after delivery. Full restoration of torsional integrity and complete scaffold resorption were achieved by 12 to 16 weeks after surgery. This biomaterial platform enables personalized bone regeneration with improved surgical handling, in vivo efficacy and safety.

Micrococcal-Nuclease-Triggered On-Demand Release of Vancomycin from Intramedullary Implant Coating Eradicates Staphylococcus aureus Infection in Mouse Femoral Canals.

Preventing orthopedic implant-associated bacterial infections remains a critical challenge. Current practices of physically blending high-dose antibiotics with bone cements is known for cytotoxicity while covalently tethering antibiotics to implant surfaces is ineffective in eradicating bacteria from the periprosthetic tissue environment due to the short-range bactericidal actions, which are limited to the implant surface. Here, we covalently functionalize poly(ethylene glycol) dimethacrylate hydrogel coatings with vancomycin via an oligonucleotide linker sensitive to Staphylococcus aureus (S. aureus) micrococcal nuclease (MN) (PEGDMA-Oligo-Vanco). This design enables the timely release of vancomycin in the presence of S. aureus to kill the bacteria both on the implant surface and within the periprosthetic tissue environment. Ti6Al4V intramedullary (IM) pins surface-tethered with dopamine methacrylamide (DopaMA) and uniformly coated with PEGDMA-Oligo-Vanco effectively prevented periprosthetic infections in mouse femoral canals inoculated with bioluminescent S. aureus. Longitudinal bioluminescence monitoring, µCT quantification of femoral bone changes, end point quantification of implant surface bacteria, and histological detection of S. aureus in the periprosthetic tissue environment confirmed rapid and sustained bacterial clearance by the PEGDMA-Oligo-Vanco coating. The observed eradication of bacteria was in stark contrast with the significant bacterial colonization on implants and osteomyelitis development found in the absence of the MN-sensitive bactericidal coating. The effective vancomycin tethering dose presented in this on-demand release strategy was >200 times lower than the typical prophylactic antibiotic contents used in bone cements and may be applied to medical implants and bone/dental cements to prevent periprosthetic infections in high-risk clinical scenarios. This study also supports the timely bactericidal action by MN-triggered release of antibiotics as an effective prophylactic method to bypass the notoriously harder to treat periprosthetic biofilms and osteomyelitis.

Impaired osteogenesis of T1DM bone marrow-derived stromal cells and periosteum-derived cells and their differential in-vitro responses to growth factor rescue.

BACKGROUND: Poor bone quality, increased fracture risks, and impaired bone healing are orthopedic comorbidities of type 1 diabetes (T1DM). Standard osteogenic growth factor treatments are inadequate in fully rescuing retarded healing of traumatic T1DM long bone injuries where both periosteal and bone marrow niches are disrupted. We test the hypotheses that osteogenesis of bone marrow-derived stromal cells (BMSCs) and periosteum-derived cells (PDCs), two critical skeletal progenitors in long bone healing, are both impaired in T1DM and that they respond differentially to osteogenic bone morphogenetic proteins (BMPs) and/or insulin-like growth factor-1 (IGF-1) rescue. METHODS: BMSCs and PDCs were isolated from Biobreeding Diabetes Prone/Worcester rats acquiring T1DM and normal Wistar rats. Proliferation, osteogenesis, and adipogenesis of the diabetic progenitors were compared with normal controls. Responses of diabetic progenitors to osteogenesis rescue by rhBMP-2/7 heterodimer (45 or 300 ng/ml) and/or rhIGF-1 (15 or 100 ng/ml) in normal and high glucose cultures were examined by alizarin red staining and qPCR. RESULTS: Diabetic BMSCs and PDCs proliferated slower and underwent poorer osteogenesis than nondiabetic controls, and these impairments were exacerbated in high glucose cultures. Osteogenesis of diabetic PDCs was rescued by rhBMP-2/7 or rhBMP-2/7 + rhIGF-1 in both normal and high glucose cultures in a dose-dependent manner. Diabetic BMSCs, however, only responded to 300 ng/nl rhBMP-2/7 with/without 100 ng/ml rhIGF-1 in normal but not high glucose osteogenic culture. IGF-1 alone was insufficient in rescuing the osteogenesis of either diabetic progenitor. Supplementing rhBMP-2/7 in high glucose osteogenic culture significantly enhanced gene expressions of type 1 collagen (Col 1), osteocalcin (OCN), and glucose transporter 1 (GLUT1) while suppressing that of adipogenic marker peroxisome proliferator-activated receptor gamma (PPARγ) in diabetic PDCs. The same treatment in high glucose culture only resulted in a moderate increase in Col 1, but no significant changes in OCN or GLUT1 expressions in diabetic BMSCs. CONCLUSIONS: This study demonstrates more effective osteogenesis rescue of diabetic PDCs than BMSCs by rhBMP-2/7 with/without rhIGF-1 in a hyperglycemia environment, underscoring the necessity to tailor biochemical therapeutics to specific skeletal progenitor niches. Our data also suggest potential benefits of combining growth factor treatment with blood glucose management to optimize orthopedic therapeutic outcomes for T1DM patients.

Age-dependent Changes in the Articular Cartilage and Subchondral Bone of C57BL/6 Mice after Surgical Destabilization of Medial Meniscus.

Age is the primary risk factor for osteoarthritis (OA), yet surgical OA mouse models such as destabilization of the medial meniscus (DMM) used for evaluating disease-modifying OA targets are frequently performed on young adult mice only. This study investigates how age affects cartilage and subchondral bone changes in mouse joints following DMM. DMM was performed on male C57BL/6 mice at 4 months (4 M), 12 months (12 M) and 19+ months (19 M+) and on females at 12 M and 18 M+. Two months after surgery, operated and unoperated contralateral knees were harvested and evaluated using cartilage histology scores and µCT quantification of subchondral bone plate thickness and osteophyte formation. The 12 M and 19 M+ male mice developed more cartilage erosions and thicker subchondral bone plates after DMM than 4 M males. The size of osteophytes trended up with age, while the bone volume fraction was significantly higher in the 19 M+ group. Furthermore, 12 M females developed milder OA than males as indicated by less cartilage degradation, less subchondral bone plate sclerosis and smaller osteophytes. Our results reveal distinct age/gender-dependent structural changes in joint cartilage and subchondral bone post-DMM, facilitating more thoughtful selection of murine age/gender when using this surgical technique for translational OA research.

Templated repair of long bone defects in rats with bioactive spiral-wrapped electrospun amphiphilic polymer/hydroxyapatite scaffolds.

Effective repair of critical-size long bone defects presents a significant clinical challenge. Electrospun scaffolds can be exploited to deliver protein therapeutics and progenitor cells, but their standalone application for long bone repair has not been explored. We have previously shown that electrospun composites of amphiphilic poly(d,l-lactic acid)-co-poly(ethylene glycol)-co-poly(d,l-lactic acid) (PELA) and hydroxyapatite (HA) guide the osteogenic differentiation of bone marrow stromal cells (MSCs), making these scaffolds uniquely suited for evaluating cell-based bone regeneration approaches. Here we examine whether the in vitro bioactivity of these electrospun scaffolds can be exploited for long bone defect repair, either through the participation of exogenous MSCs or through the activation of endogenous cells by a low dose of recombinant human bone morphogenetic protein-2 (rhBMP-2). In critical-size rat femoral segmental defects, spiral-wrapped electrospun HA-PELA with preseeded MSCs resulted in laminated endochondral ossification templated by the scaffold across the longitudinal span of the defect. Using GFP labeling, we confirmed that the exogenous MSCs adhered to HA-PELA survived at least 7 days postimplantation, suggesting direct participation of these exogenous cells in templated bone formation. When loaded with 500 ng of rhBMP-2, HA-PELA spirals led to more robust but less clearly templated bone formation than MSC-bearing scaffolds. Both treatment groups resulted in new bone bridging over the majority of the defect by 12 weeks. This study is the first demonstration of a standalone bioactive electrospun scaffold for templated bone formation in critical-size long bone defects.

Vancomycin-bearing synthetic bone graft delivers rhBMP-2 and promotes healing of critical rat femoral segmental defects.

BACKGROUND: Bone grafts simultaneously delivering therapeutic proteins and antibiotics may be valuable in orthopaedic trauma care. Previously, we developed a poly(2-hydroxyethyl methacrylate)-nanocrystalline hydroxyapatite (pHEMA-nHA) synthetic bone graft that, when preabsorbed with 400-ng rhBMP-2/7, facilitated the functional repair of critical-size rat femoral defects. Recently, we showed that pHEMA-nHA effectively retains/releases vancomycin and rhBMP-2 in vitro. The success of such a strategy requires that the incorporation of vancomycin does not compromise the structural integrity of the graft nor its ability to promote bone healing. QUESTIONS/PURPOSES: (1) To evaluate the ability of pHEMA-nHA-vancomycin composites in combination with 3-µg rhBMP-2 to repair 5 mm rat femoral segmental defects, and (2) To determine if the encapsulated vancomycin impairs the graft/rhBMP-2-assisted bone repair. METHODS: pHEMA-nHA-vancomycin, pHEMA-nHA, or collagen sponge control with/without 3-µg rhBMP-2 were press-fit in 5 mm femoral defects in SASCO-SD male rats (289-300 g). Histology, microcomputed tomography, and torsion testing were performed on 8- and 12-week explants to evaluate the extent and quality of repair. The effect of vancomycin on the temporal absorption of endogenous BMP-2 and stromal cell-derived factor-1 was evaluated by immunohistochemistry. These factors are important for bone healing initiation and stem cell recruitment, respectively. RESULTS: Partial bridging of the defect with bony callus by 12 weeks was observed with pHEMA-nHA-vancomycin without rhBMP-2 while full bridging with substantially mineralized callus and partial restoration of torsional strength was achieved with 3-µg rhBMP-2. The presence of vancomycin changed the absorption patterns of endogenous proteins on the grafts, but did not appear to substantially compromise graft healing. CONCLUSIONS: The composite pHEMA-nHA-vancomycin preabsorbed with 3-µg rhBMP-2 promoted repair of 5 mm rat femoral segmental defects. With the sample sizes applied, vancomycin encapsulation did not appear to have a negative effect on bone healing. CLINICAL RELEVANCE: pHEMA-nHA-vancomycin preabsorbed with rhBMP-2 may be useful in the repair of critical-size long bone defects prone to infections.

Three-dimensionally presented anti-fouling zwitterionic motifs sequester and enable high-efficiency delivery of therapeutic proteins.

Zwitterions are well known for their anti-biofouling properties. Past investigations of zwitterionic materials for biomedical uses have been centered on exploiting their ability to inhibit non-specific adsorption of proteins. Here, we report that zwitterionic motifs, when presented in three dimensions (e.g. in crosslinked hydrogels), could effectively sequester osteogenic bone morphogenetic protein-2 (rhBMP-2). The ionic interactions between rhBMP-2 and the 3-D zwitterionic network enabled dynamic sequestering and sustained release of the protein with preserved bioactivity. We further demonstrated that the zwitterionic hydrogel confers high-efficiency in vivo local delivery of rhBMP-2. It can template the functional healing of critical-size femoral segmental defects in rats with rhBMP-2 at a loading dose substantially lower than those required for current natural or synthetic polymeric carriers. These findings reveal a novel function of zwitterionic materials beyond their commonly perceived anti-biofouling property, and may establish 3-D zwitterionic matrices as novel high-efficiency vehicles for protein/ionic drug therapeutic delivery.