A Bioglass-Based Antibiotic (Vancomycin) Releasing Bone Void Filling Putty to Treat Osteomyelitis and Aid Bone Healing.

While the infection rate after primary total joint replacements (TJR) sits at 1-2%, for trauma-related surgery, it can be as high as 3.6 to 21.2% based on the type of trauma; the risk of reinfection after revision surgery is even higher. Current treatments with antibiotic-releasing PMMA-based bone cement/ beads and/or systemic antibiotic after surgical debridement do not provide effective treatment due to fluctuating antibiotic levels at the site of infection, leading to insufficient local antibiotic concentration. In addition, non-biodegradable PMMA does not support bone regrowth in the debrided void spaces and often must be removed in an additional surgery. Here, we report a bioactive glass or bioglass (BG) substrate-based biodegradable, easy to fabricate "press fitting" antibiotic-releasing bone void filling (ABVF-BG) putty to provide effective local antibiotic release at the site of infection along with support for bone regeneration. The ABVF-BG putty formulation had homogenously distributed BG particles, a porous structure, and showed putty-like ease of handling. Furthermore, the ABVF-BG putty demonstrated in vitro antibacterial activity for up to 6 weeks. Finally, the ABVF-BG putty was biodegradable in vivo and showed 100% bacterial eradication (as shown by bacterial cell counts) in the treatment group, which received ABVF-BG putty, compared to the infection control group, where all the rats had a high bacterial load (4.63 × 106 ± 7.9 × 105 CFU/gram bone) and sustained osteomyelitis. The ABVF-BG putty also supported bone growth in the void space as indicated by a combination of histology, µCT, and X-ray imaging. The potential for simultaneous infection treatment and bone healing using the developed BG-based ABVF-BG putty is promising as an alternative treatment option for osteomyelitis.

Nanotechnology in the Diagnosis and Treatment of Osteomyelitis.

Infection remains one of the largest threats to global health. Among those infections that are especially troublesome, osteomyelitis, or inflammation of the bone, typically due to infection, is a particularly difficult condition to diagnose and treat. This difficulty stems not only from the biological complexities of opportunistic infections designed to avoid the onslaught of both the host immune system as well as exogenous antibiotics, but also from changes in the host vasculature and the heterogeneity of infectious presentations. While several groups have attempted to classify and stage osteomyelitis, controversy remains, often delaying diagnosis and treatment. Despite a host of preclinical treatment advances being incubated in academic and company research and development labs worldwide, clinical treatment strategies remain relatively stagnant, including surgical debridement and lengthy courses of intravenous antibiotics, both of which may compromise the overall health of the bone and the patient. This manuscript reviews the current methods for diagnosing and treating osteomyelitis and then contemplates the role that nanotechnology might play in the advancement of osteomyelitis treatment.

Anti-Coagulant and Antimicrobial Recombinant Heparin-Binding Major Ampullate Spidroin 2 (MaSp2) Silk Protein.

Governed by established structure-property relationships, peptide motifs comprising major ampullate spider silk confer a balance of strength and extensibility. Other biologically inspired small peptide motifs correlated to specific functionalities can be combined within these units to create designer silk materials with new hybrid properties. In this study, a small basic peptide, (ARKKAAKA) known to both bind heparin and mimic an antimicrobial peptide, was genetically linked to a protease-resistant, mechanically robust silk-like peptide, MaSp2. Purified fusion proteins (four silk domains and four heparin-binding peptide repeats) were expressed in E. coli. Successful fusion of a MaSp2 spider silk peptide with the heparin-binding motif was shown using a variety of analytical assays. The ability of the fusion peptide to bind heparin was assessed with ELISA and was further tested for its anticoagulant property using aPTT assay. Its intrinsic property to inhibit bacterial growth was evaluated using zone of inhibition and crystal violet (CV) assays. Using this strategy, we were able to link the two types of genetic motifs to create a designer silk-like protein with improved hemocompatibility and antimicrobial properties.

Infection Responsive Smart Delivery of Antibiotics Using Recombinant Spider Silk Nanospheres.

Frequent and inappropriate usage of antibiotics has changed the natural evolution of bacteria by reducing susceptibility and increasing resistance towards antibacterial agents. New resistance mechanisms evolved in the response to host defenses and pharmaceutical interventions are threatening our ability to treat common infections, resulting in increased mortality. In the face of this rising epidemic, antibiotic drug discovery, which has long been overlooked by big pharma, is reaching a critical low. Thus, the development of an infection-responsive drug delivery system, which may mitigate multidrug resistance and preserve the lifetime of our current antibiotic arsenal, has garnered the attention of both popular science and funding agencies. The present work describes the development of a thrombin-sensitive linker embedded into a recombinant spider silk copolymer to create a nanosphere drug delivery vehicle. Recent studies have suggested that there is an increase in thrombin-like activity during Staphylococcus aureus infection; thus, drug release from this new "smart" nanosphere can be triggered in the presence of infection. A thrombin sensitive peptide (TSP) was synthesized, and the thrombin cleavage sensitivity was determined by HPLC. The results showed no cleavage of the peptide when exposed to human serum whereas the peptide was cleaved when incubated with S. aureus exudate. Subsequently, the peptide was coupled with a silk copolymer via EDC-NHS chemistry and formulated into nanospheres encapsulating antibiotic vancomycin. These nanospheres were evaluated for in vitro infection-responsive drug release and antimicrobial activity. Finally, the drug responsive nanospheres were assessed for efficacy in an in vivo septic arthritis model. Our study provides evidence that the protein conjugate was enzyme responsive and can be used to formulate targeted drug release to combat infections against multidrug-resistant bacterial strains.

Development and Processing of Novel Heparin Binding Functionalized Modified Spider Silk Coating for Catheter Providing Dual Antimicrobial and Anticoagulant Properties.

Tailored surface coatings have been used for decades to improve material performance in blood. Among different approaches, heparin based biomedical coatings have found great success in the commercial catheter market. However, they have their own limitations. Coating of a vascular device with a heparin binding peptide (HBP), which can sequester the circulating heparin, presents numerous advantages over both systemic heparin therapy and direct heparin bound surfaces. Embedding HBP in a silk biopolymer provides the mechanical integrity necessary under dynamic flow conditions to both insert the catheter and maintain proper blood flow. Furthermore, due to the similarity in structure of HBP with antimicrobial peptides, it is predicted that the fusion protein will also show antimicrobial property, a critical and unique aspect to combat catheter related blood stream infections and extend the longevity of hemodialysis catheters. To assess this hypothesis, a recombinant fusion protein (S4H4) containing both silk amino acid motifs and HBP was assessed as a coating on a silicone surface. After validating that, the protein was deposited on the surface via XPS, Raman spectroscopy, ATR and SEM imaging, antimicrobial and anticoagulant activities were evaluated. The coating was able to prevent not only planktonic bacterial growth but also prevented the growth of a biofilm. Finally, the coating had both antibacterial and anticoagulant effect simultaneously. This study proves the successful production of a silk-based biopolymer that can be embedded with a heparin-binding functionality to create a dual functional device coating that can prevent infection and thrombosis together.

An Antibiotic-Releasing Bone Void Filling (ABVF) Putty for the Treatment of Osteomyelitis.

The number of total joint replacements (TJR) is on the rise with a corresponding increase in the number of infected TJR, which necessitates revision surgeries. Current treatments with either non-biodegradable, antibiotic-releasing polymethylmethacrylate (PMMA) based bone cement, or systemic antibiotic after surgical debridement do not provide effective treatment due to fluctuating antibiotic levels at the site of infection. Here, we report a biodegradable, easy-to-use "press-fitting" antibiotic-releasing bone void filling (ABVF) putty that not only provides efficient antibiotic release kinetics at the site of infection but also allows efficient osseointegration. The ABVF formulation was prepared using poly (D,L-lactide-co-glycolide) (PLGA), polyethylene glycol (PEG), and polycaprolactone (PCL) as the polymer matrix, antibiotic vancomycin, and osseointegrating synthetic bone PRO OSTEON for bone-growth support. ABVF was homogenous, had a porous structure, was moldable, and showed putty-like mechanical properties. The ABVF putty released vancomycin for 6 weeks at therapeutic level. Furthermore, the released vancomycin showed in vitro antibacterial activity against Staphylococcus aureus for 6 weeks. Vancomycin was not toxic to osteoblasts. Finally, ABVF was biodegradable in vivo and showed an effective infection control with the treatment group showing significantly higher bone growth (p < 0.001) compared to the control group. The potential of infection treatment and osseointegration makes the ABVF putty a promising treatment option for osteomyelitis after TJR.

Using hydrodynamic focusing to predictably alter the diameter of synthetic silk fibers.

Spiders and silkworms provide a model of superior processing for multifunctional and highly versatile high-performance fibers. Mimicking the spider's complex control system for chemical and mechanical gradients has remained an ongoing obstacle for synthetic silk production. In this study, the use of hydrodynamic fluid focusing within a 3D printed biomimetic spinning system to recapitulate the biological spinneret is explored and shown to produce predictable, small diameter fibers. Mirroring in silico fluid flow simulations using a hydrodynamic microfluidic spinning technique, we have developed a model correlating spinning rates, solution viscosity and fiber diameter outputs that will significantly advance the field of synthetic silk fiber production. The use of hydrodynamic focusing to produce controlled output fiber diameter simulates the natural silk spinning process and continues to build upon a 3D printed biomimetic spinning platform.