Porous Nano-Fiber Structure of Modified Electrospun Chitosan GBR Membranes Improve Osteoblast Calcium Phosphate Deposition in Osteoblast-Fibroblast Co-Cultures.

Desirable characteristics of electrospun chitosan membranes (ESCM) for guided bone regeneration are their nanofiber structure that mimics the extracellular fiber matrix and porosity for the exchange of signals between bone and soft tissue compartments. However, ESCM are susceptible to swelling and loss of nanofiber and porous structure in physiological environments. A novel post-electrospinning method using di-tert-butyl dicarbonate (tBOC) prevents swelling and loss of nanofibrous structure better than sodium carbonate treatments. This study aimed to evaluate the hypothesis that retention of nanofiber morphology and high porosity of tBOC-modified ESCM (tBOC-ESCM) would support more bone mineralization in osteoblast-fibroblast co-cultures compared to Na2CO3 treated membranes (Na2CO3-ESCM) and solution-cast chitosan solid films (CM-film). The results showed that only the tBOC-ESCM retained the nanofibrous structure and had approximately 14 times more pore volume than Na2CO3-ESCM and thousands of times more pore volume than CM-films, respectively. In co-cultures, the tBOC-ESCM resulted in a significantly greater calcium-phosphate deposition by osteoblasts than either the Na2CO3-ESCM or CM-film (p < 0.05). This work supports the study hypothesis that tBOC-ESCM with nanofiber structure and high porosity promotes the exchange of signals between osteoblasts and fibroblasts, leading to improved mineralization in vitro and thus potentially improved bone healing and regeneration in guided bone regeneration applications.

Chitosan Membranes Stabilized with Varying Acyl Lengths Release Cis-2-Decenoic Acid and Bupivacaine at Controlled Rates and Inhibit Pathogenic Biofilm.

BACKGROUND: Adherence of complex bacterial biofilm communities to burned tissue creates a challenge for treatment, with infection causing 51% of burn victim deaths. This study evaluated the release of therapeutics from wound care biomaterials and their antimicrobial activity against pathogens Staphylococcus aureus, Acinetobacter baumannii, and Pseudomonas aeruginosa. METHODS: Electrospun chitosan membranes (ESCMs) were fabricated and acylated with chain lengths ranging from 6-10 carbons then loaded with 0.15 mg of anti-biofilm agent, cis-2-decenoic acid (C2DA), and 0.5 mg of local anesthetic, bupivacaine. RESULTS: Combinations of therapeutics released from modified ESCMs at a cumulative amount of 45-70% of bupivacaine and less than 20% of C2DA. Results from bacterial studies suggest that this combination reduced biofilm 10-fold for S. aureus, 2-fold for Acinetobacter baumannii, and 2-3-fold for Pseudomonas aeruginosa by 24 hours. Additionally, dual loaded groups reduced planktonic Staphylococcus aureus ~4-fold by 24 hours as well as Acinetobacter baumannii ~3-fold by 48 hours. CONCLUSIONS: The combination of therapeutics used has a significant role in biofilm prevention for selected strains via direct contact or diffusion in aqueous solutions.

Cis-2-Decenoic Acid and Bupivacaine Delivered from Electrospun Chitosan Membranes Increase Cytokine Production in Dermal and Inflammatory Cell Lines.

Wound dressings serve to protect tissue from contamination, alleviate pain, and facilitate wound healing. The biopolymer chitosan is an exemplary choice in wound dressing material as it is biocompatible and has intrinsic antibacterial properties. Infection can be further prevented by loading dressings with cis-2-decenoic acid (C2DA), a non-antibiotic antimicrobial agent, as well as bupivacaine (BUP), a local anesthetic that also has antibacterial capabilities. This study utilized a series of assays to elucidate the responses of dermal cells to decanoic anhydride-modified electrospun chitosan membranes (DA-ESCMs) loaded with C2DA and/or BUP. Cytocompatibility studies determined the toxic loading ranges for C2DA, BUP, and combinations, revealing that higher concentrations (0.3 mg of C2DA and 1.0 mg of BUP) significantly decreased the viability of fibroblasts and keratinocytes. These high concentrations also inhibited collagen production by fibroblasts, with lower loading concentrations promoting collagen deposition. These findings provide insight into preliminary cellular responses to DA-ESCMs and can guide future research on their clinical application as wound dressings.

Titanium coated with 2-decenoic analogs reduces bacterial and fungal biofilms.

AIMS: Due to antibiotic tolerance of microbes within biofilm, non-antibiotic methods for prevention and treatment of implant-related infections are preferable. The goal of this work is to evaluate a facile loading strategy for medium-chain fatty-acid signaling molecules 2-heptycyclopropane-1-carboxylic acid (2CP), cis-2-decenoic acid (C2DA), and trans-2-decenoic acid, which all act as diffusible signaling factors (DSFs), onto titanium surfaces for comparison of their antimicrobial efficacy. METHODS AND RESULTS: Titanium coupons were drop-coated with 0.75 mg of DSF in ethanol and dried. Surface characteristics and the presence of DSF were confirmed with Fourier Transform infrared spectroscopy, x-ray photoelectron spectroscopy, and water contact angle. Antimicrobial assays analyzing biofilm and planktonic Staphylococcus aureus, Escherichia coli, or Candida albicans viability showed that planktonic growth was reduced after 24-h incubation but only sustained through 72 h for S. aureus and C. albicans. Biofilm formation on the titanium coupons was also reduced for all strains at the 24-h time point, but not through 72 h for E. coli. Although ∼60% of the loaded DSF was released within the first 2 days, enough remained on the surface after 4 days of elution to significantly inhibit E. coli and C. albicans biofilm. Cytocompatibility evaluations with a fibroblast cell line showed that none of the DSF-loaded groups decreased viability, while C2DA and 2CP increased viability by up to 50%. CONCLUSIONS: In this study, we found that DSF-loaded titanium coupons can inhibit planktonic microbes and prevent biofilm attachment, without toxicity to mammalian cells.

Antibacterial and biocompatible polyaniline-doped titanium oxide layers.

Titanium anodization has been shown to produce crystalline oxides exhibiting photocatalytic reactions that form reactive oxygen species (ROS) when exposed to UV light. The ROS subsequently attack bacteria cells, and thus reduce bacteria attachment on titanium implant surfaces. Polyaniline (PANI) is a conductive polymer that has shown antibacterial properties when electropolymerized onto titanium. Our research group hypothesized the addition of PANI to crystalline titanium oxide surfaces would increase the available free electrons and thus increase photocatalytic activity (PCA). This research led to the development of a novel single-step anodization approach for PANI doping crystalline titanium oxide layers. The objective of the present study was to determine the proper aniline electrolyte concentration needed to maximize the PCA and reduce bacterial attachment on the formed oxides. Aniline concentrations up to 1 M were added into a 1 M sulfuric acid electrolyte. The formed oxides exhibited increased PANI surface coverage but decreased anatase and rutile crystalline titanium oxide phase formation with increasing aniline electrolyte concentrations. Despite exhibiting the lowest levels of anatase and rutile formation, the 0.75 M and 1 M aniline oxides with the greatest PANI surface coverage also exhibited the highest PCA levels. 1 M aniline oxides showed significantly higher PCA under UVA irradiation compared to oxides formed from aniline concentrations up to 0.5 M (p < 0.001). 0.75 M aniline oxides exhibited significant reductions in Staphylococcus aureus attachment with or without UVA irradiation compared to control oxides without PANI. MTT and live/dead assays confirmed cytocompatibility and nearly 100% cell viability for the PANI doped oxides.

Evaluation of Magnesium-Phosphate Particle Incorporation into Co-Electrospun Chitosan-Elastin Membranes for Skin Wound Healing.

Major challenges facing clinicians treating burn wounds are the lack of integration of treatment to wound, inadequate mechanical properties of treatments, and high infection rates which ultimately lead to poor wound resolution. Electrospun chitosan membranes (ESCM) are gaining popularity for use in tissue engineering applications due to their drug loading ability, biocompatibility, biomimetic fibrous structure, and antimicrobial characteristics. This work aims to modify ESCMs for improved performance in burn wound applications by incorporating elastin and magnesium-phosphate particles (MgP) to improve mechanical and bioactive properties. The following ESCMs were made to evaluate the individual components' effects; (C: chitosan, CE: chitosan-elastin, CMg: chitosan-MgP, and CEMg: chitosan-elastin-MgP). Membrane properties analyzed were fiber size and structure, hydrophilic properties, elastin incorporation, MgP incorporation and in vitro release, mechanical properties, degradation profiles, and in vitro cytocompatibility with NIH3T3 fibroblasts. The addition of both elastin and MgP increased the average fiber diameter of CE (~400 nm), CMg (~360 nm), and CEMg (565 nm) compared to C (255 nm). Water contact angle analysis showed elastin incorporated membranes (CE and CEMg) had increased hydrophilicity (~50°) compared to the other groups (C and CMg, ~110°). The results from the degradation study showed mass retention of ~50% for C and CMg groups, compared to ~ 30% seen in CE and CEMg after 4 weeks in a lysozyme/PBS solution. CMg and CEMg exhibited burst-release behavior of ~6 µg/ml or 0.25 mM magnesium within 72 h. In vitro analysis with NIH3T3 fibroblasts showed CE and CEMg groups had superior cytocompatibility compared to C and CMg. This work has demonstrated the successful incorporation of elastin and MgP into ESCMs and allows for future studies on burn wound applications.

Simulated large joint fluid model for evaluating intra-articular antibiotic delivery systems: initial evaluation using antibiotic-loaded calcium sulfate beads.

Introduction: Local antimicrobial delivery via calcium sulfate (CaSO 4 ) beads is used as an adjunctive treatment for periprosthetic joint infection. There is limited clinical information describing the performance of antimicrobial-loaded CaSO 4 (ALCS) in large-scale applications. We developed a simulated large joint model to study properties of eluting ALCS. Methods: The in vitro testing platform was an adapted standardized model for tribological testing of prosthetic total hips and total knees (ASTM F732). The model was 70 mL total fluid volume, 25 % bovine serum, and 75 % phosphate-buffered saline, using ISO standard 14242-1 for human synovial fluid simulation. Four brands of CaSO 4 were evaluated. Each 10 mL of CaSO 4 was loaded with 1.2 grams (g) of tobramycin and 1 g of vancomycin powders. A 35 mL bead volume, equaling 175 beads, of each product was placed in incubated flasks. The test period was 6 weeks with scheduled interval fluid exchanges. Fluid samples were tested for antibiotic and calcium concentrations and pH. Results: Antibiotic elution showed an initial burst on Day 1, followed by a logarithmic reduction over 1 week. Tobramycin fully eluted within 2.5 weeks. Vancomycin showed sustained release over 6 weeks. Calcium ion concentrations were high, with gradual decrease after 3 weeks. All four CaSO 4 products were inherently acidic. Fluid became more acidic with the addition of antibiotics primarily driven by vancomycin. Discussion: Clinicians should be cognizant of tobramycin elution burst with ALCS in large loads. The main driver of acidic pH levels was vancomycin. We propose that joint complications may result from lowered fluid acidity, and we suggest clinical study of synovial pH.

Long-Term Controlled Release of Simvastatin from Photoprinted Triple-Networked Hydrogels Composed of Modified Chitosan and PLA-PEG Micelles.

Local delivery of active agents using injectable or implantable hydrogels for tissue and bone regeneration is a promising therapy, but it remains challenging for controlling dose and duration of release. Simvastatin (SMV), a hydrophobic drug, has shown potential for osteogenic stimulation. Secure loading of hydrophobic drugs by physical interactions is particularly difficult to establish in hydrophilic polymer matrices, and their sustained release over several months for long-term regeneration has rarely been reported. Additionally, mechanical properties of hydrogels must be improved for a sufficient support while maintaining eventual biodegradability. This study assesses the effect of controlled SMV release from 3D-printed triple-network hydrogels for osteogenic stimulation and characterizes their mechanical and biological properties as an implant. SMV is loaded into polymeric micelles of polylactide/poly(ethylene glycol) triblock copolymers (PLA-PEG-PLA) and mixed with N-methacryloyl chitosan and PEG dimethacrylate to fabricate hydrogels by photo-cross-linked 3D printing. The hydrogel properties and drug release profiles have shown significant dependance on the polymer compositions. The SMV release from the triple-polymer-network hydrogel has continued for 17 weeks of observation. Cytocompatibility of hydrogels with various formulations is confirmed. The tunable triple-network hydrogels loaded with SMV provide a potential therapeutic value for bone regeneration.

In vitro evaluation of loaded chitosan membranes for pain relief and infection prevention.

Wounds resulting from surgeries, implantation of medical devices, and musculoskeletal trauma result in pain and can also result in infection of damaged tissue. Up to 80% of these infections are due to biofilm formation either on the surface of implanted devices or on surrounding wounded tissue. Bacteria within a biofilm have intrinsic growth and development characteristics that allow them to withstand up to 1,000 times the minimum inhibitory concentration of antibiotics, demonstrating the need for new therapeutics to prevent and treat these infections. Cis-2-decenoic acid (C2DA) is known to disperse preformed biofilms and can prevent biofilm formation entirely for some strains of bacteria. Additionally, local anesthetics like bupivacaine have been shown to have antimicrobial effects against multiple bacterial strains. This study sought to evaluate hexanoic acid-treated electrospun chitosan membranes (HA-ESCM) as wound dressings that release C2DA and bupivacaine to simultaneously prevent infection and alleviate pain associated with musculoskeletal trauma. Release profiles of both therapeutics were evaluated, and membranes were tested in vitro against Methicillin-resistant Staphylococcus aureus (MRSA) to determine efficacy in preventing biofilm infection and bacterial growth. Results indicate that membranes release both therapeutics for 72 hr, and release profile can be tailored by loading concentration. Membranes were effective in preventing biofilm growth but were toxic to fibroblasts when loaded with 2.5 or 5 mg of bupivacaine.

A Study of Combining Elastin in the Chitosan Electrospinning to Increase the Mechanical Strength and Bioactivity.

While electrospun chitosan membranes modified to retain nanofibrous morphology have shown promise for use in guided bone regeneration applications in in vitro and in vivo studies, their mechanical tear strengths are lower than commercial collagen membranes. Elastin, a natural component of the extracellular matrix, is a protein with extensive elastic property. This work examined the incorporation of elastin into electrospun chitosan membranes to improve their mechanical tear strengths and to further mimic the native extracellular composition for guided bone regeneration (GBR) applications. In this work, hydrolyzed elastin (ES12, Elastin Products Company, USA) was added to a chitosan spinning solution from 0 to 4 wt% of chitosan. The chitosan-elastin (CE) membranes were examined for fiber morphology using SEM, hydrophobicity using water contact angle measurements, the mechanical tear strength under simulated surgical tacking, and compositions using Fourier-transform infrared spectroscopy (FTIR) and post-spinning protein extraction. In vitro experiments were conducted to evaluate the degradation in a lysozyme solution based on the mass loss and growth of fibroblastic cells. Chitosan membranes with elastin showed significantly thicker fiber diameters, lower water contact angles, up to 33% faster degradation rates, and up to seven times higher mechanical strengths than the chitosan membrane. The FTIR spectra showed stronger amide peaks at 1535 cm-1 and 1655 cm-1 in membranes with higher concentrated elastin, indicating the incorporation of elastin into electrospun fibers. The bicinchoninic acid (BCA) assay demonstrated an increase in protein concentration in proportion to the amount of elastin added to the CE membranes. In addition, all the CE membranes showed in vitro biocompatibility with the fibroblasts.

Simvastatin loaded chitosan guided bone regeneration membranes stimulate bone healing.

BACKGROUND AND OBJECTIVE: Electrospun chitosan membranes (ESCM) modified with short-chain fatty acids have the ability to control the release of simvastatin (SMV), an anti-cholesterol drug with osteogenic potential, for guided bone regeneration (GBR) applications. This study evaluated in vivo osteogenic effects of rapid short release of SMV (4 weeks) vs long sustained release (8 weeks) from acetic anhydride (AA)-and hexanoic anhydride (HA)-modified ESCMs, respectively. METHODS: AA ESCMs loaded with 10 or 50 µg SMV and HA ESCMs loaded with 50 µg SMV were evaluated for biocompatibility and bone formation at 4 and 8 weeks, in 5 mm critical size rat calvarial defects, using histological evaluation and micro-CT analysis. RESULTS: No severe inflammatory response was noticed around the ESCMs. Less hydrophobic AA membranes showed signs of resorption by week 4 and were almost completely resorbed by week 8 whereas the more hydrophobic HA membranes resorbed slowly, remaining intact over 8 weeks. In micro-CT analysis, 10 µg SMV-loaded AA membranes did not show significant bone formation as compared to non-loaded AA membranes at either evaluation time points. 50 µg SMV-loaded AA membranes stimulated significantly more bone formation than non-loaded AA membranes by week 4 (%bone = 31.0 ± 5.9% (AA50) vs 18.5 ± 13.7% (AA0)) but showed no difference at week 8. HA membranes with 50 µg SMV showed significantly more bone formation as compared to corresponding non-loaded membranes by week 8 (%bone = 61.7 ± 8.9% (HA50) vs 33.9 ± 29.7% (HA0)), though such an effect was not significant at week 4. CONCLUSION: These results indicate that modified ESCMs may be used to control the release of SMV and promote bone healing in GBR applications.

Staphylococcal infection prevention using antibiotic-loaded mannitol-chitosan paste in a rabbit model of implant-associated osteomyelitis.

Antibiotic-loaded chitosan pastes have shown advantages in the treatment and coverage of complex musculoskeletal defects. We added mannitol, previously shown to increase antibiotic susceptibility of biofilm, to an injectable chitosan/polyethylene glycol paste for delivery of antibiotics. Ground sponges (0.85% acetic acid solution, 1% chitosan, 0% or 2% mannitol, 1% polyethylene glycol) were hydrated using phosphate-buffered saline with 10 mg/ml amikacin and 10 mg/ml vancomycin added to form pastes. We inoculated rabbit radial defects with 105 colony-forming units of Staphylococcus aureus (UAMS-1) and inserted titanium pins into the cortical bone. Groups compared included mannitol blend pastes, non-mannitol blends, antibiotic-loaded bone cement, vancomycin powder, and no treatment controls. We harvested tissue samples and retrieved the pins retrieved at 3 weeks. All antibiotic-loaded groups lowered bacterial growth and colony-forming unit counts in soft and bone tissue and on titanium pins in in vivo studies. The results indicate this biomaterial is capable of eluting active antibiotics at concentrations that reduce bacterial growth on biomaterials and tissue, which, in turn, may prevent biofilm formation. Blends of chitosan and mannitol may be useful in prevention and treatment of osteomyelitis and implant-associated infections.

A comparison of two types of electrospun chitosan membranes and a collagen membrane in vivo.

BACKGROUND: Electrospun chitosan membranes subjected to post-spinning processes using either triethylamine/tert-butyloxycarbonyl (TEA/tBOC) or butyryl-anhydride (BA) modifications to maintain nanofiber structure have exhibited potential for use in guided bone regeneration applications. The aim of this study was to evaluate ability of the modified membranes to support healing of bone-grafted defects as compared to a commercial collagen membrane. METHOD: TEA/tBOC-treated and BA-treated chitosan membranes were characterized for fiber morphology by electron microscopy, residual trifluoroacetic acid by19F NMR and endotoxin level using an endotoxin quantitation kit (ThermoScientific, US). Chitosan membranes were cut into 12 mm diameter disks. An 8 mm calvarial defect was created in each of 48 male rats and then filled with Bio-Oss (Geistlich, US) bone graft. The grafted defects were covered with either (1) TEA/tBOC-treated chitosan membrane (2) BA-treated chitosan membrane or (3) the control BioMend Extend (Zimmer Biomet, US) collagen membrane. After 3 and 8 weeks, the rats were euthanized and calvaria was retrieved for microCT and histological analyses (n = 8/group/time points). RESULTS: Both TEA/tBOC-treated and BA-treated membranes were composed of nanofibers in the ∼231 to ∼284 nm range respectively, exhibited no TFA salt residue and low endotoxin levels (≤0.1 ± 0.01 EU/membrane). All membranes supported increased bone growth from 3 weeks to 8 weeks though there was no significant difference among the membrane types. However, TEA/tBOC treated and BA treated chitosan membranes both showed significantly greater bone density (∼6% greater at 3 weeks and ∼8% greater at 8 weeks) as compared to BioMend Extend collagen membrane at both time points (p = 0.0002). CONCLUSIONS: Chitosan membranes supported better bone healing based on bone density than the collagen membrane.

Modified electrospun chitosan membranes for controlled release of simvastatin.

Chitosan nanofibrous membranes have immense potential in tissue engineering and drug delivery applications because of their increased surface area, high degree of biocompatibility, and their ability to mimic the extracellular matrix. However, their use is often limited due to their extreme hydrophilic nature causing them to lose their nanofibrous structure in vivo. In the present study, chitosan membranes were modified either by acylation reactions using fatty acids of different chain lengths or tert-butyloxycarbonyl (tBOC) protecting groups to increase the hydrophobicity of the membranes and protect the nanofibrous structure. The modified membranes were characterized using scanning electron microscopy, attenuated total reflectance Fourier transform infrared spectroscopy, water contact angle and elemental analysis to confirm the addition of the modification groups. These membranes were then evaluated to control the release of a hydrophobic osteogenic drug-simvastatin (SMV). The interaction between SMV and the polymer was determined using molecular modeling. Pure SMV and SMV loaded membranes were examined for their in vitro cytotoxicity and osteogenic potential using preosteoblast mouse bone marrow stromal cells. From results, it was evident that as the fatty acid chain length increased from two to six methylene groups, the hydrophobicity of the membranes increased (59.2 ± 8.2° to 94.3 ± 8.5° water contact angle). The amount of drug released from the membranes could be controlled by changing the amount of initial drug loaded and/or the type of modifications. After 4 weeks, for a 500 µg loading, the short chain fatty acid modified membranes released 17.8 ± 3.2% of the drug whereas a long chain fatty acid released only 4.8 ± 0.8%. Similarly, for a 50 µg loading, short chain modified membranes released more (73.3 ± 33.3%) of the loaded drug as compared to the long chain membranes (43.0 ± 3.5%). The long chain fatty acid membranes released SMV for extended time periods of up to 90 days. This data was further supported by molecular modeling, which revealed that SMV was more compatible with more hydrophobic membranes. Cell studies showed that pure SMV from 75 to 600 ng/ml range possessed osteogenic potential in a dose dependent manner and the amount of SMV released from the most hydrophobic FA treated membranes was not cytotoxic and supported osteogenic differentiation. Therefore, this study demonstrates our ability to control the release of a hydrophobic drug from modified chitosan membranes as per the clinical need.

Characterization of trimethyl chitosan/polyethylene glycol derivatized chitosan blend as an injectable and degradable antimicrobial delivery system.

Advanced local delivery systems are needed as adjunctive treatments for severe injuries with high infection rates, such as open fractures. Chitosan systems have been investigated as antimicrobial local delivery systems for orthopaedic infection but possess mismatches between elution and degradation properties. Derivatives of chitosan were chosen that have enhanced swelling ratios or tailorable degradation properties. A combination of trimethyl chitosan and poly(ethylene glycol) diacrylate chitosan was developed as an injectable local delivery system. Research objectives were elution of antimicrobials for 7 days, degradation as open fractures heal, and cytocompatibility. The derivative combination eluted increased active concentrations of vancomycin and amikacin compared to the non-derivatized chitosan paste, 6 vs. 5 days and 5 vs. 4 days, respectively. The derivative combination degraded slower than non-derivatized paste in an enzymatic degradation study, 14 vs. 3 days, which increased antimicrobial delivery duration. Cytocompatibility of the combination with fibroblast and pre-osteoblast cells exceeds the cell viability standard set in ISO 10993-5. Combination paste requires an increased ejection force of 9.40 N (vs. 0.64 N), but this force was within an acceptable injection force threshold, 80 N. These preliminary results indicate combination paste should be further developed into a clinically useful adjunctive local delivery system for infection prevention.

Magnetic stimulus responsive vancomycin drug delivery system based on chitosan microbeads embedded with magnetic nanoparticles.

Local antibiotic delivery can overcome some of the shortcomings of systemic therapy, such as low local concentrations and delivery to avascular sites. A localized drug delivery system (DDS), ideally, could also use external stimuli to modulate the normal drug release profile from the DDS to provide efficacious drug administration and flexibility to healthcare providers. To achieve this objective, chitosan microbeads embedded with magnetic nanoparticles were loaded with the antibiotic vancomycin and stimulated by a high frequency alternating magnetic field. Three such stimulation sessions separated by 1.5 h were applied to each test sample. The chromatographic analysis of the supernatant from these stimulated samples showed more than approximately 200% higher release of vancomycin from the DDS after the stimulation periods compared to nonstimulated samples. A 16-day long term elution study was also conducted where the DDS was allowed to elute drug through normal diffusion over a period of 11 days and stimulated on day 12 and day 15, when vancomycin level had dropped below therapeutic levels. Magnetic stimulation boosted elution of test groups above minimum inhibitory concentration (MIC), as compared to control groups (with no stimulation) which remained below MIC. The drug release from test groups in the intervals where no stimulation was given showed similar elution behavior to control groups. These results indicate promising possibilities of controlled drug release using magnetic excitation from a biopolymer-based DDS. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 2169-2176, 2018.

Mechanically stable surface-hydrophobilized chitosan nanofibrous barrier membranes for guided bone regeneration.

The use of chitosan based nanofiber membranes in guided bone regeneration (GBR) is limited by its uncontrolled swelling and mechanical instability in aqueous environments. This paper describes the significantly improved stability and properties of surface butyrylated chitosan nanofiber (BCSNF) membranes that greatly enhance their potential in GBR. The BCSNF membranes exhibited an overall degree of substitution of 1.61, an average diameter of 99.3 ± 33.7 nm, and a 75% decrease in swelling with an approximate doubling in suture pull out strengths as compared to unmodified fibers in aqueous environment. In a five week phosphate-buffered saline-lysozyme degradation study, it was found that the remaining mass fraction of BCSNF membranes was 11.5% more than that of unmodified fibers. In vitro, the BCSNF membranes were found to support the adhesion and proliferation of fibroblasts and were cell occulusive. In vivo, the BCSNF membranes were found to significantly improve the regeneration of a rat calvarial critical size defect in a 12 week healing period and showed better barrier function than commercially available collagen membranes with little soft tissue penetration through the membranes. Taken together, these data provide strong scientific evidence for use of BCSNF membranes in GBR applications.

Magnetic stimuli-responsive chitosan-based drug delivery biocomposite for multiple triggered release.

Stimuli-responsive biomaterials offer a unique advantage over traditional local drug delivery systems in that the drug elution rate can be controllably increased to combat developing symptomology or maintain high local elution levels for disease treatment. In this study, superparamagnetic Fe3O4 nanoparticles and the antibiotic vancomycin were loaded into chitosan microbeads cross-linked with varying lengths of polyethylene glycol dimethacrylate. Beads were characterized using degradation, biocompatibility, and elution studies with successive magnetic stimulations at multiple field strengths and frequencies. Thirty-minute magnetic stimulation induced a temporary increase in daily elution rate of up to 45% that was dependent on field strength, field frequency and cross-linker length. Beads degraded by up to 70% after 3 days in accelerated lysozyme degradation tests, but continued to elute antibiotic for up to 8 days. No cytotoxic effects were observed in vitro compared to controls. These promising preliminary results indicate clinical potential for use in stimuli-controlled drug delivery.

Evaluation of a chitosan-polyethylene glycol paste as a local antibiotic delivery device.

AIM: To investigate the efficacy of a chitosan/polyethylene glycol blended paste as a local antibiotic delivery device, particularly in musculoskeletal wounds. METHODS: Acidic (A) chitosan sponges and neutralized (N) chitosan/polyethylene glycol (PEG) blended sponges were combined in ratios of 3A:2N, 1A:1N, and 2A:3N; then hydrated with phosphate buffered saline to form a chitosan/PEG paste (CPP). Both in vitro and in vivo studies were conducted to determine the potential CPP has as a local antibiotic delivery device. In vitro biocompatibility was assessed by the cytotoxic response of fibroblast cells exposed to the experimental groups. Degradation rate was measured as the change in dry mass due to lysozyme based degradation over a 10-d period. The antibiotic elution profiles and eluate activity of CPP were evaluated over a 72-h period. To assess the in vivo antimicrobial efficacy of the CPP, antibiotic-loaded paste samples were exposed to subcutaneously implanted murine catheters inoculated with Staphylococcus aureus. Material properties of the experimental paste groups were evaluated by testing the ejection force from a syringe, as well as the adhesion to representative musculoskeletal tissue samples. RESULTS: The highly acidic CPP group, 3A:2N, displayed significantly lower cell viability than the control sponge group. The equally distributed group, 1A:1N, and the highly neutral group, 2A:3N, displayed similar cell viability to the control sponge group and are deemed biocompatible. The degradation studies revealed CPP is more readily degradable than the chitosan sponge control group. The antibiotic activity studies indicated the CPP groups released antibiotics at a constant rate and remained above the minimum inhibitory concentrations of the respective test bacteria for a longer time period than the control chitosan sponges, as well as displaying a minimized burst release. The in vivo functional model resulted in complete bacterial infection prevention in all catheters treated with the antibiotic loaded CPP samples. All experimental paste groups exhibited injectability and adhesive qualities that could be advantageous material properties for drug delivery to musculoskeletal injuries. CONCLUSION: CPP is an injectable, bioadhesive, biodegradable, and biocompatible material with potential to allow variable antibiotic loading and active, local antibiotic release to prevent bacterial contamination.

In vitro and in vivo evaluations of a novel post-electrospinning treatment to improve the fibrous structure of chitosan membranes for guided bone regeneration.

Electrospun chitosan membranes have been investigated for guided bone regeneration but are susceptible to swelling, dissolution, and loss of biomimetic nanofiber structure due to residual acid salts. A novel process was investigated for acidic salt removal from chitosan electrospun in 70% trifluoroacetic acid (TFA) by treating with triethylamine (TEA)/acetone and di-tert-butyl dicarbonate (tBOC) instead of the common Na2CO3 treatment. TFA salt removal and nanofiber structure stabilization were confirmed by EDS, FTIR, 19F NMR and electron microscopy before and after soaking in water. Membrane degradation after 4 weeks in PBS with 100 µg ml-1 lysozyme and osteoblastic proliferation were similar between TEA/tBOC-treated and Na2CO3-treated membranes. A simulated surgical tear test using surgical tacks showed that the peak tensile tear strength of the TEA/tBOC-treated chitosan membranes (62.1 ± 1.9 N mm-1) was significantly greater than a commercial polylactic acid (PLA) membrane (13.4 ± 0.4 N mm-1), similar to one commercial collagen membrane (55.3 ± 7.5 N mm-1) but lower than another commercial collagen membrane (133.9 ± 21.5 N mm-1). Rat 8 mm critical-sized calvarial defects covered with TEA/tBOC-treated chitosan membranes prevented soft tissue infiltration and supported new bone growth (15.76 ± 10.28%) similar to a commercial collagen membrane (16.08 ± 10.69%) at 12 weeks based on microCT analyses. Hence our novel TEA/tBOC process significantly improved nanofiber structure and mechanical strengths of electrospun chitosan membranes as compared to Na2CO3 treated membranes, without affecting in vitro degradation or cytocompatibility, improved membrane mechanical properties to be greater than a commercial PLA membrane and to be in range of commercial collagen membranes and supported calvarial bone defect healing similar to collagen. Thus TEA/tBOC-treated chitosan membranes exhibit many characteristics and properties that strongly support their potential for use in guided bone regeneration.