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.

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.

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.

2018 international consensus meeting on musculoskeletal infection: Summary from the biofilm workgroup and consensus on biofilm related musculoskeletal infections.

Biofilm-associated implant-related bone and joint infections are clinically important due to the extensive morbidity, cost of care and socioeconomic burden that they cause. Research in the field of biofilms has expanded in the past two decades, however, there is still an immense knowledge gap related to many clinical challenges of these biofilm-associated infections. This subject was assigned to the Biofilm Workgroup during the second International Consensus Meeting on Musculoskeletal Infection held in Philadelphia USA (ICM 2018) (https://icmphilly.com). The main objective of the Biofilm Workgroup was to prepare a consensus document based on a review of the literature, prepared responses, discussion, and vote on thirteen biofilm related questions. The Workgroup commenced discussing and refining responses prepared before the meeting on day one using Delphi methodology, followed by a tally of responses using an anonymized voting system on the second day of ICM 2018. The Working group derived consensus on information about biofilms deemed relevant to clinical practice, pertaining to: (1) surface modifications to prevent/inhibit biofilm formation; (2) therapies to prevent and treat biofilm infections; (3) polymicrobial biofilms; (4) diagnostics to detect active and dormant biofilm in patients; (5) methods to establish minimal biofilm eradication concentration for biofilm bacteria; and (6) novel anti-infectives that are effective against biofilm bacteria. It was also noted that biomedical research funding agencies and the pharmaceutical industry should recognize these areas as priorities. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res.

Ciprofloxacin and Rifampin Dual Antibiotic-Loaded Biopolymer Chitosan Sponge for Bacterial Inhibition.

Complex extremity wounds in Wounded Warriors can become contaminated with microbes, which may cause clinical outcomes resulting in amputation, morbidity, or even fatality. Local delivery of multiple or broad-spectrum antibiotics allows practicing clinicians treatment solutions that may inhibit biofilm formation. Propagation of vancomycin-resistant Staphylococcus aureus is also a growing concern. The development of vancomycin-resistant S. aureus has become a critical challenge in nosocomial infection prevention in the USA, but to date has seen little occurrence in osteomyelitis. As an alternative, locally delivered ciprofloxacin and rifampin were investigated in a preclinical model for the prevention of biofilm in complex extremity wounds with implanted fixation device. In vitro assays demonstrated ciprofloxacin and rifampin possess an additive effect against Gram-negative Pseudomonas aeruginosa and were actively eluted from a chitosan sponge based local delivery system. In an in vivo orthopedic hardware-associated polymicrobial model (S. aureus and Escherichia coli) the combination was able to achieve complete clearance of both bacterial strains. E. coli was detected in bone of untreated animals, but did not form biofilm on wires. Results reveal the clinical potential of antibiotic-loaded chitosan sponges to inhibit infection through tailored antibiotic selection at desired concentrations with efficacy towards biofilm inhibition.

Local control of polymicrobial infections via a dual antibiotic delivery system.

BACKGROUND: Contaminated traumatic open orthopedic wounds are frequently complicated by polymicrobial contamination and infection. In high-risk wounds, the standard of care comprises debridement and irrigation combined with antibiotics which can be applied directly or combined with systemic antibiotics. Recently, bioabsorbable chitosan sponges have been shown to be an effective single-agent delivery device for local antibiotics with and without negative pressure wound therapy (NPWT). Severely contaminated orthopedic wounds, however, are often complicated by polymicrobial infections, necessitating multiple antibiotic agents. As such, the purpose of this study was to determine if a chitosan sponge would provide a suitable delivery vehicle for multiple antibiotics for the treatment of a polymicrobial infection in a large animal polytraumatic extremity wound model. METHODS: A complex polytraumatic extremity wound was created in 11 adult male Boer goats. Each wound was contaminated with a bioluminescent strain of S. aureus (1 ml of 108 colony forming units/ml) and of P. aeruginosa (1 ml of 108 CFU/ml) which are genetically engineered to allow quantification with a photon-counting camera. Six hours following initial wound creation and contamination, wounds were debrided and irrigated with low-pressure normal saline. The animals were randomized into one of two treatments: wet-to-dry dressings alone or a commercially available chitosan sponge loaded with 1 g vancomycin and 1.2 g of tobramycin. Each animal was then recovered and reimaged 48 h later for total bacteria content; tissue samples were taken from the wound bed to determine relative bacterial colonization. RESULTS: All animals in the chitosan sponge group saw significant reductions in overall bacterial load of S. aureus and P. aeruginosa (p = 0.001). The bioluminescence was also significantly reduced compared to the wet-to-dry dressing group (p = 0.0001). Furthermore, whereas the antibiotic sponge group displayed near complete eradication of bacteria, the wounds treated with the wet-to-dry dressings alone displayed a significant 2-log increase in total bacteria at 48 h p = 0.0001). S. aureus was the predominant species found in the wounds, comprising 95 and 99% of all bacteria found in the chitosan sponge and wet-to-dry, respectively. CONCLUSION: Dual antimicrobial therapy loaded in a chitosan sponge is an effective way to reduce polymicrobial infections traumatic extremity wound.

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.

Blended Chitosan Paste for Infection Prevention: Preliminary and Preclinical Evaluations.

BACKGROUND: Local drug delivery devices offer a promising method for delivering vancomycin and amikacin for musculoskeletal wounds. However, current local delivery devices such as beads and sponges do not necessarily allow for full coverage of a wound surface with eluted antibiotics and do not address the need for reducing the antibiotic diffusion distance to help prevent contamination by bacteria or other microorganisms. We blended chitosan/polyethylene glycol (PEG) pastes/sponges to increase biocompatibility and improve antibiotic coverage within the wound. QUESTIONS/PURPOSES: (1) Are blended chitosan/PEG pastes biodegradable? (2) Are the blended pastes biocompatible? (3) How much force does paste require for placement by injection? (4) Will the pastes elute active antibiotics to inhibit bacteria in vitro? (5) Can the pastes prevent infection in a preclinical model with hardware? METHODS: Our blended paste/sponge formulations (0.5% acidic, 1% acidic, and acidic/neutral) along with a control neutral 1% chitosan sponge were tested in vitro for degradability, cytocompatibility, injectability tested by determining the amount of force needed to inject the pastes, elution of antibiotics, and activity tested using zone of inhibition studies. Along with these studies, in vivo models for biocompatibility and infection prevention were tested using a rodent model and an infected mouse model with hardware, respectively. By evaluating these characteristics, an improved local drug delivery device can be determined. RESULTS: All three of the paste formulations evaluated were almost fully degraded and with 6 days of degradation, the percent remaining being was less than that of the control sponge (percent remaining: control 99.251% ± 1.0%; 0.5% acidic 1.6% ± 2.1%, p = 0.002; 1% acidic 1.7% ± 1.6%, p = 0.002; acidic/neutral 2.3% ± 1.7%, p = 0.010). There was good biocompatibility because cell viability in vitro was high (control 100.0 ± 14.3; 0.5% acidic formulation at 79.4 ± 12.6, p < 0.001; 1% acidic formulation at 98.6 ± 6.1, p = 0.993; acidic/neutral formulation at 106.7 ± 12.8, p = 0.543), and in vivo inflammation was moderate (control 2.1 ± 1.2; 0.5% acidic 3.3 ± 0.2, p = 0.530; 1% acidic 2.5 ± 0.9, p = 0.657; acidic/neutral 2.9 ± 1.1, p = 0.784). Force required to inject the 0.5% acidic and 1% acidic pastes was less than the acidic/neutral paste used as a control (control 167.7 ± 85.6; 0.5% acidic 41.3 ± 10.7, p = 0.070; 1% acidic 28.0 ± 7.0, p = 0.940). At 72 hours, all paste formulations exhibited in vitro activity against Staphylococcus aureus (control 2.6 ± 0.8; 0.5% acidic 98.1 ± 33.5, p = 0.002; 1% acidic 87.3 ± 17.2, p = 0.006; acidic/neutral 83.5 ± 14.3, p = 0.010) and Pseudomonas aeruginosa (control 163.0 ± 1.7; 0.5% acidic 85.7 ± 83.6, p = 0.373; 1% acidic 38.0 ± 45.1, p = 0.896; acidic/neutral 129.7 ± 78.0, p = 0.896). Also, the paste formulations were able to prevent the infection with 100% clearance on the implanted hardware and surrounding tissue with the control being a 0.5% acidic paste group without antibiotics (control 4 × 104 ± 4.8 × 104; 0.5% acidic 0.0 ± 0.0, p value: 0.050; 1% acidic 0.0 ± 0.0, p = 0.050; acidic/neutral 0.0 ± 0.0, p = 0.050). CONCLUSIONS: The preliminary studies demonstrated promising results for the blended chitosan/PEG pastes with antibiotics provided degradability, biocompatibility, injectability, and infection prevention for musculoskeletal-type wounds. CLINICAL RELEVANCE: The preliminary studies with the chitosan paste delivered antibiotics to a contaminated musculoskeletal wound with hardware and prevented infection. More studies in a complex musculoskeletal wound and dosage studies are needed for continued development.

Evaluation of Amniotic Multipotential Tissue Matrix to Augment Healing of Demineralized Bone Matrix in an Animal Calvarial Model.

Amniotic multipotential tissue matrix (AmnioMTM) is a membrane material derived from placental tissues and rich in growth factors that have been reported to have potential in healing bone. This study hypothesized that demineralized bone matrix (DBM) supplemented with AmnioMTM would accelerate healing and bone formation as compared with DBM alone in a critical size (10 mm) rat calvarial bone defect model. Five DBM grafts and 5 DBM supplemented with AmnioMTM grafts were implanted in a 10-mm critical sized defect in 10 rats (1 implant per rat). After 4 weeks, animals were euthanized and defects evaluated by microCT and histology. There were no statistical differences in microCT data for mineral density, percent bone fill, or bone surface to volume ratios between groups, though the bone surface to volume ratio for the amnio-supplemented group suggested increased osteoid activity as compared with the DBM alone group. Histological data also indicated active osteoid activity and induced bone formation in the center of defects implanted with AmnioMTM supplemented graft as compared with DBM graft alone suggesting some potential osteoinductive potential. However, there was no significant difference at the mean percent of newly mineralized bone in the DBM group defect as compared with the AmnioMTM supplemented graft material. These data suggest that while bone formation was not increased at this early time point, the increased osteoid activity and the induction of new bone in the middle of the defect by the AmnioMTM indicates that further study is needed to assess its potential benefit to bone healing and regeneration.

Osteocompatibility of biofilm inhibitors.

The demand for infection prevention therapies has led to the discovery of several biofilm inhibitors. These inhibiting signals are released by bacteria, fungi, or marine organisms to signal biofilm dispersal or disruption in Gram-positive, Gram-negative, and fungal microorganisms. The purpose of this study was to test the biocompatibility of five different naturally-produced biofilm chemical dispersal and inhibition signals with osteoblast-like cells: D-amino acids (D-AA), lysostaphin (LS), farnesol, cis-2-decenoic acid (C2DA), and desformyl flustrabromine (dFBr). In this preliminary study, compatibility of these anti-biofilm agents with differentiating osteoblasts was examined over a 21 days period at levels above and below concentrations active against bacterial biofilm. Anti-biofilm compounds listed above were serially diluted in osteogenic media and added to cultures of MC3T3 cells. Cell viability and cytotoxicity, after exposure to each anti-biofilm agent, were measured using a DNA assay. Differentiation characteristics of osteoblasts were determined qualitatively by observing staining of mineral deposits and quantitatively with an alkaline phosphatase assay. D-AA, LS, and C2DA were all biocompatible within the reported biofilm inhibitory concentration ranges and supported osteoblast differentiation. Farnesol and dFBr induced cytotoxic responses within the reported biofilm inhibitory concentration range and low doses of dFBr were found to inhibit osteoblast differentiation. At high concentrations, such as those that may be present after local delivery, many of these biofilm inhibitors can have effects on cellular viability and osteoblast function. Concentrations at which negative effects on osteoblasts occur should serve as upper limits for delivery to orthopaedic trauma sites and guide development of these potential therapeutics for orthopaedics.

Chitosan coating to enhance the therapeutic efficacy of calcium sulfate-based antibiotic therapy in the treatment of chronic osteomyelitis.

We demonstrate that coating calcium sulfate with deacetylated chitosan enhances the elution profile of daptomycin by prolonging the period during which high concentrations of antibiotic are released. Coatings reduced initial bolus release of daptomycin by a factor of 10 to approximately 1000 µg/ml, and levels remained above 100 µg/ml for up to 10 days. Chitosan-coated and uncoated calcium sulfate implants with and without 15% daptomycin were evaluated in an experimental model of staphylococcal osteomyelitis through bacteriology scores, radiology, histopathology, and Gram staining. Significant reduction in bacteriology scores was observed for implants containing daptomycin and coated with chitosan compared with all the other groups. We confirm that the use of chitosan-coated calcium sulfate beads for local antibiotic delivery can be correlated with an improved therapeutic outcome following surgical debridement in the treatment of chronic osteomyelitis.

Effects of VEGF-loaded chitosan coatings.

Vascular endothelial growth factor (VEGF) is a powerful growth factor that promotes vascularization as well as osteoblastic differentiation and bone regeneration, all of which are key processes in the osseointegration of dental implants. Strategies to increase vascularization through delivery of VEGF may improve osseointegration, especially in patients with reduced bone healing potential. The aim of this study was to determine the potential of chitosan coatings on titanium to deliver VEGF and to support growth and matrix production of osteoblastic cells in vitro. Chitosan was chemically bonded to titanium coupons via silane-glutaraldehyde linker molecules and loaded with 0, 20, 50, or 100 ng of VEGF. Protein was released during a three day period with around 75% of VEGF (4.44, 11.37, and 22.10 ng/mL/cm(2) from the 20, 50, and 100 ng loaded levels, respectively) released during the first 12 h, and 90-95% of the VEGF released from the coatings by day 3. Saos-2 bone cells continued to proliferate over the 28-day period on the VEGF-loaded chitosan coatings in contrast to cells seeded on uncoated titanium, which plateaued after 14 days. Cells on uncoated titanium exhibited a peak in alkaline phosphatase expression at approximately 14 days, concomitant with the plateau in growth. While osteoblast-like cells on all chitosan coatings exhibited up to a 2-fold enhancement of the alkaline phosphatase activity and 10-fold increase in calcium deposition compared to uncoated controls, the incorporation of VEGF into the coatings did not enhance osteoblast matrix production over plain chitosan coatings throughout this study.

Chitosan sponges for local synergistic infection therapy: a pilot study.

BACKGROUND: Although bacterial antibiotic resistance is increasing, fewer new antibiotics are being developed to compensate. Localized delivery of synergistic antiseptics and antibiotics with a chitosan sponge device may offer an alternative infection treatment. QUESTIONS/PURPOSES: In this pilot study, we asked whether antiseptic and antibiotic combinations provided in vitro synergism against Staphylococcus aureus, whether synergism reduces cell viability, and whether their combination releases drugs at inhibitory levels. METHODS: To investigate the pharmacodynamics among three combinations of the antiseptic chlorhexidine digluconate (CHX) with the antibiotics amikacin, daptomycin, and vancomycin (VAN) (n=1), we determined the fractional inhibitory concentration (FIC) index against S aureus Cowan I. The determined synergistic combination of CHX and VAN was evaluated for cell compatibility using NIH/3T3 fibroblasts (n=3) and the drug release profile from a chitosan sponge device (n=5). RESULTS: With an FIC index<0.5, the combination of CHX+VAN exhibited synergism against S aureus. CHX concentrations≥3.91 µg/mL resulted in fibroblast viability decrease, whereas the combination of CHX+VAN did not decrease fibroblast viability until their concentrations reached ≥7.81 µg/mL. The CHX and VAN release profile, both individually and in combination, was an initial bolus with no difference between eluate concentrations after Day 5. CONCLUSIONS: CHX+VAN combination may be delivered locally by a chitosan sponge that synergistically inhibits S aureus growth. CLINICAL RELEVANCE: The use of synergism between combined antibiotic and antiseptics delivered at high local concentrations with an implanted chitosan sponge may provide a useful alternative infection treatment option.

Preliminary investigation of crosslinked chitosan sponges for tailorable drug delivery and infection control.

Local versus systemic antibiotic delivery may be an effective strategy for treating musculoskeletal infections, especially when antibiotic-resistant bacteria are present. Lyophilized uncrosslinked, genipin crosslinked, and genipin crosslinked with poly(N-isopropylacrylamide) (PNIPAM) chitosan sponges were analyzed for their in vitro degradation rate, chemical crosslinking, antibiotic uptake, elution, biologic activity, and cytotoxicity. These evaluations were pursued to determine if crosslinking with genipin could be used to create a tailorable point of care loaded sponge for local infection control. Crosslinking the chitosan sponges decreased degradation in phosphate-buffered saline from 4.48 ± 2.28 wt % remaining of the uncrosslinked sponges to 78.82 ± 1.15 and 73.87 ± 1.27 wt % remaining at week 1 for the genipin and PNIPAM/genipin crosslinked sponges, respectively. The PNIPAM/genipin crosslinked sponges exhibited the most sustained release of biologically active antibiotics, with an average antibiotic release 63% higher than uncrosslinked and 37% higher than genipin crosslinked sponges, after 96 h. No significant cytotoxic effects from sponges or eluates were exhibited with NIH 3T3 fibroblasts. These preliminary results indicate that genipin crosslinked chitosan sponges, with or without PNIPAM, have potential as local delivery systems for adjunctive therapy for infection control, especially when longer degradation periods and higher antibiotic elutions are desired.

Physical properties and in vitro evaluation of collagen-chitosan-calcium phosphate microparticle-based scaffolds for bone tissue regeneration.

Due to limitations of bone autografts and allografts, synthetic bone grafts using osteoconductive biomaterials have been designed. In this study, collagen-chitosan-calcium phosphate microparticle-based scaffolds fused with glycolic acid were compared to their counterparts without collagen in terms of degradation, cytocompatibility, porosity, and Young's modulus. It was found that 26-30% collagen was incorporated and that hydroxyapatite was present. Moreover, there were no differences between control and collagen scaffolds in degradation, cytocompatibility, porosity, and Young's modulus. In general, scaffolds exhibited 23% porosity, 0.6-1.2 MPa Young's modulus, 23% degradation over 4 weeks, and supported a four to seven fold increase in osteoblast cell number over 7 days in culture. Collagen can be incorporated into these bone graft substitute scaffolds, which show an improved degradation profile.

Preparation and functional assessment of composite chitosan-nano-hydroxyapatite scaffolds for bone regeneration.

Composite chitosan-nano-hydroxyapatite microspheres and scaffolds prepared using a co-precipitation method have shown potential for use in bone regeneration. The goal of this research was to improve the functional properties of the composite scaffolds by modifying the fabrication parameters. The effects of degree of deacetylation (DDA), drying method, hydroxyapatite content and an acid wash on scaffold properties were investigated. Freeze-dried 61% DDA scaffolds degraded faster (3.5 ± 0.5% mass loss) than air-dried 61% DDA scaffolds and 80% DDA scaffolds, but had a lower compressive modulus of 0.12 ± 0.01 MPa. Air-dried 80% DDA scaffolds displayed the highest compressive modulus (3.79 ± 0.51 MPa) and these scaffolds were chosen as the best candidate for use in bone regeneration. Increasing the amount of hydroxyapatite in the air-dried 80% DDA scaffolds did not further increase the compressive modulus of the scaffolds. An acid wash procedure at pH 6.1 was found to increase the degradation of air-dried 80% DDA scaffolds from 1.3 ± 0.1% to 4.4 ± 0.4%. All of the formulations tested supported the proliferation of SAOS-2 cells.

Osteoinductivity Assessment of BMP-2 Loaded Composite Chitosan-Nano-Hydroxyapatite Scaffolds in a Rat Muscle Pouch.

The objective of this study was to evaluate the osteoinductivity of composite chitosan-nano-hydroxyapatite scaffolds in a rat muscle pouch model. Previous in vitro characterization demonstrated the ability of the scaffolds to promote bone regeneration and as a carrier for local delivery of BMP-2. Composite microspheres were prepared using a co-precipitation method, and scaffolds were fabricated using an acid wash to adhere beads together. To determine the in vivo osteoinductivity of the scaffolds, the following groups (n = 6) were implanted into muscle pouches created in the latissimus dorsi of Sprague Dawley rats: (A) lyophilized scaffolds without rhBMP-2, (B) lyophilized scaffolds with rhBMP-2, (C) non-lyophilized scaffolds with rhBMP-2, and (D) absorbable collagen sponge with rhBMP-2 (control). Groups B, C, and D were loaded with 4 mL of a 9.0 µg/mL solution of rhBMP-2 for 48 h. The rats were sacrificed after one month and samples were analyzed for amount of residual implant material, new bone, and osteoid. Although the experimental groups displayed minimal degradation after one month, all of the scaffolds contained small amounts of woven bone and considerable amounts of osteoid. Approximately thirty percent of the open space available for tissue ingrowth in the scaffolds contained new bone or osteoid in the process of mineralization. The ability of the composite scaffolds (with and without BMP-2) to promote ectopic bone growth in vivo was demonstrated.

The characterization and optimization of injectable silicone resin particles in conjunction with dermal fibroblasts and growth factors: an in vitro study.

Minimally invasive subdermal injection of liquid silicone has been used clinically to augment the soft tissue of the foot to mitigate high pressures that cause diabetic foot ulcers. However, implant migration has been a clinical issue. The objective of this study was to assess the effects of three specific concentrations of silicone resin particles (12 mum average diameter) in conjunction with either platelet-derived growth factor (PDGF-BB) or basic fibroblast growth factor (bFGF) on fibroblast cell proliferation, collagen synthesis, cell morphology, and migration through in vitro assays and a monolayer scratch wound model. PDGF and bFGF enhanced the proliferation of fibroblasts 5.7-fold and fivefold, respectively, while the addition of silicone particles had no significant effect on proliferation. Collagen production was increased approximately twofold with the addition of bFGF and the medium concentration of particles over bFGF without particles and the PDGF groups. The addition of silicone particles had no significant effect on collagen production compared with control groups without particles. Fibroblast migration was enhanced by the addition of both PDGF and bFGF compared to controls, although slower scratch wound closure rates were observed in the presence of particles compared to controls without particles. Cell morphology suggested that particles induced cellular aggregation encircling silicone particles postwounding as well as migration into the wound area. These results suggest that silicone particles in combination with a growth factor might enhance fibroblast aggregation and implant stability, and could promote connective tissue ingrowth and implant encapsulation in the soft tissue of the diabetic foot.

Effect of growth factors in combination with injectable silicone resin particles on the biological activity of dermal fibroblasts: a preliminary in vitro study.

Injections of silicone fluid have been clinically evaluated to treat and prevent foot ulcers due to diminished plantar fat-pad in neuropathic diabetics. The objective of this study was to determine preliminary in vitro effects of an injectable form of silicone resin particles in combination with growth factors to determine the suitability of this potential therapy for prevention of diabetic foot ulcers. Basic fibroblast growth factor (bFGF), epidermal growth factor (EGF) and platelet-derived growth factor (PDGF-BB) were added to monolayer culture along with silicone resin particles (12 microm average diameter). Growth factors were also combined as follows: bFGF+PDGF-BB, EGF+PDGF-BB, and bFGF+EGF. Growth factors alone and in combination increased fibroblast proliferation, but the presence of particles did not significantly affect cellular proliferation. The addition of particles significantly increased fibronectin production 117% in the control group and 151% in the PDGF only group. Collagen production was increased with exposure to EGF and growth factor combinations, but the presence of particles did not lead to any significant differences, except an 81% increase in the bFGF group. These preliminary results suggest that a combination of PDGF and bFGF may be effective in stimulating proliferation and matrix production around injectable silicone resin particles to generate a fibrous tissue pad to alleviate the abnormal distribution of high pressures that contribute to diabetic foot ulcer formation.