development-benchtop-model-for-evaluating-compatibility-wound

Development of a Benchtop Model for Evaluating the Compatibility of Wound Dressing Materials with Negative Pressure Wound Therapy Systems

Negative pressure wound therapy (NPWT) systems facilitate wound healing by applying sub-atmospheric pressure to the wound bed, which promotes granulation ...

University of Memphis-University of Tennessee Health Science Center

Electric fields (EFs) of around 100 mV/mm are present in normal healing wounds and induce the directional migration of epithelial cells. Reepithelialization during wound healing thus may be controlled in part by this electrical signal. In this study, the early transcriptional response of human epidermal keratinocytes to EFs is examined using microarrays. Increased expression of various chemokines, interleukins, and other inflammatory response genes indicates that EFs stimulate keratinocyte activation and immune stimulatory activity. Gene expression activity further suggests that interleukin 1 is either released or activated in EFs. Expression of the chemokine CCL20 steadily increases at 100 mV/mm over time until around 8 h after exposure. This chemokine is also expressed at field strengths of 300 mV/mm-above the level of endogenous wound fields. The early effects of EFs on epithelial gene expression activity identified in these studies suggest the importance of naturally occurring EFs both in repair mechanisms and for the possibility of controlling these responses therapeutically.

Upregulation of chemokine (C-C motif) ligand 20 in adult epidermal keratinocytes in direct current electric fields.

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.

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

Cis-2 decenoic acid (C2DA) disperses biofilm in many strains of microorganisms. However, whether C2DA inhibits bacterial growth or has potential to boost the actions of antibiotics is unknown.

Cis-2-decenoic acid inhibits S. aureus growth and biofilm in vitro: a pilot study.

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.

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

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.

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

CORR Insights®: Local gentamicin delivery from resorbable viscous hydrogels is therapeutically effective.

Orthopaedic biomaterials are susceptible to biofilm formation. A novel lipid-based material has been developed that may be loaded with antibiotics and applied as an implant coating at point of care. However, this material has not been evaluated for antibiotic elution, biofilm inhibition, or in vivo efficacy.

Novel Antibiotic-loaded Point-of-care Implant Coating Inhibits Biofilm.

CORR Insights(®): D-amino acid inhibits biofilm but not new bone formation in an ovine model.

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.

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

Bone void fillers (BVFs) containing calcium sulfate, tricalcium phosphate (TCP), and hydroxyapatite can be loaded with antibiotics for infection treatment or prevention under surgeon-directed use. The aim of this study was to characterize the handling and elution properties of a triphasic BVF loaded with common antibiotics. BVF was mixed with vancomycin and/or tobramycin to form pellets, and the set time was recorded. A partial refreshment elution study was conducted with time points at 4, 8, and 24 h, as well as 2, 7, 14, 28, and 42 days. Effects on dissolution were evaluated in a 14-day dissolution study. Set time increased to over 1 h for groups containing tobramycin, although vancomycin had a minimal effect. Pellets continued to elute antibiotics throughout the 42-day elution study, suggesting efficacy for the treatment or prevention of orthopedic infections. BVF containing vancomycin or tobramycin showed similar dissolution at 14 days compared to BVF without antibiotics; however, BVF containing both antibiotics showed significantly more dissolution.

Evaluation of Antibiotic-Releasing Triphasic Bone Void Filler In-Vitro.

Complex open musculoskeletal wounds are a leading cause of morbidity worldwide, partially due to a high risk of bacterial contamination. Local delivery systems may be used as adjunctive therapies to prevent infection, but they may be nondegradable, possess inadequate wound coverage, or migrate from the wound site. To address this issue, a thermo-responsive, injectable chitosan paste was fabricated by incorporating beta-glycerophosphate. The efficacy of thermo-paste as an adjunctive infection prevention tool was evaluated in terms of cytocompatibility, degradation, antibacterial, injectability, and inflammation properties. In vitro studies demonstrated thermo-paste may be loaded with amikacin and vancomycin and release inhibitory levels for at least 3 days. Further, approximately 60% of thermo-paste was enzymatically degraded within 7 days in vitro. The viability of cells exposed to thermo-paste exceeded ISO 10993-5 standards with approximately 73% relative viability of a control chitosan sponge. The ejection force of thermo-paste, approximately 20 N, was lower than previously studied paste formulations and within relevant clinical ejection force ranges. An in vivo murine biocompatibility study demonstrated that thermo-paste induced minimal inflammation after implantation for 7 days, similar to previously developed chitosan pastes. Results from these preliminary preclinical studies indicate that thermo-paste shows promise for further development as an antibiotic delivery system for infection prevention.

Development and Evaluation of an Injectable Chitosan/β-Glycerophosphate Paste as a Local Antibiotic Delivery System for Trauma Care.

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.

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

Mannitol, a polyalcohol bacterial metabolite, has been shown to activate dormant persister cells within bacterial biofilm. This study sought to evaluate an injectable blend of mannitol, chitosan, and polyethylene glycol for delivery of antibiotics and mannitol for eradication of  biofilm. Mannitol blends were injectable and had decreased dissociation and degradation in the enzyme lysozyme compared to blends without mannitol. Vancomycin and amikacin eluted in a burst response, with active concentrations extended to seven days compared to five days for blends without mannitol. Mannitol eluted from the paste in a burst the first day and continued through Day 4. Eluates from the mannitol pastes with and without antibiotics decreased viability of established  biofilm by up to 95.5% compared to blends without mannitol, which only decreased biofilm when loaded with antibiotics. Cytocompatibility tests indicated no adverse effects on viability of fibroblasts. In vivo evaluation of inflammatory response revealed mannitol blends scored within the 2-4 range at Week 1 (2.6 ± 1.1) and at Week 4 (3.0 ± 0.8), indicative of moderate inflammation and comparable to non-mannitol pastes ( = 0.065). Clinically, this paste could be loaded with clinician-selected antibiotics and used as an adjunctive therapy for musculoskeletal infection prevention and treatment.

Article	Year
Upregulation of chemokine (C-C motif) ligand 20 in adult epidermal keratinocytes in direct current electric fields. Archives of dermatological research\| PubMed ID: 19784662	2010
Effect of growth factors in combination with injectable silicone resin particles on the biological activity of dermal fibroblasts: a preliminary in vitro study. Journal of biomedical materials research. Part B, Applied biomaterials\| PubMed ID: 19904740	2010
Cis-2-decenoic acid inhibits S. aureus growth and biofilm in vitro: a pilot study. Clinical orthopaedics and related research\| PubMed ID: 22585351	2012
Chitosan coating to enhance the therapeutic efficacy of calcium sulfate-based antibiotic therapy in the treatment of chronic osteomyelitis. Journal of biomaterials applications\| PubMed ID: 24854984	2014
Preparation and functional assessment of composite chitosan-nano-hydroxyapatite scaffolds for bone regeneration. Journal of functional biomaterials\| PubMed ID: 24956519	2012
CORR Insights®: Local gentamicin delivery from resorbable viscous hydrogels is therapeutically effective. Clinical orthopaedics and related research\| PubMed ID: 25300513	2015
Novel Antibiotic-loaded Point-of-care Implant Coating Inhibits Biofilm. Clinical orthopaedics and related research\| PubMed ID: 25604874	2015
CORR Insights(®): D-amino acid inhibits biofilm but not new bone formation in an ovine model. Clinical orthopaedics and related research\| PubMed ID: 26310678	2015
Osteoinductivity Assessment of BMP-2 Loaded Composite Chitosan-Nano-Hydroxyapatite Scaffolds in a Rat Muscle Pouch. Materials (Basel, Switzerland)\| PubMed ID: 28824147	2011
Evaluation of Antibiotic-Releasing Triphasic Bone Void Filler In-Vitro. Journal of functional biomaterials\| PubMed ID: 30248929	2018
Development and Evaluation of an Injectable Chitosan/β-Glycerophosphate Paste as a Local Antibiotic Delivery System for Trauma Care. Journal of functional biomaterials\| PubMed ID: 30322006	2018
Characterization of trimethyl chitosan/polyethylene glycol derivatized chitosan blend as an injectable and degradable antimicrobial delivery system. International journal of biological macromolecules\| PubMed ID: 30986460	2019
Characterization and Antibiofilm Activity of Mannitol-Chitosan-Blended Paste for Local Antibiotic Delivery System. Marine drugs\| PubMed ID: 31480687	2019

J Amber Jennings

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