Warren O Haggard

Publications of Warren O Haggard

• Hematoma-inspired alginate/platelet releasate/CaPO(4) composite: initiation of the inflammatory-mediated response associated with fracture repair in vitro and ex vivo injection delivery.

  Authors: Jonathan D McCanless, Lisa K Jennings, Joel D Bumgardner, Judith A Cole, Warren O Haggard

  Journal of materials science. Materials in medicine. 05/2012;

  A clinical need continues for consistent bone remodeling within problematic sites such as those of fracture nonunion, avascular necrosis, or irregular bone formations. In attempt to address suchA clinical need continues for consistent bone remodeling within problematic sites such as those of fracture nonunion, avascular necrosis, or irregular bone formations. In attempt to address such needs, a biomaterial system is proposed to induce early inflammatory responses after implantation and to provide later osteoconductive scaffolding for bone regeneration. Biomaterial-induced inflammation would parallel the early stage of hematoma-induced fracture repair and allow scaffold-promoted remodeling of osseous tissue to a healthy state. Initiation of the wound healing cascade by two human concentrated platelet releasate-containing alginate/β-tricalcium phosphate biocomposites has been studied in vitro using the TIB-71™ RAW264.7 mouse monocyte cell line. Inflammatory responses inherent to the base material were found and could be modulated through incorporation of platelet releasate. Differences in hydrogel wt% (2 vs. 8 %) and/or calcium phosphate granule vol.% (20 vs. 10 %) allowed for tuning the response associated with platelet releasate-associated growth factor elution. Tunability from completely suppressing the inflammatory response to augmenting the response was observed through varied elution profiles of both releasate-derived bioagents and impurities inherent to alginate. A 2.5-fold upregulation of inducible-nitric oxide synthase gene expression followed by a tenfold increase in nitrite media levels was induced by inclusion of releasate within the 8 wt%/10 vol.% formulation and was comparable to an endotoxin positive control. Whereas, near complete elimination of inflammation was seen when releasate was included within the 2 wt%/20 vol.% formulation. These in vitro results suggested tunable interactions between the multiple platelet releasate-derived bioagents and the biocomposites for enhancing hematoma-like fracture repair. Additionally, minimally invasive delivery for in situ curing of the implant system via injection was demonstrated in rat tail vertebrae using microcomputed tomography.

• Cis-2-decenoic Acid Inhibits S. aureus Growth and Biofilm In Vitro: A Pilot Study.

  Authors: Jessica Amber Jennings, Harry S Courtney, Warren O Haggard

  Clinical orthopaedics and related research. 05/2012;

  BACKGROUND: 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 isBACKGROUND: 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. QUESTIONS/PURPOSES: We asked whether (1) C2DA inhibited MRSA growth and biofilm, (2) antibiotics increased inhibitory effects, (3) inhibitory concentrations of C2DA were cytotoxic to human cells, and (4) effective concentrations could be delivered from a chitosan sponge drug delivery device. METHODS: Broth containing seven concentrations of C2DA and six concentrations of either daptomycin, vancomycin, or linezolid was inoculated with a clinical isolate of MRSA and added to a total of 504 coated microtiter plate wells in triplicate (n = 3) for turbidity bacterial growth and crystal violet biofilm mass quantification. We used fibroblast cell viability assays of six C2DA concentrations (n = 4) to evaluate preliminary biocompatibility. We measured the elution of C2DA from a chitosan sponge drug delivery device with two representative loading concentrations (n = 3). RESULTS: C2DA at concentrations of 500 μg/mL and above inhibited growth, while 125 μg/mL C2DA inhibited biofilm. Combination with antibiotics increased these effects. At concentrations up to 500 μg/mL, there were no cytotoxic effects on fibroblasts. Chitosan sponges loaded with 100 mg of C2DA eluted concentrations at or above biofilm-inhibitory concentrations for 5 days. CONCLUSIONS: C2DA inhibited biofilm formation by MRSA at biocompatible concentrations, with increasing biofilm reduction with added antibiotics. Elution of C2DA from a chitosan sponge can be modified through adjusting loading concentration. CLINICAL RELEVANCE: By inhibiting biofilm formation on implant surfaces, C2DA may reduce the number of infections in musculoskeletal trauma.

• Induction of the early inflammatory-mediated cellular responses of fracture healing in vitro using platelet releasate-containing alginate/CaPO(4) biomaterials for early osteoarthritis prevention.

  Authors: Jonathan D McCanless, Lisa K Jennings, Judith A Cole, Joel D Bumgardner, Warren O Haggard

  Journal of biomedical materials research. Part A. 02/2012; 100(5):1107-14.

  A significant gap exists in our understanding of subchondral and cancellous bone changes that may regulate osteoarthritis progression. Herein, we complement our prior osteochondrogenesis work withA significant gap exists in our understanding of subchondral and cancellous bone changes that may regulate osteoarthritis progression. Herein, we complement our prior osteochondrogenesis work with growth factor elution and monocyte and endothelial cell activation using two biomaterial formulations. The design of these biomaterials was inspired by the roles of a fracture hematoma, more specifically, the potential of significant cross-talk among cells and cellular factors that affect bone remodeling. Biomaterials, referred to herein as F1+ and F2+, are human concentrated platelet releasate-containing alginate/beta-tricalcium phosphate composites. F1+ has a higher calcium phosphate volume percentage and lower alginate polymer weight percent hydrogel versus F2+. The majority of releasate-derived platelet-derived growth factor eluted over 24 h for F1+ and 48 h for F2+, suggesting sustained release with an increase in alginate weight percentage. Simple monocyte and endothelial cell migration studies demonstrated 650% and 900% increases with F1+ eluate over medium alone, respectively. Induction of endothelial cell invasion over supplemented medium positive control was also shown for F2+ eluate (p = 0.03) with F1+ eluate being similar to the control. Monocyte transendothelial migration was increased over 300% and 400% for F1+ and F2+ eluates compared with medium alone, respectively. In addition, F1+ and F2+ eluates induced spontaneous endothelial tube formations similar to supplemented medium, demonstrating a well-formed network of capillary-like structures. This work demonstrated our biomaterial formulations ability to induce characteristics in vitro that parallel the in vivo behavior of fracture hematomas and potential to induce bone remodeling for the early treatment of osteoarthritic joints. © 2012 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2012.

• In vitro differentiation and biocompatibility of mesenchymal stem cells on a novel platelet releasate-containing injectable composite.

  Authors: Jonathan D McCanless, Lisa K Jennings, Judith A Cole, Joel D Bumgardner, Warren O Haggard

  Journal of biomedical materials research. Part A. 01/2012; 100(1):220-9.

  Underlying changes in subchondral and cancellous bone precede joint degeneration in spinal discs, knees, hips, and other joints. We hypothesize that an early bone remodeling intervention therapy mayUnderlying changes in subchondral and cancellous bone precede joint degeneration in spinal discs, knees, hips, and other joints. We hypothesize that an early bone remodeling intervention therapy may alleviate or postpone the later manifestation of joint failure. Utilization of a novel biomaterial for injection deliverability to the underlying subchondral and cancellous bone during the onset of osseous changes, before severe cartilaginous damage, is proposed. In this preliminary study, we introduced novel platelet releasate-containing alginate/calcium phosphate composites for this intervention therapy and evaluated them in vitro through mesenchymal stem cell biocompatibility and induction of osteochondral differentiation. DNA quantification and gene expression profiles suggested mesenchymal stem cells were directed along an osteochondral differentiation pathway, more specifically, to the immature nonhypertrophic chondrocyte phenotype. These conclusions were based on reduced mitogenic activity, a rapid upregulation followed by suppression of Runx2 transcription factor mRNA, sustained upregulation of Sox9 transcription factor mRNA and the absence of late marker expressions for both mature articular chondrocytes and osteoblasts over the course of 14 days. Initial findings in regard to the use of these materials for bone remodeling were positive and support the execution of future studies evaluating the inflammatory and angiogenic aspects of the materials. In this study, we have considered one of the many aspects of the endochondral ossification process; neovascularization and resorption of bone with an irregular physiology need to be evaluated.

• Evaluation of two sources of calcium sulfate for a local drug delivery system: a pilot study.

  Authors: Ashley C Parker, J Keaton Smith, Harry S Courtney, Warren O Haggard

  Clinical orthopaedics and related research. 05/2011; 469(11):3008-15.

  Local drug delivery has substantial potential to prevent infections compared with systemic delivery. Although calcium sulfate (CaSO(4)) has been studied for local drug delivery and two types areLocal drug delivery has substantial potential to prevent infections compared with systemic delivery. Although calcium sulfate (CaSO(4)) has been studied for local drug delivery and two types are commercially available, it is unknown whether they differentially release antibiotics. We determined the differences between two sources of CaSO(4) and the K(2)SO(4) catalyst's presence on the degradation, daptomycin elution, and activity against Staphylococcus aureus. We formed pellets from synthetic and naturally sourced (from gypsum) CaSO(4) and loaded with 5% daptomycin and 3% or 0% K(2)SO(4). We used in vitro experiments to determine the daptomycin concentration and degradation profiles over 10 days. Turbidity assays were used to evaluate the activity of the daptomycin eluates against S. aureus. All pellets exhibited a bolus release with the highest daptomycin concentration on Day 1 with the sourced CaSO(4) pellets. The synthetic CaSO(4) pellets with 3% K(2)SO(4) exhibited a slower drug release compared with the synthetic CaSO(4) pellets with 0% K(2)SO(4), which degraded and eluted daptomycin too quickly to inhibit S. aureus. Turbidity assays demonstrated that all CaSO(4) pellets inhibit S. aureus for expected lengths of time. Our preliminary in vitro data suggest differences in the degradation, elution, and activity properties between sourced and synthetic CaSO(4) pellets. The addition of K(2)SO(4) appeared beneficial when using synthetic CaSO(4). Synthetic CaSO(4) may be effective when slow degradation and longer elution times are needed. Local delivery of eluted daptomycin can be tailored through material selection and K(2)SO(4) addition.

• Chitosan coatings deliver antimicrobials from titanium implants: a preliminary study.

  Authors: Peter A Norowski, Harry S Courtney, Jegdish Babu, Warren O Haggard, Joel D Bumgardner

  Implant dentistry. 02/2011; 20(1):56-67.

  Chitosan was investigated as a coating for local delivery of antimicrobials for prevention of acute implant infection. The objectives of this study were to (1) measure the release of 2 antimicrobialsChitosan was investigated as a coating for local delivery of antimicrobials for prevention of acute implant infection. The objectives of this study were to (1) measure the release of 2 antimicrobials from chitosan coatings, (2) determine efficacy of eluted antimicrobials against bacteria, in vitro, and (3) evaluate toxicity of eluted drugs to host cells/tissues. Chitosan coatings (80.7% deacetylated, 108 kDa) containing 20% tetracycline or 0.02% chlorhexidine digluconate were bonded to titanium via silane reactions. After elution in culture medium for 7 days, eluates were tested against model pathogens Actinobacillus actinomycetemcomitans and Staphylococcus epidermidis in turbidity tests and in 24-hour cytotoxicity tests using human osteoblasts and fibroblasts. Finally, antibiotic-loaded chitosan-coated titanium pins were implanted for 7 days in muscle of Sprague-Dawley rats to evaluate the initial tissue response. Coatings released 89% of tetracycline in 7 days and 100% chlorhexidine in 2 days. Released tetracycline inhibited growth (95%-99.9%) of pathogens for up to 7 days with no cytotoxicity to human cells. Released chlorhexidine was active against pathogens for 1 to 2 days (56%-99.5% inhibition) but was toxic to cells on the first day of elution. Typical acute inflammatory response was observed to antimicrobial-loaded chitosan coatings similar to unloaded coatings. These preliminary data support the hypothesis that chitosan coatings have the potential to locally deliver antimicrobials to inhibit bacteria without being toxic to host cells/tissues and warrant additional studies to evaluate the ability of the coatings to prevent/resist infection and promote osseointegration.

• Modeling nucleus pulposus regeneration in vitro: mesenchymal stem cells, alginate beads, hypoxia, bone morphogenetic protein-2, and synthetic peptide B2A.

  Authors: Jonathan D McCanless, Judith A Cole, Steven M Slack, Joel D Bumgardner, Paul O Zamora, Warren O Haggard

  Spine. 02/2011; 36(26):2275-85.

  Using a low cell density, hypoxic, alginate-bead culture system, the effects of bone morphogenetic protein-2 (BMP-2) and synthetic peptide B2A on cell proliferation and extracellular matrix (ECM)Using a low cell density, hypoxic, alginate-bead culture system, the effects of bone morphogenetic protein-2 (BMP-2) and synthetic peptide B2A on cell proliferation and extracellular matrix (ECM) synthesis were assessed at days 0, 3, 5, and 7, using nucleus pulposus (NP)-like differentiated mesenchymal stem cells (MSCs). This is a preliminary investigation into B2A's potential adjunctive role with MSCs and BMP-2, in NP regeneration. B2A analogs, alone and in combination with BMP-2, have been shown to promote proliferation and ECM production in chondrocytes and MSCs. Articular chondrocytes and NP cells often respond in a similar manner to growth factor treatments, thus suggesting a potential role for B2A in treating disc degeneration by NP regeneration. Using the NP regeneration in vitro model (low cell density, hypoxic, alginate bead culture), B2A and BMP-2 were evaluated alone and in combination, to determine effects on proliferation and ECM synthesis in the presence of transforming growth factor-beta 3 on NP-like differentiated MSCs. B2A administration induced mild proliferation of NP-like differentiated MSCs and diminished an initial wave of low-dose BMP-2-prompted apoptosis. Individually and in combination, B2A and BMP-2 were found to inhibit transforming growth factor-beta 3-permitted collagen accumulation; levels remained similar in their presence. Both collagen I (Col I) and collagen II (Col II) were found in almost all specimens, but increased B2A levels favored Col II unlike BMP-2, which favored Col I. BMP-2 resulted in a minor reduction in aggrecan synthesis, which was unchanged by B2A. Using this in vitro model, B2A induced proliferation, continuous aggrecan synthesis, and stabilized collagen accumulation favoring Col II. These characteristics are consistent with cells of the young, healthy NP, indicating potential use of the peptide early in an MSC-based NP-regeneration therapy; whereas, BMP-2 induced apoptosis, Col I accumulation, and aggrecan production hindrance, and was found untherapeutic.

• Effect of molecular weight of chitosan degraded by microwave irradiation on lyophilized scaffold for bone tissue engineering applications.

  Authors: Marvin M Mecwan, Gabriel E Rapalo, Sanjay R Mishra, Warren O Haggard, Joel D Bumgardner

  Journal of biomedical materials research. Part A. 02/2011;

  Chitosan (CTS) is biocompatible, biodegradable, and can be formed into 3D porous structures for bone tissue engineering applications. Although studies have reported on the effects of molecular weightChitosan (CTS) is biocompatible, biodegradable, and can be formed into 3D porous structures for bone tissue engineering applications. Although studies have reported on the effects of molecular weight (MW) on CTS physicochemical properties, studies evaluating CTS biological property relationships often do not account for MW that confounds interpretation of study results. The aim of this study was to evaluate the effect of MW on CTS physicochemical and biological properties. CTS materials were treated for 6, 18, and 30 min by microwave irradiation to decrease MW without affecting deacetylation (DDA). Materials were evaluated for crystallinity using X-ray diffraction, thermal degradation using differential scanning calorimetry, water content, swelling ratio, and in vitro compatibility using Saos-2. Results showed that microwave treatments did not affect DDA but decreased MW and swelling ratio by 45.78% and 36.75%, respectively, after 30 min of microwave treatment. Microwave-treated CTS showed reduced or no crystalline peaks. Initial increase in exothermic peak temperatures with short (6 min) microwave treatment times were followed by a decrease with longer (18 and 30 min) treatment times. Cell growth over 7 days on samples was proportional to MW with the number of cells being 62% higher on CTS with the highest MW (3.71 ± 0.25 × 10(5) g/mol) when compared with the lower MW CTS (2.38 ± 0.12 × 10(5) g/mol). These results demonstrate the importance of MW of CTS to both its physicochemical characteristics and biological properties, providing researchers with another tool for the modulation and optimization of CTS for different biomedical applications. © 2011 Wiley Periodicals, Inc. J Biomed Mater Res Part A:, 2011.

• Local antibiotic delivery using tailorable chitosan sponges: the future of infection control?

  Authors: Daniel J Stinner, Scott P Noel, Warren O Haggard, J Tracy Watson, Joseph C Wenke

  Journal of orthopaedic trauma. 09/2010; 24(9):592-7.

  Local antibiotic delivery is a viable and attractive option for preventing infection. Unfortunately, the current options are limited and often necessitate surgical removal. This study evaluates theLocal antibiotic delivery is a viable and attractive option for preventing infection. Unfortunately, the current options are limited and often necessitate surgical removal. This study evaluates the ability of a biodegradable and biocompatible chitosan sponge to minimize infection by delivering local antibiotics within the wound. A complex musculoskeletal wound was created on the hindlimb of goats and contaminated with Pseudomonas aeruginosa (lux) or Staphylococcus aureus (lux) bacteria. These bacteria are genetically engineered to emit photons, allowing for quantification with a photon-counting camera system. The wounds were closed and similarly débrided and irrigated with 9 L normal saline using bulb-syringe irrigation 6 hours after inoculation. Goats were assigned to different treatment groups: a control group with no adjunctive treatment and an experimental group using a chitosan sponge loaded with either amikacin (for wounds contaminated with P. aeruginosa) or vancomycin (for wounds contaminated with S. aureus). The wounds were closed after the procedure and evaluated 48 hours after initial contamination. Serum levels of the antibiotics were also measured at 6, 12, 24, 36, and 42 hours after treatment was initiated. The wounds treated with the antibiotic-loaded chitosan sponge had significantly less bacteria than the untreated wounds (P < 0.05). The highest serum levels were 6 hours after treatment but remained less than 15% of target serum levels for systemic treatment. At study end point, all sponges were between 60% and 100% degraded. The chitosan sponges are effective delivering the antibiotic and reducing the bacteria within the wounds.

• Antibiotic-loaded chitosan film for infection prevention: A preliminary in vitro characterization.

  Authors: J Keaton Smith, Joel D Bumgardner, Harry S Courtney, Mark S Smeltzer, Warren O Haggard

  Journal of biomedical materials research. Part B, Applied biomaterials. 07/2010; 94(1):203-11.

  The growing infection rate by methicillin-resistant Staphylococcus aureus, especially with bone fracture fixation implants, is a major concern in extremity musculoskeletal wound treatment. ThisThe growing infection rate by methicillin-resistant Staphylococcus aureus, especially with bone fracture fixation implants, is a major concern in extremity musculoskeletal wound treatment. This preliminary investigation evaluates the ability of chitosan film to be loaded with daptomycin and vancomycin in the operating room, in situ loading, and applied to musculoskeletal fixation devices to lessen or prevent infection. Films with 61, 71, and 80% degrees of deacetylation (DDA) made using lactic or acetic acid solvents were analyzed for their antibiotic uptake, elution, and activity along with film swelling ratio, ultimate tensile strength, Young's modulus, adhesive strength, and degradation. Chitosan films after 1 min of rehydration were able in a simulated, clinical setting to maintain mechanical integrity and adhesive strength to be applied to bone fracture fixation devices or implant surfaces. The film percent degradation increased with DDA increasing from 61 to 80%, but film degradation rate decreased in the presence of antibiotics. Eighty percent DDA chitosan films were optimal for absorbing and eluting antibiotics. Antibiotics eluted by the films were active against Staphylococcus aureus. These findings indicate that an 80% DDA chitosan film is potentially advantageous as a clinically adjunctive treatment in musculoskeletal injuries to lessen or prevent infections.

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

  Authors: Robert M Crews, Jessica A Jennings, Jonathan McCanless, Judith A Cole, Joel D Bumgardner, Warren O Haggard

  Journal of biomedical materials research. Part B, Applied biomaterials. 04/2010; 93(1):227-35.

  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, implantMinimally 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.

• Chitosan sponges to locally deliver amikacin and vancomycin: a pilot in vitro evaluation.

  Authors: Scott P Noel, Harry S Courtney, Joel D Bumgardner, Warren O Haggard

  Clinical orthopaedics and related research. 03/2010; 468(8):2074-80.

  Open orthopaedic wounds are ideal sites for infection. Preventing infection in these wounds is critical for reducing patient morbidity and mortality, controlling antimicrobial resistance and loweringOpen orthopaedic wounds are ideal sites for infection. Preventing infection in these wounds is critical for reducing patient morbidity and mortality, controlling antimicrobial resistance and lowering the cost of treatment. Localized drug delivery has the potential to overcome the challenges associated with traditional systemic dosing. A degradable, biocompatible polymer sponge (chitosan) that can be loaded with clinician-selected antibiotics at the point of care would provide the patient and clinician with a desirable, adjunctive preventive modality. We asked (1) if an adaptable, porous chitosan matrix could absorb and elute antibiotics for 72 hours for potential use as an adjunctive therapy to débridement and lavage; and (2) if the sponges could elute levels of antibiotic that would inhibit growth of Staphylococcus aureus and Pseudomonas aeruginosa? We fabricated a degradable chitosan sponge that can be loaded with antibiotics during a 60-second hydration in drug-containing solution. In vitro evaluation determined amikacin and vancomycin release from chitosan sponges at six time points. Activity tests were used to assess the release of inhibitory levels of amikacin and vancomycin. Amikacin concentration was 881.5 microg/mL after 1 hour with a gradual decline to 13.9 microg/mL after 72 hours. Vancomycin concentration was 1007.4 microg/mL after 1 hour with a decrease to 48.1 microg/mL after 72 hours. Zone of inhibition tests were used to verify inhibitory levels of drug release from chitosan sponges. A turbidity assay testing activity of released amikacin and vancomycin indicated inhibitory levels of elution from the chitosan sponge. Chitosan sponges may provide a potential local drug delivery device for preventing musculoskeletal infections.

• A chitosan/beta-glycerophosphate thermo-sensitive gel for the delivery of ellagic acid for the treatment of brain cancer.

  Authors: Sungwoo Kim, Satoru K Nishimoto, Joel D Bumgardner, Warren O Haggard, M Waleed Gaber, Yunzhi Yang

  Biomaterials. 02/2010; 31(14):4157-66.

  We report here the development of a chitosan/beta-glycerophosphate(Ch/beta-GP) thermo-sensitive gel to deliver ellagic acid (EA) for cancer treatment. The properties of the Ch/beta-GP gels wereWe report here the development of a chitosan/beta-glycerophosphate(Ch/beta-GP) thermo-sensitive gel to deliver ellagic acid (EA) for cancer treatment. The properties of the Ch/beta-GP gels were characterized regarding chemical structure, surface morphology, and viscoelasticity. In vitro EA release rate from the EA loaded Ch/beta-GP gel and chitosan degradation rate were investigated. The anti-tumor effect of the EA loaded Ch/beta-GP gel on brain cancer cells (human U87 glioblastomas and rat C6 glioma cells) was evaluated by examining cell viability. Cell number and activity were monitored by the MTS assay. The Ch/beta-GP solution formed a heat-induced gel at body temperature, and the gelation temperature and time were affected by the final pH of the Ch/beta-GP solution. The lysozyme increased the EA release rate by 2.5 times higher than that in the absence of lysozyme. Dialyzed chitosan solution with final pH 6.3 greatly reduced the beta-GP needed for gelation, thereby significantly improving the biocompatibility of gel (p < 0.001). The chitosan gels containing 1% (w/v) of ellagic acid significantly reduced viability of U87 cells and C6 cells compared with the chitosan gels at 3 days incubation (p < 0.01, and p < 0.001, respectively).

• An Approach for determining antibiotic loading for a physician-directed antibiotic-loaded PMMA bone cement formulation.

  Authors: Gladius Lewis, Jennifer L Brooks, Harry S Courtney, Yuan Li, Warren O Haggard

  Clinical orthopaedics and related research. 02/2010; 468(8):2092-100.

  When a physician-directed antibiotic-loaded polymethylmethacrylate (PMMA) bone cement (ALBC) formulation is used in total hip arthroplasties (THAs) and total knee arthroplasties (TKAs), currentWhen a physician-directed antibiotic-loaded polymethylmethacrylate (PMMA) bone cement (ALBC) formulation is used in total hip arthroplasties (THAs) and total knee arthroplasties (TKAs), current practice in the United States involves arbitrary choice of the antibiotic loading (herein defined as the ratio of the mass of the antibiotic added to the mass of the cement powder). We suggest there is a need to develop a rational method for determining this loading. We propose a new method for determining the antibiotic loading to use when preparing a physician-directed ALBC formulation and illustrate this method using three in vitro properties of an ALBC in which the antibiotic was daptomycin. Daptomycin was blended with the powder of the cement using a mechanical mixer. We performed fatigue, elution, and activity tests on three sets of specimens having daptomycin loadings of 2.25, 4.50, and 11.00 wt/wt%. Correlational analyses of the results of these tests were used in conjunction with stated constraints and a nonlinear optimization method to determine the daptomycin loading to use. With an increase in daptomycin loading, the estimated mean fatigue limit of the cement decreased, the estimated elution rate of the antibiotic increased, and the percentage inhibition of staphylococcal growth by the eluate remained unchanged at 100%. For a daptomycin-loaded PMMA bone cement we computed the optimum amount of daptomycin to mechanically blend with 40 g of cement powder is 1.36 g. We suggest an approach that may be used to determine the amount of antibiotic to blend with the powder of a PMMA bone cement when preparing a physician-directed ALBC formulation, and highlighted the attractions and limitations of this approach. When a physician-directed ALBC formulation is selected for use in a TKA or THA, the approach we detail may be employed to determine the antibiotic loading to use rather than the empirical approach that is taken in current clinical practice.

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

  Authors: Jessica A Jennings, Robert M Crews, Joycelyn Robinson, Kelly Richelsoph, Judith A Cole, Joel D Bumgardner, Yunzhi Yang, Warren O Haggard

  Journal of biomedical materials research. Part B, Applied biomaterials. 11/2009;

  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 determineInjections 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 mum 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. (c) 2009 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 2010.

• Lyophilization to improve drug delivery for chitosan-calcium phosphate bone scaffold construct: A preliminary investigation.

  Authors: Benjamin T Reves, Joel D Bumgardner, Judith A Cole, Yunzhi Yang, Warren O Haggard

  Journal of biomedical materials research. Part B, Applied biomaterials. 06/2009;

  Lyophilization was evaluated in chitosan-calcium phosphate microspheres and scaffolds to improve drug delivery of growth factors and antibiotics for orthopedic applications. The dual delivery of anLyophilization was evaluated in chitosan-calcium phosphate microspheres and scaffolds to improve drug delivery of growth factors and antibiotics for orthopedic applications. The dual delivery of an antibiotic and a growth factor from a composite scaffold would be beneficial for treatment of complex fracture sites, such as comminuted fractures and segmental bone defects. The aim of this investigation was to increase the loading capacity of the composite by taking advantage of the increased porosity, due to lyophilization, and to produce an extended elution profile using a secondary chitosan-bead coating. The physiochemical properties of the composite were investigated, and loading and elution studies were performed with alkaline phosphatase (ALP), bone morphogenetic protein-2 (BMP-2), and amikacin. Lyophilization was found to increase the surface area of scaffolds by over 400% and the porosity of scaffolds by 50%. Using ALP as a model protein, the loading capacity was increased by lyophilization from 4.3 +/- 2.5 to 24.6 +/- 3.6 mug ALP/mg microspheres, and the elution profile was extended by a supplemental chitosan coating. The loading capacity of BMP-2 for composite microspheres was increased from 74.4 +/- 3.7 to 102.1 +/- 8.0 mug BMP-2/g microspheres with lyophilization compared with nonlyophilized microspheres. The elution profiles of BMP-2 and the antibiotic amikacin were not extended with the supplemental coating. Additional investigations are planned to improve these elution characteristics for growth factors and antibiotics. (c) 2009 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 2009.

• Composite Chitosan/Nano-Hydroxyapatite Scaffolds Induce Osteocalcin Production by Osteoblasts In Vitro and Support Bone Formation In Vivo.

  Authors: Betsy M Chesnutt, Youling Yuan, Karyl Buddington, Warren O Haggard, Joel D Bumgardner

  Tissue engineering. Part A. 04/2009;

  There is a significant clinical need to develop alternatives to autografts and allografts for bone grafting procedures. Porous, biodegradable scaffolds based on the biopolymer chitosan have beenThere is a significant clinical need to develop alternatives to autografts and allografts for bone grafting procedures. Porous, biodegradable scaffolds based on the biopolymer chitosan have been investigated as bone graft substitutes, and the addition of calcium phosphate to these scaffolds has been shown to improve the mechanical properties of the scaffold and may increase osteoconductivity. In this study, in vitro mineralization was examined for osteoblasts seeded in a porous scaffold composed of fused chitosan/nano-hydroxyapatite microspheres. Human fetal osteoblasts were cultured on composite and chitosan scaffolds for 21 days. On days 1, 4, 7, 14, and 21, total dsDNA, alkaline phosphatase, type I collagen, and osteocalcin production were measured. Total cellularity (measured by dsDNA), alkaline phosphatase, and type I collagen production were similar between the two scaffold groups. However, osteocalcin production occurred significantly earlier (day 7 vs. day 21) and was more than three times greater (0.0022 vs. 0.0068 ng/mL/ng DNA) on day 21 when osteoblasts were cultured on composite scaffolds. Osteocalcin is a marker of late osteoblastic differentiation and mineralized bone matrix formation. Therefore, the increase in osteocalcin production seen when cells were cultured on composite scaffolds may indicate that these scaffolds were superior to chitosan-only scaffolds in facilitating osteoblast mineralization. Composite scaffolds were also shown to be biocompatible and osteoconductive in a preliminary critical size rat calvarial defect study. These results demonstrate the potential of composite chitosan/nano-hydroxyapatite scaffolds to be used in bone tissue engineering.

• Preliminary in vitro evaluation of an adjunctive therapy for extremity wound infection reduction: Rapidly resorbing local antibiotic delivery.

  Authors: Stephanie R Jackson, Kelly C Richelsoph, Harry S Courtney, Joseph C Wenke, Joanna G Branstetter, Joel D Bumgardner, Warren O Haggard

  Journal of orthopaedic research : official publication of the Orthopaedic Research Society. 01/2009;

  Despite the continuing advances in treatment of open fractures and musculoskeletal wounds, infection remains a serious complication. Current treatments to prevent infection utilize surgicalDespite the continuing advances in treatment of open fractures and musculoskeletal wounds, infection remains a serious complication. Current treatments to prevent infection utilize surgical debridement and irrigation, and high doses of systemic antimicrobial therapy. The aim of this work was to evaluate, in vitro, the potential of a fast-resorbing calcium sulfate pellet loaded with an antibiotic. The pellet could be used as an adjunctive therapy at the time of debridement and irrigation to reduce bacterial wound contamination. Small pellets containing a binder and calcium sulfate were engineered to resorb rapidly (within 24 h) and deliver high local doses of antibiotic (amikacin, gentamicin, or vancomycin) to the wound site while minimizing systemic effects. Results from dissolution, elution, and biological activity tests against P. aeruginosa and S. aureus were used to compare the performance of antibiotic-loaded, rapidly resorbing calcium sulfate pellets to antibiotic-loaded crushed conventional calcium sulfate pellets. Antibiotic-loaded rapidly resorbing pellets dissolved in vitro in deionized water in 12-16 h and released therapeutic antibiotic levels in phosphate buffered saline that were above the minimal inhibitory concentration for P. aeruginosa and S. aureus, completely inhibiting the growth of these bacteria for the life of the pellet. Crushed conventional calcium sulfate pellets dissolved over 4-6 days, but the eluates only contained sufficient antibiotic to inhibit growth for the first 4 h. These data indicate that fast-resorbing pellets can release antibiotics rapidly and at therapeutic levels. Adjunctive therapy with fast-acting pellets is promising and warrants further in vivo studies. Published by Wiley Periodicals, Inc. J Orthop Res.

• Development of chitosan-ellagic acid films as a local drug delivery system to induce apoptotic death of human melanoma cells.

  Authors: Sungwoo Kim, Yongxing Liu, M Waleed Gaber, Joel D Bumgardner, Warren O Haggard, Yunzhi Yang

  Journal of biomedical materials research. Part B, Applied biomaterials. 12/2008;

  This study was designed to develop a local chemotherapy device using chitosan as a local drug carrier and ellagic acid (EA) as an anticancer drug. We fabricated chitosan-ellagic acid (Ch-EA) filmsThis study was designed to develop a local chemotherapy device using chitosan as a local drug carrier and ellagic acid (EA) as an anticancer drug. We fabricated chitosan-ellagic acid (Ch-EA) films with concentrations of 0, 0.05, 0.1, 0.5, and 1% (w/v) of EA and examined the films using Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), scanning electron microscopy (SEM), and contact angle measurement. The WM115 human melanoma cell line as a skin cancer model was used to evaluate cell response to the films with the MTS assay and apoptosis assay, and HS68 human newborn fibroblast cell line as a control. With the increase in the concentration of the EA, the composite films exhibit increasing amide and ester groups and diffraction peaks of the crystallized EA and greater surface roughness and hydrophilicity. The chitosan films with 0.5 and 1% (w/v) of EA were found to have a potent antiproliferative effect on the melanoma cells by inducing apoptotic cell death. Localized effect of composites on cell behaviors has been clearly demonstrated. Our study demonstrated that the novel Ch-EA film can be potentially used in local chemotherapy. (c) 2008 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 2009.

• Effects of material property and heat treatment on nanomechanical properties of chitosan films.

  Authors: Shervin Majd, Youling Yuan, Sanjay Mishra, Warren O Haggard, Joel D Bumgardner

  Journal of biomedical materials research. Part B, Applied biomaterials. 12/2008;

  The influence of degree of deacetylation (DDA), molecular weight (MW), and heat treatment on nanomechanical properties of three different chitosan coatings was assessed. Chitosans (2.5 wt % in 2%The influence of degree of deacetylation (DDA), molecular weight (MW), and heat treatment on nanomechanical properties of three different chitosan coatings was assessed. Chitosans (2.5 wt % in 2% acetic acid) were solution cast and bonded to glass slides, subjected to 40, 70, or 90 degrees C heat treatments for 1 h, and then allowed to air dry. Non-heat treated films were used as controls. The nanomechanical and structural properties were evaluated using quasi-static nanoindentation, X-ray diffraction (XRD), and atomic force microscopy (AFM). Nanoindentation results showed that the coating with the lowest MW and highest DDA (95.6% DDA and MW = 2.43 x 10(6) Da) had higher reduced modulus of elasticity, (E = 4.02 +/- 0.85 GPa) as compared to the other chitosans; 76.1% DDA/MW = 3.20 x 10(6) Da, E = 3.66 +/- 0.68 GPa, and 92.3% DDA/MW = 7.52 x 10(6) Da, E = 3.56 +/- 0.25 GPa; (n = 75, p < 0.05). Crystallinity index, estimated via XRD, ranged from 36.4% +/- 1% to 49.7% +/- 6.5%. AFM revealed the presence of different crystalline morphologies such as needle-like crystals, sheet-like crystals, and small spherulites. The heat treatments applied during solution casting of the chitosan coatings did not affect morphology or nanomechanical properties. In conclusion, nanomechanical properties of chitosan coatings varied with DDA and MW and may be important to cell/tissue interactions. (c) 2008 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 2009.