Warren O. Haggard

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

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 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).

Design and characterization of a novel chitosan/nanocrystalline calcium phosphate composite scaffold for bone regeneration

To meet the challenge of regenerating bone lost to disease or trauma, biodegradable scaffolds are being investigated as a way to regenerate bone without the need for an auto- or allograft. Here, we have developed a novel microsphere-based chitosan/nanocrystalline calcium phosphate (CaP) composite scaffold and investigated its potential compared to plain chitosan scaffolds to be used as a bone graft substitute. Composite and chitosan scaffolds were prepared by fusing microspheres of 500-900 microm in diameter, and porosity, degradation, compressive strength, and cell growth were examined. Both scaffolds had porosities of 33-35% and pore sizes between 100 and 800 . However, composite scaffolds were much rougher and, as a result, had 20 times more surface area/unit mass than chitosan scaffolds. The compressive modulus of hydrated composite scaffolds was significantly higher than chitosan scaffolds (9.29 +/- 0.8 MPa vs. 3.26 +/- 2.5 MPa), and composite scaffolds were tougher and more flexible than what has been reported for other chitosan-CaP composites or CaP scaffolds alone. Using X-ray diffraction, scaffolds were shown to contain partially crystalline hydroxyapatite with a crystallinity of 16.7% +/- 6.8% and crystallite size of 128 +/- 55 nm. Fibronection adsorption was increased on composite scaffolds, and cell attachment was higher on composite scaffolds after 30 min, although attachment rates were similar after 1 h. Osteoblast proliferation (based on dsDNA measurements) was significantly increased after 1 week of culture. These studies have demonstrated that composite scaffolds have mechanical properties and porosity sufficient to support ingrowth of new bone tissue, and cell attachment and proliferation data indicate composite scaffolds are promising for bone regeneration.

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

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 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.

Use of a calcium sulfate-based bone graft substitute for benign bone lesions.

Twenty-three patients with a benign bone lesion grafted with calcium sulfate, with and without demineralized bone matrix, were reviewed. At a minimum of 1 year postoperatively, 21 patients had achieved between 76% and 100% bone repair based on anteroposterior and lateral radiographs. Overall, the mean Enneking Functional Evaluation System score was 98%. Calcium sulfate is a well-tolerated, biodegradable, osteoconductive bone graft substitute. It is a reasonable alternative to autogenous bone graft for benign bone lesions.

Deacetylation of Chitosan: Material Characterization and in vitro Evaluation via Albumin Adsorption and Pre-Osteoblastic Cell Cultures

Degree of deacetylation (DDA) and molecular weight (MW) of chitosans are important to their physical and biological properties. In this study, two chitosans, HS (DDA = 73.3%) and AT (DDA = 76.8%), were deacetylated with 45% sodium hydroxide under nitrogen atmosphere at 80 °C or 90 °C for up to 120 min, to obtain two series of chitosans. The polymers produced were characterized for MW by gel permeation chromatography, DDA by titration and UV-vis methods, and crystallinity, hydrophilicity and thermal stability by X-ray diffraction, water contact angle and differential scanning calorimetry respectively. Films, made by solution casting in dilute acetic acid at ambient conditions, were evaluated for biological activity by albumin adsorption and the attachment and growth of a pre-osteoblast cell line. Chitosans with between 80–93% DDA’s (based on titration) were reproducibly obtained. Even though deacetylation under nitrogen was supposed to limit chain degradation during decetylation, MW decreased (by maximum of 37.4% of HS and 63.0% for AT) with increasing deacetylation reaction time and temperature. Crystallinity and decomposition temperature increased and water contact angles decreased with processing to increase DDA. Significantly less albumin was absorbed on films made with 93% DDA chitosans as compared with the original materials and the AT chitosans absorbed less than the HS chitosans. The cells on higher DDA chitosan films grew faster than those on lower DDA films. In conclusion, processing conditions increased DDA and influenced physicochemical and biological properties. However, additional studies are needed to unambiguously determine the influence of DDA or MW on in vitro and in vivo performance of chitosan materials for bone/implant applications.

Measurement of bone morphogenetic proteins and other growth factors in demineralized bone matrix.

Osteoinductivity of demineralized bone matrix has been attributed to bone morphogenetic proteins (BMP). Other growth factors, including insulin-like growth factor-1 (IGF-1) and transforming growth factor-beta1 (TGF-beta1), have also been detected in demineralized bone matrix. Success of bone graft substitutes containing demineralized bone matrix has been assumed to be closely associated with osteoinductivity of the demineralized bone matrix. Because of differences in bone characteristics between donors and tissue banks, confirmation and measurement of osteoinductivity may play a crucial role in predicting the success of the bone graft substitute. In the current studies, BMP-2, BMP-4, TGF-beta1, and IGF-1 were measured in demineralized bone matrix. A strong association was noted between BMP-2 and TGF-beta1 levels. A strong association was also found between BMP-2 and new bone formation in an ectopic nude rat model.

Local Antibiotic Delivery Using Tailorable Chitosan Sponges: The Future of Infection Control?

Objectives: 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 the ability of a biodegradable and biocompatible chitosan sponge to minimize infection by delivering local antibiotics within the wound. Methods: 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. Results: 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. Conclusions: The chitosan sponges are effective delivering the antibiotic and reducing the bacteria within the wounds.

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

Objective: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 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. Methods: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. Results: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. Conclusion: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.

Elution behavior of daptomycin-loaded calcium sulfate pellets: a preliminary study.

Daptomycin is a new antibiotic active against many resistant Gram-positive organisms and seems an appropriate candidate for local delivery for severe musculoskeletal infections. Calcium sulfate dihydrate as a delivery vehicle is readily resorbable, allows new bone formation, and can be combined with therapeutic agents. We compared the elution of daptomycin and tobramycin in calcium sulfate pellets over time and determined the dissolution rates of the pellets. Unlike other water-soluble antibiotics, daptomycin required special techniques to convert the calcium sulfate from a hemihydrate powder to a hardened dihydrate shape. The elution of daptomycin on day 1 (537 μg/mL/g) was greater than that from days 2, 3, 7, 10, 14, 21, and 28. The concentration fell to 153 μg/mL/g and 37 μg/mL/g on days 2 and 3, respectively, then remained at between 5 μg/mL/g and 7 μg/mL/g for the remainder of the study. The elution behavior of the daptomycin pellets differed from that of the tobramycin-containing pellets on days 1 through 3, but was similar from day 7 through day 28. Daptomycin-containing pellets dissolved more rapidly in vitro than tobramycin-containing pellets, although the importance of this more rapid dissolution in an in vivo situation is unknown. Using the techniques described in this paper, daptomycin can be incorporated within a calcium sulfate matrix.

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

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 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 microg 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 microg 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.