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Diabetic foot ulcers are particularly difficult to treat due to neuropathy and healing deficiencies. Glutathione and adenosine have been studied as possible modulators of diabetic response and would healing. The objective of this in vitro study was to measure the effects of adenosine and glutathione on fibroblast proliferation and collagen synthesis. Further, methods were explored for delivering these compounds locally through chitosan sponges. Elution concentrations of adenosine and glutathione from chitosan sponges were evaluated through HPLC, and activity was evaluated for fibroblast proliferation and viability in vitro. Furthermore, collagen production of normal and hyperglycemic fibroblasts exposed to those compounds individually and in combination was evaluated using a colorimetric collagen assay. Results indicate a dose-dependent increase in fibroblast proliferation, with exposure to concentrations up to 133.6 mg/ml adenosine and 76.8 mg/ml glutathione. Total collagen concentration determination confirmed that adenosine alone at 133.6 mg/ml significantly increases collagen production in low glucose conditions, but not to the same extent in medium and diabetic glucose conditions. Glutathione at 76.8 mg/ml did not increase collagen production compared to control or adenosine alone, and the combination attenuated increases in collagen production in low glucose conditions. There were no additive or synergistic effects of combining these two small molecule mediators of growth on fibroblast growth or collagen production. Both adenosine and glutathione were released in a burst response from chitosan sponges, and demonstrated activity in increasing proliferation. These preliminary in vitro evaluations demonstrate that the local delivery of adenosine and glutathione released from chitosan sponges may be a promising strategy for the treatment of diabetic foot ulcers under glucose conditions that are closely managed.
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Aimin, C., H. Chunlin, B. Juliang, Z. Tinyin, and D. Zhichao. 1999. Antibiotic loaded chitosan bar. An in vitro, in vivo study of a possible treatment for osteomyelitis. Clinical Orthopaedics and Related Research (366):239-47. http://www.ncbi.nlm.nih.gov/pubmed/10627741.
Bennett, S. P., G. D. Griffiths, A. M. Schor, G. P. Leese, and S. L. Schor. 2003. Growth factors in the treatment of diabetic foot ulcers. The British journal of surgery 90 (2):133-46. http://www.ncbi.nlm.nih.gov/pubmed/12555288.
Boulton, A. J. 2004. The diabetic foot: from art to science. The 18th Camillo Golgi lecture. Diabetologia 47 (8):1343-53. http://www.ncbi.nlm.nih.gov/pubmed/15309286.
Brem, H., M. S. Golinko, O. Stojadinovic, A. Kodra, R. F. Diegelmann, S. Vukelic, H. Entero, D. L. Coppock, and M. Tomic-Canic. 2008. Primary cultured fibroblasts derived from patients with chronic wounds: a methodology to produce human cell lines and test putative growth factor therapy such as GMCSF. Journal of Translational Medicine 6:75. http://www.ncbi.nlm.nih.gov/pubmed/19046453.
Brem, H., P. Sheehan, and A. J. Boulton. 2004. Protocol for treatment of diabetic foot ulcers. American Journal of Surgery 187 (5A):1S-10S. http://www.ncbi.nlm.nih.gov/pubmed/15147985.
Camera, E., and M. Picardo. 2002. Analytical methods to investigate glutathione and related compounds in biological and pathological processes. Journal of Chromatography B-Analytical Technologies in the Biomedical and Life Sciences 781 (1-2):181-206.
Demidova-Rice, Tatiana N, Michael R Hamblin, and Ira M Herman. 2012. Acute and impaired wound healing: pathophysiology and current methods for drug delivery, part 1: normal and chronic wounds: biology, causes, and approaches to care. Advances in skin & wound care 25 (7):304.
Deveci, M., R. R. Gilmont, W. R. Dunham, B. P. Mudge, D. J. Smith, and C. L. Marcelo. 2005. Glutathione enhances fibroblast collagen contraction and protects keratinocytes from apoptosis in hyperglycaemic culture. The British journal of dermatology 152 (2):217-24. http://www.ncbi.nlm.nih.gov/pubmed/15727631.
Edwards, J. L., A. M. Vincent, H. T. Cheng, and E. L. Feldman. 2008. Diabetic neuropathy: Mechanisms to management. Pharmacology and Therapeutics 120 (1):1-34.
Epstein, Franklin H, Adam J Singer, and Richard AF Clark. 1999. Cutaneous wound healing. New England journal of medicine 341 (10):738-746.
Faglia, E., G. Clerici, J. Clerissi, L. Gabrielli, S. Losa, M. Mantero, M. Caminiti, V. Curci, T. Lupattelli, and A. Morabito. 2006. Early and five-year amputation and survival rate of diabetic patients with critical limb ischemia: data of a cohort study of 564 patients. European journal of vascular and endovascular surgery : the official journal of the European Society for Vascular Surgery 32 (5):484-90. http://www.ncbi.nlm.nih.gov/pubmed/16730466.
Gentry, L. O. 1997. Management of osteomyelitis. International Journal of Antimicrobial Agents 9 (1):37-42. http://www.ncbi.nlm.nih.gov/pubmed/18611817.
Hehenberger, Karin, Johan D Heilborn, Kerstin Brismar, and Anders Hansson. 1998. Inhibited proliferation of fibroblasts derived from chronic diabetic wounds and normal dermal fibroblasts treated with high glucose is associated with increased formation of L‐lactate. Wound Repair and Regeneration 6 (2):135-141.
Hong, S. Y., H. W. Gil, J. O. Yang, E. Y. Lee, H. K. Kim, S. H. Kim, Y. H. Chung, S. K. Hwang, and Z. W. Lee. 2005. Pharmacokinetics of glutathione and its metabolites in normal subjects. Journal of Korean Medical Science 20 (5):721-6. http://www.ncbi.nlm.nih.gov/pubmed/16224142.
Ishaug, S. L., M. J. Yaszemski, R. Bizios, and A. G. Mikos. 1994. Osteoblast function on synthetic biodegradable polymers. Journal of biomedical materials research 28 (12):1445-53. http://www.ncbi.nlm.nih.gov/pubmed/7876284.
Khor, E., and L. Y. Lim. 2003. Implantable applications of chitin and chitosan. Biomaterials 24 (13):2339-49. http://www.ncbi.nlm.nih.gov/pubmed/12699672.
Kirk, J. F., G. Ritter, I. Finger, D. Sankar, J. D. Reddy, J. D. Talton, C. Nataraj, S. Narisawa, J. L. Millan, and R. R. Cobb. 2013. Mechanical and biocompatible characterization of a cross-linked collagen-hyaluronic acid wound dressing. Biomatter 3 (4). http://www.ncbi.nlm.nih.gov/pubmed/23896569.
Klabunde, R. E. 1983. Dipyridamole inhibition of adenosine metabolism in human blood. European Journal of Pharmacology 93 (1-2):21-6. http://www.ncbi.nlm.nih.gov/pubmed/6628545.
Kopal, C., M. Deveci, S. Ozturk, and M. Sengezer. 2007. Effects of topical glutathione treatment in rat ischemic wound model. Annals of Plastic Surgery 58 (4):449-55. http://www.ncbi.nlm.nih.gov/pubmed/17413890.
Lavery, L. A., D. G. Armstrong, R. P. Wunderlich, M. J. Mohler, C. S. Wendel, and B. A. Lipsky. 2006. Risk factors for foot infections in individuals with diabetes. Diabetes Care 29 (6):1288-93. http://www.ncbi.nlm.nih.gov/pubmed/16732010.
Leffler, C. C., and B. W. Muller. 2000. Influence of the acid type on the physical and drug liberation properties of chitosan-gelatin sponges. International Journal of Pharmaceutics 194 (2):229-37. http://www.ncbi.nlm.nih.gov/pubmed/10692647.
Lerman, O. Z., R. D. Galiano, M. Armour, J. P. Levine, and G. C. Gurtner. 2003. Cellular dysfunction in the diabetic fibroblast: impairment in migration, vascular endothelial growth factor production, and response to hypoxia. The American journal of pathology 162 (1):303-12. http://www.ncbi.nlm.nih.gov/pubmed/12507913.
Lippmann, H. I., A. Perotto, and R. Farrar. 1976. The neuropathic foot of the diabetic. Bulletin of the New York Academy of Medicine 52 (10):1159-78. http://www.ncbi.nlm.nih.gov/pubmed/793654.
Lipsky, B. A., A. R. Berendt, H. G. Deery, J. M. Embil, W. S. Joseph, A. W. Karchmer, J. L. LeFrock, D. P. Lew, J. T. Mader, C. Norden, and J. S. Tan. 2004. Diagnosis and treatment of diabetic foot infections. Clinical infectious diseases : an official publication of the Infectious Diseases Society of America 39 (7):885-910. http://www.ncbi.nlm.nih.gov/pubmed/15472838.
Liu, Z. J., and O. C. Velazquez. 2008. Hyperoxia, endothelial progenitor cell mobilization, and diabetic wound healing. Antioxidants & redox signaling 10 (11):1869-82. http://www.ncbi.nlm.nih.gov/pubmed/18627349.
Lu, S., W. Gao, and H. Y. Gu. 2008. Construction, application and biosafety of silver nanocrystalline chitosan wound dressing. Burns : journal of the International Society for Burn Injuries 34 (5):623-8. http://www.ncbi.nlm.nih.gov/pubmed/18226459.
Ma, C., M. A. Hernandez, V. E. Kirkpatrick, L. J. Liang, A. L. Nouvong, and Gordon, II. 2015. Topical Platelet-Derived Growth Factor vs Placebo Therapy of Diabetic Foot Ulcers Offloaded With Windowed Casts: A Randomized, Controlled Trial. Wounds : a compendium of clinical research and practice 27 (4):83-91. http://www.ncbi.nlm.nih.gov/pubmed/25855851.
Marcelo, C., M. Deveci, R. Gilmont, W. Dunham, B. Mudge, and D. Smith. 2001. Glutathione treatment accelerates diabetic wound healing. Journal of Investigative Dermatology 117 (2):420-420.
Montesinos, M. C., A. Desai, J. F. Chen, H. Yee, M. A. Schwarzschild, J. S. Fink, and B. N. Cronstein. 2002. Adenosine promotes wound healing and mediates angiogenesis in response to tissue injury via occupancy of A(2A) receptors. The American journal of pathology 160 (6):2009-18. http://www.ncbi.nlm.nih.gov/pubmed/12057906.
Moser, G. H., J. Schrader, and A. Deussen. 1989. Turnover of adenosine in plasma of human and dog blood. The American journal of physiology 256 (4 Pt 1):C799-806. http://www.ncbi.nlm.nih.gov/pubmed/2539728.
Mudge, B. P., C. Harris, R. R. Gilmont, B. S. Adamson, and R. S. Rees. 2002. Role of glutathione redox dysfunction in diabetic wounds. Wound repair and regeneration : official publication of the Wound Healing Society [and] the European Tissue Repair Society 10 (1):52-8. http://www.ncbi.nlm.nih.gov/pubmed/11983006.
Noel, S. P., H. Courtney, J. D. Bumgardner, and W. O. Haggard. 2008. Chitosan films: a potential local drug delivery system for antibiotics. Clinical Orthopaedics and Related Research 466 (6):1377-82. http://www.ncbi.nlm.nih.gov/pubmed/18421540.
Noel, S. P., H. S. Courtney, J. D. Bumgardner, and W. O. Haggard. 2010. Chitosan sponges to locally deliver amikacin and vancomycin: a pilot in vitro evaluation. Clinical Orthopaedics and Related Research 468 (8):2074-80. http://www.ncbi.nlm.nih.gov/pubmed/20352389.
Ohana, G., S. Bar-Yehuda, F. Barer, and P. Fishman. 2001. Differential effect of adenosine on tumor and normal cell growth: focus on the A3 adenosine receptor. Journal of Cellular Physiology 186 (1):19-23. http://www.ncbi.nlm.nih.gov/pubmed/11147810.
Omar, N. S., M. R. El-Nahas, and J. Gray. 2008. Novel antibiotics for the management of diabetic foot infections. International Journal of Antimicrobial Agents 31 (5):411-9. http://www.ncbi.nlm.nih.gov/pubmed/18155884.
Papanas, N., and E. Maltezos. 2008. Becaplermin gel in the treatment of diabetic neuropathic foot ulcers. Clinical Interventions in Aging 3 (2):233-40. http://www.ncbi.nlm.nih.gov/pubmed/18686746.
Parker, A. C., J. A. Jennings, J. D. Bumgardner, H. S. Courtney, E. Lindner, and W. O. Haggard. 2013. Preliminary investigation of crosslinked chitosan sponges for tailorable drug delivery and infection control. Journal of biomedical materials research. Part B, Applied biomaterials 101 (1):110-23. http://www.ncbi.nlm.nih.gov/pubmed/22997172.
Parker, A. C., J. K. Smith, B. T. Reves, J. A. Jennings, J.D. Bumgardner, and W.O. Haggard. 2014. Effects of Sodium Acetate Buffer on Chitosan Sponge Properties and In Vivo Degradation in a Rat Intramuscular Model. Journal of Biomedical Materials Research Part B-Applied Biomaterials In press.
Peplow, Philip V, and G David Baxter. 2012. Gene expression and release of growth factors during delayed wound healing: a review of studies in diabetic animals and possible combined laser phototherapy and growth factor treatment to enhance healing. Photomedicine and laser surgery 30 (11):617-636.
Porkka-Heiskanen, T., R. E. Strecker, and R. W. McCarley. 2000. Brain site-specificity of extracellular adenosine concentration changes during sleep deprivation and spontaneous sleep: An in vivo microdialysis study. Neuroscience 99 (3):507-517.
Quarles, L. D., D. A. Yohay, L. W. Lever, R. Caton, and R. J. Wenstrup. 1992. Distinct proliferative and differentiated stages of murine MC3T3-E1 cells in culture: an in vitro model of osteoblast development. Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research 7 (6):683-92. http://www.ncbi.nlm.nih.gov/pubmed/1414487.
Ramsey, S. D., K. Newton, D. Blough, D. K. McCulloch, N. Sandhu, G. E. Reiber, and E. H. Wagner. 1999. Incidence, outcomes, and cost of foot ulcers in patients with diabetes. Diabetes Care 22 (3):382-7. http://www.ncbi.nlm.nih.gov/pubmed/10097914.
Rao, N., and B. A. Lipsky. 2007. Optimising antimicrobial therapy in diabetic foot infections. Drugs 67 (2):195-214. http://www.ncbi.nlm.nih.gov/pubmed/17284084.
Rathur, H. M., and A. J. Boulton. 2007. The neuropathic diabetic foot. Nature Clinical Practice. Endocrinology and Metabolism 3 (1):14-25. http://www.ncbi.nlm.nih.gov/pubmed/17179926.
Rees, R. S., D. J. Smith, Jr., B. Adamson, M. Im, and D. Hinshaw. 1995. Oxidant stress: the role of the glutathione redox cycle in skin preconditioning. The Journal of surgical research 58 (4):395-400. http://www.ncbi.nlm.nih.gov/pubmed/7723318.
Reves, B. T., J. D. Bumgardner, and W. O. Haggard. 2013. Fabrication of crosslinked carboxymethylchitosan microspheres and their incorporation into composite scaffolds for enhanced bone regeneration. Journal of Biomedical Materials Research. Part B, Applied Biomaterials 101 (4):630-9. http://www.ncbi.nlm.nih.gov/pubmed/23359414.
Rice, G. C., E. A. Bump, D. C. Shrieve, W. Lee, and M. Kovacs. 1986. Quantitative analysis of cellular glutathione by flow cytometry utilizing monochlorobimane: some applications to radiation and drug resistance in vitro and in vivo. Cancer Research 46 (12 Pt 1):6105-10. http://www.ncbi.nlm.nih.gov/pubmed/3779630.
Roeder, B., C. C. Van Gils, and S. Maling. 2000. Antibiotic beads in the treatment of diabetic pedal osteomyelitis. The Journal of foot and ankle surgery : official publication of the American College of Foot and Ankle Surgeons 39 (2):124-30. http://www.ncbi.nlm.nih.gov/pubmed/10789104.
Shamhart, Patricia E, Daniel J Luther, Ravi K Adapala, Jennifer E Bryant, Kyle A Petersen, J Gary Meszaros, and Charles K Thodeti. 2014. Hyperglycemia enhances function and differentiation of adult rat cardiac fibroblasts 1. Canadian journal of physiology and pharmacology 92 (7):598-604.
Shearer, A., P. Scuffham, A. Gordois, and A. Oglesby. 2003. Predicted costs and outcomes from reduced vibration detection in people with diabetes in the U.S. Diabetes Care 26 (8):2305-10. http://www.ncbi.nlm.nih.gov/pubmed/12882853.
Shimegi, S. 1998. Mitogenic action of adenosine on osteoblast-like cells, MC3T3-E1. Calcified Tissue International 62 (5):418-25. http://www.ncbi.nlm.nih.gov/pubmed/9541519.
Sia, I. G., and E. F. Berbari. 2006. Infection and musculoskeletal conditions: Osteomyelitis. Best Practice and Research. Clinical Rheumatology 20 (6):1065-81. http://www.ncbi.nlm.nih.gov/pubmed/17127197.
Singh, N., D. G. Armstrong, and B. A. Lipsky. 2005. Preventing foot ulcers in patients with diabetes. JAMA : the journal of the American Medical Association 293 (2):217-28. http://www.ncbi.nlm.nih.gov/pubmed/15644549.
Smith, J. K., A. R. Moshref, J. A. Jennings, H. S. Courtney, and W. O. Haggard. 2013. Chitosan sponges for local synergistic infection therapy: a pilot study. Clinical orthopaedics and related research 471 (10):3158-64. http://www.ncbi.nlm.nih.gov/pubmed/23604649.
Ueno, H., H. Yamada, I. Tanaka, N. Kaba, M. Matsuura, M. Okumura, T. Kadosawa, and T. Fujinaga. 1999. Accelerating effects of chitosan for healing at early phase of experimental open wound in dogs. Biomaterials 20 (15):1407-14. http://www.ncbi.nlm.nih.gov/pubmed/10454012.
Valls, M. D., B. N. Cronstein, and M. C. Montesinos. 2009. Adenosine receptor agonists for promotion of dermal wound healing. Biochemical Pharmacology 77 (7):1117-24. http://www.ncbi.nlm.nih.gov/pubmed/19041853.
Veves, A., and G. L. King. 2001. Can VEGF reverse diabetic neuropathy in human subjects? The Journal of clinical investigation 107 (10):1215-8. http://www.ncbi.nlm.nih.gov/pubmed/11375408.
Victor-Vega, C., A. Desai, M. C. Montesinos, and B. N. Cronstein. 2002. Adenosine A2A receptor agonists promote more rapid wound healing than recombinant human platelet-derived growth factor (Becaplermin gel). Inflammation 26 (1):19-24. http://www.ncbi.nlm.nih.gov/pubmed/11936752.
Wu, S. C., R. T. Crews, and D. G. Armstrong. 2005. The pivotal role of offloading in the management of neuropathic foot ulceration. Current Diabetes Reports 5 (6):423-9. http://www.ncbi.nlm.nih.gov/pubmed/16316592.
Wu, S. C., V. R. Driver, J. S. Wrobel, and D. G. Armstrong. 2007. Foot ulcers in the diabetic patient, prevention and treatment. Vascular health and risk management 3 (1):65-76. http://www.ncbi.nlm.nih.gov/pubmed/17583176.
Yang, Guoguang, Richard C Crawford, and James HC Wang. 2004. Proliferation and collagen production of human patellar tendon fibroblasts in response to cyclic uniaxial stretching in serum-free conditions. Journal of biomechanics 37 (10):1543-1550.
Yilmaz, E. 2004. Chitosan: a versatile biomaterial. Advances in Experimental Medicine and Biology 553:59-68. http://www.ncbi.nlm.nih.gov/pubmed/15503447.