Doxycycline as a Modulator of Inflammation in Chronic Wounds

  Abstract: Doxycycline is a semisynthetic, chemically modified tetracycline compound that is rapidly absorbed and exerts biological effects independent of its antimicrobial activity. One such effect includes the inhibition of matrix metalloproteinases. Doxycycline has a long history as a collagenase inhibitor. This article will describe its mode of action and review its effectiveness in significantly reducing inflammation and elevated levels of proinflammatory cytokines within chronic wounds.

Introduction

  Doxycycline (DOX) is a semisynthetic, chemically modified tetracycline compound widely used to treat infections caused by both gram-negative and gram-positive microorganisms. This 4-ringed molecule was initially discovered in 1947.¹ Since then, it has been well-established that DOX is rapidly absorbed with a prolonged half-life and exerts biological effects independent of its antimicrobial activity.² One such effect includes the inhibition of matrix metalloproteinases (MMPs).^3-34 Matrix metalloproteinases are a family of zinc-dependent enzymes with the ability to degrade all components of the extracellular matrix (ECM).³⁵ Matrix metalloproteinases are produced by keratinocytes, endothelial cells, neutrophils, fibroblasts, macrophages, mast cells, and eosinophils.³⁶ There are 4 distinct subsets of enzymes that exist within the MMP family: collagenases, gelatinases, and stromelysins, plus a few membrane-bound MMPs.^37,38 Collagenases are the only enzymes in humans with the capacity to cleave the triple helix of type I, II, and III collagen, with gelatinases having the ability to cleave all other types.^38,39 Gelatinases and collagenases also have the unique ability to activate against other material within the extracellular matrix when a disruption in their balance occurs.^35,38,40,41 Inhibition of MMP activity by DOX, and other chemically modified tetracyclines (CMTs), occurs through the chelation of calcium and zinc ions.^42,43 The chelation of calcium and zinc ions allows DOX and CMTs to inhibit matrix destruction mediated by MMP activity by 3 different processes: 1) the inhibition of already active MMPs; 2) the inhibition of pro-MMP activation; and 3) the down regulation of MMP expression.^42,44 In addition, DOX and other CMTs indirectly prevent the degradation of connective tissue by protecting the host defense protein a-1 antitrypsin (a-1 PI), which inhibits leukocyte elastase. Excessive MMPs degrade and inactivate a-1 PI; the inhibition of MMPs thereby leads to higher tissue levels of a-1 PI, which reduce leukocyte elastase activity. Leukocyte elastases are responsible for the degradation and inactivation of tissue inhibitors of metalloproteinase (TIMPs). Tissue inhibitors of metalloproteinase are naturally occurring inhibitors of MMPs. Tissue inhibitors of metalloproteinase keep MMPs in balance including stromelysin (MMP-3), the only MMP that is relatively insensitive to direct inhibition by DOX.^26,44 It is important to note there is no evidence that DOX or CMTs act as TIMPs inhibitors.⁴⁵   Effect on reactive oxygen species. Free radicals are known to cause cell damage and function as inhibitory factors in the healing process.^46-48 The production of reactive oxygen species (ROS) associated with chronic wounds can originate from several potential sources.^49-52 During the healing process, various inflammatory cells — neutrophils; macrophages; fibroblasts, particularly senescent fibroblasts; and endothelial cells — are all prominent in chronic wounds, and are capable of producing ROS.^53-55 However, in chronic wounds, activated neutrophils and macrophages produce extremely large amounts of superoxides, as well as its derivatives, via the phagocytic isoform of nicotinamide adenine dinucleotide phosphate oxidases.^56-58 When polymorphonuclear neutrophils (PMNs) are recruited and activated at the wound site, they consume an increased amount of oxygen, which is converted into ROS in a process known as a “respiratory” or “oxidative” burst. This burst requires the consumption of large amounts of molecular oxygen, increasing oxygen consumption by at least 50%, and resulting in the generation of superoxide anions.⁴⁸ Most of the superoxide anions formed are converted into hydrogen peroxide,⁵⁹ which is converted into highly toxic hydroxyl radicals, creating a second source of ROS.^60-62 These highly toxic hydroxyl free radicals enhance the synthesis and activation of even more pro-MMPs and the inactivation of a-1 PI.^63-67 Doxycycline has been proven to scavenge these ROS, thereby protecting against their catabolic activities. This is partly due to its antioxidant effect on PMNs.^68,69 Doxycycline prevents the oxidative conversion of pro-MMPs in the ECM into active MMPs. This process is not dependent upon the metal-ion binding properties of DOX and other CMTs.⁷⁰   Nitric oxide (NO) is similar to ROS in that it also requires a delicate balance within the microwound environment.⁷¹ This small, gaseous free radical is involved in extracellular and intracellular neurotransmission, cell-mediated cytotoxicity, and endothelium-dependent relaxation of vascular smooth muscle.⁷² Nitric oxide is also involved in inflammatory and autoimmune-mediated tissue destruction, and is produced by the nitric oxide synthase (NOS) family of enzymes. When NO and ROS are combined, they form peroxynitrite, a potent-free radical known to cause extensive tissue destruction. Because of this, excessive amounts of NO are extremely cytotoxic to the microwound environment.⁷³ Wound infections and extended inflammation are also associated with an overproduction of NO in chronic wounds.⁷³ This over expression has been directly linked to delayed healing in both chronic venous ulcers and diabetic foot ulcers.^74,75 It is important to note that an under expression of NO has also been connected to delayed healing in diabetic foot ulcers.⁷⁵ Decreased levels of NO in diabetic foot ulcers may be correctible with L-arginine, a semiessential amino acid,⁷⁵ by using dietary supplements.^76,77 Doxycycline reduces cytokine-induced NO production by inhibiting the expression of NOS through reducing inducible NOS levels.^78-81   Another potential indirect mechanism in which DOX and other CMTs inhibit the breakdown of the ECM is through the inhibition of the proinflammatory cytokines including tumor necrosis factor (TNF-a) and interleukin-1b (IL-1b).^82-84 Chronic wound fluid has been found to contain up to a 100-fold increase in the levels of these cytokines.^85-90 Proinflammatory cytokines synthesize MMPs and suppress TIMPs,^85,87 resulting in an imbalance of the microwound environment with degradation of collagen, growth factors, and growth factor receptor sites, as well as other vital components of the ECM.^{65,84,87-88,91,92} Once growth factors are degraded, communication between the various cells participating in healing stops, and healing is delayed.⁹³ As this delay continues, the proinflammatory cytokine cascade is amplified, and wound fluid becomes absent of DNA synthesis, ultimately resulting in senescent or mitotically incompetent cells.^{52, 93,94}   Synergistic effect between doxycycline/chemically modified tetracyclines and nonsteroidal anti-inflammatory drugs. Many researchers have reported a synergistic effect between DOX/CMTs and nonsteroidal anti-inflammatory drugs (NSAIDs) in different animal and human models of disease, even though they work through different pathways. For example, in the arthritic joint, oral tetracycline uptake is increased by as much as 150% without affecting blood levels of the drug. This phenomenon facilitates the uptake into therapeutic target areas. The combination of these 2 therapies (NSAIDs and tetracycline) has significantly decreased tissue destruction and inflammation when used in rheumatoid arthritis and osteoarthritis.^95-100 Although NSAIDs do not directly affect MMP functions, researchers have found that they do potentiate the anticollagenolytic/proteolytic potential of tetracyclines by reducing edema, thereby facilitating the entry of tetracyclines into sites of inflammation.⁹⁵ There are a number of studies demonstrating the benefit of DOX and/or CMTs in the treatment of chronic wounds.^68,101-105 This synergistic effect may also be beneficial when combined with hyperbaric medicine in the treatment of a number of different wound types. For example, it may benefit patients with radiation proctitis and/or cystitis who are undergoing hyperbaric oxygen therapy for bleeding. These patients may also gain from the synergistic effect of combining DOX/CMT and NSAIDs to their treatment, as there is a significant inflammatory process involved in these radiation injuries, characterized by elevated production of MMPs in the tissue.¹⁰⁶   Pain is a frequent symptom associated with chronic wounds. Doxycycline and CMTs have a demonstrated ability to reduce pain associated with inflammation.¹⁰⁵ Evidence supports the fact that the underlying cause of chronic wound pain is, in part, related to inflammation.^106-108 A large prospective study of 758 patients found that the more chronic the duration of increased inflammation, the higher the reported wound pain (P = 0.022).¹⁰⁹ As chronic wounds begin to heal, wound pain is reduced further, suggesting a link between prolonged inflammation and pain pathways.^106-109 In 2009, a team of researchers demonstrated that ibuprofen can down-regulate proinflammatory cytokines when applied topically.¹¹⁰ This finding has led to the commercialization of ibuprofen-impregnated foam dressings for wound analgesia.^111-116 The combination therapy of DOX/CMTs with NSAIDs, which both work on different pathways, may be of value in reducing chronic wound pain as well.   The presence of hypergranulation tissue in chronic wounds is believed to result from an extended inflammatory response.^117,118 Excessive amounts of transforming growth factor-b (TGF-b) accelerate the healing response by increasing collagen deposition.^53,84,119 Few articles have been published on this subject, and the majority of those published relate to the care of equines. The predisposing factors for hypergranulation tissue formation in horses include hypoxia, infection, and trauma/pressure combined with a prolonged inflammatory reaction.^120-123 The focus of this research has been on TGF-b, which is generally acknowledged to be a cytokine with the ability to retard or accelerate granulation tissue formation.^53,124 A prolonged inflammatory phase with amplified proinflammatory cytokine leads to elevated protease activity and impaired growth factor functions, which may account for the presence of hypergranulation tissue in chronic wounds. It is interesting that TGF-b has also been identified as the key factor in the development of hypertrophic scars and keloids in humans, because both hypergranulation tissue and hypertrophic scars and keloids are associated with a prolonged inflammatory response that ends with an overproduction of connective tissue.^{124,12553,84,124,125} it may therefore decrease or inhibit the development of hypergranulation tissue in chronic wounds, and may decrease or improve hypertrophic scarring and keloid formation.   Doxycycline and chemically-modified tetracyclines as antiresorptive drugs. Doxycycline and CMT have been found to inhibit osteoclast-mediated bone resorption by inhibiting osteoclastic action and inducing apoptosis of osteoclasts.^126-131 Simultaneously, DOX and CMT enhance osteoblastic activity.^132-134 This is not surprising due to the fact that collagen is the major component of bone and represents at least 90% of its organic matrix.¹³⁵ The final process of bone remodeling and resorption is controlled by MMPs and TIMPs.136 Doxycycline and CMT are bone-seeking agents that accumulate in high concentrations in bone and act, similarly to bisphosphonates, as antiresorptive drugs.¹²⁷ Long-term use of bisphosphonates is associated with enhanced bone resorption.^137,138 When DOX/CMTs are combined with bisphosphonates, they increase the amount of bone by suppressing resorption,^127,139-141 while DOX/CMTs significantly increase the number of active osteoblastic cells.¹⁴²   The negative impact of reperfusion injury in the healing cascade is partly due to the inflammatory response of damaged tissues. Leukocytes, particularly PMNs, carried to the area upon reperfusion release a host of inflammatory factors, such as interleukins and free radicals, in response to tissue injury.^144-147 Changes in endothelial cells occurring during ischemia promote PMN binding to these cells, priming PMN synthesis of oxygen radicals and release of cytotoxic proteins which lead to vascular damage and tissue injury.^143-150 Doxycycline inhibits PMN superoxide synthesis and degranulation, and suppresses PMN-mediated red blood cells, fibroblast, and endothelial cytotoxicity, thereby providing protection from ischemia-reperfusion induced injuries.^{64,103,151-162}   Doxycycline and CMTs as modulating agents of MMPs must be used with care. Simply inhibiting MMPs in chronic wounds is not an appropriate strategy^163,164 as MMP activity is important for cytokines and chemokine production to attract cells into wound areas.^165-167 To control MMPs with anti-inflammatory drugs, they must be used during the correct phase of healing. The use of these drugs in the acute wound healing phase has been associated with a delay in healing in select clinical scenarios.⁸⁵   Although no published protocols on the dosing of DOX or CMTs in chronic wounds could be found, the majority of the articles referenced cite a subantimicrobial dose. One such drug (Periostat), administered as a 20 mg dose of DOX twice daily, is the only drug therapy approved by the United States Food and Drug Administration (FDA) for adult periodontitis as a collagenase inhibitor. A second drug (Oracea) is FDA-approved for the treatment of rosacea through its anti-inflammatory/anticollagenolytic effect. The latter is a sustained-release, 40 mg DOX tablet, with 10 mg of the 40 mg coated to delay release. Using these drugs for chronic wounds may require a loading dose to ensure there are no bacteria sensitive to the drug before starting any subantimicrobial dose. Long-term use of subantimicrobial levels of DOX has not caused resistance to the drug.^168,169

Conclusion

  Chronic wound healing requires a balance between tissue proteases and their inhibitors.^37,85,163 Doxycycline and chemically modified tetracyclines significantly reduce inflammation and elevated levels of proinflammatory cytokines, which degrade the ECM, and stimulate increased numbers of inflammatory cytokines.⁸⁵ When combined with NSAIDs, the synergistic effect may be of value in the healing of chronic wounds and reducing associated pain.^37,85 Doxycycline and CMT may also provide significant value when used in conjunction with hyperbaric medicine in diseases ranging from arterial gas embolism, to failing flaps and grafts, to reperfusion injuries. Because of the effect of DOX on reperfusion injuries, it could prove valuable in the treatment of early stage 1 pressure ulcers, diabetic wounds of the lower extremity, and early onset osteomyelitis found in chronic wounds. Evidence suggests DOX and/or CMT could improve outcomes in patients with diabetic foot ulcers, thereby reducing the risk of amputation. Furthermore, DOX and CMTs may improve the success of bioengineered tissue grafts when the microwound environment of a diabetic foot ulcer or venous ulcer is in balance, and hypoxia, edema, and infection have been corrected.   Doxycycline and CMTs have demonstrated a positive impact in the microwound environment through a variety of cellular and humeral activities. Further studies in specific disease subsets are needed to better define the precise role of this agent in the healing armamentarium.

References

1. Nelson ML. The chemistry and cellular biology of the tetracyclines. In Nelson M, Hillen W, Greenwald RA, eds. Tetracyclines in Biology, Chemistry and Medicine. Boston: Birkhauser-Verlag;2001;3-63. 2. Golub LM, Ramamurthy NS, McNamara TF, Greenwald RA, Rifkin BR. Tetracyclines inhibit connective tissue breakdown: new therapeutic implications for an old family of drugs. Crit Rev Oral Biol Med. 1991;2(3):297-321. 3. Greenwald RA. Treatment of destructive arthritic disorders with MMP inhibitors. Potential role for tetracyclines. Ann N Y Acad Sci. 1994;732:181-198. 4. Uitto VJ, Firth JD, Nip L, Golub LM. Doxycycline and chemically modified tetracyclines inhibit gelatinase A (MMP-2) gene expression in human skin keratinocytes. Ann N Y Acad Sci. 1994;732:140-151. 5. Konttinen YT, Kangaspunta P, Lindy O, et al. Collagenase in Sjogren’s syndrome. Ann Rheum Dis. 1994;53(12):836-839. 6. Cakir Y, Hahn KA. Direct action by doxycycline against canine osteosarcoma cell proliferation and collagenase (MMP-1) activity in vitro. In Vivo. 1999;13(4):327-332. 7. Suomalainen K, Sorsa T, Golub LM, et al. Specificity of the anticollagenase action of tetracyclines: relevance to their anti-inflammatory potential. Antimicrob Agents Chemother. 1992;36(1):227-229. 8. Sorsa T, Ingman T, Suomalainen K, et al. Cellular source and tetracycline-inhibition of gingival crevicular fluid collagenase of patients with labile diabetes mellitus. J Clin Periodontol. 1992;19(2):146-149. 9. Suomalainen K, Sorsa T, Ingman T, Lindy O, Golub LM. Tetracycline inhibition identifies the cellular origin of interstitial collagenases in human periodontal disease in vivo. Oral Microbiol Immunol. 1992;7(2):121-123. 10. Sorsa T, Ding Y, Salo T. et al. Effects of tetracyclines on neutrophil, gingival, and salivary collagenase. A functional and western-blot assessment with special reference to their cellular sources in periodontal diseases. Ann N Y Acad Sci. 1994;732:112-131. 11. Smith GN, Brandt KD, Hasty KA. Procollagenase is reduced to inactive fragments upon activation in the presence of doxycycline. Ann N Y Acad Sci. 1994;732: 436-438. 12. Sorsa T, Ding YL, Ingman T, et al. Cellular source, activation and inhibition of dental plaque collagenase. J Clin Periodontol. 1995;22(9):709-717. 13. Golub LM, Sorsa T, Lee HM, et al. Doxycycline inhibits neutrophil (PMN)-type matrix metalloproteinases in human adult periodontitis gingiva. J Clin Periodontol. 1995;22(2):100-109. 14. Smith GN Jr, Brandt KD, Hasty KA. Activation of recombinant human neutrophil procollagenase in the presence of doxycycline results in fragmentation of the enzyme and loss of enzyme activity. Arthritis Rheum. 1996;39(2):235-244. 15. Smith GN, Brandt KD, Mickler EA, Hasty KA. Inhibition of recombinant human neutrophil collagenase by doxycycline is pH dependent. J Rheumatol.1997;24(9):1769-1773. 16. Hanemaaijer R, Sorsa T, Konttinen Y, et al. Matrix metalloproteinase-8 is expressed in rheumatoid synovial fibroblasts and endothelial cells. Regulation by tumor necrosis factor-alpha and doxycycline. J Biol Chem. 1997;272(50):31504-31509. 17. Shlopov BV, Smith GN, Cole AA, Hasty KA. Differential patterns of response to doxycycline and transforming growth factor beta1 in the down-regulation of collagenases in ostearthritic and normal human chondrocytes. Arthritis Rheum. 1999;42:719-727. 18. Smith GN Jr, Mickler EA, Hasty KA, Brandt, KD. Specificity of inhibition of matrix metalloproteinase activity by doxycycline: relationship to structure of the enzyme. Arthritis Rheum. 1999;42(6):1140-1146. 19. Greenwald RA, Golub LM, Ramamurthy NS, Chowdhury M, Moak SA, Sorsa T. In vitro sensitivity of three mammalian collagenases to tetracycline inhibition: relationship to bone and cartilage degradation. Bone. 1998;22(1):33-38. 20. Sepper R, Prikk K, Tervahartiala T, et al. Collagenase-2 and -3 are inhibited by doxycycline in the chronically inflamed lung in bronchiectasis. Ann N Y Acad Sci. 1999;878:683-685. 21. Yu LP Jr, Smith GN, Hasty KA, Brandt KD. Doxycycline inhibits type XI collagenolytic activity of extracts from human osteoarthritic cartilage and gelatinase. J Rheumatol. 1991;18(10):1450-1452. 22. Nip LH, Uitto VJ, Golub LM. Inhibition of epithelial cell matrix metalloproteinases by tetracyclines. J Periodontal Res. 1993;28(5):379-385. 23. Hurewitz AN, Wu CL, Mancurso R, Zucker S. Tetracycline and doxycycline inhibit pleural fluid metalloproteinases. A possible mechanism for chemical pleurodesis. Chest. 1993;103(4):1113-1117. 24. Burris TS, Hugh K, Orth MW, Kuettner KE, Cole AA. The effects of doxycycline on gelatinase A and B in human cartilage explants cultures metatarsals. Transactions of the Annual Meeting of the Orthopedic Research Society. 1995; 20: 335 25. Duivenvoorden W, Hirte HW, Singh G. Use of tetracycline as an inhibitor of matrix metalloproteinase activity secreted by human bone-metastasizing cancer cells. Invasion Metastasis. 1997;17(6):312-322. 26. Jonat C, Chung FZ, Baragi VM. Transcriptional downregulation of stromelysin by tetracycline. J Cell Biochem. 1996;60(3):341-347. 27. Golub LM, McNamara TF, D’Angelo G, Greenwald RA, Ramamurthy NS. A non-antibacterial chemically modified tetracycline inhibits mammalian collagenase activity. J Dent Res. 1987;66(8):1310-1314. 28. Sorsa T, Ramamurthy NS, Vernillo T, et al. Functional sites of chemically modified tetracyclines: inhibition of the oxidative activation of human neutrophil and chicken osteoclast pro-matrix metalloproteinases. J Rheumatol. 1998;25(5):975-982. 29. Ramamurthy NS, Vernillo T, Golub LM, Rifkin BR. The effect of tetracyclines on collagenase activity in UMR 106-01 rat osteoblastic osteosarcoma cells. Res Commun Chem Pathol Pharmacol. 1990;70(3):323-335. 30. Ramamurthy NS, Vernillo AT, Greenwald RA, et al. Reactive oxygen species activate and tetracyclines inhibit rat osteoblast collagenase. J Bone Miner Res. 1993;8(10):1247-1253. 31. Zucker S, Wieman J, Lysik RM, et al. Gelatin-degrading type IV collagenase isolated from human small cell lung cancer. Invasion Metastasis. 1989;9(3):167-181. 32. Paemen L, Martens E, Norga K, et al. The gelatinase inhibitory activity of tetracyclines and chemically modified tetracycline analogues as measured by a novel microtiter assay for inhibitors. Biochem Pharmacol. 1996;52(1):105-111. 33. Seftor RE, Seftor EA, De Larco, JE, et al. Chemically modified tetracyclines inhibit human melanoma cell invasion and metastasis. Clin Exp Metastasis. 1998;16(3):217-225. 34. Maisi P, Kiili M, Raulo SM, Pirila E, Sorsa T. MMP inhibition by chemically modified tetracycline-3 (CMT-3) in equine pulmonary epithelial lining fluid. Ann N Y Acad Sci. 1999;878:675-677. 35. Woessner, JF The family of matrix metalloproteinases. Ann N Y Acad Sci. 1994;732:11-21. 36. Lobmann E, Ambrosch A, Schultz G, Waldmann K, Schiweck S, Lehnert H. Expression of matrix-metalloproteinases and their inhibitors in the wounds of diabetic and non-diabetic patients. Diabetologia. 2002;45(7):1011-1016. 37. Armstrong DG, Jude EB. The role of matrix metalloproteinases in wound healing. J Am Podiatr Med Assoc. 2002;92(1):12-18. 38. Mignatti P, Rifkin DB, Welgus HG, Parks WC. Proteinases and tissue remodeling. In: Clark, RAF, ed. The Molecular and Cellular Biology of Wound Repair. 2nd ed. New York: Plenum Press. 1996:427-474. 39. Krane SM. Clinical importance of metalloproteinases and their inhibitors. Ann N Y Acad Sci. 1994;732:1-10. 40. Murphy G, Willenbrock F, Crabbe T. et al. Regulation of matrix metalloproteinase activity. Ann N Y Acad Sci. 1994;732:31-41. 41. Smith AP. The roll of MMPs in chronic wound edema. Podiatry Today. 2003;16(8):22-26. 42. Smith GN, Hasty KA. Structure/function studies of doxycycline effects on matrix metalloproteinase activity and cartilage degeneration. In: Nelson M, Hillen W, Greenwald RA, eds. Tetracyclines in Biology, Chemistry and Medicine. Boston: Birkhauser-Verlag; 2001;283-293. 43. Ryan ME, Usman A, Ramamurthy NS, Golub LM, Greenwald RA. Excessive matrix metalloproteinase activity in diabetics: inhibition by tetracycline analogues with zinc reactivity. Curr Med Chem. 2001;8(3):305-316. 44. Ryan ME, Ashley RA. How do tetracyclines work? Adv Dent Res. 1998;12:149-151. 45. Hanemaaijer R, Visser H, Koolwijk P, et al. Inhibition of MMP synthesis by doxycycline and chemically modified tetracyclines (CMTs) in human endothelial cells. Adv Dent Res. 1998;12(2):114-118. 46. Latha B, Babu M. The involvement of free radicals in burn injury: a review. Burns. 2001;27(4):309-317. 47. Thang PT, Patrick S, Teik LS, Yung CS. Anti-oxidant effects of the extracts from the leaves of chromolaena odorata on human dermal fibroblasts and epidermal keratinocytes against hydrogen peroxide and hypoxanthine-xanthine oxidase induced damage. Burns. 2001;27(4):319-327. 48. Rabkin JM, Hunt TK. Infection and oxygen. In: Davis JC, Hunt TK, eds. Problem Wounds: The Role of Oxygen. New York: Elsevier; 1988;1-16. 49. Widgerow AD. Deconstructing the chronic wound. Wound Healing South Africa. 2009;2(1):9-11. 50. Wenk J, Foitzik A, Achterberg V, et al. Selective pick-up of increased iron by deferoxamine-coupled cellulose abrogates the iron-driven induction of matrix-degrading metalloproteinase 1 and lipid peroxidation in human dermal fibroblasts in vitro: a new dressing concept. J Invest Dermatol. 2001;116(6):833-839. 51. van den Berg AJ, Halkes SB, van Ufford HC, Hoekstra MJ, Beukelman CJ. A novel formulation of metal ions and citric acid reduces reactive oxygen species in vitro. J Wound Care. 2003;12(10):413-418. 52. Soneja A, Drews M, Malinski T. Role of nitric oxide, nitroxidative and oxidative stress in wound healing. Pharmacol Rep. 2005;57(suppl):108-119. 53. Eming SA, Krieg T, Davidson JM. Inflammation in wound repair: molecular and cellular mechanisms. J Invest Dermatol. 2007;127(3):514-525. 54. Campisi J. Replicative senescence: an old lives’ tale? Cell. 1996;84(4):497-500. 55. Mendez MV, Stanley A, Park HY, Shon K, Phillips T, Menzoian JO. Fibroblasts cultured from venous ulcers display cellular characteristics of senescence. J Vasc Surg. 1998;289(5):876-883. 56. Babior BM, Curnutte JT, McMurrich BJ. The particulate superoxide-forming system from human neutrophils. Properties of the system and further evidence supporting its participation in the respiratory burst. J Clin Invest. 1976;58(4):989-996. 57. Babior BM. Oxygen-dependent microbial killing by phagocytes (first of two parts). N Eng J Med. 1978;298(12):659-668. 58. Kalinowski L, Malinski T. Endothelial NADH/NADPH-dependent enzymatic sources of superoxide production: relationship to endothelial dysfunction. Acta Biochim Pol. 2004;51(2):459-469. 59. Hampton MB, Kettle AJ, Winterbourn CC. Inside the neutrophil phagosome: oxidants, myeloperoxidase, and bacterial killing. Blood. 1998;92(9):3007-3017. 60. Samuni A, Black CDV, Krishna CM, Malech HL, Bernstein EF, Russo A. Hydroxyl radical production by stimulated neutrophils reappraised. J Biol Chem. 1988;263(27):13797-13801. 61. Harrison JE, Shultz J. Studies on the chlorinating activity of myeloperoxidase. J Biol Chem. 1976;251(5):1371-1374. 62. Ackerman Z, Seidenbaum M, Loewenthal E, Rubinow A. Overload of iron in the skin of patients with varicose ulcers. Possible contributing role of iron accumulation in progression of the disease. Arch Dermatol. 1988;124(9):1376-1378. 63. Saarialho-Kere UK. Patterns of matrix metalloproteinase and TIMP expression in chronic ulcers. Arch Dermatol Res. 1998;290(suppl):S47-S54. 64. Weckroth M, Vaheri A, Luharanta J, Sorsa T, Konttinen YT. Matrix metalloproteinase, gelatinase and collagenase, in chronic leg ulcers. J Invest Dermatol. 1996;106(5):1119-1124. 65. Wysocki AB, Staiano-Coico L, Grinnell F. Wound fluid from chronic leg ulcers contains elevated levels of metalloproteinases MMP-2 and MMP-9. J Invest Dermatol. 1993;101(1):64-68. 66. Yeoh-Ellerton S, Stacey MC. Iron and 8-isoprostance levels in acute and chronic wounds. J Invest Dermatol. 2003;121(4):918-925. 67. Allhorn M, Lundqvist K, Schmidtchen A, Akerström B. Heme-scavenging role of alpha1-microglobulin in chronic ulcers. J Invest Dermatol. 2003;121(3):640-646. 68. Miyachi Y, Yoshioka A, Imamura S, Niwa Y. Effects of antibiotics on the generation of reactive oxygen species. J Invest Dermatol. 1986;86(4):449-453. 69. Akamatsu H, Asada M, Komura J, Asada Y, Niwa Y. Effects of doxycycline on the generation of reactive oxygen species: a possible mechanism of action of acne therapy with doxycycline. Acta Derm Venereol. 1992;72(3):178-179. 70. Ryan ME, Baker DM. Tetracycline treatment of periodontal disease: antimicrobial and non-antimicrobial mechanisms. In: Nelson M, Hillen W, Greenwald RA, eds. Tetracyclines in Biology, Chemistry and Medicine. Basel, Switzerland: Birkhauser Verlag; 2001: 237-265. 71. Schulz G, Stechmiller J. Wound healing and nitric oxide production: too little or too much may impair healing and cause chronic wounds. Int J Low Extrem Wounds. 2006;5(1):6-8. 72. Jude EB, Boulton AJ, Ferguson MW, Appleton I. The role of nitric oxide synthase isoforms and arginase in the pathogenesis of diabetic foot ulcers: possible modulatory effects by transforming growth factor beta 1. Diabetologia. 1999;42(6):748-757. 73. Moseley R, Stewart JE, Stephens P, Waddington RJ, Thomas DW. Extracellular matrix metabolites as potential biomarkers of disease activity in wound fluid: lessons learned from other inflammatory diseases? Br J Dermatol. 2004;150(3):401-413. 74. Abd-El-Aleem SA, Ferguson MW, Appleton I, et al. Expression of nitric oxide synthase isoforms and arginase in normal human skin and chronic venous leg ulcers. J Pathol. 2000;191(4):434-442. 75. Boykin JV Jr. The nitric oxide connection: hyperbaric oxygen therapy, becaplermin, and diabetic ulcer management. Adv Skin Wound Care. 2000;13(4 Pt 1):169-174. 76. Barbul A, Lazarou SA, Efon DT, Wasserkrug HL, Efron G. Arginine enhances wound healing and lymphocyte immune responses in humans. Surgery. 1990;108(2):331-336. 77. Kirk SJ, Hurson M, Regan MC, Holt DR, Wasserkrug HL, Barbul A. Arginine stimulates wound healing and immune function in elderly human beings. Surgery. 1993;114(2):155-159. 78. Kharitionov SA, Barnes PJ. Exhaled markers of inflammation. Curr Opin Allergy Clin Immunol. 2001;1(3):217-224. 79. Amin AR, Attur MG, Thakker GD, et al. A novel mechanism of action of tetracyclines: effects on nitric oxide synthases. Proc Natl Acad Sci U S A. 1996;93(24):14014-14019. 80. Hoyt JC, Ballering J, Numanami H, Hayden JM, Robbins RA. Doxycycline modulates nitric oxide production in murine lung epithelial cells. J Immunol. 2006;176(1):567-572. 81. Attur MG, Patel RN, Patel PD, Abramson SB, Amin AR. Tetracycline up-regulates COX-2 expression and prostaglandin E2 production independent of its effects on nitric oxide. J Immunol. 1999;162(6):3160-3167. 82. Golub LM, Lee HM, Ryan ME, Giannobile WV, Payne J, Sorsa T. Tetracyclines inhibit connective tissue breakdown by multiple non-antimicrobial mechanisms. Adv Dent Res. 1998;12(2):12-26. 83. Eady EA, Ingham E, Walters CE, Cove JH, Cunliffe WJ. Modulation of comedonal levels of interleukin-1 in acne patients treated with tetracyclines. J Invest Dermatol. 1993;101(1):86-91. 84. Krakauer T, Buckley M. Doxycycline is anti-inflammatory and inhibits staphylococcal endotoxin-induced cytokines and chemokines. Antimicrob Agents Chemother. 2003;47(11):3630-3633. 85. Mast BE, Schultz GS. Interactions of cytokines, growth factors, and proteases in acute and chronic wound. Wound Repair Regen. 1996;4(4):411-420. 86. Schultz G, Mast BA. Molecular analysis of the environments of healing and chronic wounds: cytokines, proteases, and growth factors. WOUNDS. 1998;10:(Suppl F):1F. 87. Tranuzzer R. et al. Epidermal growth factor in wound healing: a model for the molecular pathogenesis of chronic wounds. In: Ziegler T, Pierce G, Herndon D, eds. Growth Factors and Wound Healing: Basic Science and Potential Clinical Applications. New York: Springer; 1997:206-228. 88. Tarnuzzer RW, Schultz GS. Biochemical analysis of acute and chronic wound environments. Wound Repair Regen. 1996;4(3):321-325. 89. Yager DR, Nwomeh BC. The proteolytic environment of chronic wounds. Wound Repair Regen. 1999;7(6):433-441. 90. Trengove NJ, Langton SR, Stacey MC. Biochemical analysis of wound fluid from nonhealing and chronic leg ulcers. Wound Repair Regen. 1996;4(2):234-239. 91. Vaalamo M, Weckroth M, Puolakkainen P, et al. Patterns of matrix metalloproteinase and TIMP-1 expression in chronic and normally healing human cutaneous wounds. Br J Dermatol. 1996;135(1):52-59. 92. Yager DR, Zhang LY, Liang HX, Diegelmann RF, Cohen IK. Wound fluids from human pressure ulcers contain elevated matrix metalloproteinase levels and activity compared to surgical wound fluids. J Invest Dermatol. 1996;107(5):743-748. 93. Ovington L. Overview of Matrix Metalloprotease Modulation and Growth Factor Protection in Wound Healing. Ostomy Wound Manage. 48,6supp:3-7, 2002. 94. Telgenhoff D, Shroot B. Cellular senescence mechanisms in chronic wound healing. Cell Death Differ. 2005;12(7):695-698. 95. Lauhio A, Salo T, Ding Y, et al. In vivo inhibition of human neutrophil collagenase (MMP-8) activity during long-term combination therapy of doxycycline and non-steroidal anti-inflammatory drugs (NSAID) in acute reactive arthritis. Clin Exp Immunol. 1994;98(1):21-28. 96. Greenwald RA, Moak SA, Ramamurthy NS, Golub LM. Tetracyclines suppress matrix metalloproteinase activity in adjuvant arthritis and in combination with flurbiprofen, ameliorate bone damage. J Rheumatol. 1992:19(6):927-938. 97. Breedveld FC, Dijkmans BA, Mattie H. Minocycline treatment for rheumatoid arthritis: an open dose finding study. J Rheumatol. 1990;17(1):43-46. 98. Lauhio A, Leirisalo-Repo M, Lahdevirta J, Saikku P, Repo H. Double-blind, placebo-controlled study of three-month treatment with lymecycline in reactive arthritis, with special reference to Chlamydia arthritis. Arthritis Rheum. 1991;34(1):6-14. 99. Lee HM, Ciancio SG, Tuter G, Ryan ME, Komaroff E, Golub LM. Subantimicrobial dose doxycycline efficacy as a matrix metalloproteinase inhibitor in chronic periodontitis patients is enhanced when combined with a non-steroidal anti-inflammatory drug. J Periodontol. 2004;75(3):453-463. 100. Ramamurthy N, Leung S, Moak S, Greenwald RA, Golub L. CMT/NSAID combination increases bone CMT uptake and inhibits bone resorption. Ann N Y Acad Sci. 1993;696:420-421. 101. Ramamurthy NS, Kucine AJ, McClain SA, McNamara TF, Golub, LM. Topically applied CMT-2 enhances wound healing in streptozotocin diabetic rat skin. Adv Dent Res. 1998; 12(1): 144-148. 102. Ramamurthy NS, McClain SA, Pirila E, et al. Wound healing in aged and normal ovariectomized rats: effects of chemically modified tetracyclines doxycycline (CMT-8)on MMP expression and collagen synthesis. Ann N Y Acad Sci. 1999;878:720-723. 103. Hobeika MJ, Thompson RW, Muhs BE. Brooks PC, Gagne PJ. Matrix metalloproteinases in peripheral vascular disease. J Vasc Surg. 2007;45(4):849-857. 104. Stechmiller J, Cowan L, Schultz G. The role of doxycycline as a matrix metalloproteinase inhibitor for the treatment of chronic wounds. Biol Res Nurs. 2010;11(4):336-344. 105. Mackelfresh J, Soon S, Arbiser JL. Combination therapy of doxycycline and topical tacrollmus for venous ulcers. Arch Dermatol. 2005;141(11):1476-1477. 106. European Wound Management Association. Position document: Pain at wound dressing changes. Medical Education Partnerships LTD. https://ewma.org/fileadmin/user_upload/EWMA/pdf/Position_Documents/2002/Spring_2002__English_.pdf Published 2002. Accessed November 15, 2012. 107. World Union of Wound Healing Societies. Principles of Best Practice: Minimising pain at wound dressing-related procedures. A consensus document. Medical Education Partnerships LTD. https://www.woundsinternational.com/pdf/content_39.pdf Published 2004. Accessed November 15, 2012. 108. Krasner D. Pain management. In: Falabella AF, Kirsner RS, eds. Wound Healing. Boca Raton: Taylor & Francis; 2005. 109. Franks PJ, Moffatt CJ. Do clinical and social factors predict quality of life in leg ulceration? Int J Low Extrem Wounds. 2006;5(4):236-243. 110. Redpath M, Marques CM, Dibden C, Waddon A, Lalla R, Macneil S. Ibuprofen and hydrogel-released ibuprofen in the reduction of inflammation-induced migration in melanoma cells. Br J Dermatol. 2009;161(1):25-33. 111. Sibbald RG, Coutts P, Fierheller M, Woo K. A pilot (real-life) randomised clinical evaluation of a pain-relieving foam dressing: (ibuprofen-foam versus local best practice). Int Wound J. 2007;4(suppl 1):16-23. 112. Gottrup F, Jørgensen B, Karlsmark T, et al. Less pain with Biatain-Ibu: initial findings from a randomised, controlled, double-blind clinical investigation on painful venous leg ulcers. Int Wound J. 2007;4(suppl 1):24-34. 113. Jørgensen B, Friis G, Gottrup F. Pain and quality of life for patients with venous leg ulcers: proof of concept of the efficacy of Biatain-Ibu, a new pain reducing wound dressing. Wound Repair Regen. 2006;14(3):233-239 114. Flanagan M, Vogensen H, Haase L. Case series investigating the experience of pain in patients with chronic venous leg ulcers treated with a foam dressing releasing ibuprofen. World Wide Wounds. 2006. https://www.worldwidewounds.com/2006/april/Flanagan/Ibuprofen-Foam-Dressing.html Published April 2006. Accessed November 15, 2012. 115. Gad P, Shewale S, Drewes AM, Arendt-Nielsen, L. Effect of a local ibuprofen dressing on healing of experimentally induced laser wounds. Poster resented at: 16th Conference of the European Wound Management Association, EWMA; May 18-20, 2006; Prague, Czech Republic. 116. Romanelli M, Dini V, Polignano R, Bonadeo P, Maggio G. Ibuprofen slow-release foam dressing reduces wound pain in painful exuding wounds: preliminary findings from an international real-life study. J Dermatolog Treat. 2009;20(1):19-26. 117. Sato K, Komatsu N, Higashi N, Imai Y, Irimura T. Granulation tissue formation by nonspecific inflammatory agent occurs independently of macrophage galactose-type C-type lectin-1. Clin Immunol. 2005;115(1):47-50. 118. Shekhter AB, Berchenko GN, Nikolaev AV. [Granulation tissue: inflammation and regeneration]. Arkh Patol. 1984;46(2):20-29. 119. Uitto J, Mauviel A, McGrath J. The dermal-epidermal basement membrane zone in cutaneous wound healing. In: Clark RAF, ed. The Molecular and Cellular Biology of Wound Repair. 2nd ed. New York, NY: Plenum Press; 1988:513-547. 120. Berry DB 2nd, Sullins KE. Effects of topical application of antimicrobials and bandaging on healing and granulation tissue formation in wounds of the distal aspect of the limbs in horses. Am J Vet Res. 2003;64(1):88-92. 121. Theoret CL, Barber SM, Moyana TN, Gordon JR. Preliminary observations on expression of transforming growth factors beta 1 and beta 3 in equine full-thickness skin wounds healing normally or with exuberant granulation tissue. Vet Surg. 2002;31(3):266-273. 122. De Martin I, Theoret CL. Spatial and temporal expression of types I and II receptors for transforming growth factor beta in normal equine skin and dermal wounds. Vet Surg. 2004;33(1):70-76. 123. Roberts AB, Sporn MB. Transforming growth factor-β. In: Clark RAF, ed. The Molecular and Cellular Biology of Wound Repair. 2nd ed. New York, NY: Plenum Press; 1988:275-298. 124. Rahban SR, Garner WL. Fibroproliferative scars. Clin Plast Surg. 2003;30(1):77-89. 125. Robson MC. Cytokine manipulation of the wound. Clin Plast Surg. 2003;30(1):57-65. 126. Holmes SG, Still K, Buttle DJ, Bishop NJ, Grabowski PS. Chemically modified tetracyclines act through multiple mechanisms directly on osteoclast precursors. Bone. 2004;35(2):471-478. 127. Vernillo AT, Rifkin BR. Effects of tetracyclines on bone metabolism. Adv Dent Res. 1998;12(2):56-62. 128. Bettany JT, Wolowacz RG, Peet NM, Skerry TM, Grabowski PS. Tetracyclines induce apoptosis in osteoclasts. Bone. 2000(1):75-80. 129. Gomes BC, Golub LM, Ramamurthy NS. Tetracyclines inhibit parathyroid hormone-induced bone resorption in organ culture. Experientia. 1984;40(11):12273-1275. 130. Grevstad HJ, Bøe OE. Effects of doxycycline on surgically induced osteoclast recruitment in the rat. Eur J Oral Sci. 1995;103(3):156-159. 131. Bezerra MM, Brito GA, Ribeiro RA, Rocha FA. Low-dose doxycycline prevents inflammatory bone resorption in rats. Braz J Med Biol Res. 2002;35(5):613-616. 132. Sasaki T, Ramamurthy NS, Golub LM. Insulin-deficient diabetes impairs osteoblast and periodontal ligament fibroblast metabolism but does not affect ameloblasts and odontoblasts; response to tetracycline(s) administration. J Biol Buccale. 1990;18(3):215-226 133. Sasaki T, Kaneko H, Ramamurthy NS, Golub LM. Tetracycline administration restores osteoblast structure and function during experimental diabetes. Anat Rec. 1991;231(1):25-34. 134. Sasaki T, Ramamurthy NS, Golub LM. Tetracycline administration increases collagen synthesis in osteoblasts of streptozotocin-induced diabetic rats: a quantitative autoradiographic study. Calcif Tissue Int. 1992;50(5):411-419. 135. Exteracellular matrix biochemistry. Piez KA, Reddi, AH, eds. Amsterdam: Elsevier Science; 1984. 136. Birkedal-Hansen H, Moore WG, Bodden MK, et al. Matrix metalloproteinases: a review. Crit Rev Oral Biol Med. 1993;4(2):197-250. 137. Shane E. Evolving data about subtrochanteric fractures and bisphosphonates. N Engl J Med. 2010;362(19):1825-1827. 138. Lenart BA, Lorich DG, Lane JM. Atypical fractures of the femoral diaphysis in postmenopausal women taking alendronate. N Engl J Med. 2008;358:1304-1306. 139. Hughes DE, Wright KR, Uy HL, et al. Bisphosphonates promote apoptosis in murine osteoclast in vitro and in vivo. J Bone Miner Res. 1995;10(10):1478-1487. 140. Ramamurthy N, Bain S, Liang CT, et al. A combination of subtherapeutic doses of chemically modified doxycycline (CMT-8) and a bisphosphonates (clodronate) inhibits bone loss in the ovariectomized rat: a dynamic histomorphometric and gene expression study. Curr Med Chem. 2001;8(3):295-303. 141. El-Tonsy MM, El-Soudany K, El-Zamarany EA, Reda Berry NM. The efficacy of sub-antimicrobial dose of doxycycline with or without bisphosphonates as an adjunctive treatment in chronic periodontitis in smokers. Egypt Dent J. 2008;54(3.2):2133-2139. 142. Gomes PS, Fernandes MH. Effect of therapeutic levels of doxycycline and minocycline in the proliferation and differentiation of human bone marrow osteoblastic cells. Arch Oral Biol. 2007;52(3):251-259. 143. Jaeschke H, Farhood A. Neutrophil and kupffer cell-induced oxidant stress and ischemia-reperfusion injury in rat liver. Am J Physiol. 1991;260(3 Pt 1):G355-G362. 144. Jaeschke H, Farhood A, Smith CW. Neutrophils contribute to ischemia/reperfusion injury in rat liver in vivo. FASEB J. 1990;4(15):3355-3359. 145. Jaeschke H. Reactive oxygen and ischemia/reperfusion injury of the liver. Chem Biol Interact. 1991;79(2):115-136. 146. Itoh T, Kawakami M, Yamauchi Y, Shimizu S, Nakamura M. Effects of allopurinol on ischemia and reperfusion-induced cerebral injury in spontaneously hypertensive rats. Stroke. 1986;17(6):1284-1287. 147. Mileski WJ, Winn RK, Vedder NB, Pohlman TH, Harlan JM, Rice CL. Inhibition of CD 18-dependent neutrophil adherence reduces organ injury after hemorrhagic shock in primates. Surgery. 1990;108(2):206-212. 148. Entman ML, Michael L, Rossen RD, et al. Inflammation in the course of early myocardial ischemia. FASEB J. 1991;5(11):2529-2537. 149. Klausner JM, Paterson IS, Goldman G, et al. Postischemic renal injury is mediated by neutrophils and leukotrienes. Am J Physiol. 1989;256(5 Pt 2):F794-F802. 150. Nathan C, Srimal S, Farber C, et al. Cytokine-induced respiratory burst of human neutrophils: dependence on extracellular matrix proteins CD11/CD18 integrins. J Cell Biol. 1989;109(3):1341-1349. 151. Gabler WL, Creamer HR. Suppression of human neutrophil functions by tetracyclines. J Periodontal Res. 1991;26(1):52-58. 152. Clark WM, Lessov N, Lauten JD, Hazel K. Doxycycline treatment reduces ischemic brain damage in transient middle cerebral artery occlusion in the rat. J Mol Neurosci. 1997;9(2):103-108. 153. Kucuk A, Kabadere S, Tosun M, et al. Protective effects of doxycycline in ischemia/reperfusion injury on kidney. J Physiol Bioch em. 2009;65(2):183-191. 154. Yrjänheikki J, Keinänen R, Pellikka M, Hökfelt T, Koistinaho J. Tetracyclines inhibit microglial activation and are neuroprotective in global brain ischemia. Proc Natl Acad Sci U S A. 1998;95:15769-15774. 155. Kienle K, Rentsch M, Müller T, et al. Expression of BCL-2 in liver grafts after adenoviral transfer improves survival following prolonged ischemia and reperfusion in rat liver transplantation. Transplant Proc. 2005;37(1):439-441. 156. Clark WM, Calcagno FA, Gabler WL, Smith JR. Coull BM. Reduction of central nervous system reperfusion injury in rabbits using doxycycline treatment. Stroke. 1994;25(7):1411-1415. 157. Viappiani S, Sariahmetoglu M, Schultz R. The role of matrix metalloproteinase inhibitors in ischemia-reperfusion injury in the lever. Curr Pharm Des. 2006;12(23):2923-2934. 158. Sawicki G, Leon H, Sawicka J, et al. Degradation of myosin light chain in isolated rat hearts subjected to ischemia-reperfusion injury: a new intracellular target for matrix metalloproteinase-2. Circulation. 2005;112(4):544-552. 159. Wang W, Schulze CJ, Suarez-Pinzon WL, Dyck JR, Sawicki G, Schulz R. Intracellular action of matrix metalloproteinase-2 accounts for acute myocardial ischemia and reperfusion injury. Circulation. 2002;106(12):1543-1549. 160. Cheung PY, Sawicki G, Wozniak M, Wang W, Radomski MW, Schulz R. Matrix metalloproteinase-2 contributes to ischemia-reperfusion injury in the heart. Circulation. 2000;101(15):1833-1839. 161. Franso C, Ho B, Mulholland D, et al. Doxycycline alters vascular smooth muscle cell adhesion, migration, and reorganization of fibrillar collagen matrices. Am J Pathol. 2006;168(5):1697-1709. 162. Curci JA, Mao D, Bohner DG, et al. Preoperative treatment with doxycycline reduces aortic wall expression and activation of matrix metalloproteinase in patients with abdominal aortic aneurysms. J Vasc Surg. 2000;31(2):325-342. 163. Kahari VM, Saarialho-Kere U. Matrix metalloproteinases in the skin. Exp Dermatol. 1997;6(5):199-213. 164. Panuncialman J, Falanga V. The science of wound bed preparation. Clin Plast Surg. 2007;34(4):621-632. 165. Parks WC, Lopez-Boado YS, Wilson CL. Matrilysin in epithelial repair and defense. Chest. 2001;120(1 Suppl):36s–41s. 166. Szabo KA, Ablin RJ, Singh G. Matrix metalloproteinases and the immune response. Clin Appl Immunol Rev. 2004;4(5):295–319. 167. Naido C, Gould A, Peters J, Candy GP. Matrix metalloproteinases inhibition and antibiotics in the treatment of chronic wounds. Wound Healing South Africa. 2009;2(2):71-73. 168. Chin GA, Thigpin TG, Perrin KJ, Moldawer LL, Schultz GS. Treatment of chronic ulcers in diabetic patients with a topical metalloproteinase inhibitor, doxycycline. WOUNDS. 2003;15(10): 315-323. 169. Reddy MS, Geurs NC, Gunsolley JC. Periodontal host modulation with antiproteinase, anti-inflammatory, and bone-sparing agents. A systematic review. Ann Periodontol. 2003;8(1):12-37. The authors are from the Healogics, Inc, Jacksonville FL. Address correspondence to: James R. Wilcox, RN, BSN, ACHRN, CWCN, CFCN, CWS, WCC, DAPWCA, FCCWS 5220 Belfort Road, Suite 130 Jacksonville, Florida 32256 jrwilcox1120@yahoo.com