The Effect of Collagenase on Ischemic Wound Healing: Results of an In Vivo Study

Size: px

Start display at page:

Download "The Effect of Collagenase on Ischemic Wound Healing: Results of an In Vivo Study"

Louise Harris
5 years ago
Views:

1 Feture The Effect of Collgense on Ischemic Wound Heling: Results of n In Vivo Study Shengxin Ji, MD, PhD; Ynn Zho, MD; Michel Lw, PhD; Roert D. Glino, MD; nd Thoms A. Mustoe, MD Astrct Mny chronic wounds hve limited lood supply nd contin necrotic tissue tht must e derided. The effect of collgense, n enzymtic deriding gent, hs een studied in cute ut not in chronic wounds. The purpose of this in vivo study is to evlute the effect of collgense on wound heling in ischemic wounds. The ers of eight young New Zelnd White rits were rendered ischemic y ligtion of cudl nd centrl rteries nd derml circultion circumferentilly so oth ers were perfused only y the rostrl rtery, preserving the cudl, centrl, nd rostr veins. Three 6-mm, full-thickness derml punches were mde on the inner surfce of oth ers down to perichondrium. One er on ech rit ws treted with either collgense or petroltum ointment covered with semi-occlusive dressing; wounds on the other er of the sme rit were covered with semi-occlusive dressing only (). On post-wounding dy 8, wound smples were collected nd processed for histologicl nlysis of reepitheliliztion (epithelil gp, percentge heled, epithelil height, nd epiderml re) nd grnultion tissue formtion (pek height, grnultion tissue distnce, nd re). Within-niml comprison showed no significnt differences etween the petroltum nd wounds ut epithelil height, epiderml re, wound pek height, nd grnultion tissue distnce nd re were significntly different etween the collgense nd -treted wounds. Between-niml comprison of petroltum- nd collgensecovered wounds showed sttisticlly significnt (P <0.05) differences for the following outcomes: epithelil gp, percentge heled, epithelil height, epiderml re, wound pek height, nd grnultion tissue distnce nd re. In this ischemic wound model, outcomes for most of the vriles ssocited with heling were significntly etter in wounds covered with collgense nd semi-occlusive dressing thn in the or petroltum group. Additionl in vivo studies re wrrnted to confirm these results. Keywords: in vivo study, wound heling, ischemic rit er model, collgense, petroltum Index: Ostomy Wound Mngement 2011;57(8):20 26 Potentil Conflicts of Interest: Dr. Ji, Dr. Lw, nd Dr. Mustoe disclose they re employees of Helthpoint Biotherpeutics, Ltd, Fort Worth, TX. Dr. Glino discloses he hs received reserch grnts from Helthpoint Biotherpeutics, Ltd, Snuwve (Alphrett, GA), Smith & Nephew (St. Petersurg, FL), nd Exclird Phrmceuticls (Sn Diego, CA). Wounds tht hve filed to return to functionl nd ntomicl integrity in timely fshion re defined s chronic. 1,2 Pressure ulcers, dietic ulcers, nd venous ulcers comprise the overwhelming mjority of chronic wounds. 2 These ulcer types shre common custive fetures: the cellulr nd systemic chnges of ging, repeted ischemi-reperfusion injury, nd cteril coloniztion with resulting inflmmtory host response. 3 Although the etiologies of ll chronic wounds re multifctoril, ischemi ie, decrese in lood flow nd/or ischemi-reperfusion injury ppers to e primry cuslity. One of the mjor resons for impired wound heling is n ttenuted lood supply, which results in decrese in the concentrtion of growth fctors within the wound ed. 2 This is prticulrly true for lower extremity wounds nd/or pressure ulcers. Collgenses hve een used s n enzyme-deriding gent for chronic derml ulcers nd severe urn wounds. Although collgenses ct y degrding ntive helicl collgen firils, 4 preliminry results from niml studies show tht collgense-treted, prtil-thickness wounds in pigs heled Dr. Ji nd Dr. Zho re reserch ssocites, The Lortory for Wound Repir nd Regenertive Medicine, Division of Plstic Surgery, Deprtment of Surgery, Northwestern University, Chicgo, IL. Dr. Lw is Principl Reserch Scientist/Toxicology Professionl, Helthpoint Biotherpeutics, Ltd, Fort Worth, TX. Dr. Glino is n ssistnt professor nd Dr. Mustoe is professor, Division of Plstic Surgery, Northwestern University. Plese ddress correspondence to: Thoms A. Mustoe, The Lortory for Wound Repir nd Regenertive Medicine, Division of Plstic Surgery, Deprtment of Surgery, Northwestern University, 4th Floor, Trry, Suite 4-720, 303 Est Chicgo Avenue, Chicgo, IL ; emil: tmustoe@nmh.org. 20 ostomy wound mngement ugust

2 quicker thn -treted wounds; furthermore, significnt pro-ngiogenic ctivity ws oserved in mice with sement memrne mtrix implnts (Mtrigel, BD, Frnklin Lkes, NJ) (unpulished dt). The effects of collgense in cute wound heling hs een studied in swine ck, cute, full-thickness wound model, ut not for chronic wounds. 5 Although mny niml models re ville, only smll numer of these models ctully mimic chronic wounds; one of these is the unique impired wound heling model cused y ischemi in rit er. 6 Becuse informtion out the potentil effect of collgense on chronic wound heling is limited, the purpose of this in vivo study ws to evlute the effect of collgense on grnultion tissue formtion nd reepitheliliztion under ischemic conditions. Mterils nd Methods Eight (8) young dult New Zelnd White rits (3 to 6 months, ~3 kg; Covnce Reserch Products, Inc., Cumerlnd, VA) were cclimted, housed, nd given ccess to food nd wter d liitum under the conditions set forth in the Pulic Helth Service Guide for the Cre nd Use of Lortory Animls nd the US Deprtment of Agriculture Title 9 Animl Welfre Act nd its revisions. The study ws conducted under n pproved experimentl protocol from the Northwestern University Animl Cre nd Use Committee. Both ers in ech of the eight rits were rendered ischemic y the method descried y Ahn et l. 6 Briefly, the rit ws nesthetized with n intrmusculr (IM) injection of ketmine (45 mg/kg) nd xylzine (7 mg/kg) nd prophylcticlly treted with penicillin (50,000 units/kg, IM) efore surgery. The surgicl sites nd the dorsl surfce of the rit ers were shved with n electric clipper nd depilted with Nir (Church & Dwight Co., Inc., Lkewood, NJ). The surgicl sites then were pinted with etdine solution nd n incision ws mde to the level of re crtilge t the se of the er. Under dissecting microscope, the cudl nd centrl rteries, s well s derml circumferentil circultion, were ligted so the entire er ws perfused only y the rostrl rtery with preservtion of the cudl, centrl, nd rostrl veins to render the rit er ischemic (n importnt reson for delyed heling). This technique results in ischemi for 7 to 10 dys. In ged rits with sustined ischemi, no heling hs een known to occur up to 26 dys. 7 No truly chronic wounds hve een noted in nimls, ut this model hs een extensively chrcterized nd used to test therpeutic options for clinicl use. 6 The incision ws closed with 5-0 polypropylene suture. Three 6-mm, full-thickness derml punches then were mde on the inner surfce of oth ers down to re crtilge y removing the epidermis, dermis, nd perichondrium. One er of the rit served s nontreted er covered y trnsprent semi-occlusive dressing consisting of thin polyurethne memrne coted with lyer of n crylic dhesive Collgense nd ischemic wounds Ostomy Wound Mngement 2011;57(8):20 26 Key Points Reserchers compred the effect of collgense ointment to untreted nd petroltum ointment in n ischemic rit er model. Within-niml comprisons showed no difference in the vriles ssessed etween petroltum-treted nd wounds, ut some outcomes in the collgense treted wounds were etter thn their respective. Collgense-covered wounds exhiited some etter outcomes thn wounds in the nimls treted with petroltum. Additionl studies re needed to confirm these in vivo results. (Tegderm dressing, 3M, St. Pul, MN). The wounds in the other er of the sme rit were treted with 0.05 ml of either collgense ointment (Sntyl Ointment, Helthpoint Biotherpeutics, Ltd, Forth Worth, TX pproximtely 1.23 g collgense in petroltum-sed ointment) or petroltum lone, nd covered y the sme semi-occlusive dressing. The sme gent (either petroltum or collgense) ws used on ll three wounds (1 cm to 1.5 cm prt) on the treted er. Ech rit received one tretment in one er nd no tretment in the other er. All nimls were scrificed t post-wounding dy 8. Tissue from ll the wounds ws collected nd nlyzed. The tissue of ech wound ws eqully split into two hlves. One hlf ws processed for histologicl nlysis nd the other hlf ws stored t -80 º C or colder for future western lot nlysis or RNA extrction. The collgense nd petroltum test products were provided y Helthpoint Biotherpeutics, Ltd (Fort Worth, TX) in individully seled, coded, nd leled luminum tues. The tues were stored t room temperture. Histologicl nlysis. The tissue from hlf of the circulr wound tken for histologicl nlysis ws suject to routine prffin emedding nd sectioning. A 4-μm cross-section ws otined through the center of ech circulr wound. Tissues were stined with hemotoxylin nd eosin (H&E) for microscopic exmintion nd mesurement. Histologicl nlyses included reepitheliliztion in terms of the epithelil gp (EG) nd the percentge heled (PH); kertinocyte prolifertion in terms of the epithelil height (EH) nd the epiderml re (EA); nd the formtion of grnultion tissue in terms of the pek height, grnultion tissue distnce, grnultion tissue re, nd the grnultion tissue re per µm wound (see Tle 1). All mesurements were performed using Nikon microscope nd ccompnying imging softwre t 2X nd 10X mgnifiction. ugust 2011 ostomy wound mngement 21

3 Feture Tle 1. Histologicl mesurement terms nd descriptions Term nd Arevition Epithelil gp (EG) Percentge heled (PH) Epithelil height (EH) Pek height Epiderml re (EA) Grnultion tissue distnce Grnultion tissue re Grnultion tissue re per µm wound Description Becuse the mesured wound size mong slides ws not identicl due to differences in cutting sections or slightly off-center in some smples, the rtio of 6 mm (the originl wound size) to the mesured wound size ws used to djust the rw dt, with the exception of PH nd epiderml re per µm wound nd grnultion tissue re per µm wound mesurements, which lredy were djusted. Dt nd sttisticl nlysis. There were 12 wounds for ech of the collgense- nd petroltum-treted groups nd their respective s. To detect difference etween the collgense or petroltum tretment nd its own groups, the dt were compred using two-tiled pired t- tests (SAS Institute Inc., Cry, NC). Anlysis of vrince (ANOVA) with Tukey s test ws used to compre outcomes of the collgense or petroltum tretment groups from different nimls while ling for differences etween nimls. P <.05 ws considered significnt in ll nlyses. Results Wound reepitheliliztion. No differences in epithelil gp nd percentge heled were oserved etween the collgense-treted ischemic nd nontreted wounds Distnce etween the tip of the new epiderml tongue on the left side nd tht on the right side of the wound or the length of unepithelilized wound re when hrvested t post-wounding dy 8 The rtio of EG over the distnce (dimeter of the wound) etween nick (or the edge of left side grnultion tissue if no nick is visile) nd nick (or the edge of right side grnultion tissue if no nick is visile) of the wound Averge epithelil height of oth the left nd right sides of the wound. Becuse the thickness of newly formed epidermis vries throughout, the height of newly formed epidermis ws mesured t two points nd verged Thickness of the wound tissue t its thickest point, including grnultion tissue nd overlying epidermis. Two mesurements re mde one on the left nd one on the right side of wound nd verge clculted s pek height of the whole wound Quntifiction of epiderml re over the 1-mm wide wound re Distnce etween the nick (or edge of the wound on dy 0) to the most dvnced tip of the grnultion tissue ingrowth (tongue) in the wound. Two mesurements re mde one on the left nd one on the right side of wound nd verge clculted s grnultion tissue distnce of the whole wound Sum of the re of grnultion tissue mesured from the left nd right sides of the wound using Nikon imging softwre (NIS-Elements BR 2.30) Averge mount of grnultion tissue re per µm wound (EG: 1,123 ± 389 µm versus 1,779 ± 497 µm, P =.4; PH:.81 ±.06 versus.70 ±.08, P =.4). No significnt difference in reepitheliliztion mesures ws found etween petroltum-treted nd nontreted s (EG: 3,858 ± 754 µm versus 2,707 ± 583 µm, P =.3; PH:.36 ±.13 versus.55 ±.10, P =.3). However, etween-niml comprison of collgense- nd petroltum-treted wounds showed significnt differences (EG collgense 1,123 ± 389 µm versus 3,858 ± 754 µm petroltum group, P =.004 nd PH collgense 81% ±.06 versus 36% ±.13 petroltum group, P =.004) (see Figures 1 nd 2). Kertinocyte prolifertion, s mesured y epithelil thickness nd re, ws significntly higher in collgense-treted wounds thn either of the respective (untreted ischemic er) or the petroltum-treted wounds (EH: 165 ± 11 µm versus 117 ± 8 µm or 79 ± 18 µm, P =.004 or <.0001; EA: 152 ± 6 µm 2 versus 98 ± 8 µm 2 or 66 ± 16 µm 2, P <.0001 or <.0001). However, no significnt difference ws noted in kertinocyte prolifertion etween petroltum-treted wounds nd their own nontreted s (EH: 79 ± 18 µm versus 124 ± 15 µm, P =.10; EA: 66 ± 16 µm 2 versus 100 ± 10 µm 2, P =.10) (see Figures 3 nd 4). 22 ostomy wound mngement ugust

Feture Figure 1. Adjusted epithelil gp verges nd stndrd error of the men (n=12 in ech group). Own comprison P = 0.4 for collgen/collgen nd P = 0.

4 for collgen/collgen nd P = 0.3 for petroltum/petroltum Between-niml comprison (collgense/petroltum) P = 0.004 Grnultion tissue formtion.

Pek height mesured 684 ± 28 µm versus 535 ± 42 µm or 442 ± 56 µm, P =.002 or.0006. Grnultion tissue distnce mesured 1,451 ± 110 µm versus 989 ± 111 µm or 713 ± 170 µm, P =.02 or.0002.

Grnultion tissue re per µm wound ws 132 ± 20 µm 2 versus 68 ± 14 µm 2 or 48 ± 21 µm 2, P =.007 or.003).

5 Feture Figure 1. Adjusted epithelil gp verges nd stndrd error of the men (n=12 in ech group). Own comprison P = 0.4 for collgen/collgen nd P = 0.3 for petroltum/petroltum Between-niml comprison (collgense/petroltum) P = Figure 2. Averge percentge heled nd stndrd error of the men (n=12 in ech group). Own comprison P = 0.4 for collgen/collgen nd P = 0.3 for petroltum/petroltum Between-niml comprison (collgense/petroltum) P = Grnultion tissue formtion. Averge tissue pek height, grnultion tissue distnce, nd tissue re were higher in the collgense thn in the collgense- nd petroltumtreted wounds. Pek height mesured 684 ± 28 µm versus 535 ± 42 µm or 442 ± 56 µm, P =.002 or Grnultion tissue distnce mesured 1,451 ± 110 µm versus 989 ± 111 µm or 713 ± 170 µm, P =.02 or Grnultion tissue re ws 790,907 ± 118,010 µm 2 versus 407,288 ± 85,867 µm 2 or 285,596 ± 127,739 µm 2, P =.007 or.003. Grnultion tissue re per µm wound ws 132 ± 20 µm 2 versus 68 ± 14 µm 2 or 48 ± 21 µm 2, P =.007 or.003). Yet no significnt difference ws noted regrding grnultion tissue formtion etween petroltum-treted wounds nd their own nontreted s (pek height: 442 ± 56 µm versus 451 ± 40 µm, P = Figure 3. Averge epitelil height nd stndrd error of the men (n=12 in ech group). Own comprison P = for collgen/ collgen nd P = 0.1 for petroltum/petroltum Between-niml comprison (collgense/petroltum) P < Figure 4. Averge epiderml re per µm length of wound nd stndrd error of the men (n=12 in ech group). Own comprison P = for collgen/ collgen nd P = 0.1 for petroltum/petroltum Between-niml comprison (collgense/petroltum) P < ; grnultion tissue distnce: 713 ±170 µm versus 852 ± 60 µm, P =.50; grnultion tissue re: 285,596 ± 127,739 µm 2 versus 317,127 ± 62,178 µm 2, P =.80; nd grnultion tissue re per µm wound: 48 ± 21 µm 2 versus 53 ± 10 µm 2, P =.80) (see Figures 5 through 8). Discussion Pierce nd Mustoe 8 descried the sic principles of optiml wound heling to include minimizing tissue dmge, dériding nonvile tissue, mximizing tissue perfusion nd oxygention, ensuring proper nutrition, nd providing moist wound-heling environment. Most preclinicl study designs certin conditions, such s minimizing tissue dmge (y exclusion from study) nd proper nutrition. 24 ostomy wound mngement ugust

Figure 5. Averge pek height of wounds nd stndrd error of the men (n=12 for ech group). Own comprison P = 0.002 for collgen/ collgen nd P = 0.

0006 Collgense is n enzymtic deridement gent nd lthough this study did not focus on mintining moist wound environment, ll wounds (including ) were covered with semi-occlusive dressing.

6 Figure 5. Averge pek height of wounds nd stndrd error of the men (n=12 for ech group). Own comprison P = for collgen/ collgen nd P = 0.9 for petroltum/petroltum Between-niml comprison (collgense/petroltum) P = Collgense is n enzymtic deridement gent nd lthough this study did not focus on mintining moist wound environment, ll wounds (including ) were covered with semi-occlusive dressing. The oserved incresed rte of reepitheliliztion in the collgense-treted compred to Collgense nd ischemic wounds Figure 6. Averge grnultion tissue distnce nd stndrd error the men (n=12 in ech group) Own comprison P = 0.02 for collgen/ collgen nd P = 0.5 for petroltum/petroltum Between-niml comprison (collgense/petroltum) P = the petroltum-treted wounds is similr to tht oserved in nother study, 5 lthough the in the ltter study is dry guze. However, following clinicl nd lortory studies, Ghdilly et l 19 found tht pplying petroltum did not completely occlude the epidermis of cetone-treted skin in ugust 2011 ostomy wound mngement 25

Feture Figure 7. Averge grnultion tissue re nd stndrd error of the men (n=12 in ech group) Own comprison P = 0.007 for collgen/ collgen nd P = 0.

003 oth humns nd mice; the uthors concluded tht petroltum permetes throughout the epidermis interstices to llow recovery of the skin rrier function.

7 Feture Figure 7. Averge grnultion tissue re nd stndrd error of the men (n=12 in ech group) Own comprison P = for collgen/ collgen nd P = 0.8 for petroltum/petroltum Between-niml comprison (collgense/petroltum) P = oth humns nd mice; the uthors concluded tht petroltum permetes throughout the epidermis interstices to llow recovery of the skin rrier function. Hence, petroltum ffects wound heling despite eing viewed s inert. 20 In the present study, collgense ointment ws significntly more effective compred to tretment with petroltum for the following wound heling vriles ssessed: reduction in wound epithelil gp, percentge heled, epithelil height, epiderml re, pek tissue height, grnultion tissue distnce, nd grnultion tissue re. When compred with their own respective wounds (n ischemic er with no ointment pplied), no significnt differences were oserved etween the petroltum nd petroltum wounds; wheres, epithelil height, epiderml re, pek height, grnultion tissue distnce, grnultion tissue re, nd grnultion tissue re per µm wound were significntly different etween the collgense nd collgense- wounds. Differences etween collgense nd wounds in epithelil gp nd percentge heled were not significnt. Additionl in vivo studies including direct petroltum or using other wound heling models re needed to confirm these results. Conclusion In this ischemic wound model, wounds treted with topicl deriding gent (collgense) covered with semi-occlusive dressing hd etter wound heling-relted outcomes thn wounds covered with semi-occlusive dressing lone (untreted ) or covered with petroltum. Additionl preclinicl studies re wrrnted nd clinicl studies to confirm the potentil effects of collgense ointment on heling chronic wounds re needed. n Acknowledgment The uthors re grteful to Helthpoint Biotherpeutics, Ltd, Fort Worth, TX, for its support nd cknowledge Renée Crstens for medicl writing contriutions. Figure 8. Averge grnultion tissue re per µm nd stndrd error of the men (n=12 in ech group) Own comprison P = for collgen/ collgen nd P = 0.8 for petroltum/petroltum Between-niml comprison (collgense/petroltum) P = References 1. Telgenhoff D, Shroot B. Cellulr senescence mechnisms in chronic wound heling. Cell Deth Differ. 2005;12(7): Eglstein WH, Flng V. Chronic wounds. In: Brul A (ed). The Surgicl Clinics in North Americ: Wound Heling. Phildelphi, PA: WB Sunders Co.;1997: Mustoe TA. Understnding chronic wounds: unifying hypothesis on their pthogenesis nd implictions for therpy. Am J Surg. 2004;187(5A):65S 70S. 4. Collgense, Clostridium histolyticum collgense (E.C ) In: Worthington V (ed). Worthington Enzyme Mnul, Enzymes nd Relted Biochemicls. Lkewood, NJ: Worthington Biochemicl Corportion;1993: Riley KN, Hermn IM. Collgense promotes the cellulr responses to injury nd wound heling in vivo. J Burn Wound. 2005;4: Ahn ST, Mustoe TA. Effects of ischemi on ulcer wound heling: new model in the rit er. Ann Plst Surg. 1990;24(1): Bonomo SR, Dvidson J, Tyrone JW, Lin X, Mustoe TA. Enhncement of wound heling y hyperric oxygen nd trnsforming growth fctor-3 in new chronic wound model in ged rits. Arch Surg. 2005;135(10): Pierce GF, Mustoe TA. Phrmcologic enhncement of wound heling. Ann Rev Med. 1995;46: Ayello EA, Cuddign JE. Deridement: ling the necrotic cellulr urden. Adv Skin Wound Cre. 2004;17(2): Brdley M, Cullum N, Sheldon T. The deridement of chronic wounds: systemtic review. Helth Technol Assess. 1999;3(17 Pt 1):iii Brookens T. Secrets of wound deridement yield improved outcomes nd incomes. Poditr Mnge. 1997(June): Flell AF. Deridement nd wound ed preprtion. Dermtol Ther. 2006;19(6): Rmundo J, Gry M. Enzymtic wound deridement. J WOCN. 2008;35(3): Steed DL. Deridement. Am J Surg. 2004;187(5A):71S 74S. 15. Adms SB Jr, Sesn VJ, Esley ME. Wound heling gents. Foot Ankle Clin. 2006;11(4): Eglstein WH, Dvis SC, Mehle AL, Mertz PM. Optiml use of n occlusive dressing to enhnce heling. Arch Dermtol. 1998;124(3): Eglstein WH, Mertz PM, Flng V. Occlusive dressings. Am Fm Phys. 1987;35(3): Eglstein WH. Effect of occlusive dressings on wound heling. Clinics Dermtol. 1984;2(3): Ghdilly R, Hlkier-Sorensen L, Elis PM. Effects of petroltum on strtum corneum structure nd function. J Am Acd Dermtol. 2001;26(3 Pt 2): Eglstein WH, Mertz PM. Inert vehicles do ffect wound heling. J Investig Dermtol. 1980;74(2): ostomy wound mngement ugust

EFFECTS OF AN ACUTE ENTERIC DISEASE CHALLENGE ON IGF-1 AND IGFBP-3 GENE EXPRESSION IN PORCINE SKELETAL MUSCLE

Swine Dy 22 Contents EFFECTS OF AN ACUTE ENTERIC DISEASE CHALLENGE ON IGF-1 AND IGFBP-3 GENE EXPRESSION IN PORCINE SKELETAL MUSCLE B. J. Johnson, J. P. Kyser, J. D. Dunn, A. T. Wyln, S. S. Dritz 1, J.