Influence of papain urea copper chlorophyllin ... - Wiley Online Library

Wound Repair and Regeneration

Influence of papain urea copper chlorophyllin on wound matrix remodeling Dale Telgenhoff, PhD1; Kan Lam, BSc2; Sarah Ramsay, MS1; Valerie Vasquez, MS1; Kristine Villareal, MS3; Paul Slusarewicz, PhD1; Paul Attar, PhD2; Braham Shroot, PhD1 1. DFB Pharmaceuticals, San Antonio, Texas 2. Bridge PTS, San Antonio, Texas, and 3. Healthpoint Ltd., Fort Worth, Texas

Reprint requests: Dale Telgenhoff, PhD, 318 McCullough, San Antonio, TX 78215. Tel: 210 396-5128; Fax: 210 533-5423; Email: [email protected] Manuscript received: February 6, 2007 Accepted in final form: April 2, 2007 DOI:10.1111/j.1524-475X.2007.00279.x

ABSTRACT The purpose of this study was to examine the dermal and epidermal alterations associated with wound healing in wounds treated with papain urea copper chlorophyllin (PUC), papain-urea, copper chlorophyllin, or urea base ointment and compare these with moist wound care using a porcine full-thickness infected wound model. All the wounds were evaluated postsurgery for erythema, transepidermal water loss, microscopic morphology, and changes in protein expression. Examination of stained paraffin sections revealed an increase in the number of keratinocytes present in the epidermis of the PUC and papain-treated pigs, relative to moist control. This increase in keratinocyte number corresponded to an increase in the movement of the keratinocytes into the underlying dermis in the form of rete pegs. In the dermis, there appeared to be an increase in blood vessel formation, collagen I deposition, and mature collagen in the papain and PUC treated tissues. The quality of healing appears to be enhanced based on the number of keratinocytes present in the epidermis, the extensive rete peg formation, the increase in vasculature, and the increase in collagen birefringence.

Wound healing normally proceeds in a timely, sequential manner and can be divided into four phases: inflammation, granulation, reepithelialization, and tissue remodeling.1,2 When this scheme is disrupted, chronic wounds develop. Defined by their inability to heal without clinical intervention, this intervention typically begins with debridement, which can be surgical, mechanical, enzymatic, or autolytic.3,4 Surgical debridement is the most rapid but is highly invasive. Mechanical debridement is similar in that it involves a skilled professional; however, it is much less discriminate and may remove or damage both viable and nonviable tissue.4 Enzymatic debridement utilizes proteases to degrade necrotic tissue, thus allowing healthy tissue to migrate into the wound area. Autolytic debridement is a process that occurs naturally in a moist wound, involving phagocytic cells and proteinases, and for a number of reasons fails to occur in the chronic wound environment.5 One enzymatic debridement agent is the enzyme papain, obtained from the ripening fruit of Carica papaya. Papain is an archetypal cysteine proteinase, whose family includes chymopapain, caricain, bromelain, actinidin, and ficin.6 Papain digests necrotic tissue by liquefying eschar,7 thus facilitating the migration of viable cells from the wound edge into the wound cavity. Papain is also useful in reducing the bacterial burden, decreasing exudates, and increasing granulation tissue formation.8 The addition of urea (a chaotropic agent) in the papain formulation facilitates the action of papain by solubilizing proteins.9,10 Certain papain formulations also contain a copper chlorophyllin complex, which has been shown clinically to promote the formation of healthy granulation tissue, control inflammation, and decrease odor.11–13 c 2007 by the Wound Healing Society Wound Rep Reg (2007) 15 727–735

Although there exists clinical evidence detailing the effectiveness of the papain urea copper chlorophyllin (PUC) treatment,8,11 there is very little information detailing how each individual component functions at the wound site. It has been argued that certain enzymes could inhibit wound healing by degrading viable proteins or healthy tissue;14 however, PUC combination was shown to only degrade denatured collagen, with no effect on viable tissue collagen or elastin.15 The purpose of this study was to examine the effects of the PUC in an animal model of acute wound healing and to compare the effects with the same formulation either without papain, copper chlorophyllin, or both. These formulations are compared with moist dressings (standard of wound care control) using morphological and biochemical differences to further elucidate the effects of each treatment.

CD CK DAB H&E IFN-g IL PAS PUC TEWL TNF-a USP

Cluster of differentiation Cytokeratin Diaminobenzidine Hematoxylin and eosin Interferon gamma Interleukin Periodic acid-Schiff Papain urea copper chlorophyllin Trans-epidermal water loss Tumor necrosis factor a United States Pharmacopeia convention

727

Papain urea chlorophyllin on wound remodeling

Telgenhoff et al.

Table 1. Concentrations of United States Pharmacopeia convention (USP) papain and sodium copper chlorophyllin in treatments

Papain-urea copper chlorophyllin (PUC) ointment Papain in base ointment Chlorophyllin in base ointment Base ointment

Papain (%) Chlorophyllin (%) Urea (%) 7.9

1

7.9 0.0

0.5

0.0 0.5

10

10 10

* p < 0.05 ** p < 0.01 50

Water Loss (g/m /h)

Treatment

60

*

40

* **

30

20

10

0.0

0.0

10 0

1

Papain concentration in PUC ointment based on activity units, formulation contains 521,700 USP units per gram of ointment.

MATERIALS AND METHODS Animals

Five female Yorkshire cross specific-pathogen free commercially raised 8-week-old pigs (20–25 kg) were kept in separate cages on 12-hour light cycles with ad libitum water and 500 g of Lab Diet mini-pig HF grower (PMI, Brentwood, MO) per day. This study conformed to the Guide for the Care and Use of Laboratory Animal (National Research Council) and was approved by local IACUC (Protocol # 03-09). The pigs were anesthetized with isoflurane and 20 full-thickness wounds were created on the dorsum with a 2 cm trephine. Following hemostasis, three isolates of bacteria were used to inoculate each wound. The organisms used were Pseudomonas aeruginosa, Fusobacterium sp., and coagulase-negative Staphylococcus aureus (University of Calgary Culture Collection). The inoculating procedure was described previously.16 Fentanyl patches (25 mg/hour) were applied to the dorsum of pigs in unwounded sites to relieve pain. Treatments

Inoculation was followed by application of either 0.5 mL of commercially available PUC ointment (Panafils Ointment, Lot# UBFN-1, Exp. 02/06, Healthpoint Ltd., Ft. Worth, TX) per wound site or the 0.5 mL of each of the

No Wound

PUC

Papain

Chlorophyllin

Base

Moist Control

Figure 1. Effect of various treatments on transepidermal water loss of healed wounds. Evaporimeter measurements were used to assess barrier function at the conclusion of the study (day 21). Water loss was significantly higher in all treatments compared with unwounded skin. Papain, chlorophyllin, and papain urea copper chlorophyllin (PUC) treatments all showed a decrease in water loss compared with moist control and base ointment. p-Values indicate significance compared with control, error bars indicate 1 SEM.

components of the same formulation lacking papain, copper chlorophyllin, or both (Table 1). All of the treatments (including PUC) were prepared in a hydrophilic base ointment composed of purified water, USP; propylene glycol, USP; white petrolatum, USP; stearyl alcohol, NF; polyoxyl 40 stearate, NF; sorbitan monostearate, NF; boric acid, NF; chlorobutanol (anhydrous), NF as a preservative; and sodium borate, NF. One formulation was applied to all of the wounds on one side of each pig, a separate treatment to all of the wounds on the other side. The wounds were wrapped with saline moistened gauze, covered with moisture-retentive blue pads (Tyco Healthcare, Mansfield, MA), and wrapped with Transpore tape (3M Healthcare, St. Paul, MN). Six-millimeter punch biopsies were obtained from the center of different wounding sites at various time points (0, 1, 4, 8, 14, and 21 days). The biopsies were cut in half longitudinally and either frozen in OCT medium (Sakura, Torrance, CA) or frozen in Eppendorf tubes in liquid nitrogen for Western blot analysis.

Figure 2. Histology of healed wounds following various treatments. Hematoxylin and eosin-stained tissue sections were examined following 21 days of treatment. Note the extensive rete pegs in the papain (E) and papain urea copper chlorophyllin (F) treatments (arrows), compared with the unwounded control (A) moist control (B), base ointment (C), and chlorophyllin (D) treatments. Scale bar50.5 mm.

728

c 2007 by the Wound Healing Society Wound Rep Reg (2007) 15 727–735

Telgenhoff et al.


Figure 3. Periodic acid-Schiff (PAS) staining for carbohydrates. Microscope images of 21 day postwounding pig tissue stained with PAS stain for carbohydrates reveal a thick, intact basement membrane in the unwounded pig tissue (A, arrow). The basement membrane is present in all other treatments (moist control, B; base, C; chlorophyllin, D; papain, E; papain urea copper chlorophyllin, F; arrows); however, it is not as thick as the unwounded control. Scale bar580 mm.

Duplicate punch biopsies from parallel sites were fixed in 4% formaldehyde and embedded in paraffin for sectioning and staining. The pigs were visually graded throughout the experiment for erythema using a visual scale. At day 21, the pigs were anesthetized and transepidermal water loss (TEWL) measurements were obtained using a DermaLab evaporimeter (Cortex Technology, Hadsurd, Denmark). The pigs were euthanized as per an IACUC-approved protocol. Histology

Figure 4. Effect of various treatments on the collagen content of healed wounds. Microscope images of 21 day postwounding pig tissue stained with Verhoeff’s stain for collagen reveal unwounded tissue (A) contains more collagen (pink) present than the wounded sections treated with moist dressings (B), base ointment (C), or chlorophyllin (D). The papain (E) and papain urea copper chlorophyllin (PUC) (F) treatments display an increase in collagen staining compared with moist control. Intensity measurements were used to quantify staining (G); asterisks indicate significance (p < 0.01). Bar50.6 mm, error bars indicate 11 SEM.

Paraffin-embedded tissues were cut at 4 mm thickness and stained with either hematoxylin and eosin (H&E), Verhoeff’s stain for collagen,17 or periodic acid-Schiff (PAS) for carbohydrates (EM Sciences, Hatfield, PA). Sections were examined for morphology, epidermal migration, thickness, and extent of rete pegs. Dermal areas were examined for inflammation and contraction. Additional sections were stained with Picrosirius Red (Sigma, St. Louis, MO) following the method described by Junqueira et al.18 and examined using cross-polarization optics to visualize birefringent collagen. Frozen sections were also cut at 6 mm thickness and used for immunohistochemistry.

Markers examined were cytokeratins (CKs) 6, 10, and 14 (mouse anti-human, 1 : 200, Abcam, Cambridge, MA), and CD31 (mouse anti-pig, 1 : 500, Serotec, Raleigh, NC). All antibodies were cross reactive in pig. Antibodies were prepared in tris-buffered saline (Pierce, Rockford, IL), pH 7.4. Secondary antibodies were horseradish peroxidase labeled (goat anti-mouse, 1 : 1,000, Bio-Rad, Hercules, CA), and were detected with either Vector Red or diaminobenzidine (DAB) Vectastain kits (Vector Labs,


729


Telgenhoff et al.

Figure 5. Effect of various treatments on collagen birefringence. Cross-polarization imaging of picrosirius red-stained pig skin sections shows more red birefringence (thick collagen fibers) in the unwounded tissue (A) compared with green birefringence (thin collagen fibers). The moist control (B) shows very little birefringence even 21 days after wounding. The base ointment (C) shows increased birefringence, which is even more prevalent in the chlorophyllin (D)- and papain (E)-treated tissues. The papain urea copper chlorophyllin (PUC) treatment (F) induced the highest degree of birefringent collagen, with both green and red birefringence present. Intensity measurements were used to quantify yellowred (G) or green (H) birefringence; asterisks indicate significance (p < 0.01). Bar50.4 mm, error bars indicate 11 SEM.

Burlingame, CA). Slides were examined on an Olympus BX41 microscope (Olympus, Tokyo, Japan) using a Spot Insight digital camera (Diagnostic Instruments, Sterling Heights, MI) and Metafluor Imaging software (Molecular Devices, Sunnyvale, CA). Vessel counts were performed using the linescan feature of this software, with four random linescans per microscopic image. 730

RESULTS There was no clinically apparent difference in edema, granulation, or eschar in any of the treatments (data not shown) when compared with the control. There was an increase in erythema in the papain-treated pigs, which continued until the wound was reepithelialized. TEWL c 2007 by the Wound Healing Society Wound Rep Reg (2007) 15 727–735

Telgenhoff et al.


Figure 6. Effect of various treatments on wound vascularization. Cluster of differentiation 31 staining was used to examine blood vessel formation and persistence at various time points. All three treatments resulted in an increase in blood vessels at days 4 and 21, compared with base ointment and moist control. Scale bar5100 mm.

readings were elevated in all treatments (including control) compared with unwounded skin adjacent to wounded sites (p < 0.002 for all). However, TEWL was decreased in PUC, papain, and chlorophyllin treatments when compared with moist control and significantly decreased compared with base ointment (p < 0.03 for all) (Figure 1). Microscopic examination (based on mononuclear cell and neutrophil inflammatory infiltrates) of the H&Estained slides showed no apparent differences in inflammation at any of the time points examined. Reepithelialization was first evidenced in the sections at day 14, and was complete in all treatments by day 21. The papain and PUC sections showed an increase in epidermal thickness, and a large increase in both the depth and number of rete pegs formed (Figure 2). Hyperproliferation and deep rete pegs were not seen in the base ointment, chlorophyllin, or moist control treatments. PAS staining revealed that all treatments had an intact basement membrane, although in none of the treatments was the basement membrane as developed as unwounded control sections (Figure 3). The PUC treatment also showed an increase in vasculature and a decrease in cellularity in the dermis. Verhoeff’s-stained sections showed a large decrease in dermal collagen (pink in tissue sections) in all wounded tissues compared with unwounded control (Figure 4). Papain and PUC sections appeared to contain a greater amount of collagen in the dermis compared with other treatments at day 21. This increase could also be seen in the sections stained with Sirius red (Figure 5). The Sirius-red stained sections also displayed more red birefringence (thick fibers) in the papain and PUC-treated sections compared with the green birefringence (thin fibers) in the chlorophyllin and base ointment treatments, and very little birefringence in the moist control. Immunohistochemistry was also performed on frozen sections to further examine the blood vessel formation. Both CD31 (Figure 6) and von Willebrand’s Factor (not shown) staining showed an increase in blood vessels at days 4 and 21 in the papain-, chlorophyllin-, and PUCtreated tissues (Figure 7). At day 14, none of the blood vessel counts were significantly different from the moist control. In the epidermis, the CK were examined immunohistologically at day 21. CK14, normally present only in the basal layer, was found in the first several layers of the moist control, base ointment, and chlorophyllin-treated tissues (Figure 8, first row). Exclusive basal layer staining was only observed in the papain and PUC treatments. CK6 was high in all treatments, with a slight decrease in c 2007 by the Wound Healing Society Wound Rep Reg (2007) 15 727–735

the chlorophyllin and PUC treatments (Figure 8, second row). CK10 is normally found only in the differentiating layers of the epidermis, as was seen in the PUC-treated sections (Figure 8, third row). There was a decrease in CK10 in the base ointment and papain-treated sections, and a large decrease in the moist control and chlorophyllin sections.

DISCUSSION Devitalized tissue over the area of a wound poses a major impediment to wound healing. The removal of necrotic tissue is essential in wound-bed preparation for the treatment of chronic wounds.8 This impediment is multifactorial; the eschar enhances bacterial growth, reduces the host’s ability to prevent infection, delays the formation of granulation tissue, and delays the reepithelialization process.8,16 Enzymatic debridement is one of the options for the removal of wound eschar and is most useful in large wounds when surgical techniques cannot be utilized. The purpose of this study was to determine the efficacy of the PUC ointment and compare this with each of the components of the same formulation lacking papain, copper chlorophyllin, or both and moist wound care using an infected acute porcine full-thickness wound model. Because the model used was an acute wound that heals fairly rapidly without intervention, even with bacterial insult,16 the rate of reepithelialization was not the focus of this study. In addition, as this was not a chronic wound, the main goal of this study was to examine the interactions of PUC components with dermal and epidermal cell types. A more thorough single variable study on chronic wounds in humans is required to fully understand PUC function on debridement and inflammation. PUC ointment has been shown to have no detrimental or adverse effects and does not increase pain,8,14 and there exist multiple clinical examples of PUC accelerating healing in chronic wounds.4,19 We primarily chose this model to examine the effects on the quality of wound healing and the differences between the individual components of the PUC formulation. Our study demonstrates that wounds treated with PUC more closely resemble normal pig skin (with the exception of the deep rete ridges) than do wounds treated with moist control or base ointment. Evaporimeter readings on day 21 revealed a decrease in TEWL (which correlates with an improvement in barrier function)20,21 in wounds treated with PUC, papain, or chlorophyllin when compared with 731


A

Telgenhoff et al.

Average Vessel Counts (Day 4) 10.0

Average Count/Random Linescan

9.0 8.0 7.0 6.0 5.0 4.0 3.0 2.0 1.0 0.0 Moist

B

Base

Chlorophyllin

Papain

PUC



7.0 6.0 5.0 4.0 3.0 2.0 1.0 0.0 Moist

C

Base

Chlorophyllin

Papain

PUC



4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 Moist

Base

Chlorophyllin

Papain

PUC

Figure 7. Vessel counts at 4, 14, and 21 days after wounding. Intensity measurements were used to quantify staining seen in Figure 7 for days 4 (A), 14 (B), and 21 (C). Random line scans for staining intensity were quantified and averaged; p-values are based on t-tests comparing treatments with moist control. pValues indicate significance compared with control; error bars indicate 1 SEM.

732

moist control or base ointment. TEWL values and rete pegs return to normal levels after 6 months (data not shown), presumably through wound remodeling and a return to normal epidermal differentiation. The enhancement of the barrier function in the treated tissues could be explained by a return to normal keratinocyte differentiation more rapidly than moist control, as evidenced by normal keratin distribution. Wounding the epidermis results in a change in keratin distribution from keratins 5 and 14 in the basal layer and 1 and 10 in the suprabasal layers to 6, 16, and 17 in the proliferating, migratory keratinocytes.22 The moist control, base ointment, and to some extent the chlorophyllin-treated wounds displayed suprabasal keratin 14 staining and delayed onset of keratin 10 staining at day 21, even though reepithelialization occurred by day 14. Only in the PUC and papain treatments was a normalized keratin expression observed with keratin 14 only in the basal cells and keratin 10 throughout the differentiating layers. Keratin 6 was also decreased in the PUC-treated wounds by day 21 when compared with all the other treatments, again indicating a more rapid return to normal tissue distribution following treatment. The rete pegs present in the papain- and PUC-treated wounds at day 21 recall psoriatic epidermis, as is the prevalence of the enhanced microvasculature.23,24 Psoriasis has been described as a persistent wound-healing response due to epidermal hyperplasia, increased levels of keratin 16, and increased vasculature.25,26 Although papain- and PUC-treated wounds possess some aspects of the appearance of psoriatic skin, they differ in that neither treatment elevated the number T cells, whose presence is characteristic of psoriatic lesions. It is possible that the psoriasis-associated cytokines are released during the initial wounding and then propagated by papain through cell activation.27,28 Early in the wound response (4–7 days), all treatments displayed an increase in rete peg formation (data not shown), but the response was sustained in the papainand PUC-treated wounds. The depth of the rete pegs and apparent tortuosity of these appendages are similar to areas of skin that experience increased mechanical stress (such as the palm) and these structures may help anchor the epidermis to the underlying dermis.29–31 PAS staining for the basement membrane zone between the epidermis and dermis confirmed the presence of an intact basement membrane in all treatments, indicating that the rete pegs examined are anchored into the dermis. Establishing an intact vasculature is important in wound healing in order to supply the wound with immune cells to combat infection and growth factors to speed healing. Vessels first appear as buds as endothelial cells migrate into the hemostatic clot and begin to multiply and form new capillaries.32,33 At day 4, there were significantly more capillaries in the chlorophyllin-, papain-, and PUC-treated sections compared with base ointment and moist control. By day 14 the vessel counts were similar in all treatments; then, at day 21 the papain, chlorophyllin, and PUC treatments again revealed elevated vessel counts. It is possible that the day 4 counts were skewed due to irritation, which was decreased by day 14. However, it does not explain the increase in the three treatments at day 21. Possible mechanisms for increased vasculature include a release of growth factors or angiogenic peptides from the hemostatic plug, or a more thorough digestion of the necrotic tissue, c 2007 by the Wound Healing Society Wound Rep Reg (2007) 15 727–735

Telgenhoff et al.


Figure 8. Effect of various treatments on epidermal cytokeratin expression. Sections of treated wounds at day 21 were stained with antibodies against cytokeratins 14, 6, and 10 in the epidermis. Cytokeratins 14 and 10 display a normal tissue distribution only in the papain- and papain urea copper chlorophyllin (PUC)-treated wounds. Cytokeratin 6 is still present in all treatments, but is decreased in the chlorophyllin and PUC treatments. Scale bar5100 mm.

that creates a more permissive area for angiogenesis to occur; however, further experimentation is required to determine the cause of the increased angiogenesis. The enzyme treatments also appear to have an effect on granulation tissue formation and collagen deposition. The papain-, chlorophyllin-, and PUC-treated wounds displayed increases in collagen at day 21 beyond the moist control or the base ointment treatments as seen in the Verhoeff’s-stained tissue sections. Further, these treatments appear to promote the formation of more mature collagen fibers as seen in the cross-polarization images from the Sirius red-stained sections. Collagen I stains red to orange with Sirius red, while collagen III tends to be green to yellow.34 In the unwounded dermis, collagen I is predominant, while in the wounded dermis the initial collagen deposited is collagen III.35 This ratio then reverses during wound remodeling, although collagen I predominance never reaches prewounding levels.36 In this study, we have shown an increase in collagen I-like fibers (based on birefringence) in the papain-, chlorophyllin-, and PUC-treated wounds. This may indicate a role for chlorophyllin in the switch from immature to mature collagen fiber formation.

The results of this study are summarized in Table 2. The PUC treatment increased the barrier properties of the epidermis, blood vessel formation and maintenance, rete peg presence and depth, and mature collagen formation. It appears that the presence of sodium copper chlorophyllin in this formulation acts to decrease inflammation as the papain-urea by itself was inflammatory in the early phase of wound healing. This finding is consistent with previous studies describing chlorophyllin’s anti-inflammatory properties.11,12 In addition to debridement, the enzyme papain also seems to be effective in promoting rete peg development, collagen formation, and blood vessel production. All of these endpoints may be the result of a decrease in resistance to cell and surrounding tissue migration into the wound site through the digestive functions of the enzyme. It is surprising to note the effect on epidermal keratins, but this may also be the result of the wound site recovering its normal function in the acute wound situation. It may be that the enzyme is stimulating the release of growth factors or matrikines either through its proteolytic activity or by binding to receptors such as the protease-activated receptor-1.37,38 This is the first comprehensive study of the

Table 2. Summary of treatment effects

Barrier (TEWL) Erythema Moist control Base ointment Chlorophyllin Papain-urea

Papain-urea copper chlorophyllin

High TEWL

Low

Cytokeratins

Abnormal distribution Medium TEWLLow Abnormal distribution Medium Low Abnormal TEWLn distribution High at day Normal distribution Medium 4, of 10 and 14 TEWLn low by day 14 Medium Low Normal distribution TEWLn of 10 and 14

Rete pegs

Vasculature

Dermal collagen

None

Normal

Very low

Increased

Normal

Low

None

Moderate

Large increase

Increased, maintainedn Increased, maintainedn

Large increase

Increased, maintainedn

Very highn

High

TEWL, trans-epidermal water loss. n Significance compared to moist control. c 2007 by the Wound Healing Society Wound Rep Reg (2007) 15 727–735

733


matrix remodeling properties of a clinically effective wound product. Our future research will focus on the applicability of these findings to chronic wounds to identify key biological markers and targets for advanced woundhealing therapeutics.

ACKNOWLEDGMENTS The authors would like to thank The Air Force Research Laboratory, Human Effectiveness Directorate at Brooks City Base, San Antonio, TX, for their assistance with the animals and tissue processing.

REFERENCES 1. Mulder GD, Vande Berg JS. Cellular senescence and matrix metalloproteinase activity in chronic wounds. Relevance to debridement and new technologies. J Am Podiatr Med Assoc 2002; 92: 34–7. 2. Mustoe T. Understanding chronic wounds: a unifying hypothesis on their pathogenesis and implications for therapy. Am J Surg 2004; 187: 65S–70S. 3. Falanga V, Brem H. Wound bed preparation for optimal use of advanced therapuetic products. In: Falanga V, editor. Cutaneous wound healing. London: Martin Dunitz Ltd., 2001: 457–68. 4. Alvarez O, Fernandez-Obregon A, Rogers R, Bergamo L, Masso J, Black M. Chemical debridement of pressure ulcers: a prospective, randomized, comparitive trial of collagenase and papain/urea formulations. Wounds 2000; 12: 15–25. 5. Kerstein MD. Moist wound healing: the clinical perspective. Ostomy Wound Manage 1995; 41: 37S–44S; discussion 5S. 6. Almeida PC, Nantes IL, Rizzi CC, Judice WA, Chagas JR, Juliano L, Nader HB, Tersariol IL. Cysteine proteinase activity regulation. A possible role of heparin and heparin-like glycosaminoglycans. J Biol Chem 1999; 274: 30433–8. 7. Schultz GS, Sibbald RG, Falanga V, Ayello EA, Dowsett C, Harding K, Romanelli M, Stacey MC, Teot L, Vanscheidt W. Wound bed preparation: a systematic approach to wound management. Wound Repair and Regeneration 2003; 11: 1–28. 8. Falanga V. Wound bed preparation and the role of enzymes: a case for multiple actions of therapeutic agents. Wounds 2002; 14: 47–57. 9. Schindler J, Jung S, Niedner-Schatteburg G, Friauf E, Nothwang HG. Enrichment of integral membrane proteins from small amounts of brain tissue. J Neural Transm 2006; 113: 995–1013. 10. Singh SM, Panda AK. Solubilization and refolding of bacterial inclusion body proteins. J Biosci Bioeng 2005; 99: 303–10. 11. Brett D. Chlorophyllin a healer? A hypothesis for its activity. Wounds 2005; 17: 190–5. 12. Tao H, Sun Z, Liu M, Wen X. Synthesis of zinc chlorophyllin a and its preliminary clinical application. Hua Xi Yi Ke Da Xue Xue Bao 1990; 21: 341–3. 13. Chernomorsky SA, Segelman AB. Biological activities of chlorophyll derivatives. NJ Med 1988; 85: 669–73. 14. Hebda PA, Flynn KJ, Dohar JE. Evaluation of the efficacy of enzymatic debriding agents for removal of necrotic tissue and promotion of healing in porcine skin wounds. Wounds 1998; 10: 83–96.

734

Telgenhoff et al.

15. Hebda PA, Lo CY. The effects of active ingredients of standard debriding agents—papain and collagenase—on digestion of native and denatured collagenous substrates, fibrin and elastin. Wounds 2001; 13: 190–4. 16. Wright JB, Lam K, Buret AG, Olson ME, Burrell RE. Early healing events in a porcine model of contaminated wounds: effects of nanocrystalline silver on matrix metalloproteinases, cell apoptosis, and healing. Wound Repair Regen 2002; 10: 141–51. 17. Kiernan JA. Histological & histochemical methods: theory and practice. 2nd ed. Oxford: Pergamon Press, 1990. 18. Junqueira LC, Montes GS, Sanchez EM. The influence of tissue section thickness on the study of collagen by the Picrosirius-polarization method. Histochemistry 1982; 74: 153–6. 19. Melano E, Rodriguez HL, Carrillo R Jr., Dillon L. The effects of Panafil when using topical negative pressure to heal an infected sternal wound. J Wound Care 2004; 13: 425–6. 20. Gioia F, Celleno L. The dynamics of transepidermal water loss (TEWL) from hydrated skin. Skin Res Technol 2002; 8: 178–86. 21. Tagami H, Kobayashi H, Zhen XS, Kikuchi K. Environmental effects on the functions of the stratum corneum. J Investig Dermatol Symp Proc 2001; 6: 87–94. 22. Freedberg IM, Tomic-Canic M, Komine M, Blumenberg M. Keratins and the keratinocyte activation cycle. J Invest Dermatol 2001; 116: 633–40. 23. Grossman RM, Krueger J, Yourish D, Granelli-Piperno A, Murphy DP, May LT, Kupper T, Sehgal P, Gottlieb A. Interleukin 6 is expressed in high levels in psoriatic skin and stimulates proliferation of cultured human keratinocytes. Proc Natl Acad Sci USA 1989; 86: 6367–71. 24. Kyriakides TR, Tam JW, Bornstein P. Accelerated wound healing in mice with a disruption of the thrombospondin 2 gene. J Invest Dermatol 1999; 113: 782–7. 25. Nanney LB, Stoscheck CM, Magid M, King LE Jr. Altered [125I] epidermal growth factor binding and receptor distribution in psoriasis. J Invest Dermatol 1986; 86: 260–5. 26. Bhawan J, Bansal C, Whren K, Schwertschlag U. K16 expression in uninvolved psoriatic skin: a possible marker of pre-clinical psoriasis. J Cutan Pathol 2004; 31: 471–6. 27. Riewald M, Ruf W. Protease-activated receptor-1 signaling by activated protein C in cytokine-perturbed endothelial cells is distinct from thrombin signaling. J Biol Chem 2005; 280: 19808–14. 28. Van den Steen P, Rudd PM, Dwek RA, Van Damme J, Opdenakker G. Cytokine and protease glycosylation as a regulatory mechanism in inflammation and autoimmunity. Adv Exp Med Biol 1998; 435: 133–43. 29. Badylak SF, Record R, Lindberg K, Hodde J, Park K. Small intestinal submucosa: a substrate for in vitro cell growth. J Biomater Sci Polym Ed 1998; 9: 863–78. 30. Montagna W, Kligman AM, Carlisle KS. Atlas of normal human skin. New York: Springer-Verlag, 1992. 31. Compton CC, Gill JM, Bradford DA, Regauer S, Gallico GG, O’Connor NE. Skin regenerated from cultured epithelial autografts on full-thickness burn wounds from 6 days to 5 years after grafting. A light, electron microscopic and immunohistochemical study. Lab Invest 1989; 60: 600–12. 32. Mehendale F, Martin P. The cellular and molecular events of wound healing. In: Falanga V, editor. Cutaneous wound healing. London: Martin Dunitz Ltd, 2001: 15–38. 33. Galiano RD, Tepper OM, Pelo CR, Bhatt KA, Callaghan M, Bastidas N, Bunting S, Steinmetz HG, Gurtner GC. Topical vascular endothelial growth factor accelerates diabetic wound healing through increased angiogenesis and by c 2007 by the Wound Healing Society Wound Rep Reg (2007) 15 727–735

Telgenhoff et al.

mobilizing and recruiting bone marrow-derived cells. Am J Pathol 2004; 164: 1935–47. 34. Cuttle L, Nataatmadja M, Fraser JF, Kempf M, Kimble RM, Hayes MT. Collagen in the scarless fetal skin wound: detection with picrosirius-polarization. Wound Repair Regen 2005; 13: 198–204. 35. Ihlberg L, Haukipuro K, Risteli L, Oikarinen A, Kairaluoma MI, Risteli J. Collagen synthesis in intact skin is suppressed during wound healing. Ann Surg 1993; 217: 397–403.



36. Wolman M, Kasten FH. Polarized light microscopy in the study of the molecular structure of collagen and reticulin. Histochemistry 1986; 85: 41–9. 37. Telgenhoff D, Shroot B. Further evidence of multifunctionality in the keratinocyte: the endothelial protein C receptor. J Investigative Dermatol 2005; 125: xviii–xix. 38. Tran KT, Lamb P, Deng JS. Matrikines and matricryptins: implications for cutaneous cancers and skin repair. J Dermatol Sci 2005; 40: 11–20.

735