Venous Ulcers: Pathophysiology and Treatment Options—Part 2

Part 1

Grafting

    When dressings and medical management fail or when rapid healing is essential, skin grafting is an excellent alternative. The first recorded skin grafts were performed in India and Egypt. More recently, skin grafting has been used to treat recalcitrant venous leg ulcers as well as large or slow-healing wounds. Currently, several types of grafts are available for venous ulcers. These grafts can be categorized based on the source of the donor tissue, the thickness of the donor tissue, and whether the graft is cultured in a laboratory.²

    Grafts are categorized as autografts or allografts depending on the source of the donor tissue. Autografts are grafts taken from the patient’s own skin, usually from the thigh, abdomen, or buttocks.² Autologous skin grafts are not rejected, and donor tissue is usually readily available.

    Allografts are taken from the skin of another person; of these, cadaveric allografts are the most commonly used.¹⁰² These grafts must be changed frequently because of rejection — periodic removal of the allografts debrides the wound bed. In addition, allografts promote healing and reduce the possibility of surface infection. These grafts are chemically treated to prevent bacterial contamination, but the possibility of viral contamination exists.¹⁰³

    Grafts also may be categorized as either full- or partial- (split) thickness.² Full-thickness skin grafts (FTSG) consist of all the epidermis and all the dermis; split-thickness skin grafts (STSG) contain all the epidermis and part of the dermis. Compared to STSG, FTSG are thicker, more durable, prevent wound contracture, and offer better cosmetic results. They make poorer grafts for leg ulcers because more tissue is necessary to re-vascularize, which decreases the graft’s chance for survival.

    Conversely, split-thickness skin grafts (STSG) require less tissue to re-vascularize and, therefore, the chance for graft survival is greater.² Types of STSG include pinch grafts, punch grafts, and shave grafts, which are harvested freehand, as well as dermatome-harvested grafts. The advantage of using a dermatome is the production of uniformly sized grafts.¹⁰⁴

    Split-thickness skin grafts are typically expanded via a meshing technique (see Figures 5 and 6).^2,105 By creating interstices in the original graft, the graft’s size may be expanded by 1.5 to nine times the original size; therefore, less tissue needs to be harvested and larger areas can be treated. These interstices also allow blood and exudates to escape from the wound; thus, improving graft survival. Split-thickness skin grafts adhere well to the graft bed and give reasonable cosmetic results.^106,107

    Numerous reports cite use of STSG for venous ulcers. Millard et al¹⁰⁸ grafted 41 venous ulcers with pinch skin grafts; 74% of the ulcers healed after grafting. The authors proposed that this approach is a simple, inexpensive, and effective way to treat ulcers, which also offers long-term benefits. Ahnlide et al’s¹⁰⁹ study of pinch grafting for venous ulcers also showed healing, although at a lower rate than Millard’s study.

    In Kirsner et al’s¹¹⁰ study of meshed STSG for various lower extremity ulcers, a greater than 90% graft take and 70% complete ulcer healing was achieved in hospitalized patients. After a mean outpatient follow-up of 11 months, 52% of the grafts remained healed and 26% exhibited partial breakdown. Schmeller et al¹¹¹ reported using meshed STSG after debriding the ulcer site and surrounding lipodermatosclerotic skin to fascia with a Shink dermatome. After 1 week post graft, an 80% success rate was reported; after 3 months, 79%; and long-term success (an average of 32 months for 12 of the 13 ulcers and 20 months for 11 of the 13 ulcers) was 88%. In addition to healing, improvement in laser Doppler flow and transcapillary and intracutaneous O₂ tension was noted at 3 months. Randomized controlled trials proving the efficacy of autologous skin grafts have not been undertaken.

    Cultured grafts. Cultured grafts are created from cells that are harvested, cultured, and expanded in a laboratory.⁶⁷ Limited availability of donor tissue and the need for repeated grafting procedures fostered the development of cultured grafts. It usually takes about 3 weeks to produce cultured grafts. If the patient’s own cells are used, the graft is referred to as a cultured autograft. Alternatively, if the donor cells are obtained from another individual, the graft is termed a cultured allograft. Using cultured epidermal autografts (CEA) for the treatment of leg ulcers and burns, Limova et al¹¹² reported 80% of recalcitrant venous ulcers healed in 5.7 weeks. Hefton et al¹¹³ also reported rapid healing, relief from pain, and permanent covering of venous ulcers with the use of CEAs. In this study, four of the six ulcers treated healed in 21 to 35 days and three of those four ulcers remained healed for 2 years; the other one recurred in 2 months.

    Cultured epidermal allografts have been used as a temporary covering for both acute and chronic wounds.¹¹⁴ In a study by Leigh et al,¹¹⁵ 51 patients with either venous or rheumatoid ulcers previously unresponsive to STSG were treated with fresh cultured epidermal allografts. After placement, healing occurred from the previously indolent edges. Because of the difficulties in coordinating the use of fresh allografts, Bolivar-Flores et al¹¹⁶ sought to determine the feasibility and efficacy of using frozen cultured epidermal allografts for venous ulcers. Ten patients were treated with frozen cultured allografts that were thawed immediately before use and applied every 2 weeks until complete healing was achieved. All wounds healed within 31 weeks. Teepe et al¹¹⁷ treated 43 patients with 47 venous ulcers in a randomized, controlled trial comparing cryopreserved cultured epidermal allografts with compression to hydrocolloid dressings with compression. No significant difference was noted in percentage of healing or decrease in pain. Interestingly, the 27 ulcers that did not heal were given the alternative treatment and the results showed a higher percentage of healing in the ulcers treated first with the hydrocolloid dressing and then with the allografts. It was postulated that hydrocolloid dressings may improve the wound bed, allowing improved allograft take.

    Several years ago, the FDA approved the use of Apligraf® (Graftksin, Organogenesis, Canton, Mass.), a cultured, bilayered skin equivalent, for the treatment of venous ulcers (see Figure 7).¹⁸ This off-the-shelf skin product contains living epidermal cells (keratinocytes) and dermal cells (fibroblasts) derived from neonatal foreskin. The fibroblasts are seeded in a bovine type 1 collagen matrix. The product is thought to stimulate healing through growth factor and cytokine replacement, providing a moist environment and immediate wound coverage. However, Phillips et al¹¹⁸ demonstrated that the skin equivalent does not permanently remain on the wound. In a series of patients treated for venous leg ulcers with this skin equivalent, the product’s DNA was undetectable by polymerase chain reaction by 8 weeks.

    The bilayered skin equivalent has been shown to be superior to compression therapy for recalcitrant venous ulcers. In a controlled clinical trial published by Falanga et al,^67,119 patients with venous ulcers received either compression or compression plus the bilayered skin equivalent. A greater percentage of patients healed with the skin equivalent (63% versus 49%) and in less time (61 versus 181 days). Among venous ulcer patients, those with refractory wounds (defined as wound duration greater than 1 year), the skin equivalent was especially effective with a significantly higher complete closure rate when used with compression than compression alone (47% versus 19%).¹²⁰ Also, patients showed significantly higher closure rates at all points in the study (6 weeks, 8 weeks, 12 weeks, and 6 months). Thus, the authors determined that this skin equivalent was 60% more effective at wound closure at any time point; after 6 months, wounds were twice as likely to completely close. Brem et al¹²¹ in an uncontrolled study demonstrated that the use of skin equivalent plus compression led to complete closure of long-standing ulcers 70% of the time. While the bilayered skin equivalent’s efficacy has been shown in various studies, its cost has limited its use. However, Kirsner et al¹²² have demonstrated cost benefits for use with refractory venous ulcers compared to the historical use of Unna boots.

    Orcel® (Ortec International, New York, NY) is a bilayered cellular matrix consisting of a type I bovine collagen sponge onto which human allogeneic keratinocytes and fibroblasts are seeded.¹²³ The fibroblasts are grown on the porous side, and the keratinocytes are grown on the non-porous side of the sponge. These allogeneic cells are cultured from neonatal foreskin. The cellular matrix product contains an absorbable biocompatible matrix that provides temporary protection and a favorable environment for host cell migration. It provides a number of growth factors, such as FGF1, GMCSF, MCSF, IL1a and b, IL6, KGF1, PDGF-AB, TFG alpha and beta, VEGF, and HGF. The cultured skin is devoid of Langerhan’s cells, melanocytes, macrophages, lymphocytes, vessels, or follicles. A 12-week, controlled phase III study¹²⁴ using this product in the treatment of hard-to-heal venous leg ulcers demonstrated a 64% improvement compared to standard care using compression therapy for those ulcers. Among participants, 59% treated with the cellular matrix product achieved complete closure, while only 36% of patients treated with standard care achieved complete closure.
 

    Optimizing graft success. Grafts may fail for several reasons. Compliance with dressing changes, leg elevation, and compression stockings will help prevent graft failure.^2,19,110 Other reasons for failure include underlying comorbid factors such as diabetes mellitus, protein malnutrition, and vitamin C deficiency. A deficit of local fibrin deposits may inhibit graft adherence.² Fibrinolytic abnormalities may lead to microthrombi deposition within the dermal vessels, resulting in graft failure. Medications such as steroids, chemotherapeutics, anticoagulants, nonsteroidal anti-inflammatories, and aspirin also may interfere with graft survival, as may other factors such as alcohol use, cigarette smoking, and local viral infections, although these remain to be studied in a rigorous fashion.

Sclerotherapy

    Sclerotherapy, or the injection of a sclerosing agent into the vein, has been used not only for venous ulcers but also varicose veins and esophageal varices. In a case series, Takeda et al¹²⁵ reported that three out of 11 patients with venous ulcers treated with sclerotherapy healed by 1 month after treatment, while seven out of 11 healed within 2 months. Diagnosing incompetent perforators is critical when healing venous ulcers with this procedure. The effectiveness of sclerotherapy is limited because many incompetent perforators are not identified.

    Queral et al¹²⁶ performed a study comparing patients treated with Unna boots and sclerotherapy with Sotradecol® (Elkin-Sinn Inc., St. Davids, Pa.) to patients treated only with Unna boots. Portable continuous wave Doppler and manual palpation were used to detect the incompetent channels. Complete healing was significantly faster with the combination of Unna boots and sclerotherapy (4.3 versus 6.1 weeks, P <0.05).

Subfascial Endosdopic Perforator Surgery

    In many patients with venous disease, abnormal perforating veins are present. Surgical approaches have been created to correct these veins. In the past, ligation of these veins was accomplished via a long open incision down the leg and through the indurated skin.^127,128 Incompetent veins were ligated with electrocoagulation or metal clips and scissor division.¹²⁷ Pre-operative duplex scanning is essential to diagnosis obstruction and incompetence in the venous system.¹²⁹ Also, thromboembolism prophylaxis is necessary.

    More recently, subfascial endoscopic perforator surgery (SEPS) has been suggested as an alternative to both treat and prevent venous ulcers by addressing perforating veins. Several series have reported rapid healing, diminished morbidity, and better cosmetic results when compared to the open procedure.¹²⁹ In a case series study conducted by Rhodes et al,¹³⁰ SEPS was shown to improve calf muscle pump activity, reduce venous incompetence, and help heal all active ulcers. Kolvenbach et al¹³¹ in a case series demonstrated improvement in the clinical parameters of venous insufficiency in 86% of the ulcers studied. Minor complications after surgery, such as hematomas or cellulites, were reported in 21% of participants. Gloviczki et al¹³² suggested that ablation of superficial reflux and deep venous obstruction with SEPS improves healing. Lee et al¹²⁹ combined SEPS with saphenous vein ligation and reported that 58% of ulcers healed in 6 weeks, with only 11% infection rate post surgery. Recently, Proebstle et al¹³³ published the findings in a series of patients using tumescent anesthesia instead of general anesthesia in 67 legs from 51 patients — 79% of legs were treated successfully with only tumescent anesthesia consisting of prilocaine 200 mg, suprarenine 1 mg, and sodium bicarbonate 840 mg in 1,000-mL 0.9% saline. A Klein pump was used to inject the anesthesia from below the knee to above the ankle in the medial leg; 20 minutes elapsed between placement of the anesthesia and the start of the procedure. However, five patients required additional intravenous analgesia (tramadol 100 mg with trifluoperazine 10 mg or midazolam 2 mg with piritramid 2 mg) and the procedure had to be stopped in four other patients due to extreme pain. They attributed this need for additional anesthesia to excess subcutaneous fat and increased body mass index. Also, administering tumescent anesthesia to patients with lipodermatosclerosis was difficult because of the extensive induration present. In the authors’ opinion, these patients would best be served with the use of general anesthesia. Tumescent anesthesia was effective in the vast majority of patients and its use reduced the risk of DVT, allowed increased mobility post surgery, and did not carry the risks associated with general anesthesia.

    However, in some cases, ulcers recur, suggesting SEPS may be a treatment but not a cure for venous insufficiency. Rhodes et al¹³⁴ reported that ulcer recurrence was associated with venous outflow obstruction. Multilevel deep venous reflux and size of the ulcer (>2 cm) were found to delay healing. Bianchi et al¹³⁵ showed that ulcer recurrence after SEPS was associated with prior trauma to the area and the presence of greater saphenous vein reflux and deep venous obstruction. However, randomized trails have not been performed.

Recombinant Growth Factors

    It is believed that venous ulcers do not heal because the environment of the wound bed is disturbed due to the long-standing effects of CVI.¹³⁶ Therefore, artificial stimuli in the form of exogenous growth factors have been used on these ulcers.

    Platelet-derived wound healing factor. Reutter et al¹³⁶ conducted a controlled study comparing the effects of topical platelet-derived wound healing factor (PDWHF) to placebo cream. The platelet-derived wound healing factor did not yield any significant clinical advantage. However, capillary density and transcutaneous oxygen measurement increased in the PDWHF ulcers, suggesting PDWHF has neoangiogenic abilities.

    Coerper et al¹³⁷ evaluated PDWHF on recalcitrant venous ulcers greater than 6 months’ duration and showed higher healing rates than with placebo cream. The overall healing rate was 77%. Other platelet products, such as platelet releasate, platelet lysate, and autologous cryopreserved platelets, have been used topically to treat patients with venous ulcers; however, all failed to demonstrate significant healing rates when compared to placebo.^138-140

    Granulocyte-macrophage colony stimulating factor. Granulocyte-macrophage colony stimulating factor (GM-CSF) has been used successfully in treating venous ulcers. Jaschke et al¹⁴¹ reported complete healing in 90.4% of ulcers treated with topical GM-CSF and compression dressings in an uncontrolled study. Mean healing time was 19 weeks without significant side effects. After 40 weeks, no recurrence of the healed ulcers was noted. Borbolla-Escoboza et al¹⁴² performed an open study involving the single weekly perilesional injection of 300 mcg of GM-CSF into the ulcer bed. After 4 weeks, eight out of 10 ulcers had healed and two out of 10 ulcers decreased 21% in size. Only one patient reported moderate pain with the injections. Da Costa et al¹⁴³ compared perilesional injection of 200 mcg of GM-CSF, perilesional injection of 400 mcg of GM-CSF, and placebo injection. Each patient received four perilesional injections each week for 4 weeks or until the ulcer had healed. Of the ulcers treated with 400 mcg, 61% were healed, compared to 57% of the ulcers treated with 200 mcg dose and 19% of the ulcers treated with the placebo. After 6 months, no recurrence was noted in ulcers that had healed.

    Human keratinocyte growth factor. Human keratinocyte growth factor (KGF) has been shown to promote proliferation and migration of keratinocytes and stimulate granulation tissue formation by the chemoattraction of fibroblasts.¹⁴⁴ Robson et al¹⁴⁴ published their findings from a multicenter, randomized, double-blind, placebo-controlled trial comparing the topical application of HKGF (Repifermin®, Human Genome Sciences, Rockville, Md.) at 20 µg/cm² plus compression, HKGF 60 µg/cm² plus compression, and placebo plus compression. They found more patients achieved 75% closure with HKGF, especially those wound that were ≤15 cm² or ≤18 months’ duration. Also, patients treated with placebo experienced more bacterial contaminations and more pain. However, a subsequent larger trial did not demonstrate improved healing.

    Human epidermal growth factor. Human epidermal growth factor (EGF) has been shown to enhance wound healing in donor sites of patients who had STSG harvested.¹⁴⁵ Falanga et al¹⁴⁶ reported on 44 patients with venous ulcers treated in a randomized, double-blind, placebo-controlled trial comparing EGF plus compression to placebo plus compression. They found no significant difference in healing between the two groups (35% versus 11%). However, a greater trend toward healing and a greater decrease in size of the ulcers in the HEGF treated group were noted.

Novel Therapies

    Wound healing is a complex process involving the interactions of various cell types, cytokines, and growth factors. One cell of particular importance is the keratinocyte, which when deficient leads to delayed healing. Cultured keratinocyte grafts, which were previously discussed, are effective but may be difficult to obtain and use. Because the outer root sheath of anagen scalp hairs contain keratinocytes with high proliferative capacity and are easy to procure, Limat et al¹⁴⁷ cultured these scalp hairs with autologous serum and coated them with human dermal fibroblasts to create epidermal equivalents (EE). Using a case series study design, these EE were applied to 50 leg ulcers, both venous and venous/arterial, most of which had failed prior autologous split-thickness skin grafting. After 8 weeks, 70% of the wounds were re-epithelialized and 32% were completely healed. Patients also noted a significant relief of their pain following the placement of EE.

    Chronic wounds have shown many changes characteristic of cellular senescence, which may indicate a deficiency of stem cells for wound repair. Because bone marrow contains stem cells capable of differentiating into non-hematopoetic tissue important to organ repair and wound healing, Badiavas et al¹⁴⁸ studied the use of bone marrow cell grafting in acutely wounded mice. Next, they grafted autologous bone marrow cells onto four patients with chronic wounds of greater than 1-year duration that had failed bioengineered skin and autologous STSGs.¹⁴⁹ All patients healed with no recurrence.

Conclusion

    Chronic venous ulcers are a common and debilitating condition afflicting a significant portion of the population. They are difficult to treat. Standard therapy includes compression therapy with or without oral medication. Several successful systemic treatments have been reported in the literature, including pentoxifylline, aspirin, and micronized purified flavenoids. In refractory cases, skin grafts and other surgical approaches to correcting venous disease are therapeutic alternatives. Both autologous split-thickness skin grafts and cultured skin grafts have shown success in healing venous ulcers. The latter (tissue-engineered skin) has been found in randomized trials to be superior to compression alone. Other surgical options to consider in patients with venous ulcers are sclerotherapy and subfascial endoscopic perforator surgery. Also, in some instances topical growth factors have been effective in healing venous ulcers — the rationale is that chronic wounds have been shown to possess diminished amounts of “normal” growth factors compared with acute wounds. Finally, recent reports using autologous scalp hairs to create epidermal equivalents and the use of bone marrow grafting to supply stem cells to the wound bed offer intriguing future research and therapeutic possibilities.

1. Lazarus GS, Cooper DM, Knighton DR, et al. Definitions and guidelines for assessment of wounds and evaluation of healing. Arch Dermatol. 1994;130:489–493.

2. Kirsner RS, Eaglstein WH, Kerdel FA. Split-thickness skin grafting for lower extremity ulceration. Dermatol Surg. 1997;23:85–91.

3. Picascia DD, Roenigk HH. Surgical management of leg ulcers. Adv Dermatol Surg. 1987;5:303–312.

4. Samson HS, Showalter DP. Stockings and the prevention of recurrent venous ulcers. Dermatol Surg. 1996;22:373–376.

5. Vanhoutte PM, Corcaud S, De Montrion C. The demographics of venous disease of the lower limbs. Angiology. 1997;48:557–558.

6. Ruckley CV. Socioeconomic impact of chronic venous insufficiency and leg ulcers. Angiology. 1997;48:67–69.

7. Colgan MP, Dormandy JA, Jones PW, et al. Oxypentifylline treatment of venous ulcers of the leg. BMJ. 1990;300:972–975.

8. Coon WW, Willis PW III, Keller JB. Venous thromboembolism and other venous disease in the Tecumseh Community Health Study. Circulation. 1973;58:839–846.

9. Alguire PC, Mathes BM. Chronic venous insufficiency and venous ulceration. JGIM. 1997;12:374–383.

10. Dalen JE, Paraskos JA, Ockene IS, et al. Venous thromboembolism: scope of the problem. Chest. 1986;89:370S-373S.

11. Callam MJ, Ruckley CV, Harper DR, Dale JJ. Chronic ulceration of the leg: extent of the problem and provision of care. BMJ. 1985;290:1855–1856.

12. Chaetle TR, Scott HJ, Scurr JH, Coleridge Smith PD. White cells, skin blood flow and venous ulcers. Br J Dermatol. 1991;125:288–290.

13. Scott TE, LaMorte WW, Gorin DR, Menzoian JO. Risk factors for chronic venous insufficiency: a dual case-control study. J Vasc Surg. 1995;22:622–628.

14. Olin JW, Beusterien KM, Childs MB, Seavey C, McHugh L, Griffiths RI. Medical costs of treating venous stasis ulcers: evidence from a retrospective cohort study. Vasc Med. 1999;4:1–7.

15. Dinner MI, Peters CR. Surgical management of the ulcers on the lower limbs. J Dermatol Surg Oncol. 1978;4:696–699.

16. Welch HJ, Young CM, Semegran AB, et al. Duplex assessment of venous reflux and chronic venous insufficiency: the significance of deep venous reflux. J Vasc Surg. 1996;24:755–762.

17. White JV, Katz ML, Cisek P, Kreithen J. Venous outflow of the leg: anatomy and physiologic mechanism of the plantar venous plexus. J Vasc Surg. 1996;24:819–824.

18. Norgren L. Chronic venous insufficiency — a well known disorder with many question marks. Angiology. 1997;48:23–26.

19. Ruffieux PH, Hommel I, Saurat JH. Long-term assessment of chronic leg ulcer treatment by autologous skin grafts. Dermatology. 1997;195:77–80.

20. Lawrence MB, McIntire LV, Eskin SG. Effect of flow on polymorphonuclear leukocyte/endothelial cell adhesion. Blood. 1987;70:1284–1290.

21. Coleridge Smith PD, Scurr JH, Dormandy JA. Causes of venous ulceration: a new hypothesis. Br Med J. 1988;296:1693–1727.

22. Scott HJ, Coleridge Smith PD, Scurr JH. Histologic study of white blood cells and their association with lipodermatosclerosis and venous ulceration. Br J Surg. 1991;78:210–211.

23. Browse NL, Burnand KG. The cause of venous ulceration. Lancet. 1982;ii:243–254.

24. Franzeck UK, Bollinger A, Huch R, Huch A. Transcutaneous oxygen tension and capillary morphologic characteristics and density in patients with chronic venous incompetence. Circulation. 1984;70:806–811.

25. Landis EM. Microinjection studies of capillary blood pressure in human skin. Heart. 1930;15:404–453.

26. Pietra GG, Szidon JP, Leventhal MM, Fishman AP. Hemoglobin as a tracer in hemodynamic pulmonary edema. Science. 1969;166:1643–1646.

27. Burnand KG, Whimster I, Naidoo A, Browse NL. Pericapillary fibrin in the ulcer bearing skin of the leg: the cause of lipodermatosclerosis and venous ulceration. BMJ. 1982;285:1071–1072.

28. Danielpour D, Sporn MB. Differential inhibition of transforming growth factor beta-1 and beta-2 activity by alpha-2 macroglobulin. J Biol Chem. 1990;265:6973–6977.

29. Falanga V, Eaglstein WH. The “trap” hypothesis of venous ulceration. Lancet. 1993;341:1006–1008.

30. Malanin K. About the pathophysiology of venous leg ulceration. JAAD. 2002;47:157–158.

31. Labropoulos N, Giannoukas AD, Nicolaides AN, et al. The role of venous reflux and calf muscle pump function in nonthrombotic chronic venous insufficiency. Arch Surg. 1996;131:403–406.

32. Labropoulos N, Delis K, Nicolaides AN, et al. The role of the distribution and anatomic extent of reflux in the development of signs and symptoms in chronic venous insufficiency. J Vasc Surg. 1996;23:504–510.

33. Subcommittee on Reporting Standards. Classification of CVI. 1994. Available at: www.phlebology.com.

34. Phillips TJ, Machado F, Trout R, Porter J, Olin J, Falanga V. Prognostic indicators in venous ulcers. JAAD. 2000;43:627–630.

35. Margolis DJ, Berlin JA, Strom B. Risk factors associated with the failure of a venous ulcer to heal. Arch Dermatol. 1999;135:920–926.

36. Skene AI, Smith JM, Dore CJ, Charlett A, Lewis DJ. Venous leg ulcers: a prognostic index to predict time to healing. Br Med J. 1992;305:1119–1121.

37. Mayberry JC, Moneta GL, Taylor LM, Porter JM. Fifteen years’ results of ambulatory compression therapy. Surgery. 1991;109:575–581.

38. Valencia IC, Falabella A, Kirsner RS, Eaglstein WH. Chronic venous insufficiency and venous leg ulceration. J Am Acad Dermatol. 2001;44:401–421.

39. Phillips T, Stanton B, Provan A, Lew R. A study of the impact of leg ulcers on quality of life: Financial, social, and psychological implications. J Am Acad Dermatol. 1994;31(1):49–53.

40. Freidman SA. The diagnosis and medical management of vascular ulcers. Clin Dermatol. 1990;8(3/4):30–48.

41. Nelzen O, Bergqvist D, Lindhagen A. Leg ulcer etiology- a cross sectional population study. J Vasc Surg. 1991;14:557–564.

42. Browse NL, Clemenson G, Lea Thomas M. Is the postphlebitic leg always postphlebitic? Relation between phlebographic appearances of deep vein thrombosis and late sequelae. Br Med J. 1980;281:1167–1170.

43. Sibbald RG. An approach to leg and foot ulcers: a brief overview. Ostomy Wound Manage. 1998;44:28-32.

44. Falanga V. Venous ulceration. J Dermatol Surg Oncol. 1993;19:764–771.

45. Falanga V. Venous ulceration. WOUNDS. 1996;8(3):102–108.

46. Maessen-Visch MB, Koedam MI, Hamulyak K, Neuman HAM. Atrophie blanche. Int J Dermatol. 1999;38:161–172.

47. Kirsner RS, Pardes JB, Eaglstein WH, Falanga V. The clinical spectrum of lipodermatosclerosis. J Am Acad Dermatol. 1993;28:623–627.

48. Phillips TJ, Dover JS. Leg ulcers. J Am Acad Dermatol. 1991;25(6, Pt.1):965–987.

49. Phillips TJ. Successful methods of treating leg ulcers: the tried and true plus the novel and new. Postgrad Med. 1999;105:159–179.

50. McGee SR, Boyko EJ. Physical examination and chronic lower-extremity ischemia. A critical review. Arch Intern Med. 1998;158:1357–1364.

51. Scriven JM, Hartshorne T, Bell PR, et al. Single visit venous ulcer assessment clinic: the first year. Br J Surg. 1997;84:334–336.

52. Lopez A, Phillips TJ. Venous ulcers. WOUNDS. 1998;10149–157.

53. McGukin M, Stineman M, Goin J, et al. Draft guideline: diagnosis and treatment of venous leg ulcers. Ostomy Wound Manage. 1996;42:48–78.

54. Labropoulos N, Leon M, Geroulakos G, et. Al. Venous hemodynamic abnormalities in patients with leg ulceration. Am J Surg. 1995;169:572–574.

55. Thibault PK. Duplex examination. Dermatologica. 1995;21:77–82.

56. Grayson ML, Gibbons GW, Balogh K, Levin E, Karchmer AW. Probing to bone in infected pedal ulcers: a clinical sign of underlying osteomyelitis in diabetic patients. JAMA. 1995;273:721–723.

57. Ackroyd JS, Young AE. Leg ulcers that do not heal. Br Med J. 1983;286:207–208.

58. Hansson C, Andersson E. Malignant skin lesions on the legs and feet at a dermatological leg ulcer clinic during five years. Acta Derm Venereol. 1997;78:147–148.

59. Kramer SA. Compression wraps for venous ulcer healing: a review. J Vasc Nurs. 1999;17:89–97.

60. Cherry GW, Hoffman D, Cameron J, Poore SM. Bandaging in the treatment of venous ulcers: an European view. Ostomy Wound Manage. 1996;42:13S–18S.

61. Partsch H. Compression therapy of the legs. J Dermatol Surg Oncol. 1991;17:799–805.

62. Fletcher A, Cullum N, Sheldon TA. A systematic review of compression treatment for venous leg ulcers. BMJ. 1997;315:576–560.

63. Staudinger P, Weiss RA. Compression therapy: low or short stretch bandage and graduated compression stockings for leg edema. Dermatol Surg. 1995;106.

64. Hafner J, Botonakis I, Burg G. A comparison of multilayer bandage systems during rest, exercise, and over 2 days of wear time. Arch Dermatol. 2000;136:857–863.

65. Spence RK, Cahall E. Inelastic versus elastic leg compression in chronic venous insufficiency: a comparison of limb size and venous hemodynamics. J Vasc Surg. 1996;24:783–787.

66. Maune J, Giordano J. Experience with open-heeled Unna boot application technique. J Vasc Nurs. 1997;15:63–72.

67. Trent JF, Kirsner RS. Tissue engineered skin: Apligraf, a bi-layered living skin equivalent. International J Clin Pract. 1998;52:408–413.

68. Gould DJ, Campbell S, Newton H, Duffelen P, Griffin M, Harding EF. Setopress vs. Elastocrepe in chronic venous ulceration. Br J Nursing. 1998;7:66–73.

69. Williams C. Profore four-layer bandage system. Br J Nurs. 1996;5:1075–1076.

70. Falanga V. Venous ulceration. In: Krasner D, Kane D, eds. Chronic Wound Care, Second Edition. Wayne, Pa.: Health Management Publications, Inc.;1997:165–171.

71. Falanga V, Eaglstein WH. Compression. In: Falanga V, Eaglstein WH. Leg and Foot Ulcers: A Clinician’s Guide. UK: Martin Dunitz Ltd;1995:144–165.

72. Partsch H. Compression therapy of the Legs. J Dermatol Surg Oncol. 1991;17:799–805.

73. Richmond DM, O’Donnell TF, Zelikovski A. Sequential pneumatic compression for lymphedema. Arch Surg. 1985;120:1116–1119.

74. Cullum N, Nelson EA, Fletcher AW, Sheldon TA. Compression for venous leg ulcers (Cochrane Review). Cochrane Database Syst Rev. 2000;3:CD000265.

75. Johnson G, Kupper C, Farrar DJ, Swallow RT. Graded compression stockings. Arch Surg. 1982;117:69–72.

76. Sigel B, Edelstein AL, Savitch L, et al. Type of compression for reducing venous stasis. Arch Surg. 1975;110:173–175.

77. Sigel B, Edelstein AL, Felix WR, et al. Compression of the deep venous system of the lower leg during inactive recumbency. Arch Surg. 1973;106:38–43.

78. Lewis CE, Antoine J, Mueller C, et al. Elastic compression in the prevention of venous stasis. Am J Surg. 1976;132:739–743.

79. Scurr JH, Ibrahim SZ, Faber RG, et al. The efficacy of graduated compression stockings in the prevention of deep vein thrombosis. Br J Surg. 1977;64:371–373.

80. Holford CP. Graded compression for preventing deep venous thrombosis. Br Med J. 1976;2:969–970.

81. Barnes RW, Brand RA, Clarke W, et al. Efficacy of graded compression anti-embolism stockings in patients undergoing total hip arthroplasty. Clin Orthop Rel Res. 1978;132:61–67.

82. Wilkins RW, Stanton JR. Elastic compression stockings in the prevention of pulmonary embolism. NEJM. 1953;248:1087–1090.

83. Nielsen PG, Madsen SM, Stromberg L. Treatment of chronic leg ulcers with a hydrocolloid dressing. Acta Derm Venerol. 1989;152 (suppl):1–12.

84. Eaglstein WH, Falanga V. Chronic wounds. Surg Clin North Am. 1997;77:689–700.

85. Mulder GD, Reis TM. Venous ulcers: pathophysiology and medical therapy. Am Fam Physician. 1990;42:1323–1330.

86. Fitzpatrick TB, Johnson RA, Wolff K, Polano MK, Suurmond D. Leg ulcers. Color Atlas and Synopsis of Clinical Dermatology. 1997;486–491.

87. Layton AM, Ibbotson SH, Davies JA, Goodfield MJD. Randomised trial of oral aspirin for chronic venous leg ulcers. Lancet. 1994;344:164–165.

88. Puchmayer V. Modern therapeutic trends in the conservative treatment of ischemic disease of the lower extremities. Vnitrni Lekarstvi. 1995;41:204–206.

89. Ratner D. Skin grafting. Dermatol Clinics. 1998;16:75–90.

90. Angelkort B, Kiesewetter H. Influence of risk factors and coagulation phenomenon on the fluidity of blood on chronic arterial occlusive disease. Scand J Clin Lab Invest. 1984;41:185–188.

91. Ambrus JL, Stadler S, Kulaylat M. Hemorrhagic effects of metabolites of pentoxifylline (Trental). J Med. 1995;26:65–75.

92. Belcaro G,Cesarone MR, Nicolaides AN, DeSanctis MT, Incandela L, Geroulakos G. Treatment of venous ulcers with pentoxifylline: a 6-month, randomized, double-blind, placebo-controlled trial. Angiology. 2002;53:45–48.

93. Falanga V, Fujitani RM, Diaz C, et al. Systemic treatment of venous leg ulcers with high doses of pentoxifylline: efficacy in a randomized, placebo-controlled trial. Wound Repair Regen. 1999;7:208–213.

94. DeSanctis MT, Belcaro G, Cesarone MR, et al. Treatment of venous ulcers with pentoxifylline: a 6-month randomized, double-blind, placebo controlled trial. Angiology. 2002;53:49–52.

95. Jull A, Waters J, Arroll B. Pentoxifylline for treatment of venous leg ulcers: a systematic review. Lancet. 2002;359:1550–1554.

96. Ibbotson SH, Layton AM, Davies JA, Goodfield MJD. The effect of aspirin on haemostatic activity in the treatment of chronic venous leg ulceration. Br J Dermatol. 1995;132:422–426.

97. Lyseng-Williamson KA, Perry CM. Micronized purified flavonoid fraction. Drugs. 2003;63:71–100.

98. Bergan JJ, Schmid-Schonbein GW, Takase S. Therapeutic approach to chronic venous insufficiency and its complications: place of Daflon® 500 mg. Angiology. 2001;52:43–50.

99. Ramelet AA. Clinical benefits of Daflon® 500 mg in the most severe stages of chronic venous insufficiency. Angiology. 2001;52:49–64.

100. Guilhou JJ, Fevrier F, Debure C. Benefits of a 2-month treatment with a micronized purified flavonoidic fraction on venous ulcer healing: a randomized double blind controlled versus placebo trial. Int J Microcirc Clin Exp. 1997;17:21–26.

101. Glinski W, Chodynicka B, Roszkiewicz J. The beneficial augmentative effect of micronized purified flavonoid fraction on the healing on leg ulcers: an open multicenter controlled randomized study. Phlebology. 1999;14:151–157.

102. Morris PJ, Bondoc C, Burke JF. The use of frequently changed skin allografts to promote healing in the nonhealing infected ulcer. Surgery. 1966;60:13–19.

103. Kealey GP. Disease transmission by means of allograft. J Burn Care Rehab. 1997;18:10S–11S.

104. Skouge JW. Techniques for split thickness skin grafting. J Dermatol Surg Oncol. 1987;13:841–849.

105. Kirsner RS, Falanga V. Techniques of split-thickness skin grafting for lower extremity ulcerations. J Dermatol Surg Oncol. 1993;19:779–783.

106. Davison PM, Batchelor AG, Lewis-Smith PA. The properties and uses of non-expanded machine meshed skin grafts. Br J Plastic Surg. 1986;39:462–468.

107. Fatah MF, Ward CM. The morbidity of split-skin graft donor sites in the elderly: the case for mesh-grafting the donors. Br J Plastic Surg. 1984;37:184–190.

108. Millard LG, Roberts MM, Gatecliffe M. Chronic leg ulcers treated by the pinch graft method. Br J Dermatol. 1977;97:289–295.

109. Ahnlide I, Bjellerup M. Efficacy of pinch grafting in leg ulcers of different aetiologies. Acta Derm Venereol. 1997;77:144–145.

110. Kirsner RS, Mata SM, Falanga V, Kerdel FA. Split-thickness skin grafting of leg ulcers. Dermatol Surg. 1995;21:701–703.

111. Schmeller W, Gaber Y, Gehl HB. Shave therapy is a simple, effective treatment of persistent venous leg ulcers. JAAD. 1998;39:232–238.

112. Limova M, Mauro T. Treatment of leg ulcers with cultured epithelial autografts: clinical study and case reports. Ostomy Wound Manage. 1995;41:48–50.

113. Hefton JM, Caldwell D, Biozes DG, Balin AK, Carter DM. Grafting of skin ulcers with cultured autologous epidermal cells. JAAD. 1986;14:399–405.

114. Phillips TJ. New skin for old: developments in biological skin substitutes. Arch Dermatol. 1998;134:344–349.

115. Leigh IM, Purkis PE, Navsaria HA, Phillips TJ. Treatment of chronic venous ulcers with sheets of cultured allogenic keratinocytes. Br J Dermtol. 1987;117:591–597.

116. Bolivar-Flores YJ. Kuri-Harcuch W. Frozen allogeneic human epidermal cultured sheets for the cure of complicated leg ulcers. Dermatol Surg. 1999;25:610–617.

117. Teepe RGC, Roseeuw DI, Hermans J, et al. Randomized trial comparing cryopreserved cultured epidermal allografts with hydrocolloid dressings in healing chronic venous ulcers. JAAD. 1993;29:982–988.

118. Phillips TJ, Manzoor J, Rojas A, et al. The longevity of a bilayered skin substitute after application to venous ulcers. Arch Dermatol. 2002;138:1079–1081.

119. Falanga V, Margolis D, Alvarez O, et al. Rapid healing of venous ulcers and lack of clinical rejection with an allogeneic human skin equivalent. Arch Dermatol. 1998;134:293–300.

120. Falanga V, Sabolinski M. A bilayered living skin construct (Apligraf®) accelerates complete closure of hard to heal venous ulcers. Wound Rep Reg. 1999;7:201–207.

121. Brem H, Balledux J, Sukkarieh T, Carson P, Falanga V. Healing of venous ulcers of long duration with a bilayered living skin substitute: results from a general surgery and dermatology department. Dermatol Surg. 2001;11:915–919.

122. Kirsner RS, Fastenau J, Falabella A, Valencia I, Long R, Eaglstein WH. Clinical and economic outcomes with Graftskin for hard to heal venous leg ulcers: a single center experience. Dermatol Surg. 2002;28:81–82.

123. Martin LM, Kirsner RS. Use of a meshed bilayered cellular matrix to treat a venous ulcer. Adv Skin Wound Care. 2002;15:260–264.

124. Orcel® press release December 2003. Ortec International. Data on file.

125. Takeda Y, Agui T, Tanaka K, Okuzawa M, Tanigawa N. Sclerotherapy with a ligation of incompetent veins for a stasis ulcer due to varix cruris: minimal invasive therapy for varix cruris. Surg Today. 1999;29:1154–1157.

126. Queral LA, Criado FJ, Lilly MP, Rudolphi D. The role of sclerotherapy as an adjunct to Unna’s boot for treating venous ulcers: a prospective study. J Vasc Surg. 1990;11:572–575.

127. Murray JD, Bergan JJ, Riffenburgh RH. Development of open-scope subfascial perforating vein surgery: lessons learned from the first 67 cases. Ann Vasc Surg. 1999;13:372–377.

128. Gloviczki P. Subfascial Endoscopic perforator vein surgery: indications and results. Vasc Med. 1999;4:173–180.

129. Lee QWH, Chan ACW, Lam YH, et al. Early outcomes after subfascial endoscopic perforator surgery (SEPS) and saphenous vein Surgery in chronic venous insufficiency. Surg Endosc. 2001;15:737–740.

130. Rhodes JM, Gloviczki P, Canton L, Heaser TV, Rooke TW. Endoscopic perforator vein division with ablation of superficial reflux improves venous hemodynamics. J Vasc Surg. 1998;28:839–847.

131. Kolvenbach R, Ramadan H, Schwierz E. Redone endoscopic perforator surgery: feasibility and failure analysis. J Vasc Surg. 1999;30:720–726.

132. Gloviczki P, Bergan JJ, Rhodes JM, Canton LG, Harmsen S, Ilstrup DM. Mid-term results of endoscopic perforator vein interruption for chronic venous insufficiency: lessons learned from the North American subfascial endoscopic perforator surgery registry. The North American Study Group. J Vasc Surg. 1999;29:489–502.

133. Proebstle TM, Bethge S, Barnstedt S, Kargl A, Knop J, Sattler G. Subfascial endoscopic perforator surgery with tumescent local anesthesia. Dermatol Surg. 2002;28:689–693.

134. Rhodes JM, Gloviczki P, Canton LG, Rooke T, Lewis BD, Lindsey JR. Factors affecting clinical outcome following endoscopic perforator vein ablation. Am J Surg. 1998;176:162–167.

135. Bianchi C, Ballard JL, Abou-Zamzam AM, Teruya TH. Subfascial endoscopic perforator vein surgery combined with saphenous vein ablation: results and critical analysis. J Vasc Surg. 2003;38:67–71.

136. Reutter H, Bort S, Jung MF, et al. Questionable effectiveness of autologous platelet growth factors (PDWHF) in treatment of venous ulcers of the leg. Hautarzt. 1999;50:859–865.

137. Coerper S, Koveker G, Flesch I, Becker HD. Ulcus cruris venosum: surgical debridement, antibiotic therapy and stimulation with thrombocytic growth factors. Langenbecks Arch Chir. 1995;380:102–107.

138. Stacey MC, Mata SD, Trengove NJ, Mather CA. Randomized double-blind placebo controlled trial of topical autologous platelet lysate in venous ulcer healing. Eur J Vasc Endovasc Surg. 2000;20:296–301.

139. Krupski WC, Reilly LM, Perez S. A prospective randomized trial of Autologous platelet-derived wound healing factors for treatment of chronic nonhealing wounds: a preliminary report. J Vasc Surg. 1991;14:526–536.

140. Senet P, Bon FX, Benbunan M, et al. Randomized trial and local biological effect of autologous platelets used as adjuvant therapy for chronic venous leg ulcers. J Vasc Surg. 2003;38:1342–1348.

141. Jaschke E, Zabernigg A, Gattringer C. Recombinant human granulocyte-macrophage colony stimulating factor applied locally in low doses enhances healing and prevents recurrence of chronic venous ulcers. Int J Dermatol. 1999;38:380–386.

142. Borbolla-Escoboza JR, Maria-Aceves R, Lopez-Hernandez MA, Collados-Larumbe MT. Recombinant human granulocyte-macrophage colony stimulating factor as treatment for chronic leg ulcers. Rev Invest Clin. 1997;49:449–451.

143. Da Costa RM, Ribeiro Jesus FM, Aniceto C, Mendes M. Randomized, double-blind, placebo-controlled, dose-ranging study of granulocyte-macrophage colony stimulating factor in patients with chronic venous leg ulcers. Wound Repair Regen. 1999;7:17–25.

144. Robson MC, Phillips TJ, Falanga V, et al. Randomized trial of topically applied Repifermin (recombinant human keratinocyte growth factor 2) to accelerate wound healing in venous ulcers. Wound Rep Reg. 2001;9:347–352.

145. Brown GL, Nanney LB, Griffin J, et al. Enhancement of wound healing by topical treatment with epidermal growth factor. NEJM. 1989;321:76–79.

146. Falanga V, Eaglstein WH, Bucalo B, Katz MH, Harris B, Carson P. Topical use of human recombinant epidermal growth factor (h-EGF) in venous ulcers. J Dermatol Surg Oncol. 1992;18:604–606.

147. Limat A, French LE, Blal L, Saurat JH, Hunziker T, Salomon D. Organotypic cultures of autologous hair follicle keratinocytes for the treatment of recurrent leg ulcers. JAAD. 2003;48:207–214.

148. Badiavas EV, Abedi M, Butmarc J, Falanga V, Quesenberry P. Participation of bone marrow derived cells in cutaneous wound healing. J Cell Physiol. 2003;196:245–250.

149. Badiavas EV, Falanga V. Treatment of chronic wounds with bone marrow derived cells. Arch Dermatol. 2003;139:510–516.