A Closer Look At The Research On Bilayered Living Cell Therapy

George Liu, DPM, FACFAS, and John Steinberg, DPM, FACFAS


Diabetic foot ulcers are among the many complications encountered with poorly controlled diabetes mellitus. Approximately 15 percent of all patients with diabetes will experience an ulcer in their lifetimes.1,2 Additionally, 85 percent of all nontraumatic lower extremity amputations are preceded by a preventable ulceration.3,4
   Diabetic foot ulcerations pose a considerable economic burden. In 1995, Medicare spent $1.5 billion on diabetic lower extremity ulcers.5 One retrospective analysis found that foot ulcer management averaged up to $27,987 two years from the time of diagnosis.6 Harrington, et al., suggested that if healing rates could increase by 9 percent at a 20-week time period, the cost savings would average $189 per episode.5
   Problem chronic wounds are defined as wounds that do not heal or respond to standard treatment. Prolonged wound exposure increases the risk for bacterial infection.Wound healing in patients with diabetes is physiologically impaired. Hyperglycemia delays phagocytosis and the migration of inflammatory cells pertinent to the acute phase of wound healing.7 Nonenzymatic glycosylation, altered endothelial cell proliferation and impaired collagen deposition prolong the inflammatory phase of wound healing, causing the wound to linger in the chronic stage.
   In chronic venous ulcerations, investigators have found alterations of the wound tissue phenotype that cause an unresponsiveness to specific growth factors needed to promote wound healing.8,9 In addition, chronic wounds often have abnormal expression of individual growth factors needed to stimulate granulation.
   While one may address the primary etiology of ulcerations with offloading for diabetic foot ulcerations and compression therapy for venous leg ulcerations, abnormal intrinsic wound physiology may still persist in prevention of progressive wound healing. Several advances address stagnant wound healing and these advances include recombinant human platelet-derived growth factors, collagen-alginate dressings and growth factors.10-15 These modalities have produced variable stimulation of wound healing.
   Over the years, more bioactive cellular analogs similar to skin were needed to stimulate healing effectively in diabetic wounds that were larger, deeper and chronic in duration. One of the few advances in human living cell therapy was cultured epidermal human keratinocytes, which were procured from a full-thickness biopsy of the patient’s own skin. One would cultivate the autogenic keratinocytes and subsequently apply them later to the wound in the form of sheets. Researchers believed this living allograft stimulated chronic wounds through the delivery of cytokines.16
   While the autogenic keratinocytes demonstrated encouraging success with venous stasis wounds, clinical trials were limited as the process of harvesting and culturing autologous keratinocytes required several weeks.17,18 Another major disadvantage of a keratinocyte sheet was its inherent fragility due to the absence of a dermal layer, which caused difficulty in the application to wounds.19 Evidence suggested that a dermal graft component was necessary to provide a collagen structural lattice and fibroblasts which play an important role in wound healing.20-22

Key Insights On The Makeup And Mechanisms Of Action For Bilayered Living Cell Grafts

   Apligraf (Organogenesis), also referred to as graftskin or human skin equivalent (HSE), is a living, bilayered skin substitute with an epidermal layer consisting of viable human keratinocytes and a dermal layer composed of viable human fibroblasts in a bovine type I collagen lattice. There are four components of the graft: epidermal keratinocytes, the stratum corneum, allogenic dermal fibroblasts and the extracellular matrix. Both keratinocytes and fibroblasts are harvested and cultured from a donor human neonatal foreskin.
   Though Apligraf is histologically similar to human skin, this skin equivalent is absent of endothelial, melanocytes, eccrine glands and Langerhans cells, which can contribute to host rejection.23 The collagen lattice provides a matrix to promote cellular ingrowth and mechanical stability to the graft.
   In addition to providing a structural scaffold for wound healing, both epidermal keratinocytes and dermal fibroblasts produce growth factors, cytokines and regulatory factors that physiologically stimulate wound healing.24 Apligraf releases wound healing factors, such as interleukin (IL-1, IL-3, IL-5, IL-6, IL-8), fibroblast growth factors and transforming growth factors α and Β.25-27 Apligraf closely resembles the biology and histology of human skin.
   Various researchers have suggested that bilayered cell therapy has several mechanisms of action. Some have proposed that the occlusive biologic barrier dressing allows secondary intention wound healing.28,29
   Other investigators have noted that the biologic medium provides wound stimulation via growth factors, cytokines and matrix materials.27,29,30 Various studies have also suggested that bilayered cell therapy serves as a graft substitute for vascularization and integration.29-31

What The Literature Says About The Efficacy Of Apligraf For Venous Stasis Ulcers

   In 1998, Falanga, et al., reported the first multicenter, prospective randomized trial on the application of Apligraf for venous stasis ulcerations.23 They compared 129 patients treated with compression therapy alone to 146 patients who received Apligraf. They found that patients treated with the human skin equivalent had a 54 percent improved chance for wound healing in comparison to the control group. In groups that had ulcers greater than six months in duration, patients treated with a human skin equivalent healed 98 days quicker in comparison to patients treated with compression alone.
   In 1999, Falanga and Sabolinski reported a prospective, multicenter, randomized, controlled trial evaluating the efficacy of Apligraf on venous ulcerations greater than one year in duration.32 One hundred and twenty patients participated with 72 randomized to Apligraf application with compression therapy and 48 randomized to compression alone. At six weeks, ulcers in the Apligraf group were demonstrating three times higher closure rates in comparison to those of the compression group. Furthermore, at six months, 47 percent of the Apligraf group achieved complete closure in comparison to 19 percent in the compression group.
   In a prospective, randomized, controlled clinical trial, Sabolinski, et al., assessed the treatment of venous stasis ulcerations that had been present in 233 patients for six to 12 months.27 The study compared 127 patients treated with Apligraf and compression to 106 patients treated with compression alone. At six months, Apligraf patients demonstrated a faster rate of wound closure achieved at 57 days in comparison to 181 days for compression alone. Additionally, 64 percent achieved complete wound closure with Apligraf in comparison to 44 percent in the control group.

What The Research Reveals About Apligraf And Diabetic Foot Ulcers
   In a pivotal multicenter, randomized, prospective trial, Veves, et al., applied Apligraf to 112 patients with noninfected chronic ischemic plantar diabetic ulcerations and compared them to a control group of 96 patients treated with gauze moistened with normal saline.33
   At 12 weeks, 56 percent of the Apligraf group achieved statistically significant healing in comparison to 38 percent healing with the control group. These authors found that diabetic foot ulcers treated with Apligraf were two times more likely to achieve healing in comparison to ulcers treated with routine care.
   As part of a multicenter, prospective, randomized trial, Pham, et al., compared moistened saline gauze to a human skin equivalent in the treatment of diabetic foot ulcers.34 Application of the human skin equivalent each week for four consecutive weeks reduced wound closure time to 38.5 days in comparison to 91 days in those treated with moistened saline gauze.
   In a multicenter, prospective, randomized, controlled, comparative trial in 2002, Sams, et al., examined the healing rates of diabetic foot ulcers treated with Apligraf and standard dressing with moist saline gauze.35 At 12 weeks, 56 percent of patients with diabetic foot ulcers were healed in comparison to 38 percent healed in the control group.
   Brem, et al., reported a prospective, nonrandomized case series of 23 patients with 41 wounds (20 diabetic foot ulcerations and 21 sacral/hip decubitus lesions).They treated patients with Apligraf after excisional debridement of the wound.36 Eighty-five percent of all diabetic foot ulcers healed in an average of 42 days.
   Studies evaluating wound healing with Apligraf have demonstrated no clinical or immunologic evidence of host rejection. Clinical observations reveal no erythema, pain or degradation of the allograft on human skin.23,34,37
   Additionally, there was no serologic evidence of immunogenic reactions (allospecific T cells, HLA-cytolytic antibodies, anti-bovine type-I collagen antibodies) to antigens present on the keratinocytes, fibroblasts or bovine collagen found in subjects treated with Apligraf.23,29,37,38

Is There Clinical Proof Of Bioactivity With Apligraf Or Is It Just Another Expensive Dressing?

   In a multicenter, prospective, randomized study of venous ulcerations, Falanga, et al., found that in patients with ulcers less than six months in duration, Apligraf treatment was only as effective as employing compression therapy alone.23
   However, in patients with venous ulcers greater than six months in duration, Apligraf treated legs healed statistically quicker than the compression group (92 days versus 190 days for complete healing). This finding supported suggestions that Apligraf may provide biologic stimulation to chronic problem wounds.
   Another multicenter, prospective, randomized study evaluated the efficacy of Apligraf in the treatment of venous ulcers greater than one year in duration. At the six-month follow-up, the study authors found 47 percent of the Apligraf treated group achieved complete healing in comparison to 19 percent in the compression group.32
   Whether the living HSE incorporates (engraftment) or only acts as a biologic dressing to provide wound stimulation has been a topic of interest since successful clinical trials were published. In other words, how does Apligraf work to heal wounds? Clinical studies have suggested that autogenous skin grafts not only behave as replacement tissue but as a conduit to provide biologic activity to recipient sites.26
   Accordingly, recent studies have also suggested that Apligraf acts as a temporary bioactive graft, delivering growth factors to stimulate wound healing. In an investigative study, Hu, et al., evaluated the persistence of Apligraf and basement membrane on healing skin graft donor sites.39
   Using HLA antigen typing, investigators were able to identify a limited pres- ence of Apligraf keratinocytes and fibroblasts up to the fourth week after application, but found no evidence of the Apligraf DNA after the fourth week. Immunohistochemistry techniques to identify the presence of Apligraf basement membrane components did not demonstrate the residual presence of Apligraf basement membrane at the fourth week.
   Other studies evaluating the persistence of Apligraf on healing wounds also demonstrated the presence of Apligraf DNA only up to four to six weeks.40,41 The short persistence of Apligraf on the healing wounds suggests a gradual replacement of the allographic cells with host tissue.39 This phenomenon is referred to as silent rejection.
   Badiavas, et al., examined the grafthost interaction of Apligraf treated wounds via biopsy two weeks after application.42 Histologic examination found degeneration of the Apligraf collagen and dermal cells, and the presence of excess mucin indicated a mutual stimulatory interaction between the host wound bed and allograft.This finding indicates that the Apligraf fibroblasts may serve as a living tissue that stimulates host and graft cells to produce excess ground substance, which elicits a wound healing response.
   Matrix metalloproteinases (MMPs) contribute to proteolytic activity and cellular turnover. Chronic nonhealing wounds have lower levels of tissue inhibitors of matrix metalloproteinases (TIMPs), allowing uninhibited activity of matrix metalloproteinases. This causes delayed wound healing.43 Apligraf produces TIMPs and fibronectins, reestablishing balance to the deficient matrix and altered proteinase activity one sees in chronic wounds.This stimulates matrix formation and keratinocyte migration to the wound.
   In mathematical modeling of wound healing that compared Apligraf identified hyaluronan to other wound variables — such as platelet-derived growth factor, collagen density and fibroblast density — researchers found that Apligraf identified hyaluronan was the essential component responsible for healing in the diabetic model.44 In chronic diabetic wounds, inflammatory macrophages are in overabundance over repair macrophages.45 Waugh and Sharratt proposed that Apligraf restores hyaluronan levels in the wound, allowing monocytes to differentiate into repair macrophages and re-initiate the wound healing process.44

In Summary

   Use of a HSE does not substitute for appropriate wound debridement, which researchers have noted is a crucial and independent factor contributing to diabetic foot ulcer healing.46 Additionally, metaanalysis standard wound care revealed that 24 percent of diabetic neuropathic ulcers heal at 12 weeks and this increased to 31 percent at 20 weeks with standard moist dressings alone.47
   A systematic review of randomized controlled trials on wound dressing for chronic venous stasis ulceration found one significant trial demonstrating ulcer healing with Apligraf to be superior to Tegapore (63 percent to 48 percent respectively).48 In 2006, Cavorsi, et al., developed an algorithm for the practical use of Apligraf in the treatment of diabetic foot ulcerations based on a compilation of professional society guidelines and wound care clinical trials.49
   Current guidelines recommend the application of Apligraf if a diabetic foot ulcer is not responding to standard wound care including debridement and offloading for three weeks. Further studies are currently in progress as researchers are evaluating the histologic activity of HSE to host wound beds.
   Various researchers have shown in prospective, randomized, controlled, comparative trials that bilayered cell therapy effectively accelerates and stimulates healing in both chronic venous leg ulcers and diabetic foot ulcerations. Furthermore, allograft bilayered cell therapy provides biologic activity to stimulate wound healing without the problems of immunologic rejection. The use of HSE can effectively reduce the time of ulcer healing, thereby reducing complications of wound infection and the relative rate of amputation.
Dr. Liu is a Clinical Associate Professor in the Department of Orthopaedics at the University of Texas Health Science Center at San Antonio, Texas. Dr. Liu currently practices at the Austin Diagnostic Clinic multispecialty group in Austin,Texas.
Dr. Steinberg is an Assistant Professor in the Department of Plastic Surgery at the Georgetown University School of Medicine in Washington,D.C.








1. Palumbo P and Melton L. Peripheral vascular disease and diabetes. In Diabetes in America, pp. XV 1-21. Edited by Harris M and Hamman R, XV 1-21,Washington DC,U.S. Govt. Printing Office, 1985.
2. Reiber G, Boyko E and Smith D. Lower extremity foot ulcers and amputations in diabetes. In Diabetes in America, pp. 409-428. Edited by Harris M, Cowie C, Stern M, Boyko E, Reiber G and Bennett P. 409-428, Washington DC, National Institutes of Health, 1995.
3. Apelqvist J, Ragnarson-Tennvall,G, Persson U, and Larsson J. Diabetic foot ulcers in a multidisciplinary setting.An economic analysis of primary healing and healing with amputation. J Intern Med, 235(5): 463-71, 1994.
4. Pecoraro RE, Reiber GE and Burgess EM. Pathways to diabetic limb amputation. Basis for prevention. Diabetes Care, 13(5): 513-21, 1990.
5. Harrington C, Zagari MJ, Corea J and Klitenic J. A cost analysis of diabetic lower-extremity ulcers. Diabetes Care, 23(9): 1333-8, 2000.
6. Ramsey SD, Newton K, Blough D, McCulloch DK, Sandhu N, Reiber GE and Wagner EH. Incidence, outcomes, and cost of foot ulcers in patients with diabetes. Diabetes Care, 22(3): 382-7, 1999.
7. Fahey TJ 3rd, Sadaty A, Jones WG 2nd, Barber A, Smoller B and Shires GT. Diabetes impairs the late inflammatory response to wound healing. J Surg Res, 50(4): 308-13, 1991.
8. Hasan A, Murata H, Falabella A, Ochoa S, Zhou L, Badiavas E and Falanga V. Dermal fibroblasts from venous ulcers are unresponsive to the action of transforming growth factorbeta 1. J Dermatol Sci, 16(1): 59-66, 1997.
9. Stanley AC, Park HY, Phillips TJ, Russakovsky V and Menzoian JO. Reduced growth of dermal fibroblasts from chronic venous ulcers can be stimulated with growth factors. J Vasc Surg, 26(6): 994-9; discussion 999-1001, 1997.
10. Knight EV, Oldham JW, Mohler MA, Liu S and Dooley J.A review of nonclinical toxicology studies of becaplermin (rhPDGF-BB). Am J Surg, 176(2A Suppl): 55S-60S, 1998.
11. LeGrand EK. Preclinical promise of becaplermin (rhPDGF-BB) in wound healing. Am J Surg, 176(2A Suppl): 48S-54S, 1998.
12. Donaghue VM, Chrzan JS, Rosenblum BI, Giurini JM, Habershaw GM and Veves A. Evaluation of a collagen-alginate wound dressing in the management of diabetic foot ulcers. Adv Wound Care, 11(3): 114-9, 1998.
13. Gough A, Clapperton M, Rolando N, Foster AV, Philpott-Howard J and Edmonds ME. Randomised placebo-controlled trial of granulocyte- colony stimulating factor in diabetic foot infection. Lancet, 350(9081): 855-9, 1997.
14. Richard JL, Parer-Richard C, Daures JP, Clouet S,Vannereau D, Bringer J, Rodier M, Jacob C and Comte-Bardonnet M. Effect of topical basic fibroblast growth factor on the healing of chronic diabetic neuropathic ulcer of the foot. A pilot, randomized, double-blind, placebo-controlled study. Diabetes Care, 18(1): 64-9, 1995.
15. Steed DL.The role of growth factors in wound healing. Surg Clin North Am, 77(3): 575-86, 1997.
16. McKay IA and Leigh IM. Epidermal cytokines and their roles in cutaneous wound healing. Br J Dermatol, 124(6): 513-8, 1991.
17. Leigh IM, Purkis PE, Navsaria HA and Phillips TJ.Treatment of chronic venous ulcers with sheets of cultured allogenic keratinocytes. Br J Dermatol, 117(5): 591-7, 1987.
18. Phillips TJ, Kehinde O, Green H and Gilchrest BA.Treatment of skin ulcers with cultured epidermal allografts. J Am Acad Dermatol, 21(2 Pt 1): 191-9, 1989.
19. Rennekampff HO, Kiessig V and Hansbrough JF. Current concepts in the development of cultured skin replacements. J Surg Res, 62(2): 288-95, 1996.
20. Clark RA. Basics of cutaneous wound repair. J Dermatol Surg Oncol, 19(8): 693-706, 1993.
21. Mian E, Martini P, Beconcini D and Mian M. Healing of open skin surfaces with collagen foils. Int J Tissue React, 14 Suppl: 27-34, 1992.
22. Palmieri B. Heterologous collagen in wound healing: a clinical study. Int J Tissue React, 14 Suppl: 21-5, 1992.
23. Falanga V, Margolis D,Alvarez O,Auletta M, Maggiacomo F,Altman M, Jensen J, Sabolinski M and Hardin-Young J. Rapid healing of venous ulcers and lack of clinical rejection with an allogeneic cultured human skin equivalent. Human Skin Equivalent Investigators Group. Arch Dermatol, 134(3): 293-300, 1998.
24. Spiekstra SW, Breetveld M, Rustemeyer T, Scheper RJ and Gibbs S.Wound-healing factors secreted by epidermal keratinocytes and dermal fibroblasts in skin substitutes.Wound Repair Regen, 15(5): 708-17, 2007.
25. Falanga V, Isaacs C, Paquette D, Downing G, Kouttab N, Butmarc J, Badiavas E and Hardin- Young J.Wounding of bioengineered skin: cellular and molecular aspects after injury. J Invest Dermatol, 119(3): 653-60, 2002.
26. Kirsner RS, Falanga V and Eaglstein WH.The biology of skin grafts. Skin grafts as pharmacologic agents. Arch Dermatol, 129(4): 481-3, 1993.
27. Sabolinski ML,Alvarez O,Auletta M, Mulder G and Parenteau NL. Cultured skin as a ‘smart material’ for healing wounds: experience in venous ulcers. Biomaterials, 17(3): 311-20, 1996.
28. Brain A, Purkis P, Coates P, Hackett M, Navsaria H and Leigh I. Survival of cultured allogeneic keratinocytes transplanted to deep dermal bed assessed with probe specific for Y chromosome. Br Med J, 298(6678): 917-9, 1989.
29. Eaglstein WH and Falanga V.Tissue engineering and the development of Apligraf a human skin equivalent. AdvWound Care, 11(4 Suppl): 1-8, 1998.
30. Phillips TJ. New skin for old: developments in biological skin substitutes. Arch Dermatol, 134(3): 344-9, 1998.
31. Eaglstein WH and Falanga V.Tissue engineering and the development of Apligraf, a human skin equivalent. Cutis, 62(1 Suppl): 1-8, 1998.
32. Falanga V and Sabolinski M.A bilayered living skin construct (APLIGRAF) accelerates complete closure of hard-to-heal venous ulcers. Wound Repair Regen, 7(4): 201-7, 1999.
33. Veves A, Falanga V,Armstrong DG and Sabolinski ML. Graftskin, a human skin equivalent, is effective in the management of noninfected neuropathic diabetic foot ulcers: a prospective randomized multicenter clinical trial. Diabetes Care, 24(2): 290-5, 2001.
34. Pham HT, Rosenblum BI, Lyons TE, Giurini JM, Chrzan JS, Habershaw GM and Veves A. Evaluation of a human skin equivalent for the treatment of diabetic foot ulcers in a prospective, randomized, clinical trial.Wounds, 11(4): 79-86, 1999.
35. Sams HH, Chen J and King LE. Graftskin treatment of difficult to heal diabetic foot ulcers: one center’s experience. Dermatol Surg, 28(8): 698-703, 2002.
36. Brem H, Balledux J, Bloom T, Kerstein MD and Hollier L. Healing of diabetic foot ulcers and pressure ulcers with human skin equivalent: a new paradigm in wound healing. Arch Surg, 135(6): 627-34, 2000.
37. Eaglstein WH, et al.Acute excisional wounds treated with a tissue-engineered skin (Apligraf). Dermatol Surg, 25(3): 195-201, 1999.
38. Falabella AF,Valencia IC, Eaglstein WH and Schachner LA.Tissue-engineered skin (Apligraf) in the healing of patients with epidermolysis bullosa wounds. Arch Dermatol, 136(10): 1225-30, 2000.
39. Hu S, Kirsner RS, Falanga V, Phillips T and Eaglstein WH. Evaluation of Apligraf persistence and basement membrane restoration in donor site wounds: a pilot study.Wound Repair Regen, 14(4): 427-33, 2006.
40. Griffiths M, Ojeh N, Livingstone R, Price R and Navsaria H. Survival of Apligraf in acute human wounds. Tissue Eng, 10(7-8): 1180-95, 2004.
41. Phillips TJ, Manzoor J, Rojas A, Isaacs C, Carson P, Sabolinski M,Young J and Falanga V. The longevity of a bilayered skin substitute after application to venous ulcers. Arch Dermatol, 138(8): 1079-81, 2002.
42. Badiavas EV, Paquette D, Carson P and Falanga V. Human chronic wounds treated with bioengineered skin: histologic evidence of hostgraft interactions. J Am Acad Dermatol, 46(4): 524-30, 2002.
43. Vaalamo M, Leivo T and Saarialho-Kere,U. Differential expression of tissue inhibitors of metalloproteinases (TIMP-1, -2, -3, and -4) in normal and aberrant wound healing. Hum Pathol, 30(7): 795-802, 1999.
44. Waugh HV and Sherratt JA. Modeling the effects of treating diabetic wounds with engineered skin substitutes.Wound Repair Regen, 15(4): 556-65, 2007.
45. Loots MA, Lamme EN, Zeegelaar J, Mekkes JR, Bos JD and Middelkoop E. Differences in cellular infiltrate and extracellular matrix of chronic diabetic and venous ulcers versus acute wounds. J Invest Dermatol, 111(5): 850-7, 1998.
46. Steed DL, Goslen JB, Holloway GA, Malone JM, Bunt TJ and Webster MW. Randomized prospective double-blind trial in healing chronic diabetic foot ulcers. CT-102 activated platelet supernatant, topical versus placebo. Diabetes Care, 15(11): 1598-604, 1992.
47. Margolis DJ, Kantor J and Berlin JA. Healing of diabetic neuropathic foot ulcers receiving standard treatment.A meta-analysis. Diabetes Care, 22(5): 692-5, 1999.
48. O’Donnell TF Jr. and Lau J.A systematic review of randomized controlled trials of wound dressings for chronic venous ulcer. J Vasc Surg, 44(5): 1118-25, 2006.
49. Cavorsi J,Vicari F,Wirthlin DJ, Ennis W, Kirsner R, O'Connell SM, Steinberg J and Falanga V. Best-practice algorithms for the use of a bilayered living cell therapy (Apligraf) in the treatment of lower-extremity ulcers. Wound Repair Regen, 14(2): 102-9, 2006.



Add new comment