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Human chronic wounds treated with bioengineered skin: Histologic evidence of host-graft interactions
Human chronic wounds treated with bioengineered skin: Histologic evidence of host-graft interactions Evangelos V. Badiavas, MD, PhD,a,b Dana Paquette, MD,a Polly Carson, CWS,a and Vincent Falanga, MDa Providence, Rhode Island Bioengineered skin is being used to successfully treat a variety of wounds. Randomized controlled clinical trials have shown that a living bilayered skin construct (BSC), consisting of human neonatal keratinocytes and fibroblasts in a collagen matrix, was able to accelerate complete closure of both venous and diabetic ulcers. BSC was particularly effective in difficult-to-heal wounds of long duration. In patients treated with BSC, no obvious signs of gross clinical rejection were observed. Testing of these treated patients showed no BSC-specific immune response and no immune response to bovine collagen or alloantigens expressed on keratinocytes and fibroblasts. However, very little is known about the histologic changes that occur after BSC has been placed on human wounds. We report our preliminary histologic observations in this uncontrolled study of a cohort of 11 patients with 14 wounds treated with BSC in whom biopsy specimens of the grafted sites were obtained at least 2 weeks after application of the construct. The etiology of these ulcers varied from arterial or venous disease to an extensively and poorly healing burn wound. Histologically, thickening of the grafted bioengineered skin was seen in all samples where residual BSC could be identified. Mucin deposition was noted in the dermal layer of the wounds and BSC in 13 of the 14 specimens examined. Unexpectedly, and in spite of good clinical outcome, 4 of the 14 specimens exhibited a foreign body–like granulomatous response. There was no history of prior exposure to BSC in the 4 patients who had a granulomatous response. These early histologic observations suggest that stimulatory interactions develop between BSC and the wound. The consistently found deposition of mucin may point to a fetal pattern of wound repair associated with the neonatal cells in BSC. (J Am Acad Dermatol 2002;46:524-30.)
T
he development of a bilayered skin construct (BSC) has led to the Food and Drug Administration (FDA)–approved use of bioengineered skin in chronic venous ulcers and, more recently, in neuropathic diabetic foot ulcers. BSC has been reported to be especially beneficial in difficultto-heal ulcers of a year’s duration or more.1 From the Department of Dermatology and Skin Surgery, Roger Williams Medical Center, Boston University School of Medicinea and the Department of Pathology, Brown University School of Medicine.b Funding sources: National Institutes of Health research grants RO1 AR46557-01A1 and RO1 AR42935-06A1 and unrelated research grants from Organogenesis (Canton, Mass) and Novartis Pharmaceutical Corporation (East Hanover, NJ). Conflict of interest: None. Accepted for publication June 11, 2001. Reprint requests: Evangelos Badiavas, Department of Pathology, Roger Williams Medical Center, Brown University School of Medicine, 825 Chalkstone Ave, Providence, RI 02908. Copyright © 2002 by the American Academy of Dermatology, Inc. 0190-9622/2002/$35.00 + 0 16/1/120534 doi:10.1067/mjd.2002.120534
524
Although clinical evidence of rejection of the construct has not been reported,2 the persistence of BSC and its interaction with the host histologically have not been adequately described. Lack of knowledge about how the host and allogeneic cells interact is in part due to clinicians’ reluctance to perform biopsies on wounds, which has actually been shown to be quite safe. In clinical studies performed with BSC, patients treated with BSC demonstrated no antibody response to bovine type 1 collagen or bovine serum proteins and no anti-HLA antibodies to human dermal fibroblasts or human epidermal cells. Patients also did not exhibit T-cell–specific responses to bovine collagen, human fibroblasts, or human epidermal cells.2 Although these data attempt to address the issue of classical skin graft rejection, they do not provide us with information about the fate of BSC once it is applied to a wound or its interaction with the wound to cause healing. These skin substitutes do not appear to work only by coverage of the wound or by
Badiavas et al 525
J AM ACAD DERMATOL VOLUME 46, NUMBER 4
Fig 1. A, Biopsy specimen taken from yellow exudate material (week 2). Biopsy site is at arrow. B, BSC before application (hematoxylin and eosin stain). C, BSC, week 2. Biopsy reveals a swollen dermal layer (hematoxylin and eosin stain). D, BSC, week 2. Mucin deposition is noted in swollen dermis (PAS/alcian blue stain).
a prolonged and complete graft take. We have attempted to examine some of these questions by performing a histologic analysis of patients receiving BSC. Only treated sites were analyzed in this uncontrolled study. Graftskin is the generic name for a bioengineered BSC consisting of type I bovine collagen and viable dermal fibroblasts and keratinocytes derived from neonatal foreskin. The product is delivered “fresh” with an expiration date 5 days after its manufacture. Graftskin is produced by Organogensis (Canton, Mass) and licensed by Novartis Pharmaceutical Corporation (East Hanover, NJ). We used graftskin because it is readily available commercially, but we hope our studies shed light on bioengineered skin in general.
MATERIAL AND METHODS Graftskin (as described previously) was the BSC used. Informed consent was obtained and punch biopsy specimens from patients receiving BSC were
obtained by standard procedures. Specimens were first fixed in 10% formalin and then processed in a Sakura Tissue-Tek VIP automatic processor (Torrance, Calif). The specimens were then paraffin embedded, cut on a Leica microtome(Wetzlar, Germany), and stained for initial examination with hematoxylin and eosin. Analysis of ground substance or mucin formation was performed by alcian blue staining (pH 2.5) with or without periodic acid–Schiff (PAS) staining. PAS staining was carried out in the presence of diastase. In total, 14 biopsy specimens from 11 patients were examined. All biopsy specimens were obtained from 2 to 6 weeks after the application of BSC.
RESULTS Table I shows the demographics of the patients treated with BSC, their diagnoses, and the timing of the biopsy after BSC application. Fig 1 shows the histology of BSC before grafting. BSC was removed from the agar culture transport package in which it
526 Badiavas et al
J AM ACAD DERMATOL APRIL 2002
Fig 2. BSC 2 weeks after application. A, Hematoxylin and eosin–stained section. e, Migrating epithelium; g, granulation tissue; BSC, remnant of bilayered skin equivalent. Arrow indicates a multinucleated giant cell. B, Punch biopsy site can be seen at center of wound. Meshed BSC can be seen overlapping wound edge. C, Alcian blue–stained section. BSC, Remnant of bilayered skin equivalent; PD, patient’s dermis (wound bed). Note that mucin is present in both BSC and patient’s dermis.
arrived and was placed in formalin for routine histologic processing. Before grafting, little to no mucin deposition can be demonstrated by staining with alcian blue. Clinically, a common early feature after grafting was the development of a yellow exudate-like material in the vicinity of the applied BSC (Fig 1). Clinically, it was not clear whether BSC, viable or not, was present. A biopsy specimen of this yellow material was obtained and it was found to be BSC with a thickened and swollen appearing dermis. Alcian blue staining revealed that much of the dermal swelling was due to mucin deposition in the BSC dermis. This dermal swelling could be demonstrated even in areas of BSC dermis where collagen and many of the dermal cells were degenerated (Fig 2). Evidence of mucin deposition was demonstrated by mucin depo-
sition in 13 of the 14 biopsy specimens examined. Mucin was observed both in the patient’s dermis and in the dermal material from the grafted bioengineered skin (Fig 2). In contrast, the yellow material often covering chronic wounds usually contains fibrin, necrotic tissue, and both intact and degraded inflammatory cells but not mucin. A granulomatous response, indicated by the presence of multinucleated histiocytes, was noted in 4 of the biopsy specimens (Figs 2 and 3). This granulomatous response did not correlate with a poor clinical outcome because all wounds exhibiting this response eventually healed. The granulomatous response was not associated with prior exposure to BSC. In some cases, degenerated BSC could be seen adjacent to granulation tissue (Fig 2), indicating stimulation of the wound bed.
J AM ACAD DERMATOL VOLUME 46, NUMBER 4
Badiavas et al 527
Fig 3. BSC, week 2. A, Clinical appearance of wound before biopsy. Meshed BSC can be seen overlapping wound. B, Hematoxylin and eosin–stained section. At interface of bioengineered skin dermis and patient’s dermis, there is a granulomatous reaction containing multinucleated giant cells.
An apparent graft take occurred in a wound that was extensively debrided. This apparent take is identified from a histologic perspective in that the BSC has persisted on the wound and remained viable. It does not indicate that the wound is healed at this point. Despite the persistence of BSC in this wound, a small granulomatous reaction was also noted (Fig 4). However, the clinical outcome was good, leading to complete healing.
DISCUSSION As keratinocyte sheets became available for grafting, it was found that obvious clinical rejection of allogeneic cells was not a significant problem. This presumed lack of rejection may have been due to the fact that the cultured keratinocytes had lost some of their immunologic properties.3 Experimentally, cultured allogeneic keratinocytes have been shown to inhibit the response to alloantigen for both naive T cells and anti-CD3–activated T cells.4 This inhibition does not require cell-to-cell contact and is thought to be due to soluble factors secreted by keratinocytes. The partial inhibition of this activity by transforming growth factor-β antibody implicates transforming growth factor-β in at least part of this phenomenon. Other cytokines are also likely to be involved in this inhibition. In other studies, allogeneic keratinocytes, fibroblasts, and smooth muscle cells failed to induce naive T-cell stimulation despite induction of major histocompatibility class II expression by interferon gamma; however, allogeneic endothelial cells did stimulate T cells.5 Grafts consisting of human allogeneic ke-
ratinocytes and fibroblasts in an organotypic culture system (such as BSC) might then take advantage of the limited immune stimulation observed by these studies. In an effort to analyze the immune stimulatory effects of BSC, both BSC and human neonatal foreskin were applied to both naive and humanized SCID mice.6 Both BSC and neonatal foreskin engrafted to the naive SCID mice. In humanized SCID mice, the neonatal foreskin was rejected, whereas the BSC engrafted. This effect did not change even when BSC was treated with interferon-γ to induce major histocompatibility complex class II expression. Vascularization of BSC by the host (ie, host endothelial cells) after grafting did not seem to increase immune stimulation. It seemed that the transplanted endothelial cells in the foreskin likely led to rejection. This report supports the limited capabilities of human allogeneic keratinocytes and fibroblasts to stimulate an immune response and suggests that the endothelial cells should come from the host.6 Another possibility for the limited rejection seen with bioengineered skin equivalents such as BSC is that host cells eventually replace the allogeneic cells delivered to a wound. Keratinocyte grafts from autologous sources are replaced over time by growth of surrounding cells, and it is possible that allogeneic cells could behave in a similar fashion.7 Replacement, rather than rejection, could be the fate of these grafted allogeneic cells. Clinically, BSC has been extensively studied in the treatment of chronic wounds.1,2 In these studies there was no reported innate or specific immune
528 Badiavas et al
J AM ACAD DERMATOL APRIL 2002
Fig 4. BSC, 3 weeks. A, Clinical appearance of wound. Arrow indicates biopsy site. Meshed BSC can be seen overlapping wound. B, Hematoxylin and eosin–stained section. Extensively debrided wound with evidence of graft take. Asterisks indicate fibrinous zone at interface between BSC and patient’s dermis. BSC dermis is swollen. Arrow indicates a multinucleated giant cell within BSC. C, Alcian blue–stained section shows mucin in both patient’s and BSC dermis. Asterisks indicate interface between BSC and patient’s dermis.
reaction to the bioengineered skin. In this report there was also no clinical evidence of graft rejection or graft-versus-host disease. In addition to the immunologic interactions between BSC and the patient, the mechanism of action and healing that occurs with bioengineered skin also remains largely undetermined. In our histologic analysis of patients treated with BSC, we have made several observations that address the interactions between BSC and the patient’s wound. Mucin deposition, noted in most specimens, indicates production of ground substance. Although healing wounds treated by other modalities produce small amounts of mucin, we have observed a significantly greater amount of mucin in the wound bed dermis of BSC-treated wounds. In addition, mucin
deposition was observed in both the patients’ (wound bed) dermis and the dermis of the bioengineered skin. This implies an interaction and stimulation of both patient and bioengineered dermis to produce ground substance. The presence of granulation tissue near the BSC dermis also indicates a stimulation of the patient’s (wound bed) dermis by bioengineered material. Although it is easy to deduce from previous clinical data2 that the BSC stimulates the wound bed, it is interesting to note that we show a reciprocal stimulation of the BSC dermis to produce ground substance. This finding implies that the BSC may act in vivo, at least for a short while, as a living tissue with the ability to respond to its environment. Given that BSC may respond and change in the in vivo setting, it is impor-
Badiavas et al 529
J AM ACAD DERMATOL VOLUME 46, NUMBER 4
Table I. Demographics of patients treated with BSC Patient No.
Age (y)
Prior graftskin
1 2 2 2 3 4 5 6
69 73 73 73 66 80 86 103
No No No No Yes No No No
7 2 8
84 74 90
No Yes No
9 10 11
53 56 73
No No No
Diagnosis
Time of biopsy
Biopsy site
Granulomatous response
Mucin deposition
4 wk 2 wk 2 wk 3 wk 2 wk 2 wk 2 wk 2 wk
Right thigh Dorsum of left foot Left heel Left heel Left leg Left lower leg Buttocks Anterior tibia
ND ND ND ND ND Present ND Present
Present Present No increase detected Present Present Present Present Present
3 wk 2 wk 3 wk
Left posterior tibia Left tibia Left anterior shin
Present ND Present
Present Present Present
3 wk 2 wk 6 wk
Left leg Left leg Left lateral ankle
ND ND ND
Present Present Present
Epidermolysis bullosa Diabetic Diabetic Diabetic Venous Venous Thermal burn Poorly healing surgical wound (excision of a primary squamous cell carcinoma) Venous Diabetic Mixed arterial and venous Venous Diabetic Venous
ND, Not detected.
tant to study this bioengineered material in vivo to clearly ascertain its mechanism of action. It is quite possible that other substances besides mucin (eg, growth factors) might be produced by BSC when it is placed in the wound bed. Swelling of BSC was commonly observed on histologic examination and may not be related to loss in viability of bioengineered skin or diminished clinical effect. Clinically, this swelling may be seen as a yellow material and should be recognized by those treating patients with bioengineered skin. It does not represent infection, as shown previously. In addition to swelling, a granulomatous response was observed in 4 specimens. The granulomatous response was observed as the presence of multinucleated giant cells at the interface of the BSC dermis and the patient’s wound bed dermis or as the presence of scattered multinucleated histiocytes within the BSC dermis alone. Oddly, the BSC dermis appeared viable (with intact fibroblasts) where multinucleated cells were observed within the BSC dermis. The presence of the granulomatous response did not correlate with a poor clinical outcome. There was no correlation of a granulomatous reaction with prior exposure to bioengineered skin. This granulomatous reaction may represent an innate reaction to the presence of a foreign material and supports the idea that this allogeneic material is replaced and does not engraft. Granulomatous infiltrates have been noted with wound dressings8 and keratinocyte sheet grafting.9
In pig wounds treated with hydrocolloid dressings, we and others8 have observed granulomatous infiltrates. Infiltrates associated with hydrocolloid dressings could be due to foreign substances, possibly dressing material, deposited in the wound.8 These reactions occurred in the deeper portions of the wound, below an area of granulation tissue. In our experience with pig wounds, re-absorption of partially debrided endogenous necrotic material could also lead to these changes. Granulomatous infiltrates in hydrocolloid-treated chronic human wounds have not been established.10 The granulomatous infiltrate associated with keratinocyte sheet grafting has been reported particularly when these epidermal grafts were placed over a cadaver allograft dermis.9 Our findings differ in that the granulomatous infiltrate we noted occurred at the interface of the BSC dermis and the wound bed and was also observed in viable material with living cells, unlike cadaver allograft. REFERENCES 1. Falanga V, Sabolinski M. A bilayered living skin construct (Apligraft) accelerates complete closure of hard-to-heal venous ulcers. Wound Repair Regen 1999;7:201-7. 2. Falanga V, Margolis D, Alvarez O, Auletta M, Maggiacomo F, Altman M, et al. Rapid healing of venous ulcers and lack of clinical rejection with an allogeneic cultured human skin equivalent. Human Skin Equivalent Investigators Group. Arch Dermatol 1998;134:293-300. 3. Hefton JM, Amberson JB, Biozes DG,Weksler ME. Loss of HLA-DR expression by human epidermal cells after growth in culture. J Invest Dermatol 1984;83:48-50. 4. Laning JC, Isaacs CM, Hardin-Young J. Normal human ke-
530 Badiavas et al
ratinocytes inhibit the proliferation of unprimed T cells by TGFbeta and PGE2, but not IL-10. Cell Immunol 1997;175:16-24. 5. Theobald VA, Lauer JD, Kaplan KA, Baker KB, Rosenberg M. “Neutral allografts”—lack of allogeneic stimulation by cultured human cells expressing MHC class I and class II antigens. Transplantation 1993;55:128-33. 6. Briscoe DM, Dharnidharka VR, Isaacs C, Downing G, Prosky S, Shaw P, et al. The allogeneic response to cultured human skin equivalent in the hu-PBL-SCID mouse model of skin rejection. Transplantation 1999;67:1590-9. 7. Phillips TJ, Kehinde O, Green H, Gilchrest BA. Treatment of skin ulcers with cultured epidermal allografts. J Am Acad Dermatol 1989;21:191-9.
J AM ACAD DERMATOL APRIL 2002
8. Young SR, Dyson M, Hickman R, Lang S, Osborn C. Comparison of the effects of semi-occlusive polyurethane dressings and hydrocolloid dressings on dermal repair. 1. Cellular changes. J Invest Dermatol 1991;97:586-92. 9. 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. 10. Phillips TJ, Palko MJ, Bhawan J. Histologic evaluation of chronic human wounds treated with hydrocolloid and nonhydrocolloid dressings. J Am Acad Dermatol 1994;30:61-4.
T
he development of a bilayered skin construct (BSC) has led to the Food and Drug Administration (FDA)–approved use of bioengineered skin in chronic venous ulcers and, more recently, in neuropathic diabetic foot ulcers. BSC has been reported to be especially beneficial in difficultto-heal ulcers of a year’s duration or more.1 From the Department of Dermatology and Skin Surgery, Roger Williams Medical Center, Boston University School of Medicinea and the Department of Pathology, Brown University School of Medicine.b Funding sources: National Institutes of Health research grants RO1 AR46557-01A1 and RO1 AR42935-06A1 and unrelated research grants from Organogenesis (Canton, Mass) and Novartis Pharmaceutical Corporation (East Hanover, NJ). Conflict of interest: None. Accepted for publication June 11, 2001. Reprint requests: Evangelos Badiavas, Department of Pathology, Roger Williams Medical Center, Brown University School of Medicine, 825 Chalkstone Ave, Providence, RI 02908. Copyright © 2002 by the American Academy of Dermatology, Inc. 0190-9622/2002/$35.00 + 0 16/1/120534 doi:10.1067/mjd.2002.120534
524
Although clinical evidence of rejection of the construct has not been reported,2 the persistence of BSC and its interaction with the host histologically have not been adequately described. Lack of knowledge about how the host and allogeneic cells interact is in part due to clinicians’ reluctance to perform biopsies on wounds, which has actually been shown to be quite safe. In clinical studies performed with BSC, patients treated with BSC demonstrated no antibody response to bovine type 1 collagen or bovine serum proteins and no anti-HLA antibodies to human dermal fibroblasts or human epidermal cells. Patients also did not exhibit T-cell–specific responses to bovine collagen, human fibroblasts, or human epidermal cells.2 Although these data attempt to address the issue of classical skin graft rejection, they do not provide us with information about the fate of BSC once it is applied to a wound or its interaction with the wound to cause healing. These skin substitutes do not appear to work only by coverage of the wound or by
Badiavas et al 525
J AM ACAD DERMATOL VOLUME 46, NUMBER 4
Fig 1. A, Biopsy specimen taken from yellow exudate material (week 2). Biopsy site is at arrow. B, BSC before application (hematoxylin and eosin stain). C, BSC, week 2. Biopsy reveals a swollen dermal layer (hematoxylin and eosin stain). D, BSC, week 2. Mucin deposition is noted in swollen dermis (PAS/alcian blue stain).
a prolonged and complete graft take. We have attempted to examine some of these questions by performing a histologic analysis of patients receiving BSC. Only treated sites were analyzed in this uncontrolled study. Graftskin is the generic name for a bioengineered BSC consisting of type I bovine collagen and viable dermal fibroblasts and keratinocytes derived from neonatal foreskin. The product is delivered “fresh” with an expiration date 5 days after its manufacture. Graftskin is produced by Organogensis (Canton, Mass) and licensed by Novartis Pharmaceutical Corporation (East Hanover, NJ). We used graftskin because it is readily available commercially, but we hope our studies shed light on bioengineered skin in general.
MATERIAL AND METHODS Graftskin (as described previously) was the BSC used. Informed consent was obtained and punch biopsy specimens from patients receiving BSC were
obtained by standard procedures. Specimens were first fixed in 10% formalin and then processed in a Sakura Tissue-Tek VIP automatic processor (Torrance, Calif). The specimens were then paraffin embedded, cut on a Leica microtome(Wetzlar, Germany), and stained for initial examination with hematoxylin and eosin. Analysis of ground substance or mucin formation was performed by alcian blue staining (pH 2.5) with or without periodic acid–Schiff (PAS) staining. PAS staining was carried out in the presence of diastase. In total, 14 biopsy specimens from 11 patients were examined. All biopsy specimens were obtained from 2 to 6 weeks after the application of BSC.
RESULTS Table I shows the demographics of the patients treated with BSC, their diagnoses, and the timing of the biopsy after BSC application. Fig 1 shows the histology of BSC before grafting. BSC was removed from the agar culture transport package in which it
526 Badiavas et al
J AM ACAD DERMATOL APRIL 2002
Fig 2. BSC 2 weeks after application. A, Hematoxylin and eosin–stained section. e, Migrating epithelium; g, granulation tissue; BSC, remnant of bilayered skin equivalent. Arrow indicates a multinucleated giant cell. B, Punch biopsy site can be seen at center of wound. Meshed BSC can be seen overlapping wound edge. C, Alcian blue–stained section. BSC, Remnant of bilayered skin equivalent; PD, patient’s dermis (wound bed). Note that mucin is present in both BSC and patient’s dermis.
arrived and was placed in formalin for routine histologic processing. Before grafting, little to no mucin deposition can be demonstrated by staining with alcian blue. Clinically, a common early feature after grafting was the development of a yellow exudate-like material in the vicinity of the applied BSC (Fig 1). Clinically, it was not clear whether BSC, viable or not, was present. A biopsy specimen of this yellow material was obtained and it was found to be BSC with a thickened and swollen appearing dermis. Alcian blue staining revealed that much of the dermal swelling was due to mucin deposition in the BSC dermis. This dermal swelling could be demonstrated even in areas of BSC dermis where collagen and many of the dermal cells were degenerated (Fig 2). Evidence of mucin deposition was demonstrated by mucin depo-
sition in 13 of the 14 biopsy specimens examined. Mucin was observed both in the patient’s dermis and in the dermal material from the grafted bioengineered skin (Fig 2). In contrast, the yellow material often covering chronic wounds usually contains fibrin, necrotic tissue, and both intact and degraded inflammatory cells but not mucin. A granulomatous response, indicated by the presence of multinucleated histiocytes, was noted in 4 of the biopsy specimens (Figs 2 and 3). This granulomatous response did not correlate with a poor clinical outcome because all wounds exhibiting this response eventually healed. The granulomatous response was not associated with prior exposure to BSC. In some cases, degenerated BSC could be seen adjacent to granulation tissue (Fig 2), indicating stimulation of the wound bed.
J AM ACAD DERMATOL VOLUME 46, NUMBER 4
Badiavas et al 527
Fig 3. BSC, week 2. A, Clinical appearance of wound before biopsy. Meshed BSC can be seen overlapping wound. B, Hematoxylin and eosin–stained section. At interface of bioengineered skin dermis and patient’s dermis, there is a granulomatous reaction containing multinucleated giant cells.
An apparent graft take occurred in a wound that was extensively debrided. This apparent take is identified from a histologic perspective in that the BSC has persisted on the wound and remained viable. It does not indicate that the wound is healed at this point. Despite the persistence of BSC in this wound, a small granulomatous reaction was also noted (Fig 4). However, the clinical outcome was good, leading to complete healing.
DISCUSSION As keratinocyte sheets became available for grafting, it was found that obvious clinical rejection of allogeneic cells was not a significant problem. This presumed lack of rejection may have been due to the fact that the cultured keratinocytes had lost some of their immunologic properties.3 Experimentally, cultured allogeneic keratinocytes have been shown to inhibit the response to alloantigen for both naive T cells and anti-CD3–activated T cells.4 This inhibition does not require cell-to-cell contact and is thought to be due to soluble factors secreted by keratinocytes. The partial inhibition of this activity by transforming growth factor-β antibody implicates transforming growth factor-β in at least part of this phenomenon. Other cytokines are also likely to be involved in this inhibition. In other studies, allogeneic keratinocytes, fibroblasts, and smooth muscle cells failed to induce naive T-cell stimulation despite induction of major histocompatibility class II expression by interferon gamma; however, allogeneic endothelial cells did stimulate T cells.5 Grafts consisting of human allogeneic ke-
ratinocytes and fibroblasts in an organotypic culture system (such as BSC) might then take advantage of the limited immune stimulation observed by these studies. In an effort to analyze the immune stimulatory effects of BSC, both BSC and human neonatal foreskin were applied to both naive and humanized SCID mice.6 Both BSC and neonatal foreskin engrafted to the naive SCID mice. In humanized SCID mice, the neonatal foreskin was rejected, whereas the BSC engrafted. This effect did not change even when BSC was treated with interferon-γ to induce major histocompatibility complex class II expression. Vascularization of BSC by the host (ie, host endothelial cells) after grafting did not seem to increase immune stimulation. It seemed that the transplanted endothelial cells in the foreskin likely led to rejection. This report supports the limited capabilities of human allogeneic keratinocytes and fibroblasts to stimulate an immune response and suggests that the endothelial cells should come from the host.6 Another possibility for the limited rejection seen with bioengineered skin equivalents such as BSC is that host cells eventually replace the allogeneic cells delivered to a wound. Keratinocyte grafts from autologous sources are replaced over time by growth of surrounding cells, and it is possible that allogeneic cells could behave in a similar fashion.7 Replacement, rather than rejection, could be the fate of these grafted allogeneic cells. Clinically, BSC has been extensively studied in the treatment of chronic wounds.1,2 In these studies there was no reported innate or specific immune
528 Badiavas et al
J AM ACAD DERMATOL APRIL 2002
Fig 4. BSC, 3 weeks. A, Clinical appearance of wound. Arrow indicates biopsy site. Meshed BSC can be seen overlapping wound. B, Hematoxylin and eosin–stained section. Extensively debrided wound with evidence of graft take. Asterisks indicate fibrinous zone at interface between BSC and patient’s dermis. BSC dermis is swollen. Arrow indicates a multinucleated giant cell within BSC. C, Alcian blue–stained section shows mucin in both patient’s and BSC dermis. Asterisks indicate interface between BSC and patient’s dermis.
reaction to the bioengineered skin. In this report there was also no clinical evidence of graft rejection or graft-versus-host disease. In addition to the immunologic interactions between BSC and the patient, the mechanism of action and healing that occurs with bioengineered skin also remains largely undetermined. In our histologic analysis of patients treated with BSC, we have made several observations that address the interactions between BSC and the patient’s wound. Mucin deposition, noted in most specimens, indicates production of ground substance. Although healing wounds treated by other modalities produce small amounts of mucin, we have observed a significantly greater amount of mucin in the wound bed dermis of BSC-treated wounds. In addition, mucin
deposition was observed in both the patients’ (wound bed) dermis and the dermis of the bioengineered skin. This implies an interaction and stimulation of both patient and bioengineered dermis to produce ground substance. The presence of granulation tissue near the BSC dermis also indicates a stimulation of the patient’s (wound bed) dermis by bioengineered material. Although it is easy to deduce from previous clinical data2 that the BSC stimulates the wound bed, it is interesting to note that we show a reciprocal stimulation of the BSC dermis to produce ground substance. This finding implies that the BSC may act in vivo, at least for a short while, as a living tissue with the ability to respond to its environment. Given that BSC may respond and change in the in vivo setting, it is impor-
Badiavas et al 529
J AM ACAD DERMATOL VOLUME 46, NUMBER 4
Table I. Demographics of patients treated with BSC Patient No.
Age (y)
Prior graftskin
1 2 2 2 3 4 5 6
69 73 73 73 66 80 86 103
No No No No Yes No No No
7 2 8
84 74 90
No Yes No
9 10 11
53 56 73
No No No
Diagnosis
Time of biopsy
Biopsy site
Granulomatous response
Mucin deposition
4 wk 2 wk 2 wk 3 wk 2 wk 2 wk 2 wk 2 wk
Right thigh Dorsum of left foot Left heel Left heel Left leg Left lower leg Buttocks Anterior tibia
ND ND ND ND ND Present ND Present
Present Present No increase detected Present Present Present Present Present
3 wk 2 wk 3 wk
Left posterior tibia Left tibia Left anterior shin
Present ND Present
Present Present Present
3 wk 2 wk 6 wk
Left leg Left leg Left lateral ankle
ND ND ND
Present Present Present
Epidermolysis bullosa Diabetic Diabetic Diabetic Venous Venous Thermal burn Poorly healing surgical wound (excision of a primary squamous cell carcinoma) Venous Diabetic Mixed arterial and venous Venous Diabetic Venous
ND, Not detected.
tant to study this bioengineered material in vivo to clearly ascertain its mechanism of action. It is quite possible that other substances besides mucin (eg, growth factors) might be produced by BSC when it is placed in the wound bed. Swelling of BSC was commonly observed on histologic examination and may not be related to loss in viability of bioengineered skin or diminished clinical effect. Clinically, this swelling may be seen as a yellow material and should be recognized by those treating patients with bioengineered skin. It does not represent infection, as shown previously. In addition to swelling, a granulomatous response was observed in 4 specimens. The granulomatous response was observed as the presence of multinucleated giant cells at the interface of the BSC dermis and the patient’s wound bed dermis or as the presence of scattered multinucleated histiocytes within the BSC dermis alone. Oddly, the BSC dermis appeared viable (with intact fibroblasts) where multinucleated cells were observed within the BSC dermis. The presence of the granulomatous response did not correlate with a poor clinical outcome. There was no correlation of a granulomatous reaction with prior exposure to bioengineered skin. This granulomatous reaction may represent an innate reaction to the presence of a foreign material and supports the idea that this allogeneic material is replaced and does not engraft. Granulomatous infiltrates have been noted with wound dressings8 and keratinocyte sheet grafting.9
In pig wounds treated with hydrocolloid dressings, we and others8 have observed granulomatous infiltrates. Infiltrates associated with hydrocolloid dressings could be due to foreign substances, possibly dressing material, deposited in the wound.8 These reactions occurred in the deeper portions of the wound, below an area of granulation tissue. In our experience with pig wounds, re-absorption of partially debrided endogenous necrotic material could also lead to these changes. Granulomatous infiltrates in hydrocolloid-treated chronic human wounds have not been established.10 The granulomatous infiltrate associated with keratinocyte sheet grafting has been reported particularly when these epidermal grafts were placed over a cadaver allograft dermis.9 Our findings differ in that the granulomatous infiltrate we noted occurred at the interface of the BSC dermis and the wound bed and was also observed in viable material with living cells, unlike cadaver allograft. REFERENCES 1. Falanga V, Sabolinski M. A bilayered living skin construct (Apligraft) accelerates complete closure of hard-to-heal venous ulcers. Wound Repair Regen 1999;7:201-7. 2. Falanga V, Margolis D, Alvarez O, Auletta M, Maggiacomo F, Altman M, et al. Rapid healing of venous ulcers and lack of clinical rejection with an allogeneic cultured human skin equivalent. Human Skin Equivalent Investigators Group. Arch Dermatol 1998;134:293-300. 3. Hefton JM, Amberson JB, Biozes DG,Weksler ME. Loss of HLA-DR expression by human epidermal cells after growth in culture. J Invest Dermatol 1984;83:48-50. 4. Laning JC, Isaacs CM, Hardin-Young J. Normal human ke-
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ratinocytes inhibit the proliferation of unprimed T cells by TGFbeta and PGE2, but not IL-10. Cell Immunol 1997;175:16-24. 5. Theobald VA, Lauer JD, Kaplan KA, Baker KB, Rosenberg M. “Neutral allografts”—lack of allogeneic stimulation by cultured human cells expressing MHC class I and class II antigens. Transplantation 1993;55:128-33. 6. Briscoe DM, Dharnidharka VR, Isaacs C, Downing G, Prosky S, Shaw P, et al. The allogeneic response to cultured human skin equivalent in the hu-PBL-SCID mouse model of skin rejection. Transplantation 1999;67:1590-9. 7. Phillips TJ, Kehinde O, Green H, Gilchrest BA. Treatment of skin ulcers with cultured epidermal allografts. J Am Acad Dermatol 1989;21:191-9.
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8. Young SR, Dyson M, Hickman R, Lang S, Osborn C. Comparison of the effects of semi-occlusive polyurethane dressings and hydrocolloid dressings on dermal repair. 1. Cellular changes. J Invest Dermatol 1991;97:586-92. 9. 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. 10. Phillips TJ, Palko MJ, Bhawan J. Histologic evaluation of chronic human wounds treated with hydrocolloid and nonhydrocolloid dressings. J Am Acad Dermatol 1994;30:61-4.