Papers

Epidermal differentiation and dermal changes inhealing following treatment of surgical woundswith sheets of cultured allogeneic keratinocytes

S R Myers, H A Navsaria, A N Brain, P E Purkis, I M Leigh

AbstractAims-To establish the structural changesthat occur in deep surgical wounds en-grafted with allogeneic sheets, their timecourse and inter-relation.Methods-Deep surgical wounds followingshave excision of tattoos (down to deepdermislsubcutaneous fat) were treatedwith sheets of sex mismatched allogeneickeratinocytes in 19 patients and then bi-opsied weekly until wound healing wascomplete. More superficial surgicalwounds-that is, 20 standard skin graftdonor sites, were biopsied at seven to 10days (all healed) following application ofkeratinocyte allografts. All biopsy speci-mens were examined with a large panel ofmonoclonal antibodies to keratins, en-velope proteins, basement membranecomponents, and to extracellular matrixcomponents.Results-The hyperproliferative keratinpair K6/16 was expressed in all wounds,for up to six weeks in keratinocyte grafteddeep wounds, and up to six months in splitthickness skin grafted wounds.Conclusions-Keratins 6 and 16 have notbeen detected in normal skin, althoughthe relevant mRNA has. This raises thepossibility of regulation at a post-tran-scriptional level allowing a rapid responseto injury with cytoskeletal changes thatmay aid cell migration. This keratin pairoffers the most sensitive marker for alteredepidermis following wounding.(J7 Clin Pathol 1995;48:1087-1092)

Keywords: Wound healing, keratinocyte, keratins, al-lograft.

Department ofExperimentalDermatology, RoyalLondon Hospital,London El 1BB

Correspondence to:Mr S R Myers.

Accepted for publication13 March 1995

The development of keratinocyte culture tech-niques in the 1970s and early 1980s meant thata given population of cells could be expandedfrom a small skin biopsy specimen into a

large area of cultured keratinocytes by serialpassaging.' The potential use of this tissueexpansion technique for skin grafts was re-

cognised and such cultured grafts are now inclinical and commercial use.23 Earliest reportsof keratinocyte grafting came from bums

centres. Patients with very extensive burns weretreated successfully with sheets of culturedautologous keratinocytes (keratinocyte auto-grafts).' The experience has gradually extendedto involve multiple bums centres in USA, Eur-ope and Japan. In 1983 Hefton et al' reportedthe use of keratinocyte allografts in the treat-ment of initially three, and subsequently 22patients with bums.6 Clinical "take" and goodresults were observed, and speculation to ex-plain this phenomenon was based on the knownloss of antigen presenting Langerhans cells inthe cultures, suggesting that keratinocyte al-lografts might not be rejected.7 However, cul-tured keratinocytes retain the ability to expressclass II antigens on their surface in response to-y-interferon and tumour necrosis factor makingthis somewhat unlikely.8 Similarly, good clinicalresults were reported for allogeneic ker-atinocyte grafts applied to donor sites, wherewounds were healed by one week.9'- Severalother centres reported apparent take of ker-atinocyte allografts in bums. In order to deter-mine whether keratinocytes truly survivedtransplantation, we performed shave excisionof a series of tattoos'2 in patient volunteers.The tattoos were excised to deep dermis ordermis-to-fat junction and then treated withsheets of sex mismatched cultured allogeneickeratinocytes. Biopsy specimens were taken atweekly intervals and examined by in situ hy-bridisation with a probe to the Y chromosome(phY2 1). The biopsy specimens showed noevidence of survival of donor keratinocytes, butdid show rapid wound healing with recipientcells from the wound edge (appendages hadbeen excised).

Studies of keratinocytes grafted in a trans-plantation chamber onto nude mice show thatdeep dermis does not support full epidermaldifferentiation,'3 the keratinocytes remainingpoorly differentiated and expressing simple epi-thelial phenotype when close to deep sub-cutaneous connective tissue. In view of thepossibility that the depth of the wound in thetattoo study was influencing the attachment ofthe allogeneic keratinocytes, so that biologicalnon-attachment rather than immunological re-jection was causing the failure of trans-plantation, the study was repeated in moresuperficial wounds (routine surgical split thick-

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Table 1 Summary of antibodies used

Antibody Target antigen Reference

LLO17 Keratin 1 Leigh et al'66B10 Keratin 4 van Muijen et al"7LL020 Keratin 6 Lane et al'8RCK105 Keratin 7 Ramaekers et al'9LE41 Keratin 8 Lane"0LH2 Keratin 10 Leigh et al'6IC7 Keratin 13 van Muijen et al'7LLOO1 Keratin 14 Purkis et al'LH8 Keratin 14 Purkis et al2'LL025 Keratin 16 Lane et al'8K3 Keratin 17 Guelstein et al55C46 Keratin 17 (+ 7) Bartek et a 13LE61 Keratin 18 Lane20LP2K Keratin 19 Stasiak et al"4PCO Collagen IV Eurodiagnostics, UKLH7 2 Collagen VII Leigh et al25HMFG2 Mucin Burchell and Taylor-

Papadimitriou26sm3 Mucin Burchell and Taylor-

Papadimitriou26

ness skin graft donor sites). All donor sites werehealed at seven to 10 days, but there was no

evidence of allograft survival. Thus, the depthof the wound was not influential and ker-atinocyte allografts do not survive trans-plantation in either superficial or deep surgicalwounds.Although keratinocyte allografts do not sur-

vive transplantation, their use has highlightedthe fact that autologous and allogeneic ker-atinocytes can have effects on wound healingapart from take which may be due to synthesisof cytokines14 and extracellular matrix proteins.The identification and characterisation ofthesefactors may give rise to recombinant productswhich can be used in wound healing by in-corporation into dressings. Autologous cells,however, are required for graft take.

Keratins are major structural proteins of allepithelial cell types. Their profile appears to beclosely related to the differentiated state of a

tissue.'5 The biopsy specimens from the ker-atinocyte allograft survival studies were usedto examine the time course ofchanges in keratinexpression and other markers of differentiationand basement membrane synthesis duringwounding.

MethodsFULL THICKNESS WOUNDS: TATTOO EXCISION

Nineteen patients (13 women and six men)requesting removal of decorative tattoos were

offered treatment with sheets of cultured allo-geneic keratinocytes, and gave their informedconsent. The tattoos were excised by serialkeratotome excision until all pigment was re-

moved. All wounds removed the deep dermiswith hair follicles, and some reached sub-cutaneous fat. Wounds were treated with sheetsof sex mismatched cultured allogeneic ker-atinocytes and were examined and biopsiedweekly until they were healed (three weeks).All biopsy specimens were snap frozen in liquidnitrogen and stored at - 70°C. Occasionalsamples were taken following grafting at in-tervals up to six months.

PARTIAL THICKNESS WOUNDS: DONOR SITES

Twenty patients (all women) receiving splitthickness skin grafts for chronic leg ulcers were

offered keratinocyte allografts to their donorsites as an alternative to paraffin dressing, andgave their informed consent. The wounds were

examined and biopsied at seven or 10 days.

CONTROL WOUNDSDonor sites for split thickness skin grafts re-

ceiving conventional dressings (paraffin gauze)were biopsied at 10 days following grafting.Split thickness skin grafted wounds were alsobiopsied as controls where possible.

All the wounds were studied by routine im-munofluorescence techniques using the mono-clonal antibodies listed in table 1, directedagainst keratin polypeptides, basement mem-

brane proteins, and epithelial mucins.

ResultsThe results are summarised in table 2.

Table 2 Summary of keratin expresion in regenerating wounds

Excised tattoo beds (weeks post surgery) Donor sites Split grafts

Keratin One Two Three Seven days Ten days Seven days Six months

K1(LL017) SB SB SB SB SB SB SBK4(6B10)K6(LL020) SB/B SB SB SB SB SB SBK7(RCK105)K8(LE41)KIO(LH2) SB SB SB SB SB SB SBK13(IC7)K14(LL001) Pan Pan B+SB Pan Pan Pan PanK14(LH8) B B B B B B BK16(LL025) SB SB SB SB SB SB SBK17(E3) SB SB SB SB SBK7/17(C46) SB SB SB SB SBK18(LE61)K19(LP2K)Pan-keratin (AE1) Pan Pan SB Pan Pan Pan PanCollagen VII(LH7-2) + + + + +Collagen IV(LH7) + + + + +Anti-involucrin EB SB SB + + + +HMFG2 + (+)sm3 (+)

Pan= panepidermal; SB suprabasal; EB= epibasal; B =basal.

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Wound healing after treatment with cultured allogeneic keratinocytes

Figure 1 Keratin 1 staining with LL017 two weeks after cultured keratinocyteallografting of an excised tattoo bed.

Figure 2 Keratin 10 staining with LH2 two weeks after cultured keratinocyteallografting of an excised tattoo bed.

Figure 3 Keratin 6 staining with LL020 two weeks after cultured keratinocyteallografting of an excised tattoo bed.

WOUND HEALING FOLLOWING KERATINOCYTEALLOGRAFTING OF EXCISED TATTOO BEDS (FULLTHICKNESS) AND DONOR SITES (PARTIALTHICKNESS)Post-graft biopsy specimens showed a normalappearing epidermis with a flattened dermo-epidermal junction. Compact hyperkeratosisand a thickened granular layer, three to fourcells deep, was seen. Six months after ker-atinocyte grafting, the stratum comeum hada normal basket-weave appearance and thegranular layer was six cells thick. The epidermisappeared normal and the dermoepidermaljunction remained flat with little evidence ofrete peg formation. In the donor site woundsthe epidermis was better differentiated at oneweek to 10 days, with comification and someparakeratosis. The split thickness skin graftsshowed a convoluted dermoepidermal junctionand an epidermis of essentially normal ap-pearance. An increased dermal mixed cell in-filtrate, however, was observed compared withnormal skin.

KERATIN EXPRESSION IN FULL THICKNESS ANDPARTIAL THICKNESS WOUNDSWherever stratified epidermis was seen, thesuprabasal expression of keratins 1 (fig 1) and10 (fig 2) was found, commencing in the epi-basal layer. This occurred at all time pointsof tattoo and donor site biopsy. The basallyrestricted epitopes on keratins 5 and 14 werealso detected within a single basal cell layer.This was easier to delineate than normal in theabsence of rete pegs and dermal papillae. Theantibody LLOO1, showing the full presence ofkeratin 14 throughout the epidermis, reactedwith all epidermal cells in all wounds with noheterogeneity of expression. The reaction wasseen throughout the epidermis in first weekbiopsy specimens of deep wounds, but was lostin the stratum corneum three weeks after tattooremoval.

Hyperproliferation keratinsKeratins 6 (fig 3) and 16 (fig 4) could bedetected suprabasally at all time points aftersurgery in the tattoos and donor sites. Keratin6 was also expressed in basal cells. There wasno loss of keratin 6/16 expression three weeksafter tattoo removal when the epithelium ap-peared to be normalising morphologically. Thefew samples from later time points showed that,using the weak monoclonal antibody LMM3,keratin 16 expression was lost at six to eightweeks. Using the strong monospecific anti-peptide antibody directed against keratin 16,however, expression of this keratin persisted forlonger, although there were insufficient biopsyspecimens to determine the exact time of dis-appearance. In split thickness skin grafts keratin16 expression was still present six months aftersurgery. Keratin 17 was coexpressed supra-basally with keratins 6 and 16.

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1090 Myers, Nasaria, Brain, Purkis, Leigh

Mucosal keratins and simple epithelial keratinsThere was no evidence of the simple epithelialkeratins 7, 8, 18, 19, or mucosal keratins 4/13 in any interfollicular site at any time afterwounding. The donor site specimens retainingappendages acted as internal controls for the

O_.--c;*}-} t tre -simple epithelial keratins, as their luminal sweatgland cells stained positively. Mucosal samplesfrom gingiva acted as positive controls for ker-atin 4/13 staining.

INVOLUCRIN EXPRESSIONIn the tattoo wounds at one week involucrin__,;, - ;v;^< -- >staining was present in epibasal cells at the cellperiphery, but at two weeks it was reverting toitS normal position at the periphery of cells inthe upper stratum spinosum. At three weeks

Lthe position and staining were comparable withFigure 4 Keratin 16 staining with LL025 two weeks after cultured keratinocyte normal skin.allografting of an excised tattoo bed.

MUCINSEpithelial mucins could be detected mainly atone week after tattoo excision. The stainingwas focally suprabasal and was polarised withinthe cell to the upper border. Antibody HMFG2stained suprabasal tattoo biopsy specimens twoto three cell layers above the basal cell com-partment, but not into the stratum corneum,with small amounts of suprabasal staining attwo weeks and none at three weeks (fig 5).No staining was seen in the donor site biopsyspecimens. The antibody sm3 reacts with thecore protein of the HMFG2 antigen and isusually only exposed during altered glyco-sylation such as during tumorigenesis. Thisantigen could be detected in a limited numberof suprabasal keratinocytes in the tattoo biopsywounds at one week only, and within the popu-lation of HMFG2 positive cells.

Figure 5 HMFG2 staining one week after cultured keratinocyte allografting of anexcised tattoo bed. BASEMENT MEMBRANE STRUCTURE

Wherever the wounds were epithelialised, allbasement membrane proteins could be de-tected immunohistochemically at the dermo-epidermal junction. Type IV collagen was

present in vascular and epidermal basementmembranes in the tattoo biopsy specimenstaken at one week, but the staining was raggedand more diffuse than normal skin. There wasalso a high level of dermal staining distinctfrom the basement membrane zone, but spreaddiffusely throughout the connective tissue,which was reproducible and clearly not arte-factual. This was thought to be associated withrapid tissue remodelling. Type VII collagenreaction was limited to epidermal basementmembranes and was seen at all epithelial sites(fig 6). Unfortunately, no wound edges couldbe seen as the biopsied areas were uniformlyepithelialised, so the relation ofbasement mem-brane zone synthesis to wound edge couldnot be determined. Some diffuse staining ofconnective tissue was seen in the tattoo biopsy

Figure 6 Collagen VII staining with LH 7-2 two weeks after cultured keratinocyte specimens taken at one week, but this hadallografting of an excised tattoo bed. disappeared by two weeks.

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Wound healing after treatment with cultured allogeneic keratinocytes

DiscussionIn the deep wounds the appendages were re-moved as was most of the dermis. The contourand general morphology were strikinglyflattened and the epidermal healing must havecome from the cut edge ofthe wound. However,the changes in keratin expression were identicalwith changes found in interfollicular epidermisin more superficial wounds. Because of theform of treatment, biopsy specimens onlyshowed epidermis with an intact basementmembrane and changes in the advancingtongue against mesenchyme were not seen.

This study confirms that keratin 16, as de-tected by monoclonal antibodies, is normallyexpressed in wounds with keratin 6: the hy-perproliferative keratin pair,27 although keratin6 expression precedes that of keratin 16. Thesekeratins are constitutively expressed in certainstratified squamous mucosal epithelia2" and theskin of palm and sole (areas subjected to re-peated minor trauma), but have not been foundbiochemically in normal epidermis of othersites external to the outer hair root sheath andjunctional region.29 They have been dem-onstrated suprabasally in the epidermal hy-perproliferation seen in psoriasis and otherepidermal pathology including benign tu-mours, such as keratoacantomas and warts.27Our study has shown that keratin 16 persistsin keratinocyte grafted deep wounds for up tosix weeks, and up to six months in split thick-ness skin grafted wounds. Although keratins 6and 16 have not been detected in normal skin,Stoler et al0 have found the relevant mRNAspresent in normal skin. This raises the pos-sibility that regulation is occurring at a post-transcriptional level, allowing a rapid responseto injury with changes in the keratinocyte cy-toskeleton that may aid cell migration. In thissense the keratin 6/16 pair might be consideredmucoregenerative rather than hyperpro-liferative. The fact that this expression persistsfor weeks to months suggests a continuingprocess of migration, or an additional, moreprolonged and undetermined advantage re-sulting from that expression.The normal suprabasal keratins in skin (1/

10) were also found in wound cells. There wastherefore clear evidence of suprabasal co-expression of the differentiation keratins 1 and10 and the hyperproliferative keratins 6 and 16in the wound healing epidermis in the samecells. This does not, however, exclude a quant-itative reduction in expression of keratins 1/10as seen on gels in epidermal hyperproliferationin psoriasis."

Glycosylation of membrane glycoproteins isassociated with epidermal differentiation, andthis process is altered in wound healing andhyperproliferation such as psoriasis. The ex-pression of human milk fat globule mucins inskin has not been reported except in retinoidtreated skin, but presumably reflects this knownaltered termination of glycosylation.

Studies of the regenerating basement mem-brane zone in wounds both in animal modelssuch as the primate and rodent, and in the fewhuman studies performed, have shown that thecomponents are not synthesised synchronously.

The emergence of laminin, fibronectin andcollagen VII lags behind the re-epithelialisationwhich is associated with the presence ofbullouspemphigoid antigen (BPA) and collagen IV.This may reflect the likelihood that BPA andcollagen IV provide a framework onto whichother basement membrane zone componentsattach. Type VII collagen probably requiresformation of a neodermis rather than granu-lation tissue to provide an attachment (or an-choring) plaque. Anchoring fibrils were foundto be slow in appearance and maturation inprevious studies of keratinocyte graftedwounds.32

Following keratinocyte grafting the healingskin expresses the hyperproliferative phenotypethroughout the first three weeks after grafting,despite epithelialisation having occurred. Thus,these markers are the most sensitive for alteredepidermis following wounding. This has somesignificance to both the production of com-posite skin grafts in the laboratory, and to theclinical grafting of partial thickness defects. Inthe former case, although cultured keratinocytesheets express the keratin 6/16 pair, the ex-pression appears subjectively decreased to vary-ing degrees in composite grafts, in a reciprocalfashion to the differentiation keratin 1/10 pair.Quantification of keratin 6/16 expression may,therefore, become a measure of the quality ofsuch composites. One might envisage a degreeof expression which represents a compromisebetween the differentiation required for graftstability on the one hand, and the regenerativecapacity capable of providing the necessarysignals (for example, cytokines) for graft vas-cularisation and complete healing without scarson the other. In the latter case quantificationof the keratin 6/16 pair in partial thicknesswounds by punch biopsy might offer somemore objective assessment of the need for splitthickness skin grafting.

In cultured keratinocyte grafted wounds thedermoepidermal junction remains flat and, al-though a basement membrane is laid downhelping to anchor keratinocytes, they are stilllikely to be vulnerable to shearing forces.Clearly, split thickness skin grafted woundshave an advantage as they maintain the con-volution of the dermoepidermal junction, eventhough hyperproliferative keratins appear topersist longer than keratinocyte graftedwounds. The time course of expression ofregulatory cytokines will be of considerableinterest.

Mr S R Myers is generously sponsored by the Restoration ofAppearance and Function Trust (RAFT) at Mount VernonHospital, Northwood, Middlesex.

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