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F.B. Niessen - On the nature of hypertrofic scars

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Samenvatting

Hoofdstuk 1

Littekenvorming is noodzakelijk. Wondgenezing is nodig voor de bescherming van het individu tegen infectie en ter voorkoming van overmatig vochtverlies via de huid. Een teveel aan genezing echter kan leiden tot excessief littekenweefsel in de vorm van hypertrofische littekens en keloïden en lijkt onnodig. De oorzaak daarvan weten we tot op heden niet. Daarom zijn er vele, niet afdoende, therapieën voorhanden en kan de vorming van een overmaat aan littekenweefsel nog steeds niet worden voorkomen. Voor nader onderzoek naar deze processen ontbreekt een experimenteel diermodel, omdat alleen mensen hypertrofische littekens en keloïden maken.

Het doel van dit proefschrift is tweeledig. Ten eerste, het vinden van een betrouwbaar humaan wondgenezingmodel waarin bij de mens de wondgenezing op een gestandaardiseerde wijze kan worden onderzocht en waarin hypertrofische en normale littekens binnen hetzelfde individu kunnen worden vergeleken. Ten tweede, het verkrijgen van meer inzicht in de afwijkende wondgenezing processen die littekenhypertrofie en/of keloïd veroorzaken.

Hoofdstuk 2

Om de ontspoorde wondgenezing te bestuderen is het eerst noodzakelijk de normale wondgenezing te beschrijven. Wondgenezing bestaat uit drie elkaar overlappende fasen: ontsteking, proliferatie of granulatie en remodellering, waarbij de hemostase tot de ontstekingsfase wordt gerekend. De laatste fase, het uitrijpen en remodelleren van een litteken, is normaal gesproken na ongeveer een jaar voltooid.

Genezing ontstaat doordat specifieke cellen in en om het wondgebied mediatoren produceren en uitscheiden waarop andere cellen reageren met mobilisatie, proliferatie, de vorming van nieuwe genezingsmediatoren of de productie van dermale matrixcomponenten. In eerste instantie wordt een tijdelijke wondmatrix gevormd. Deze is rijk aan fibronectine, hyaluronzuur en collageen type 3, voornamelijk geproduceerd door fibroblasten. Tijdens de remodellering rijpt de wondmatrix uit en neemt de relatieve hoeveelheid van collageen type 1 toe.

Dit hoofdstuk beschrijft de mediatoren en receptoren die een rol spelen bij de genezing van de huid. Er wordt uitgebreid ingegaan op de groeifactoren die geproduceerd worden door thrombocyten, neutrofiele granulocyten, macrophagen, fibroblasten, keratinocyten, Langerhanscellen, T-lymphocyten, mestcellen en endotheelcellen. Deze groeifactoren zijn van belang bij de sturing van de genezingsprocessen en spelen een belangrijke rol bij het ontstaan van hypertrofische littekens en keloïden.

Hoofdstuk 3

In gepredisponeerde, bij voorkeur donker-gekleurde, mensen kan met name presternaal, op de schouders en het bovenste deel van de rug, een teveel aan littekenweefsel ontstaan na operatie, verwonding of verbranding. Hypertrofische littekens zijn verheven boven het huidniveau en blijven binnen de grenzen van de oorspronkelijke huidlaesie, terwijl keloïden boven het huidniveau zijn verheven maar buiten de grenzen van de oorspronkelijke laesie groeien. Hypertrofische littekens ontstaan meestal drie tot vier weken na een verwonding van de huid terwijl keloïden drie maanden tot meer dan een jaar na verwonding kunnen ontstaan.

De  behandeling van deze littekens is tot op heden nog puur symptomatisch en in de meeste gevallen onvoldoende.

Om een goede therapie te vinden en een behandeling ter voorkoming van overmatig littekenweefsel is het noodzakelijk te weten hoe en op welk moment de genezing ontspoort. In dit hoofdstuk wordt de huidige kennis omtrent littekenhypertrofie en keloïdvorming gerangschikt naar analogie van de processen beschreven in hoofdstuk 2. Het lijkt erop dat niet één proces, maar dat een aantal elkaar opeenvolgende wondgenezingsprocessen gestoord zijn geraakt, waarbij immunologische cellen als Langerhanscellen, T-lymfocyten, mestcellen en keratinocyten d.m.v. groeifactoren fibroblasten stimuleren tot een overmatige littekenvorming en/of een verminderde afbraak.

Voorzover bekend grijpt geen van de huidige therapieën duidelijk aan op de oorzakelijke mechanismen van littekenhypertrofie en geen van hen laat een afdoende respons zien of een verlaging van het recidief percentage. Het meest toegepast wordt de intralesionale behandeling met corticosteroïden, al dan niet in combinatie met chirurgische technieken.

Hoofdstuk 4

Een dierexperimenteel model is niet voorhanden. Wil een littekentherapie op een adequate wijze worden getest dan zijn er twee vergelijkbare littekens nodig binnen dezelfde patiënt en moeten deze littekens zich bij voorkeur bevinden op een voor hypertrofie gepredisponeerde plaats op het lichaam. De littekens van een mamma-reductie zijn daarvoor ideaal. Ze kunnen voor onderzoek worden onderverdeeld in vier gedeelten: links en rechts, mediaal en lateraal.

Bij 129 vrouwen die een mamma-reductie ondergingen werden de littekens onderzocht. Na drie maanden heeft 64,3% van de patiënten in één of meer littekengedeelten hypertrofie, na zes maanden daalt dit tot 56,6% en na een jaar heeft nog steeds 35,3% van de patiënten minimaal één hypertrofisch litteken over. Indien de 516 (129 x 4) littekengedeelten in totaal worden bekeken dan is de incidentie van hypertrofie 39,0% na drie maanden hetgeen daalt tot 17,4% na een jaar. In het littekenmodel worden geen keloïden gezien.

Patiënten die niet roken, sneller bruin worden of ergens allergisch voor zijn vormen in dit model meer littekenhypertrofie ten opzichte van de controle groep (zie hoofdstuk 9).

Echoonderzoek, Laser-doppler flow-meting en kleurmeting van de littekens kunnen in dit model alle significant hypertrofie van normaal onderscheiden en tonen hun waarde als objectieve parameters om de littekenvorming te beoordelen.

Hoofdstuk 5

De relatie tussen littekenhypertrofie en allergie, mestcellen en Langerhanscellen is eerder beschreven in de literatuur. Deze onderzoeken waren over het algemeen niet gestandaardiseerd. Ook in het onderzoek beschreven in hoofdstuk 9 lijken patiënten met allergie gepredisponeerd te zijn voor de vorming van hypertrofische littekens. Deze gegevens waren echter gebaseerd op een globale anamnese. Een uitgebreide enquête die werd teruggestuurd door 77% van de 129 patiënten toont geen relatie aan tussen littekenhypertrofie en contactallergie, rhinitis of asthma.

Uit de laterale inframammaire littekens werden biopsieën genomen, waarvan coupes werden gesneden en gekleurd. Expressie van antilichamen tegen groeifactoren werden op verschillende plaatsen van het litteken gescoord. In de epidermis basaal en suprabasaal, in de dermis perivasculair en in de dermale matrix. De mestcellen werden subepidermaal en middermaal gescoord, de Langerhanscellen als percentage van het oppervlak van de epidermis. De drie maanden oude littekens konden verdeeld worden in normaal (NN) of hypertrofisch (HH/HN). De hypertrofische littekens werden verder onderverdeeld in littekens die hypertrofisch blijven (HH) of normaal worden (HN) na twaalf maanden. Het aantal coupes was als volgt verdeeld: negen coupes van normale littekens na drie maanden en vijf coupes na twaalf maanden, zes coupes na zowel drie als twaalf maanden van hypertrofische littekens die hypertrofisch blijven en acht respectievelijk zeven coupes van hypertrofische littekens die normaal worden.

Het aantal subepidermale en middermale mestcellen toont geen relatie met de vorming van hypertrofische littekens. Het aantal epidermale Langerhanscellen daarentegen blijkt sterk significant gerelateerd aan littekenhypertrofie (p < 0,001). Interleukine-4, met name geproduceerd door mestcellen, is bekend om zijn stimulatie van fibroblasten tot de vorming van collageen. Deze relatie wordt ook in dit onderzoek gevonden; de hypertrofische littekens tonen een significant verhoogde suprabasale expressie van IL-4 (p < 0,05). Gamma-interferon kan deze stimulatie van IL-4 remmen, maar laat geen verschillen zien tussen hypertrofische en normale littekens.

Hoofdstuk 6

Onderzoek naar littekenhypertrofie wordt meestal gefocused op de neodermis, waarschijnlijk omdat dit deel van het litteken het meest opvallende aspect vertoont. Maar het zijn juist de gebieden tussen de mazen van een mesh-graft (waar de epidermis ontbreekt) die hypertrofisch worden.

In de studie naar epidermale processen tijdens littekenvorming worden in de hypertrofische littekens afwijkingen gevonden ten opzichte van normale littekens. Bij littekenhypertrofie wordt een toegenomen acanthose gezien (p < 0,001), de keratinocyten delen meer (p = 0,03)  en rijpen abnormaal uit (p = 0,02) zoals bewezen door een verhoogde expressie van respectievelijk Ki-67 antigen en cytokeratine 16 in de keratinocyt. Interessant is de bevinding dat een toegenomen proliferatie en een verhoogde CK 16-expressie bij hypertrofische littekens na drie maanden voorspelbare waarde hebben bij het al of niet hypertrofisch blijven van het litteken.

Hoofdstuk 7

Groeifactoren spelen een belangrijke rol bij de vorming en ombouw van de dermale matrix. Zij kunnen fibroblasten aanzetten tot de productie van collagenen en proteoglycanen of stimuleren de aanmaak van metalloproteinasen en collagenasen die de matrix afbreken.

TGF-β, PDGF en bFGF zijn geassocieerd met collageenaanmaak, IL-1α, IL-1β en TNF-α zijn geassocieerd met collageenafbraak.

Scoring van de groeifactorexpressie in de epidermis en dermis laat een significant verlaagde epidermale expressie van IL-1α zien in de hypertrofische littekens zowel drie en twaalf maanden post-operatief (p = 0,02/0,01). PDGF daarentegen toont een verhoogde expressie in de dermis na drie maanden (p = 0,04) en in de epidermis en dermis van de hypertrofische littekens twaalf maanden post-operatief (p < 0,01/0,02). De andere groeifactoren laten geen significante verschillen zien.

Hoofdstuk 8

Ontstekingscellen spelen een rol bij het ontstaan van littekenhypertrofie. Na een chirurgische interventie wordt de huid gesloten met een hechtdraad. Deze draad is een vreemd lichaam en zal in de huid een ontstekingsreactie veroorzaken met als gevolg meer of minder littekenweefsel. De omvang van de reactie hangt onder andere af van het soort en het type hechtmateriaal en de hoogte waarop deze wordt geïmplanteerd. Een gevlochten draad geeft meer reactie dan een draad uit één stuk en een oppervlakkig gelegen hechtdraad geeft meer reactie dan een dieper gelegen hechting.

Bij de afdelingen plastische chirurgie in de Academische Ziekenhuizen van Utrecht en Nijmegen werden mamma-reductie littekens gehecht met oplosbaar multi-filament Vicryl-rapide. Een nieuwe oplosbare mono-filament hechtdraad met een langere treksterkte, Monocryl, kwam op de markt.

Uit het vergelijkende onderzoek in het mamma-reductie model blijkt dat Monocryl (n = 28) significant smallere littekens geeft, die minder hypertrofie laten zien en minder lang rood blijven dan Vicryl-rapide (n = 53).

Hoofdstuk 9

Een nieuwe, veelbelovende behandeling van hypertrofische littekens en keloïden is de applicatie van siliconenmaterialen. Alhoewel niet alle littekens hun volume verliezen, geven deze materialen forse verlichting van de littekenklachten bij patiënten.

Kunnen siliconensheets en -gels littekenhypertrofie voorkomen?

Wanneer deze drie dagen na de operatie op de helft van een inframammair litteken worden geappliceerd, 24 uren per dag gedurende drie maanden, dan geven siliconensheets (n = 68) of -gels (n = 61) geen verminderde incidentie van littekenhypertrofie ten opzichte van de onbehandelde zijde van het borstlitteken. Na zes en twaalf maanden daarentegen tonen de behandelde littekens zelfs meer hypertrofie dan de onbehandelde littekens (p < 0,01/0,02).

Spaarzame gegevens uit de recente literatuur tonen wel een profylactische werking wanneer pas twee tot drie weken na de operatie met de behandeling wordt begonnen.

Hoofdstuk 10

Het proefschrift leidt tot een nieuwe theorie over de vorming van littekenhypertrofie welke in de toekomst verder bewezen zal moeten worden.

Na verwonding van de huid probeert het lichaam zich zo snel mogelijk te beschermen tegen invloeden van buitenaf. Gestuurd vanuit de dermale matrix vormen keratinocyten vrij snel een nieuwe beschermende epidermale laag. Daaronder wordt tijdelijk granulatieweefsel gevormd. Indien de epidermis een sufficiënte bescherming biedt volgt er een signaal naar de dermis, waarop de remodellering en uitrijping van de dermale matrix kunnen starten.

In de hypertrofische littekens is de epidermis afwijkend en is zijn functie mogelijk gestoord. Er zijn meer Langerhanscellen aanwezig en de keratinocyten delen vaker en rijpen abnormaal uit. Beide celtypen werken samen in de immunologische afweer van de huid. Een verstoorde afweer biedt een inadequate bescherming met als gevolg geen, of een verlaat signaal naar de dermis wat leidt tot het uitblijven of vertragen van de remodellering. Een hypertrofisch litteken kan ontstaan.

Een verlaagde hoeveelheid IL-1a zorgt voor een verlengde aanwezigheid van Langerhanscellen en een verminderde collageenafbraak. Toegenomen IL-4 en PDGF veroorzaken een verhoogde collageen aanmaak. De aanwezigheid echter van groeifactoren zegt nog niets over hun exacte origine.

Dit proefschrift toont afwijkingen in de epidermis van hypertrofische littekens. Of de oorzaak daarvan in de epidermis zelf of in de dermis is gelegen zal verder moeten worden onderzocht.

De toegankelijkheid van de epidermis daarentegen biedt de mogelijkheid om de gestoorde epidermale-dermale interactie bij hypertrofische littekens te beïnvloeden middels applicatie van medicijnen. Op deze wijze kan mogelijk littekenhypertrofie worden voorkomen. Voor onderzoek naar de epidermale-dermale interactie is het mamma-reductie model uitermate geschikt.

Chapter 10

General discussion and future perspectives

The skin is our contact with and protection against the outside world. It interacts with the environment by touching, feeling, tasting and creating, but unfortunately , it is sometimes damaged by external violence. Then, our sensory interface is disrupted, whereupon it will repair itself as soon as possible to restore its important protective function against micro-organisms and excessive water loss.1-4 These repair mechanisms are very complex, but well balanced, and restore the skin’s protective function quickly; first by creating a scab, and second by epidermal and dermal scar tissue formation. Sometimes though, an overdrive response may occur, leading to the formation of excessive scars like hypertrophic scars and keloids. They lack flexibility and give rise to severe functional and cosmetic problems, which hinder a normal interaction with the environment.

The causative factors of these scars are still unknown. Early wound healing research was mainly focused on the description of clinical features of scar tissue. Only the last decades studies were published regarding growth factor influence and scarless foetal wound repair, that clarified some of the basic mechanisms of excessive scar tissue formation.4 Unfortunately, this premature knowledge has not yielded new satisfying wound healing therapies that provide normal or scarless tissue repair. Until now, therapies just aim at reduction of scar tissue, but are not based on visions about the causative disturbed wound healing mechanisms, and therefore not able to prevent the development of excessive scar tissue.5,6

For a better understanding of the origin of excessive scars, the current knowledge about hypertrophic scarring and keloid formation is reviewed in chapter 3 of this thesis, and placed into the light of the successive wound healing processes as described in chapter 2. This rearrangement of knowledge furthers the insight about ‘the exact point’ where normal healing ends and excessive healing begins, and will help to focus on the processes that can ultimately be influenced to prevent and treat excessive scar tissue formation.

It is difficult to find a satisfying wound healing model to investigate the processes leading to hypertrophic scar and keloid formation because, and this is very interesting, only humans suffer from excessive scarring.7,8 Unfortunately, most wound healing studies are performed in vitro or in animal studies, of which the results can not always be extrapolated to the human situation, and most human research is done on hypertrophic scars in burn patients. In addition,

most of the human scar studies compare scars from a different age and different sites on the body, and most of the time these excessive scars were compared with normal skin instead of normal scar tissue. Furthermore, these studies generally lack a proper standardisation and, more importantly, they describe static events whereas wound healing is a highly dynamic proces.9

The scope of this thesis is twofold. First, to find a reliable human wound healing model in which normal and excessive scars can be compared at the same site of the human body and in which it is possible to standardise the dynamics of the wound healing processes as much as possible. Second, to get more insight in ‘the exact point’ where normal wound healing becomes excessive healing in order to find a better treatment against the development of hypertrophic scars and keloids.

The inframammary scars following breast reduction surgery are used as a human wound healing model. This model proved to be very satisfactory in studying and comparing hypertrophic scar and normal scar formation within the same patient. Furthermore, this model makes it possible to investigate different scar treatment modalities with the patient as her own control. To be able to perform this research it is necessary that the breast reduction technique results in anchor-like or at least lateral inframammary scars. Researching scar tissue formation with objective indicators like B-scan ultrasound, laser Doppler flowmetry and colour measurements has shown its value in this model. All significantly differentiate between hypertrophic and normal scars. In future research therefore it is not necessary to use all these indicators. We recommend the usage of just B-scan ultrasound, because it provides information about the intradermal height of the scar, making the clinical feature whether or not the scar is raised above skin level less important in distinguishing hypertrophic from normal scars.         

This thesis shows that it is very important to take scar samples of the same wound healing age, because hypertrophic as well as normal scars exhibit very different histopathologic features both at three and twelve months following surgery. Probably, it is also important to take scar samples from the same spot on the body, because excessive scar formation has a highly regional susceptibility.10,11 It is obvious that the wound healing process is a dynamic process and to investigate these dynamics it is necessary to obtain several scar samples at different wound healing ages. Unfortunately, we have only taken scar samples at three and twelve months following surgery, which just resembles the scar tissue dynamics regarding the remodelling phase. According to the literature, hypertrophic scars remain in an earlier wound healing stage and lack an adequate maturation as shown by the persistence of an abundant amount of young scar tissue.12,13 In future research it is important therefore to obtain scar samples of the earlier wound healing phases as well, especially around three and four weeks post-operatively, because in our opinion, clinically, this is the moment excessive scarring appears to begin.

It is likely that scar formation follows the successive wound healing processes, but that each scar in each person has its own time scale. When we compare scars of the same age every scar is more or less in a different stage of the wound healing process, depending on the kind of scar tissue, the patient, and the site on the body we are dealing with. So, when trying to find and use a reliable wound healing model we must be aware that there still are inter- and intra-individual scar differences. The inframammary scars after breast reduction surgery provide a good scar model in dealing with these problems, because it gives the opportunity to research different scar types on the same spot of the body within the same patient. Because it has been shown that the incidence of excessive scarring is equal in both sexes, and there appear to be no indications that wound healing in females differs from wound healing in males,14 it seems to be no problem that only female patients are researched in this model.

Although hypertrophic scars and keloids were thought to resemble different expressions of a gliding scar tissue scale, their clinical and histopathological behavior as well as their therapeutic opportunities differ to such an extend that it is highly questionable if this is true.13 The wound healing model is perfect for studying hypertrophic scar formation, but unfortunately no keloids were found, not even in the dark-skinned patients. Research on keloids therefore needs another wound healing model, such as research on earlobe keloids for example.

The second part of the thesis tries to provide more insight into ‘the exact point’ where normal wound healing ends and excessive healing starts in the breast reduction research model. It deals with the seven questions stated in the introduction.

   1. Which patient is predisposed for the formation of excessive scar tissue? (chapter 4 and 9)

This question is mainly answered by: black people. Some surgeons will add “red hair” and “fair skinned people”, as well. The first is true. Although only a few dark skinned patients were included in our research group, the results show that easily tanning patients suffer a higher incidence in scar hypertrophy than non-tanning patients. The fact that dark skinned people show a higher incidence in excessive scar tissue formation, is probably related with their ease of tanning.15,16 The amount and behavior of melanocytes that play an active

role in the epidermal immunological net,17,18 can therefore be involved in hypertrophic scar tissue or keloid formation.

The latter is not true. Fair skinned people develop scars that are more reddish, which lasts for a longer period, but they do not exhibit a higher incidence of hypertrophic scar formation during research.

Patients who smoke show a lower incidence of scar hypertrophy than non-smokers. This can be the result of several processes. Apart from an increased amount of collagen, hypertrophic scars also contain an increased amount of scar tissue water,19,20 probably due to the higher amount of hyaluronic acid.21,22 The nicotine in cigarettes is responsible for blood vessel constriction,23 and can lead to a reduction of tissue oedema. Furthermore, smoking impedes the collagen production during wound healing,24 and nicotine reduces the ability of the immunological system to overreact in defence reactions and can inhibit immunological processes involved in hypertrophic scarring.23,25

Another interesting feature was the observation that patients suffering from allergies showed a higher tendency in hypertrophic scar formation.(chapter 9) Both in allergic skin reactions and hypertrophic scars red, oedematous and itching skin lesions are observed, which suggests the possibility of shared pathogenetic mechanisms. More extensive research by questionnaire, however, showed us that neither contact allergy nor rhinitis or asthma is related with scar hypertrophy.(chapter 5) For a more accurate proof of this relationship blood research and cutaneous tests should be done. It remains possible that cells involved in allergic, cell mediated immune responses may also play a role in the pathogenesis of hypertrophic scar and keloid formation. An abundant presence of Langerhans cells, CD4 T-lymphocytes and macrophages was found in active post-burn hypertrophic scars, compared with mature hypertrophic and normotrophic scars.26

    * Are mast cells and Langerhans cells involved in the formation of hypertrophic scars? (chapter 5).

It is known that both mast cells and Langerhans cells are involved in allergic reactions. In the literature mast cells are associated with the development of excessive scar tissue as well.27-29 Their granules contain several factors that can stimulate fibroblasts to produce collagen and other matrix products.29-32 But according to our research, when scars of the same age are taken and, probably important also, when they are taken from the same part of the body,33,34 no difference in mast cell numbers is found between hypertrophic and normal scars. Other authors in recent publications came to the same conclusion.35,36 Moreover, the absence of fibrotic skin lesions in patients with mastocytosis, and the lack of fibrosis in many neoplastic tissues infiltrated with mast cells,28 show that skin fibrosis not necessarily develops in the presence of an increased number of mast cells. It is also possible that mast cells play a more important role during the inflammatory phase of wound healing, whereas this research was dealing with the remodelling phase.

Langerhans cells, on the other hand, are found in significantly higher numbers in the epidermis of hypertrophic scars compared with normal scars,37 as shown in the research. Like mast cells, they are involved in allergic reactions, can bind IgE and are important in processing and presenting antigens to T-lymphocytes.38,39 T-lymphocytes in turn are involved in tissue fibrosis 40,41 and CD4 cells are especially found in higher numbers in post-burn hypertrophic scars.26 They contain several lymphokines that can induce tissue matrix production and are possibly important in the development of excessive scar tissue. An overview of the scars in this study though, did not show any differences in T-lymphocyte numbers. But their subsets were not differentiated during this research and will be investigated in the near future. Whether or not Langerhans cells directly influence fibroblasts during ECM turn over, or if they activate certain T-lymphocyte subsets first that in turn stimulate fibroblasts to produce matrix products, has to be clarified by future research as well.

    * Are there differences in epidermal behavior between hypertrophic and normal scars. (chapter 6)

The results described in this thesis show that the changes due to hypertrophy are not con­fined to the dermal compartment, but also include the epidermis. In this respect three interesting observations were made. First, three-month-old hypertrophic scars showed more acanthosis than non-hypertrophic scars of the same age, irrespective of whether they remain hypertrophic or become normal scars. Second, epidermal abnormalities with respect to differentiation persist up to three months as witnessed by the expression of cytokeratin 16 (CK16). At three months, hyper­trophic scars that remain hypertrophic showed significantly more CK16 expression than normal scars (that remained normal after twelve months). Moreover, hypertrophic scars at three months that remain hyper­trophic showed significantly more CK16 than hypertrophic scars that would flatten in the futu­re. And third, staining for Ki-67 antigen, a marker for cell proliferation, revealed an increase in basal keratinocyte growth fraction in three-month-old hypertrophic scars compared to non-hypertrophic scars. At three months, hypertrophic scars that remain hypertrophic tend to be more hyper­proliferative than the ones that become normal after twelve months. Recently, some other authors published research that supports these results.42,43

The keratinocytes of hypertrophic scars show a different behavior than keratinocytes from normal scars.  According to this research however, it is still unclear whether the observed epidermal activation is a mere consequence of the hypertrophic scar formation, or causally involved in the pathogenic process, because it is known that both keratinocytes and fibroblasts can influence each other during wound healing.44-46 IL-1 especially proved to be important in this interaction 47,48 and has shown its importance in the studies as well. In the breast reduction scar model research with medication that inhibits keratinocyte proliferation will be done in the near future to try to answer this question.

Keratinocytes and Langerhans cells serve as accessory cells in the epidermal immunological network and are also known to influence each other.17,49-51 It is not clear if the activated keratinocytes are responsible for the observed increased number of epidermal Langerhans cells, or that these Langerhans cells induce the keratinocyte activation. Both cells are capable of influencing fibroblast extracellular matrix production by the release of their cytokines and can be responsible for hypertrophic scar formation. To further elucidate these mechanisms it is necessary to obtain and investigate scar tissue samples from the inflammation and granulation processes during wound healing. Further proof of the epidermal involvement in hypertrophic scar formation is shown by the fact that the epidermal differences seen at three months following surgery disappear after scar maturation at twelve months.

    * Do hypertrophic scars show different cytokine profiles compared with normal scars? (chapter 7)

Many papers have been published regarding growth factor influence on wound healing processes. TGF-b1 and PDGF especially are associated with excessive scar tissue production 13 and have proven to be successful for the acceleration of wound healing in animal models.52,53 This research, however, does not show an overexpression of TGF-b1 in hypertrophic scars, probably because it plays a more important role during the earlier phases of wound healing.54,55 In this role TGF-b1 can also be responsible for the development and increased appearance of epidermal Langerhans cells we have found.56-58 PDGF on the other hand shows a higher expression in both epidermis and dermis of hypertrophic scars in this research, which is in concordance with the literature.59,60 New is the discovery of differences in epidermal IL-1a and IL-4 expression, without differences in the dermal expression of these cytokines. This suggests an important epidermal signalling pathway during hypertrophic scar formation. IL-1a, produced by Langerhans cells and keratinocytes, is found significantly reduced in the epidermal area of the hypertrophic scars, and is probably responsible for an insufficient collagen breakdown in the dermis.61,62 IL-1a also proved to induce Langerhans cell differentiation and migration to the dermis.63-65 Decreased levels of this cytokine, due to abnormal functioning keratinocytes 42,66 therefore, can be responsible for a prolonged epidermal Langerhans cell presence and a continuous hyperreactive state, responsible for scar tissue oedema.67,68 An insufficient reciprocal interaction between keratinocytes and Langerhans cells has also proved to provide chemotaxis for T-lymphocytes, which can further modulate the ongoing immune responses involved in excessive scar tissue formation.39,69 IL-4 on the other hand is found significantly

increased in the epidermal area of the hypertrophic scars. It has a role in hypersensitivity reactions 70 by switching the B-lymphocytes to IgE production,71-74 and by enhancing IgE receptor expression on Langerhans cells.75 Fibroblasts in turn express IL-4 receptors, and can be stimulated to produce overabundant extracellular matrix collagen and proteoglycans after IL-4 stimulation.72,76-78 The importance of IL-4 is also shown by the fact that an overexpression of IL-4 is associated with abnormal keratinocyte activation and proliferation,79,80 which is seen in the hypertrophic scars as well.42,81

Research on cytokines is very difficult because an immunohistochemical proof of their existence in scar biopsies will tell us nothing about their actions. A low concentration for example can have opposite effects compared with a high concentration of the same growth factor, while its mode of action during the inflammatory phase can be completely opposite during the remodelling phase. TGF-b for example stimulates ECM production in the early phases of wound healing, but regulates collagen breakdown during remodelling.82-85 The investigated cytokines show mainly epidermal differences. However, this does not proof with certainty that these cytokines are produced in the epidermis, and moreover it will not give total evidence that the increased epidermal expression will have an increased dermal effect on fibroblast matrix turnover. It also can indicate an increased reaction of the epidermis on dermal events. Nevertheless, this descriptive research at least strongly indicates that the epidermis is involved in hypertrophic scar formation. This will give the opportunity to interfere with the interaction between epidermis and dermis by the application of drugs onto the skin, to influence hypertrophic scar tissue formation.

    * Is it possible to predict whether or not a hypertrophic scar at three months following surgery will remain hypertrophic, or will become normal at twelve months? (chapter 6, 7 and 9)

It is of clinical importance to predict whether or not a hypertrophic scar will become normal. In case an hypertrophic scar will flatten it is not necessary to treat the existing scar and is it justified to wait for the natural course the scar will exhibit. The objective indicators B-scan ultrasound, laser Doppler flowmetry and colour measurements that were used at three months unfortunately could not predict the scars behavior during maturation at twelve months. For more information it is necessary to take a scar biopsy. As mentioned before, the abnormal differentiation of keratinocytes, as witnessed by cytokeratin 16 expression, will predict the clinical outcome at twelve months. A higher expression of CK 16 is significantly associated with a persistent scar hypertrophy.

From the cytokines we have investigated in hypertrophic scars, an increased suprabasal epidermal IL-1a expression at three months post-operatively provides a significantly higher chance of losing the hypertrophic appearance during maturation.

    * What is the influence of suture materials on the formation of hypertrophic scars? (chapter 8)

Suture materials act as foreign bodies in the human skin and will attract inflammatory cells. They are responsible for an inflammatory reaction leading to redness, raised temperature and oedema of the sutured tissue. This reaction at the wound site is more extensive in case of absorbable compared with non-absorbable sutures, multifilamentous compared with monofilamentous, non-flexible compared with flexible sutures and/or superficial compared with deep dermis implantation of the suture.86,87 In the experiments absorbable monofilamentous Monocryl, which has less affinity with micro-organisms compared with absorbable multifilamentous Vicryl-rapide,86,88 show a reduced tissue reaction, as proved by a decreased scar blood flow three and six months postoperatively. As mentioned before, the increased tissue reaction most likely causes the higher incidence of hypertrophic scarring and the significantly increased intradermal height found in this study. The deeper an absorbable suture is buried the less tissue reaction it will give.87 An unequal suturing depth can be responsible for the capricious appearance hypertrophic scars can exhibit. It is important therefore not to suture too superficially and to keep an equal distance to the skin surface while suturing.

    * Can silicone materials be used for the prevention of hypertrophic scar formation? (chapter 9)

It is well known that occlusive therapy has positive effects on wound healing and stimulates the reepithelialization of an open wound.89,90 It is also well known empirically, but insufficiently investigated, that therapeutic occlusion with silicone materials reduces the volume of hypertrophic scars in most of the cases.91-93 In some articles in the literature it is described as well that the prophylactic usage of silicone gels and sheets show a lower incidence of scar hypertrophy compared with untreated scars. The mechanism of action however remains elusive too.92,94,95 Unexpectedly, in contrast with these results, this study does not show a prevention of hypertrophic scar development after the prophylactic usage of silicone materials. At three months following surgery the incidence of scar hypertrophy is equal in the silicone treated and micropore supported scars. At six and twelve months however the treated scars even show a surprisingly increased incidence of scar hypertrophy compared with the untreated scars. Assuming that the studies described in the literature are performed according to good scientific standards, the different results are most likely due to the moment the silicone application starts. The main difference with our research is the onset of application. In the studies of Ahn, Fulton, Cruz-Korchin and their co-workers, they started the prophylactic therapy at the remodelling phase two or three weeks postoperatively, whereas in this research the silicone application started during the inflammatory phase, three days following operation. In chapter 9 it is suggested that silicone materials reduce the non-immunological stimulation of mast cells leading to a reduced oedema, responsible for a flattening of hypertrophic scar tissue, which is possibly the cause for the different results. But during later research it is found out that there are no differences in mast cell numbers between hypertrophic and normal scars. Although the number of cells is not directly related with the mast cell activity, it is not likely that the unequal results are explained by mast cell involvement.(chapter 5)

Because little previous research has been done it is very difficult to explain why a prophylactic usage of silicone materials at three days following surgery will fail and a usage at two to three weeks following surgery will have its positive effect on the development of hypertrophic scars. It is known that the occlusion and hydration, not the silicone, provides the effect of silicone therapy,96-99 and it has been found  that its therapeutic effect is not because of pressure, the difference in oxygen tension and temperature, or silicone leakage.100,101 An important finding is the increased hydration of the stratum corneum of the skin after silicone therapy.96,97,101

An explanation for the prophylactic failure in our study is highly speculative, but some facts can be mentioned. Hypertrophic scars are build up by an excess of extracellular matrix and an excess of tissue fluid. In our opinion silicone materials mainly have their effect on the amount of tissue water as proved by a quick reduction in scar volume the moment application starts, and the quick return to the original volume in case the therapy did not last long enough. The fact that silicones show inferior results on more mature scars containing less tissue water provides further proof of this assumption. It is thought that the silicone materials function as a sort of epidermal barrier which provides tissue resilience, vasoconstriction and a reduction in tissue water.91,102 Instead of a reduced stimulation of mast cells, as previously supposed, it is possible that tissue resilience has its effect on the epidermal Langerhans cells or keratinocytes. Occlusion widens the epidermal intracellular space and activates Langerhans cells to lower their epidermal number and increase their dermal number,103-105 which can affect their cytokine release.106,107 How this will influence the inflammatory effects of IL-1 and TNF-a 108 is not yet clear. Hydration of keratinocytes, on the other hand, induce the release of mediators that inhibit fibroblast proliferation and reduce their collagen and glycosaminoglycan synthesis.46,97,109 This can have a long term effect on extracellular matrix production. An epidermal induced inhibition of fibroblasts is in concordance with the theory that the epidermis is involved in hypertrophic scar formation and is supported by unpublished results which show a significantly reduced keratinocyte proliferation after silicone application. However, to further complicate the questions raised by occlusive therapy, occlusion has also shown an increased mean level of IL-8, bFGF, and GMCF mRNA,99 that in contrast stimulates inflammation and matrix production.108 On the other hand, in the same clinical study on hypertrophic scars a decreased mean level of TGF-b and fibronectin mRNAs was found, responsible for a reduced extracellular matrix production.99

It remains strange though that immediate application of silicone materials will not have this effect later during wound repair. It is possible that when epidermal cells sense an insufficient barrier function they remain activated and continue to signal the dermis to produce young scar tissue. The absence of a signal necessary to start the remodelling of young granulation tissue into silent mature scar, can result in hypertrophic scar formation. In early wound repair epidermal cell proliferation and differentiation is induced by dermal matrix fibroblasts, while during later phases the epidermal cells in turn signal the fibroblasts to influence their matrix production.45,46,110 The reciprocal interaction from epidermis to dermis can probably not be influenced by silicone application in early wound repair, but can have its extracellular matrix modulating effect only during the later phases. When occlusion starts at two to three weeks following surgery a new epidermis has been formed,111 and the epidermal cells are capable to influence the fibroblasts. If the epidermis is used to an existing hydrated milieu, in case the application starts at three days following surgery, there seems to be no effect on this reciproque interaction. After the removal of the protective silicone barrier the epidermis is triggered and provides a counter regulating effect on extracellular matrix production, which can be the explanation for the higher incidence in scar hypertrophy at six and twelve months in this study.

Finally, occlusion stimulates keratinocyte proliferation in partial thickness wounds where the entire surface epithelium and papillary dermis is removed,89 whereas an opposite effect was seen in tape-stripped skin from which only the stratum corneum had been removed.90 Furthermore, disruption of the epidermal barrier function showed an increase in the epidermal synthesis of free fatty acids, sphingolipids and cholesterol and an epidermal hyperplasia by which the epidermis repairs the defects in barrier function. Artificial barrier repair by latex occlusion prevented the increase in both lipids and epidermal hyperplasia 112 and blocked the normalisation of the function during research.113 So, the kind of wound dealing with and the moment occlusion starts can have different effects. More research is necessary to clarify these confusing results. For example prolonged occlusion has shown to reduce IL-1a mRNA levels in mice,114,115 contrary to the expectation that an increased level of IL-1a will reduce hypertrophic scar formation.116

Conclusion

Wound healing in susceptible individuals can result in the development of hypertrophic scar tissue, characterised by an excessive deposition of tissue collagen, water, fibronectin and proteoglycans in the extracellular matrix.12 But, despite advances in biochemical and histopathological knowledge, the exact mechanism of excessive scarring remains unknown. Probably because of the striking amount of dermal tissue in these scars, most of the literature is focused on dermal processes to elucidate the pathogenesis of hypertrophic scars. In our clinical practice, however, we have never seen the development of hypertrophic scars out of striae, in which the dermis is traumatised but the epidermal continuity is still maintained. On the contrary, when a meshed skin graft is used in burn patients, the holes within the mesh where the new epidermis will develop are especially prone to hypertrophic scar formation. Furthermore, it has been reported that deep second degree burn wounds, in which less epithelial tissue remains, are more prone to hypertrophic scar formation compared with superficial second degree burn wounds.117 This raised the question whether or not specific epidermal processes are as important in the formation of excessive scar tissue as the dermal events are.

When the skin integrity is disrupted, keratinocytes migrate over the wound bed to protect the body as soon as possible against infection and water loss, whereas the dermal tissue is patched up temporarily by granulation tissue. We surmise that if the wound is closed by this cell migration and the barrier function is complete, keratinocytes will return to a quiescent stage and will be responsible for a signal to the dermis, which slows down the dermal repair processes and provides the onset of extracellular matrix remodelling from young to mature scar tissue. In case of hypertrophic scar formation this signal seems to be lacking or is at least inadequate. The fact that completion of the reepithelialization process, at about three weeks following injury,111,118-120 correlates well with the onset of hypertrophic scar formation,12,121 and the fact that epithelial-mesenchymal interactions seem important in scarless foetal healing,122 supports this contention. We have found that hypertrophic scars are accompanied by an increased epidermal acanthosis as well as an abnormal keratinocyte proliferation and differentiation, compared with normal scars.81 In addition, cytokine expression in scar tissue show a difference between the epidermal cytokine-profiles in hypertrophic compared with normal scars. In the inframammary breast reduction scars epidermal IL-1a showed a

significantly lower, and epidermal PDGF and IL-4 a significantly higher expression in hypertrophic scars compared with normal scars, both at three and twelve months following surgery. The dermal expression on the other hand showed just a higher expression of PDGF. Even more interesting was the observation that the number of epidermal Langerhans cells was significantly increased in hypertrophic scars. Keratinocytes and Langerhans cells are known to act as an immunological signalling and protecting interface between the external environment and the organism.17 In theory, when epidermal cells sense an insufficient barrier function they remain activated, and continue to signal to the dermis, resulting in hypertrophic scar formation. A sufficient protecting interface, on the other hand, is responsible for an adequate interaction and provides the onset of remodelling young granulation tissue into mature scar.45,46,48

This thesis gives rise to a switch in paradigm, that would focus on epidermal processes in addition to dermal abnormalities in hypertrophic scar formation. This ultimately can lead to elucidation of the basic mechanisms of excessive scar formation and can open new avenues for therapy and prevention in the near future.123

The surgeon removes his gloves, the operation is done. Meanwhile the assistant closes the remaining wound with the best of his knowledge and most of his care, and the days after tomorrow he will start his immunomodulating application therapy to provide an optimal condition for the upcoming wound healing events.

Deventer 1999,

Frank Niessen

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