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Artificial dermis and cellular aspects of scar formation

E Middelkoop, AJ van den Bogaerdt, MM Ulrich Montpellier, 1st Scar Meeting, 29March ­ 1 April, 2006

Abstract

Full thickness wounds of a substantial size, such as burns or larger chronic wounds require skin transplantation in order to reach wound closure in an acceptable time frame. Even then, these wounds heal with considerable wound contraction and scarring. It is the general consensus that the lack of dermal tissue in split thickness transplants is of importance in these processes. In order to improve the quality of healing, skin and dermal replacements have been developed for a number of years. Dermal substitutes can be subdivided into acellular materials such as Alloderm®, Integra® or TransCyte®, or cellular materials such as Dermagraft® and Hyalograft®. In the latter materials, allogeneic or autologous fibroblasts are seeded into three dimensional matrices, which are transplanted onto the wound surface. These tissue engineered substitutes serve as optimal matrices to allow keratinocytes to grow out on top of the substitutes from the wound margins, or as improved wound bed for an epidermal component. However, the exact role and function of the cells present in these matrices is hardly known. Therefore, we investigated different cellular sources to be used in tissue engineered dermal substitutes. Scars are characterized by excess collagen accumulation. This may result from increased collagen synthesis, decreased degradation or a combination of both. Recently, an enzyme that is involved in collagen crosslinking in fibrotic tissue, lysyl hydroxylase (LH2) was identified (1). This enzyme is normally active in bone and cartilage, but hardly in skin. We found that in scar tissue as well as in subcutaneous fat, this enzyme is highly expressed; this is in contrast to the dermis, where the enzyme is hardly expressed (2). Cells were isolated and characterized from dermal tissue, subcutaneous fat and eschar (debris from burn wounds, discarded during surgical treatment). Cell cultures were established, and fibroblast phenotypes were investigated by FACS analysis and immunohistochemistry. The tissues were analysed for mRNA expression of collagen 1 and III, a-smooth muscle actin and the enzyme LH2.

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Dermal fibroblasts contained fewer myofibroblasts than the other cell populations, and showed limited contraction of collagen matrices in vitro (3). Expression of mRNA for asmooth muscle actin, collagen I and III and LH2 were all higher in scar tissue and subcutaneous fat tissue than in dermal tissue. In conclusion, the 'scar-like' profile of fibroblasts derived from subcutaneous fat suggests a role for these cells in scar formation.

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Introduction

The scar problem Scar formation results when the wound healing cascade does not progress optimally. This may be related to abnormalities in growth factor, cytokine, proteolytic and cellular profiles (4). The level of scarring is associated with a number of processes among which are: the depth of injury (5), bacterial contamination and related extended time span of inflammation (6), the rate of re-epithelialisation (7), the presence of mechanical forces in the wound (8), the persistence of myofibroblasts in the wound (9), anatomical location of the wound, age of the patient and genetic predisposition (10). Although there is a general paucity of data on the prevalence of (hypertrophic) scarring after burn wounds, several authors conclude that a deeper burn wound represents a greater risk of developing a hypertrophic scar than a more superficial burn wound (11). In spontaneously healed burn wounds, Deitch et al (7) found an incidence of hypertrophic scarring varying from 15% of burned sites in White patients versus 30% in Black patients. In grafted sites, which would generally be full thickness burns, the prevalence of hypertrophic areas was as high as 75% in children (both Black and White) and 50% in Black adults versus only 7% in White adults (12). In addition to wound depth, these data already point to two further characteristics of the patient population that are important in determining the outcome of healing: the age of the patient and their genetic predisposition. Our own data (13) indicate an incidence of hypertrophy (defined as having a score of 1 or greater in the relevant item 'thickness' of the Vancouver Scar Scale) in 30­52.5 % of patients treated for partial thickness (mainly scald) burns, which was reduced to 17.5 to 32.5% if only severe hypertrophy (defined as hypertrophy in 10% of the study burn area) was counted. Due to the many different parameters that might influence the prevalence of hypertrophic scarring, it is extremely difficult to compare data from different studies in a retrospective study. Therefore at this point in time, we do not possess an overview of data on scar risk assessment based on hypertrophic scar epidemiology for different categories of burns and patient groups. Nevertheless, we do need such baseline data in order to be able to judge the relevant outcome of new treatment regimes, such as the use of tissue engineered skin and artificial skin substitutes.

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Skin substitutes Skin substitutes can be divided according to several criteria: dermal versus full skin substitutes, biological versus synthetic materials, acellular versus cellular materials and the latter category can be subdivided further into substitutes based on allogeneic or autologous cells. Several reviews on many different products have appeared in the scientific press over the last few years, nevertheless, only a few products have actually succeeded in conquering a piece of the wound treatment market. The products that are being used more or less routinely for wound treatment nowadays are: Alloderm®, Integra® and cadaver skin (in different conservation techniques) as acellular materials, and Dermagraft® (marketed until recently), Hyalograft® and Apligraf® as cellular dermal and full skin substitutes, respectively. The basic principles and characteristics of skin substitution and relevant materials have been described already some decades ago in a series of papers by Yannas et al (14­17). Some of the very basic requirements for engineered skin are: ability to allow epidermal coverage (restore barrier function of the skin) provide dermal matrix (a biodegradable template for synthesis and deposition of neodermal tissue) presence or influx of cells that will function as dermal cells and produce dermal tissue rather than scar tissue. The basics of dermal substitution go back as far as the experiments by Bell et al (18), using reconstituted collagen gels populated by fibroblasts. Three-dimensional matrices were introduced and characterized by Yannas & Burke (14), which eventually led to the development of Integra® artificial skin. Dermal substitutes function as an optimized wound bed to support outgrowth of keratinocytes from the wound margins, or from an epidermal component such as a skin graft, cultured skin or an artificial epidermal component containing autologous or allogeneic keratinocytes. Despite the fact that beneficial effects on wound healing and (chronic) wound closure have been described (19­22), the exact role and function, necessary concentration and cellular phenotype of the cells in these dermal substitutes are basically unknown (3, 23).

Methods

Tissues Dermal and subcutaneous fat tissue was obtained from healthy donors during abdominal dermo-lipectomy. Large blood vessels, glandular tissue and fascia were discarded. Hypertrophic scar tissue was obtained during reconstructive surgery. In some cases, burn eschar was harvested at excision of the burn wound on average 16 days post burn (range 4

5­35). These tissues were cleared from visibly denatured areas and large blood vessels. Part of the tissue was frozen in liquid nitrogen for RNA isolation. The remaining tissues were used for cell isolation as described before (3). Matrix contraction Cells from different sources were seeded into a non-crosslinked collagen­elastin hydrolysate matrix (Suwelack Skin & Health Care) at 100,000 cells/cm2. Contraction was followed by planimetry for 18 days. Flow cytometry Freshly isolated as well as cultured cells were harvested and 100,000 cells from each batch were labeled with various antibodies as indicated in Table 1 and analysed in a FACS flow cytometer. Forward scattering, sideward scattering, FITC fluorescence and propidium fluorescence were recorded as described before (Arch). RNA analysis RNA was prepared from the tissues as well as from cultured cells with TRIzol reagent (Invitrogen Life Technologies, Breda, Nl) according to the manufacturer's instructions. Total RNA was reversely transcribed into cDNA, which was then used in specific real-time PCR reactions using molecular beacons for each specific target expression product (1,2). mRNA levels for alpha-smooth muscle actin, collagen I and III and the enzyme lysyl hydroxylase (LH2), which is involved in collagen crosslinking, were determined versus a housekeeping gene beta2-microglobulin.

Results

Cells isolated from subcutaneous fat and burn eschar contracted the three-dimensional matrix more than cells isolated from dermis (Fig 1). Immediately after cell isolation from the tissues, more than 95% of the cells were stained positive with the vimentin marker, as would be expected. However, 37% of the cells from dermal tissue versus 50% from subcutaneous fat tissue and 20% of cells from burn eschar were positive for the fibroblast marker (AS02). Myofibroblasts, characterized by the presence of alpha-smooth muscle actin, accounted for 23% of the cells from subcutaneous fat, which was significantly higher than in dermal tissue (8%).

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The majority of cells present in burn eschar at this early time point was positive for the granulocyte marker CD16 (74%). These cells did not remain present during culture: after 14 days, cells bearing CD16 accounted for no more than 0.5% of the cells. After 14 days in culture, the proportion of aSMA positive cells was stable at 3% of cells from dermis, remained high in cells from subcutaneous fat (40%) and now was also high in cells from burn eschar (38%). Both were significantly higher than in cells from dermis (p<0.003). (Fig 2). a-SMA mRNA expression was significantly higher in cells isolated from subcutaneous fat and also in cells derived from scar tissue (Fig 3). Extracellular matrix production, as monitored by mRNA expression of collagen type I and III, was significantly elevated in cells derived from subcutaneous fat and from scar versus cells from dermal origin (Fig 4). Finally, the enzyme LH-2, which is involved in crosslinking of collagen and is normally expressed predominantly in cartilage and bone, was now expressed at a much higher level in subcutaneous fat and scar tissue (Fig 5).

Discussion

In a porcine wound model for skin substitution, we studied the effects of using fibroblastseeded dermal substitutes versus acellular dermal substitutes. Reduced wound contraction, reduced numbers of myofibroblasts and better quality of the dermal tissue were associated with seeding of higher numbers of autologous fibroblasts in the substitutes (23, 24). We investigated some of these aspects in more detail by studying skin regenerative properties, such as extracellular matrix synthesis and remodeling by different cellular sources of cells to be used in tissue engineered dermal substitutes. Dermal tissue, subcutaneous fat tissue and burn eschar were used as cell sources. Cells were isolated from these tissue and the cellular profiles determined immediately upon isolation and after 14 days of culturing (3). Cells isolated from subcutaneous fat were more contractile in collagen gels, less supportive of keratinocyte migration (25l), and more asmooth muscle actin positive cells were detected in these cultures (3). Also fibroblasts

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isolated from scar or subcutaneous fat tissue contained higher mRNA expression levels for a-smooth muscle actin than fibroblasts isolated from dermal tissue. Expression levels of mRNA for collagen type I and III were determined for fibroblasts isolated from dermal, subcutaneous fat and scar fibroblasts. Collagen synthesis seemed to be increased in the latter two cell populations, as compared with dermal fibroblast cultures, consistent with findings that scar tissue is characterized by a higher collagen content than normal skin (26). Furthermore, the enzyme LH-2 that is normally expressed at a high level in cartilage and bone and at a low level in normal skin was recently found to be highly expressed in fibrotic tissue (2). This enzyme potentially leads to a highly crosslinked collagen molecule, which may be less accessible for remodeling and degradation (27). The depth and size of a wound may ultimately determine the source of cells that are recruited to the wound site and are responsible for the deposition of new extracellular matrix. In a partial thickness wound the cells will mainly be recruited from the remaining surrounding dermis. The cellular phenotype will therefore be the phenotype present in the dermal tissue and will possess the best characteristics for optimal repair. In a full thickness wound, however, there are few dermal fibroblasts left, therefore recruitment will take place involving other tissues such as the peripheral blood, subcutaneous fat and other underlying tissues. In this paper we have demonstrated that the latter do not possess optimal characteristics for dermal repair. The activity of these cells in the repair process will most likely lead to scar formation. In conclusion, we can state that improvements are warranted in the function of dermal templates. Such improvements could come from new scaffold materials, but also the phenotype and function of cells involved in such templates are important. Future research should aim to try and control the function of the cells involved in tissue repair. Acknowledgements The authors gratefully acknowledge the contribution of Michelle Verkerk, Linda Reijnen and Marcel Vlig from the Association of Dutch Burn Centres (ADBC) to the work described in this paper: This work was financially supported by the Dutch Burn Foundation.

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References

1. van der Slot AJU, Zuurmond AM, Bardoel AF, Wijmenga C, Pruijs HE, Sillence DO, Brinckmann J, Abraham DJ, Black CM, Verzijl N, DeGroot J, Hanemaaijer R, TeKoppele JM, Huizinga TW, Bank RA. Identification of PLOD2 as telopeptide lysyl hydroxylase, an important enzyme in fibrosis. J Biol Chem. 2003 Oct 17;278(42):40967-72. Epub 2003 Jul 24. 2. van der Slot AJ, Zuurmond AM, van den Bogaerdt AJ, Ulrich MM, Middelkoop E, Boers W, Karel Ronday H, DeGroot J, Huizinga TW, Bank RA. Increased formation of pyridinoline cross-links due to higher telopeptide lysyl hydroxylase levels is a general fibrotic phenomenon. Matrix Biol. 2004 Jul;23(4):251-7. 3. Van den Bogaerdt AJ, van Zuijlen PPM, van Galen MJM, Lamme EN, Middelkoop E. The suitability of cells from different tissues to be used in tissue engineered skin substitutes. Arch Dermatol Res 2002; 294: 135-42. 4. Hardy MA. The biology of scar formation. Phys Ther 1989; 69: 1014-1024. 4. Muir IFK. Control of fibroblast activity in scars: a review. Eur J Plastic Surg 1998; 21:1-7. 5. Klein DG, Fritsch DE, Amin SG. Wound infection following trauma and burn injuries. Crit Care Nurs Clin North Am 1995; 7: 627-642. 6. Deitch EA, Wheelahan TM, Rose MP, Clothier J, Cotter J. Hypertropic burn scars and keloids: a review. Plast Reconstr Surg 1999; 104: 1435-1458. 7. Hinz B, Mastrangelo D, Iselin CE, Chaponnier C, Gabbiani G. Mechanical tension controls granulation tissue contractile activity and myofibroblast differentiation. Am J Pathol 2001; 159: 1009-1020. 8. Tomasek JJ, Gabbiani G, Hinz B, Chaponnier C, Brown RA. Myofibroblasts and mechanoregulation of connective tissue remodeling. Nat Rev Mol Cell Biol 2002; 3: 349363. 9. Niessen FB, Spauwen PH, Schalkwijk J, Kon M. On the nature of hypertrophic scars and keloids: a review. Plast Rec Surg 1999; 104: 1435-1458. 10. Bombaro KM, Engrav LHH, Carrougher GGJ, Wiechman SA, Faucher L, Costa BA, Heimbach DM, Rivara FP, Honari S. What is the prevalence of hypertrophic scarring following burns ? Burns 2003; 29: 299-302. 11. McDonald WS, Deitch EA. Hypertrophic skin grafts in burned patients: a prospective analysis of variables. J Trauma 1987; 27: 147-150. 12. Vloemans AF, Soesman AM, Suijker M, Kreis RW, Middelkoop E. A randomised clinical trial comparing a hydrocolloid-derived dressing and glycerol preserved allograft skin in the management of partial thickness burns. Burns 2003; 29:702-710. 13. Yannas IV, Burke JF. Design of an artificial skin. I. Basic design principles. J Biomed Mater Res. 1980; 14: 65-81. 8

14. Yannas IV, Burke JF, Gordon PL, Huang C, Rubenstein RH. Design of an artificial skin. II. Control of chemical composition.J Biomed Mater Res. 1980;14:107-132. 15. Dagalakis N, Flink J, Stasikelis P, Burke JF, Yannas IV. Design of an artificial skin. Part III. Control of pore structure.J Biomed Mater Res. 1980;14:511-528. 16. Orgill DP, Yannas IV. Design of an artificial skin. IV. Use of island graft to isolate organ regeneration from scar synthesis and other processes leading to skin wound closure. J Biomed Mater Res. 1998; 39: 531-535. 17. Bell E, Ivarsson B, Merrill C. Production of a tissue-like structure by contraction of collagen lattices by human fibroblasts of different proliferative potential in vitro. Proc Natl Acad Sci USA 1979; 76: 1274-1278. 18. Duinslaeger LA, Verbeken G, Vanhalle S, Vanderkelen A. Cultured allogeneic keratinocyte sheets accelerate healing compared to Op-site treatment of donor sites in burns. J Burn Care Rehabil. 1997; 18: 545-551. 19. Wood FM, Kolybaba ML, Allen P. The use of cultured epithelial autograft in the treatment of major burn wounds: Eleven years of clinical experience. Burns. 2006; 32: 538-544. 20. Marston WA, Hanft J, Norwood P, Pollak R, Dermagraft Diabetic Foot Ulcer Study Group. The efficacy and safety of Dermagraft in improving the healing of chronic diabetic foot ulcers: results of a prospective randomized trial. Diabetes Care. 2003; 26:1701-1705. 21. Veves A, Falanga V, Armstrong DG, Sabolinski ML, Apligraf Diabetic Foot Ulcer Study. Graftskin, a human skin equivalent, is effective in the management of noninfected neuropathic diabetic foot ulcers: a prospective randomized multicenter clinical trial. Diabetes Care. 2001;24: 290-295. 22. Lamme, E.N., Van Leeuwen, R.T.J., Brandsma, K., Van Marle, J., Middelkoop, E. Higher numbers of autologous fibroblasts in an artificial dermal substitute improve tissue regeneration and modulate scar tissue formation. J. Pathol. 2000; 190, 595-603. 23. Lamme, E.N., van Leeuwen, R.T.J., Mekkes, J.R., Middelkoop, E. Allogeneic fibroblasts in dermal substitution induce inflammatory responses and interfere with dermal tissue regeneration. Wound Repair and Regeneration, 2002; 10(3), 152-160. 24. El-Ghalbzouri A, Van den Bogaerdt AJ, Kempenaar J, Ponec M. Human adipose tissuederived cells delay re-epithelialization in comparison with skin fibroblasts in organotypic skin culture.Br J Dermatol. 2004; 150: 444-454. 25. Garner WL, Karmiol S, Rodriguez JL, Smith DJ Jr, Phan SH. Phenotypic differences in cytokine responsiveness of hypertrophic scar versus normal dermal fibroblasts. J Invest Dermatol. 1993;101: 875-879. 26. Van den Bogaerdt AJ, van der Slot A, Ulrich MMW, Zuurmond A, Bank RA, Middelkoop E. Altered collagen crosslinking by subcutaneous fat fibroblasts during wound healing. Wound Rep Regen 2003; 11: A28.

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Table 1. The cell types and their respective antibodies used in the flow cytometer Cell type · · · · · · Mesenchymal cells Fibroblasts Myofibroblasts Monocytes/macrophages Granulocytes Keratinocytes Antibody for flow cytometry anti-vimentin (vim) AS02 antibody (AS02) anti-a-smooth muscle actin (a-SMA) anti-CD14 (CD14) anti-CD16 (CD16) anti-pan-cytokeratin (CK)

A

B

Fig. 1. Matrix contraction after 18 days by dermal fibroblasts (A) and cells derived from subcutaneous fat (B).

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Day 0 100 Positivity [%] 75 50 25 0 Vim AS02 aSMA CK CD14 CD16 Cell markers

Eschar Sub. Fat Spl.Dermis

Day 14

100

Positivity [%]

75 50 25 0

Eschar Sub. Fat Spl.Dermis

Vim

AS02

aSMA

CK

CD14

CD16

Cell markers

Fig 2. Detection of various cell types by the respective antibodies at days 0 and 14 after isolation of the cells from the tissues as determined by FACS analysis.

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Ratio to 2M expression +/- SEM

300 250 200 150 100 50 0

dermal FB fat FB scar FB

Mann-Whitney, significance level at P< 0.05

* *

Fig. 3. -smooth muscle actin mRNA expression in cells isolated from dermal, subcutaneous fat or scar tissue.

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Ratio to 2M expression +/- SEM

25

20

* * * *

* *

Collagen I Collagen III

15

10

5

0

dermal FB

fat FB

scar FB

Mann-Whitney, P< 0.05

Fig. 4. mRNA analysis of collagen I and III.

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0,25

0,2 Ratio to b2M +/- SEM

0,15

0,1

0,05

0 dermal FB Fat FB Scar FB

Mann-Whitney, P< 0.05

Fig. 5. LH-2 mRNA expression in fibroblasts from normal skin, subcutaneous fat and scar tissue.

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