The redistribution of collagen in expanded pig skin

The redistribution of collagen in expanded pig skin

0007-1??6’9O,OOJi ~rrmh ~ournul of Plasm Surgery ( 1990). 43. 565-570 ~‘a 1990 The Trustees of Brmsh Assocmtion of Plastic Surgeons The redistributi...

561KB Sizes 37 Downloads 89 Views

0007-1??6’9O,OOJi

~rrmh ~ournul of Plasm Surgery ( 1990). 43. 565-570 ~‘a 1990 The Trustees of Brmsh Assocmtion of Plastic Surgeons

The redistribution K.R. KNIGHT, Microsurgery

J. J. MCCANN,

Research

Centre,

of collagen C. A. VANDERKOLK, St Vincent’s

Hospital,

in expanded

0565.510.00

pig skin

S. A. COE and B. McC. O’BRIEN

Melbourne,

Australia

Summary-Silicone tissue expanders were inserted subcutaneously in the buttocks of nine young pigs and gradually inflated to maximum capacity over 5 weeks. On the control side the expanders were left uninflated. Island buttock flaps were then raised, the expanders removed and the flaps spread into the same sites for 10 days. The tissue was harvested. Area measurements and full thickness skin biopsies were taken 10 days after flap inset in order to study the changes in collagen composition and isotypes in the skin layers. Ten days after inset of the flap the expanded skin had a mean 47% increase in surface area, was 9% thinner (from surface to implant), mostly due to thinning of the subcutaneous zone, but was not significantly different in water content, relative to the control skin. The expanded skin had a significant 9.3% increase (p
has been rapidly incorporated into plastic surgery procedures over the past decade. It has the major advantages of producing extra skin where this is in short supply and provides a thinner type of skin particularly suitable for head and neck defects. Light microscopic studies of expanded guinea pig skin from 7 to 36 weeks have revealed that, in the expanded skin compared with normal skin, the thickness of the epidermis was unchanged whereas the dermis and panniculus carnosus became progressively thinner. A capsule layer rich in collagen formed adjacent to the expander. Overall there was a net gain of new tissue (Austad et a/., 1982). At the electron microscopic level, the dermis and capsule were found to have active fibroblasts, some with intracellular collagen, suggestive of new collagen synthesis. Large bundles of compacted collagen were seen in the dermis, subcutaneous tissue and capsule, suggesting that some existing collagen was stretched and flattened. A few myofibroblasts, commonly seen in granulation tissue, were observed in the early stages of capsule formation (Pasyk et (II.. 1981). Recently our laboratory has reported changes in the area and thickness of epidermis and dermis (VanderKolk et al., 1988) as well as viability (McCann ef al., 1988) of expanded axial pattern Sott tissue expansion

565

skin flaps in pigs. Following 5 weeks’ expansion and prior to flap elevation, the skin had gained a 63% increase in area compared with controls in which the expander was inserted but not inflated. However, skin flaps based on this expanded tissue were only 300,/, larger than controls immediately after flap elevation and inset. Dermal and cellular non-keratinised epidermal layers thickened markedly in expanded skin compared with control skin. Following elevation and inset of flaps for up to 4 months, both the dermis and epidermis thickened in expanded and control flaps (VanderKolk et al., 1988). In the second study (McCann et al., 1988) a comparison was made of the viable areas of nine flaps which were marked by a grid as 18 x 10 cm before expansion. This article represents an extension of the McCann et al. experiments. Biopsies of skin were taken at the time of sacrifice of the pig, i.e. at the end of the 5 weeks expansion and 10 days after inset of the flap. (Johnson et a/. (1988) previously reported no significant change in the collagen composition of the expanded pig skin in the first 10 days after cessation of expansion.) The changes in collagen content and isotypes in the dermis and subcutaneous zone (including the capsule), together with the respective thickness of these layers. were

566

BRITISH JOURNAL

determined. The purpose was to assess the redistribution of this important structural connective tissue protein following skin expansion. A preliminary account of these findings has been published (VanderKolk et al., 1987).

Materials and methods Operative technique and measurement

of viable area

Nine adolescent pigs previously described by McCann et al. (1988) were used for this study. The operative technique and measurement of flap area after expansion are also described in that article. Collection of biopsies and estimates of skin thickness Ten days after flap inset, full thickness skin biopsies to the depth of the capsule were taken from the centre of each flap for the studies detailed below. The average thickness (surface to capsule) was estimated under the microscope with a measuring grid in the eyepiece. The method for the determination of epidermal plus dermal thickness by light has been previously published microscopy (VanderKolk et al., 1988). An estimate of the thickness of the capsule plus subcutaneous fat was therefore obtained by subtraction of the (epidermis + dermis) thickness from the total thickness. Rectangular segments of approximately 1 x 1 cm, measured accurately, were divided by visual examination into dermis plus epidermis and subcutaneous fat plus capsule. Collagen content of skin biopsies Tissue specimens were rinsed in saline, blotted dry on filter paper and wet weight determined. A weighed portion of each specimen was heated at 50°C for 5-7 days to a constant dry weight. Ten mg of each dried specimen was hydrolysed for 24 hours in 6 M hydrochloric acid and aliquots neutralised and assayed in duplicate for hydroxyproline content according to the method of Stegemann and Stalder (1967). Hydroxyproline is an amino acid almost specifically found in collagen; elastin contains much smaller amounts of this amino acid and is a minor protein in skin compared with collagen. Collagen isotypes in pig skin Segments of skin of approximately 0.5 g wet weight were diced into 1 mm cubes. Each specimen was digested three times at 22°C with gentle stirring in 0.2 M acetic acid containing 0.1 mg/ml of pepsin (Sigma Chemical Co., St Louis, MO, USA) for 24

OF PLASTIC SURGERY

hours. Each extract was collected by centrifugation at 1700 x g (pepsin-soluble collagen) for 30 minutes. The pepsin-soluble extracts were pooled and the collagen was precipitated at 4°C by slow addition of an equal volume of 5 M sodium chloride. The precipitated collagen was collected by centrifugation for 10 minutes, dialysed exhaustively with distilled water and freeze dried. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) III/I ratios of collagen isotypes were determined by SDS-PAGE. Freeze-dried samples of pepsin-soluble collagen were dissolved in Laemmli sample buffer (Laemmli, 1971) without reducing agent and at a concentration of approximately 5 mg/ml. Samples (30 ~1) were applied to homogenous gels containing 5% acrylamide, electrophoresed under delayed reducing conditions (McCoy et al., 1984) and stained with Coomassie blue dye (Sear et al., 198 1). Each sample was examined with a scanning densitometer (Quick Scan Jr, Helena Laboratories, Beaumont, Texas, USA) and the III/I ratios determined from the areas under the peaks of the delayed al(II1) band, compared with the al(I) and a2(1) bands. Results Area of the skin after expansion The biopsies analysed in this section have been taken from viable sections of the flap, although a small amount of necrosis was present at the distal edge of all flaps. The area of the expanded flap at the time of sacrifice was found to increase by a mean of 46.7% (SEM 9.0) compared with the control flap (McCann et al., 1988), based on the tattooed grid. Water content (Table 1) Results were expressed as a percentage dry weight of the wet weight. There were no significant changes

Percentage dry weight of wet weight for pig skin biopsies

Table 1

Dermis + epidermis Subcutaneous fat+capsule Full thickness None of these changes SEM in parentheses.

Control

E.rpanded

Dtflerence

30.7(1.0) 40.1 (4.5) 34.9 (2.5)

30.7(1.1) 39.6 (5.6) 35.0 (3.2)

0 -0.5 f0.1

was statistically

significant

(p < 0.05)

THE REDISTRIBUTION

OF COLLAGEN

IN EXPANDED

in hydration of the tissue after tissue expansion. For the dermis+epidermis, both expanded and controls were 30.7%; for the subcutaneous fat and capsule, 39.60/d in the expanded and 40.1’4 in controls; for full thickness biopsies, 35.0% in the expanded and 34.9”/0 in control skin biopsies. Skin thickness (Table 2) After 5 weeks of expansion the full thickness biopsies had reduced from 0.55 cm to 0.50 cm (p ~0.01; paired t test). The most significant thinning occurred in the subcutaneous fat + capsule zone, 0.33 cm to 0.25 cm (p
Table 2

hydroxyproline content were almost identical, with only 2.8% more in expanded compared with control specimens. Collagen distribution within the tissue The distribution of collagen, i.e. hydroxyproline, may be expressed either per unit area or per unit volume (Johnson et al., 1988). In this study, the collagen per surface area (Table 4) and the collagen density (Table 5) were not significantly different in expanded compared with control biopsies, nor in the various layers of the tissues. There is, however, an overall net gain of collagen, comparing a piece of expanded tissue with an equivalent piece of control tissue with the expander inserted but not inflated. Using the combined data of all previous tables, an average 1 cm’ biopsy of control tissue to the depth of the capsule will contain 11.73 mg hydroxyproline. Given that there is an increased area (on average) of 46.7:; in the expanded tissue, this same piece of tissue after 5 weeks’ expansion would then have 17.63 mg hydroxyproline, a net gain of 50.3%. Changes in the collagen types (Table 6) Approximately 70% of the total tissue collagen was solubilised by pepsin digestion. This constitutes a representative proportion of the collagen present since much of the remaining portion woulcl be insoluble collagen with mature intermolecular crosslinks. There was slightly more Type III collagen relative

Mean thickness in cm of specimens of expanded and control

Dermis + epidermis Subcutaneous fat + capsule Full thickness *Paired t test. tUsing the data of VanderKolk SEM in parentheses.

Table 3

567

PIG SKIN

Hydroxyproline

Dermis+epidermis Subcutaneous fat + capsule Full thickness *Paired t test. NS not significant. SEM in parentheses.

tissue

Conrrol

E.\rpanded

D~@wnce

.Sign~ficanee*

0.22 (o.ol)t 0.33 (0.04) 0.55 (0.5)

0.25 (o.ol)t 0.25 (0.04) 0.50 (0.04)

+ 13.20, - 22.69, - 8.60,

p
et (I/. (1988).

in mg/g wet weight of tissue Control

E.\panded

Dif

29.0 ( I .2) 14.0(1.8) 21.4(1.3)

3 I .7 (I .4) 12.5 (1.4) 22.0 (0.7)

+ 9.30, -10.7’, + 2.8O,

feWtlCt2 Signi$cuncr* p
568

BRITISH JOURNAL

Table 4 Hydroxyproline biopsy (mg/cm2)

content

Control Dermis + epidermis Subcutaneous fat+capsule Full thickness SEM in parentheses. None of these changes

per surface E.xpanded

area of D@erence

8.32 (0.55) 8.96 (0.42) +7.7x 3.41 (0.56) 3.06 (0.47) - 12.8% 11.73(1.11) 12.02(0.89) +1.6%

was statistically

significant.

Table 5 Collagen density: hydroxyproline unit volume of biopsy (mg/cm3)

content

per

Control

Expanded

Difference

Dermis + epidermis Subcutaneousfat+capsule Full thickness

40.0 (2.6) 10.1 (1.7) 21.5 (2.0)

37.6 (1.8) 11.7(1.0) 24.0 (1.8)

-6.0% +15.8x +11.6x

SEM in parentheses. None of these changes

was statistically

significant.

Distribution of collagen T ---- T ypes i and ‘I1 in unexpanded and expar iaea ’ ’ pig skin ’

Table 6

Dermis + epidermis Subcutaneous fat+capsule

Control

Expanded

Dlrerence

22.1 (1.8) 21.3 (1.4)

19.6(1.2) 20.6 (2.6)

-2.6 -0.6

Results are expressed as a percentage of Type III collagen total Types I + III collagens. SEM in parentheses. These differences were not statistically significant.

of the

to Type I collagen in control skin compared with expanded skin, for the two zones investigated, although neither of these changes was statistically significant (Table 6).

Discussion The finding of increased deposits of collagen in expanded pig skin compared with unexpanded control skin poses several questions. Are existing cells increasing their biosynthetic rate or are more cells migrating to the zones in which tissue expansion is greatest? What is the nature of the stimulus for new tissue formation? The majority of the collagen in normal skin is located in the dermis with small amounts in the epidermis and subcutaneous layers (Weber et al., 1984). A considerable portion of the collagen will also be present in the capsule which forms around the expanders (Pasyk et al., 1982) and has also been

OF PLASTIC SURGERY

found to encapsulate silicone breast prostheses (McCoy et al., 1984). In our experiments capsular collagen was found to be present (and in equivalent amounts) in both control and expanded skin specimens. Preliminary histological analysis of the biopsies used in this study also demonstrated small but variable amounts of capsule (Hickey and Knight, unpublished data). The dermis of the expanded skin was the major source of increased collagen deposition. Pasyk et al. (1982), studying the collagen in expanded guinea pig skin in the electron microscope, observed both compacted preexisting collagen fibres as well as collagen fibres of variable diameter, the latter probably representative of newly synthesised collagen. The main cell type responsible for collagen synthesis is the fibroblast with more minor contributions from smooth muscle cells, myofibroblasts (Majno et al., 1971; Pasyk et al., 1982) and other mesenchymal cells. Fibroblasts, with more synthetically active organelles, have been observed in larger numbers in the expanded dermis compared with the normal dermis (Pasyk et al., 1982). Furthermore, there is evidence that stretched skin in guinea pigs has a more rapid turnover of thymidine indicative of increased mitosis (Francis and Marks, 1977). A similar study performed in stretched mouse skin also noted increased mitotic activity determined by histology (Squier, 1980). Thus, tissue expansion may give rise to increased cell numbers as well as a generalised increase in biosynthetic activity. Increased collagen synthesis and deposition has two other precedents. Obese people have stretched skin with increased amounts of collagen, presumably an attempt to maintain the normal skin thickness (Black et al., 1971). Similar changes have also been observed histologically in the stretched skin of pregnant rats (Muller, 1951), suggesting a possible role for hormones (such as oestrogen) and maybe cell growth factors. Thus the tensile forces on the skin during tissue expansion may be sufficient on their own to result in the increased collagen deposition in the dermis observed here, although the hormonal response as a result of the stretching “injury” to the skin may also be a contributory factor. Johnson et al. (1988) recently studied collagen during tissue expansion in the pig. Tissue was expanded for 6 weeks and analyses of normal skin, sham treated skin (expander inserted but not inflated) and expanded skin were performed every 6 weeks up to 36 weeks. Epidermal thickening and

THE REDISTRIBUTION

OF COLLAGEN

IN EXPANDED

569

PIG SKIN

dermal thinning were observed. The former was probably due to the natural growth of the pigs to maturity. The latter was contrary to our findings; we observed most of the thinning in the subcutaneous layers. The increase in area immediately after expansion of 597; was comparable to our study (477;) and the finding of an identical collagen density in the sham and expanded skin concurs with our results. Thus both studies demonstrate a theoretical gain in tissue (50% in our study) in the dermal layer in response to tissue expansion. One aspect of de not’o collagen synthesis which has not been investigated previously in skin expansion studies is the composition of collagen types. In normal skin there is a characteristic ratio of Types III to I collagens which remains relatively constant throughout the reticular layer of the dermis and the subcutaneous fat, sometimes slightly enriched with Type III collagen in the papillary dermis (Weber et al., 1984). The stretching of the skin. particularly overstretching, may injure the tissue causing a response similar to that of wound healing. In skin in the early stages of wound healing in children, there is a relative increase in Type III relative to Type I collagen (Gay et al., 1978). Furthermore, the presence of myofibroblasts in the early stages of tissue expansion (Pasyk et al., 1982) and in granulation tissue in the early stages of wound healing (Majno et al., 1971) suggests some parallels between these two quite different events. In this study, however, no significant differences were found between (a) control and expanded tissue and (b) the two zones of skin investigated in these experiments. Changes in collagen types, if any, during skin expansion may have occurred at an earlier stage of expansion when remodelling of the extracellular matrix was taking place. C‘onclusion The most likely explanation for the increased deposition of collagen found in expanded pig skin is that stretching of the skin resulted in increased synthesis of collagen by fibroblasts and/or an increased migration of fibroblasts into the dermis. This produced additional matrix molecules in an attempt to restore the normal dermal thickness.

Acknowledgements The technical assistance of Miss Diana Lepore as well as the staffof the ExperimentalSurgical and Medical Research Service of St Vincent’s Hospital is gratefully acknowledged. Thanks too

to the secretarial staff of the Microsurgery Research Centre for typing the drafts of this article. The work was completed with financial assistance from St Vincent’s Hospital, Melbourne.

References Austad, E. D., Pasyk, K. A., McClatchey, K. D. and Cherry, G. W. (1982). Histomorphologic evaluation of guinea pig skin and soft tissue after controlled tissue expansion. P/~~>:ficanti Reconsrrucrite Surgery. IO, 704. Black, M., Bottoms, E. and Schuster, S. ( 1971). Skin collagen and thickness in simple obesity. Brirbh Mrdrc 01 Journal. 4. 149. Francis, A. J. and Marks, R. (1977). Skm stretching and epidermopoiesis. Britkh Journal o/ Eprinwrf~rl P~lh~/q.v. 58, 35. Gay, S., Viljanto, T., Raekallio, J. and Pentinnen, R. (1978). Collagen types in early phases of wound healing in children Actor Chirurgia Scandinatica. 144,205. Johnson, P. E., Kernahan, D. A. and Baver, B. S. C1988). Dermal and epidermal response to soft tissue expanslan in the pig Plastic and Reconstructice Surgery, 81. 390. Laemmli, U. K. (1971). Cleavage of structural proteins during the assembly of the head of bacteriophage T4 .Yarurr, 227, 680. McCann, J. J., Mitchell, G. M., O’Brien, B. McC. and VanderKolk, C. A. (1988). Comparative viability of expanded and unexpanded axial pattern skm flaps in pigs British Journal u/’ Plastic Surgery. 41, 294. McCoy, B. J., Person, P. and Cohen, I. K. (19X4). Collagen production and types in fibrous capsules around hreast implants. Plastic and Reconstructire Surgey,~. 13. 924. Majno, C., Gabbiani, G., Hirschel. B. J., Ryan, G. 8. and Statkov. P. R. (1971). Contraction of granulation IIssue ill I?tro: similarity to smooth muscle. Science, 173.54X. Muller, T. (1951). The effect of estrogen on the loose connective tissue of the albino rat. AnatomicalRecord, 3. 355. Pasyk, K. A., Austad, E. D., McClatchey, K. D. and Cherry, G. W. C1982). Electron microscope evaluation of gumea pug skin and soft tissues expanded with self-inflating silicone Implant. Pla.rtic and Rrconsiructiw Surgery. 70. 31. Sear, C. H. J., Jones, C. J. P., Knight, K. R. and Grant, M. E. ( 198 1). Elastogenesis and microfibriliar glycoprotein synthesis by hovlne ligamentum nuchae cells in culture C~vznrctitr Tixwe Research, 8, 167,

Squier, C. A. (1980). The stretching of mouse skin v1 riw: effect on epidermal proliferation and thickness. J~>rrmcr/cjf h~srigariw

Dermcrtolog~.

74, 68.

Stegemann, H. and Stalder, K. (1967). Determination of hydroxyproline. C/inica Chimica Acta. 18. 167. VanderKolk, C. A., McCann, J. J., Knight, K. R. and O’Brien, B. McC. (1987). Some further characteristics of expanded tissue. Clinic.~ in Plwtic

Surgrr~~. 14. 447.

VanderKolk, C. A., McCann, J. J., Mitchell, G. M. and O’Brien, B. McC. (1988). Changes in area and thickness of expanded and unexpanded axial pattern skin flaps in pigs British Journuiqf’Plasric Surgery. 41. 284. Weber, L., Kirsch, E., Muller, P. and Krieg, T. (1984). C’ollagen type distribution and macromolecular organization ofconnective tissue in different layers of human skin J~rruol (?/ Inwstignfiw Dermatology, 82. 156.

570 The Authors Kenneth, R. Knight, PhD, Senior Research Officer, Microsurgery Research Centre. John J. McCann, FRCSI, Senior Registrar in Plastic Surgerv. Cork Regional Hospital, Eire; foymerly Research Fellow, Microsurgery Research Centre. Craig A. VanderKolk, MD, Plastic Surgeon, Johns Hopkins Hospital, Baltimore, USA; formerly Research Fellow, Microsurgery Research Centre. Serena A. Coe, BSc, Research Assistant, Microsurgery Research Centre.

BRITISH JOURNAL

OF PLASTIC SURGERY

Bernard McC. O’Brien, CMG, BSc, MD, MS, FRCS, FRACS, FACS(Hon), FRCSEd(Hon), FRCSI(Hon), Director, Microsurgery Research Centre, St Vincent’s Hospital, Melbourne.

Requests for reprints to: Dr K. R. Knight, Microsurgery Research Centre, St Vincent’s Hospital, Victoria Parade, Fitzroy, Victoria 3065, Australia. Paper received 25 August 1989 Accepted 4 January 1990.