Interactions of vascular smooth muscle cells with collagen matrices

Clinical

Materials

7 (1991),247-252

f Vascular Smooth Muscle atrices N. Kearney Yorkshire

Regional

Tissue

Bank,

Pinderfields

General

Hospital,

Wakefield,

West Yorkshire

Fl 4DG,

UK

avies Cardiac

Research

Unit,

Killingbeck

Department

of Biochemistry,

Department

of Mechanical

(Received

11 November

Hospital,

University

Engineering,

Leeds, West Yorkshire

LS14 6UQ

of Leeds, Leeds, West Yorkshire

University

1990 ; sent for revision

UK

LS2 9JT, UK

of Leeds, Leeds, West Yorkshire

10 January

1991; accepted

29 January

LS2 9JT, U 1991)

Abstract: As a first step to producing a viable blood vessel graft, smooth muscle cell (SMC) populated collagen matrices were fabricated with type I collagen extracted from rat tail tendons. The rate at which these matrices were reorganised was determined by the reduction in surface area with time. Porcine and bovine SMC shared an equal ability to contract collagen matrices 1 x lo5 cells consistently causing a 99% reduction in upper surface area of a matrix fabricated with 0.5 mg/ml collagen over a period of 26 days. This property did not appear to be affected by the time in culture of the SMC, i.e. passage number. Using a range of collagen concentrations (0.25-1.00 mg/ml), the time to maximum contraction was shown to be proportional to collagen content. Increasing seeding density (5 x 104-5 x lo5 per matrix) had the effect of shortening contraction time. The final surface area, however, was not dependent on either of these parameters: all matrices eventually underwent a 98-99% decrease in upper surface area. A histological study highlighted common features indicative of the remodelling process: the formation of cavities, accumulation of cells at the periphery of the matrix, and the formation of collagen fibres.

INTRODUCTION In view of the increasing demand for a readily available small-calibre vascular prosthesis with a patency rate comparable to, or better than that of autologous material, the authors are currently evaluating the feasibility of constructing a viable blood vessel graft. Bell and co-wor ers have developed a system1 Clinical

Marerids

Q267-6605/91/$03.50

0

1991

whereby cultured cells are su in a collagen solution. Following fibrilloge this solution, the cells become enmeshed in tbe interstices of the matrix. After a few hours the cells disperse and become elongated. A number of cell types, including fibroblasts and smooth muscle cells (SMC), are capable of subsequently reorganising and remodelling the matrix, this being by a reduction in matrix size. The proces contraction is

247 Elsevier Science Publishers Ltd, England

S. L. Haynes et al.

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a dynamic one, requiring viable cells, culture medium containing serum and collagen,l and results in the formation of a tissue-like structure of considerable mechanical strength. This system not only has an application in the modelling of cellular function in vitro, but also has potential in the area of tissue replacement. The interactions of dermal fibroblasts with collagen are well documented,1-7 particularly in connection with the construction of a ‘skin equivalent,‘s-11 and the mechanism by which fibroblasts remodel collagen gels has been characterised in some detail. Collagen matrix contraction has been shown to involve the attachment of collagen fibrils to the cell surface via either fibronectin6 or collagen specific receptors.4 Physical activity of fibroblasts, such as spreading, extension and retraction of cytoplasmic processes, and translocation, produces tractional forces bringing the attached collagen fibrils closer together and eventually compacting them.l-l1 Synthesis of proteins and glycoproteins5,6 may also be involved in the remodelling process. In contrast with dermal fibroblasts, the interaction of SMC with collagen is less well characterised. In the studies that have been reported so far, a certain amount of controversy exists over the effectiveness of SMC in matrix contraction. Ehrlich et a1.12 have found SMC to be ineffective in matrix contraction, whereas other authors13 reported that they were more proficient than fibroblasts. The purpose of the present study was to evaluate the ability of porcine and bovine SMC at different seeding densities to reorganise collagen matrices fabricated with a range of collagen concentrations. MATERIALS AND METHODS Tissue culture plasticware was obtained from Gibco (Paisley, UK) or Sterilin (Hounslow, UK). Culture media and newborn calf serum (NBCS) were purchased from Gibco and foetal calf serum (FCS) from Tissue Culture Services (Slough, UK). Unless otherwise stated, all other reagents were obtained from Sigma Chemical Co. (Poole, UK). SMC were isolated by a modification of the explant technique described by Ross.14 Minced tissue from the media of the aortic root of porcine and bovine hearts was placed in 25 cm2 flasks in 1 ml Dulbecco’s modified Eagle’s medium (DMEM) containing 20% FCS. The tissue was incubated under standard conditions, i.e. 37 “C and an at-

mosphere of 5 % CO,/95 % air, until outgrowth of cells was observed. The resulting culture was grown to confluence and routinely cultured in DMEM containing 10% serum after the first passage. Porcine cells were grown with FCS, bovine cells with NBCS. The identity of the cells was confirmed by northern blotting, using a cDNA probe for aactin, a cytoskeletal protein specific to muscle cells.15 Type I collagen was extracted from rat tail tendons by the method of Rowling et aZ.ll The protein content of the acid solubilised collagen solution was quantified by the Biuret meth0d.l” SMC populated collagen matrices were prepared in sterile 85 mm bacteriological petri dishes. The following were added to each dish: 6 ml DMEM (2 x concentrated, serum-free); 1.5 ml NBCS; 1.5 ml SMC suspension in DMEM and 4.5 ml acidsolubilised collagen solution containing 38-l 5 mg protein. Addition of 1.5 ml 0.1 M NaOH induced fibrillogenesis of the collagen which, following incubation at 37 “C, was completed within 30 min. Matrices were incubated at 37 “C in 5 % CO,/95 % air and the medium was renewed every 4 days. Reorganisation and remodelling of the matrix was assessed by matrix contraction and histology for matrices with different SMC numbers and collagen concentrations. Contraction was measured as the mean decrease in diameter and subsequently transformed to surface area using rcr2. These data were transformed to arc sin, and subjected to a two-way analysis of variance.l’ Comparisons of individual means were made using an a posteriori technique. l’ The means together with the computed minimum significant differences (MSD) were back transformed to percentages and plotted graphically. Histology was performed on matrices at different stages of contraction. Fixed 10 pm sections, stained with Haematoxylin (H) and/or Picro Sirius Red (PSR), were examined using a Vickers M 17 microscope with transmitted light and crossed polar filters. Ilford Pan F film was used for photography. ESULTS Both porcine and bovine SMC were effective in the contraction of collagen matrices, 1 x lo5 cells causing a 99 % reduction in the upper surface area of a matrix fabricated with O-5mg/ml collagen (Fig. 1). Porcine aortic SMC (PASMC) and bovine aortic SMC (BASMC) of low passage number (PASMC 6th passage, BASMC 5th passage) showed no significant differences in contraction proficiency.

249

Smooth muscle cell populated collagen matrices

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TIME (days)

TIME I days1

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Fig. 1. Comparison

of collagen contraction abilities of porcine and bovine cells at different passage numbers. Matrices fabricated with 0.5 mg/ml collagen, were seeded with 1 x lo5 cells: BASMC p5 (O), BASMC p14 (0) and PASMC p6 (a). Bars represent half minimum significant differences. Statistically significant differences occur where bars do not overlap.

The same was true for bovine cells of low and high passage number (BASMC 5th passage, BASMC 14th passage). Figure 2 shows the effect of varying collagen concentration on the contraction of matrices by BASMC. Analysis of variance demonstrated significant differences in matrix area with respect to collagen concentration @ < 0.01) and time (p < O-01). In general, the time required for maximum contraction was directly proportional’to collagen content, although there was a degree of overlap between the 0~75 and 1.00 mg/ml collagen concen-

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I Fig. 2. Effect of varying collagen content on matrix contraction by bovine SMC. Matrices seeded with 1 x lo5 BASMC were fabricated with 0.25 (O), 0.5 (a), 0.75 (A) and 1.0 (m) mg/ml. Statistically significant differences occur where bars do not overlap. TIME (days

18

TIME,

days1 (b)

Fig. 3. Effect of varying BASMC seeding density on collagen

matrix contraction. Matrices were fabricated with (a) 0.5 mg/ml collagen and (b) 1.00 mg/ml collagen a seeded with 5 x IO4 (O), 7 x 104 (@), 1 x loj (ci), 2 x IO5 ( and 5 x 1Oj (A) bovine SMC per matrix. Statistically significant differences occur where bars do not overlap.

trations. All matrices underwent a 98-99 % reduction in upper surface area, the extent of final contraction being independent of collagen content. The effect of varying SMC seeding nsity on matrix contraction is illustrated in Fig. Experiments were set up using 5 seeding 7x 104; 1 x 105; 2x IO5 and 5x 1 matrix, and two collagen concentra (Fig. 3(a)) and 1.00 mg/ml (Fig. 3(b)). The same trends were seen for both collagen concentrations, the time to maximum contraction proportional to seeding density. maximum contraction times for matrices fabricated with a high collagen content (1 mgs/ml) were longer than for low collagen content (O-5mgjml) matrices. All matrices reached the same final u area irrespective of initial seeding de~sity~ ECM

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Fig. 4. Matrix sections stained with H-PSR after (A) 8 days and (B) 50 days in culture. Matrices were fabricated with @5 mg/ml collagen and seeded with 5 x lo5 BASMC per matrix. Compaction of collagen fibres (f) is accompanied by formation of cavities (c).

Histology revealed a marked difference in matrix structure when comparing a matrix in the early stages of contraction (day 8) with a completely reorganised matrix (day 50). Figure 4 shows the initial fibrillar structure (Fig. 4(A)) becoming more fibrous (Fig. 4(B)), accompanied by the appearance of cavities. The formation of collagen fibres is more evident when examining these sections under polarised light (Fig. 5). Collagen fibres, enhanced with PSR, are highly birefringent under polarised 1ight.l’ The greater light intensity in Fig. 5(B) shows that the fibrils had condensed into thicker organised fibres. These fibres appear to be randomly orientated and to have a non-uniform distribution, a dense area being seen at the periphery of the matrix. The change in distribution of cells with time is illustrated in Fig. 6. Cells are evenly distributed throughout the matrix at day 8 (Fig. 6(A)) but are found more predominantly at the periphery by day 50 (Fig. 6(B)).

Fig. 5. Matrix sections stained with H-PSR after (A) 8 days and (B) 50 days in culture, examined using polarised light. Matrices were fabricated with 0.5 mg/ml collagen and seeded with 5 x lo5 BASMC per matrix. The increase in birefringence intensity of brightness is indicative of collagen fibre formation. The fibres appear to be randomly orientated and to have a non-uniform distribution.

DISCUSSION

The data presented here show clearly that both porcine and bovine vascular SMC are able to contract rat collagen matrices-a phenomenon previously noted.13 Ehrlich et a1.l’ reported that SMC were ineffective at matrix contraction, attributing this to a deficiency in cell spreading and elongation. This anomaly may be attributed to the different method of collagen extraction used; Ehrlich et al. used pepsin-extracted collagen as opposed to acid-solubilised collagen. Pepsin cleaves the telopeptides from the collagen molecules thereby interfering with fibrillogenesis. ls In agreement with Bell et al.,13 the authors of the present paper have found SMC to be more effective at matrix contraction than dermal fibroblasts cell foI cell. For example, in the present study 1 x 10’ SMC

Smooth muscle cell populated collagen matrices

Fig. 6. (B) 50 mg/ml Evenly

Matrix sections stained with H after (A) 8 days and days in culture. Matrices were fabricated with 0.5 collagen and seeded with 5 x 1Oj BASMC per matrix. distributed SMC at day 8 are located at the periphery (p) of the matrix by day 50.

caused a 99 % decrease in area of matrix fabricated with 7.7 mg collagen over 26 days, compared with an 84% decrease by the same number of dermal fibroblasts.” Unlike SMC, fibroblasts show a direct etween extent of contraction and relationship diameter is dependent on The mechanism by which SMC contract collagen matrices is similar to that observed for fibroblasts. Histology demonstrated the translocation of cells accompanied by collagen fibre formation. The change in cell distribution may be the result of either cell migration and/or proliferation of cells at the surface of the matrix. This accumulation of cells at the periphery of the matrix has been previously observed with fibroblasts.2,3 The appearance of cavities within the matrix is possibly due to the high collagenolytic activity of the SMC. Although collagenase inhibitors are present in serum, SMC may be producing collagenase in excess of the inhibitor concentration over the 4-day period prior

2.51

to medium replenishment. Colla enase activity has been implicated in dermal equivalent ~emodell~ng’ by fibroblasts, and its presence has also been demonstrated for SMC grown on a collagen matrix. ‘O The collagen discs used in t provide a simple model system interaction of SMC with colla assessed and quantified. Wit developing a small diameter has now progressed onto a cy which will be subject to cycli reported here show the contractio by SMC in collagen discs cause in matrix area. In a cylindrical system, as there is a central obturator determining the diameter of the cylinder, matrix contraction can only affect wall thickness and length. Taking into account this and other possible differences, these ~re~irni~a~y data still provide useful information. The equal e with which early and later passage cells re collagen is encouraging. This indicates relatively small number of cells, as harvested from a biopsy, may be expanded into usable sized cultures without loss of contraction efficiency. The rudimentary tissue collagen matrix reorganisation ho entirely suitable for graft fabrication in its present form. It is not clear at this stage corporation of fibroblasts as well as advantageous to the matrix strut however that the mechanical material may be improved by i~~~e~cing collagen fibre orientation. The presence of cavities in the final biomaterial is undesirable as the tissue would be susceptible to aneurysm formation and leakage. This problem may be overcome by rna~i~ulati~~ collagenase inhibitor concentrations and/or incorporating a ground substance (glycosami glycans) into the matrix structure.

The author would like to thank assistance with cDNA probing tee H. Wright for histological evalu was funded by the National Hea REFERENCE 1. Bell, E., Ivarrson, B. & Merrill, C., PYOC.Natl. Acad Sci. USA, 76 (1979) 1274-8. 2. Sarber, R., Hull, B., Merrill, C., Soranno, T. & Bell, E., Mech. Ageing Dev., 17 (1981) 107-17. 18.2

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3. Allen, T. D. & Schor, S. L., J. Ultrastruct. Res., 83 (1983) 205-19. 4. Grinnell, F. & Lamke, C. R., J. Cell Sci. 66 (1984) 51-63. 5. Guidry, C. & Grinnell, F., J. Cell Sci., 79 (1985) 67-81. 6. Gillery, P., Maquart, F.-X. & Borel, J.-P., Exp. Cell Res., 167 (1986) 29-37. 7. Guidry, C. & Grinnell, F., Collagen Related Res., 6 (1986) 515-29. 8. Rowling, P. J. E., Raxworthy, M. J. & Kearney, J. N., Biochem. Sot. Trans., 16 (1988) 326. 9. Bell, E., Ehrlich, H. P., Buttle, D. J. & Nakatsuji, T., Science, 211 (1981) 10524.

10. Bell, E., Sher, S., Hull, B., Merrill, C., Rosen, S., Chamson, A., Asselineau, D., Dubertret, L., Coulomb, B., Lapiere, C., Nusgens, B. & Neveux, Y., J. Invest. Dermatol., 81 (1983) 2s-10s. 11. Rowling, P. J. E., Raxworthy, M. J., Wood, E. J., Kearney, J. N. & Cunliffe, W. J., Biomaterials, 11 (1990) 181-5.

12. Ehrlich, H. P., Griswold, T. R. & Rajaratnam, J. B. M., Exp. Cell Res., 164 (1986) 15462. 13. Wang, S. Y., Merrill, C. & Bell, E., Mech. Ageing Dev., 44

(1988) 127-41. 14. Ross, R., J. Cell Biol., 56 (1974) 172-186. 15. Kocher, 0. & Gabbiani, G., Dzfirentiation,

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A. G., Bardawill, C. J. & David, M. M., J. Biol, Chem., 17 (1949) 751-66. 17. Sokal, R. R. & Rohlf, F. J. Biometry: The Principles and Practise of Statistics in Biological Research, W. H. Freeman & Co., San Francisco, 1981. 18. Haynes, S. L., Wright, H. M., Kearney, J. N., Davies, G. A., In Micro 90: Trans. Roy. Microsc. Sot., ed. H. Y. Elder. Adam Hilger, New York, 1990, pp. 729-32. 19. Asselineau, D. & Pnmieras, M., Br. J. Dermatol., 111 (1984) 219-22. 20. Delvos, U., Gajdusek, C., Sage, H., Harker, L. A. & Schwartz, S. M., Lab. Znvest., 46 (1982) 61-72.