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Modeling dermal granulation tissue with the linear fibroblast-populated collagen matrix: A comparison with the round matrix model
Journal of Dermatological Science (2006) 41, 97—108
www.intl.elsevierhealth.com/journals/jods
Modeling dermal granulation tissue with the linear fibroblast-populated collagen matrix: A comparison with the round matrix model Mark J. Eichler, Mark A. Carlson * Department of Surgery, University of Nebraska Medical Center and the Omaha VA Medical Center, 4101 Woolworth Avenue, Omaha, NE 68105, Nebraska, USA Received 15 July 2005; received in revised form 2 September 2005; accepted 13 September 2005
KEYWORDS Fibroblast-populated collagen matrix; Wound healing; Granulation tissue; Mechanical tension; Actin cytoskeleton
Summary Background: Wound contraction typically is not symmetrical; for example, a squareshaped wound will not yield a square scar. Interestingly, the round fibroblast-populated collagen matrix has been used as a model of wound contraction, even though contraction in this model is mostly symmetrical. Objective: We wanted to compare the round versus linear fibroblast-populated collagen matrix to see which would be a better model of dermal granulation tissue. Methods: Gross and microscopic morphology, contraction kinetics, cytoskeletal architecture, and apoptotic and proliferative indices were compared between the round versus the linear fibroblast-populated collagen matrix. A rat excisional wound model was used as an in vivo standard of healing. Results: The rate of contraction was similar between the two models, although the mode of contraction was grossly asymmetric in the linear while remaining symmetric in the round model. Cellular survival and proliferation were both dependent on matrix attachment in both models; this was analogous to the attachment-dependence of granulation tissue. In the attached (restrained) condition, the level of cellular organization was higher in the linear than in the round matrix; the tissue architecture of the linear matrix, moreover, mimicked that of the excisional wound model. Conclusion: The round versus linear fibroblast-populated collagen matrix displayed a similar proliferative and survival response to matrix attachment. The latter model, however, demonstrated tissue organization with attachment and asymmetrical contraction after detachment analogous to that of the in vivo wound model. The linear
Abbreviations: BrdU, 5-bromo-20 -deoxyuridine; FBS, fetal bovine serum; LFPCM, linear fibroblast-populated collagen matrix; PI, propidium iodide; RFPCM, round fibroblast-populated collagen matrix; TUNEL, terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling * Corresponding author. Tel.: +1 402 346 8800x5371; fax: +1 402 977 5672. E-mail address: [email protected] (M.A. Carlson). 0923-1811/$30.00 # 2005 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jdermsci.2005.09.002
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fibroblast-populated collagen matrix appears to be the better model of dermal granulation tissue. # 2005 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.
1. Introduction Mechanical forces regulate survival, proliferation, gene expression, and other processes in a number of cell types [1], including dermal fibroblasts in a three-dimensional collagen matrix [2,3]. The latter has been utilized as a model of granulation tissue in dermal wound healing [4]. Typically, the fibroblastpopulated collagen matrix (also known as lattice or gel) is pipeted into a culture well, which yields a disklike (i.e., round) configuration [5]. In a restrained (also known as attached) matrix, the subsequent fibroblast-dependent prestress that develops is fairly evenly distributed around the circular perimeter of the matrix. If the mechanical restraint is removed from such a matrix (i.e., it is detached from the culture well), then the early result is symmetrical contraction–—in other words, the matrix initially shrinks into a smaller circle. In actual excisional wounds, however, contraction typically is not a symmetrical process. For example, a square wound can contract in one direction and produce a linear scar [4,6]. Previous experimentation with a rat excisional wound model demonstrated that survival and proliferation of the cells within the granulation tissue was acutely dependent on attachment of its extracellular matrix to the dermal edge [7—9]. Thus, the dependence of cell fate on matrix attachment appears to be similar in the in vivo wound model versus the round collagen matrix [4]. Casual inspection of these two models, however, revealed that their gross and microscopic morphology was dissimilar. Considering the apparent morphologic disparity between the round fibroblast-populated collagen matrix and our in vivo wound model, we wanted to determine if an alternative (that is, a linear [10—12]) collagen matrix model would be a better morphologic approximation of granulation tissue. This concern for a better morphologic equivalent of granulation tissue stems from our interest in the regulation of cellular survival and proliferation in wound healing. As stated above, mechanical attachment of the extracellular matrix is a proven regulator of wound tissue; another potential regulator is cell shape. This has been shown to have crucial influence on cellular survival in vitro [3,13]. Experiments to dissect out cell shape—cell fate relation-
ships in granulation tissue presumably would be facilitated by a model that was a reasonable morphologic equivalent. So, our intent with the studies herein was to characterize the relationship of cell fate with respect to matrix attachment in the linear collagen matrix (not previously done), and to determine whether the linear matrix model was indeed a closer morphologic mimic of granulation tissue than the round matrix model.
2. Materials and methods 2.1. Reagents Vitrogen (bovine dermal collagen) was from Angiotech BioMaterials Corporation (formerly Cohesion Technologies). Anti-BrdU antibody was from Zymed. TUNEL kits (In Situ Cell Death Detection Kit, Fluorescein) were purchased from Roche. Alexa Fluor1 488 phalloidin was from Molecular Probes. Fetal bovine serum was from Invitrogen. All other reagents were purchased from Sigma or Life Technologies.
2.2. Cell culture The use of human fibroblasts in this research was approved by our Institutional Review Board. Primary dermal fibroblasts were cultured from neonatal foreskin specimens and maintained as previously described [14,15]. All experiments were performed on cells derived from one foreskin specimen; this strain of cells initially was expanded to passage 7, and then frozen down. To perform an experiment, a vial of these passage 7 cells was thawed, plated, and then expanded to passage 9; collagen matrices then were prepared from the passage 9 cells. The days of the week upon which thawing, splitting, freezing, etc. was performed was kept constant.
2.3. Collagen matrices The protocol for setting-up round collagen matrices has been described [14,15]. Round matrices were incubated for 72 h in the attached state prior to the initiation of an experiment. For the linear matrix model, a 900 ml aliquot of the same solution of cells (106 ml1) in bovine collagen (1.5 mg/ml) that was
Round vs. linear fibroblast-populated collagen matrix
used to generate round matrices was pipeted into the linear gel casting apparatus (not shown; details available upon request), and allowed to polymerize at 37 8C for 2 h. After polymerization, each linear matrix was carefully transferred into the incubation chamber (Fig. 2G); medium (10% FBS in DMEM with 1 mg/ml ascorbic acid) then was added to cover the matrices. Linear matrices were incubated for 24 h in the attached state prior to the initiation of an experiment.
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The excisional wounding model in the rat has been previously described [4,7].
2.7. Statistical analysis Numerical data are represented with the mean standard deviation. Unpaired groups of data were analyzed with analysis of variance and the unpaired t-test, with p = 0.05 as the level of significance.
2.4. Histologic analysis The techniques of TUNEL and BrdU immunohistochemistry on cytospin preparations of collagen matrices follows previous descriptions [9,14,15]. For the BrdU labeling, matrices were incubated with 10 mM BrdU for 4 h prior to the cytospin preparation.
2.5. Phalloidin labeling and directionality index (DI) The technique of phalloidin-labeling follows a previous description [16]. In the present work, intact collagen matrices were fixed in 3% paraformaldehyde in PBS for 20 min at 22 8C prior to staining with fluorescently labeled phalloidin. Matrices then were imaged with a Zeiss LSM 410 confocal laser scanning microscope. Subsequent, analysis was done with the directionality index, a softwarebased method which can quantify the overall directionality (i.e., polarity or orientation) of a digital image of complex biological structures. The index is calculated with a freeware program called ImageJ (http://rsb.info.nih.gov/ij/) with a Java plugin called SurfCharJ (http://home.online.no/gary. c/IJ/SurfCharJ.htm) [17]. The details and mathematical descriptors of the directionality index have been described in detail elsewhere [16]. As an example of the directionality index, consider a histologic section of striated muscle cut parallel to the fibers. Almost all of the fibers in such an image will be oriented along a single vector; such an image will yield an index of approaching one or zero, which indicates a highly polarized directionality. In contrast, consider a histologic section of liver cut in a random section. Although the liver is highly organized, the hepatocytes are oriented along multiple vectors. The directionality index in this case would approach 0.5, indicating very little polarization of directionality.
2.6. Excisional wound model The use of animals in this research was approved by our Institutional Animal Care and Use Committee.
Fig. 1 Morphology of wound contraction in a rat excisional model. (A) Overhead photographs of a 2 cm 2 cm excisional wound on the dorsum of a rat at the time of wounding (postwounding day 0) and 18 days later. Arrow = cephalad; scale is in cm. (B) H&E micrographs of granulation tissue from the rat excisional model (postwounding day 6), sectioned in the transverse vs. sagittal plane. The dotted line in each diagram indicates the orientation of the corresponding section; the open rectangle represents the wound (in the same orientation as shown in A). Arrows = direction of wound contraction; bar = 50 mm.
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3. Results 3.1. Polarization of granulation tissue cells in vivo during wound contraction It has been documented by others [6,18] and observed by us [4,7—9,16] that wound contraction in various excisional wound models is asymmetrical; for example, a 4 cm2 square-shaped incision on the upper dorsum of the rat contracted almost entirely along a transverse vector, which left the animal with a vertically oriented linear scar (Fig. 1A). In other words, a square-shaped wound did not yield a squareshaped scar. The pattern of wound contraction shown in Fig. 1 has been consistent in over 500 animals wounded in similar fashion in our laboratory. Examination of the microscopic morphology of the wound in Fig. 1A revealed a high degree of polarization of the cells in the granulation tissue (Fig. 1B). The predominant orientation of the cells (mostly fibroblasts, although a cell-specific marker was not employed) was parallel to the direction of wound contraction, as indicated by the transverse section in Fig. 1B. Cutting perpendicular to the direction of contraction (i.e., a sagittal section) resulted in most cells being cut across their long axis, which was consistent with the orientation shown in the transverse section.
3.2. Gross morphology and contractile properties of the round versus the linear fibroblast-populated collagen matrix The gross morphology and contractile properties of the round fibroblast-populated collagen matrix have been reviewed [5,19], but they are reiterated here for the sake of comparison. Primary dermal fibroblasts were mixed with soluble collagen and pipeted into a culture well. The collagen solution underwent polymerization and formed a gel-like material (also known as collagen matrix or lattice); a lateral view of a freshly polymerized matrix is shown in Fig. 2A. Fig. 2 Gross morphology of round vs. linear fibroblastpopulated collagen matrices. (A) Round attached matrix, 1 h after pipeting into culture well (i.e., just prior to adding medium); edge view (postfixation). (B) Round attached matrix, 3 h after pipeting into culture well (i.e., 2 h after addition of serum-supplemented medium); bar = 2 mm. (C) Round attached matrix after 3 days of incubation with serum-supplemented medium; top view (postfixation). (D) Round matrix of (C), 1 h after mechanical release from the culture dish; bar = 2 mm. (E) Plot of attached matrix contraction (i.e., decrease in matrix height) which was shown in (A and B). Each data point in this and subsequent plots represents the mean S.D. of four measurements. (F) Plot of stressed round matrix contraction (i.e., decrease in matrix diameter) which was shown in (C and D). (G) Linear gel incubation chamber (see text for dimensions).
(H) Pylon apparatus (top) shown with temporary matrix support fitting (bottom). (I) Linear fibroblast-populated collagen matrix (nonfixed) in the pylon apparatus after polymerization and prior to immersion in medium; matrix is suspended between two tabs of Velcro1. (J) Three linear matrices shown incubating in medium within the chamber; extent of one matrix is marked with a bracket. (K) Linear matrix fixed in the attached state after 48 h of incubation in serum-supplemented medium; matrix is stretched between the two Velcro1 tabs. (L) Linear matrix similar to (K), but mechanically released from one Velcro1 tab and incubated for an additional hour (bar = 5 mm); arrowhead = contracted matrix. (M) Plot of stressed linear matrix contraction (i.e., decrease in matrix length) which was shown in (K and L).
Round vs. linear fibroblast-populated collagen matrix
In the presence of serum, the fibroblasts reorganized the collagen fibers, and the matrix contracted in the vertical direction (compaction), as shown by the interval photograph in Fig. 2B. A time course of vertical contraction is plotted in Fig. 2E; this compaction was cell-dependent, and most of it occurred within several hours after application of medium. If a compacted, prestressed matrix was physically detached (released) from the tissue culture plastic, then lateral contraction ensued (Fig. 2C and D) until the matrix area reached a steady state at 10—20% of the prerelease area; most of this contraction occurred in the first hour and also was cell-dependent (see plot in Fig. 2F). The incubation chamber for the linear fibroblastpopulated collagen matrix was constructed of methyl methacrylate polymer (Lucite1) and consisted of a 5 cm (height) 7 cm 7 cm rectangular well with tracks cut into the bottom for holding the pylon apparatus (Fig. 2G). The latter (Fig. 2H) consisted of inverted-U-shaped section of Lucite; two tabs of Velcro1 were glued on opposing sides of the pylons. There also was an associated interlocking section of Lucite (lower portion of Fig. 2H), which was used to support the collagen matrix during the polymerization phase, and then was removed. A freshly polymerized linear fibroblast-populated collagen matrix is shown suspended between the two Velcro anchorage points in Fig. 2I. The pylon apparatus containing the polymerized matrix was placed into the well, medium was added, and compaction analogous to that of the round matrix (see Fig. 2A and B) ensued. Instead of flattening into a thin disk, however, the linear matrix underwent compaction along its midsection, resulting in a slight ‘‘hourglass’’ [12] appearance (Fig. 2J). The development of mechanical tension (prestress) in the long axis of the linear matrix was confirmed by releasing one end of the matrix from its pylon (Fig. 2K and L), which resulted in matrix shortening (plotted in Fig. 2M) analogous to the release induced contraction of the round matrix (Fig. 2C and D).
3.3. Microscopic morphology of the round versus linear fibroblast-populated collagen matrix The microscopic morphology of the round fibroblastpopulated collagen matrix has been described [20], but is shown here for the sake of comparison. Fibroblasts in the attached round matrix had an elongated, bipolar morphology, and were parallel to the plane of the matrix (i.e., in the plane of mechanical tension), as illustrated by sectioning parallel and perpendicular to the plane of the matrix (Fig. 3A and B). The orientation of the cells within the plane of
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the round matrix appeared to be random (Fig. 3A), indicating that the tension was distributed in randomly oriented 3608 vectors. This conclusion was consistent with the relatively uniform shape that the round matrix maintained during contraction (see Fig. 2C and D above). In contrast, the fibroblasts in the attached linear matrix were aligned parallel to the long axis of the matrix, as shown by longitudinal and transverse sections (Fig. 3C and D). One day after release, however, the fibroblasts in both the round and linear matrix became more rounded and lost their orientation (Fig. 3E and F); there was not much difference in microscopic morphology in the round versus linear matrix after release. The microscopic characteristics apparent from the H&E histology were reinforced by fluorescent staining of the actin cytoskeleton of cells within the linear matrix (Fig. 4). The actin fibers within the attached linear matrix (Fig. 4A and B) were aligned along the long axis of the matrix (i.e., parallel to the vector of mechanical tension). One day after release of the linear matrix (Fig. 4C and D), the alignment was lost and the cells adopted a dendritic appearance [21].
3.4. Quantification of cellular orientation in the fibroblast-populated collagen matrix The subjective morphometric differences apparent between the micrographs of the round versus linear matrix in Fig. 3 may be quantified with the directionality index [16]. This index converts a digital image into an array of ‘‘facets.’’ The direction of each facet can be graphed in a polar plot, such as shown in Fig. 5B—D. The plot in Fig. 5B, for example, was derived from the facet transformation of the phalloidin image in Fig. 4A. The relative polarity of the plot in Fig. 5B (i.e., the directionality index) can be calculated by measuring the area bounded by the plot which falls into defined sectors [16]. For highly polarized images, the directionality index will approach 1 or 0; for nonpolarized images, the index will approach 0.5. As expected, the directionality index for the attached linear matrix was close to 1 (Fig. 5B and E), while the index for the released linear matrix was closer to 0.5 (Fig. 5C and E). For comparison, the directionality index for the attached round matrix (Fig. 5A) also was close to 0.5 (Fig. 5D and E). This latter finding indicates that there was little polarization (i.e., no cell alignment) within the plane of the attached round matrix, even though the individual cells within this matrix were highly polarized. The directionality index also can be calculated on individual cells, as shown in Fig. 5F and G. The images of attached (polarized) versus
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Fig. 3 Microscopic morphology of round vs. linear fibroblast-populated collagen matrices; H&E stained paraffin sections. (A) Round attached matrix, sectioned in the frontal plane. Inset in (A—D) illustrates the histologic plane; bar = 50 mm (same for A, C, E, and F). (B) Round attached matrix, sectioned in the transverse plane; bar = 10 mm. (C) Linear attached matrix, sectioned in the frontal plane. (D) Linear attached matrix, sectioned in the transverse plane; bar = 20 mm. (E) Round released matrix, sectioned in random plane. (F) Linear released matrix, sectioned in random plane.
released (dendritic) cells in these figure panels were extracted from Fig. 4B and D, respectively. The resultant value of the directionality index reflected the relative polarization of each cell.
3.5. Proliferative and apoptotic indices in the round versus linear fibroblastpopulated collagen matrix Previous work demonstrated that attachment of the fibroblast-populated collagen matrix promoted survival and proliferation of the embedded cells, while detachment (also known as release or stress-relaxation) induced apoptosis and quiescence [15,22,23]. After a comparison of the gross and microscopic
morphology of the round versus linear matrix models, we wanted to determine if the cell cycle and survival response to matrix anchorage was similar in the round versus linear matrix. To perform this comparison, cytospin preparations of cells isolated after enzymatic degradation of the matrix underwent either TUNEL or BrdU immunohistochemistry; an example of the latter is shown in Fig. 6A. As expected, the BrdU-labeling index was greater (by about one order of magnitude) in the attached round matrix compared to the released round matrix (Fig. 6B), which confirmed that matrix attachment supports fibroblast proliferation. Matrix attachment also supported fibroblast proliferation in the linear matrix model (Fig. 6B). Consistent with
Round vs. linear fibroblast-populated collagen matrix
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Fig. 4 Confocal microscopic images of intact fibroblast-populated collagen matrices stained with fluorescently labeled phalloidin and propidium iodide. (A) Attached matrix; bar = 100 mm. Fluorescent images in this and subsequent panels represent phalloidin (PI) and merged signal. Double-headed arrow = orientation of poles (i.e., direction of force generation). (B) Higher power view of the attached matrix; bar = 20 mm. (C) Released matrix; same scale as (A). (D) Higher power view of the released matrix; same scale as (B). Arrowheads: individual cells used for analysis in Fig. 5F and G.
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published results [15,22,23], matrix detachment induced apoptosis in the round matrix (Fig. 6C); the TUNEL-positivity rate was about an order of magnitude greater in the released compared to the attached condition. Similar to the round matrix model, matrix detachment in the linear model produced a marked increase in the apoptotic index (Fig. 6C).
3.6. Alternative models of the linear fibroblast-populated collagen matrix During development of the linear fibroblast-populated collagen matrix model technical problems arose with fragility of the system. The collagen polymerization phase for the linear matrix required a watertight casting mold (not shown) in which the pylon apparatus and the associated support (Fig. 2H) temporarily were contained. The solubilized collagen was pipeted into this mold, polymerization ensued, and the pylon apparatus then was transferred to the Lucite incubation well (Fig. 2G). Unfortunately, the collagen solution had a propensity to leak out of the mold prior to polymerization. In addition, because of the relatively small surface area of attachment to the Velcro tabs, the attached collagen matrices had a propensity to detach prematurely from either pylon; incubating these gels beyond 72—96 h was difficult because of this premature detachment. The multiple manipulations required for the matrix set-up also increased the likelihood of bacterial contamination. In general, less than half of a given group of attached linear matrices would survive intact beyond 72 h. In an effort to make the linear matrix model more robust, an alternative ‘‘square-well’’ system was devised (Fig. 7A), in which two opposing sides of the well were lined with Velcro. This system is analogous to that of a commercially available linear matrix casting system (Tissue TrainTM Culture System; Flexcell International Inc., Hillsborough, NC), except that the system in Fig. 7 provided a wide base of matrix attachment at each pole, and also allowed a greater matrix volume (1000 ml versus 225 ml, [12]).
Fig. 5 Directionality analysis of actin cytoskeleton in the fibroblast-populated collagen matrix. (A). Section of attached round matrix taken in the frontal plane (see Fig. 3A), stained with phalloidin-AF488, and imaged with conventional fluorescent microscopy. Bar = 50 mm. (B) Azimuthal plot of attached linear matrix after facet transformation [16] of the digital image in the left panel of Fig. 4A. The directionality index (DI) [16] derived from
this plot also is shown. (C) Azimuthal plot with DI for the image of the linear released matrix in the left panel of Fig. 4C. (D) Azimuthal plot with DI for the image of the round attached matrix in Fig. 5A. (E) Bar blot of mean DI S.D. for linear attached vs. linear released vs. round attached matrix (four microscopic fields analyzed per condition); *p < 0.001 compared to linear attached, ANOVA, and unpaired t-test. (F) An individual cell from the attached vs. released matrix, extracted from Fig. 4B and D (see arrowheads therein). (G) Azimuthal plots with the derived DI of the images in (F).
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Fig. 7 Alternative linear matrix models. (A) Square well, overhead, and oblique views; scale in cm. (B) Overhead view of collagen matrix in a square well after 96 h incubation; bar = 5 mm. (C) Linear matrix with mobile poles after 24 h incubation; bar = 5 mm. (D) Linear matrix with mobile poles after 96 h incubation.
Fig. 6 Proliferation and cell death data for the round vs. linear fibroblast-populated collagen matrix. (A) Cytospin preparations of attached vs. released round collagen matrices (labeled with 10 mM BrdU for 4 h prior to processing) underwent BrdU immunocytochemistry with PI counterstaining. (B) BrdU incorporation data (mean S.D.) of attached vs. released matrices from the round and linear models. Each mean was calculated from 4 images, an example of which was shown in (A); *p < 0.05 compared to attached, unpaired t-test. Each experiment was performed a minimum of three times. (C) Same as (B), except displaying TUNEL data.
The tight-fitting seams of the square-well system in Fig. 7 did not allow leakage of the soluble collagen that occurred with the linear apparatus of Fig. 2. The square apparatus also did not require matrix transfer into another incubation chamber; the square well was placed into a 35 mm culture dish, and remained there for the duration of an experiment. The simple set-up of the square matrix decreased the amount of manipulation and subsequent contamination risk. Furthermore, the increased surface area of attachment (Fig. 7B) eliminated the premature detachment problem; matrices could be incubated in the attached state for >96 h without difficulty. Examination of microscopic morphology confirmed that the cellular alignment of the square-well linear matrix model was similar to that shown in Fig. 2 (data not shown); i.e., the cells were aligned along the axis that ran between the two Velcro poles. We also devised the ‘‘mobile pole’’ linear matrix (Fig. 7C and D) in order to simulate the dynamics of the excisional wound model in Fig. 1. This model variant had a similar configuration and set-up procedure to the linear apparatus shown in Fig. 2, except that the pylons could move freely and independently within the tracking groove of the incubation well (Fig. 2G). As the matrix contracted, it would pull the pylons closer together, analogous to the dermal edges of a contracting wound. The contraction demonstrated in Fig. 7C and D occurred over a 3 days period.
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4. Discussion An advantage of the linear fibroblast-populated collagen matrix model [10—12] was that it provided a three-dimensional model of fibroblast—collagen interactions in which there was uniformity of cellular orientation. This uniformity should facilitate the study of cell survival, proliferation, gene expression, etc. with respect to cell shape and mechanical loading. An advantage to the particular model shown in Fig. 2I, compared to other linear matrix systems, is that it was a true two-pole system; the matrix in Fig. 2I made no physical contact with a solid substrate other than at the Velcro-coated pylons. These two small attachment points minimized the confounding contribution of the broad attachment surface that characterized the round matrix model. This arrangement made the linear matrix a more ‘‘pure’’ model of mechanical loading compared to the round matrix; that is, the force vectors within the former were defined by one axis, not two. As expected, the survival and proliferative response of the fibroblasts with respect to matrix attachment were similar in the linear versus the round matrix model (see Fig. 6). This is reassuring evidence that fibroblast biology in the linear matrix is similar to that in the round matrix. The disadvantages of the linear matrix model depicted in Fig. 1 were (1) it was somewhat cumbersome to set-up compared to the round model; (2) the freshly pipeted collagen solution tended to leak out of the matrix mold prior to polymerization; (3) repeated manipulation increased the risk of bacterial contamination of the apparatus; (4) the relatively small Velcro anchorage points were insufficient to restrain the matrix for extended periods. These concerns were addressed by the employment of a ‘‘square-well’’ linear matrix system shown in Fig. 7A. The main disadvantage to the square-well apparatus was that it was not a pure two-pole system like that in Fig. 2I. The matrix in a square well not only was in contact with the Velcro anchorage points, but it sat on the Lucite floor of the well and touched the two walls that were not covered with Velcro. In practice, this may not be much of a concern, however, because the matrix did not appear to adhere well to the Lucite. The attached matrix in the square well (Fig. 7B) attained the ‘‘wasp-waist’’ or ‘‘hourglass’’ appearance of pylon linear matrix, indicating that lateral contact of the matrix with the bare Lucite did not have much influence on matrix morphology. Previous investigators have described linear-type fibroblast-populated collagen matrix models which developed prestress between two poles; the intent of these studies was to quantify mechanical force generated or to study the effect of extrinsic force on fibroblast biology [10,11]. Our intent with a linear-
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type matrix model was to develop a model to study the relationships between cell shape, mechanical loading, and the biological fate/function of individual cells in a three-dimensional culture system. Recent evidence has indicated that cell shape and mechanical loading are important regulators of cell fate and function [3,13]. In order to study these entities, we believed that it would be helpful to have a model which approximated the polarized morphology of the in vivo excisional wound tissue. The best approximation of wound contraction in this study may have been the mobile pole model shown in Fig. 7C and D. This model combined the polarity of fixed pylon model of Fig. 2I with the dynamic characteristics of a contracting wound. The conventional restrained fibroblast-populated collagen matrix model also will contract when released [5], but this contraction occurs over minutes to hours, not days to weeks that typify in vivo wound contraction. Unrestrained collagen matrices can be set-up to contract over a period of days, but only by decreasing the cellular density to a level, that is, several orders of magnitude less than that typically found in granulation tissue [2,24]. It is debatable how faithful such models are to in vivo wound contraction. The mobile pole system (Fig. 7C and D), however, uses a relatively high concentration of fibroblasts (though still lower than what exists in vivo [24]) in a matrix that contracts over a period of days to a week, depending on the force of resistance provided by the anchoring blocks. In our preliminary investigation, however, this model has been somewhat difficult to utilize because of variability in the degree and speed of contraction. The mobile pole model will require further refinement. Software-aided morphometric analysis of the cytoskeleton has been described [25,26]. In the present paper, a measure of cytoskeletal polarity (the directionality index [16]) was used to demonstrate that cytoskeletal organization of both single cells and cell clusters could be quantified. The directionality index showed that the cytoskeletal organization was greater in the attached linear versus the released linear matrix, which confirmed the visual impression (Figs. 4 and 5). Interestingly, while the individual cells in the attached round matrix were highly polarized, the directionality index of this matrix did not indicate polarization at the tissue level (Fig. 5). If the attached round matrix was sectioned in the plane of mechanical tension (Fig. 3A), then the elongated fibroblasts had a fairly random orientation, indicating dispersal of mechanical tension in a 3608 arc around the matrix perimeter. This is in contrast to the attached linear matrix, which when sectioned in a plane parallel to the axis of mechanical tension (Fig. 3C), demonstrated high polarity at the tissue
Round vs. linear fibroblast-populated collagen matrix
level, i.e., nearly all the elongated fibroblasts were parallel to the direction of tension. The ability to quantify organization at both the cell and tissue level in the linear matrix model should enable future study of cell shape—cell fate relationships in the threedimensional matrix. The elongated, polarized morphology of fibroblasts in a mechanically restrained (attached) collagen matrix has been associated with promotion of survival and the cell cycle [4]. In contrast, the ‘‘rounded’’ morphology (as described with H&E staining of paraffin sections) has been associated with induction of apoptosis and quiescence. In situ imaging of the cytoskeleton in the fibroblast-populated collagen matrix) with confocal microscopy (as was done in Fig. 4), however, revealed that this ‘‘rounded’’ morphology actually was dendritic [27]. This morphologic switch (from elongate to dendritic) that occurred with the transition from mechanically attached to release was not associated with universal cell death or quiescence. In other words, detachment of a mechanically restrained matrix induced cell death in some, but not all, of the indwelling fibroblasts (see Fig. 6). Conversely, there was ongoing, albeit low, apoptotic activity in the mechanically restrained matrix. There was an analogous situation with BrdU labeling; that is, not all cells in a mechanically restrained matrix were cycling, and not all cells in a detached matrix became quiescent. The mechanical state of the collagen matrix apparently plays a permissive role in the outcome of fibroblast survival and proliferation. There likely are other factors, such as responsiveness to growth factors [28] that influence cell fate in the fibroblast-populated collagen matrix. Growth factors were constantly present in the models of this manuscript, as the incubation medium was supplemented with 10% FBS. It has been shown that the activity of growth factor receptors in fibroblasts is influenced by the mechanical state of the matrix in the round collagen matrix model [28]. Previous investigation with the round fibroblastpopulated collagen matrix model has demonstrated differences in signal transduction between the attached (also known as stressed, or anchored, or restrained) versus the detached (also know as stressreleased) state. The phosphatidylinositol 3-kinase/ Akt pathway in attached matrices is upregulated via a b1-integrin/focal adhesion kinase-dependent mechanism [14,29,30]. The PI-3-kinase/Akt pathway has been implicated in promoting survival and cell cycling in a range of cell types [31]. In addition, the extracellular-regulated kinase/mitogen activated protein kinase pathway (Erk/MAPK) is activated in attached matrices, providing another signal for DNA synthesis [32]. Release of stressed matrices results in
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an early induction of c-fos [33], which, as a component of the AP-1 transcription complex, is a prototypical responder to cellular stress that regulates proliferation and survival [34]. cDNA microarray analysis of mechanically stressed versus unstressed fibroblasts in a collagen matrix demonstrated upregulation of 60 known genes in the former and 64 in the latter. Some of the upregulated genes in stressed matrices included cyclin D3, VEGF, plasminogen activator inhibitors, TIMPs, collagen a1, g-tubulin, asmooth muscle actin, vinculin, and integrin-linked kinase [3]. It was concluded that stressed fibroblasts had a synthetic phenotype compared to their unstressed counterparts. The signal transduction differences in attached versus stress-released linear matrices have yet to be investigated, but considering the similarity in mechano-regulated proliferation and survival in the round versus linear models, it might be assumed that the mechanotransduction pathways also are similar. The linear fibroblast-populated collagen matrix model provides an environment in which there is relative uniformity of cellular alignment along an axis of mechanical tension. This characteristic imitates what occurs with in vivo excisional healing, as was shown in Fig. 1. For reasons which currently are not well understood, some (if not most) wounds will have a predominant axis of contraction, resulting in alignment of the cells within the granulation tissue (i.e., an enhanced level of tissue organization). This axis of contraction is reproduced by the linear matrix model. Moreover, the attached versus released linear matrix model represent opposite extremes for a number of descriptors, including cellular morphology, tissue organization, cellular survival and proliferation, and so on. The extent of the biologic differences between the attached versus released condition should facilitate the study of these differences. Using methods to augment or disrupt signaling pathways involved with survival and/or proliferation, and then studying the effects on the cytoskeleton (and also by performing the inverse) should uncover dependencies of cell shape and cell fate. This in turn should lead to avenues to explore regulation of the healing response.
Acknowledgements The authors would like to acknowledge the technical assistance of Chris Hansen and Amy Prall. Supported by a grant from the NIH (K08 GM00703) to MAC. MJE was supported by a Summer Research Stipend from the University of Nebraska College of Medicine. The authors also would like to thank Janice Taylor of the
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Confocal Laser Scanning Microscope Core Facility at the University of Nebraska Medical Center, which is supported by the Nebraska Research Initiative, for providing assistance with confocal microscopy.
References [1] Katsumi A, Orr AW, Tzima E, Schwartz MA. Integrins in mechanotransduction. J Biol Chem 2004;279:12001—4. [2] Fluck J, Querfeld C, Cremer A, Niland S, Krieg T, Sollberg S. Normal human primary fibroblasts undergo apoptosis in three-dimensional contractile collagen gels. J Invest Dermatol 1998;110:153—7. [3] Kessler D, Dethlefsen S, Haase I, Plomann M, Hirche F, Krieg T, et al. Fibroblasts in mechanically stressed collagen lattices assume a ‘‘synthetic’’ phenotype. J Biol Chem 2001; 276:36575—85. [4] Carlson MA, Longaker MT. The fibroblast-populated collagen matrix as a model of wound healing: a review of the evidence. Wound Repair Regen 2004;12:134—47. [5] Grinnell F. Fibroblasts, myofibroblasts, and wound contraction. J Cell Biol 1994;124:401—4. [6] Grillo HC, Watts GT, Gross J. Studies in wound healng: I. Contraction and wound contents. Ann Surg 1958;148:145—52. [7] Carlson MA, Longaker MT, Thompson JS. Granulation tissue regression induced by musculocutaneous advancement flap coverage. Surgery 2002;131:332—7. [8] Carlson MA, Longaker MT, Thompson JS. Wound splinting regulates granulation tissue survival. J Surg Res 2003;110: 304—9. [9] Carlson MA, Thompson JS. Wound splinting modulates granulation tissue proliferation. Matrix Biol 2004;23:243—50. [10] Brown RA, Prajapati R, McGrouther DA, Yannas IV, Eastwood M. Tensional homeostasis in dermal fibroblasts: mechanical responses to mechanical loading in three-dimensional substrates. J Cell Physiol 1998;175:323—32. [11] Delvoye P, Wiliquet P, Leveque JL, Nusgens BV, Lapiere CM. Measurement of mechanical forces generated by skin fibroblasts embedded in a three-dimensional collagen gel. J Invest Dermatol 1991;97:898—902. [12] Garvin J, Qi J, Maloney M, Banes AJ. Novel system for engineering bioartificial tendons and application of mechanical load. Tissue Eng 2003;9:967—79. [13] Chen CS, Mrksich M, Huang S, Whitesides GM, Ingber DE. Geometric control of cell life and death. Science 1997;276: 1425—8. [14] Carlson MA, Longaker MT, Thompson JS. Modulation of FAK, Akt, and p53 by stress release of the fibroblast-populated collagen matrix. J Surg Res 2004;120:171—7. [15] Grinnell F, Zhu M, Carlson MA, Abrams JM. Release of mechanical tension triggers apoptosis of human fibroblasts in a model of regressing granulation tissue. Exp Cell Res 1999;248:608—19. [16] Carlson MA, Thompson JS. Wound matrix attachment regulates actin content and organization in cells of the granulation tissue. Wound Repair Regen 2005;13:84—92.
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[17] Chinga G, Gregersen O, Dougherty B. Paper surface characterisation by laser profilometry and image analysis. J Microsc Anal 2003;84:5—7. [18] Watts GT. Wound shape and tissue tension in healing. Br J Surg 1959;47:555—61. [19] Ehrlich HP. The fibroblast-populated collagen lattice. A model of fibroblast collagen interactions in repair. Methods Mol Med 2003;78:277—91. [20] Tomasek JJ, Haaksma CJ, Eddy RJ, Vaughan MB. Fibroblast contraction occurs on release of tension in attached collagen lattices: dependency on an organized actin cytoskeleton and serum. Anat Rec 1992;232:359—68. [21] Grinnell F. Fibroblast biology in three-dimensional collagen matrices. Trends Cell Biol 2003;13:264—9. [22] Niland S, Cremer A, Fluck J, Eble JA, Krieg T, Sollberg S. Contraction-dependent apoptosis of normal dermal fibroblasts. J Invest Dermatol 2001;116:686—92. [23] Fringer J, Grinnell F. Fibroblast quiescence in floating or released collagen matrices: contribution of the ERK signaling pathway and actin cytoskeletal organization. J Biol Chem 2001;276:31047—52. [24] Wilson L, Fathke C, Isik F. Tissue dispersion and flow cytometry for the cellular analysis of wound healing. Biotechniques 2002;32:548—51. [25] 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—20. [26] Lichtenstein N, Geiger B, Kam Z. Quantitative analysis of cytoskeletal organization by digital fluorescent microscopy. Cytometry 2003;54A:8—18. [27] Grinnell F, Ho CH, Tamariz E, Lee DJ, Skuta G. Dendritic fibroblasts in three-dimensional collagen matrices. Mol Biol Cell 2003;14:384—95. [28] Lin Y-C, Grinnell F. Decreased level of PDGF-stimulated receptor autophosphorylation by fibroblasts in mechanically relaxed collagen matrices. J Cell Biol 1993;122:633—72. [29] Tian B, Lessan K, Kahm J, Kleidon J, Henke C. Beta 1 integrin regulates fibroblast viability during collagen matrix contraction through a phosphatidylinositol 3-kinase/Akt/protein kinase B signaling pathway. J Biol Chem 2002;277: 24667—75. [30] Xia H, Nho RS, Kahm J, Kleidon J, Henke CA. Focal adhesion kinase is upstream of phosphatidylinositol 3-kinase/Akt in regulating fibroblast survival in response to contraction of type I collagen matrices via a beta 1 integrin viability signaling pathway. J Biol Chem 2004;279:33024—34. [31] Brazil DP, Yang ZZ, Hemmings BA. Advances in protein kinase B signalling: AKTion on multiple fronts. Trends Biochem Sci 2004;29:233—42. [32] Rosenfeldt H, Grinnell F. Fibroblast quiescence and the disruption of ERK signaling in mechanically unloaded collagen matrices. J Biol Chem 2000;275:3088—92. [33] Rosenfeldt H, Lee DJ, Grinnell F. Increased c-fos mRNA expression by human fibroblasts contracting stressed collagen matrices. Mol Cell Biol 1998;18:2659—67. [34] Bossis G, Malnou CE, Farras R, Andermarcher E, Hipskind R, Rodriguez M, et al. Down-regulation of c-Fos/c-Jun AP-1 dimer activity by sumoylation. Mol Cell Biol 2005;25:6964—79.
www.intl.elsevierhealth.com/journals/jods
Modeling dermal granulation tissue with the linear fibroblast-populated collagen matrix: A comparison with the round matrix model Mark J. Eichler, Mark A. Carlson * Department of Surgery, University of Nebraska Medical Center and the Omaha VA Medical Center, 4101 Woolworth Avenue, Omaha, NE 68105, Nebraska, USA Received 15 July 2005; received in revised form 2 September 2005; accepted 13 September 2005
KEYWORDS Fibroblast-populated collagen matrix; Wound healing; Granulation tissue; Mechanical tension; Actin cytoskeleton
Summary Background: Wound contraction typically is not symmetrical; for example, a squareshaped wound will not yield a square scar. Interestingly, the round fibroblast-populated collagen matrix has been used as a model of wound contraction, even though contraction in this model is mostly symmetrical. Objective: We wanted to compare the round versus linear fibroblast-populated collagen matrix to see which would be a better model of dermal granulation tissue. Methods: Gross and microscopic morphology, contraction kinetics, cytoskeletal architecture, and apoptotic and proliferative indices were compared between the round versus the linear fibroblast-populated collagen matrix. A rat excisional wound model was used as an in vivo standard of healing. Results: The rate of contraction was similar between the two models, although the mode of contraction was grossly asymmetric in the linear while remaining symmetric in the round model. Cellular survival and proliferation were both dependent on matrix attachment in both models; this was analogous to the attachment-dependence of granulation tissue. In the attached (restrained) condition, the level of cellular organization was higher in the linear than in the round matrix; the tissue architecture of the linear matrix, moreover, mimicked that of the excisional wound model. Conclusion: The round versus linear fibroblast-populated collagen matrix displayed a similar proliferative and survival response to matrix attachment. The latter model, however, demonstrated tissue organization with attachment and asymmetrical contraction after detachment analogous to that of the in vivo wound model. The linear
Abbreviations: BrdU, 5-bromo-20 -deoxyuridine; FBS, fetal bovine serum; LFPCM, linear fibroblast-populated collagen matrix; PI, propidium iodide; RFPCM, round fibroblast-populated collagen matrix; TUNEL, terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling * Corresponding author. Tel.: +1 402 346 8800x5371; fax: +1 402 977 5672. E-mail address: [email protected] (M.A. Carlson). 0923-1811/$30.00 # 2005 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jdermsci.2005.09.002
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fibroblast-populated collagen matrix appears to be the better model of dermal granulation tissue. # 2005 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.
1. Introduction Mechanical forces regulate survival, proliferation, gene expression, and other processes in a number of cell types [1], including dermal fibroblasts in a three-dimensional collagen matrix [2,3]. The latter has been utilized as a model of granulation tissue in dermal wound healing [4]. Typically, the fibroblastpopulated collagen matrix (also known as lattice or gel) is pipeted into a culture well, which yields a disklike (i.e., round) configuration [5]. In a restrained (also known as attached) matrix, the subsequent fibroblast-dependent prestress that develops is fairly evenly distributed around the circular perimeter of the matrix. If the mechanical restraint is removed from such a matrix (i.e., it is detached from the culture well), then the early result is symmetrical contraction–—in other words, the matrix initially shrinks into a smaller circle. In actual excisional wounds, however, contraction typically is not a symmetrical process. For example, a square wound can contract in one direction and produce a linear scar [4,6]. Previous experimentation with a rat excisional wound model demonstrated that survival and proliferation of the cells within the granulation tissue was acutely dependent on attachment of its extracellular matrix to the dermal edge [7—9]. Thus, the dependence of cell fate on matrix attachment appears to be similar in the in vivo wound model versus the round collagen matrix [4]. Casual inspection of these two models, however, revealed that their gross and microscopic morphology was dissimilar. Considering the apparent morphologic disparity between the round fibroblast-populated collagen matrix and our in vivo wound model, we wanted to determine if an alternative (that is, a linear [10—12]) collagen matrix model would be a better morphologic approximation of granulation tissue. This concern for a better morphologic equivalent of granulation tissue stems from our interest in the regulation of cellular survival and proliferation in wound healing. As stated above, mechanical attachment of the extracellular matrix is a proven regulator of wound tissue; another potential regulator is cell shape. This has been shown to have crucial influence on cellular survival in vitro [3,13]. Experiments to dissect out cell shape—cell fate relation-
ships in granulation tissue presumably would be facilitated by a model that was a reasonable morphologic equivalent. So, our intent with the studies herein was to characterize the relationship of cell fate with respect to matrix attachment in the linear collagen matrix (not previously done), and to determine whether the linear matrix model was indeed a closer morphologic mimic of granulation tissue than the round matrix model.
2. Materials and methods 2.1. Reagents Vitrogen (bovine dermal collagen) was from Angiotech BioMaterials Corporation (formerly Cohesion Technologies). Anti-BrdU antibody was from Zymed. TUNEL kits (In Situ Cell Death Detection Kit, Fluorescein) were purchased from Roche. Alexa Fluor1 488 phalloidin was from Molecular Probes. Fetal bovine serum was from Invitrogen. All other reagents were purchased from Sigma or Life Technologies.
2.2. Cell culture The use of human fibroblasts in this research was approved by our Institutional Review Board. Primary dermal fibroblasts were cultured from neonatal foreskin specimens and maintained as previously described [14,15]. All experiments were performed on cells derived from one foreskin specimen; this strain of cells initially was expanded to passage 7, and then frozen down. To perform an experiment, a vial of these passage 7 cells was thawed, plated, and then expanded to passage 9; collagen matrices then were prepared from the passage 9 cells. The days of the week upon which thawing, splitting, freezing, etc. was performed was kept constant.
2.3. Collagen matrices The protocol for setting-up round collagen matrices has been described [14,15]. Round matrices were incubated for 72 h in the attached state prior to the initiation of an experiment. For the linear matrix model, a 900 ml aliquot of the same solution of cells (106 ml1) in bovine collagen (1.5 mg/ml) that was
Round vs. linear fibroblast-populated collagen matrix
used to generate round matrices was pipeted into the linear gel casting apparatus (not shown; details available upon request), and allowed to polymerize at 37 8C for 2 h. After polymerization, each linear matrix was carefully transferred into the incubation chamber (Fig. 2G); medium (10% FBS in DMEM with 1 mg/ml ascorbic acid) then was added to cover the matrices. Linear matrices were incubated for 24 h in the attached state prior to the initiation of an experiment.
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The excisional wounding model in the rat has been previously described [4,7].
2.7. Statistical analysis Numerical data are represented with the mean standard deviation. Unpaired groups of data were analyzed with analysis of variance and the unpaired t-test, with p = 0.05 as the level of significance.
2.4. Histologic analysis The techniques of TUNEL and BrdU immunohistochemistry on cytospin preparations of collagen matrices follows previous descriptions [9,14,15]. For the BrdU labeling, matrices were incubated with 10 mM BrdU for 4 h prior to the cytospin preparation.
2.5. Phalloidin labeling and directionality index (DI) The technique of phalloidin-labeling follows a previous description [16]. In the present work, intact collagen matrices were fixed in 3% paraformaldehyde in PBS for 20 min at 22 8C prior to staining with fluorescently labeled phalloidin. Matrices then were imaged with a Zeiss LSM 410 confocal laser scanning microscope. Subsequent, analysis was done with the directionality index, a softwarebased method which can quantify the overall directionality (i.e., polarity or orientation) of a digital image of complex biological structures. The index is calculated with a freeware program called ImageJ (http://rsb.info.nih.gov/ij/) with a Java plugin called SurfCharJ (http://home.online.no/gary. c/IJ/SurfCharJ.htm) [17]. The details and mathematical descriptors of the directionality index have been described in detail elsewhere [16]. As an example of the directionality index, consider a histologic section of striated muscle cut parallel to the fibers. Almost all of the fibers in such an image will be oriented along a single vector; such an image will yield an index of approaching one or zero, which indicates a highly polarized directionality. In contrast, consider a histologic section of liver cut in a random section. Although the liver is highly organized, the hepatocytes are oriented along multiple vectors. The directionality index in this case would approach 0.5, indicating very little polarization of directionality.
2.6. Excisional wound model The use of animals in this research was approved by our Institutional Animal Care and Use Committee.
Fig. 1 Morphology of wound contraction in a rat excisional model. (A) Overhead photographs of a 2 cm 2 cm excisional wound on the dorsum of a rat at the time of wounding (postwounding day 0) and 18 days later. Arrow = cephalad; scale is in cm. (B) H&E micrographs of granulation tissue from the rat excisional model (postwounding day 6), sectioned in the transverse vs. sagittal plane. The dotted line in each diagram indicates the orientation of the corresponding section; the open rectangle represents the wound (in the same orientation as shown in A). Arrows = direction of wound contraction; bar = 50 mm.
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3. Results 3.1. Polarization of granulation tissue cells in vivo during wound contraction It has been documented by others [6,18] and observed by us [4,7—9,16] that wound contraction in various excisional wound models is asymmetrical; for example, a 4 cm2 square-shaped incision on the upper dorsum of the rat contracted almost entirely along a transverse vector, which left the animal with a vertically oriented linear scar (Fig. 1A). In other words, a square-shaped wound did not yield a squareshaped scar. The pattern of wound contraction shown in Fig. 1 has been consistent in over 500 animals wounded in similar fashion in our laboratory. Examination of the microscopic morphology of the wound in Fig. 1A revealed a high degree of polarization of the cells in the granulation tissue (Fig. 1B). The predominant orientation of the cells (mostly fibroblasts, although a cell-specific marker was not employed) was parallel to the direction of wound contraction, as indicated by the transverse section in Fig. 1B. Cutting perpendicular to the direction of contraction (i.e., a sagittal section) resulted in most cells being cut across their long axis, which was consistent with the orientation shown in the transverse section.
3.2. Gross morphology and contractile properties of the round versus the linear fibroblast-populated collagen matrix The gross morphology and contractile properties of the round fibroblast-populated collagen matrix have been reviewed [5,19], but they are reiterated here for the sake of comparison. Primary dermal fibroblasts were mixed with soluble collagen and pipeted into a culture well. The collagen solution underwent polymerization and formed a gel-like material (also known as collagen matrix or lattice); a lateral view of a freshly polymerized matrix is shown in Fig. 2A. Fig. 2 Gross morphology of round vs. linear fibroblastpopulated collagen matrices. (A) Round attached matrix, 1 h after pipeting into culture well (i.e., just prior to adding medium); edge view (postfixation). (B) Round attached matrix, 3 h after pipeting into culture well (i.e., 2 h after addition of serum-supplemented medium); bar = 2 mm. (C) Round attached matrix after 3 days of incubation with serum-supplemented medium; top view (postfixation). (D) Round matrix of (C), 1 h after mechanical release from the culture dish; bar = 2 mm. (E) Plot of attached matrix contraction (i.e., decrease in matrix height) which was shown in (A and B). Each data point in this and subsequent plots represents the mean S.D. of four measurements. (F) Plot of stressed round matrix contraction (i.e., decrease in matrix diameter) which was shown in (C and D). (G) Linear gel incubation chamber (see text for dimensions).
(H) Pylon apparatus (top) shown with temporary matrix support fitting (bottom). (I) Linear fibroblast-populated collagen matrix (nonfixed) in the pylon apparatus after polymerization and prior to immersion in medium; matrix is suspended between two tabs of Velcro1. (J) Three linear matrices shown incubating in medium within the chamber; extent of one matrix is marked with a bracket. (K) Linear matrix fixed in the attached state after 48 h of incubation in serum-supplemented medium; matrix is stretched between the two Velcro1 tabs. (L) Linear matrix similar to (K), but mechanically released from one Velcro1 tab and incubated for an additional hour (bar = 5 mm); arrowhead = contracted matrix. (M) Plot of stressed linear matrix contraction (i.e., decrease in matrix length) which was shown in (K and L).
Round vs. linear fibroblast-populated collagen matrix
In the presence of serum, the fibroblasts reorganized the collagen fibers, and the matrix contracted in the vertical direction (compaction), as shown by the interval photograph in Fig. 2B. A time course of vertical contraction is plotted in Fig. 2E; this compaction was cell-dependent, and most of it occurred within several hours after application of medium. If a compacted, prestressed matrix was physically detached (released) from the tissue culture plastic, then lateral contraction ensued (Fig. 2C and D) until the matrix area reached a steady state at 10—20% of the prerelease area; most of this contraction occurred in the first hour and also was cell-dependent (see plot in Fig. 2F). The incubation chamber for the linear fibroblastpopulated collagen matrix was constructed of methyl methacrylate polymer (Lucite1) and consisted of a 5 cm (height) 7 cm 7 cm rectangular well with tracks cut into the bottom for holding the pylon apparatus (Fig. 2G). The latter (Fig. 2H) consisted of inverted-U-shaped section of Lucite; two tabs of Velcro1 were glued on opposing sides of the pylons. There also was an associated interlocking section of Lucite (lower portion of Fig. 2H), which was used to support the collagen matrix during the polymerization phase, and then was removed. A freshly polymerized linear fibroblast-populated collagen matrix is shown suspended between the two Velcro anchorage points in Fig. 2I. The pylon apparatus containing the polymerized matrix was placed into the well, medium was added, and compaction analogous to that of the round matrix (see Fig. 2A and B) ensued. Instead of flattening into a thin disk, however, the linear matrix underwent compaction along its midsection, resulting in a slight ‘‘hourglass’’ [12] appearance (Fig. 2J). The development of mechanical tension (prestress) in the long axis of the linear matrix was confirmed by releasing one end of the matrix from its pylon (Fig. 2K and L), which resulted in matrix shortening (plotted in Fig. 2M) analogous to the release induced contraction of the round matrix (Fig. 2C and D).
3.3. Microscopic morphology of the round versus linear fibroblast-populated collagen matrix The microscopic morphology of the round fibroblastpopulated collagen matrix has been described [20], but is shown here for the sake of comparison. Fibroblasts in the attached round matrix had an elongated, bipolar morphology, and were parallel to the plane of the matrix (i.e., in the plane of mechanical tension), as illustrated by sectioning parallel and perpendicular to the plane of the matrix (Fig. 3A and B). The orientation of the cells within the plane of
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the round matrix appeared to be random (Fig. 3A), indicating that the tension was distributed in randomly oriented 3608 vectors. This conclusion was consistent with the relatively uniform shape that the round matrix maintained during contraction (see Fig. 2C and D above). In contrast, the fibroblasts in the attached linear matrix were aligned parallel to the long axis of the matrix, as shown by longitudinal and transverse sections (Fig. 3C and D). One day after release, however, the fibroblasts in both the round and linear matrix became more rounded and lost their orientation (Fig. 3E and F); there was not much difference in microscopic morphology in the round versus linear matrix after release. The microscopic characteristics apparent from the H&E histology were reinforced by fluorescent staining of the actin cytoskeleton of cells within the linear matrix (Fig. 4). The actin fibers within the attached linear matrix (Fig. 4A and B) were aligned along the long axis of the matrix (i.e., parallel to the vector of mechanical tension). One day after release of the linear matrix (Fig. 4C and D), the alignment was lost and the cells adopted a dendritic appearance [21].
3.4. Quantification of cellular orientation in the fibroblast-populated collagen matrix The subjective morphometric differences apparent between the micrographs of the round versus linear matrix in Fig. 3 may be quantified with the directionality index [16]. This index converts a digital image into an array of ‘‘facets.’’ The direction of each facet can be graphed in a polar plot, such as shown in Fig. 5B—D. The plot in Fig. 5B, for example, was derived from the facet transformation of the phalloidin image in Fig. 4A. The relative polarity of the plot in Fig. 5B (i.e., the directionality index) can be calculated by measuring the area bounded by the plot which falls into defined sectors [16]. For highly polarized images, the directionality index will approach 1 or 0; for nonpolarized images, the index will approach 0.5. As expected, the directionality index for the attached linear matrix was close to 1 (Fig. 5B and E), while the index for the released linear matrix was closer to 0.5 (Fig. 5C and E). For comparison, the directionality index for the attached round matrix (Fig. 5A) also was close to 0.5 (Fig. 5D and E). This latter finding indicates that there was little polarization (i.e., no cell alignment) within the plane of the attached round matrix, even though the individual cells within this matrix were highly polarized. The directionality index also can be calculated on individual cells, as shown in Fig. 5F and G. The images of attached (polarized) versus
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Fig. 3 Microscopic morphology of round vs. linear fibroblast-populated collagen matrices; H&E stained paraffin sections. (A) Round attached matrix, sectioned in the frontal plane. Inset in (A—D) illustrates the histologic plane; bar = 50 mm (same for A, C, E, and F). (B) Round attached matrix, sectioned in the transverse plane; bar = 10 mm. (C) Linear attached matrix, sectioned in the frontal plane. (D) Linear attached matrix, sectioned in the transverse plane; bar = 20 mm. (E) Round released matrix, sectioned in random plane. (F) Linear released matrix, sectioned in random plane.
released (dendritic) cells in these figure panels were extracted from Fig. 4B and D, respectively. The resultant value of the directionality index reflected the relative polarization of each cell.
3.5. Proliferative and apoptotic indices in the round versus linear fibroblastpopulated collagen matrix Previous work demonstrated that attachment of the fibroblast-populated collagen matrix promoted survival and proliferation of the embedded cells, while detachment (also known as release or stress-relaxation) induced apoptosis and quiescence [15,22,23]. After a comparison of the gross and microscopic
morphology of the round versus linear matrix models, we wanted to determine if the cell cycle and survival response to matrix anchorage was similar in the round versus linear matrix. To perform this comparison, cytospin preparations of cells isolated after enzymatic degradation of the matrix underwent either TUNEL or BrdU immunohistochemistry; an example of the latter is shown in Fig. 6A. As expected, the BrdU-labeling index was greater (by about one order of magnitude) in the attached round matrix compared to the released round matrix (Fig. 6B), which confirmed that matrix attachment supports fibroblast proliferation. Matrix attachment also supported fibroblast proliferation in the linear matrix model (Fig. 6B). Consistent with
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Fig. 4 Confocal microscopic images of intact fibroblast-populated collagen matrices stained with fluorescently labeled phalloidin and propidium iodide. (A) Attached matrix; bar = 100 mm. Fluorescent images in this and subsequent panels represent phalloidin (PI) and merged signal. Double-headed arrow = orientation of poles (i.e., direction of force generation). (B) Higher power view of the attached matrix; bar = 20 mm. (C) Released matrix; same scale as (A). (D) Higher power view of the released matrix; same scale as (B). Arrowheads: individual cells used for analysis in Fig. 5F and G.
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published results [15,22,23], matrix detachment induced apoptosis in the round matrix (Fig. 6C); the TUNEL-positivity rate was about an order of magnitude greater in the released compared to the attached condition. Similar to the round matrix model, matrix detachment in the linear model produced a marked increase in the apoptotic index (Fig. 6C).
3.6. Alternative models of the linear fibroblast-populated collagen matrix During development of the linear fibroblast-populated collagen matrix model technical problems arose with fragility of the system. The collagen polymerization phase for the linear matrix required a watertight casting mold (not shown) in which the pylon apparatus and the associated support (Fig. 2H) temporarily were contained. The solubilized collagen was pipeted into this mold, polymerization ensued, and the pylon apparatus then was transferred to the Lucite incubation well (Fig. 2G). Unfortunately, the collagen solution had a propensity to leak out of the mold prior to polymerization. In addition, because of the relatively small surface area of attachment to the Velcro tabs, the attached collagen matrices had a propensity to detach prematurely from either pylon; incubating these gels beyond 72—96 h was difficult because of this premature detachment. The multiple manipulations required for the matrix set-up also increased the likelihood of bacterial contamination. In general, less than half of a given group of attached linear matrices would survive intact beyond 72 h. In an effort to make the linear matrix model more robust, an alternative ‘‘square-well’’ system was devised (Fig. 7A), in which two opposing sides of the well were lined with Velcro. This system is analogous to that of a commercially available linear matrix casting system (Tissue TrainTM Culture System; Flexcell International Inc., Hillsborough, NC), except that the system in Fig. 7 provided a wide base of matrix attachment at each pole, and also allowed a greater matrix volume (1000 ml versus 225 ml, [12]).
Fig. 5 Directionality analysis of actin cytoskeleton in the fibroblast-populated collagen matrix. (A). Section of attached round matrix taken in the frontal plane (see Fig. 3A), stained with phalloidin-AF488, and imaged with conventional fluorescent microscopy. Bar = 50 mm. (B) Azimuthal plot of attached linear matrix after facet transformation [16] of the digital image in the left panel of Fig. 4A. The directionality index (DI) [16] derived from
this plot also is shown. (C) Azimuthal plot with DI for the image of the linear released matrix in the left panel of Fig. 4C. (D) Azimuthal plot with DI for the image of the round attached matrix in Fig. 5A. (E) Bar blot of mean DI S.D. for linear attached vs. linear released vs. round attached matrix (four microscopic fields analyzed per condition); *p < 0.001 compared to linear attached, ANOVA, and unpaired t-test. (F) An individual cell from the attached vs. released matrix, extracted from Fig. 4B and D (see arrowheads therein). (G) Azimuthal plots with the derived DI of the images in (F).
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Fig. 7 Alternative linear matrix models. (A) Square well, overhead, and oblique views; scale in cm. (B) Overhead view of collagen matrix in a square well after 96 h incubation; bar = 5 mm. (C) Linear matrix with mobile poles after 24 h incubation; bar = 5 mm. (D) Linear matrix with mobile poles after 96 h incubation.
Fig. 6 Proliferation and cell death data for the round vs. linear fibroblast-populated collagen matrix. (A) Cytospin preparations of attached vs. released round collagen matrices (labeled with 10 mM BrdU for 4 h prior to processing) underwent BrdU immunocytochemistry with PI counterstaining. (B) BrdU incorporation data (mean S.D.) of attached vs. released matrices from the round and linear models. Each mean was calculated from 4 images, an example of which was shown in (A); *p < 0.05 compared to attached, unpaired t-test. Each experiment was performed a minimum of three times. (C) Same as (B), except displaying TUNEL data.
The tight-fitting seams of the square-well system in Fig. 7 did not allow leakage of the soluble collagen that occurred with the linear apparatus of Fig. 2. The square apparatus also did not require matrix transfer into another incubation chamber; the square well was placed into a 35 mm culture dish, and remained there for the duration of an experiment. The simple set-up of the square matrix decreased the amount of manipulation and subsequent contamination risk. Furthermore, the increased surface area of attachment (Fig. 7B) eliminated the premature detachment problem; matrices could be incubated in the attached state for >96 h without difficulty. Examination of microscopic morphology confirmed that the cellular alignment of the square-well linear matrix model was similar to that shown in Fig. 2 (data not shown); i.e., the cells were aligned along the axis that ran between the two Velcro poles. We also devised the ‘‘mobile pole’’ linear matrix (Fig. 7C and D) in order to simulate the dynamics of the excisional wound model in Fig. 1. This model variant had a similar configuration and set-up procedure to the linear apparatus shown in Fig. 2, except that the pylons could move freely and independently within the tracking groove of the incubation well (Fig. 2G). As the matrix contracted, it would pull the pylons closer together, analogous to the dermal edges of a contracting wound. The contraction demonstrated in Fig. 7C and D occurred over a 3 days period.
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4. Discussion An advantage of the linear fibroblast-populated collagen matrix model [10—12] was that it provided a three-dimensional model of fibroblast—collagen interactions in which there was uniformity of cellular orientation. This uniformity should facilitate the study of cell survival, proliferation, gene expression, etc. with respect to cell shape and mechanical loading. An advantage to the particular model shown in Fig. 2I, compared to other linear matrix systems, is that it was a true two-pole system; the matrix in Fig. 2I made no physical contact with a solid substrate other than at the Velcro-coated pylons. These two small attachment points minimized the confounding contribution of the broad attachment surface that characterized the round matrix model. This arrangement made the linear matrix a more ‘‘pure’’ model of mechanical loading compared to the round matrix; that is, the force vectors within the former were defined by one axis, not two. As expected, the survival and proliferative response of the fibroblasts with respect to matrix attachment were similar in the linear versus the round matrix model (see Fig. 6). This is reassuring evidence that fibroblast biology in the linear matrix is similar to that in the round matrix. The disadvantages of the linear matrix model depicted in Fig. 1 were (1) it was somewhat cumbersome to set-up compared to the round model; (2) the freshly pipeted collagen solution tended to leak out of the matrix mold prior to polymerization; (3) repeated manipulation increased the risk of bacterial contamination of the apparatus; (4) the relatively small Velcro anchorage points were insufficient to restrain the matrix for extended periods. These concerns were addressed by the employment of a ‘‘square-well’’ linear matrix system shown in Fig. 7A. The main disadvantage to the square-well apparatus was that it was not a pure two-pole system like that in Fig. 2I. The matrix in a square well not only was in contact with the Velcro anchorage points, but it sat on the Lucite floor of the well and touched the two walls that were not covered with Velcro. In practice, this may not be much of a concern, however, because the matrix did not appear to adhere well to the Lucite. The attached matrix in the square well (Fig. 7B) attained the ‘‘wasp-waist’’ or ‘‘hourglass’’ appearance of pylon linear matrix, indicating that lateral contact of the matrix with the bare Lucite did not have much influence on matrix morphology. Previous investigators have described linear-type fibroblast-populated collagen matrix models which developed prestress between two poles; the intent of these studies was to quantify mechanical force generated or to study the effect of extrinsic force on fibroblast biology [10,11]. Our intent with a linear-
M.J. Eichler, M.A. Carlson
type matrix model was to develop a model to study the relationships between cell shape, mechanical loading, and the biological fate/function of individual cells in a three-dimensional culture system. Recent evidence has indicated that cell shape and mechanical loading are important regulators of cell fate and function [3,13]. In order to study these entities, we believed that it would be helpful to have a model which approximated the polarized morphology of the in vivo excisional wound tissue. The best approximation of wound contraction in this study may have been the mobile pole model shown in Fig. 7C and D. This model combined the polarity of fixed pylon model of Fig. 2I with the dynamic characteristics of a contracting wound. The conventional restrained fibroblast-populated collagen matrix model also will contract when released [5], but this contraction occurs over minutes to hours, not days to weeks that typify in vivo wound contraction. Unrestrained collagen matrices can be set-up to contract over a period of days, but only by decreasing the cellular density to a level, that is, several orders of magnitude less than that typically found in granulation tissue [2,24]. It is debatable how faithful such models are to in vivo wound contraction. The mobile pole system (Fig. 7C and D), however, uses a relatively high concentration of fibroblasts (though still lower than what exists in vivo [24]) in a matrix that contracts over a period of days to a week, depending on the force of resistance provided by the anchoring blocks. In our preliminary investigation, however, this model has been somewhat difficult to utilize because of variability in the degree and speed of contraction. The mobile pole model will require further refinement. Software-aided morphometric analysis of the cytoskeleton has been described [25,26]. In the present paper, a measure of cytoskeletal polarity (the directionality index [16]) was used to demonstrate that cytoskeletal organization of both single cells and cell clusters could be quantified. The directionality index showed that the cytoskeletal organization was greater in the attached linear versus the released linear matrix, which confirmed the visual impression (Figs. 4 and 5). Interestingly, while the individual cells in the attached round matrix were highly polarized, the directionality index of this matrix did not indicate polarization at the tissue level (Fig. 5). If the attached round matrix was sectioned in the plane of mechanical tension (Fig. 3A), then the elongated fibroblasts had a fairly random orientation, indicating dispersal of mechanical tension in a 3608 arc around the matrix perimeter. This is in contrast to the attached linear matrix, which when sectioned in a plane parallel to the axis of mechanical tension (Fig. 3C), demonstrated high polarity at the tissue
Round vs. linear fibroblast-populated collagen matrix
level, i.e., nearly all the elongated fibroblasts were parallel to the direction of tension. The ability to quantify organization at both the cell and tissue level in the linear matrix model should enable future study of cell shape—cell fate relationships in the threedimensional matrix. The elongated, polarized morphology of fibroblasts in a mechanically restrained (attached) collagen matrix has been associated with promotion of survival and the cell cycle [4]. In contrast, the ‘‘rounded’’ morphology (as described with H&E staining of paraffin sections) has been associated with induction of apoptosis and quiescence. In situ imaging of the cytoskeleton in the fibroblast-populated collagen matrix) with confocal microscopy (as was done in Fig. 4), however, revealed that this ‘‘rounded’’ morphology actually was dendritic [27]. This morphologic switch (from elongate to dendritic) that occurred with the transition from mechanically attached to release was not associated with universal cell death or quiescence. In other words, detachment of a mechanically restrained matrix induced cell death in some, but not all, of the indwelling fibroblasts (see Fig. 6). Conversely, there was ongoing, albeit low, apoptotic activity in the mechanically restrained matrix. There was an analogous situation with BrdU labeling; that is, not all cells in a mechanically restrained matrix were cycling, and not all cells in a detached matrix became quiescent. The mechanical state of the collagen matrix apparently plays a permissive role in the outcome of fibroblast survival and proliferation. There likely are other factors, such as responsiveness to growth factors [28] that influence cell fate in the fibroblast-populated collagen matrix. Growth factors were constantly present in the models of this manuscript, as the incubation medium was supplemented with 10% FBS. It has been shown that the activity of growth factor receptors in fibroblasts is influenced by the mechanical state of the matrix in the round collagen matrix model [28]. Previous investigation with the round fibroblastpopulated collagen matrix model has demonstrated differences in signal transduction between the attached (also known as stressed, or anchored, or restrained) versus the detached (also know as stressreleased) state. The phosphatidylinositol 3-kinase/ Akt pathway in attached matrices is upregulated via a b1-integrin/focal adhesion kinase-dependent mechanism [14,29,30]. The PI-3-kinase/Akt pathway has been implicated in promoting survival and cell cycling in a range of cell types [31]. In addition, the extracellular-regulated kinase/mitogen activated protein kinase pathway (Erk/MAPK) is activated in attached matrices, providing another signal for DNA synthesis [32]. Release of stressed matrices results in
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an early induction of c-fos [33], which, as a component of the AP-1 transcription complex, is a prototypical responder to cellular stress that regulates proliferation and survival [34]. cDNA microarray analysis of mechanically stressed versus unstressed fibroblasts in a collagen matrix demonstrated upregulation of 60 known genes in the former and 64 in the latter. Some of the upregulated genes in stressed matrices included cyclin D3, VEGF, plasminogen activator inhibitors, TIMPs, collagen a1, g-tubulin, asmooth muscle actin, vinculin, and integrin-linked kinase [3]. It was concluded that stressed fibroblasts had a synthetic phenotype compared to their unstressed counterparts. The signal transduction differences in attached versus stress-released linear matrices have yet to be investigated, but considering the similarity in mechano-regulated proliferation and survival in the round versus linear models, it might be assumed that the mechanotransduction pathways also are similar. The linear fibroblast-populated collagen matrix model provides an environment in which there is relative uniformity of cellular alignment along an axis of mechanical tension. This characteristic imitates what occurs with in vivo excisional healing, as was shown in Fig. 1. For reasons which currently are not well understood, some (if not most) wounds will have a predominant axis of contraction, resulting in alignment of the cells within the granulation tissue (i.e., an enhanced level of tissue organization). This axis of contraction is reproduced by the linear matrix model. Moreover, the attached versus released linear matrix model represent opposite extremes for a number of descriptors, including cellular morphology, tissue organization, cellular survival and proliferation, and so on. The extent of the biologic differences between the attached versus released condition should facilitate the study of these differences. Using methods to augment or disrupt signaling pathways involved with survival and/or proliferation, and then studying the effects on the cytoskeleton (and also by performing the inverse) should uncover dependencies of cell shape and cell fate. This in turn should lead to avenues to explore regulation of the healing response.
Acknowledgements The authors would like to acknowledge the technical assistance of Chris Hansen and Amy Prall. Supported by a grant from the NIH (K08 GM00703) to MAC. MJE was supported by a Summer Research Stipend from the University of Nebraska College of Medicine. The authors also would like to thank Janice Taylor of the
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Confocal Laser Scanning Microscope Core Facility at the University of Nebraska Medical Center, which is supported by the Nebraska Research Initiative, for providing assistance with confocal microscopy.
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