Ann Thorac Surg 2003;75:1450-1456
© 2003 The Society of Thoracic Surgeons
Original article: cardiovascular
Development of abnormal tissue architecture in transplanted neonatal rat myocytes
Peter Whittaker, PhDa*,
Jochen Müller-Ehmsen, MDb,
Joan S. Dow, BSc,
Larry H. Kedes, MDd,
Robert A. Kloner, MD, PhDc,e
a Department of Emergency Medicine, University of Massachusetts Medical School, Worcester, Massachusetts, USA
b Department of Internal Medicine, University of Cologne, Cologne, Germany
c Heart Institute, Good Samaritan Hospital, Los Angeles, California, USA
d Institute for Genetic Medicine Los Angeles, CA, USA
e Department of Medicine, University of Southern California, Los Angeles, California, USA
Accepted for publication December 4, 2002.
* Address reprint requests to Dr Whittaker, University of Massachusetts Medical School, Department of Emergency Medicine, 55 Lake Avenue North, Worcester, MA 01655, USA
e-mail: peter.whittaker{at}umassmed.edu
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Abstract
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BACKGROUND: Most myocardial cell transplant studies focus on demonstration of improved function; however, such improvement depends on the development of appropriate tissue structure. Thus, our aim was to assess the architectural changes that occurred after cell transplant into normal and infarcted myocardium.
METHODS: Male neonatal cells (1 to 2 days old) were injected into the left ventricular free wall of adult female rats. The tissue was examined 0 to 1 days and 1 to 2, 4 to 6, and 12 weeks later in noninfarcted hearts and 6 months after transplant into infarcts. In histologic sections, we assessed the cells’ retardation of polarized light (to measure development of contractile elements), two-dimensional cell orientation, cell nuclear morphology, and collagen content.
RESULTS: The transplant cells’ retardation of polarized light gradually increased to 81% of that of host cells after 6 months (p < 0.001). The transplant cells were disorganized and although their nuclei increased in size, they always had a rounded appearance. Collagen content in the transplant was 210% to 430% higher than in host tissue (p < 0.01). In addition, scar collagen always separated transplant and host cells.
CONCLUSIONS: One architectural feature, the rounded nuclei, provided a distinctive marker to identify transplanted cells. Nevertheless, the transplants’ inhibited muscle development together with disorganization, separation from the host muscle, and a substantial increase in collagen resulted in a structure unlikely to play an active role in systolic function.
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Introduction
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The focus of most myocardial cell transplant studies has been the demonstration of improved function [1–3]. Although functional enhancement is the ultimate goal, the intimate association between structure and function mandates the documentation of a structure compatible with improvement in contractile performance. If transplanted cells are to contribute to active myocardial function, there are at least two architecture-related goals that must be achieved: (1) differentiation and development from the stem, fetal, or neonatal cell types used to become full-sized adult cells; and (2) the organization of transplanted cells into a structure duplicating, or at least similar to, that which existed originally [4]. Thus, in this retrospective analysis, we sought to assess the architectural changes that occurred in transplanted tissue and focused on cell development and organization.
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Material and methods
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We examined tissue from two studies, described elsewhere [5, 6], that enabled us to follow the time-course of transplant tissue development. These experiments were approved by the hospital’s Animal Care and Use Committee and were performed in accordance with the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (National Institutes of Health publication 85 to 223, revised 1985). Male neonatal cells (1 to 2 days old; 0.8 to 5.0 x 106 determined by the yield after isolation) were injected into the left ventricular free wall of adult female hearts (Fischer 344 rats). In one study, the recipient hearts were normal and the tissue was examined 0 to 1 days (n = 2), and 1 to 2 weeks (n = 5), 4 to 6 weeks (n = 5), and 12 weeks (n = 2) after transplantation. In the second study, using the same preparation methods, cells (n = 10) or culture medium (n = 10) were injected into infarcted myocardium 1-week after permanent coronary artery occlusion and the tissue examined 6-months later.
We measured the following structural variables in both the transplant and host tissue: (1) the cells’ retardation of polarized light (to assess the development of contractile elements within the cells); (2) two-dimensional cell orientation; (3) cell nuclear morphology; and (4) collagen content.
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Retardation of polarized light
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Retardation (
) is described by the equation, = d (ne - no), where d is sample thickness, the term (ne - no) is the birefringence, and ne and no are the refractive indices of the orthogonal components into which linearly polarized light resolves on passing through anisotropic materials [7]. This variable provides information on the cell’s structure; for example, thermal injury decreased retardation by denaturizing contractile elements resulting in a reduction of (ne - no) [8]. In contrast, addition of calcium increased retardation in biopsy sections because cell contraction increased the thickness (d) of contractile elements within the section [9]. We measured retardation in cells from picrosirius red-stained sections by de Sénarmont’s method [7–9]. The cells were viewed with monochromatic light (
= 546 nm), aligned at 45 degrees to the transmission axes of the polarizing filters and a quarter-waveplate compensator inserted below the upper polarizing filter, which was then rotated until the cells appeared dark. The retardation (), measured in nanometers, was calculated from the formula; = (rotation angle x )/180 [8, 9]. Ten retardation measurements were made in longitudinally sectioned transplant cells and in host tissue. To eliminate the potential influence of staining variation (sections from the two studies were stained at different times), we expressed the average transplant cell retardation as a percent of that of host cells. This relative retardation will increase with transplant cell development if there is an increase in contractile material.
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Cell orientation
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We used the muscle’s optical properties to measure two-dimensional orientation [10, 11]. When viewed with linearly polarized light, birefringent materials appear bright except when their optic axis is parallel to the transmission axis of the microscope’s polarizing filters at which angle they are dark (the extinction angle). Muscle’s optic axis is parallel to the cell’s long axis [7]. Therefore, the extinction angle corresponds to the cell’s orientation. We made 50 orientation measurements on longitudinally sectioned cells in both the transplant and host tissue (mid-myocardial layer) of each sample from picrosirius red-stained sections. Orientation data analysis was performed using methods developed to assess periodic data [12]. For each region, the mean orientation angle was calculated relative to the tangent at the measurement site (the circumferential direction). We also calculated the angular deviation (the equivalent of standard deviation) of each distribution; the greater the alignment, the smaller the angular deviation.
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Nuclear morphology
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Qualitative examination of transplant cells indicated that their nuclei appeared round. Therefore, we assessed nuclear morphology in the transplant, in host tissue, in the infarct of hearts that did not receive a transplant, and in sham-operated hearts. Images obtained at 40x magnification from hematoxylin–eosin stained sections were displayed on a monitor and planimetry was used to trace nuclear profile. For each region, we analyzed 
25 nuclei and calculated the average area, perimeter length, and shape, which was assessed using a form factor, FF = 4
A/P2, where A is the area and p the perimeter. For a circular nucleus, p = 2r, A = r2, and FF = 1. At the other extreme, a long, thin nucleus has a large perimeter and FF tends to zero.
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Collagen content
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Collagen content was measured within the transplant and in host myocardium from picrosirius red-stained sections [13]. A monochrome, blue-filtered brightfield image (consisting of dark collagen fibers and bright muscle) was digitally subtracted from a circularly polarized monochrome image of the same field (consisting of bright collagen and bright muscle). To ensure that muscle was eliminated, its brightness in the blue-filtered image was adjusted (by manipulation of the lamp voltage) so that it exceeded the brightness in the polarized image. This subtraction removed muscle and interstitial space to yield an image of gray collagen on a black background. Thus, collagen content was determined as the number of gray pixels and expressed as a percent of the total pixels in the field. Three fields (total area 
36,000 µm2) were analyzed for each region in each sample.
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Statistics
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Statistical analysis of the two protocols was done separately. For noninfarcted hearts, comparisons between transplant and host tissue and between different transplant durations were performed by ANOVA followed by pair-wise comparisons using Tukey’s test. For infarcted hearts, comparison between transplant and host tissue were performed by t-tests. All values are expressed as mean ± standard error and were considered to differ significantly if p value was less than 0.05.
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Results
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The location of cells injected into normal myocardium was identified by a band of fibrosis (a result of the injection-associated injury) completely surrounding the transplant (Fig 1B),
whereas cells injected into infarcts were identified by regions of substantially increased wall thickness and by the presence of muscle cells outside of the subendocardial regions where host cells can sometimes survive
(see Figs 1 and 2 in Müller-Ehmsen and coworkers [6]). Although transplanted cells had well-defined cross-striations at 2 weeks (Fig 1B), the cells were smaller than host cells. Cell size increased at 6 months; however, the lack of coherent alignment and the presence of extensive fibrosis were revealed when the tissue was observed with polarized light (Fig 1).
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Fig 1. Micrographs of picrosirius red-stained sections viewed with circularly polarized light. Muscle cells appear green with cross-striations and collagen fibers appear green, yellow, or orange depending upon their thickness; myocardium distant from the infarct at 6 months (A), transplant tissue at (B) 2 weeks and (C) 6 months. In host tissue muscle cells are highly aligned, and the relatively small amount of collagen present is predominantly aligned parallel to the muscle. Although transplanted cells developed cross-striations after 2 weeks, they appeared small and disorganized. The collagen at the top and bottom of figure B is scar tissue associated with the injection. The transplant (central region) contains thinner collagen fibers. At 6 months, the cells and collagen fibers have increased in size; however, both lack the host tissue’s coherent organization. (Bar = 20 µm in all panels.)
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Fig 2. Retardation of transplant cells expressed as a percent of the host cells’ retardation. Although there was a progressive increase, transplant cell retardation never attained that of host cells.
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Retardation of polarized light
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The transplant cells’ relative retardation increased progressively (Fig 2), indicating an increase in the amount of contractile material. Nevertheless, at 6 months, transplant cell retardation remained less than that of host cells, 8.7 ± 0.3 nm versus 10.8 ± 0.3 nm (p < 0.0001; relative retardation, 81%).
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Cell orientation
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Figure 3
depicts the average angular deviation of the orientation distributions for each group (because transplant cells at 0 to 1 days were round, they have no specific orientation and were excluded from analysis). In host tissue, the cell orientation distributions had small angular deviations, consistent with muscle’s normal coherent alignment (Fig 1A). In contrast, the transplant cell orientation distributions had larger angular deviations (p < 0.05 vs host), consistent with disarray (Figs 1B and 1C). Furthermore, the mean transplant cell orientation in infarcts was significantly different from circumferential (difference 14 degrees ± 4 degrees; p < 0.05), whereas mean orientation in midmyocardial host cells did not differ from circumferential (3 degrees ± 1 degree).
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Fig 3. Average angular deviation of muscle cell orientation distributions obtained from transplant and mid-myocardial host tissue. Distributions from transplant cells had higher angular deviations than host cells (p < 0.05), an indicator of disarray. The 12 week group was excluded from statistical analysis because of the small sample size.
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Nuclear morphology
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We reported previously an increase in nuclear area from 20 µm2 at 0 to 1 days to 35 µm2 at 1 to 12 weeks, which was significantly less than the 50 µm2 in host cells [5]. In these samples, the transplant cell nuclear form factor decreased slightly from 0.88 ± 0.01 at 0 to 1 days to 0.83 ± 0.01 at 12 weeks (Fig 4),
which indicated that although transplant nuclei enlarged they maintained a round shape (Fig 5).
In host cells, form factors ranged from 0.55 ± 0.01 to 0.49 ± 0.01 (Fig 4), consistent with elongated nuclear profiles (Fig 5).
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Fig 4. Form factors for transplant and host cell nuclei. The transplant cells’ form factors were greater than that of host cells (p < 0.001), indicating round versus elongated profiles.
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Fig 5. Micrographs illustrating cell nuclear shape differences (hematoxylin & eosin staining). (A) Cells from a sham-operated heart reveal archetypal elongated nuclei (white arrows). (B and C) Cells 6 months after transplant into an infarct have rounded nuclei (white arrows). Fibroblasts (white arrowheads) are also present. (Bar = 20 µm in all panels.)
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Six months in infarcted hearts did not significantly change form factor values (Fig 4); however, nuclear changes did occur. The transplant cell nuclear area increased (45 ± 1 µm2) and no longer differed statistically from that of sham hearts (51 ± 2 µm2); however, smaller perimeter lengths relative to host cells (26 ± 1 µm vs 41 ± 1 µm; p < 0.001) maintained higher form factor values.
We also examined cell nuclear profile within control infarcts. The nuclei of these surviving subendocardial cells were rounded (FF = 0.84 ± 0.01); however, they had smaller areas (20 ± 1 µm2) and perimeters (17 ± 1 µm) than transplanted cells (p < 0.0001), consistent with oblique sectioning.
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Collagen content
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Collagen content was much higher within the transplant (p < 0.01) versus host tissue (Figs 1 and 6).
However, because the host tissue’s collagen content had, as would be expected 6 months after myocardial infarction, also increased versus the 0 to 1 day group (8.7% ± 0.6% vs 4.4% ± 0.2%) the actual increase within the infarct transplant versus normal tissue was 424%. In addition, the fibers were less coherently aligned in the transplant than in host tissue (Fig 1).
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Fig 6. Collagen content expressed as a percent of area analyzed. No value was calculated for the 0 to 1 day transplant period. Collagen content was always higher in the transplant than host tissue (p < 0.01; 12 week group excluded from statistical analysis).
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Comment
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Transplanted neonatal cells develop from a small and rounded appearance into elongated, cross-striated myocytes [5]. In the current study, we extended our initial observation to an evaluation of the developmental time-course and an examination of the transplant’s structural organization. Although there was evidence of progressive development and organization, we found that the tissue architecture remained abnormal. Specifically, transplanted cells lacked the coherent organization of normal muscle, the cells’ size and density of contractile material was less than normal, and their nuclei retained a rounded appearance. Furthermore, the transplanted cells were located in a highly fibrotic environment. These architectural features were found after transplantation into both normal and infarcted tissue; however, in this retrospective study, we did not determine whether or not the status of the host tissue influenced outcome.
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Retardation of polarized light
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We attribute the transplant cells’ lower relative retardation, even after 6 months, to the presence of less contractile material than normal. However, whether this indicates very slow development or that transplanted cells never fully develop is unknown. The transplant’s isolation may restrict access to endogenous signaling mechanisms or growth factors required for development. Two additional factors could contribute to lower retardation. A birefringent material’s retardation also depends upon three-dimensional orientation; the maximum occurs when the material is longitudinally sectioned and deviation from this plane decreases retardation [8]. It is also possible that lower relative retardation was the result of host cell hypertrophy. However, we do not believe that these factors played a role because we avoided obliquely sectioned cells and because there was no increase in septal thickness (sham 1190 ± 70 µm; transplant 1030 ± 60 µm; media injected 1070 ± 50 µm; p = NS). Relative retardation is a structural rather than a functional measurement; therefore the progressive increase in retardation found does not guarantee improved function, but rather indicates increased potential for function enhancement because of an increase in contractile elements.
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Cell orientation
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Even though the initial cell injection formed an apparently homogeneous bubble, the subsequent organization was, despite considerable disarray, never random. That something other than a random structure developed from such beginnings is consistent with an aligning influence. Collagen fibers within infarcts are coherently organized and generally align in the same direction as the muscle that previously occupied the space [14]. Such organization was attributed to guidance from the preexisting collagen matrix and the influence of systolic wall deformation [15]. However, these factors did not appear to align tissue within the transplant because the collagen fibers were disorganized and the mean cell orientation was not circumferential. The bubblelike constitution of the transplant initially isolates it from surrounding tissue, eliminating guidance from a preexisting collagen network and limiting transmission of systolic deformation signals. Instead, the eventual "semiorganized" transplant muscle may be simply the result of packing constraints to accommodate developing cells within the confines of a relatively rigid collagenous cocoon. Nevertheless, in other situations such disarray is associated with dysfunction [16] and electrical conduction disturbances [17].
Other factors that influence organization relate to the circumstances of muscle contraction. For example, muscle disarray occurred in fetal hearts transplanted into the anterior eye chamber of adult rats [18]. This beating tissue developed vascular and neural connections to the iris, and even though the cells differentiated and enlarged, they were disorganized and had round nuclei. The graft had no ventricular lumen and did not pump against a pressure gradient and hence hemodynamic stresses appeared to influence cell alignment. In human hearts, disarray developed in papillary muscle after the chordae tendineae that connected them to the valve were severed during mitral replacement surgery [19]. The subsequent lack of tension during systole resulted in isotonic muscle contraction, which led to atrophy and disarray. Conversely, isometric contraction is also thought to cause disarray [20], and thus altered stress distribution appears to influence cell organization.
Whatever the cause of disarray, the transplanted cells were always less coherently organized than host tissue. In addition, transplant and host cells were always separated by collagen and so, even if the organization problems were resolved, synchronous contraction of the two populations would be compromised. We note that cell disarray and round nuclei are apparent, but not discussed, in other rat studies; eg, in micrographs obtained 8 weeks after fetal and neonatal cell transplant into infarcts [21, 22].
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Nuclear morphology
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Although their histologic area profile eventually increased to equal that of normal cells, transplanted cell nuclei retained a rounded shape. The explanation for such morphology and its functional consequences are unknown; however, it has been proposed that shape changes may, through mechanical forces, play a role in regulation of gene expression [23]. Nevertheless, this distinctive feature may be useful as a marker. All transplant studies face the problem of differentiating host and transplanted cells. In our studies, injection of male cells into female hearts enabled a polymerase chain reaction (PCR) method to detect a specific male gene [5]; however, such methods are relatively complex and are incompatible with subsequent histologic examination of that tissue. In contrast, measurement of nuclear area and perimeter length are relatively simple. One potential confounding factor for this differentiation approach is the presence within the infarct of host cells that survived the coronary occlusion. In our control group, nuclei from these cells, located in the subendocardium, appeared round because they were obliquely sectioned. Nevertheless, they were readily distinguished from transplant cell nuclei because they were considerably smaller than all but the 0 to 1 day group.
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Collagen content
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Collagen content within the transplant appeared to decrease over time (Fig 6); however, this represents a relative rather than an absolute decrease because the calculation method expressed content as a percent of the field. Therefore, as muscle size increased, the relative collagen content could decrease even with an absolute increase. This possibility is supported by the observation of increased collagen fiber thickness from 2 weeks to 6 months (Fig 1). Fibroblasts can function within the initially hypoxic transplant environment and so probably have a head-start over the muscle, whose maturation may be delayed until a blood supply develops. Our injectate was 81% ± 2% muscle together with a mixture of fibroblasts and endothelial cells. This potentially large number of fibroblasts was probably responsible for the high collagen content; however, there was most likely also a contribution from host fibroblasts, especially at the periphery of the transplant. Removal of fibroblasts from the injectate could limit fibrosis. Nevertheless, this approach may be undesirable because too little collagen could be as detrimental as too much. Furthermore, collagen organization and amount may determine muscle alignment; for example, neonatal myocytes achieved a parallel organization when cultured on an aligned collagen substrate, whereas culture on a disorganized collagen substrate resulted in muscle disarray [24].
Increased collagen could have consequences for the transplanted muscle’s function; for example, collagen’s stiffness impedes both systolic contraction and diastolic relaxation [25], and increased interstitial collagen creates barriers to intercellular electrical signal propagation [17, 26]. Thus, transplant tissue structure resembles viable myocardium at the edge of an infarct [14], which is frequently either dyskinetic or akinetic and can provide an arrhythmogenic substrate [17].
The failure of previous studies to report increased collagen can be explained by differences in the transplant or staining protocols. Although the limitations of hematoxylin–eosin staining for detecting collagen are known, trichrome-based methods are considered specific for collagen and have been used in transplant studies [2, 3, 21]. Nevertheless, we have demonstrated that trichrome substantially underestimates collagen content when compared with the use of picrosirius red and polarized light [13]. We attribute the latter’s specificity to its reliance on a physical property of collagen, its birefringence, rather than trichrome’s poorly understood staining mechanism influenced by many factors [13].
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Cell survival: an additional architectural goal
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Although the minimum number of cells necessary to enhance function is unknown and although survival in the absence of appropriate development and organization might be of questionable value, some level of survival is clearly necessary for success [4]. We did not directly assess survival; nevertheless, our structural study does provide insight. In the infarcted hearts, the largest transplant was 5x1 mm in cross-section and, because it appeared in only one slice, was no more than 2-mm deep (the slice thickness). Adult male rat myocytes are 140-µm long with a diameter of 20 µm [27]. However, the transplant cells were smaller, and if we take the 80% value suggested by the retardation measurements, the number of cells required to occupy the observed volume would be 44x62x123 
336,000. In this case, with an initial injection of 2.4x106 cells, survival would be 
15%. For the smaller transplant regions found in other hearts, survival would be much less. These data are in contrast to results obtained by PCR (60% survival) [6], probably because of the PCR method’s inability to distinguish between cell types.
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Conclusions
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Although transplanted neonatal cells developed sarcomeres and enlarged, our results indicate that there remains room for considerable improvement of their architectural organization. Specifically, cellular disarray together with retarded development, separation from host cells, and elevated collagen content indicate that although the transplant met, at least partially, some of the development and organization goals required for enhanced myocardial function, it did not meet them all [4]. Indeed, in the infarct study, although paradoxical systolic bulging was reduced by cell transplant (a likely consequence of increased wall thickness), there was no conclusive evidence for active enhancement of contractile function [6]. Therefore, consistent with the architecture, the cells appeared to play a passive rather than an active role. The prospect of engineering the tissue to correct these organizational deficiencies is an intriguing possibility.
An essential component of myocardium’s structure is its anisotropy. It seems optimistic to expect that transplantation of an isotropic cellular mixture will, without intervention, result in the development of the required anisotropic structure. However, because many transplant studies report enhanced function, it is possible that methodologic differences explain our failure to generate an appropriate structure; for example, the timing and transplantation method in addition to the number, distribution, and cell type are factors that influence outcome. Nevertheless, no study has systematically evaluated these variables, nor has any study yet supported function data with quantitative assessment of tissue structure. Thus, even though neonatal cell transplantation is unlikely ever to have clinical application, the development of appropriate tissue architecture will be a requirement of all cell transplantation methods.
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Acknowledgments
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We thank Seda Dzhandzhapanyan for assistance with histology. PW is an Established Investigator of the American Heart Association. This study is also supported by the National Institutes of Health (61488 to RAK and 52771 to LHK) and the Deutsche Forschungsgemeinschaft (DFG Mu 1469/1–1 to JM-E).
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References
|
|---|
- Orlic D., Kajstura J., Chimenti S., et al. Bone marrow cells regenerate infarcted myocardium. Nature 2001;410:701-705.[Medline]
- Jain M., DerSimonian H., Brenner D.A., et al. Cell therapy attenuates deleterious ventricular remodeling and improves cardiac performance after myocardial infarction. Circulation 2001;103:1920-1927.[Abstract/Free Full Text]
- Rajnoch C., Chachques J.-C., Berrebi A., et al. Cellular therapy reverses myocardial dysfunction. J Thorac Cardiovasc Surg 2001;121:871-878.[Abstract/Free Full Text]
- Thurmond F.A., Cronin E.M., Williams R.S. A deadly game of musical chairs: survival of cells transplanted for myocardial repair. J Mol Cell Cardiol 2001;33:883-885.[Medline]
- Müller-Ehmsen J., Whittaker P., Kloner R.A., et al. Survival and development of neonatal rat cardiomyocytes transplanted into normal adult myocardium. J Mol Cell Cardiol 2002;34:107-116.[Medline]
- Müller-Ehmsen J., Peterson K.L., Kedes L., et al. Rebuilding a damaged heart: long-term survival of transplanted neonatal rat cardiomyocytes after myocardial infarction and effect on cardiac function. Circulation 2002;105:1720-1726.[Abstract/Free Full Text]
- Módis L. Organization of the extracellular matrix: a polarization microscopic approach. Boca Raton: CRC Press, 1991.
- Whittaker P., Zheng S.M., Patterson M.J., et al. Histologic signatures of thermal injury: applications in transmyocardial laser revascularization and radiofrequency ablation. Lasers Surg Med 2000;27:305-318.[Medline]
- Chayen J., Bitensky L., Braimbridge M.V., Darracott-Cankovic S. Increased myosin orientation during muscle contraction: a measure of cardiac contractility. Cell Biochem Funct 1985;3:101-114.[Medline]
- Whittaker P., Canham P.B. Demonstration of quantitative fabric analysis of tendon collagen using two-dimensional polarized light microscopy. Matrix 1991;11:56-62.[Medline]
- Whittaker P., Romano T., Silver M.D., Boughner D.R. An improved method for detecting and quantifying disarray in hypertrophic cardiomyopathy. Am Heart J 1989;118:341-349.[Medline]
- Batschelet E. Circular statistics in biology. London: Academic Press, 1981.
- Whittaker P., Kloner R.A., Boughner D.R., et al. Quantitative assessment of myocardial collagen with picrosirius red staining and circularly polarized light. Basic Res Cardiol 1994;89:397-410.[Medline]
- Whittaker P., Boughner D.R., Kloner R.A. Analysis of healing after myocardial infarction using polarized light microscopy. Am J Pathol 1989;134:879-893.[Abstract]
- Zimmerman S.D., Karlon W.J., Holmes J.W., et al. Structural and mechanical factors influencing infarct scar collagen organization. Am J Physiol 2000;278:H194-H200.
- Karlon W.J., McCulloch A.D., Covell J.W., et al. Regional dysfunction correlates with myofiber disarray in transgenic mice with ventricular expression of ras. Am J Physiol 2000;278:H898-H906.
- Horvath G., Racker D.K., Goldberger J.J., et al. Electrophysiological and anatomic heterogeneity in evolving canine myocardial infarction. Pacing Clin Electrophysiol 2000;23:1068-1079.[Medline]
- Bishop S.P., Anderson P.G., Tucker D.C. Morphological development of the rat heart growing in oculo in the absence of hemodynamic work load. Circ Res 1990;66:84-102.[Abstract/Free Full Text]
- Pirolo J.S., Hutchins G.M., Moore W., et al. Myocyte disarray develops in papillary muscles released from normal tension after mitral valve replacement. Circulation 1982;66:841-846.[Free Full Text]
- Bulkley B.H., Weisfeldt M.L., Hutchins G.M. Isometric cardiac contraction. A possible cause of disorganized myocardial pattern of idiopathic subaortic stenosis. N Engl J Med 1977;296:135-139.[Abstract]
- Reinecke H., Zhang M., Bartosek T., et al. Survival, integration, and differentiation of cardiomyocyte grafts. A study in normal and injured rat hearts. Circulation 1999;100:193-202.[Abstract/Free Full Text]
- Li R.-K., Mickle D.A., Weisel R.D., et al. Natural history of fetal rat cardiomyocytes transplanted into adult rat myocardial scar tissue. Circulation 1997;96(Suppl II):II-179-II-186.
- Maxwell C.A., Hendzel M.J. The integration of tissue structure and nuclear function. Biochem Cell Biol 2001;79:267-274.[Medline]
- Simpson D.G., Terracio L., Terracio M., et al. Modulation of cardiac myocyte phenotype in vitro by the composition and orientation of the extracellular matrix. J Cell Physiol 1994;161:89-105.[Medline]
- Weber K.T. Cardiac interstitium in health and disease: the fibrillar collagen network. J Am Coll Cardiol 1989;13:1637-1652.[Abstract]
- Spach M.S., Boineau J.P. Microfibrosis produces electrical load variations due to loss of side-to-side cell connections: a major mechanism of structural heart disease arrhythmias. Pacing Clin Electrophysiol 1997;20:397-413.[Medline]
- Bai S., Campbell S.E., Moore J.A., et al. Influence of age, growth, and sex on cardiac myocyte size and number in rats. Anat Rec 1990;226:207-212.[Medline]
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Abstract
Introduction
