| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
J Thorac Cardiovasc Surg 1999;118:36-49
© 1999 Mosby, Inc.
SURGERY FOR ACQUIRED CARDIOVASCULAR DISEASE |
From The Departments of Surgery of the Washington, DC, Veterans Affairs Medical Center, Georgetown University Medical Center, Washington, DC, Brown University, Providence, RI, and The Collis Cardiac Surgical Research Laboratory of the Lifespan Academic Medical Center, Providence, RI.
Supported in part by research grants from The American Heart Association and Lifenet.
Address for reprints: Richard A. Hopkins, MD, Chief, Cardiothoracic Surgery, Brown University School of Medicine, The Miriam Hospital, 164 Summit Ave, Providence, RI 02906.
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and methodsResultsDiscussionReferences |
|---|
–smooth muscle actin. After cryopreservation, viable cell numbers were always markedly reduced at all ischemic intervals and damage to both soluble extracellular matrix components and cell ultrastructure was increased. At all time and processing points, however, some retention of matrix secretory and cellular contractile capabilities was observed among the surviving cells. After the extended periods of preharvest ischemia (2-24 hours) followed by processing, a restitution of functioning cells was accomplished by means of whole-leaflet incubation in 15% fetal bovine serum. |
Introduction |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
1 It has been suggested that the cells residing within the cusp matrix interstitium are important to posttransplantation durability, theoretically because of retained interstitial cellular functions (matrix refurbishing and cuspal remodeling). 2-5 Conversely, others have reasoned that any significance of this cell viability may be more as a marker of "gentle" valve preservation. 6 Both intuition and a number of clinical studies suggest that gentle treatment of donor leaflet interstitial cells has improved durability, and cryogenic preimplantaton protocols have therefore been designed specifically with the intent of maintaining postthaw viability of matrix cells (but not endothelial cells). 7 Importantly, no long-term studies in either human beings or animals have been published that show a posttransplantation proliferation of donor leaflet cells to suggest that in vivo resuscitation of the cell population can or does occur.
We previously developed a model of valve harvest and cryogenic storage that closely resembles clinical practice, and with it we evaluated leaflet interstitial cellular metabolism with respect to fluxes in adenine nucleotides. 8-12 These studies showed a graduated nucleotide depletion in leaflet cells. We additionally characterized a porcine aortic leaflet myofibroblast cell line. 13 In previous studies we correlated the apparent damage reflected by reductions in the components of the adenine nucleotide pool in cryopreserved aortic valves (both porcine 8 and human 14 ) with reductions in leaflet cell densities and altered functional (synthetic) and ultrastructural features consistent with reversible and irreversible cell injury. Because cryopreservation-associated injury has been reported to be reversible in other eukaryotic systems, 15 this study examined whether a modified organ culture technique could restore leaflet cell population and function. We tested our hypothesis that the retention of even a small residual population of viable cells would allow proliferation to restore constitutive cell density and cell function in both single-cell suspensions and intact leaflets.
|
Materials and methods |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
7 We suspect that harvesting, transport, and processing may result in leaflets with substantially injured interstitial cells. 12 For this study 3 experimental groups were designed to examine variable preharvest ischemia, variable preharvest ischemia followed by completed processing (disinfection, cryopreservation, storage, and thawing), and variable preharvest ischemia and processing followed by an organ culture period of valve "resuscitation."
Valve harvest protocol
Mediastinal contents including the heart, great arteries (aorta to ligamentum arteriosum), thymus, and portions of the pericardium were cleanly procured from 24 female 8-month-old Yorkshire swine at a local abattoir. Each was obtained within 40 minutes after electrical stunning followed by exsanguination, with the myocardium usually fibrillating at the time of recovery. At retrieval the specimens were allocated into sets of 6 to sustain varying periods of ischemia (2, 6, 12, or 24 hours, including the 40 minutes in situ), during which the tissue was maintained in sterile organ procurement bags (Steri-Drape Isolation Bag; 3M Health Care, St Paul, Minn) at room temperature. These intervals were arbitrarily selected to simulate conditions in a cooling cadaver with variable times until actual organ harvest. At the end of 2, 6, 12, or 24 hours, the aortic roots were disarticulated from the hearts, the conduits were rinsed in sterile phosphate-buffered saline solution (PBS; Life Technologies, Inc, Rockville, Md), and leaflets were prepared as previously described. 16
Group allocation
To control for variability among the animals each separate leaflet of the 3 from every valve was assigned to assay at 1 of the 3 experimental intervals: group 1, ischemia only; group 2 , ischemia plus disinfection plus cryopreservation plus thaw; and group 3, ischemia plus disinfection plus cryopreservation plus thaw plus resuscitation. At the conclusion of the designated 2-, 6-, 12-, or 24-hour ischemic period the group 1 leaflets were removed (n = 6 leaflets/ischemic period, 1 from each of the 24 valves). All valves (now bicuspid minus the group 1 leaflets) were then rinsed in sterile PBS and immersed in a 4°C solution of antibiotics reconstituted in Roswell Park Memorial Institute medium 1640 (Life Technologies): 240 µg/mL cefoxitin, 120 µg/mL lincomycin, 100 µg/mL polymyxin B, and 50 µg/mL vancomycin. 7
After 24 hours of this cold disinfection the valves were rinsed again in sterile PBS and cryopreserved. 17 The 24-hour group 2 leaflets (ischemic for 2, 6, 12, or 24 hours, disinfected, and cryopreserved) were then thawed, excised from their anuli, and assayed. Simultaneously, each group 3 leaflet was excised from its anulus and placed into a 35-mm2 culture well containing M199 medium (Life Technologies) supplemented with 15% heat-inactivated fetal bovine serum (FBS; Life Technologies). We previously showed that this concentration of FBS is strongly mitogenic to porcine aortic leaflet interstitial cells. 12,13 These "restoring" valves were incubated in a humidified atmosphere of 95% air and 5% carbon dioxide at 37°C for 8 days and then assayed.
Viability, proliferation, and contraction
Preparation of dispersed cells. At the time of assay for each leaflet in each of the 3 groups and at all 4 ischemic points the central third of each cusp (beginning at the nodule of Arantius and ending at a point 3 to 5 mm from the base) was excised longitudinally from the base. All 72 leaflets from the 24 valves (n = 6 valves/ischemic period, 4 ischemic periods/group) were used in the viability assessments. The excised segments were removed, dried, weighed on a Mettler balance (Mettler-Toledo International, Inc, Greifensee, Switzerland), rinsed in chilled PBS, and placed into M199 medium with 15% heat-inactivated FBS and 0.08% collagenase (lyophilized, prepared from Clostridium histolyticum; Life Technologies). Antibiotics (100 U/mL penicillin G, 100 µg/mL streptomycin, and 5 µg/mL amphotericin B) were added to each tube and each tube was incubated in a humidified atmosphere of 95% air and 5% carbon dioxide at 37°C.
Viability. After 24 hours in collagenase suspensions were centrifuged twice for 2 minutes at 25g with an intervening wash in sterile PBS. Cells were resuspended in M199 medium and the aliquots from each suspension were counted by hemocytometer, with Trypan blue dye exclusion to determine viability. Counts were performed by 2 separate investigators blinded to the group identity of the leaflet and were normalized as cells per milligram of wet weight of leaflet segment.
Proliferation. We assessed changes in the ability of aortic leaflet interstitial cells to proliferate after ischemia and completed processing with cells dispersed from the 2- and 24-hour group 1 and 2 leaflets, anticipating that any decreases (or increases) in proliferation potential would be demonstrable at the ischemic extremes. After deriving the viability counts for each experimental interval at each ischemic time, aliquots from the digestates of the 2- and 24-hour group 1 and 2 leaflets were separately resuspended in M199 medium supplemented with 15% FBS. These were then counted by hemocytometer and plated in quadruplicate on plastic, 24-well, 2-cm 2 surface area plates (Multiwell; Becton Dickinson and Company, Franklin Lakes, NJ) at a density of 2 x 104 cells/well. After a 24-hour period of recovery in M199 medium supplemented with 15% FBS, the cultures were deprived of serum for 24 hours and 4 random wells per plate were counted with a Coulter counter (Coulter Corporation, Hialeah, Fla). These numbers were averaged to establish baseline counts for growth curves. The remaining wells had M199 medium supplemented with 15% FBS administered and growth curves were derived from harvest (0.25% trypsin–ethylenediaminetetraacetic acid) and quantitation at 24, 48, 72, and 96 hours after serum administration. Trypsin-digested cells were examined under the light microscope before counting to ensure that single cells, rather than multicellular aggregates, were present. Accuracy of quantitation was verified through periodic visual counting with a hemocytometer.
Cellular contractility. To verify that the ability of leaflet interstitial cells to contract is maintained throughout ischemia, processing, and resuscitation, we employed the method of Harris and colleagues. 17,18 The preparations were stimulated with 10–7 mol/L l-epinephrine, 3.2 x 10–7 mol/L bradykinin, 3.2 x 10–7 mol/L angiotensin II, 110 µmol/L carbachol, and 110 µmol/L isoproterenol (INN: isoprenaline sulphate). 12,13 For each administration, 10 to 50 cells were observed continuously for 40 minutes. Photographs were taken under the phase-contrast microscope (Nikon Inc, Melville, NY) before and immediately after each compound was applied and at the time of response.
Cellular and matrix protein
Preparation of leaflets and immunohistochemical examination. For the whole-leaflet preparations, 4 leaflet portions were washed several times in PBS (with calcium and magnesium ion, hereafter PBS++) fixed in 4% buffered para formaldehyde, and processed for paraffin embedding. The embedded tissue was then sectioned to between 4 and 5 µm in thickness, mounted on standard glass microscope slides, and maintained in a 4°C refrigerator until use. The slides had the paraffin removed in PBS++ and were incubated for 20 minutes in a blocking serum (5% goat serum and 1% bovine serum albumin in PBS). After washing in PBS (without calcium and magnesium ion, hereafter PBS–– ) the sections were placed in a humidified 5% carbon dioxide incubator at 37°C for 60 minutes and each was administered 1 of the primary antibodies in PBS–– (supplemented with 1% bovine serum albumin) for 2 hours. After washing the secondary antibodies (antimouse immunoglobulin G–fluorescein isothiocyanate conjugate; Sigma, St Louis, Mo) were then applied at 1:100 dilution and cells were subsequently incubated for 45 minutes at 37°C in a humidified 5% carbon dioxide incubator. After washing the slides were mounted with Fluoromount G (Fisher). Sections were examined with an epifluorescence microscope (Nikon Diaphot) equipped with a blue filter and photographed (TMAX 3200; Eastman Kodak Company, Rochester, NY). As control preparations several sections were incubated in PBS–– with secondary antibody (no primary antibody) to detect any fluorescence resulting from nonspecific binding of secondary antibody. In addition, sections were viewed in the absence of either primary or secondary antibody to demonstrate any autofluorescence.
Antisera. The following primary antibodies were used at empirically derived dilutions: (1) 1:28 monoclonal anti–-smooth muscle actin (A-2547; Sigma), (2) 1:100 monoclonal antivimentin (V-6630; Sigma), (3) 1:200 monoclonal anti–chondroitin sulfate (C-8035; Sigma), and (4) 1:100 monoclonal anti–cellular fibronectin (F-6140; Sigma). No nuclear counterstains were used. The antiserum to a–smooth muscle actin has been extensively characterized as monospecific to smooth muscle cells. 19 No cross-reaction with fibroblast or endothelial actin isomers (stress fibers) has been found.
Leaflet ultrastructure (preparation and electron microscopy)
Five cusp segments from valves at the 2- and 24-hour ischemic time points of each group were used to qualitatively study ultrastructural and cellular effects of ischemia and processing, as well as to assess histologic status of the leaflets after resuscitation. The central third from each of the 5 cusps per interval was dissected and fixed in phosphate-buffered 2% paraformaldehyde and 1% glutaraldehyde solution (pH 7.4). Thin sections were washed in buffered sucrose solution (0.1 mol/L phosphate buffer, 5% sucrose solution) and postfixed at 4°C in phosphate-buffered 2% osmium tetroxide solution. Ultrathin longitudinal and cross sections from 2 selected areas of the semithick sections devoid of artifactual damage were mounted on 300-mesh copper grids (6 grids/area). These specimens were enhanced with uranyl acetate and lead citrate for 1 to 2 minutes 20 and viewed under the transmission electron microscope (Zeiss EM 10CA; Carl Zeiss, Inc, Thornwood, NY) at 80 kV. Photographs of densely populated (
4 cells/field) fields were then taken with electron microscopy film (Kodak ESTAR Thick Base 4489).
|
Results |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
|
Group 3, resuscitation. Proliferative capabilities were assessed from leaflets sustaining 2 and 24 hours of preharvest ischemia to separate any alterations in mitotic potential among the cells surviving cryopreservation caused by increasing doses of preprocessing ischemia. Fig 2 depicts growth curves of cells obtained directly from leaflets at each ischemic interval and indicates that each group contained cells that retained the ability to proliferate in vitro in growth media supplemented with 15% FBS. Viable cell counts after resuscitation returned in cell population to levels equivalent to those obtained from the minimally ischemic unprocessed leaflets. Toluidine blue–stained sections from the restored leaflets showed cellularity similar to that noted in the briefly ischemic leaflets; nuclei were rounded with visible nucleoli and few pyknotic nuclei were observed. The time pattern of this repopulation, determined by vital dye exclusion counting of digested leaflets on each day of the resuscitation period, is depicted in the inset of Fig. 2.
|
21 clusters of pure smooth muscle cells with complete basal laminae, subplasmalemmal plaques, intracellular myofilaments, and few intracellular organelles were noted. After 24 hours of ischemia (24-hour group 1) only rare cells appeared completely uninjured. In many of the clearly injured cells the plasma membrane was largely intact but the nuclear membrane showed extensive separation of the inner and outer layers. Many nuclei that showed this feature assumed a crescent appearance (Fig. 3).
|
|
|
|
|
|
–smooth muscle actin were also altered with each experimental interval (Fig. 9).A pattern of a gradual reduction in binding continued through the successive intervals. In the 24-hour group 2 leaflets immunofluorescence was undetectable. After resuscitation the labeling returned to a high-intensity distribution in the lamina ventricularis, with scattered localization in the deeper leaflet segments. Occasional focal areas of hyperplasia with strongly positively staining cells were noted throughout the leaflet.
|
Fibronectin was present in leaflets through all experimental intervals (Fig. 10).In the 2 hours of preharvest ischemia–only valves (2-hour group 1), fibronectin was localized to the aortic leaflet interstitial cells throughout the leaflet. Decreases in binding were noted with successive experimental intervals. The 2-hour group 1 leaflets did possess more fibronectin, but the immunofluorescence remained strong even in the maximally processed 24-hour group 2 leaflets. After resuscitation (2- and 24-hour group 3) an increased binding indicative of nascent fibronectin synthesis was evident.
|
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
2-6 In the native state this population has been shown to synthesize extracellular leaflet matrix proteins in response to specific stress-strain stimuli, probably contributing to the remarkable durability of undiseased valve leaflets. 4 Accordingly, the objectives of human valve cryopreservation have been not only to store and subsequently implant a hydraulically favorable prosthesis but also to transplant capacity for growth, repair, and remodeling through the gentle preservation of donor cell viability. The biophysics and cellular physiology of cryogenic storage have been well studied in many tissues and single-cell suspensions 22; however, extrapolation from data derived from single-cell preparations cannot be applied to cardiac valve leaflets, in part because 3-dimensional structures do not generally sustain the requisite uniform tissue freeze necessary for maximum postthaw viability, despite the use of cryoprotective agents.
In previous studies conducted by our group adenine nucleotide pool components extracted from porcine valve leaflets exposed to staggered intervals of the standard homograft processing and cryopreservation protocol were quantified with high-performance liquid chromatography. 8-12,14,15 These analyses indicated that a marked energy store depletion took place, particularly during disinfection and cryopreservation itself, and that most cells if not dead were metabolically stunned. 11 Although protein losses were also noted during processing, there are few data regarding the fates of specific protein components. These studies also showed that despite marked high-energy phosphate depletion small quantities of energy stores were measurable as a nucleotide reserve, indicative of a resilient subset of leaflet interstitial cells that were present in even fully processed porcine leaflets sustaining the maximum 24 hours of preharvest ischemia. 8-13 We also found a similar subgroup within the stunned leaflet myofibroblast population in human cryopreserved valves. 11 Our current results suggest that only a small population of such retained hardy cells may be required as an inoculum on which to base restoration of interstitial cell populations.
The interstitial cells of mitral valve leaflets have been characterized by several groups to possess features similar to smooth muscle cells. 23,24 Our laboratory was the first to demonstrate that interstitial cells from semilunar valves were also rich in smooth muscle–specific -actin, vimentin, and were able to secrete both fibronectin and chondroitin sulfate. These cells are capable of contractile and relaxive responses when challenged with physiologic stimuli, as manifested by wrinkle formation when plated on flame-polymerized silicone 18,25 (that is, they are myofibroblasts).
In this study we followed changes in these identifying characteristics through the intervals of ischemia and processing and then attempted a restitution to the "native" state by means of a period of organ culture that we termed resuscitation. Resuscitation was essentially cellular bioengineering after thaw through mitotic stimulation of the residual population that had survived processing. FBS at 15% concentration has been demonstrated in our laboratory to be the most efficient additive for these cells when compared with individual cytokines such as endothelium-derived relaxing factor and platelet-derived growth factor. 12 Presumably this is due to the combination of various growth factors (known and unknown) present in FBS as opposed to single additives. Thus although data from our own series of studies as well as other laboratories suggest that only a few viable and functioning interstitial cells survive through transplantation, this study demonstrated that the reduction in population of living healthy cells, as well as any depletion of matrix proteins in the interstitium, could be reversed by in vitro cellular engineering to generate a more densely populated and functional leaflet before transplantation.
This study does not address the issue of whether such restored but genetically unmodified donor cell populations remain functional for a prolonged period after transplantation. It is possible that although cellular elements are beneficial initially in restoring leaflet functional properties they may render the valve more antigenic, ultimately resulting in greater rejection and inflammatory responses by the host and thus reducing durability, as has been suggested in neonatal recipients. These results suggest the feasibility of the alternate and appealing strategy of repopulating with immunologically compatible or genetically altered cells.
As a first step we used the standard viability assay of trypan blue dye exclusion, an indicator of functional plasma membrane integrity. 26 The trypan blue test by itself is not a perfect viability assay because it cannot predict future viability or indicate origin and mitotic potential of cells, and it may be susceptible to denominator errors. Nevertheless, it is a routinely used assay in cell culture laboratories for comparison of cell viability between comparably managed cell cultures. Other types of cell labeling techniques (eg, bromodeoxyuridine and genetic markers) have additional advantages and would be mandatory for long-term implant studies. Viable cell numbers do not appear to change significantly in the clinical harvest period between 2 and 24 hours post mortem. After processing, and without relationship to preharvest ischemia, viable cell density always falls precipitously. We therefore believe that the agreement between simple, visual viability assays (trypan blue), quantitative electron microscopy, and sensitive, precise metabolic analyses (high-pressure liquid chromatography) demonstrates fairly conclusively that the cellular population decreases during preimplantation processing. 9,11
On ultrastructural examination, leaflets that had been ischemic for 24 hours showed overt subcellular changes despite continued ability to exclude trypan blue. 26,27 We have also noted similar effects on human leaflet cells in cryopreserved specimens harvested after varying durations of ischemia and that such changes appeared to evolve in a time-dependent manner. 16 Conversely, plasma membranes themselves were often intact, perhaps attesting to the means by which the cells continued to exclude the vital dye at 24 hours. In addition, these responses seen with the electron microscope serve to emphasize the difficulties of real-time viability assays. Cells may show no evidence of damage by a given indicator, whereas damage is overt according to another.
We suspect that the near ablation of chondroitin sulfate through 24 hours of ischemia is probably due to simple leeching and should be expected to occur during any interval of valve suspension. At present, all donor valves in the United States are subjected to hypothermic and prolonged immersion in an antibiotic solution.
The group 2 leaflets were specifically designed to examine the matrix and cells shortly after thawing. Clinically this is a time when certain measures could be performed in the operating room to ensure minimal osmolysis and rapid rewarming. Theoretically these precautions are prudent because transient cell dysfunction and osmotic fragility have been noted in many cell types to be characteristic of the thaw interval. 15,28,29 In the situation of valve cryopreservation, our studies indicate not only that this dysfunction is a consideration but also that there is a severe reduction in potentially viable cells. The reductions in monoclonal antibody binding to the contractile intracellular –smooth muscle actin and the intermediate filament vimentin, as well as to pericellular fibronectin, were in agreement with our viability assessments at each stage. We therefore conclude that current cardiac cryopreserved homografts possess a markedly altered interstitium with a reduced and compromised cell population and a matrix that has been stripped of many soluble components. In even the most rigorously stressed valves (24-hour group 2), however, some cells remain that could be considered "normal" by several parameters. This occasional instance of morphologic integrity was accompanied by retained functional abilities. Collagenase digestates of both 2- and 24-hour group 2 leaflets demonstrated mitosis to be a spared process. The specialized cellular function of contraction was not obviated through processing. In collagenase digestates of leaflets from each group, cells retained a baseline tonus and could still be induced to contract or relax in response to challenge.
In this study we reasoned that perhaps the small subset of viable and functional leaflet interstitial cells could be stimulated to repopulate the virtually barren collagen lattice of a cryopreserved valve leaflet. This hypothesis was tested with the resuscitated leaflets of group 3. A complete restoration of the numeric population was shown to be achievable during a period of 8 days with simple organ culture in 15% FBS. The rate of this repopulation may be appreciated by examining Fig. 2
, in which weighed leaflets were digested in collagenase each day and the cells were counted. The growth curve was similar in morphologic appearance to that seen in leaflet interstitial cells in culture with 15% FBS.
In any organ culture setting the possibility of a purely fibroblastic overgrowth must be ruled out. By using the ultrastructural features, protein identification, and verification of contractile responses to physiologic agents we were able to characterize the restored population as phenotypically similar to the original population. The group 3 leaflets were not simply segments of collagen repopulated with standard connective tissue fibroblasts. They were restored and functional semilunar cusps that showed evidence of a normal matrix. The cells retained synthetic capabilities, contractile function, and mitotic potential, and they could be anticipated to be capable of growth, repair, and remodeling. Whether these potential activities would continue to be expressed after transplantation will require validation by long-term implant studies.
The function of these interstitial myofibroblast cells in vivo remains undefined. We and others have previously speculated on an interaction between contractile properties and the synthesis of extracellular matrix in an environment of varying hemodynamic stresses and have conjectured that this may be central to the remarkable durability of native aortic valves. 12,30 These studies show that this specific interstitial cell population in cryopreserved leaflets may be restored in vitro after thaw. Such techniques may be extrapolated to other seeding strategies as well as to donor cell restoration.
|
References |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
This article has been cited by other articles:
|
|
 |
|
  R. Hopkins, H. Gitter, J. Stave, A. Bert, and M. Atalay Stable partial dehiscence of aortic homograft inserted freehand by using the subcoronary intra-aortic root noncoronary sinus Ross scallop inclusion technique J. Thorac. Cardiovasc. Surg., January 1, 2008; 135(1): 214 - 216. [Full Text] [PDF]
|
|
|
|
 |
|
 K. Schenke-Layland, J. Xie, S. Heydarkhan-Hagvall, S. F. Hamm-Alvarez, U. A. Stock, K. G.M. Brockbank, and W. R. MacLellan Optimized Preservation of Extracellular Matrix in Cardiac Tissues: Implications for Long-Term Graft Durability Ann. Thorac. Surg., May 1, 2007; 83(5): 1641 - 1650. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Schenke-Layland, N. Madershahian, I. Riemann, B. Starcher, K.-J. Halbhuber, K. Konig, and U. A. Stock Impact of Cryopreservation on Extracellular Matrix Structures of Heart Valve Leaflets. Ann. Thorac. Surg., March 1, 2006; 81(3): 918 - 926. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. A. Rice and L. A. Leinwand Skeletal myosin heavy chain function in cultured lung myofibroblasts J. Cell Biol., October 13, 2003; 163(1): 119 - 129. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. J. F. Baskett, M. A. Nanton, A. E. Warren, and D. B. Ross Human leukocyte antigen-DR and ABO mismatch are associated with accelerated homograft valve failure in children: implications for therapeutic interventions J. Thorac. Cardiovasc. Surg., July 1, 2003; 126(1): 232 - 238. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Murakawa, J. Nakajima, M. Ono, A. Murakami, Y. Suematsu, and S. Takamoto Allogenicity of cryopreserved human fibroblasts: Cryopreservation does not downregulate the allogenicity of fibroblasts making up the matrices of allografts J. Thorac. Cardiovasc. Surg., October 1, 2000; 120(4): 712 - 719. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||
