This Article Abstract Full Text (PDF) Supplemental Data for this article All Versions of this Article: jhc.5A6824.2006v1 54/6/673    most recent Alert me when this article is cited Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Rippstein, P. Articles by O'Brien, E. R. Search for Related Content PubMed PubMed Citation Articles by Rippstein, P. Articles by O'Brien, E. R. Social Bookmarking                      What's this?

Comparison of Processing and Sectioning Methodologies for Arteries Containing Metallic Stents

Peter Rippstein, Melanie K. Black, Marie Boivin, John P. Veinot, Xiaoli Ma, Yong-Xiang Chen, Paul Human, Peter Zilla and Edward R. O'Brien

Core Pathology Laboratory (PR) and Division of Cardiology, Vascular Biology Laboratory (XM,Y-XC,ERO), University of Ottawa Heart Institute, Ottawa, Ontario, Canada; Department of Laboratory Medicine, The Ottawa Hospital, Ottawa, Ontario, Canada (MB,JPV); and Christian Barnard Division of Cardiothoracic Surgery, Cape Heart Center, University of Cape Town, Cape Town, South Africa (MKB,PH,PZ)

Correspondence to: Peter Rippstein, ART, MLT, Core Pathology Laboratory, University of Ottawa Heart Institute, 40 Ruskin Street, Rm H2102, Ottawa, Ontario, Canada K1Y 4W7. E-mail: prippstein{at}ottawaheart.ca

	Summary

Top Summary Introduction Materials and Methods Results Discussion Literature Cited

 
The histological study of arteries with implanted metallic scaffolding devices, known as stents, remains a technical challenge. Given that the arterial response to stent implantation can sometimes lead to adverse outcomes, including the re-accumulation of tissue mass within the stent (or in-stent restenosis), overcoming these technical challenges is a priority for the advancement of research and development in this important clinical field. Essentially, the task is to section the stent–tissue interface with the least amount of disruption of tissue and cellular morphology. Although many methacrylate resin methodologies are successfully applied toward the study of endovascular stents by a variety of research laboratories, the exact formulations, as well as subsequent processing and sectioning methodology, remain largely coveted. In this paper, we describe in detail a methyl methacrylate resin–embedding methodology that can successfully be applied to tungsten carbide blade, as well as saw and grinding sectioning methods and transmission electron microscopy. In addition, we present a comparison of the two sectioning methodologies in terms of their effectiveness with regard to morphological, histochemical, and immunohistochemical analyses. This manuscript contains online supplemental material at http://www.jhc.org. Please visit this article online to view these materials. (J Histochem Cytochem 54:673–681, 2006)

Key Words: stent • artery • restenosis • methyl methacrylate • resin • immunohistochemistry

	Introduction

Top Summary Introduction Materials and Methods Results Discussion Literature Cited

 
THE PAST DECADE has witnessed a marked increase in the use of stents for the treatment of obstructive vascular disease (Froeschl et al. 2004). Stents are metallic scaffolds that are inserted on a balloon catheter into a blood vessel and, with balloon expansion, are deployed against the vessel wall to restore patency (Supplemental Figure 1 ). Renarrowing of stents, or in-stent restenosis (ISR) is a major drawback of this medical technology, occurring in 5–35% of cases (Moses et al. 2003; Sketch et al. 2005). Although coating of stents with polymers that elute a drug that may help prevent cell growth and/or inflammation has somewhat attenuated this problem, it is estimated that in North America alone, 100,000 patients per year will continue to develop ISR (Chu and Waksman 2005). Therefore, attenuating the frequency of ISR remains an important goal of vascular interventional research. Unfortunately, our understanding of the arterial reaction to stent implantation is incomplete—primarily due to technical limitations in the histological study of stented vascular tissue (Froeschl et al. 2004). Conventional paraffin embedding and sectioning procedures have demonstrated limitations with regard to their application to the histological study of the vascular effects of endovascular stent implantation. In most cases, these procedures require the complete removal of the metal stent prior to tissue processing, thereby disrupting normal vascular architecture and, in particular, the stent–tissue interface. To accurately observe the vascular response, it is imperative that the stent–tissue interface be sectioned with the least amount of disruption of tissue and cellular morphology. To this end, it is now common practice to employ methacrylate resins as the embedding media of choice (Chen et al. 2004; Ma and O'Brien 2004).

View larger version (76K): [in this window] [in a new window]   Figure 1 Equipment and procedure for cutting tungsten carbide (TC) (A–C) and saw and grinding (SG) (D–F) sections. (A) Tissue sections (4–5-µm) were cut on an automated Leica RM2255 rotary microtome utilizing a D-profile TC blade. In a 42C water bath, 0.1% aqueous Elmer's glue solution and 30% methanol were used for floating and promoting adherence of the sections to slides. (B) The sections were picked up on 3 aminopropyltriethoxysaline-treated slides, stretched, covered with a thin plastic film, and gently flattened with a rubber roller. (C) Stacked slides were pressed together with a spring clamp. (D) Clean, dry blocks were glued onto polysine slides. (E) Slides with blocks were secured in the slide holder of the Isomet saw (Buehler). Sections (100-µm) were cut with a wafering diamond blade (12.7 mm x 0.4 mm; Buehler). (F) Thick sections were ground on the Metaserve 2000 grinder (Buehler) using two grades of water paper. Bar = 20 mm.

	Materials and Methods

Top Summary Introduction Materials and Methods Results Discussion Literature Cited

 
Stented human coronary arteries from patients were obtained at necropsy and processed as follows.

Tissue Processing
Coronary arteries were removed from the heart, fixed in 10% neutral buffered formalin (NBF) for >24 hr, decalcified in 10% formic acid, and subjected to radiography to localize the stent(s). Decalcification of the entire artery is routinely performed at The Ottawa Hospital to accurately assess adjacent atherosclerotic plaques at either end of the stent. The isolated stented vascular segments were then transferred to 70% ethanol before being processed and embedded in MMA resin (for schedule details, see Supplemental Table 1) (Erben 1997). The following solutions were used to make the resin mixture: Solution I [60 ml MMA (Marivac, Inc.; Montreal, Canada), 35 ml BMA (Marivac), 5 ml methylbenzoate, and 1.2 ml polyethylene glycol 400]; Solution II (100 ml of Solution I, 0.4 g benzoyl peroxide); Solution III (100 ml of Solution I, 0.8 g benzoyl peroxide), and a polymerization resin mixture (400 µl of N,N-dimethyl-P- toluidine added to 100 ml of Solution III and stirred for a few minutes before use). All resin solutions were kept at 4C and stirred for at least 1 hr before use.

Infiltrated stented segments were carefully placed in an upright position in 20-ml polypropylene vials (Leica Instuments GmbH; NuBloch, Germany), which were completely filled with the polymerization mixture to exclude air, then tightly capped and polymerized at –18 to –20C. The presence of oxygen within the embedding vials can ultimately cause bubble formation, thereby inhibiting adequate polymerization and adversely affecting subsequent sectioning technique. This can, however, be prevented either by bubbling nitrogen gas into the tissue-embedded, resin-filled vials or by embedding the tissue segments in vials containing prepolymerized resin bases or plateaus (Erben 1997).

Depending upon specimen size, other suitable molds include glass vials (Fisher Scientific; Nepean, Ontario, Canada), polypropylene microcentrifuge tubes (Fisher Scientific), Eppendorf tubes (Fisher Scientific), or Beem capsules (Marivac). Following adequate polymerization, the specimens were removed by either cutting the ends off the polypropylene vials or, alternatively, breaking the glass vials and pushing the specimens out. The polymerized blocks were then shaped and trimmed of excess plastic with a grinder and sandpaper prior to sectioning.

Rotary Microtome and TC Blade Method of Tissue Sectioning
Tissue sections of a 4–5-µm thickness were generated on an automated Leica RM2255 rotary microtome utilizing a D-profile TC blade (Leica). Because MMA resin is hydrophobic, sectioning was facilitated by moistening both the specimen block and the knife edge with 30% methanol. In a 42C water bath, cut sections were picked up with forceps and first floated onto the surface of a solution of 30% methanol for stretching and then transferred onto the surface of a 0.1% aqueous Elmer's glue solution to promote adherence of the section to the slide (Figure 1A). The sections were then picked up on 3'aminopropyltriethoxysaline-treated slides (Sigma-Aldrich; Oakville, Canada), stretched again with chloroform vapors, covered with a thin plastic film, and gently flattened with a rubber roller (Figure 1B). Finally, the slides were carefully stacked, pressed together with a spring clamp, and dried in an oven at 42C for a period of 2 days (Figure 1C).

SG Method of Tissue Sectioning
A Buehler IsoMet 5000 high-speed precision saw (Buehler; Dusseldorf, Germany) was used to section the stented specimens at a cutting speed of 3200 rpm with a feed advance of 12 mm/min. Continual water cooling was employed throughout sectioning, and a cooling agent (Isomet Plus lubricant, at a ratio of 9:1) was added to reduce heat caused by friction. The surfaces of the resin blocks were prepared for sectioning by grinding on a Metaserve 2000 grinder (Buehler) using fine 600-grit water paper to remove irregular surfaces in the block. Clean, dry blocks were glued onto polysine slides using cyanoacrylate super glue by placing a small drop of glue on the block surface and holding it firmly in position on the slide for 20 sec (Figure 1D). Care must be taken to avoid irregular pressure, because this will result in the formation of bubbles underneath the section. Slides were secured in the slide holder of the Isomet saw, and 100-µm sections were cut (Figure 1E). A wafering diamond blade (12.7 mm x 0.4 mm; Buehler) was used for this purpose. A dressing stone was used to maintain a clean blade edge. Thick sections were ground on the Metaserve 2000 grinder (Buehler) using two grades of water paper (Buehler) (Figure 1F). Coarse 600-grit paper was used to obtain the required thickness, followed by a polishing with 1200-grit paper to remove any scratches. A fine residue of sanding grains was sometimes noted, and attempts to remove the residue using isotonic baths, mild detergents, or polishing were unsuccessful. A final thickness of 8–10 µm was achieved.

Histochemistry
After undergoing deplasticization at room temperature (RT), tissue specimens sectioned using the TC blade method were stained with Gill 3 hematoxylin (Fisher) and eosin (H and E), Movat pentachrome, Masson trichrome, Azan, and Verhoeff's Van Gieson dyes according to conventional methodologies (Bancroft and Gamble 2001). For deplasticization, the sections were brought through solutions of 2-methoxyethylacetate for 20 min thrice, acetone for 5 min twice, and distilled water for 5 min twice.

In our experience, deplasticization of SG sections was often unsuccessful because of the reaction of the solvents with the cyanoacrylate glue used to affix the sections onto the slides, thereby resulting in the sections becoming detached from the slides. A limited number of stains were made possible without prior removal of the resin, and these included Mayer's H and E and Verhoeff's elastin. For H and E staining of SG sections, air-dried slides were placed on a hot plate at 60C and covered with Mayer's hematoxylin (Sigma-Aldrich; Steinheim, Germany) for 12 min, taking care not to let the slides dry out. Slides were then rinsed and blued in running tap water for 30 min before further staining with eosin/phloxine (1:1) on a hot plate at 60C for 1 min, rinsing in water, and finally mounting in Glycergel (DakoCytomation; Glostrop, Denmark). For Verhoeff's elastin staining, slides were immersed in Verhoeff's solution (20 ml alcoholic hematoxylin, 8 ml 10% ferric chloride, 8 ml Lugol's iodine) for 20 min at RT, rinsed in tap water, and mounted in Glycergel (DakoCytomation) (Figure 2 ).

View larger version (107K): [in this window] [in a new window]   Figure 2 Histochemical staining. (A,B) Hematoxylin and eosin (H and E) stain (TC sections); (C,D) H and E-phloxine stain (undeplasticized SG sections); (E) Movat pentachrome stain (TC section); (F) Azan stain (TC section); (G) Verhoeff Van Gieson elastin stain (TC section); (H) Verhoeff Van Gieson elastin stain (undeplasticized SG section). Bar = 200 µm.

View larger version (135K): [in this window] [in a new window]   Figure 3 Immunohistochemistry was performed by applying various antibodies onto deplasticized TC sections and undeplasticized SG sections. (A) HAM56 (TC section); (B) vWF (TC section); (C) SMA (TC section); (D) smooth muscle -actin (SMA) (SG section); (E) routine transmission electron microscopy (TC section); (F) immunogold analysis with 15 nm gold-labeled anti-SMA (TC section). Anti-SMA-labeled, dense bodies are indicated by arrows. Arrowheads define the border of the cell membrane. Bar = 200 µm except where otherwise specified.

View larger version (123K): [in this window] [in a new window]   Figure 4 Combination of immunofluorescence and histochemistry. The double immunofluorescence was performed with rabbit anti-proliferating cell nuclear antigen (PCNA) (green) and mouse anti-SMA (red) antibodies (A), rabbit anti-PCNA (green) and mouse anti-macrophage (CD68, red) antibodies (C), respectively. After obtaining a photomicrograph, the same section was stained with Masson's trichrome (B), and H and E (D), respectively. Both PCNA- and SMA-immunopositive cells are indicated by arrows in A and B. The CD68-immunopositive, PCNA-immunonegative cells are indicated by arrowheads in C and D. A single PCNA-immunopositive, CD68-immunonegative cell is indicated by an arrow in C and D. The inserted photos in the bottom left corner of B and D are the same sections shown at low power. The red stars indicate the former locations of displaced stent struts. Bar = 20 µm.

Transmission Electron Microscopy
Two 10% NBF-fixed human coronary artery specimens were processed in the outlined MMA resin mixture for both routine TEM and immuno-electron microscopy (IEM) analyses. Post-fixation with 1% osmium tetroxide was omitted for the specimens designated for IEM. Thin tissue sections (60–90 nm) were cut on a Reichert Ultracut E ultramicrotome using a diamond knife (Diatome; Hatfield, PA) and mounted on formvar-coated nickel grids (Marivac). Tissue sections for routine TEM were counterstained with 3% aqueous uranyl acetate (Marivac) for 30 min and Reynolds' lead citrate (Marivac) for 5 min. In contrast, tissue sections for IEM analysis were placed onto drops of 1% BSA in PBS for 30 min at RT, and, without rinsing, the sections were placed onto drops of the primary antibody, polyclonal anti-SMA (DakoCytomation; 1:100, diluted in 0.1% BSA-PBS) before being incubated overnight at 4C. After rinsing in PBS, the sections were placed onto drops of a secondary 15 nm gold-conjugated goat anti-rabbit IgG (EY Laboratories, San Mateo, CA; 1:100) for 1 hr at RT. Gentle rinsing in distilled water was followed with counterstaining in aqueous 3% uranyl acetate and Reynolds' lead citrate. The stained sections were then examined by TEM (JEOL 1230; JEOL, Peabody, MA) equipped with software from Advanced Microscopy Techniques (Danvers, MA).

	Results

Top Summary Introduction Materials and Methods Results Discussion Literature Cited

 
Sectioning
Thin tissue sections were consistently produced with a TC knife. The main concern with regard to the TC knife method was the potential for blade damage and resultant scoring of tissue sections. In contrast, production of SG sections proved to be more laborious and technically challenging. Because the SG sections are considerably thicker, specimen depletion was also a concern. Typically, only one or two SG sections were produced per block.

Morphology
Although both sectioning methods yielded sections of acceptable morphological integrity, fine histological detail was more readily observed in the thinner TC sections (Figure 2). Section thickness proved variable with the SG method because of uneven section surfaces, as compared with consistently uniform thickness with the TC method. Sectioning artifacts such as scoring and folding were not present in SG sections but occurred with a noticeable frequency in TC sections. Stent struts consistently remained in situ for SG sections but were often displaced or lost within TC sections (Figure 2).

Histochemical Staining
The range of histochemical staining methods applicable to SG sections was limited because of the incompatibility of the deplasticization solvent and the glue utilized to fix the tissue sections to the slides. Staining was therefore accomplished without deplasticization and limited to H and E and Verhoeff's elastin stains (Figure 2). In contrast, TC sections could be deplasticized and accommodated a broader range of histochemical stains such as H and E, Movat, Azan, Masson, Orcein/Resorcin Fuchsin, and Verhoeff's Van Gieson (Figure 2).

Immunohistochemical Labeling
The range of immunocytochemical stains applicable to SG sections was limited to SMA for reasons previously described. TC sections were deplasticized and stained for specific markers such as SMA, vWF, and HAM56 (Figure 3). Moreover, we devised a novel method of combining fluorescent immunolabeling and histochemical staining on the same slide. For example, we immunolabeled specimens with a fluorescently tagged antibody to PCNA, obtained a fluorescent photomicrograph, and then performed a Masson's trichrome stain on the same section before repeating light photomicrographs of the same tissue section (Figure 4). This combined approach allowed us to specifically localize proliferating cells in relation to various components of the extracellular matrix of these lesions.

TEM and Gold Labeling
The MMA resin proved conducive for the performance of both routine TEM and immunogold analyses. For example, 15 nm gold-labeled anti-SMA antibody was successfully used to immunolabel smooth muscle cells (Figure 3).

	Discussion

Top Summary Introduction Materials and Methods Results Discussion Literature Cited

 
With the escalating global use of stents to treat patients with obstructive coronary artery disease, the problem of ISR is of paramount importance. Although drug-coated stents may reduce the frequency of ISR, the overall incidence of ISR is still estimated to be 9%, and cost may prevent the widespread use of these bioprostheses (Waksman 2004). Currently, there is much interest in better understanding the vascular response to stent implantation, as well as identifying bona fide therapeutic targets to prevent ISR. Unfortunately, not all laboratories are adequately equipped or have sufficient experience to engage in the intricacies of sectioning, staining, and immunolabeling vascular tissues that contain metallic bioprostheses. In this manuscript, we review methodologies and outcomes of two sectioning methods for resin-embedded arteries that contain metallic stents.

The SG method consistently produces intact tissue sections without displacement of stent struts or scoring or folding of tissue. Moreover, it is superior to the TC method when stents made of more durable alloys, such as nitinol, are encountered or when longitudinal sections of stented arteries are required. Nonetheless, there are important limitations to note about the SG method. First, there is significant tissue depletion (kerf loss) with the SG technique, because only one or two sections can be produced per block. Second, because the minimum thickness of sections produced with SG is 8–10 µm, the quality of morphological studies may be inferior to that of the TC approach. Third, to produce multiple SG sections is laborious and certainly more technically challenging than producing TC sections. Fourth, the initial equipment setup for the SG method is considerably more expensive than that necessary for performing the TC method (refer to Supplemental Table 2).

The TC method consistently yields thin serial sections that are suitable for both histochemical and immunohistochemical staining of deplasticized sections (Figure 2). Although occasional scoring and folding, together with stent strut displacement, do occur in TC sectioning, in our experience, this method clearly provides superior morphological and immunolabeling detail at the tissue–stent interface. For example, using this technique, we have carefully examined stented vessels from animal arteries and determined the efficacy of novel anti-inflammatory therapies that reduce stent neointima formation (Chen et al. 2004; Ma and O'Brien 2004). This method has also provided accurate information concerning the status of human coronary stents for both forensic and hospital autopsies performed over the past 8 years at The Ottawa Hospital. Of note, others have used the SG technique utilizing Technovit 4000 glue (Heraeus-Kulzer; Friedrichsdorf, Germany) to adhere tissue sections onto slides and have obtained seemingly very acceptable immunolabeling results (Brasen et al. 2001; Rammelt et al. 2004) This has so far proven unsuccessful in our hands.

In summary, the described MMA embedding protocol adequately accommodates both the TC and SG methods of sectioning. Although either method of sectioning can be effectively utilized to evaluate the histopathological features of resin-embedded vascular stents, the TC method allows thinner sections to be used and a wider range of histochemical staining and immunolabeling procedures to be readily performed. Future studies of the vascular response to a variety of current and newer stent materials (e.g., magnesium biodegradable stent) will provide important clues to the pathogenesis of ISR and may allow the formulation of a durable scaffolding device for the growing number of patients that require percutaneous revascularization.

	Acknowledgments

 
These methods were initially developed during P.R.'s former affiliation with The Ottawa Hospital Department of Pathology and Laboratory Medicine in collaboration with M.K.B. and under the supervision of J.P.V. The project was supported by an operating grant and research chair (UOP 36383, URC 57093) held by E.R.O. and jointly funded by the Canadian Institutes of Health Research and Medtronic. The Heart and Stroke Foundation of Ontario program grant no. 5275 funded some of the laboratory equipment used in this study. The method of combination of immunofluorescence and histochemistry was developed by Y-X.C., postdoctoral fellow, under the supervision of E.R.O.

The authors are indebted to Jordana Laporte for administrative support and preparation of this manuscript.

	Footnotes

 
Received for publication August 31, 2005; accepted January 9, 2006

	Literature Cited

Top Summary Introduction Materials and Methods Results Discussion Literature Cited

 

Bancroft JD, Gamble M (2001) Theory and Practice of Histological Technique. 5th ed. New York, Churchill Livingstone

Brasen JH, Kivela A, Roser K, Rissanen TT, Niemi M, Luft FC, Donath K, et al. (2001) Angiogenesis, vascular endothelial growth factor and platelet-derived growth factor-BB expression, iron deposition, and oxidation-specific epitopes in stented human coronary arteries. Arterioscler Thromb Vasc Biol 21:1720–1726[Abstract/Free Full Text]

Chen YX, Ma X, Whitman S, O'Brien ER (2004) Novel antiinflammatory vascular benefits of systemic and stent-based delivery of ethylisopropylamiloride. Circulation 110:3721–3726[Abstract/Free Full Text]

Chu WW, Waksman R (2005) Contemporary use of drug-eluting stents. Curr Treat Options Cardiovasc Med 7:35–46

Erben RG (1997) Embedding of bone samples in methylmethacrylate: an improved method suitable for bone histomorphometry, histochemistry, and immunohistochemistry. J Histochem Cytochem 45:307–313[Abstract/Free Full Text]

Froeschl M, Olsen S, Ma X, O'Brien ER (2004) Current understanding of in-stent restenosis and the potential benefit of drug eluting stents. Curr Drug Targets Cardiovasc Haematol Disord 4:103–117[CrossRef][Medline]

Ma X, O'Brien ER (2004) Antagonism of the alpha4 integrin subunit attenuates the acute inflammatory response to stent implantation yet is insufficient to prevent late intimal formation. J Leukoc Biol 75:1016–1021[Abstract/Free Full Text]

Malik N, Gunn J, Holt CM, Shepherd L, Francis SE, Newman CMH, Crossman DC, et al. (1998) Intravascular stents: a new technique for tissue processing for histology, immunohistochemistry, and transmission electron microscopy. Heart 80:509–516[Abstract/Free Full Text]

Moses JW, Leon MB, Popma JJ, Fitzgerald PJ, Holmes DR, O'Shaughnessy C, Caputo RP, et al., for the SIRIUS Investigators (2003) Sirolimus-eluting stents versus standard stents in patients with stenosis in a native coronary artery. N Engl J Med 349:1315–1323[Abstract/Free Full Text]

Rammelt S, Manthey S, Zwipp H (2004) Immunohistochemical characterization of the bone-implant interface on MMA-embedded cutting and grinding sections. Eur Cell Mater 7(suppl 1):82

Rogers C, Parikh S, Seifert P, Edelman ER (1996) Endogenous cell seeding: remnant endothelium after stenting enhances vascular repair. Circulation 94:2909–2914[Abstract/Free Full Text]

Sketch J, Ball M, Rutherford B, Popma JJ, Russell C, Kereiakes DJ (2005) Evaluation of the Medtronic (Driver) cobalt-chromium alloy coronary stent system. Am J Cardiol 95:8–12[Medline]

Theuns HM, Bekker H, Fokkenrood H, Offerman E (1993) Methyl-methacrylate embedding of undecalcified rat bone and simultaneous staining for alkaline and tartrate resistant acid phosphatase. Bone 14:545–550[Medline]

van Beusekom HMM, Whelan DM, van de Plas M, van der Giessen WJ (1996) A practical and rapid method of histological processing for examination of coronary arteries containing metallic stents. Cardiovasc Pathol 5:69–76

Waksman R (2004) Antagonist's view: will cost constraints affect the widespread use of DES? http://www.cardiosource.com/expertopinions/hottopics/article.asp?paperID=74

CiteULike    Complore    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this?

This article has been cited by other articles:

				P. Zilla, P. Human, M. Wolf, W. Lichtenberg, N. Rafiee, D. Bezuidenhout, N. Samodien, C. Schmidt, and T. Franz Constrictive external nitinol meshes inhibit vein graft intimal hyperplasia in nonhuman primates J. Thorac. Cardiovasc. Surg., September 1, 2008; 136(3): 717 - 725. [Abstract] [Full Text] [PDF]

				S. Rousselle and J. Wicks Preparation of Medical Devices for Evaluation Toxicol Pathol, January 1, 2008; 36(1): 81 - 84. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Supplemental Data for this article All Versions of this Article: jhc.5A6824.2006v1 54/6/673    most recent Alert me when this article is cited Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Rippstein, P. Articles by O'Brien, E. R. Search for Related Content PubMed PubMed Citation Articles by Rippstein, P. Articles by O'Brien, E. R. Social Bookmarking                      What's this?

Guidelines | Subscriptions | About | exPRESS - Current - Archive | Business Information | Contact