Pregnancy-Induced Remodeling of Collagen...

Pregnancy-Induced Remodeling of Collagen Architecture and Content

in the Mitral Valve

CAITLIN M. PIERLOT,1 J. MICHAEL LEE,1,2 ROUZBEH AMINI,3 MICHAEL S. SACKS,4 and SARAH M. WELLS1

1School of Biomedical Engineering, Dalhousie University, 5981 University Avenue, Halifax, NS B3H 3J5, Canada; 2Departmentof Applied Oral Sciences, Dalhousie University, Halifax, NS, Canada; 3Department of Biomedical Engineering, The Universityof Akron, Akron, OH, USA; and 4Center for Cardiovascular Simulation, Institute for Computational Science and Engineering

and the Department of Biomedical Engineering, The University of Texas at Austin, Austin, TX, USA

(Received 14 April 2014; accepted 23 July 2014; published online 8 August 2014)

Associate Editor Jane Grande-Allen oversaw the review of this article.

Abstract—Pregnancy produces rapid, non-pathological vol-ume-overload in the maternal circulation due to the demandsof the growing fetus. Using a bovine model for humanpregnancy, previous work in our laboratory has shownremarkable pregnancy-induced changes in leaflet size andmechanics of the mitral valve. The present study sought torelate these changes to structural alterations in the collage-nous leaflet matrix. Anterior mitral valve leaflets wereharvested from non-pregnant heifers and pregnant cows(pregnancy stage estimated by fetal length). We measuredchanges in the thickness of the leaflet and its anatomic layersvia Verhoeff-Van Gieson staining, and in collagen crimp(wavelength and percent collagen crimped) via picrosirius redstaining and polarized microscopy. Collagen concentrationwas determined biochemically: hydroxyproline assay fortotal collagen and pepsin-acid extraction for uncrosslinkedcollagen. Small-angle light scattering (SALS) assessedchanges in internal fiber architecture (characterized by degreeof fiber alignment and preferred fiber direction). Pregnancyproduced significant changes to collagen structure in themitral valve. Fiber alignment decreased 17% with an 11.5�rotation of fiber orientation toward the radial axis. Collagenfiber crimp was dramatically lost, accompanied by a 53%thickening of the fibrosa, and a 16% increase in totalcollagen concentration, both suggesting that new collagen isbeing synthesized. Extractable collagen concentration waslow, both in the non-pregnant and pregnant state, suggestingearly crosslinking of newly-synthesized collagen. This studyhas shown that the mitral valve is strongly adaptive duringpregnancy, with significant changes in size, collagen contentand architecture in response to rapidly changing demands.

Keywords—Mitral valve, Collagen remodeling, Pregnancy,

Collagen fiber architecture.

INTRODUCTION

Dramatic increases in blood volume and cardiacdimensions occur during pregnancy24,40,53 as thematernal circulation accommodates the developingplacental circulation. In humans, blood volumeincreases by approximately 40% during gestation,triggering left-ventricular hypertrophy and enlarge-ment of the atrial and ventricular chambers.11,24,26,40,53

Heart valve annular dilatation also occurs duringpregnancy, with the orifice area of the mitral valve inhumans increasing by up to 12%.40 Annular geometrystrongly affects leaflet tissue stresses in vivo, particu-larly in the central region of the leaflet.3,43 Thus,annular dilation causes a substantial increase in theradius of curvature of the closed leaflet and, by the lawof Laplace, the tension across the closed valve is sig-nificantly increased. Furthermore, this rise in mem-brane tension is much greater than the rise in orificearea. Finite element models predict that an 18%increase in orifice area (similar to that observedin pregnancy) would result in a 40–50% increase intensile stress in the anterior mitral valve leaflet, and amore than 60% increase in tensile stress in the belly ofthe leaflet.31 Interestingly, mitral valve regurgitation,generally due to mitral valve prolapse with incompleteleaflet coaptation, is relatively uncommon in preg-nancy.9,41 This might be explained by the mitral valve’slarge functional reserve: a surplus of the coaptationsurface area that allows annular expansion withoutdevelopment of regurgitation.36,40 Another possibleexplanation, however, is that the mitral valve leafletsundergo adaptive remodeling during pregnancy.

To investigate this possibility, we previously exam-ined the changes in bovine mitral valve dimensions and

Address correspondence to Sarah M. Wells, School of Biomedi-

cal Engineering, Dalhousie University, 5981 University Avenue,

Halifax, NS B3H 3J5, Canada. Electronic mail: [email protected]

Annals of Biomedical Engineering, Vol. 40, No. 10, October 2014 (� 2014) pp. 2058–2071

DOI: 10.1007/s10439-014-1077-6

0090-6964/14/1000-2058/0 � 2014 Biomedical Engineering Society

2058

mechanical properties during pregnancy.53 A bovinemodel was used due to important similarities tohumans in singleton birth deliveries, hemodynamicchanges and hormone levels,28,34,39,49 and gestationalperiod.6,18 As well, the stage of bovine pregnancy canbe estimated using the crown-to-rump length of thefetus.19 That study showed, for the first time, signifi-cant adaptive remodeling of the bovine mitral valveduring pregnancy, with striking increases in leaflet sizeand chordal attachments, as well as a biphasic changein leaflet extensibility.53 Leaflets enlarged uniformly inboth the radial and circumferential directions, result-ing in a 33% increase in leaflet area, with a surprising25% increase in the number of chordae tendineaeattachments into the ventricular surface. It is interest-ing that leaflet mechanical properties did not change intime-step or direction with leaflet geometry. Instead,leaflet extensibility rapidly decreased by 30% (leftwardshift in the tension-stretch curve) in early pregnancy,then, remarkably, increased linearly over the remain-der of gestation, returning to pre-pregnant mechanicsby late pregnancy.

The results of our previous study showed thatremodeling of the mitral valve anterior leaflet duringpregnancy involves two phases. In early pregnancy,leaflet extensibility decreases as it enlarges, attaining itsnew anatomical/dimensional set-point correspondingto the increased orifice area. In late pregnancy, theenlarged leaflet dimensions are maintained whileextensibility then increases (largely in the radial direc-tion), back to pre-pregnant values by term. The triggerfor this remodeling may be the increased stress in theleaflet that must accompany its enlargement. Latepregnancy may then represent the phase where thevalve leaflet becomes ‘‘entrenched’’ at increaseddimensions and, as it remodels toward normalizingleaflet stress, re-attains its pre-pregnancy mechanicalproperties. The reversal of extensibility (i.e., anincrease back to pre-pregnant values) in late pregnancycould contribute to the maintenance of coaptation, andmay be the mechanism via which mitral regurgitation isprevented in pregnancy, despite the large increase invalve orifice area.9,41 Such 2-stage remodeling could bea result structural changes that are confined to—ordistinct in—early vs. late pregnancy. For instance, theexpansion and decreased stiffness of the leaflet in earlypregnancy may be a result of leaflet thinning and/orincreased collagen fiber splay, while the stiffening ofthe leaflet in late pregnancy may result from leafletthickening/increased content and/or crosslinking ofcollagen. This may correspond to an initial plastic or‘‘permanent set’’ adaptation followed by a true‘‘growth’’ phase.

Thus, the purpose of the present study was thus torelate changes in leaflet size and mechanical properties

(assessed in our previous study) to changes in thestructural components of the leaflet, with a particularfocus on collagen. Given the complex interplaybetween leaflet dimension and mechanical propertiesduring mitral valve remodeling, detailed histologicaland biochemical analyses were called for. Expansion ofthe leaflet with a biphasic change in extensibility couldinvolve changes in the thickness of the leaflet or it’sindividual layers, collagen fiber architecture, and/orcollagen content. Thus, we sought to (i) determinechanges in collagen fiber orientation (alignment andpreferred direction), collagen crimp, thickness of theleaflet and its individual layers, and collagen content(total and acid/pepsin-soluble) during pregnancy, andto (ii) relate these changes to the previously observedpregnancy-induced changes in leaflet size andmechanical properties.53

METHODS

Tissue Harvest and Sample Preparation

All protocols for the harvesting of bovine tissueswere approved by the University Committee on Lab-oratory Animals (UCLA) at Dalhousie University.The hearts of young female cattle were purchased asfood from a local abattoir (Armstrong Food ServicesLimited, Kingston, Nova Scotia, Canada) immediatelyfollowing slaughter. Hearts were collected from heifers(female cattle of sexual maturity which had never beenpregnant), and from pregnant cows. To assess gesta-tional age in pregnant cattle, fetal crown-rump lengthwas measured, with a full term (278–290 days gesta-tion) bovine fetus measuring approximately 100 cm.19

Pregnant animals ranged in gestational age from61 days (9 cm fetal length) to full term (98 cm fetallength).

All cattle were under 30 months of age, and due toimposed federal food regulations at Canadian Abatt-oirs, animals were inspected by a federally licensedmeat inspector such that any animals exhibiting signsof illness were excluded from collection (CanadianFood Inspection Agency, CFIA). Cattle were dividedinto two groups: non-pregnant (NP), and pregnant (P)based on their reproductive status at slaughter.

Since the mitral valve displays differing structuralproperties between leaflets, with higher stresses expe-rienced in vivo by the anterior leaflet,29,30 all experi-ments used this leaflet. It was excised as close to thevalve root as possible. Leaflets were washed withHanks’ physiological solution (pH 7.4) including 6 mg/L trypsin inhibitor (lyophilisate), and an antibiotic–antimycotic agent containing 10,000 units/mL of Pen-icillin G, 10 mg/mL streptomycin sulphate, and 25 lg/

Collagen Architecture and Content in the Mitral Valve 2059

mL amphotericin B (Sigma-Aldrich Canada, Oakville,ON, Canada).

Small-Angle Light Scattering

The SALS protocol has been detailed previ-ously.42,45,54 Briefly, a non-polarized 4 mW HeNe laser(k = 632.8 nm) is passed through the valve leaflet tis-sue. Scattering of photons from internal fiber structureproduces a light intensity distribution (I(/)), whichcaptures the distribution of fiber angles within thetissue. The degree of fiber orientation is proportionalto the width of the intensity distribution and isexpressed via the orientation index (OI): the anglerange (from 0� to 180�) that contains one half of thetotal area under the I(/) distribution and therebyrepresents 50% of the total fibers (Fig. 1). To clarifythe physical interpretation for this index, a normalizedorientation index (NOI) was calculated and exclusivelyused in the present study:

NOI ¼ 90� �OI

90�� 100%

NOI thus ranges from 0% (an isotropic distribution) to100% (all fibers uniformly aligned to the same

direction) fiber network, providing an indicator of thedegree of fiber alignment.4,21,25,33 High NOI values arecharacteristic of highly oriented fiber networks (nar-row distribution peak), and low NOI values indicateless oriented, more random networks (broad distribu-tion peak). The centroid of the fiber distributionobtained from the SALS data (/c) is indicative of thepreferred fiber direction (/p) of the fiber network,although rotated by 90� from that direction.

Complete mitral valve leaflets from 4 non-pregnantheifers and 5 pregnant cows were fixed from fresh byimmersion in 10% neutral buffered formalin for a mini-mum of 72 h, then prepared for small-angle light scat-tering (SALS) analysis of tissue fibrous structure. Toincrease translucency of tissues for optical SALS mea-surements, specimens were dehydrated and cleared in agraded glycerol/water solution (formalin fi etha-nol fi water fi glycerol), then stored in a final solu-tion of 100% glycerol for transport to University ofPittsburgh for SALSmeasurement. The entire leaflet wasscanned and the resulting data separated into threeregions using data from five different areas (Fig. 2): (i)Free-edge (regions 1 and 2), (ii) Belly (region 3), and (iii)Fixed-edge (regions 4 and 5). Comparison of the scat-tered light intensity distribution (NOI), and preferred

FIGURE 1. Schematic exhibiting contributions of collagen crimp to scattered light distribution (I(/) and orientation index (OI) asmeasured by SALS: (a) small-scale, local changes in fiber orientation due to crimp, (b) corresponding broad intensity peak with alarge OI (small NOI), (c) straightened or stretched fiber, and (d) corresponding narrow intensity peak with small OI (large NOI).Alternating light and dark bands depicted in (a) and (d) represent the periodic arrangement of collagen fibers (D-bands). This figurewas modified with permission from Liao et al.,33 Figure 9, page 85.

PIERLOT et al.2060

fiber direction (/p), between non-pregnant and pregnantanimals was used to determine whether collagen fiberreorientation is occurring in pregnancy.

Histological Analysis

For histological analysis, valves were collected fromanother set of animals. Leaflets from 22 animals (9non-pregnant heifers and 13 pregnant cows) weredivided to produce two symmetric halves of the leaflet

(Fig. 3), both fixed from fresh in 10% neutral bufferedformalin for a minimum of 72 h, dehydrated in 70%ethanol, embedded in paraffin, sectioned into 5 lmserial sections, mounted on slides, and dried at 57 �Cfor a minimum of 24 h. Embedding and sectioning wasperformed by the Histology Research Services Labo-ratory (Faculty of Medicine, Dalhousie University).Circumferential cross-sections were cut from one halfof the leaflet for picrosirius red (PR) staining toexamine collagen alignment and crimp. Radial cross-sections were cut in the other half of the leaflet forVerhoeff-Van Gieson (VVG) staining to identify leafletlayering (Verhoeff-Van Gieson Elastin Staining Kit,Polysciences, Inc., Washington, PA) (Fig. 3). Eachradial section contained the complete leaflet cross-section from the free edge to the fixed edge. For bothstaining protocols, sections were deparaffinized inxylene and rehydrated in graded ethanol/water solu-tions. For picrosirius red staining protocol, circumfer-ential sections were stained for 1 h with 0.1% PicrosiriusRed solution, followed by several water washes. ForVerhoeff-Van Gieson staining, radial sections werestained for 1 h with Verhoeff’s solution, differentiated in2% ferric chloride for 1 min, followed by several waterwashes. All stained slides were then dehydrated in eth-anol and cleared in xylene for mounting.

Collagen Crimp Analysis

Images were taken using a Nikon Eclipse E600 lightmicroscope equipped with a polarizer and a 10 MPAmScope digital camera. Collagen crimp was charac-terized by two measures: (i) crimp length, peak-to-peakmeasurement of crimp period, and (ii) the percentageof leaflet area which was crimped. For both measures,6 adjacent images were taken at 409 objective mag-nification along the circumferential direction, spanning2 mm of the belly region of the leaflet, and analyzedusing ImageJ software (National Institutes of Health).For crimp length measurements, a line was drawnacross the centerline of each image (through the centerof the image, aligned to the principal crimp direction)and every distinguishable crimp length along that linewas recorded (Fig. 4). For crimped area measure-ments, a grid (0.0003 mm2/square) was placed on eachimage. The number of grid points in contact withcrimped tissue, as well as the total number of gridpoints in contact with tissue (crimped or uncrimped)was recorded. The ratio of crimped-to-total grid pointswas then used to calculate the area percentage of theleaflet occupied by crimped. Both crimp length andcrimped area were averaged across all 6 images. Blin-ded measurements were made by three observers, withexcellent agreement between observer counts.

FIGURE 2. Image of an isolated heifer mitral valve leaflet:orientation index for SALS analysis was examined in fiveregions of the anterior mitral valve leaflet: two nearer the free-edge (regions 1 and 2), two nearer the fixed edge (regions 4and 5), and one centered in the belly region (region 3) of theleaflet. Ruler scale is in cm.

FIGURE 3. Histological sectioning: each leaflet was dis-sected along the dotted lines to divide the leaflet in half andexpose the belly region for sectioning. 5 lm serial sectionswere cut starting at the dotted lines to obtain circumferentialsections for picrosirius red (PR) staining to examine collagenalignment and crimp, and radial sections for Verhoeff-VanGieson (VVG) staining to identify leaflet layering and layerthicknesses. Ruler scale is in cm.


Leaflet and Layer Thicknesses

VVG stained sections were photographed at lowmagnification (using a 59 or 109 objective) along theentire length of the valve leaflet, using a Zeiss Axioplan2 Imaging Microscope (Cellular & Molecular DigitalImaging Facility, Faculty of Medicine, DalhousieUniversity) and analyzed using AxioVision software(Release 4.8.1). ImageJ imaging software pluginsMosaicJ51 and TurboReg50 were used to stitch to-gether one large mosaic image for each sample. Anarray of lines was placed on each mosaic such that 25vertical lines transected the full thickness of the tissuealong the length of the leaflet (Fig. 5). Thicknessmeasurements, of the full leaflet as well as of eachlayer, were taken at each gridline. From these mea-surements the following parameters were extracted:total thickness, fibrosa thickness, spongiosa thickness,

and atrialis thickness. Thickness measurements werethen averaged across the length of the leaflet for eachvalve sample, producing these same four parametersfor each individual valve leaflet.

Collagen Content

For each biochemical assay, valves were collectedfrom yet another set of animals: 25 hearts (10 non-pregnant heifers and 15 pregnant cows) for hydroxy-proline collagen assay, and 24 hearts (9 non-pregnantheifers, and 15 pregnant cows) for Sircol collagenassay. Heart valve leaflets for both collagen assayswere excised, wrapped in cheesecloth soaked in Hanks’physiological solution, placed immediately in the286 �C freezer, and stored until they could be tested.The first set of frozen leaflets were freeze-dried for aminimum of 52 h. Dry samples of approximately10 mg dry weight, from the belly region of the leaflet,were weighed and recorded. Total collagen content wasestimated using the hydroxyproline assay described byWoessner.55 Assay product was read using a micro-plate reader (Synergy HT, Bio-Tek Instruments Inc.,Winooski, Vermont) to measure dye absorbance at awavelength of 561 nm. Comparison of sample absor-bance to a generated standard absorbance curve wasused to quantify the hydroxyproline present in eachleaflet sample. The quantity of collagen in each sample,normalized to dry weight, was determined by dividingthe quantity of hydroxyproline by 0.1277, based on astudy by Keeley et al.27

The Sircol Collagen Assay Kit (Biocolor Ltd., Car-rickfergus, UK: Accurate Chemical & Scientific Corpo-ration, Westbury, NY, USA) was used to assess thecontent of acid and pepsin-soluble collagen. Thisextractable form of collagen is often interpreted to benewly laid down collagen that is not sufficiently cross-linked into the network to resist solubilization. For thisstudy, the second set of frozen leaflets were thawed anddissected to take samples from the belly of the leaflet, andweighed prior to testing. All samples were assayed induplicate for extractable collagen, and compared to a set

FIGURE 4. Collagen crimp measurement: Crimp lengthmeasurements in each image were taken along a line passingthrough the center of the image and normal to the crimpdirection (A), and averaged across all 6 adjacent images.Crimped area measurements were taken at each intersectionof gridlines (B), as the ratio of crimped-to-total grid points(grid 5 0.0003 mm2/square) across consecutive images, tocalculate the area percentage of the leaflet occupied by crimp.

FIGURE 5. Leaflet and layer thickness measurements: an array of 25 vertical lines, normal to the leaflet surface, was placed oneach mosaic of the complete leaflet section, with lines transecting the full thickness of the tissue along the length of the leaflet,from the fixed edge to the free edge. Thickness measurements, of the full leaflet as well as each layer (fibrosa, spongiosa, andatrialis), were taken at each line and then averaged across the length of the leaflet for each valve sample.

PIERLOT et al.2062

of blanks and standards. Following the assay, a micro-plate reader (Synergy HT, Bio-Tek Instruments Inc.,Winooski, Vermont) was used to measure the dyeabsorbance of each prepared plate at 555 nm.Dryweightmeasures of extractable collagen were calculated usingthe wet/dry weights from each valve.

Statistical Analysis

All results are expressed as themean ± standard errorof the mean (SEM), with the n value representing thenumber of animals per group.All data is divided into twogroups: NP (non-pregnant), and P (pregnant). Statisticalcomparisonsweremade using t test comparisons betweenthe two groups. To evaluate changes in any parameter asa function of pregnancy duration, data were plotted as afunction of gestational age and fitted with a least-squareslinear regression. The effect of pregnancy duration wasalso assessed by sub-dividing the pregnant animals intoearly pregnant (EP) and late pregnant (LP) groupsaccording to fetal crown-to-rump length: cows carryingfetuses of crown-to-rump length <45 cm (0–169 days)were classified as EP, and those carrying fetuses >55 cm(193–270 days) were classified as LP—the mid-preg-nancy group of 45–55 cm (169–193 days) was omitted inthis comparison to obtain a more accurate separation ofearly and late groups. A one-way ANOVA was per-formed, followed by Tukey honestly significant differ-ence comparisons among the three groups (NP, EP, andLP). For analysis of the SALS data, statistical compari-sons were made in each of the three regions of the valveleaflet. All statistics presented are two-tailed t test com-parisons between the non-pregnant and pregnant groups.For each of SALS, histology, hydroxyproline, and Sircolassays, tissuewas collected froma separate set of animals,therefore statistics are unpaired.

Results were considered significant for p< 0.05.Outliers were defined as data points falling outside of thefollowing range: [25th quartile 2 1.5 9 (interquartilerange)] to [75th quartile + 1.5 9 (interquartile range)],where interquartile range = 75th quartile 2 25th quar-tile. These data points were excluded from further ana-lysis. Two outliers were removed from P % crimp, oneremoved from P leaflet thickness, one removed from NPatrialis thickness, and one NP removed from total col-lagen content. All statistical analyses were performedusing JMP Statistical Software (Version 10.0, SASInstitute, Inc., Cary, NC).

RESULTS

We originally hypothesized that the bovine anteriormitral valve leaflet remodels during pregnancy by (i)‘‘biphasic’’ structural changes that occur in early

pregnancy that are reversed in late pregnancy, or (ii) acombination of structural changes that are confined toearly or late pregnancy. Surprisingly, none of our struc-tural measurements from the current study showed sig-nificant changes associated with pregnancy duration(fetal length or early vs. late pregnancy), as in our earlierstudy,53 nor were any changes biphasic, as previouslyseen with pregnancy-induced changes in leaflet extensi-bility.53 Instead, changes in all of our structural mea-surements were monotonic upward or downward duringpregnancy. Data obtained from all pregnant animalswere therefore grouped and significant differences weredetected vs. data from non-pregnant animals. To enabledirect comparisons, data from the present study and fromour previous study are presented here only as as meanvalues for pregnant (P) and non-pregnant (NP) animals.

Small-Angle Light Scattering

The collagen fiber architecture of entire bovine -mitral valve leaflets were mapped, focusing on mea-surements of the NOI and preferred fiber direction(Fig. 6). Fiber alignment of the leaflets decreasedduring pregnancy in the belly region of the leaflet only,as indicated by a significant 20% decrease (p = 0.0174)in the normalized orientation index (NOI) from non-pregnant (NOI = 45.8 ± 0.5%) to pregnant (NOI =

36.5 ± 1.9%) (Fig. 7). The orientation indices in thefree edge and fixed edge regions were unchanged inpregnancy (Table 1). The preferred fiber direction (/p)was referenced to the circumferential axis of the leafletfrom the SALS distribution centroid (/c) as /c—90 .This is the direction plotted as the small arrows inFig. 6. To describe the rotation of preferred fiberdirection away from the circumferential direction, theabsolute value of the preferred fiber direction wascalculated as:

/p ¼ /c � 90�j j

The collagen fiber bundles of the non-pregnantgroup were initially closely aligned along the circum-ferential direction of the leaflet (/p = 1.8� ± 1.1�), butrotated slightly toward the radial axis of the leaflet inpregnancy (/p = 13.3� ± 2.9�) (p = 0.0072, Fig. 8), inthe belly region only.

Together, this radial rotation of fibers and thedecrease in fiber alignment, correspond to a largerangular deviation of collagen fibers from the preferredfiber direction (i.e., increased fiber splay). In the freeedge and fixed edge regions of the leaflets from thenon-pregnant group, collagen fiber bundles werealigned away from the circumferential direction at15.5� ± 2.6� (free edge) and 15.8 ± 4.3� (fixed edge)


respectively. This structure was unchanged in preg-nancy (Table 1).

Collagen Crimp

Although the entire valve leaflet was examined forstructural changes in the collagen network using theSALS technique, alterations were found only in thebelly region of the leaflet. Analysis of picrosirius red-stained samples was therefore conducted in the bellyregion to determine how changes in collagen crimpwere contributing to the apparent increased fiber

alignment in that region (Fig. 9). A summary of allstructural, mechanical, and thermal data obtained forthe belly region of the bovine anterior mitral valveleaflet from our present and previous studies53 areshown in Table 2. Collagen crimp results showed aremarkable 186% increase in crimp length(p = 0.0079) from non-pregnant (22.9 ± 1.2 lm) topregnant (65 ± 15 lm) (Table 2; Fig. 10a). Thisincrease in crimp length was accompanied by a 45%decrease in the percentage of leaflet area occupied bycrimped tissue (p = 0.0116) from non-pregnant(40.2 ± 6.0%) to pregnant (22.8 ± 3.0%) (Table 2;Fig. 10b).

Leaflet and Layer Thickness

The mitral anterior leaflet thickness was unchangedduring pregnancy. However, considering the layers sep-arately, the atrialis became 46% thinner (p = 0.0447),while the fibrosawas 53%thicker (p = 0.0372) (Table 2).The spongiosa thickness was not significantly changed inpregnancy.

Collagen Content

Alongside the increase in fibrosal thickness, mitralvalve total collagen content (concentration byhydroxyproline assay) increased by 16% (p = 0.0256),from non-pregnant (56.7 ± 3.0% dry wt.) to pregnant(66.0 ± 3.3% dry wt.) (Table 2). This suggests thatcollagen was preferentially laid down in the fibrosa.The concentration of acid/pepsin soluble (perhapsnewly-synthesized) collagen was very low in non-pregnant animals (0.6 ± 0.1% dry wt.), and was sur-prisingly unchanged with pregnancy (Table 2). Watercontent was unchanged in pregnancy.

FIGURE 6. Color maps of fiber alignment (normalized orientation index, NOI) and preferred fiber direction (/p; indicated by arrowdirection) from the excised anterior mitral valve leaflet for (a) non-pregnant (NP) and (b) pregnant (P) animals. In the belly region ofnon-pregnant animals, the collagen fiber network was moderately well aligned (green) with a very uniformly, circumferentiallyaligned preferred fiber direction (horizontal vectors). In the belly region of pregnant animals, fiber alignment is reduced (purple-blue) and there is a loss of the very uniform fiber direction (rotated vectors).

FIGURE 7. Regional normalized orientation index (in per-cent) of the anterior mitral valve leaflet from non-pregnant(open bars; n 5 4) and pregnant (filled bars; n 5 5) animals.Bars indicate mean 6 SE. Statistical comparisons were madein each region (free edge, belly, and fixed edge), between non-pregnant and pregnant groups using two-tailed t test com-parisons of groups. Columns labeled with an asterisk showeda statistically significant change with pregnancy.

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DISCUSSION

Pregnancy is a physiological condition characterizedby rapid and dramatic increases in blood volume andcardiac dimensions, thereby resulting in significantincreases in valve leaflet tension. In our previousstudy,53 we showed, for the first time, significantpregnancy-induced adaptive remodeling of the anteriorleaflet of the bovine mitral valve, with large increases inleaflet size and a biphasic change (an increase, thendecrease) in leaflet extensibility. Leaflets enlarged uni-formly in both the circumferential and radial direc-tions, resulting in a 40% increase in leaflet area.Collagen denaturation temperature decreased 2.4 �C,indicative of significant collagen remodeling, while

significant increases in HIT load decay for control (t1/2-control) and treated (t1/2-treated) samples signifiedincreases in both mature and total crosslinkingrespectively. These findings raised the question of howstructural and material factors, such as changes inleaflet thickness and collagen architecture, might becontributing to the mechanisms of pregnancy-inducedremodeling of the mitral valve. The present studysought to answer that question.

Despite the large and rapid increases in leaflet areaduring pregnancy, the gross collagen fiber architecturewas largely maintained across the leaflet. However,significant changes in collagen fiber orientation andalignment were observed in the belly region, wherethere was a decrease in fiber alignment (NOI decreased17%), with an 11.5� rotation of the preferred fiberdirection away from the circumferential axis (towardthe radial axis). These structural changes wereaccompanied by a remarkable loss in collagen fibercrimp, with the percentage area occupied by crimpnearly halved, and the crimp length nearly doubling.Accompanying these changes was a 46% thinning ofthe atrialis, and a 53% thickening of the fibrosa, with a16% increase in total collagen content. We’ve previ-ously shown that the collagen denaturation tempera-ture, a measure of thermal stability, decreases inpregnancy. This, combined with the leaflet areaexpansion, fibrosa thickening, and increased collagencontent, together signify that new collagen is beingsynthesized. Curiously, our results indicate that verylittle newly-synthesized, immature collagen was presentin native conditions and during pregnancy. Acid/pep-sin-soluble collagen remained low and only a smallproportion of immature crosslinking was evident.

As a whole, these results demonstrate that preg-nancy provokes rapid remodeling of collagen in thebelly of the mitral valve anterior leaflet. It is surprisingthat none of our structural components paralleled thebiphasic changes which we observed in leaflet biaxialextensibility.53 Instead, the observed structural changes

TABLE 1. Changes in small-angle light scattering (SALS) measurements by leaflet region with pregnancy.

Region Non-pregnant (4) Pregnant (5) DNOI p value

NOI (%)

Free edge 42.5 ± 2.0 41.3 ± 1.0 – 0.6221

Belly 43.8 ± 0.5 36.5 ± 1.9 fl 0.0174

Fixed edge 44.6 ± 1.1 46.7 ± 2.5 – 0.4944

/p (�)Free edge 15.5 ± 2.6 17.3 ± 3.0 – 0.3294

Belly 1.8 ± 1.1 13.3 ± 2.9 › 0.0072

Fixed edge 15.8 ± 4.3 13.1 ± 1.9 – 0.7034

Values are means ± SE of regional normalized orientation index (NOI) and preferred fiber direction (/p) relative to the circumferential direction, for

animals in the non-pregnant (NP; n = 4), and pregnant (P; n = 5) groups. Statistical comparisons in each region of the leaflet were made between

NP and P groups for each parameter using two-tailed t test comparisons and are presented with the corresponding p value.

FIGURE 8. Regional preferred fiber direction (absolute anglein degrees) of the anterior mitral valve leaflet from non-preg-nant (open bars; n 5 4) and pregnant (filled bars; n 5 5) ani-mals. Bars indicate mean 6 SE. The direction of /p here isreferenced to the circumferential axis. Statistical comparisonswere made in each of the five regions, between non-pregnantand pregnant groups using two-tailed t test comparisons ofgroups. Columns labeled with an asterisk showed a statisti-cally significant change with pregnancy.


FIGURE 9. Histological sections taken along the circumferential direction under polarized light microscopy in the belly region ofthe anterior mitral valve leaflet for non-pregnant (NP) and pregnant (P) animals. In non-pregnant animals, the collagen fibernetwork was highly undulated, covered almost entirely with crimped fibers. In pregnant animals, crimp length was greatlyincreased and the undulations had virtually disappeared. Leaflet circumferential and thickness axes are indicated by arrows.

TABLE 2. Summary of all changes in mitral anterior leaflet with pregnancy.

Non-pregnant Pregnant D p value

SALS measurements (belly)

NOI (%) 43.8 ± 0.5(4) 36.5 ± 1.9(5) fl 0.0174

/p (degrees) 1.8 ± 1.1(4) 13.3 ± 2.9(5) › 0.0072

Crimp measurements

Crimp length (lm) 22.9 ± 1.2(9) 65 ± 15(13) › 0.0079

Crimped area (%) 40.2 ± 6.0(9) 22.8 ± 3.0(11) fl 0.0116

Leaflet dimensions

Thickness (lm) 1170 ± 75(9) 1194 ± 59(11) – 0.4011

Radial lengtha (cm) 2.5 ± 0.1(11) 3.0 ± 0.1(15) › 0.0016

Circumferential lengtha (cm) 5.1 ± 0.1(11) 5.9 ± 0.2(15) › 0.0008

Areaa (cm2) 11.1 ± 0.6(11) 15.6 ± 0.9(15) › 0.0003

Layer thickness (lm)

Atrialis 540 ± 130(9) 833 ± 76(11) › 0.0372

Spongiosa 431 ± 85(9) 346 ± 76(11) – 0.4694

Ventricularis 221 ± 40(8) 135 ± 22(10) fl 0.0447

Collagen content (% dry wt.)

Total collagen 56.7 ± 3.0(9) 66.0 ± 3.3(13) › 0.0256

Extractable collagen 0.6 ± 0.1(5) 0.6 ± 0.1(9) – 0.6638

Water (% wet wt.) 81.4 ± 0.7(10) 80.6 ± 0.6(12) – 0.4228

Leaflet stretch

Areal stretcha 2.37 ± 0.09(7) 2.11 ± 0.11(12) biphasic 0.0439

kRpeak a 1.67 ± 0.06(9) 1.67 ± 0.06(13) – 0.9721

kCpeak a 1.37 ± 0.03(7) 1.26 ± 0.03(14) biphasic 0.0153

HIT measurements

Tda (�C) 68.6 ± 0.2(10) 66.3 ± 0.2(22) fl <0.0001

t1/2 controla (h) 8.6 ± 1.8(10) 13.6 ± 1.6(19) › 0.0144

t1/2 treateda (h) 11.5 ± 2.5(7) 17.1 ± 1.7(19) › 0.0039

Immature crosslinking indexa 1.6 ± 0.2(7) 1.3 ± 0.1(17) – 0.4385

Values are means ± SE of normalized orientation index (NOI), preferred fiber direction (/p), crimp length, leaflet crimped area, total leaflet

thickness, radial leaflet length, circumferential leaflet length, leaflet area, fibrosa, spongiosa, and atrialis thickness, total collagen content

(hydroxyproline assay), extractable (acid/pepsin-soluble) collagen (Sircol assay), water content, areal stretch, peak radial stretch ratio, peak

circumferential stretch ratio, denaturation temperature (Td), and halftime of load decay (t1/2), for animals in the non-pregnant (NP), and

pregnant (P) groups. Statistical comparisons were made between NP and P groups for each parameter using t test comparisons and are

presented with the corresponding p value. n values for the pregnancy groups are presented in brackets for each measurement.aData from Wells et al.53 for (i) NP animals and (ii) all pregnant animals.

PIERLOT et al.2066

suggest that leaflet extensibility should have beenmonotonically reduced from non-pregnant to pregnantanimals. There must be, therefore, some other mech-anism occurring in pregnancy, which modulates leafletbiomechanics.

Changes in collagen fiber alignment and orientationwere observed in the central belly region of the valveleaflet in pregnancy, however no significant changeswere found in the fixed edge and free edge regions.First of all, the fixed edge regions of the valve leafletare reported to be loaded almost uniaxially, radial tothe valve annulus.13,14,17 As the valve orifice expands inpregnancy, the loading direction would be relativelyunchanged. Thus significant reorientation of fibers isnot unexpected here. When addressing the free edge ofthe leaflet though, one must consider architecture andleaflet function. While the fibrosa is the primary load-bearing layer due to its dense network of circumfer-entially aligned collagen fibers, it extends from theannulus through only two-thirds of the leaflet. It iscompletely absent along the free edge.35 The mitralvalve, in particular, has a great deal of redundant tis-sue in the closed valve position (0.5–1.0 cm inhumans), between the coaptation line and free edge ofthe leaflet (zone of apposition).47 Therefore the freeedge region of the leaflet would not be subjected to thesame pregnancy-induced increase in tensile stressesexperienced by the leaflet between the coaptation lineand the fixed edge (or the annulus). The absence ofaltered collagen alignment in the free edge in preg-nancy is therefore not surprising, but for a differentreason from that in the fixed edge.

While the SALS orientation index reflects changesin the large-scale splay of the collagen bundles, in somecases it can also be affected by the small-scale, localchanges in fiber orientation due to waviness or crimp.The latter effect is shown in Fig. 1a, where local fiberorientation varies along the length of a crimped fiber,resulting in a broad intensity peak (Fig. 1b) with asmall NOI, indicating a low degree of fiber alignment.By contrast, straight fibers (Fig. 1c) will generate asingle, narrow intensity peak (Fig. 1d) with a largeNOI, indicating a high degree of fiber alignment. Thus,while the SALS technique provides a simple, accuratemeasure of the collagen fiber angular distribution inthe tissue, it is unable to distinguish the contributionsof large-scale splay and small-scale crimp. For thisreason, previous studies on heart valve leaflets haveperformed paired measurements of crimp length alongwith SALS orientation index. Those studies capturedincreases in fiber orientation (increased NOI) that weredominated by decreases in crimp with both (i) cyclicloading20,54 and (ii) stress-state during fixation.25,46 Inparticular, pooled data from those studies demon-strated that an increase in crimp length is associatedwith narrowing of the SALS scattered light plot, asevidenced by an overall trend toward increasing NOI(Fig. 11).

In keeping with this approach, we also examinedchanges in collagen crimp length using polarized lightmicroscopy. Collagen fiber crimp was assessed only inthe belly region of the leaflet for two reasons: (i) it wasonly in this region where the SALS OI data was alteredduring pregnancy, and (ii) we wished to relate changesin crimp to changes in the equibiaxial extensibility thatwe observed in this region during pregnancy.53 Themitral leaflet underwent a significant loss of collagencrimp during pregnancy (Fig. 10), where crimp length

FIGURE 10. Collagen crimp length and percentage of leafletarea occupied by crimped (% Area Crimped) of the anteriormitral valve leaflet from non-pregnant (open bars; n 5 9) andpregnant (filled bars; n 5 13) animals. (a) collagen crimplength (in lm). (b) leaflet crimped area (in %). Bars indicatemeans 6 SE. Statistical comparisons were made betweennon-pregnant and pregnant groups using two-tailed t testcomparisons of groups. Columns labeled with an asteriskshowed a statistically significant change with pregnancy.

FIGURE 11. Increases in crimp length (overall loss of crimp)are associated with an increase in alignment (NOI), as evi-denced by previous studies of bioprosthetic heart valvesusing paired measurements of crimp length and SALSNOI.20,25,46,54 Data points are mean values from each study.


more than doubled while the percent area occupied bycrimp was nearly halved. This change is concurrentwith leaflet expansion in the circumferential direction(Fig. 12). Despite the significant loss in crimp, theoverall collagen fiber orientation (NOI) decreasedduring pregnancy, indicating that increased collagenfiber splay away from the circumferential axis over-whelmed the decrease in crimp.

Altered composition of the extracellular matrix,particularly the increase in collagen content (expressedas percent dry weight), demonstrates that pregnancy isassociated with the growth of new matrix. While totalleaflet thickness was unchanged, the fibrosa became53% thicker—likely due to the accumulation of col-lagen—and the atrialis thinned by 46%. Interestingly,these changes do not parallel those seen in mitral valveremodeling in heart failure, despite the fact that bothconditions produce elevated stresses in the mitralleaflet.22,23,30,38,53 While the length and collagen con-tent increase in valve leaflets during heart failure, theyalso thicken and lose water content. This is differentthan what we observe in pregnancy. Thus, the patho-logical remodelling of valve leaflets associated withheart failure is likely distinct from changes in thoseleaflets during pregnancy.

Results from the current and previous study53

together demonstrate that new collagen is synthesizedin the leaflet during pregnancy. It is curious, therefore,that the Sircol assay used in the present study showedthat little acid/pepsin-soluble collagen was present inthe native bovine mitral anterior leaflet, and that thisamount remained unchanged during pregnancy. Thissuggests limited presence of uncrosslinked ‘‘new’’ col-lagen. This result is in agreement with HIT crosslink-ing profiles observed in pregnancy.53 In such analyses,the ratio of t1/2-treated/t1/2-control is used as an indi-cator of the ratio of total (mature plus immature) tomature crosslinking, and is thus referred to as theimmature crosslinking index.2,53 This index provides ameasure of the degree of collagen turnover in a tis-sue.1,2,5,10,48 Interestingly, the immature crosslinkingindex in the mitral valve has not been shown to changeduring pregnancy, and is significantly lower than thevalues reported for the ovine pericardium37 and ovine

aorta52 during development. This result again suggeststhat mature, crosslinked collagen dominates and thischaracteristic is unchanged in pregnancy. It is possiblethat, in pregnancy, valve collagen is crosslinked morequickly after synthesis and assembly: a hypothesisworthy of further investigation.

This study and its antecedent53 have demonstratedboth structural and mechanical alterations to mitralvalve tissues in pregnancy, providing insight into themechanisms of valve remodeling. Functionally, theseresults indicate that as the valve leaflet enlarges inpregnancy, there is a drastic loss of crimp, which isassociated with reduced extensibility, namely througha loss of circumferential stretch. Collagen crimp andfiber orientation have been shown to contribute totissue extensibility in heart valve and pericardial tis-sues.8,20,32,54 Under in vitro cyclic loading there is (i) anincrease in crimp length as collagen fibers un-crimp(straighten) in the direction of loading, and (ii) rota-tion of the fibers towards the preferred direction(direction of loading), together causing an increase inSALS NOI (fiber alignment). However, in pregnancy,we have reported a decrease in fiber alignment. Weattribute the decrease in SALS NOI to the rotation offibers toward the radial direction, increasing theangular splay (i.e., wider distribution of angles) of thecollagen bundles. This fiber rotation and loss of crimpmay be associated with the concurrent radial leafletlengthening that also occurs in pregnancy. In fact,stretch-induced fiber reorientation toward of thedirection of the applied tensile force has been welldocumented throughout the literature.7,8,12,15,16,44

Finite element (FE) models of the biaxially loadedaortic valve leaflet indicate circumferential fiber rota-tion, and an increase in fiber alignment16 as in cyclicloading—completely the opposite of our model. Thesedifferences may be attributed to Driessen’s assumptionthat valve leaflet dimensions do not change. Quite thecontrary, we have reported tremendous increases inleaflet dimensions in pregnancy, indicating not only theturnover of tissue, but also tissue growth to accom-modate the expanding valve orifice. Future FE modelsof fiber re-orientation may therefore need to includethe possibility of leaflet enlargement.

FIGURE 12. Schematic representing the changes in collagen fiber architecture of the bovine mitral valve anterior leaflet duringpregnancy. There is an increase in the crimp length, a decrease in the overall presence of crimp, and rotation of the fibers towardsthe radial direction, increasing fiber splay and resulting in circumferential lengthening.

PIERLOT et al.2068

Interestingly, the changes in collagen fiber archi-tecture and content did not occur in synchrony withthe leaflet expansion and extensibility shift reportedpreviously.53 Alterations in normalized orientationindex, preferred fiber direction, crimp length, layerthickness, and collagen content were only detectedbetween non-pregnant and pregnant animals, whereasleaflet expansion and loss of extensibility occurredlargely in early pregnancy. This suggests that in earlypregnancy, the valve leaflet has enlarged through whatis likely a passive expansion and, as a consequence, haslost any reserve of physiological extensibility. Never-theless, the expansion of the valve leaflet allows thevalve to maintain complete coaptation, even under theannular expansion of early pregnancy. The return ofbiaxial extensibility in late pregnancy must indicateadaptive tissue remodeling. Surprisingly, however,there is no detectable recovery of collagen crimp in latepregnancy. Further studies to evaluate structuralalterations through pregnancy, particularly on thebiphasic timeline of early and late pregnancy, wouldgreatly improve our understanding of the contribu-tions to the structural and mechanical leaflet propertiesunder the triggers of volume loading and orificeexpansion.

While this study has taken the first step to begin toexamine structural changes in the collagen fiber net-work, research is still needed both in the investigationof hormonal triggers as well as changes in biochemicalcomposition (such as elastin, proteoglycans, and gly-cosaminoglycans) and crosslinking profiles. Particu-larly, the evaluation of differences in the degree andmaturity of crosslinking in pregnant and non-pregnantanimals could be assessed through thermomechanical(HIT, DSC, etc.) and biochemical methods in parallelwith biomechanical measurements of time-dependentviscoelastic materials properties, which are highlydependent on the degree of crosslinking in a tissue.Future work must also involve performing non-equi-biaxial mechanical testing to (i) assess axial cross-coupling: a factor that would be expected to decreasegiven the increased splay of the fiber architecture, and(ii) to inform a constitutive model of the leaflet and itsremodeling process. Furthermore, the inclusion ofother heart valve types (aortic, pulmonary, and tri-cuspid) could provide insight into valve-specificmechanisms of adaptive remodeling.

ACKNOWLEDGMENTS

The authors thank Lucas Tedesco (Department ofBioengineering, University of Pittsburgh) for per-forming SALS data acquisition, Patricia Colp

(Department of Pathology, Dalhousie University) forsharing expertise in histological staining techniques,Maxine Langman (Department of Applied Oral Sci-ences, Dalhousie University) for providing biochemicaltechnical expertise, as well as O.H. Armstrong for thesupply of bovine tissues.

CONFLICT OF INTEREST

None.

REFERENCES

1Aldous, I. G., J. M. Lee, and S. M. Wells. Differentialchanges in the molecular stability of collagen from thepulmonary and aortic valves during the fetal-to-neonataltransition. Ann. Biomed. Eng. 38:3000–3009, 2010.2Aldous, I. G., S. P. Veres, A. Jahangir, and J. M. Lee.Differences in collagen cross-linking between the fourvalves of the bovine heart: a possible role in adaptation tomechanical fatigue. Am. J. Physiol. Heart Circ. Physiol.296:H1898–H1906, 2009.3Amini, R., C. E. Eckert, K. Koomalsingh, et al. On thein vivo deformation of the mitral valve anterior leaflet:effects of annular geometry and referential configuration.Ann. Biomed. Eng. 40:1455–1467, 2012.4Amini, R., C. A. Voycheck, R. E. Debski. A method forpredicting collagen fiber realignment in non-planar tissuesurfaces as applied to the glenohumeral capsule duringclinically relevant deformation. J. Biomech. Eng. 236:031003-1–031003-8, 2013.5Avery, N. C., and A. J. Bailey. Enzymic and non-enzymiccross-linking mechanisms in relation to turnover of colla-gen: relevance to aging and exercise. Scand. J. Med. Sci.Sports 15:231–240, 2005.6Bergsjø, P., D. W. Denman, H. J. Hoffman, and O. Meirik.Duration of human singleton pregnancy. A population-based study. SOBS 69:197–207, 1990.7Billiar, K. L., and M. S. Sacks. A method to quantify thefiber kinematics of planar tissues under biaxial stretch.J Biomech. 30:753–756, 1997.8Broom, N. D. The stress/strain and fatigue behaviour ofglutaraldehyde preserved heart-valve tissue. J. Biomech.10:707–724, 1977.9Campos, O., J. Andrade, J. Bocanegra, et al. Physiologicmultivalvular regurgitation during pregnancy: a longitu-dinal Doppler echocardiographic study. Int. J. Cardiol.40:265–272, 1993.

10Cannon, D. J., and P. F. Davison. Aging, and crosslinkingin mammalian collagen. Exp. Aging Res. 3:87–105, 1977.

11Clapp, III, M. D., J. Ford, and M. D. Capeless. Cardiovas-cular function before, during, and after the first and sub-sequent pregnancies. Am. J. Cardiol. 80:1469–1473, 1997.

12Clark, R. E., and E. H. Finke. Scanning and lightmicroscopy of human aortic leaflets in stressed and relaxedstates. J. Thorac. Cardiovasc. Surg. 67:792–804, 1974.

13Cochran, R. P., K. S. Kunzelman, C. J. Chuong, et al.Nondestructive analysis of mitral valve collagen fiber ori-entation. ASAIO Trans. 37:M447–M448, 1991.

14Driessen, N. J., R. A. Boerboom, J. M. Huyghe, et al.Computational analyses of mechanically induced collagen


fiber remodeling in the aortic heart valve. J. Biomech. Eng.125:549–557, 2003.

15Driessen, N. J. B., C. V. C. Bouten, and F. P. T. Baaijens.Improved prediction of the collagen fiber architecture inthe aortic heart valve. J. Biomech. Eng. 127:329–336, 2005.

16Driessen, N. J. B., M. A. J. Cox, C. V. C. Bouten, andF. P. T. Baaijens. Remodelling of the angular collagen fiberdistribution in cardiovascular tissues. Biomech. Model.Mechanobiol. 7:93–103, 2008.

17Driessen, N. J. B., G. W. M. Peters, J. M. Huyghe, et al.Remodelling of continuously distributed collagen fibres insoft connective tissues. J. Biomech. 36:1151–1158, 2003.

18Estergreen, V. L., O. L. Frost, W. R. Gomes, et al. Effect ofovariectomy on pregnancy maintenance and parturition indairy cows. J. Dairy Sci. 50:1293–1295, 1967.

19Evans, H. E., and W. O. Sack. Prenatal development ofdomestic and laboratory mammals: growth curves, externalfeatures and selected references. Zentralbl Veterinarmed C2:11–45, 1973.

20Fulchiero G. J., S. M. Wells, M. S. Sacks. Alterations incollagen fiber crimp morphology with accelerated cyclicloading and transvalvular pressure fixation in porcine aorticvalves. Proceedings of the Second Joint EMBS/BMESConference. Houston, TX, USA, pp. 1248–1249, 2002.

21Gilbert, T. W., M. S. Sacks, J. S. Grashow, et al. Fiberkinematics of small intestinal submucosa under biaxial anduniaxial stretch. J. Biomech. Eng. 128:890–898, 2006.

22Grande-Allen, K. J., J. E. Barber, K. M. Klatka, et al.Mitral valve stiffening in end-stage heart failure: evidenceof an organic contribution to functional mitral regurgita-tion. J. Thorac. Cardiovasc. Surg. 130:783–790, 2005.

23Grande-Allen, K. J., R. P. Cochran, P. G. Reinhall, andK. S. Kunzelman. Mechanisms of aortic valve incompe-tence: finite-element modeling of Marfan syndrome.J. Thorac. Cardiovasc. Surg. 122:946–954, 2001.

24Hunter, S., and S. Robson. Adaptation of the maternalheart in pregnancy. Br. Heart J. 68:540–543, 1992.

25Joyce, E. M., J. Liao, F. J. Schoen, et al. Functional col-lagen fiber architecture of the pulmonary heart valve cusp.Ann. Thorac. Surg. 87:1240–1249, 2009.

26Kametas, N. A., F. McAuliffe, J. Hancock, et al. Maternalleft ventricular mass and diastolic function during preg-nancy. Ultrasound Obstet. Gynecol. Off. J. Int. Soc. Ultra-sound Obstet. Gynecol. 18:460–466, 2001.

27Keeley, F. W., J. D. Morin, and S. Vesely. Characterizationof collagen from normal human sclera. Exp. Eye Res.39:533–542, 1984.

28Kindahl, H., B. Kornmatitsuk, and H. Gustafsson. Thecow in endocrine focus before and after calving. Reprod.Domest. Anim. 39:217–221, 2004.

29Kunzelman, K. S., R. P. Cochran, C. Chuong, et al. Finiteelement analysis of the mitral valve. J. Heart Valve Dis.2:326–340, 1993.

30Kunzelman, K., D. Quick, and R. Cochran. Altered col-lagen concentration in mitral valve leaflets: biochemicaland finite element analysis. Ann. Thorac. Surg. 66:S198–S205, 1998.

31Kunzelman, K. S., M. S. Reimink, and R. P. Cochran.Annular dilatation increases stress in the mitral valve anddelays coaptation: a finite element computer model. Car-diovasc. Surg. 5:427–434, 1997.

32Langdon, S. E., R. Chernecky, C. A. Pereira, et al. Biaxialmechanical/structural effects of equibiaxial strain duringcrosslinking of bovine pericardial xenograft materials.Biomaterials 20:137–153, 1999.

33Liao, J., L. Yang, J. Grashow, and M. S. Sacks. Therelation between collagen fibril kinematics and mechanicalproperties in the mitral valve anterior leaflet. J. Biomech.Eng. 129:78–87, 2007.

34Longo, L. D. Maternal blood volume and cardiac outputduring pregnancy: a hypothesis of endocrinologic control.Am. J. Physiol. 245:R720–R729, 1983.

35McCarthy, K. P., L. Ring, and B. S. Rana. Anatomy of themitral valve: understanding the mitral valve complex inmitral regurgitation. Eur. J. Echocardiogr. 11:i3–i9, 2010.

36Muresian, H. The clinical anatomy of the mitral valve.Clin. Anat. 22:85–98, 2009.

37Naimark, W. A., S. D. Waldman, R. J. Anderson, et al.Thermomechanical analysis of collagen crosslinking in thedeveloping lamb pericardium. Biorheology 35:1–16, 1998.

38Quick, D. W., K. S. Kunzelman, J. M. Kneebone, andR. P. Cochran. Collagen synthesis is upregulated in mitralvalves subjected to altered stress. ASAIO J. 43:181–186,2006.

39Reynolds, M. Measurement of bovine plasma and bloodvolume during pregnancy and lactation. Am. J. Physiol.175:118–122, 1953.

40Robson, S. C., S. Hunter, R. J. Boys, and W. Dunlop.Serial study of factors influencing changes in cardiac outputduring human pregnancy. Am. J. Physiol. 256:H1060–H1065, 1989.

41Robson, S. C., D. Richley, R. J. Boys, and S. Hunter.Incidence of Doppler regurgitant flow velocities duringnormal pregnancy. Eur. Heart J. 13:84–87, 1992.

42Sacks, M. S. Small-angle light scattering methods for softconnective tissue structural analysis. In: Encyclopedia ofBiomaterials and Biomedical Engineering, Vol. 2, edited byG. E. Wnek, and G. L. Bowlin. New York: InformaHealthcare USA Inc., 2004, pp. 2450–2463.

43Sacks, M. S. Biomechanics of engineered heart valvetissues. Conference Proceedings: Annual InternationalConference of the IEEE Engineering in Medicine andBiology Society IEEE Engineering in Medicineand Biology Society Conference, Vol. 1, pp. 853–854,2006.

44Sacks, M. S., C. J. Chuong, and R. More. Collagen fiberarchitecture of bovine pericardium. ASAIO J. 40:M632–M637, 1994.

45Sacks, M., D. Smith, and E. Hiester. A small angle lightscattering device for planar connective tissue microstruc-tural analysis. Ann. Biomed. Eng. 25:678–689, 1997.

46Sacks, M. S., D. B. Smith, and E. D. Hiester. The aorticvalve microstructure: effects of transvalvular pressure.J. Biomed. Mater. Res. 41:131–141, 1998.

47Silbiger, J. J., and R. Bazaz. Contemporary insights intothe functional anatomy of the mitral valve. Am. HeartJ. 158:887–895, 2009.

48Sims, T. J., N. C. Avery, and A. J. Bailey. Quantitativedetermination of collagen crosslinks. Methods Mol. Biol.139:11–26, 2000.

49Strek, M. Critical Illness in Pregnancy. In: Principles ofCritical Care, Vol. 3, edited by J. Hall, G. Schmidt, and L.Wood. New York: McGraw Hill Professional, 2005,pp. 1593–1614.

50Thevenaz, P., U. E. Ruttimann, and M. Unser. A pyramidapproach to subpixel registration based on intensity. IEEETrans. Imag. Proc. 7:27–41, 1998.

51Thevenaz, P., and M. Unser. User-friendly semiautomatedassembly of accurate image mosaics in microscopy.Microsc. Res. Tech. 7:135–146, 2007.

PIERLOT et al.2070

52Wells, S. M., S. L. Adamson, B. L. Langille, and J. M. Lee.Thermomechanical analysis of collagen crosslinking in thedeveloping ovine thoracic aorta.Biorheology 35:399–414, 1998.

53Wells, S. M., C. M. Pierlot, and A. D. Moeller. Physio-logical remodeling of the mitral valve during pregnancy.AJP Heart Circul. Physiol. 303:H878–H892, 2012.

54Wells, S. M., T. Sellaro, and M. S. Sacks. Cyclic loadingresponse of bioprosthetic heart valves: effects of fixationstress state on the collagen fiber architecture. Biomaterials26:2611–2619, 2005.

55Woessner, J. F. Determination of hydroxyproline in connec-tive tissues.Methodol Connect Tissue Res 23:227–234, 1984.


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