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ontrol of Cryopreservation Procedures on Blood Vessels Using Fiber-Ray Diffraction
. Pérez Campos, M.C. Saldías, W. Silva, D. Machin, L. Suescun, R. Faccio, A.W. Mombrú, and. Alvarez
ABSTRACT
Aim. We sought to determine variations in fiber organization at the molecular level usingx-ray diffraction analyses on human blood vessel specimens after cryopreservationprocesses.Materials and Methods. Diffractometric profiles were performed on aortic and carotidcryopreserved-thawed vessel samples (CVS) versus the same fresh vessel samples (FVS).X-ray diffraction was performed on vascular tissues from 17 cadaveric donors afterinformed consent. Measurements utilized a Seifert Scintag PAD-II powder diffractometerwith CuKa radiation; � � 1.5418 Å. Scans were evaluated in the 5° to 60° range in theta�2theta mode, in the 5° to 60° range in 2-theta, with steps 0.1° and 10 seconds per step.Ten aortic and 8 carotid diffractometric profiles were analyzed, using differential plani-metric surfaces measured under x-ray diffraction curve. Diffractographic profiles wereanalyzed according to intervals based upon the ages of the donors. An ordering profilecoefficient (OPC) was obtained as the quotient between the differential planimetric surface(DPS) of FVS versus CVS vessel ordering diffraction.Results. There was a decreased ordering profile according to age: older donors showedless ordering than younger ones. Clear peaks at d-spacing of 2.86 Å and 2.15 Å (2-theta �31.3° and 42.0°, respectively) were always confirmed despite the different profiles ofsamples. OPC showed a higher ordering profile among the CVS than FVS: 70% aortas and62.5% carotids.Conclusion. The cryopreserved-thawed procedure does not damage the fibrillar organi-
zation of vessels.bviod
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OWADAYS, cardiovascular human pathology is oneof the greatest challenges in public health manage-
ent. Mortality is increasing in many countries despiteducational programs concerning sanitary hygiene, diet,nd pharmacologic interventions. Surgical procedures offerhe only tool to solve arterial circulatory troubles of ob-tructive wall atheromatous lesions: venous autograft, andynthetic Dacron or e-PTFE prosthesis bypasses with orithout surface modifications of the blood stream. Complexomposites with autologous venous or synthetic prosthesesr bioabsorbable materials in addition to tissue bankingllografts as fresh or cryopreserved arterial vessel1,33 haveeen obtained from multiorgan cadaveric donors basedpon International Standards of quality and safety.2,3
All these therapeutic tools display biomechanical or
emodynamic mismatches. Together with adverse meta- g041-1345/08/$–see front matteroi:10.1016/j.transproceed.2008.02.025
68
olic conditions in the patients, they may lead to a wideariety of functional graft behaviors including laminar flownterruption, intimal hyperplasia, and late vascular graftcclusion. These local and systemic pathophysiologic con-itions affect patency rates.The best graft behavior is achieved by grafts that are close
From the Instituto Nacional de Donación y Trasplantes, MSPac. Medicina, Udelar; and the Laboratorio de Cristalografia,stado Solido y Materiales, Detema, Facultad de Quı́mica -delar. Montevideo, Uruguay.Supported by CSIC and Pedeciba.Address reprint requests to Héctor Pérez Campos, MD, Insti-
uto Nacional de Donación y Trasplante, Hospital de Clı́nicas, Avtalia s/N°, 4° Piso Montevideo, Uruguay. E-mail: hpc55ster@
mail.com© 2008 by Elsevier Inc. All rights reserved.360 Park Avenue South, New York, NY 10010-1710
Transplantation Proceedings, 40, 668–674 (2008)
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o the functional native arterial vessel with fewer hemody-amic mismatch.4–6 The most common failures, includingseudointimal hyperplasia, stenosis, and thrombotic occlu-ion, are directly linked to mechanical dysfunctions andellular/humoral host responses in relation to blood flowontact with a nonbiological endoluminal surface, such as inn artificial prosthesis.1
Cryopreserved vessels are an interesting option as arte-ial substitutes. The use of banked vascular allograft tissuess increasing, mainly in relation to postoperative complica-ions with artificial grafts, particularly infections when au-ografts are impossible and allografts are the only option.7,8
he structural stromal composition, including collagenicnd elastic properties have a central role in the patency ratend functional biomechanical behavior of cryopreserved/efrosted allografts. Thus the damage induced by cyo-reservation/defrosting protocols applied for vascular tissueanking must be assessed based upon the cryopreservantedia, cooling rate program, cryo storage, and the subse-
uent defrosting schedule. Many cellular and extracellularnjuries of arterial walls have been described which canhange the viscoelastic parameters and other functionalesponses of the parietal structure.9,10 Extracellular damages associated with important deterioration of metallopro-einases,11 which produces functional and structural conse-uences on stromal biopolymers. Rendal-Vazquez et al12
howed light and nonsignificant modifications in pig aorticlastic fibres using standard optic microscopy (OM), aicture that was not described by other authors in dogemoral arteries.13
In the early 1980s, Shepherd and Dawber14 observed badesults when cryosurgery was applied to 17 patients to treateloid skin scar healing. Only 2 patients showed significantcar reduction, while 15 experiencd no improvement sug-esting collagenic matrix endurance to cryogenic tempera-ures. Later search showed no damage to skin collagenicbers among pigs treated with cryosurgical protocols.15
In the biochemical field, earlier works from Levit16,17
ostulated that cryo damage mechanisms occurred at theiomolecular level of vegetable protoplasma proteins. Thisuthor postulated that during the frost and later defrostrocesses changes occurred in electrochemical functionalisulfide bonds that stabilize tertiary protein configurations.ccording to this postulate, after cryo injury functionalonds transform their electrochemical unions to nonfunc-ional covalent disulfide bonds with irreversible changes inertiary profiles. In additions, water efflux through semiper-eable cellular membranes during the cooling process
nduces intracellular dehydration and osmotic contraction,hile the extra cellular ice front and osmotic pressure of the
upercooled liquid compartment grows. The spatial modi-cation of volumes affects the intra- and extracellularrchitecture, shortening the distance between intermem-rane layers and generating “repulsion hydration forces”etween polar hydrophilic molecular surfaces.18 This phe-omenon promotes chemical intermolecular equilibrium
isplacement, which could explain Levit’s postulates con- perning molecular tertiary protein modifications by changesn bond configurations.
In the biomechanical field, many works show excellent initro behavior of arterial cryopreserved defrosted tis-ues,19–21 although other authors have reported disappoint-ng results about this topic.10,22,23 From a morphostructuraloint of view, the recent papers of Schenke-Layland etl24,25 analyzed fresh, vitrified, and frozen cryopreservedorcine cardiac valves in relation to extracellular matrixECM) preservation.
Conventional histology, electron microscopy, and mul-iphoton imaging to obtain autofluorescence and second-armonic generation (SHG) images have been performedn cardiac tissues. Frozen leaflets of cardiac valves showevere loss of ECM structures compared with fresh oritrified controls, particularly on SHG images. Laser-in-uced autofluorescence imaging has emphasized substan-ial ultrastructural deterioration and disintegration of mostollagen structures.
X-ray diffraction is perhaps the most important techniqueor structural determination of solid specimens. Whenpplied to crystalline samples, the average structure can beefined to a resolution up to 0.6 Å. Thus, detailed structuralnformation can be obtained leading to the determinationf the molecular configuration in relation to the intermo-
ecular distance and the stereochemical angle conforma-ion. However, when the specimens to be studied are notrystalline, but rather exhibit low ordering such as polymersr fibers, the available information is limited. Because there
s no long-range order that yields sharp peaks with highntensity, no atomic coordinates have been found, andherefore, no bond lengths or angles can be determined.owever, this technique is still useful to give at least rough
nformation such as the degree of ordering of the speci-ens.Bragg’s Law, the theoretical background of these tech-
iques, had established that for a given x-ray angularncidence, the characteristic crystalline spacing “d” deter-
ines an interference dispersion where emerging diffractedeams become constructive:
n� � 2dsin(�) (1)
here n is an integer, � is the x-ray monochromaticavelength, d is the distance between the planes of therystalline net, and � (theta) is an angular value between thencidental x-ray and the considered crystalline plane.
In the early 1950s, Pauling and Corey26 explored the usef x-ray diffraction on collagen, detecting a wide peak
ocated at approximately 2.86 A (2 theta � 31.3° when usingadiation). They suggested that this peak showed the amideroups of the polypeptide chain to be in the cis configura-ion. They based their research on the limited informationielded by x-ray diffraction analyses on fibers.
Our research followed the same idea, applying thisechnique to tissue banking of blood vessel, which are richn collagen and elastin. We compared fiber diffraction
atterns between fresh and cryopreserved specimens. We
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xpected that this technique by studying the internal order-ng of the fibrillar stroma present in the vessels could beseful to check the primary quality of the tissues. Ifryopreservation damaged a vessel, the effect should beetected by fiber x-ray diffraction, due to decreased order-
ng, revealed by a loss in signal. On the contrary, if theryopreservation process was correctly applied, the internalrdering of the fibers should not be decreased and there-ore the diffraction pattern should show wide, intenseeatures. Taking into account these statements, the aim ofhe present work was to apply an analytical model to lookor fibrillar changes at the molecular level induced byryopreservation during vascular tissue banking procedures.
ATERIAL AND METHODSrocurement Protocol of Vascular Tissues From Multiorganadaveric Donors
ascular tissue procurements were performed according to theuides of the transplant program of the Donation and Transplantational Institute (INDT) of Uruguay. All procedures of vascular
issue procurement and processing conformed to the ethical andafety concerns for therapeutic use, including written consent (Law4005/1971 and Law 17668/2003). General exclusion criteria wereonsistent with the International Atomic Energy Agency.3
Seventeen cadaveric donors were selected after informed con-ent procedures according to transplant program of INDT ofruguay.2 The donor ages was ranged from 18 to 60 years (mean,
5.5 � 11.8 years). There were 47.5% male and 52.5% femaleonors. A 4-cm-long thoracic aorta or carotid was harvested fromonors using aseptic technique. After harvesting, carotid arteriesnd thoracic aorta were washed with saline solution and stored at°C. In a laminar flow cabinet we dissected 1-cm rings of thoracicorta and carotids and longitudinally excised 1-cm2 surface seg-ents of these vascular tissues. Proximal and distal extremes werearked with surgical (00000) thread. Intimal and adventitial facesere also identified. Every fresh vascular sample (FVS) measuringcm2 was labelled and stored in saline solution at 4°C for 24 hoursefore x-ray diffraction (DETEMA) at the same temperature. Theemaining fresh vessel segments (3-cm long) were labelled and
Fig 1. Diffractometer Seifert Scintag PAD II.
ryopreserved.
ryopreserved/Defrosted Protocol
resh vascular tissues were cryopreserved according to the follow-ng procedure. Cryopreserved vascular samples (CVS) immersedor 30 minutes in a final volume of 85 cc cryopreservant solution at0°C, containing culture medium (RPMI 1640) 85%; humanlbumin solution (20%) 5%; and dimethylsulfoxide 10%, in aryo-resistant bag (Joisten and Kettenbaum, D51429, Bereischladbach, Mod.011342) were sealed using a thermal machine in a
aminar flow cabinet (Microflow, Laminar Flow Work Station,DH Ltd, Andover, Hants, England; SP.10.5.AA; Fahy et al.34).
rogrammed cryopreservation was performed in a Controlled Ratereezing System (Model 9000, Gordinier Electronics, Inc., Ros-ville, Mich). The modified cooling rate protocol28 from Pegg et alas applied: First, a slow, programmed cooling rate achieved aean value of 1°C/min to �90°C. Second, a rapid cooling rate was
btained by the immediate transfer of the bag to the gas phase ofliquid nitrogen compartment (�142°C). Frozen arterial speci-ens were stored for 30 days at �142°C in vapor liquid nitrogen32
Mark III, Temperature and Liquid Level Controller, Taylor,harton, Theodore, Ala).The defrost protocol was a two-stage rewarming process derived
lso from Pegg et al.28 First, slow warming by transferring the bagrom nitrogen gas phase to the room temperature at 20°C over 30inutes. Second, rapid warming by immersing the bag in a water
ath at 40°C until completely defrosted. The cryoprotectant liquidas gradually removed in four 10-minutes steps by immersion in
apering concentrations of DMSO (10%, 5%, 2.5%, and 0%) at0°C over a total time of 40 minutes.27 A 1 cm2 surface of eachascular defrosted segment was prepared under the same condi-ions as FVS. CVS were then sent to the x-ray diffraction labora-ory.
-Ray Diffraction Procedures Protocols
easurements were performed with a Seifert Scintag PAD-IIowder diffractometer (Laboratorio de Cristalografia, Estado Só-
ido y Materiales, DETEMA, Facultad de Quı́mica, Universidad dea Republica, Montevideo, Uruguay) using CuK� radiation (� �.5418 Å; Fig 1). We performed Scans in the 5° to 60° range in-theta, with measurements steps of each 0.1°, with 10 seconds pertep. Figure 2 shows the schematic design of the technical proce-
Fig 2. Schematic design of diffractometer principle.
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ure. Data were collected for longitudinal and transverse configu-ations, exposing the inner and the outer layers of the vessel of theame donor for FVS and CVS. Each normalized diffractometricrofile pair was codified and recorded in an electronic data base.e analyzed 10 aortic and 8 carotid diffractometric profiles with
ifferential planimetric patterns measured under an x-ray diffrac-ion curve, comparing FVS and CVS curves for trend changes inhe wall collagen stromal after thawing. Figure 3 shows theiffractometric profile from an 18 year old male donor comparingVS and CVS records.
ig 4. Diffractographic profiles FVS (blue), CVS (red) from aorticascular tissue of a 34-year-old male donor. Blue arrows: Irdering diffractometric intensity FVS values, in punctual angular
ncidence for [(2-theta(°)]; red arrows: I ordering diffractometricntensity CVS values, in punctual angular incidence for [(2-
ig 3. Dirffractographic profile from an 18-year-old male donorf thoracic aorta, FVS and CVS. x values: angular incidence ofuK� [(2-theta(°)] according to Bragg’s law (black arrows in1.3° and 42°, 2-theta(°) ordering picks values). y values: order-
ng diffractometric intensity in normalized units. References: redrofile � FVS; blue profile � CVS; scanner incidence � intimal
ransverse.
fheta(°)]; scanner incidence � intimal transverse.
nterpretation of Results
iffractometric profile were obtained for each pair from a singleonor for qualitative and quantitative analyses.The qualitative analysis compared profiles to a close homologous
rterial pair. The quantitative analysis examined planimetric sur-aces under each diffractometric profile, because these surfaces arefunction of y axes ordering intensity.The subtraction of CVS planimetric surface minus FVS planimetric
urface yielded a differential planimetric surface (DPS) between bothomologous donor samples. DPS graphic profiles were obtained fromnalysis of data obtained from the Seifert Scintag PAD-II powderiffractometer. Its mathematical expression is given by:
� (↑� ↓ ) � ICVS-FVS[2-theta (°)] � DPS (2)
here 1 represents ordering diffractometric intensity for y axesalues of FVS in one point in 2-theta (°); 2 represents orderingiffractometric intensity for y axes values of CVS in the same point
n 2-theta (°); ICVS-FVS is the difference between each respectiverdering diffractometric intensity for y axes value at the same point
n 2-theta (°); and [(2 � theta (°)] is each point in x axes between0° and 60° of angular incidence (Fig 4). The subtraction of bothatching profiles yielded DPS, whose I values are �0 for all pointshen ICVS � IFVS; I values are below 0 for all points when ICVS �
FVS (Fig 5). Graphic subtraction profiles were edited by Seifertcintag PAD-II powder diffractometer. DPS graphic edited lead toalculation of the ordering profile coeficient (OPC) according toollowing expression:
OPC ��DPS
�DPS(3)
here �DPS represents the positive planimetric surfaces values
ig 5. Diffractographic profile through the different 2-thetaalues of carotid from a 58-year-old male donor. References:lue � FVS; red � CVS Scanning incidence: inner transversal;PS profile; Positive values for ordering CVS � ordering FVS;nd Negative values for ordering CVS � ordering FVS.
rom DPS graphics in Figure 5; and �DPS represents the negative
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lanimetric surfaces values from DPS graphics in Figure 5. Thisatio is always above 0 and its values are greater than 1 when CVShow a higher value ICVS than FVS one; and is above 0 but less thanwhen FVS have a higher value IFVS than CVS one.
iffractometric Scanning of Vascular Samples
ll aortic and carotid donor samples were scanned for both FVSnd CVS by qualitative analysis. Scanning was performed usingongitudinal and transverse orientation analyses of intimate anddventitial surfaces of 10 aortic and 8 carotid sample Diffractomet-ic profiles were analyzed according to intervals of ages of donors.
ESULTS
tandard qualitative observations from the visual analysis ofiffractometric profiles showed that the patterns of all diffrac-ion intensities through the various 2-theta values maintainedimilar forms. Regardless of the kind of vessel, the peaks at
ig 6. Diffractographic profiles from a 30-year-old donor mefrosted, blue; scanner incidence, inner longitudinal (LI), inner
-spacing (Bragg Law) 2.86 Å and 2.15 Å (2-theta � 31.3° and t
2.0°, respectively) were always confirmed despite the differentrofiles of the curves (Fig 3). There were differences in therofiles of cryopreserved and fresh probands, also from theame donor (Fig 6). At 20° (2-theta value), a slight and broadise of the diffractometric curve was seen, as vitreous amor-hous structures usually show (Fig 3, inner square). A clearecrease in ordering profiles was shown in the distribution ofges; older donors contrasted with the highest ordering inounger ones (Fig 7 and 8).
Standard quantitative Observation from OPC analysis ofiffractometric profiles in aortic samples are shown in TableThe OPC values in carotid samples are shown in Table 2According to the OPC calculation, 70% of probands
howed higher organization profiles in cryopreservedortic than fresh tissue; 62.5% of probands showedigher organization profiles in cryopreserved carotid
Vessel, descending thoracic aorta; fresh red; cryopreserved,versal (TI), external longitudinal (LE), external transversal (TE).
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ISCUSSION
hese results for allograft vascular tissue banking con-rmed Pauling’s communication26 concerning a wide andtrong peak at d-spacing 2.86 Å (2-theta � 31.3°) forollagen structures, thus validating this analytical method.his phenomena and the other peak at d-spacing 2.15 Å
2-theta � 42°) allow us to identify both patterns for human
ig 7. Diffractographic profiles from three arterial health do-ors: 18 years old (red), 19 years old (blue), 46 years old (green),nd one 60 years old (yellow) with severe arterial pathologyabdominal disecant aneurism aorta). Vessels: fresh thoracicescending aortas; Scanner incidence: inner transverse. Note
he severe decrease of ordering intensity profiles through agesnd pathologies (60 year old with vascular illness).
ig 8. Diffractographic profiles from all 17 vascular donorested according to ages ranking. Vessels: fresh thoracic de-cending aortas; scanner incidences: inner transversal. Peaksrdering intensities in d spacing � 2,15 A (white squares) and� 2,86 (black squares). Note the clear decrease of ordering
rofiles shown in the distribution of ages.L
ortic and carotid arterial structures because of its repeti-ive character.
The vascular cryopreservation-defrost procedures did notodify collagen structure, taking into account both d-
pacing peak phenomena patterns. This observation vali-ates our vascular tissue banking procedures for therapeu-ic purposes. Nevertheless, all FVS diffractometric profilesere different with respect to CVS diffractometric ones,
ndependent of the donor, vessel type, or side scan inci-ence. All of our observations led us to propose that thelow cooling procedure during cryopreserved vascular tissueanking, reproduces molecular kinematics and thermody-amic phenomena of water crystallization in nature. In fact,or aqueous solvent–solute systems, the first molecularucleation of water crystallization is a random event; there-ore, the final solid spatial structures also have a randomonfiguration.
Solution supercooling under hyperosmolar conditions iswell-defined phenomenon that forms amorphous vitreous
olidification, because the high viscosity promotes molecu-ar arrest.27,29 All these physical molecular situations areresent in the stroma of cryopreserved vascular tissue andould explain the broad profiles at 20° in 2-theta as anxpression of vitreous solid amorphous production duringhe cooling program.31
The higher diffractometric profiles of CVS related toVS observed by OPC value analysis, shows collagenolymeric behavior during our cryopreserved-defrostedrotocol. Collagen fibers were not damaged, but acquired aew high ordering molecular composition, showing repeti-ive peaks that defined a “pattern identification” of human
Table 1. OPC Values for Descending Thoracic Aorta (n � 10)
Donor Code Vessel OPC Values
SM 12011982 DTA 0.313707518SM 12011982 DTA 1.186177383DRJ 30091974 DTA 1.861302269DRJ 30091974 DTA 2.110162156FB 10071954 DTA 4.223204562FB 10071954 DTA 1.52072386PB 15021967 DTA 28.0915473PB 15021967 DTA 0.498631776PHE07071964 DTA 3.930986183PHE07071964 DTA 0.959981063
Abbreviation: DTA, descending thoracic aorta.
Table 2. OPC Values for Carotids (n � 8)
Donor Code Vessel OPC Values
DRJ 30091974 Carotid 1.363512199DRJ 30091974 Carotid 0.903835643DRJ 30091974 Carotid 0.830101158DRJ 30091974 Carotid 0.739290625NML 22101947 Carotid 2.000088308NML 22101947 Carotid 1.411527536IMJ 24041976 Carotid 1.601447791
IMJ 24041976 Carotid 1.575402375
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iopolymeric collagen of vascular tissues, independent ofhether they were in a fresh or cryopreserved condition.ollagen cryoresistance has already been reported by Shep-ferd and Dawber.14 Our previous work about the biome-hanical behavior of vascular tissues processed in INDTonfirmed the quality of the cryopreserved-defrosted prod-ct.5,20,30 Finally, the clear decrease in ordering profileshown in the distribution of ages validates the use of theiffractometric technique.In conclusion, procurement, cryopreservation, and de-
rosting of vascular tissue did not damage fibrillar collagentroma. The results showed structural changes in cryopre-erved defrosted probands but without any modification inunctional biomechanical behavior, according to our previ-us data.
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unctional en aortas de cerdo criopreservadas. Estudio preliminar.ngiologia 56:107, 2004tu
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