prospects in lyophilization of bovine pericardium

Prospects in Lyophilization of Bovine Pericardium

*Adolfo A. Leirner, †Virgílio Tattini Jr, and †Ronaldo N.M. Pitombo

*Center of Biomedical Technology of the Heart Institute (Incor); and †Department of Biochemical and PharmaceuticalTechnology, Pharmaceutical Science School, University of São Paulo, São Paulo, Brazil

Abstract: Almost 30 years after the introduction of heartvalve prostheses patients worldwide are benefiting fromthe implant of these devices. Among the various types ofheart valves, the ones made of treated bovine pericardiumhave become a frequently used replacement of the heart’snative valve. Lyophilization, also known as freeze-drying, isan extremely useful technique for tissue storage for surgicalapplications. This article gives a brief overview on thecurrent bovine pericardium lyophilization development,

including the chemical modification to improve physical–chemical characteristics and the advanced technologiesused to guarantee a high-quality product. It was shown thatlyophilization process can be successfully applied as amethod of bovine pericardium preservation and also as atechnological tool to prepare new materials obtained bychemical modification of native tissues. Key Words: Bo-vine pericardium—Heart valves—Freeze-drying—Ramanspectroscopy—Thermal analysis.

In cardiovascular surgery field, almost 30 yearsafter the introduction of heart valve prosthesespatients worldwide are benefiting from the implant ofclose to 300 000 of these devices each year. It is esti-mated that approximately half are mechanical andhalf are biological tissue, suggesting a shift towardincreasingly greater usage of tissue valves over thelast decade.The ones made of treated bovine pericar-dium, among the several existing kinds, have becomea frequently used replacement of the heart’s nativevalve (1).

From the socio-economic point of view bovinepericardium tissue prostheses possess numerousadvantages: the raw material costs are minimal,because the materials are obtained from organs andanimal tissues rejected by slaughterhouses; the utili-zation of bioprostheses foregoes the use of antico-agulants; the morbidity is decreased through thereduced incidence of hemorrhagic episodes; thenecessity of frequent coagulation tests is eliminated.

Nonetheless, they are less durable and frequentlyrequire reoperation.

LYOPHILIZATION OF BIOLOGICALTISSUES—INTRODUCTION

Lyophilization, also known as freeze-drying, is anextremely useful technique for tissue storage for sur-gical applications. The purpose of freeze-drying bio-logical tissue either homografts or heterografts is thebanking of implants for use in human and veterinarysurgery. The primary object of lyophilization is topreserve biological material without damage, byfreezing the enclosed water and then removing theice by sublimation. It combines the advantages ofboth freezing and drying to obtain a more favorablestate of preservation. Lyophilization reduces theproblems of storage and distribution of frozen tissuedue to its dry form. It has been used for preservationof bioprostheses and in tissue engineering methods(2–4).

In clinical practice other advantages of freeze-drying include decreased surgical time and morbidity,assessment of the graft size and shape prior to opera-tion, and storage of tissue grafts in operating rooms(5).

Lyophilization does not possess an inherent steril-izing action although the equipment used can be

doi:10.1111/j.1525-1594.2009.00712.x

Received July 2008.Address correspondence and reprint requests to Dr. Ronaldo

N.M. Pitombo, Department of Biochemical and PharmaceuticalTechnology, Pharmaceutical Science School, University of SãoPaulo, Ave. Lineu Prestes, 580, CEP: 055508-900, São Paulo, SP,Brazil. E-mail: [email protected]

Artificial Organs33(3):221–229, Wiley Periodicals, Inc.© 2009, Copyright the AuthorsJournal compilation © 2009, International Center for Artificial Organs and Transplantation and Wiley Periodicals, Inc.

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sterilized and operated under aseptic conditions.However, the lyophilized material allows gamma rayssterilization and the application of other processessuch as microwave irradiation or ultraviolet light foreffective sterilization to eliminate microorganisms,including viruses, when the material is destined formedical or surgical applications (6).

Although lyophilization is a sophisticated andhighly developed technology, it is far from being fullyestablished, so that each new product justifies adetailed investigation.Seeking the final process aimedat its full development and utilization demands amultidisciplinary approach, combining researchers inmany areas of knowledge (biology, biochemistry,medicine, and physics). Freeze-drying is a multistageoperation that stabilizes biomaterials by meansof four main procedures: freezing, sublimation orprimary drying, desorption or secondary drying, andfinal storage. As a drying technology, it belongs to athird generation of driers that was developed at first toovercome structural damage to the material.

In the biological area, lyophilization, which at itsbeginning was restricted to human plasma and anti-biotics, is now used to preserve sensitive biochemicalas well as highly sophisticated biotechnological prod-ucts, especially in products where the recovery oftheir full biological activity is linked to preserva-tion of the structure of active substances (usuallyproteins).

Although a dried product with the highest qualitycan be obtained by lyophilization, the process is stillslow and expensive. The scientific and engineeringunderstanding of the freeze-drying process hasincreased significantly due to theoretical and experi-mental studies aimed at determining the freeze-drying conditions for the product of interest throughmodeling the process, thermal characterizationanalysis, process analytical technology, and freeze-drying control parameters. In this regard it is neces-sary to know more about the effects of each step ofthe lyophilization process on the involved structures.The use of this knowledge in the design and opera-tion of the freeze-dryers will allow operation offreeze-drying processes at optimum conditions that

may lower the cost and operation time and improvethe quality of the resulting process.

The design of lyophilization process can have asignificant impact on tissue stability. The cycle mustbe robust and efficient to reduce structural damageand consequently preserve the natural properties ofthe tissue. Freezing rate and temperature, thermaltreatment method, drying rate, and finally the mois-ture content must be analyzed step-by-step in orderto improve the stability during storage.

BIOLOGICAL TISSUES—STRUCTUREAND TREATMENTS

Biological tissues normally used in the manufac-ture of prostheses are mainly composed of collagen.The structure of water surrounding collagen has beenstudied by a wide variety of techniques; for example,hydrothermal isometric tension, and differentialscanning calorimetry (DSC) (7). Water plays a veryimportant role in maintaining the conformation ofcollagen molecules and the mechanical properties ofcollagen fibrils (8).

It is commonly accepted that in the simplest repre-sentation, water associated with collagen can bedivided into three types: structural, bound, and free orbulk water.Structural water is believed to stabilize thetriple helix by participating in the H-bond backbone.The second fraction corresponds to hydrogen-bondedwater between triple helices and between themicrofibrils. The third fraction consists of free waterbetween the microfibrils and fibrils (9).

To improve the material’s resistance severalchemical and physical reticulation processes havebeen developed. Nowadays, the most employedmethod is reticulation by glutaraldehyde (GA) (Fig.1), although GA reticulation is believed to inducecalcification “in vivo” leading to valve failure (10).GA-treated bovine pericardium (GABP) has beenused extensively to construct heart valve bioprosthe-ses, patches, and to shape conduits. GA stabilizesthe collagen structure, prevents tissue digestion byenzymes or bacteria, and reduces the antigenicity ofthe material (11).

FIG. 1. Collagen reticulation by glutaral-dehyde.

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Several efforts have been made to decrease thecalcium deposition in valve prostheses: heparincoating; hyaluronic acid coating; changes in reticula-tion methods like ultraviolet radiation, photo-chemical cross-link (12), and utilization of epoxidemolecules derived of epichloridrine (13).

FREEZE-DRYING—BASIC CONCEPTS

Freeze-drying is a technique where freezable wateris removed from a frozen material first by sublima-tion followed by desorption of the unfrozen water,under reduced pressure. The freezing stage is consid-ered a critical step because it influences the ice crystalsize and, consequently, the primary and secondarydrying stages (14,15). In the primary drying, the icecrystal sizes must be large enough to obtain the short-est drying cycle time. However, in the secondarydrying, the sizes must be smaller to offer a largespecific surface area of the dried matrix in orderto improve desorption of unfrozen water from thesurface pores of the amorphous matrix (16).Thefreezing step is a key step because it fixes the icecrystal structure (shape, dimensions) and, conse-quently the sublimation time, which is the longestphase of the whole process and the pores specificsurface area—which is sometimes a key factor duringthe secondary drying period—and finally the rehy-dration time. In order to optimize the whole freezing-drying cycle, firstly, optimal ice crystal sizes must bepursued to minimize the operating costs related tothe whole duration of these drying steps (16).

The immobilization caused by the freezing stepprevents the migration of nonvolatile molecules onthe drying boundary, resulting in retention of form,and large surface area of lyophilized tissue. Anannealing treatment just after the freezing stepduring a few hours by reheating the product, gen-erally confers a certain homogenization to theice crystal size distribution. Thermal treatmentby annealing has been studied by many researchers(17–19).

One key parameter that has been identified ascrucial to understanding the lyophilization process isthe glass transition temperature, or Tg′, of a givenproduct. Generally, the process temperature is setbelow the Tg′ of the product during primary drying inorder to avoid the “collapse” during lyophilization.Collapse during freeze-drying is defined as the loss ofthe microstructure that was established by freezingdue to the viscous flow of amorphous material duringprimary drying (sublimation of ice) or secondarydrying (desorption of bound water). Collapse is abehavior that is characteristic of amorphous systems

arising from glass-transition-associated mobility (20).In addition to the glass transition temperature, themagnitude of the change in heat capacity (DCp) atTg′ along with the occurrence of any recrystallizationevents may have a major effect on the successfulavoidance of the collapse of the product duringprocessing. Thus, an analytical technique is required,which yields accurate, sensitive, and reproducibledata on Tg′ and recrystallization transformations inthe sub-ambient temperature regions. This informa-tion is valuable in the generation of a freeze-driedproduct, which does not exhibit any signal of matrixshrinkage due to collapse phenomenon (21).

Differential scanning calorimetry provides ameans of addressing the key issues surrounding theproduction of a successful lyophilized material. Itprovides the necessary high degree of sensitivity,resolution, and stable sub-ambient performance nec-essary to observe the weak glass transition(s), andrecrystallization events associated with productsundergoing lyophilization. For simple aqueous solu-tions, glass transition temperature was shown to takeplace 1–2°C below the collapse temperature (Tc)(22). Tc is also often considered close to the glasstransition temperature, which in turn is usually mea-sured by DSC (23,24). Collapse temperatures aremeasured by direct microscopic observation in astage at low temperature and low pressure simulatingfreeze-drying conditions. Actually, a temperature,above which the “mobility” of the system is observedon an experimental time scale, characterizes the glasstransition.

However, Tc cannot be considered as identical tothe glass transition temperature. Glass transitionis measured in closed systems after freezing (andbefore melting), whereas collapse is a dynamic phe-nomenon taking place during freeze-drying (25).

To assure long-term stability of the dried pericar-dium, the glass transition temperature must exceedthe planned storage temperature.

The residual moisture must be sufficiently low toavoid the presence of solvent water, which acts as aplasticizer of the amorphous phase.

The goal of the process is to provide the highest Tcpossible to maintain the protein stability. The effectof collapse on lyophilized bovine pericardium isshown in Fig. 2.

MOLECULAR PHENOMENA IN BOVINEPERICARDIUM—LINKING

Collagen is the main structure protein of connec-tive tissue, including new blood vessels, skin, bone,and membranes. Pericardial tissue is composedmainly of collagen type I (26).

LYOPHILIZATION OF BOVINE PERICARDIUM 223


Raman spectroscopy is a very powerful toolfor studying the structure and molecular inter-actions of many complex molecules. This methodwas successfully used for studying the mechanismof GA–collagen cross-linking. One of the greatestadvantages of this technique is its ability to provideinformation about the structure and interactions ofbiomolecules in their microenvironment withinintact cells and tissue. The technique is not destruc-tive and does not require homogenization, extrac-

tion or the use of dyes, labels, or other contrast-enhancing agents. Most of proteins can undergoqualitative and quantitative changes under variousstress conditions (e.g., freezing, drying, and storage).Because Raman spectroscopy offers the possibilityto examine these changes in situ, several authorshave studied the spectra of various collagenousbiological substances such as calcified tissue (27),bovine Achilles tendon (28), and GA-treated peri-cardium tissue (9).

FIG. 2. Scanning electron microscopy (SEM) micrographs of lyophilized bovine pericardium.



The collagen structure itself shows heat sensibilitywhen exposed to higher temperatures. At tempera-tures above 50°C the physical structure starts toshow shrinkage signals. This phenomenon is alsoknown as denaturation temperature. When the col-lagen reaches this specific temperature, it loses oneof the main properties of a tissue: its elasticity. Thetemperature at which denaturation—and henceshrinkage—under constant load begins is termed the“shrinkage temperature,” a term often applied to thecollagen denaturation temperature, even if measuredby other means. A typical procedure involves clamp-ing the sample to the load cell of a tensiometer fol-lowed by heating of the sample in a water or salinebath at a rate of 0.5–1°C/min. When the tissue con-tracts, the tension increases rapidly and the tempera-ture at the onset of this contraction is defined asthe shrinkage temperature. The limitations of thehydrothermal method are that it measures onlydimensional changes on a macroscopic scale, has amaximum workable temperature of 100°C, and if thesamples are preloaded with weights to keep themstraight, the load may retard contraction of the speci-men and give an erroneous shrinkage temperature.

Several reports in the last decade have utilizedDSC as an alternative method to determine theshrinkage temperature of tanned biological tissues(29–34). DSC is a well-developed analytic toolused for measurement of transitions and associatedenthalpies in polymers and other chemical systems.For collagenous materials, one pan contains a smallsample of tissue in a bathing solution while the otherpan contains—as a blank—an equivalent volumeof solution only. The two pans’ temperatures areincreased, and a difference in heat flow between thetwo pans indicates a transition-dependent enthalpyin the tissue. At the denaturation temperature, atleast one peak is observed and the heat flow/

temperature curve is recorded as well as the enthalpyof the transition calculated from the area under thepeak.

Freeze-drying microscopy is a very useful tool tomeasure the crystallization, collapse, and eutectictemperatures of pharmaceutical solutions intendedto freeze-dry. The temperature of the stage is thenmanipulated in order to identify the point at whichthe product will collapse or melt. This process can beviewed either through the microscope, or with a PCusing the camera and image capture software sup-plied with the system. The equipment has the capac-ity to heat the sample up to 100°C but it has neverbeen used to study the denaturation event. In orderto verify the efficiency of freeze-drying microscopy inmeasuring the denaturation temperature, Tattini andcoworkers (35) performed DSC and freeze-dryingmicroscopy measurements on each of two samplematerials: one natural (fresh bovine pericardium)and the other one of interest for cardiovasculardevices (bovine pericardium cross-linked with GA).The measurements were performed to comparethe results of shrinkage temperature obtained byDSC with the results obtained by freeze-dryingmicroscopy. Figures 3 and 4 show the DSC andderivative DSC (DDSC) curves from bovine pericar-dium untreated and treated with GA cross-linked,respectively. The shrinkage of bovine pericardium isaccompanied by the absorption of heat, giving rise toan endothermic peak on the DSC curve. The areaunder the peak is directly proportional to theenthalpy change DH (enthalpy energy) while itsheight is a measure of the heat capacity (i.e., dH/dT)of the transition. The temperature of the transitioncan be defined as either the onset or the peakmaximum. The peak temperature values used toreport the shrinkage temperature generally displayed

FIG. 3. DSC/DDSC curves obtained in dynamic N2 atmosphere(50 mL/min) and heating rate 10°C/min of the untreated bovinepericardium sample.

FIG. 4. DSC/DDSC curves obtained in dynamic N2 atmosphere(50 mL/min) and heating rate 10°C/min of GA cross-linked bovinepericardium sample.



less deviation than the extrapolated onset value.According to Loke and Khor (36), they recommendthat the peak temperature value should be used asthe shrinkage temperature. The untreated bovinepericardium tissue presented an endothermic peakthat is directly related to the shrinkage of the mate-rial at 57°C (Fig. 3). The same thermal event wasdetected for the GA cross-linked bovine pericardium(Fig. 4) but at a higher temperature (60°C) whencompared with the untreated tissue.

The enthalpic energy (DH) involved during thedenaturation event was different between thesamples too. The GA cross-linked bovine pericar-dium showed a fivefold increase on the DH valuewhen compared with untreated bovine pericardium.

This difference could be explained due to the strongnet structure originated between collagen fibersafter the GA treatment. Freeze-drying microscopyallowed observation of the shrinkage of bovinepericardium tissue; both treated and untreated, atapproximately 60°C as in Figs. 5 and 6, respectively.The results showed that shrinkage temperature ofbovine pericardium tissue obtained from freeze-drying microscopy is quite similar to that obtainedfrom DSC curves. The freeze-drying microscopy canbe used as a very useful tool to determine the shrink-age temperature from biological tissues composedmainly of collagen fibers.

Various experiments have been conducted (per-sonal data) using freeze-drying microscopy to

FIG. 5. Freeze-drying microscope photo-graphs of GA bovine pericardium at differ-ent temperatures: (A) 30.5°C; (B) 40.8°C;(C) 49.9°C; (D) 59.9°C. Heating rate of10°C/min.

FIG. 6. Freeze-drying microscope photo-graphs of untreated bovine pericardiumat different temperatures: (A) 30.9°C; (B)40.4°C; (C) 50.5°C; (D) 60.5°C. Heatingrate of 10°C/min.



observe the Tc of various tissues but, probably, due tophysical characteristics of the samples, we have notobserved any sign of loss of the tissue structure (col-lapse) during freeze-drying.

We have studied the structural changes of nativebovine pericardium after freeze-drying using two dif-ferent freezing protocols: 2°C/min and 30°C/min (37).Alterations both on the intensities and positions of theRaman spectrum peaks were observed. Considerablealterations on the intensity were observed at 939/cm,apeak position related to the C-C a-helix stretch.Also,at 1004/cm, the intensity of the peak was changed.Thispeak is related to the C-C phenylalanine group.According to Leikin and coworkers (38), this peak isrelated to the loss of bulk water from the collagenstructure. It was observed that annealing during slowfreezing protocol maintained the Amide III band(1241–1272/cm) related to the C-N stretch and pre-served the CH3–CH2 binding formation. Also, themaintenance of the peptide carbonyl stretching on theAmide I band (1655–1667/cm) was noticed.

The changes in the freeze-drying time using differ-ent freezing protocols were observed. The annealingstep decreases the freeze-drying time for the samplesunder slow freezing and increases the freeze-dryingtime for the samples which used a fast freezingprotocol. Apparently, the thermal treatment wasresponsible for the reduction in the drying time dueto the increase in the size and distribution of icecrystals. These changes in the interstitial frozenregion alter the sublimation rate in the material (18).It was verified, also by dew point data, that freeze-drying using slow freezing without annealing was theunique freeze-drying run, which showed secondarydrying. Probably, this behavior occurred due to thelarge amount of glassy water formed between the icecrystals in the interstitial region compared with theother freeze-drying runs. Comparing all freeze-drying protocols used in this work, the slow freezingwith annealing presented the best results when com-pared with the other protocols. It maintained the sec-ondary structure mainly on the Amide I and IIIbands, presented the shortest freeze-drying cycle andthe lowest water content in the tissue, optimizing thestorage conditions. However, the annealing step usingthe conditions cited was not enough to increase theTg′ of the maximally freeze-concentrated tissue. Itwas shown that bovine pericardium tissue has enoughphysical support to avoid the shrinkage during thefreeze-drying above maximal product temperature inthe primary drying (Fig. 2). Hence, it follows thatfreeze-drying will not damage bovine pericardiumstructure and consequently the performance offreeze-dried bovine pericardium as a biomaterial.

INFLUENCE OF FREEZE-DRYING INANTIMINERALIZATION METHODS

The finding that GA promotes calcification has ledto new fixation strategies that limit mineralizationand optimally preserve molecular structure. Theypreserve low thrombogenicity and improve durabil-ity, but the risk of late structural failure remains.

The most promising preventive strategies haveincluded binding of calcification inhibitors to GA-fixed tissue, removal or modification of calcifiablecomponents, modification of GA fixation, and use oftissue cross-linking agents other than GA.

Alternative tissue substitutes for bovine pericar-dium have been introduced. The assembly of thevalve and individual leaflets has been altered toimprove flow patterns; and stent materials have beenmodified or eliminated to reduce both stress andpressure gradients (39).

The effect of lyophilization on cytotoxicity andresidual aldehyde concentration of GA-treated wasassessed (39). Cytotoxicity was measured by incu-bating a pericardium sample from each group insaline and assessing the eluant’s influence on cellulargrowth. Residual aldehydes were measured by high-performance liquid chromatography. Although bothgroups’ eluants exhibited some cytotoxicity, theeluant from group A was less cytotoxic, with a cyto-toxicity index (IC50 [%]) of 41%. Group B eluantsall had marked cytotoxic effects; cell growth was24.15% of the negative controls at the most diluteeluant concentration (6.25%). The mean residualGA level was less in group A than in group B(2.36 � 0.11 and 9.90 � 3.70 g/L, respectively; n = 3,P < 0.05), but residual formaldehyde levels did notdiffer. These results demonstrate that compared withconventional GABP, lyophilized pericardium is lesscytotoxic, with fewer residues. It was shown that lyo-philization does not alter the mechanical propertiesof GABP. Thus, this process may be useful inimproving the characteristics of implantable devicesmade of GABP, possibly leading to lower calcifica-tion rates.

Various antimineralization methods have beeninvestigated to overcome the high rate of clinicalfailure of GA-fixed heart valve bioprostheses. Formost of those, stable binding to the fixed tissue is vitalfor long-term efficacy, because the treated valve willoften be stored after treatment for months, some-times years.

Some researchers (40–45), using in vivo models,observed that biomaterials cross-linked with epoxidecompounds showed less calcification in comparisonwith those fixed with GA.



Other potential properties of epoxy compoundfixation, including preservation of natural mechanicalproperties, induction of nonthrombogenic surfaces,and reduction of antigenicity and immunogenicity,have also been reported (45–47).

Figure 7 shows the schematic collagen reticulationby using phenethylamine-diepoxide treatment. It wasinspired by the idea of using a bifunctional epoxidethat, besides cross-linking the tissue through theiramino and sulfhydryl groups also increases the quan-tity of nonpolar, noncharged groups. The sampleswere previously lyophilized in relation to the chemi-cal treatment (13).

From the results, it is possible to infer that thelyophilization associated with the epoxide treatmentis a potential route of bovine pericardium biopro-sthetic heart valves preparation. This hypothesis issustained by the fact that nonlyophilized samplesshowed calcium phosphate deposits, in contrast withthe lyophilized ones.This is a field of study with manypotential applications. The lyophilization of bovinepericardium before chemical treatments with cross-link agents such as epoxy compounds may be analternative to the conventional calcification preven-tion methods, but further investigations are recom-mended to check if the same behavior is found in alllyophilized systems.

CONCLUSION

We have found that the lyophilization process canbe successfully applied as a method of bovine peri-cardium preservation and also as a technological toolto prepare new materials obtained by chemical modi-fication of native tissues.

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