Micro- and nanostructured hydroxyapatite-collagen microcarriers for bone tissue-engineering applications.

Novel hydroxyapatite (HA)-collagen microcarriers (MCs) with different micro/nanostructures were developed for bone tissue-engineering applications. The MCs were fabricated via calcium phosphate cement (CPC) emulsion in oil. Collagen incorporation in the liquid phase of the CPC resulted in higher MC sphericity. The MCs consisted of a porous network of entangled hydroxyapatite crystals, formed as a result of the CPC setting reaction. The addition of collagen to the MCs, even in an amount as small as 0.8 wt%, resulted in an improved interaction with osteoblast-like Saos-2 cells. The micro/nanostructure and the surface texture of the MCs were further tailored by modifying the initial particle size of the CPC. A synergistic effect between the presence of collagen and the nanosized HA crystals was found, resulting in significantly enhanced alkaline phosphatase activity on the collagen-containing nanosized HA MCs.

Energy consumption as a predictor test of the durability of a biological tissue employed in cardiac bioprosthesis.

The mechanical behavior of the young bull pericardium in a fatigue test has been studied. This material is a similar tissue to those used in valve leaflet construction for a cardiac bioprosthesis. The consumed energy on each test was evaluated and afterwards used as a predictor of the biomaterial strength. Two-hundred and nine samples were tested to cyclical fatigue. The cut-off point to determine the sample quality was whether or not they resisted at least 4500 cycles. Only 22 samples withstood over that point (10.52%). The samples were classified according to their fatigue behavior in excellent, undefined and unsuitable. By using as a reference the consumed energy in the first 25 cycles, we could distinguish correctly (between 93.2 and 96.1%) the unsuitable material and most of the excellent (between 78.1 and 95.2%). From the rejected material 77% was really detachable and from the accepted, only 50% was excellent, with an equal methodology. The receiver operating characteristics curve was employed to establish decision levels when selecting samples, being 0.85 the best area (theoretical maximum value of 1). It is concluded that the energy wasted is a good predictor of the strength of the tissue. More than 90% of the unsuitable material and 50% of the excellent material (5% of all the material) is detected with this method.

Influence of the suture in the propagation of tears in calf pericardium employed in the construction of cardiac bioprostheses.

The tearing of the fibers of biomaterials employed in implants or bioprostheses leads to early the failure of these devices. The purpose of this study was to determine the force necessary to propagate a tear in a biological tissue, calf pericardium, when sutured. We analyzed the outcome of 230 trials. There was a loss of resistance to tearing in samples sutured edge-to-edge as compared to unsutured control samples. This loss was not observed when the suture was preceded by an intact or protective zone. The values corresponding to the tearing force for an overlapping suture, especially when sewn with Gore-Tex(R), were higher than those obtained in controls. This study confirms the deleterious effect of the edge-to-edge suture, which can be minimized by protecting the suture, and the excellent behavior of the overlapping suture.

Biochemical and mechanical behavior of ostrich pericardium as a new biomaterial.

We have performed a comparative analysis of glutaraldehyde-preserved ostrich pericardium, as a novel biomaterial, with bovine pericardium. The biochemical characteristics (histology, water content, amino acid composition, and collagen and elastin contents), mechanical properties, and in vivo calcification in a subcutaneous rat model were examined. Ostrich pericardium is slightly thinner and shows a higher water content (70+/-2% vs. 62+/-2%) than bovine pericardium. Additionally, ostrich pericardium presents 1.6-fold lower elastin content and a lower percentage of collagen in reference to the total protein content (68+/-2% vs. 76+/-2%). However, ostrich pericardium shows better mechanical properties, with higher tensile stress at rupture (32.4+/-7.5 vs. 11.5+/-4.6) than calf pericardium. In vivo calcification studies in a rat subcutaneous model show that ostrich pericardium is significantly less calcified than bovine pericardium (23.95+/-13.30 vs. 100.10+/-37.36 mg/g tissue) after 60 days of implantation. In conclusion, glutaraldehyde-stabilized ostrich pericardium tissue shows better mechanical properties than calf tissue. However, calcium accumulation in implanted ostrich tissue is still too high to consider it a much better alternative to bovine pericardium, and anticalcification treatments should be considered.

Tissue heart valve mineralization: Review of calcification mechanisms and strategies for prevention.

Attempts to replace diseased human valves with prostheses began more than 30 yrs ago. Heart valve prostheses can be broadly classified into mechanical prostheses (made out of non-biological materials) and bioprostheses made out of biological tissue. Biological valves are made from animal tissue bovine pericardium and porcine valves. The use of these tissues became commercially available after the introduction of the glutaraldehyde (GA) fixation technique. GA reacts with tissue proteins to form inter- and intramolecular crosslinks, resulting in improved durability. The advantage of bioprostheses compared with mechanical valves is the freedom from thromboembolism; and therefore, the avoidance of long-term anticoagulation therapy. These prostheses are preferable in elderly people and in patients who do not tolerate anticoagulants. However, tissular calcification and primary tissue failure (caused by the mechanical stress) are the main unresolved problems. The causes of calcification are numerous and, to date, a satisfactory solution to this question has not been found, although chemical treatments with metal cations, diphosphonates and treatments eliminating phospholipids have proved to mitigate calcification. In addition, alterna-tive approaches to GA chemical treatment fixation are being proposed to provide the tissue with greater resistance to this process. Studies are under way using polyepoxy compounds, derivates of amino oleic acid (AOA), agents such as diphenylphosphorylazide, carbodiimide, amino acids etc. Further improvements in fixation techniques, as well as in bioprosthesis design (stentless valves) are being made to improve the durability and functional characteristics of bioprosthetic heart valves. The development of a biomaterial capable of withstanding calcification and mechanical stress, while being as durable as mechanical prostheses, would convert the bioprostheses into the replacement of choice by eliminating the need for anticoagulation therapy.

Comparative study of the mechanical behaviour of a cyanoacrylate and a bioadhesive.

We compared the mechanical resistance of 18 samples of calf pericardium bonded with a 100 mm2 overlap, by two types of glues: a cyanoacrylate (Loctite 4011) and a bioadhesive (BioGlue). Comparative tensile testing was also carried out in 40 paired samples, 20 bonded with the cyanoacrylate and 20 unbonded controls. The findings at rupture showed a greater resistance of the calf pericardium glued with cyanoacrylate, with a mean tensile strength of 0.15 MPa vs. 0.04 MPa for the biological glue (p= 0.000). They also demonstrated a loss of resistance of the samples bonded with cyanoacrylate when compared with that of the unbonded other halves of the pairs: 0.20 MPa and 0.27 MPa vs. 19.47 MPa and 24.44 MPa (p < 0.001). The method of selection by means of paired samples made it possible to establish the equations that relate the stress and strain, or deformation, with excellent coefficients of determination (R2). These equations demonstrate the marked elastic behaviour of the bonded samples. Moreover, these findings show the cyanoacrylate to be superior to the biological glue, leading to the examination of the compatibility, inalterability over time and mechanical behaviour of the cyanoacrylate in sutured samples, as well as the study of the anisotropy of the biomaterial when bonded with a bioadhesive.

Resistance to tensile stress of a bioadhesive utilized for medical purposes: Loctite 4011.

Sutures are the materials presently employed to secure and give shape to the valve leaflets of cardiac bioprostheses. Their high resistance and low degree of elasticity in comparison with the calf pericardium of which the leaflets are made generates internal stresses that contribute to the failure of the bioprostheses. Biological adhesives are bonding materials that have begun to be utilized in surgery, although there is a lack of experience in their use with inert tissues or bioprostheses. We report our study of Loctite 4011, a biological glue composed of a cyanoacrylate that has been employed for medical purposes, in which samples of pericardium bonded with this adhesive were subjected to uniaxial tensile stress. The samples were glued in such a way as to leave an overlap of 1 cm2 between the surfaces of the tissue. The series included 83 samples: 12 tested 24 h after bonding, 17 after 45 days, 17 after 90 days, 19 after 106 days and 18 after 152 days. The samples subjected to deferred trials were preserved using three types of chemical substances: glutaraldehyde, glycerol or saline plus antibiotics. The mean resistance to rupture of the series tested 24 h after gluing was 0.15 MPa (1.47 machine kg). This resistance remained nearly unchanged, regardless of the preservation solution employed, for at least 152 days, the time at which the study ended. The stress-strain curves demonstrated a high degree of elasticity throughout the 152 days, a finding that was not influenced by the preservation solution. This adhesive showed a considerable resistance to tensile stress, although probably insufficient to replace sutures. However, it maintained a surprisingly high degree of elasticity in the samples. Perhaps the time has come to combine these two elements, sutures and adhesives, to improve the elasticity of the structure without a loss of resistance, and increase the durability of bioprostheses.

Comparison of the mechanical behaviors of biological tissues subjected to uniaxial tensile testing: pig, calf and ostrich pericardium sutured with Gore-Tex.

The purpose of this study was to compare the mechanical behavior of calf pericardium, pig pericardium and ostrich pericardium when subjected to tensile testing. Tensile stress was applied to 108 tissue samples, 36 of each type of tissue, until rupture. Groups of three adjacent strips measuring 12 x 2 cm(2) were cut longitudinally. Each group consisted of an unsutured center sample, or control, and the two contiguous samples, that on the right sutured with Gore-Tex at a 90 degrees angle with respect to the longitudinal axis and that on the left sewn with the same suture material at 45 degrees angle. The sutured samples showed a statistically significant loss of resistance (p<0.001) when compared with the corresponding unsutured tissue. The mean stresses at rupture for sutured ostrich pericardium were 21.81 and 20.81 MPa in the samples sewn at 45 degrees and 90 degrees, respectively, higher than those corresponding to unsutured calf and pig pericardium, 14.0 and 11.49 MPa, respectively, at rupture. The analysis of the stress/strain curve shows a smaller difference between sutured and unsutured ostrich pericardium than those observed in the other two biomaterials. These results demonstrate that, in addition to its greater resistance, ostrich pericardium also presents a less pronounced interaction with the suture material. Its capacity to absorb the shearing stress produced by the suture is greater. This report also confirms that the method of selection using paired samples ensures their homogeneity and makes it possible to predict the behavior of a sample by determining that of the other half of the pair.

Gelatinases in soft tissue biomaterials. Analysis of different crosslinking agents.

Chemical modification of pericardium-based cardiac valves tends to reduce the relatively high degree of biodegradation and calcification of the implanted bioprostheses. We analysed the tissue properties of pericardium from young calves and pigs after crosslinking with different agents (glutaraldehyde. diphenylphosphorylazide (DDPA), 1-ethyl-3,3-dimethyl-aminopropyl-carbodiimide (EDAC)) and when exposed to anticalcification treatments (chloroform/methanol or ethanol) prior to glutaraldehyde (GA) crosslinking. Protein extraction after tissue homogenisation in the presence of detergents showed that crosslinking using GA or DPPA was much more effective. The amounts of protein extracted from these two groups of chemically modified pericardium were significantly lower: the other modified tissues presented only a slight reduction when compared with untreated tissue. Matrix metalloproteinases- (MMP) 2 and 9 were detected in native pericardium from calf and pig by zymography. While the MMP-9/MMP-2 activity ratio was close to 1 in pig pericardium, it was 8.5-fold higher in bovine tissue. Crosslinking with GA and with DPPA almost completely abolished gelatinase activities, even when equal amounts of solubilised protein were loaded onto the zymograms. Anticalcification treatments followed by GA crosslinking or treatment with EDAC were not as effective in reducing gelatinase activities; but, interestingly, a relative reduction of MMP-9 versus MMP-2 was detected. The presence of these gelatinase activities in pericardium may contribute to the in vivo degradability of pericardium-based cardiac valves.

The telescoping suture--Part 1: Does this technique improve the mechanical behavior of a biomaterial?: Calf pericardium.

The authors study the mechanical behavior of calf pericardium employed in the construction of cardiac valve leaflets when subjected to telescoping suture, followed by tensile stress until rupture. One hundred twenty pericardial tissue samples were employed, 60 cut from root-to-apex and another 60 cut in transverse direction. Each of these two groups consisted of 12 control samples that were left unsutured and four sets of 12 samples each that were rejoined by telescoping suture using silk, Prolene, nylon or Gore-Tex., and subjected to tensile stress. At the rupture of the sutured tissues, the tensile stress of the suture materials ranged between 57.54 MPa for the series sewn lengthwise with Gore-tex and 114.08 MPa for the series sewn crosswise with silk. At these levels of stress, the deformation of the suture thread was much less marked than that of the calf pericardium, and internal stresses were produced that were difficult for the biomaterial to absorb. There was a loss of real load in all the sutured series when the observed resistance to rupture, expressed in kilograms, was compared with the estimated value. This loss of resistance did not invalidate the telescoping suture technique since the resistance to rupture was still much greater than that associated with suturing the two edges of the cut pericardium together. This report confirms the deleterious role of the shear force generated in the pericardium by the suture.

The telescoping suture--Part II: A novel method to improve the mechanical behavior of a new biomaterial: ostrich pericardium.

Ostrich pericardium, sutured using a telescoping or overlapping technique, was studied to determine its mechanical behavior. From each of 12 pericardial sacs, four contiguous strips were cut longitudinally, from root to apex, and another four contiguous strips were cut in transverse direction. One of the strips in each set of four was used as an unsutured control and the remaining three were sutured by overlapping 0.5 cm of the tissue and sewing with Gore-tex, Prolene or Pronova. These 96 samples were then subjected to tensile testing along their major axes until rupture. The tensile stresses recorded in the suture materials at the moment tears appeared in the pericardium ranged between 55.99 MPa and 70.23 MPa for Gore-tex in samples cut in the two directions. Shear stress became ostensible at 56 MPa, with clearly evident tears. However, microfracture of the collagen fibers must be produced at much lower stress levels. The comparison of the resistance in kilograms (machine-imposed), without taking into account the sections in which the load was applied, demonstrated only a slight loss of load when the telescoping suture was employed in ostrich pericardium samples. Ostrich pericardium may continue to be an alternative biological material for the construction of heart valve leaflets.

Ostrich pericardium, a biomaterial for the construction of valve leaflets for cardiac bioprostheses: mechanical behaviour, selection and interaction with suture materials.

The mechanical behavior of ostrich pericardium was studied for the purpose of assessing its utility in the construction of bioprosthetic cardiac valve leaflets. The tissue was tested biaxially using a hydraulic simulator that subjected it to increasing stress until rupture. One hundred eighty trials were performed, 36 with unsutured pericardium and four series of 36 trials each with pericardium sutured with silk, Prolene, nylon or Gore-Tex. The samples were tested in pairs from three different pericardial regions. One sample from each pair (the predictive specimen) was assessed according to morphological and mechanical criteria, while the other (the predicted or selectable specimen) was subjected only to morphological analysis. The findings show that ostrich pericardium treated with glutaraldehyde according to standard methods has an excellent resistance to rupture in biaxial testing, withstanding stresses of up to 100 MPa, and never lower than 30 MPa. Its resistance to rupture is lowered by suturing, a loss that is less pronounced when silk sutures are used. The results with Gore-Tex are very homogeneous and the elastic behavior of the pericardium/suture unit appears to be similar to that of unsutured tissue, suggesting that the interaction between the two biomaterials is minor. Similar results were observed in the series sutured with Prolene and nylon. The use of paired samples makes it possible to closely estimate the mechanical behavior of the tissue in a given zone by determining that of its mate. The statistical study shows that this estimation is not conditioned by the suture employed, thus validating this approach and providing more precise criteria for tissue selection.

Chemical treatment and tissue selection: factors that influence the mechanical behaviour of porcine pericardium.

Calcification and mechanical failure are the major causes of the loss of cardiac bioprostheses. The chemical treatments used to stabilize the tissue employed are considered to play a fundamental role in the development of these two phenomena, although the problem is multifactorial and the underlying causes are yet to be fully identified. Currently, there is an ongoing search for chemical treatments capable of reducing or eliminating the process of calcification while preserving the mechanoelastic characteristics of the tissue. One of the approaches to this effort is the elimination of the phospholipid component from the biological tissue employed in prosthesis construction. There is evidence that this component may be responsible for the precipitation of calcium salts. The present study compares two delipidating chemical treatments involving chloroform/methanol and sodium dodecyl sulfate (SDS) with the use of glutaraldehyde (GA) alone. For this purpose, porcine pericardial tissue was subjected to tensile strength testing employing a hydraulic simulator. A total of 234 samples were studied 90 treated with GA, 72 treated with chloroform/methanol and 72 treated with SDS. The mean breaking strength was significantly higher in the samples treated with GA (between 43.29 and 63.01 MPa) when compared with those of tissue treated with chloroform/methanol (29.92-42.30 MPa) or with SDS (13.49-19.06 MPa). In a second phase of the study, selection criteria based on morphological and mechanical factors were applied to the pericardial membranes employing a system of paired samples. The mathematical analysis of the findings in one fragment will aid in determining the mechanical behavior of its adjacent twin sample. In conclusion, the anticalcification chemical treatments tested in the experimental model conferred a lesser mechanical resistance than that obtained with GA. On the other hand, the utilization of paired samples was found to be useful in the prediction of the mechanical behavior of porcine pericardial tissue. Nevertheless, in order for our method of selection to be considered the most adequate approach, it will be necessary to validate these findings in dynamic studies involving a real, functional model.

A new method for selecting calf pericardium for use in cardiac bioprostheses on the basis of morphological and mechanical criteria.

The durability of existing calf pericardium bioprostheses is limited by phenomena such as mechanical stress and calcification, the factors most frequently implicated in valve failure. Varying the preferred direction of the collagen fibers influences the mechanical behavior of the pericardial membrane. Given this possible variation, a strict control of the selection of the biomaterial employed in the construction of valve leaflets is essential, but a reliable method of selection has yet to be established. This study describes the development of a new system of in vitro selection involving a hydraulic simulator that reproduces the mechanical behavior of pericardial membranes subjected to the stress of continuous flow. By combining morphological criteria such as thickness and homogeneity with those of mechanical behavior, and by selecting paired samples from different parts of the pericardium, we obtained excellent mathematical fits. Linear regression analysis provided the mode of predicting the tensile strength in a given sample when this value had been determined in its twin. The upper zones of calf pericardium, corresponding to either right or left ventricle but at a distance from ligamentous structures, showed the best mean results at rupture (60 MPa) and permitted the most reliable prediction. The expected stress for an elongation of 30% was 1.12 MPa, as was previously observed, with a 95% confidence interval of between 1.11 and 1.14 MPa. These trials, together with the careful selection of the pairs, should help to establish definitive selection criteria.

Porcine pericardial membrane subjected to tensile testing: preliminary study of the process of selecting tissue for use in the construction of cardiac bioprostheses.

The durability of cardiac bioprostheses is limited fundamentally by structural failure due to mechanical fatigue and calcification. In the present report, we analyze, using an in vitro hydraulic simulator to test tensile strength, the mechanical behavior of porcine pericardium for the purpose of establishing the criteria for selecting the biomaterial, taking into account both morphological criteria (thickness and homogeneity of the specimens) and mechanical criteria (stress at breaking point), using the epidemiological model of paired samples. The stress at breakage was found to range widely from 24.07 MPa to 100.29 MPa, although we observed no statistically significant differences when comparing the mean results in the different regions and zones of the pericardium being studies. The application of the selection criteria in the present series resulted in an excellent mathematical fit in terms of the stress/elongation (R2 > 0.95), making it possible to establish, by means of linear regression, the prediction of the tensile strength in one zone on the basis of the values observed in its paired specimen.

Calcification and identification of metalloproteinases in bovine pericardium after subcutaneous implantation in rats.

The purpose of this study was to evaluate the influence of two anticalcification pre-treatments (chloroform/methanol and ethanol) and serum conditioning of glutaraldehyde-crosslinked bovine pericardium on the calcification degree and the presence of gelatinase activities in a subcutaneous implantation model in rats. Regarding calcification of the implants, glutaraldehyde control treatments showed a significatively higher calcification degree than pericardium treated with anticalcification reagents. Serum conditioning of glutaraldehyde treated tissues did not influence the calcification degree; moreover, no differences were found in these samples with the time of implantation (30 and 90 days). On the other hand, anticalcification treatments resulted in a very significant decrease in the calcium content in the implanted membranes. Gelatinase activities were detected by gelatin zymography in almost all the implanted samples. However, control tissues with and without serum conditioning showed less gelatinase activities than those samples pre-treated with anticalcification treatments. Metalloproteinase (MMP-2) activity was detected in all the samples analyzed but a higher expression of MMP-9 was detected in those implants treated with chloroform/methanol and ethanol. Additional gelatinase activities showing lower molecular weight than MMP-2 were also detected in both anticalcification treated samples. The presence of these gelatinase activities is probably due to host cellular infiltrates and could contribute to the biomaterial degradation.

Uniaxial and biaxial tensile strength of calf pericardium used in the construction of bioprostheses: biomaterial selection criteria.

Using morphological and mechanical criteria and applying a method involving paired samples that is widely employed in epidemiology, we obtained an excellent prediction of the mechanical behavior of the calf pericardium used in the construction of cardiac bioprostheses. The method of selection employed in this study may be a highly useful tool for guaranteeing the mechanical resistance of calf pericardium, with a very low level of error.

Resorption and calcification of chemically modified collagen/hyaluronan hybrid membranes.

The objectives of this study were to characterize collagen/hyaluronan hybrid membrane before and after different chemical crosslinking, as well as to investigate the impact of the treatment method on resorption and calcification processes by in vivo tests. The kinetics study of resorption of collagen/hyaluronan membranes showed that after 28 days the membranes were completely resorbed. The modification of the membranes with glutaraldehyde comparing to the similar modification of pericardial tissue conducts higher calcification. Treatment with hexamethylene diisocyanate together with poly(oxyethylene) improved mechanical properties but the resorption process was similar. Additional treatments with diphenylphosphorylazide and 1-ethyl-3-(3-dimethylamino-propyl) carbodiimide (EDAC) resulted in prolongation of the resorption time. Calcification was suppressed in case of treatment with EDAC.

Assessment of pericardium in cardiac bioprostheses. A review.

Cardiac valve bioprostheses are assessed in terms of their present and future clinical utility. The problems concerning durability basically involve early failure due to tears in the valve leaflets and late failure mainly associated with calcification of the biological tissue. New strategies for selection and chemical treatment of the biomaterials employed are analyzed, and the available knowledge regarding their mechanical behavior is reviewed. It is concluded that the durability of these devices, and thus their successful use in the future, depends on the knowledge of the interactions among the different biomaterials of which they are composed, the development of new materials, and the engineering design applied in their construction.

Influence of different chemical cross-linking treatments on the properties of bovine pericardium and collagen.

The use of biological materials in the construction of bioprostheses requires the application of different chemical or physical procedures to improve the mechanical performance of the material without producing any undesirable effects. A number of cross-linking methods have been tested in biological tissues composed mainly of collagen. The basis for most of them is the use of glutaraldehyde (GA), which acts on the Lys or Hyl residues. We have studied the effects of alternative chemical treatments: diphenylphosphorylazide (DPPA) and ethyldimethylaminopropyl carbodiimide (EDAC). Their mechanism of action is based on the activation of the carboxyl groups, which then permits their cross-linking to amino groups. As a control, we employed conventional treatment with GA, applying it to bovine pericardium and collagen membranes removed from bovine pericardium. The analysis of the Lys and Hyl residues showed that DPPA and EDAC produced 50% of the chemical change provoked by GA. This value was even lower in the trials with collagen. In terms of the resistance to collagenase degradation, chemical cross-linking with GA provided much greater protection in both materials (3.81 +/- 3.47 nmol of amino acid/mg dry tissue for pericardium and 4.41 +/- 1.13 nmol of amino acid/mg dry tissue for collagen). Treatment with DPPA also protected pericardium (13.11 +/- 6.57 nmol amino acid/mg dry tissue) although the values for collagen was lower (50.0 +/- 32.4 nmol amino acid/mg dry tissue). Treatment with EDAC was much less protective than the other two chemical reagents (43.28 +/- 17.4 and 55.85 +/- 14.57 nmol amino acid/mg dry tissue for pericardium and collagen, respectively). The degree of tissue calcification after implantation of the chemically treated materials into young rats was considerably greater for GA and DPPA (32.9 +/- 18.8 and 36.3 +/- 13.3 mg g(-1) dry tissue, respectively) than with EDAC (18.0 +/- 7.2 mg g(-1) dry tissue; P < 0.001). After 60 days of implantation, the values for GA and EDAC were higher(124.1 +/- 31.3 and 124.6 +/- 21.0 mg g(-1) dry tissue, respectively) versus 34.6 +/- 19.2 mg g(-1) dry tissue for DPPA. There were no significant differences in collagen levels in samples treated with GA or EDAC after 30 days of implantation, although both groups showed significant differences when compared with DPPA-treated samples (P < 0.001). After 60 days of implantation, there were no significant differences among these three treatments in terms of the calcium accumulated on samples.