2.1.

Specimen Preparation

Fresh bovine pericardia were obtained from an abattoir. Extraneous fat or muscle was removed and sections with heavy vasculature or attached ligaments were discarded. The fresh tissue was stored in chilled phosphate buffered saline (PBS, pH 7.4) until the time of treatment with HSHS (proprietary storage solution, Sulzer Carbomedics, Incorporated), glutaraldehyde, ethanol, or octanol.

2.2.

Rat Subcutaneous Implantation

The rat subcutaneous model for assessment of tissue calcification has been described previously.²⁵ Briefly, 1.5-cm² samples of tissue were implanted subcutaneously in three-week-old Sprague-Dawley rats. After 60 days of implantation, the rats were euthanized and the tissue specimens withdrawn and immediately placed in formalin. Calcium and phosphorus levels in the tissues were determined by elemental analysis. Histological sections were prepared, stained with von Kossa’s stain for phosphate, and evaluated for mineral content. Specimens were also subjected to differential scanning calorimetry and Raman spectroscopy studies.

2.3.

Elemental Analysis

Retrieved implanted tissue specimens were digested in concentrated HCl and analyzed by inductively couple plasma/atomic emission spectroscopy. The group of implanted glutaraldehyde-fixed pericardia averaged 259.7±82.5 mg/g Ca, 130.3±39.9 mg/g P, and a von Kossa score of 2.5 (on a scale of 0 to 5), indicating extensive mineralization. The average for the glutaraldehyde/ethanol-fixed, implanted pericardia and the glutaraldehyde/octanol/ethanol-fixed, implanted pericardia were 3.7±1.2 mg/g and 30.5±69.6 mg/g Ca, respectively, 0.0±0.0 mg/g and 15.6±36.4 mg/g P, respectively, and von Kossa scores of 0.7 and 1.2 (no mineral detected). Specimens chosen for Raman spectroscopy were representative of the average for each respective group.

2.4.

Differential Scanning Calorimetry

All tissues were hydrated in PBS for 24 hours prior to differential scanning calorimetry analyses. The fresh pericardium stored in the HSHS solution had a shrink temperature of 61.8°C. The glutaraldehyde-, glutaraldehyde/ethanol- and the glutaraldehyde/octanol/ethanol-fixed, nonimplanted tissues had shrink temperatures of 88.4, 87.4, and 86.4°C, respectively. All fixed tissues had higher shrink temperatures than the fresh tissue, indicative of an increased number of cross-links in the fixed tissues. The postglutaraldehyde treatments did not result in significant changes in the shrink temperature.

2.5.

Raman Spectroscopy

Specimens representative of the various treatment production lots were chosen for Raman spectroscopic analysis. One specimen representative of each group was chosen for Raman spectroscopy. All specimens were placed in two consecutive 1-h immersions of PBS and then soaked in PBS overnight before spectroscopic analysis. Three nonimplanted specimens were examined: a fresh pericardium, a glutaraldehyde-fixed pericardium, and a glutaraldehyde/ethanol-fixed pericardium. Three implanted specimens were also examined: a glutaraldehyde-fixed pericardium, a glutaraldehyde/ethanol-fixed pericardium, and a glutaraldehyde/octanol/ethanol-fixed pericardium. Raman transects (lines of point spectra) were acquired on the smooth areas of the bovine pericardium. The instrument design has been described previously.³⁰ Briefly, a 785-nm diode laser (SDL, Incorporated, San Jose, California, 80 mW at specimen) was used as the excitation source. The laser light was focused to a 1.5-μm spot on the specimen using a 10×/0.50 NA objective (Zeiss, Fluar Series, Thornwood, New York) mounted on a BH-2 microscope frame (Olympus, Incorporated, Melville, New York). The Raman scatter was focused into a near infrared-optimized axial transmissive spectrograph (Kaiser Optical Systems, Incorporated, Holospec f/1.8i) fitted with a 25-μm slit (3 to 4 cm⁻¹ spectral resolution) and dispersed onto either a liquid nitrogen-cooled, back-thinned charge-coupled device (CCD) camera (Roper Scientific, MASD, San Diego, California) or else a thermoelectrically cooled, back-thinned, deep-depletion camera (Andor Technology, Belfast, Ireland). A motorized motion stage (New England Affiliated Technologies, NEAT, Danaher Precision Systems, Salem, New Hampshire) moved the specimen at 5-μm increments under the laser spot to provide 10-μm spatial resolution. The transects ranged in length from 500 to 750 μm.

2.6.

Data Analysis

Data were processed using MATLAB software (MathWorks, Incorporated, Natick, Massachusetts) via both vendor-provided and locally written scripts. Data were preprocessed by removing cosmic ray spikes (typically 1 or 2 per spectrum) and by subtracting the dark current. For factor analyses, the datasets were divided into two spectral subregions. The factor analysis consisted of a principal components analysis followed by manual rotation of only the nonnoise eigenvectors using nonnegativity and band shapes as constraints.^{31 32} The resulting linear combinations of the original eigenvectors are referred to as factors; each factor describes the spectral signature of a particular tissue component or a background component. Each factor has a corresponding score. The score describes the location and relative amount of that factor in the original dataset.

Center-of-gravity calculations were also used to examine the pericardia spectra. The advantage of using center-of-gravity measurements to examine changes in the amide I band is that they probe small changes in the overall band contour rather than just changes in the peak maximum. Prior to calculating the center of gravity of the amide I band, the amide I region was baselined using a quadratic baseline and then offset to zero. The center of gravity of the amide I envelope (1580 to 1700 cm⁻¹) was then calculated using Eq. (1):

3.1.

Raman Spectra

The Raman spectra revealed the presence of an apatitic phosphate, similar to early deposited mineral in bone tissue,¹⁰ in the implanted glutaraldehyde-fixed pericardium [Fig. 1(b)]. The spectra from the rest of the implanted and nonimplanted pericardia contained only a collagen-dominated protein signature. A representative single-point spectrum from the nonimplanted glutaraldehyde-fixed specimen is shown in Fig. 1(a). Comparison of the amide I regions of the glutaraldehyde-fixed, nonimplanted pericardium spectrum and the glutaraldehyde-fixed, implanted pericardium spectrum shows that there is a change in the collagen structure (Fig. 1, boxed area). The nonimplanted specimen spectrum has an amide I envelope with two readily visible bands at 1635 and 1660 cm⁻¹. However, in the implanted specimen, the ratio of the 1635 to the 1662 cm⁻¹ intensity has decreased. A slight shoulder on the 1690 cm⁻¹ side of the implanted specimen is also present. Similar results were seen in the spectra of tissues treated with the other fixation techniques.

Figure 1

Raw, single-point Raman spectra reveal the presence of an apatitic lattice in the glutaraldehyde-fixed, implanted specimen. (a) The glutaraldehyde-fixed, nonimplanted specimen has a collagen-dominated spectrum (C-C backbone stretches, 814 and 935 cm⁻¹; hydroxyproline, 853 and 872 cm⁻¹; proline, 918 cm⁻¹; phenylalanine, 1000 cm⁻¹; phosphate (nonapatitic), 1031 cm⁻¹; amide III, 1242 and 1272 cm⁻¹; CH ₂ wag, 1447 cm⁻¹; and amide I, 1635 and 1660 cm⁻¹). (b) The glutaraldehyde-fixed, implanted specimen has formed an apatitic phosphate (ν₁, 959 cm⁻¹) on the collagen matrix [same assignments as for (a)]. Changes in the amide I envelope are highlighted in the boxed area. Spectra have been offset for clarity.

3.2.

Factor Analysis

3.2.2.

Implanted specimens

The factor analysis of the glutaraldehyde-fixed and implanted specimen transect revealed the presence of four nonnoise factors: a carbonate-containing apatitic phosphate mineral factor [Fig. 2(b), PO ₄ ³⁻ν₁ 959 cm ⁻¹, PO ₄ ³⁻ν₃ 1030 cm ⁻¹, and CO ₃ ²⁻ν₁ 1070 cm ⁻¹] , a collagenous protein signature [Fig. 2(a), amide III, 1242 cm⁻¹; CH ₂ wag, 1447 cm⁻¹; and amide I, 1664 cm⁻¹], and two background factors (not shown). The score, which describes the relative amount and location of a factor along the transect, shows that the relative amount of collagen was fairly even [Fig. 2, score (a)], but the mineral score shows an appreciable increase in mineral content after 160 μm along the transect [Fig. 2, score (b)]. While the mineral deposition was irregular along the transect, no evidence for chemical changes in the collagen or the mineral was found. Separate factor analyses of the glutaraldehyde/ethanol- and the glutaraldehyde/octanol/ethanol-fixed implanted specimen transects revealed the presence of three nonnoise factors for each of the specimens. The only nonbackground factor was a collagenous protein; no mineral was found.

Figure 2

Tissue factors and scores from the glutaraldehyde-fixed, implanted specimen reveal that mineral deposition was uneven over the surface of the pericardium. (a) The proteinaceous factor is mostly composed of collagen, while (b) the mineral factor contains bands from an apatitic phosphate lattice (ν₁, 959 cm⁻¹) with a slight amount of carbonate (ν₁, 1070 cm⁻¹). A score relates the amount of its corresponding factor at each point. Two nonnoise background factors were omitted for clarity.

3.3.

Center-of-Gravity Calculations

Center-of-gravity calculations revealed that there was a slight difference in the contour of the amide I envelope of the nonimplanted pericardia. The fresh and the glutaraldehyde/ethanol-fixed specimens have similar center of gravities, while the glutaraldehyde-fixed pericardium tissue has a center of gravity at a lower frequency (Fig. 3). There was little difference in the amide I envelope center of gravity within the implanted pericardia; however, the implanted tissue and the nonimplanted have center of gravities that differ by about 10 cm⁻¹ (Fig. 3).

Figure 3

Center-of-gravity measurements for the nonimplanted and implanted specimens. The amide I center-of-gravity measurements are higher for the implanted specimens than the nonimplanted specimens. The statistical uncertainties are equal to one standard deviation.

4.1.

Mineralization

The glutaraldehyde-fixed specimen was the only fixed pericardium specimen that mineralized on implantation. A lightly carbonated apatitic mineral, similar to that found in bone tissue, was deposited unevenly on the surface of the pericardium. The uneven mineral deposition could be due to local irregularities in the tissue or changes in the collagen structure; further studies are needed to better elucidate the composition and structure of these mineral nucleation sites. Glutaraldehyde fixation followed by either 80 ethanol or 40 ethanol/5 octanol treatment prevented mineralization during the 60 days of implantation. These results are consistent with the findings of previous studies that glutaraldehyde-fixed tissues will calcify once placed in vivo, while those cross-linked by glutaraldehyde and treated with ethanol mixtures (>50) do not.^{4 5 6} Interestingly, using FTIR spectroscopy, Vyavahare and coworkers demonstrated that there was significant calcification of porcine aortic valves after treatment with glutaraldehyde and 40 ethanol.⁴ In this work, the pericardium treated with a 40 ethanol/5 octanol mixture showed no evidence of calcification.

4.2.

Amide I Center-of-Gravity Measurements