Directional porosity of porcine small-intestinal submucosa

B. K. Ferrand,’ K. Kokini,’+ S. F. Badylak? L. A. Geddes? M. C. Hiles? and R. J. Morffj lSchool of Mechanical Engineering and, ZHillenbrand Biomedical Engineering Center, Purdue University, W. Lufayette, IN 47907; and Eli Lilly G. Co., Indianapolis, IN 46285

Small-intestinal submucosa (SIS) has been shown to be a promising biomaterial for vascular graft applications. This study examines the directionality property of SIS porosity using 35 SIS specimens from 13 pigs. In addi- tion, the effects of the weight of the donor pig, pre- conditioning of 13 additional SIS specimens, and the duration of the test of five additional SIS specimens on such porosity are reported. The porosity from serosal to mucosal direction was found to be four times greater than the porosity in the opposite direction. The weight of the donor pig was not found to be an important factor

in SIS porosity. Preconditioning served to increase the average serosal porosity index at 120 mm Hg static water pressure from 2.99 to 8.33 mL/(min cm’). The porosity in the mucosal direction was not affected by precondition- ing. Porosity in both directions decreased with increasing test duration. The directionality property of SIS porosity may be an important factor in its success as a vascular graft. The term ’porosity’ is used throughout this article, but current standards also refer to the term ’permeability’ to describe the passage of liquid through a vascular graft. 0 1993 John Wiley & Sons, Inc.

INTRODUCTION

The porosity of vascular graft materials has been reported to be an important physical property and a determinant of implant success.I The rate of neovas- cularization of a graft material appears to be related to its porosity. Wesolow? in his discussion of the need for standards in vascular graft materials, described a direct correlation between the porosity of the graft material and adherence of the graft to the connective tissue capsule which forms around it. The term ’poros- ity’ is used in this article, but current standards also refer to the term ’permeability’ as a measure of the passage of liquid through a vascular graft material.

Porosity is defined here as the flow of water through the wall per unit of time and per unit of surface area for a constant pressure. For most materials the porosity has a strong dependence on the applied pressure; therefore, a porosity value is associated with a pressure. Wesolowslu’ defined the porosity index for vascular graft materials as the flow of water through 1 cm2 of the material, in milliliters per minute, for a pressure of 120 mm H$.

*To whom correspondence should be addressed at: 1288 Mechanical Engineering Building, Purdue University, West Lafayette, Indiana 47907-1288.

Small-intestinal submucosa (SIS) prepared from pig intestine has been shown to function well as a bio- material for use as a vascular graft.3 Hiles et al.4 described the porosity of SIS and its dependence on pressure and tensile stress. These measurements were made on the mucosal (luminal) surface only. Preliminary findings in our laboratory suggest that SIS porosity depends strongly on the sample orientation; i.e., if SIS is subjected to water pressure on the serosal (abluminal) side, a different porosity index will be obtained than if the SIS sample is subjected to water pressure on the mucosal (luminal) side. No other biological or synthetic material has been reported to exhibit this directionality in porosity. In the present study, the porosity properties of SIS in both directions were quantified. Variables tested were the directional- ity, donor-pig weight, stress history (preconditioning), and the test duration.

MATERIALS AND METHODS

All studies herein were performed in accordance with the Guide for the Care and Use of Laboratory animal^.^ In addition, this study was approved by the Purdue University Animal Care and Use Committee.

Journal of Biomedical Materials Research, Vol. 27, 1235- 1241 (1993) 0 1993 John Wiley & Sons, Inc. ccc 0021-9304/93/101235-07

1236 FERRAND ET AL.

Preparation of SIS

Sections of porcine jejunum were harvested within 10 min following euthanasia and immediately placed in 0.9% saline solution. Sections were cut into 10-20- cm lengths and the SIS was prepared for use as a vascular graft as described by Badylak et a1.6 A brief summary of this preparation follows.

All mesenteric tissues were removed from the out- side of the small intestine. The intestine was then everted and the tunica mucosa abraded from the tube using a longitudinal wiping motion with a scalpel handle and moistened gauze. A natural separation of the superficial tunica mucosa from the deeper stratum compactum layer resulted. The tube was then everted to its original orientation. The serosa and tunica muscularis were then gently removed from the outer surface of the intestinal tube using the same abrasion technique. The remaining thin (0.1 mm wall thickness), whiteish, translucent tube consisted of the stratum compactum and muscularis mucosa of the tunica mucosa. Following this preparation, the SIS was rinsed with saline and placed in a 5% neomycin sulfate solution until it was used as a vascular graft. Samples of this material were used for the porosity tests conducted in the present study.

h

n

Porosity tests were performed in the following man- ner. A 10-cm specimen was cut open to obtain a rectangular sheet for placement in the testing ap- paratus described below. Deionized (DI) water at room temperature (25°C) was used for all tests. All specimens were stored at approximately 5°C prior to testing.

Preconditioning

It has been shown by Fung7 that biological mate- rials such as SIS, when subjected to several cycles of tensile loading and unloading, attain a state in which little hysteresis is observed upon further loading and unloading. This process is called preconditioning. Al- though the effects of preconditioning on the mechan- ical behavior of SIS have been discussed elsewhere,' the effect of preconditioning on porosity has not been investigated and is addressed in the present article.

The preconditioning apparatus is shown in Figure 1. Preconditioning consisted of subjecting tubular SIS samples to an internal pressure cycle that varied from 0-100 mm Hg. Three pressure cycles were de- termined to be sufficient essentially to eliminate hys- teresis in SIS"' , therefore, three pressure cycles were

I ! Figure 1. transducer, TD-transducer driver, PS-power supply, DD-digital display.

Preconditioning apparatus: DI-water reservoir, h-adjustable pressure head, SIS-SIS specimen, PT-pressure

DIRECTIONAL POROSITY OF SIS 1237

applied to each SIS sample. The pressure was varied by raising and lowering the water reservoir by means of a pulley and cable. Pressure was monitored with a pressure transducer (Cobe, Inc., Lakewood, CO) connected to an amplifier and display system.

trials of specified time, and the flow was collected in a graduated cylinder to measure volume.

Experimental design

Directionality Testing apparatus

The porosity-testing apparatus, shown in Figure 2, was constructed of Plexiglas. Its design specifications were consistent with those described in the Amer- ican National Standard for Vascular Graft Prosthe- ses (ANSI/AAMI VP20),9 which recommends a fixed orifice of 0.5-1 cm2 area. A 0.7 cm2 section of SIS was subjected to a static pressure head defined by the difference in height between the deionized water reservoir level and the specimen (see Fig. 3). The mon- itoring orifice allowed direct measurement of pressure with the pressure transducer. Specimens placed in the apparatus were subjected to the static pressure for

Thirty-five specimens of SIS from 13 pigs, ranging in weight from 220-625 lb, were tested at a pressure of 120 mm Hg. The flow for each specimen was collected for 3 min; the flow direction (serosal or mucosal) was recorded. The porosity-testing apparatus was then turned over and flow collected in the opposite direc- tion. A total of 91 data points were obtained.

The order of the tests was varied (i.e., serosal first for one test, mucosal first for the next test on the same specimen, or vice versa) to determine whether the flow direction order affected porosity. The serosal flow was tested first for 50 of the 91 data points. The mucosal flow was tested first for 40 of the 91 data

Figure 2. Porosity testing apparatus.

1238 FERRAND ET AL.

Figure 3. cylinder, PT-pressure transducer, TD-transducer driver, PS-power supply, DD-digital display.

Porosity setup: DI-water reservoir, h-adjustable pressure head, PA-porosity testing appartus, gc-graduated

points. The test order was not recorded for one data point.

The SIS specimens were of sufficient size to allow up to four tests to be performed at different locations on each specimen. Of the 35 specimens, a single test was performed on six, two tests were performed on 12, three tests were performed on seven, and four tests were performed on 10 specimens.

Effect of preconditioning

Thirteen preconditioned samples of SIS from five animals were cut into sheets and subjected to the same porosity test as the nonpreconditioned SIS, just described. A total of 30 data points were obtained in the serosal direction and 28 data points were obtained in the mucosal direction.

Effect of test duration

The testing procedure was modified to investigate the dependence of SIS porosity on the test duration. A preconditioned SIS sample was subjected to 120 mm Hg continuously for 53 min. Flow was collected for 3 min at 10-min intervals, yielding six data points per

test. The testing device was then turned over and the test was repeated with flow in the opposite direction. Each sample was therefore subjected to a duration test in both flow directions. A total of five samples were tested. The sample orientation, test order, flow value at each time interval, and donor pig weight were recorded for each test.

STATISTICAL ANALYSIS

The raw data were converted to porosity index values by dividing the measured flow by the flow area (0.7 cm2) and then dividing by the individual test time (3 min), noting the static pressure (120 mm Hg). All statistical calculations were performed using SAS software package. The alternative hypotheses were rejected unless the P value for a given test was less than the rejection (alpha) level of .05.

For the directionality experiment! analysis of vari- ance was performed with respect to flow direction order. For example, the mean, standard deviation, and standard error were calculated for the initial serosal data points, i.e., those that were recorded

DIRECTIONAL POROSITY OF SIS 1239

prior to the mucosal data point for the same test location. Likewise, the mean and standard error were calculated for the subsequent serosal data points, i.e., those recorded immediately after the mucosal data point for the same test location. A Student-Newman- Kuel test was used to compare these two means. The null hypothesis was that no difference existed between initial and subsequent data. Identical calcu- lations were performed for the mucosal data.

A regression model was used to fit the serosal data to determine whether the serosal porosity was dependent on the test location. A Student-Newman- Keul test for significant difference among the means at each test location (1-4) was performed. These calculations were repeated for the mucosal data.

A regression model was applied to the data to test the hypothesis that porosity in either flow direc- tion is independent of donor pig weight. A Student- Newman-Keul test for significant difference among means versus pig weight was performed with an alpha of .05.

The mean, standard deviation, and standard error of all serosal data were calculated for comparison with the mucosal data. A standard two-tailed statistical test was used to compare the serosal and mucosal mean values. The null hypothesis was that no difference existed between serosal and mucosal porosities.

The mean, standard deviation, and standard error were calculated for preconditioned SIS in each di- rection. A Student-Newman-Keul test was used to determine the significance of difference between pre- conditioned SIS and the previously determined values for non-preconditioned SIS for each flow direction. The null hypothesis was that no difference existed in either direction between preconditioned SIS porosity and non-preconditioned SIS porosity.

The time tests were analyzed differently from the method used for the directionality and precondi- tioning experiments. For each 53-min test in each direction, the data points at each 10-min interval were normalized by dividing by the initial porosity value obtained during the first 3 min of the test. A regression model was used to determine the effect of increasing test duration. A Student-Newman-Keul test

was used to determine the significance of difference between the porosities at each subsequent time interval.

Outlying data points (those lying at last three stan- dard deviations from the mean) were eliminated. Only four data points were eliminated from the direction- ality data. Two data points wee eliminated from the preconditioning data. No data were eliminated from the duration tests. The means, standard deviations and standard error were recalculated prior to any data comparisons.

RESULTS

The results of the directionality tests and the pre- conditioning tests are summarized in Table I. The normalized test duration results are shown in Table 11. Test order information was not recorded for one data point. This datum was not used in the calculations of initial and subsequent means but was included in the calculations of total means. Statistical comparisons of the results in Tables I and I1 are made in Table 111.

These comparisons indicate that test order, test lo- cation, and pig weight did not significantly influence porosity in either the serosal or mucosal direction. These results allowed the data to be pooled and the average serosal porosity to be compared with the aver- age mucosal porosity. This directionality comparison revealed that serosal porosity is significantly greater than mucosal porosity. Preconditioning is shown to increase serosal porosity, but not mucosal porosity. Test duration is a significant factor on both porosity values.

DISCUSSION AND CONCLUSIONS

The average porosity index of SIS was approxi- mately four times greater in the serosal direction than in the mucosal direction. Preconditioning increased this ratio of serosal porosity to mucosal porosity to 12. Directional porosity is an unusual property and may play an important role in the success of SIS as a

TABLE I Directionality and Preconditioning Summary

Data Group N Mean Standard Deviation Standard Error

Initial serosal 47 3.15 2.16 0.31 Subsequent serosal 39 2.73 1.69 0.27 Initial mucosal 39 0.54 0.45 0.07 Subsequent mucosal 47 0.58 0.53 0.07 Serosal total 87 2.99 1.94 0.21 Mucosal total 87 0.57 0.51 0.05 Precond. serosal 28 8.33 4.05 0.76 Precond. mucosal 26 0.69 0.37 0.07

All porosity index units = mL/(sq. cm X min) at 120 mm Hg. Resolution = 0.024 mL/(sq. cm xmin).

1240 FERRAND ET AL.

TABLE I1 Normalized Duration Testing Summary

Percent of Initial Porosity at Time (min)

Direction Initial Porosity 0 10 20 30 40 50 ~

Serosal mean 6.57 100 79.6 63.4 55.7 56.5 51.1 std. dev. 4.24 0 13.9 16.7 14.0 18.8 19.1 std. err. 1.98 0 6.23 7.48 7.01 8.40 8.54

Mucosal mean 0.65 100 77.3 60.7 66.5 57.2 52.4 std. dev.. 0.41 0 13.9 4.95 18.8 6.06 13.8 std. err. 0.19 0 6.2 1.81 8.40 2.71 2.62

vascular graft. The low mucosal porosity might pre- vent graft leakage, while the higher serosal porosity may allow greater tissue ingrowth.

While it is difficult to determine the cause of the directional porosity property of SIS, a model is pro- posed herein that may prove useful in future studies. The model suggests that the mucosal surface structure (stratum compactum) contains a valve-like mecha- nism that restricts flow when the mucosal surface is subjected to pressure, but opens up (i.e., allows greater flow) when the serosal surface (muscularis mucosa of the tunica mucosa) is subjected to pressure.

Hiles et a1.8 reported the failure strength of SIS increases with pig weight, while SIS porosity was found to be independent of pig weight. Future studies directly comparing how SIS physical properties, such as porosity and strength, vary with donor pig weight may be helpful in determining the directional porosity mechanism.

Preconditioned SIS has a significantly higher mean porosity index in the serosal direction than does non- preconditioned SIS. This is difficult to explain, but may be due to the stress applied during precondi- tioning resulting in permanent deformation in the structure of SIS. It is possible that the preconditioning stress increased the size of the porosity pathway (i.e., pores), and the viscoelasticity characteristic of biolog- ical materials prevented complete recovery. Further studies are required to evaluate this possibility.

Preconditioning did not significantly increase the average mucosal porosity index. It may be possible to explain this phenomenon with the valve-like model of SIS. Preconditioning may increase the overall porosity path size as previously stated, yet the proposed valve mechanisms may not be altered enough to allow a sig- nificantly greater mucosal flow after preconditioning. However, a significant change in mucosal porosity due to preconditioning might not have been detected due to a limitation of the testing procedure. The cylinder used for flow measurement was graduated in 0.1 mL increments. During a typical 3-min test, the av- erage mucosal flow was approximately 1.0 mL. Some flow may have been lost due to evaporation, wetting of the Plexiglas tube, and the graduated cylinder. Another variable that was difficult to control precisely was the initial filling of the graduated cylinder to the 1.0 mL level. These uncertainties should behave in a random fashion, affecting all measurements equally, but accuracy and precision may be affected enough that comparison of average porosity indices less than 1 mL/(min cm2) may not be realistic. Future studies will include modification of the test apparatus and procedure to confirm or reject the conclusion that mucosal porosity is not significantly affected by pre- conditioning.

Figure 4 is a plot of the means at each time interval for each flow direction. The general trend is that porosity for both flow directions decreases with time.

TABLE I11 Statistical Comparison Summary

Test Parameters P Conclusions

Test order Serosal Dir.

Test location Serosal Dir.

Pig weight Serosal Dir.

Directionality Average Preconditioning Serosal Dir.

Test duration Serosal Dir.

Mucosal Dir.

Mucosal Dir.

Mucosal Dir.

Mucosal Dir.

Mucosal Dir.

.31

.69

.76

.42

.95

.58

.0001 ,0001 .25 ,0001 .0001

No apparent relationship No apparent relationship No apparent relationship No apparent relationship No apparent relationship No apparent relationship Serosal porosity is greater than mucosal Precond. increases serosal porosity No apparent relationship Initial porosity is greater than subsequent porosities in both directions

DIRECTIONAL POROSITY OF SIS 1241

A Serasal Direction (100% - 6.57)

+ Mucosal Direcoon (lW% - 0.647)

M

I 401 20

01 . I I 0 10 20 30 40 50

Tune (mmutes)

Figure 4. Porosity duration results.

The average percent of initial flow after 50 min was significantly less than at 10 min for the serosal direc- tion. The average percent of initial flow after 40 min was significantly less than at 10 min for the mu- cosal direction. This decrease in porosity, regardless of sample orientation, may indicate that SIS under- goes some time-dependent deformation of structure that decreases porosity as the time of applied static pressure is prolonged.

This study was supported by Eli Lilly & Co., Indianapolis, Indiana.

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Received August 3, 1992 Accepted May 6, 1993