Our objective in this study was to compare an adapted version of a previously described method of whole globe inflation mechanical testing with traditional uniaxial strip testing. We found that tracking surface strain markers at the posterior sclera is feasible in porcine eyes and likely applicable to eyes from humans or other species (as porcine scleral rigidity is similar to human scleral rigidity).(Asejczyk-Widlicka & Pierscionek, 2008
As was expected, significant differences were seen between the results of the uniaxial and whole globe inflation tests. Though not directly comparable, since uniaxial data is in the form of stress-strain and whole globe inflation data is presented as IOP-strain, trends and unique features from either test can still be related, and the thin-walled pressure vessel assumption can aid in rough comparisons. The differences seen are attributed to a relatively high degree of artifactual behavior resultant from testing conditions, primarily from the uniaxial test. Care was taken to ensure that testing condition effects were minimized or at least well defined. For example, a ~4.5 to 1 length to width ratio was used for uniaxially tested strips to minimize Saint-Venant related issues (i.e. non-uniform stress application near the source of the load). Nevertheless, some boundary conditions are inevitably unavoidable and their effects are difficult to control, such as tissue gripping. Waldman found that bovine pericardia tissue properties were significantly affected by gripping technique.(Waldman & Lee, 2002
) To better understand the implications of artifacts from the testing conditions in the uniaxial tests, the following table () briefly explains the importance of each in terms of their potential effects.
Testing Conditions / Artifacts Summary for Uniaxial Testing
A closer look at the load-deformation curves helps reveal the practical meaning of differences between the two tests studied. Load-deformation curves are typically broken down into three regions; first, the initial “toe” region where the load-deformation relationship is exponential, next, the linear region, and last, the nonlinear region which ends in tissue rupture. The initial region before the elbow is also called the elastic region (reversible deformation), and this region is of high significance because it represents the physiologic range in which tissues normally operate.(Fung, 1993
) Whole globe inflation curves had an obvious elbow in the curve that occurred early in the loading. Alternately, curves from uniaxial testing had gradually rising trajectory, with less of an obvious initial elbow. This difference seems reasonable because in whole globe inflation testing, scleral tissue was being stretched uniformly and becoming stiff relatively quickly; whereas in scleral strips, compression in the perpendicular direction allowed for more distensibility and higher strains in the loading direction. Such a difference has substantial implications for clinical applications. For instance, an investigation using uniaxial tests to look at the dose-response relationship between a collagen crosslinker and scleral stiffness may report findings irrelevant to the physiologic portion of the load-deformation curve. The differences seen in the relative stiffness values at 1% strain and peak load between our two tests add merit to this discussion. While stiffness values were calculated at similar numeric points along the stress-strain curve in both tests, the actual location along the curve in terms of region (exponential, linear, or non-linear) must be considered as well. Without knowing the geometry of a complete curve (i.e. tested to rupture) it is difficult to definitively delineate the regions; however, our data reveals markedly different curve shapes within a localized range. This aids in explaining the differences seen in our scleral stiffness data at 1% strain and peak load between the two tests. The region, and location along a region, from which stiffness values are calculated plays a role in the relative differences seen in stiffness. Clinically, this becomes important when analyzing the effects of treatments that alter the mechanical properties of any soft tissues, such as the sclera.
Recently, investigators studying the mechanics of sclera and other tissues have begun to emphasize the shortcomings of uniaxial strip testing and hence the need for more physiologic multiaxial testing. Bass et al compared biaxial and uniaxial testing of human annulus fibrosus and concluded that uniaxial methods cannot be used to predict multiaxial stress behaviors in vivo.
(Bass, Ashford, Segal, & Lotz, 2004
) Multiaxial testing in the form of globe inflation specifically for ophthalmic research has also recently shown signs of rapid growth. Elsheikh and Anderson performed a comparative study of corneal strip versus corneal inflation tests and detailed the many inherent deficiencies in uniaxial testing due to the flattening of an originally curved specimen, a concern for uniaxial testing of sclera as well.(Elsheikh & Anderson, 2005
) Myers et al have developed methods of evaluating the response to globe inflation for bovine and mouse sclera.(Myers et al., 2010
; Myers, Coudrillier, Boyce, & Nguyen, 2010
)(Myers et al., 2010
; Myers, Coudrillier et al., 2010
) Their studies looked at issues such as where displacement was most pronounced in the peripapillary region of globes, and provided information to better characterize the viscoelastic properties of the sclera. Thornton et al performed a study demonstrating the clinical relevance of inflation testing by looking at the effect of IOP on the ONH and surrounding sclera using crosslinking, with results relevant to mitigation of glaucoma.(Thornton, Dupps, Roy, & Krueger, 2009
) Bisplinghoff et al have focused on trauma to whole globes with interest in globe rupture pressure, dynamic behavior, developing prediction tools, etc.(Bisplinghoff, McNally, Brozoski, & Duma, 2008
; Bisplinghoff, McNally, & Duma, 2009
; Bisplinghoff, McNally, Manoogian, & Duma, 2009
) The cornea has also been the subject of globe inflation testing—Boyce et al have looked at full-field deformation of bovine cornea to gain better understanding of the tissue’s non-linear behavior as well as deformation behavior across the cornea.(Boyce, Grazier, Jones, & Nguyen, 2008
) In using inflation tests rather than traditional uniaxial strip tests, insights gained in the lab are more easily translatable to the clinic, as demonstrated by several of these examples.
This study was able to show simple yet improved whole globe inflation testing incorporating aspects of methods reported previously.(M. J. Girard, Downs, Burgoyne, & Suh, 2008
; Ku & Greene, 1981
) By mounting the eye with a skewer-type needle at the equator, inserting a pressurizing cannula in the anterior chamber, and placing fiducial markers on the surface of the sclera, we were able to calculate actual localized circumferential strains and correlate IOP with multiaxial scleral deformation. Previously, varying pitfalls in biaxial experiments have limited the strength of comparing uniaxial and biaxial or inflation tests. Eilaghi and co-authors performed planar biaxial testing of human eyes, which introduces many of the same problems as traditional uniaxial testing such as straightening a naturally curved specimen and fraying the collagen fibers from cutting the sample.(Eilaghi et al., 2010
) In Ku and Greene’s study, IOP was correlated with only the anterior-posterior diameter of the eye, which is a less accurate method than looking at localized strains (i.e. deformation patterns may not be uniform.(Ku & Greene, 1981
) Elsheikh and Anderson performed a comparative study of uniaxial and inflation tests of corneal samples, though whole eyes were not used, creating the potential for errors due to tissue mounting.(Elsheikh & Anderson, 2005
Our study is distinct in that it compares basic mechanical features of scleral tissue between traditional uniaxial strip testing and whole globe inflation testing using paired eyes and localized strain measurements. We found that the set-up time and experimental time needed to perform whole globe inflation testing was similar to that of uniaxial testing.
Our data revealed some degree of variability within a relatively homogeneous population of eyes. Whole globe inflation testing sometimes identified the sagittal direction as stiffer, whereas other times the transverse direction appeared stiffer. The average of the sagittal to transverse peak strain was close to one (1.16 ± 0.49), showing that on average the stiffness was similar. However, the coefficient of variation was 0.49/1.16 = 42%. Additionally, the average of the sagittal to transverse peak stiffness (at max strain) was also close to one (1.19 ± .34), but with a coefficient of variation of 0.34/1.19 = 28%. This may be a consequence of the fact that fibers are predominantly oriented circumferentially to the ONH in the immediate peripapillary region.(Greene, 1980
) Thus, it is not surprising to observe a degree of variation within the ten eye pairs that were tested. Importantly, this data provides information about the anisotropy of scleral tissue that is difficult to obtain from uniaxial tests. It should be noted that preliminary data showed no statistically significant difference in mechanical parameters from paired eyes (n = 5 pairs), when both eyes were tested using the whole globe inflation method (sagittal, transverse peak strains (p = 0.77, 0.78)). These results help assure that variation between uniaxial and whole globe inflation was primarily a response of the testing environment and individual animal differences, not from variation within pairs.
Directly comparing the biaxial scleral data from our study and from prior reports(Eilaghi et al., 2010
; M. J. Girard et al., 2008
; Ku & Greene, 1981
; Mattson et al., 2010
) is difficult because of methodological, analytical, and species differences. Compared to Ku and Greene who performed whole globe inflation testing in rabbit eyes, our IOP-strain curves of porcine globes were about twice as stiff as that of the rabbit’s. Girard and co-authors studied porcine eyes but only inflated the posterior half of the sclera, clamped at the equator onto a customized apparatus, and compared to our results found four times more strain in the 5-45 mmHg region.(M. J. Girard et al., 2008
) We believe this discrepancy can be attributed to the difference in inflation method. Also, these investigators kept their tissue samples in buffered saline, which can cause swelling and likely affected tissue mechanics.(Greene & McMahon, 1979
) Mattson and co-authors used a similar whole globe inflation testing setup to ours, but studied shape restoration and creep behavior of eyes from two- to three-week old and adult rabbits, making comparisons to our porcine stress-strain data challenging (younger eyes generally demonstrate higher elasticity).(Mattson et al., 2010
) Eilaghi’s group performed planar biaxial tests of human tissue, and IOP-strain data from our study (though different in species) falls within a similar range to theirs.(Eilaghi et al., 2010
Similar to this study, Bass and coauthors found uniaxial tests to produce considerably higher strains than biaxial tests in human annulus fibrosis.(Bass et al., 2004
) However, in the study by Elsheikh and coauthors looking at the cornea, uniaxial tests had lower strains than inflation tests.(Elsheikh & Anderson, 2005
) Differences in the degree of anisotropy may be the cause of such discrepancies, as the mechanical properties of tissues vary based on the orientation of their components (i.e. fibers).(Elsheikh & Anderson, 2005
Though the whole globe inflation method circumvented many challenges inherent in uniaxial testing, one limitation should be mentioned. The pressurizing needle was inserted into the aqueous of the anterior chamber of the eye because the vitreous in the posterior chamber was viscous enough such that it clogged the cannulating needle. The concern with the tip’s position in the anterior chamber is whether the pressure adequately equalized between the anterior and posterior chamber. Preliminary tests using a tonometer verified consistent pressures throughout the globe. Currently, we are investigating the effects of storage method and length of storage time on tissue properties, again by utilizing paired eyes, to expand on the work reported by Girard and co-authors.(M. Girard, Suh, Hart, Burgoyne, & Downs, 2007
) Our preliminary data as well as the work by Girard and co-authors show that storage time (and temperature), within the tested ranges, minimally affect mechanical properties of the sclera.
Due to the testing artifacts inherent in uniaxial tests, a direct comparison of mechanical properties with a near physiologic test such as our whole globe inflation test is challenging, partly because it is difficult to even know how much of a uniaxial test represents tissue properties and how much represents bias from the testing environment. While Waldman found that their suturing and clamping techniques resulted in considerably different mechanical properties of bovine pericardia, they go on to claim that it is likely neither method actually reflects true in situ
tissue properties.(Waldman & Lee, 2002
) Their claim can be generalized to other related testing conditions, and consequently, it becomes difficult to define a set of standards that should be followed for all research groups looking at scleral mechanics. Our described method presents minimal boundary conditions, near physiologic loading, can be used for any region of the sclera (and potentially the cornea), is relatively simple to perform, and offers more information than uniaxial testing. A comparison between the tests is important in demonstrating differences between methodologies, but even more important is that it brings about an awareness of the underlying mechanisms of such differences.