The present study showed that differences existed in the collagen fiber alignment and biaxial biomechanical behavior of ECM scaffold materials derived from the porcine urinary bladder depending upon the layer from which the scaffold was obtained and the processing to which the scaffold was subjected. In general, the porcine urinary bladder was shown to have preferred collagen fiber alignment and a greater modulus in the longitudinal axis of the tissue. This collagen fiber alignment was consistent with previous findings in the rat bladder that showed longitudinal alignment of the smooth muscle and an increased stiffness of the full thickness bladder in the longitudinal direction [21
UBML and UBS showed very similar collagen fiber distributions, but the biomechanical behavior was quite different with a longer toe region and lower modulus observed for UBS over the range of loads applied. This result was in contrast to a previous study in which multilaminate devices of UBM and UBS were subjected to ball burst testing [13
]. In that case, the UBS multilaminate scaffold was significantly stronger than the UBM multilaminate scaffolds. The preparation of a multilaminate scaffold also changed the mechanical properties of the scaffold by altering the fibrous structure of each individual layer [13
]. The differences observed in the present study may have been a result of aggressive scraping that was done to remove the tunica submucosa, tunica muscularis externa, and tunica serosa from the UBM that may have reoriented and reduced the crimping of collagen within the tissue. At greater loads, it is possible that UBS would still reach a greater modulus and ultimate strength than UBM.
The comparisons between UBML and UBMC showed that the methods of preparation have an effect upon the resulting collagen fiber alignment and biomechanical behavior of the tissue. UBML and UBMC showed very different collagen fiber distributions, UBML showing high longitudinal alignment and UBMC showing a more homogeneous collagen fiber distribution. However, the longitudinal biomechanical properties for UBML and UBMC were very similar for the loading conditions applied, and the circumferential biomechanical behavior for UBMC approached the longitudinal biomechanical behavior. The process of scraping the tissue circumferentially rotated a substantial population of collagen fibers from the longitudinal direction towards the circumferential direction, and the results clearly showed that those fibers retained the new orientation. Moreover, the fact that the longitudinal biomechanical behavior did not substantially change suggested that there was a population of collagen “struts” that were relatively fixed in the longitudinal direction and served to limit the longitudinal elongation of the bladder.
The biomechanical behavior and/or collagen fiber alignment were evaluated for a number of other ECM scaffolds in previous studies. The results for the present study compared well with the results of similar analyses performed with SIS-ECM [2
]. SIS-ECM also possessed a preferred collagen fiber alignment along the longitudinal axis of the tissue, and was composed of two populations of collagen fibers that were each offset by approximately 30° from the longitudinal axis. No evidence of a bimodal collagen distribution was observed for ECM derived from UBM in the present study, but this may have been due to the relatively large beam diameter used in this study. Previous studies with SIS-ECM have also shown a substantial degree of collagen fiber motility in response to uniaxial loading and strip biaxial loading, especially in the hydrated state [3
]. SIS-ECM is typically delaminated by applying pressure along the longitudinal axis of the intestine, so processing may have contributed to the relatively uniform collagen fiber alignment. This explanation would support the concept that circumferential scraping of the UBM led to large fiber rotations.
In contrast to SIS-ECM and the urinary bladder ECM studied in the present study, it was recently found that cholecyst-derived ECM (CEM) possessed a preferred collagen fiber orientation offset by approximately 65° from the longitudinal axis (i.e., from the neck to the fundus) [23
]. Using the preferred fiber direction as the principal direction for mechanical testing [23
], the biomechanical behavior for CEM was similar to UBMC in terms of the stress-strain relationship and the weakly anisotropic behavior. There was no specific mention of whether the direction of delamination was controlled in the preparation of CEM.
The results of the present study have direct implications for the fabrication of medical devices from ECM scaffolds. It has been reported that the mechanical behavior of SIS-ECM is dependent upon the segment of the small intestine from which the SIS is harvested [24
]. It is possible that these differences may be a function not only of biologic variability of the tissue, but also process variability. It is therefore important to monitor the direction and magnitude of pressure that was applied to the tissue. These findings could also factor into the design of an ECM device. A recent study compared the mechanical behavior of several commercially available ECM derived devices indicated for rotator cuff repair to determine how they compared to the mechanical behavior of the infraspinatus tendon [1
]. In all cases, the mechanical behavior of the scaffold was inferior to that of the tendon, with a lower stiffness and strength. One of the devices evaluated was Restore (DePuy Orthopaedics, Warsaw, IN), a device composed of 10 layers of SIS-ECM that is configured such that 2 layers of ECM are aligned every 72° to give the device an isotropic mechanical behavior. Based on the present results, it is possible that an ECM scaffold could be processed in such a way that it may more closely mimic the biomechanical properties of the tissue the scaffold is intended to replace. Using UBM as an example, for designing a device for tendon repair, the material could be scraped longitudinally and then laminated such that the longitudinal axis of the bladder is aligned for all of the layers, providing a transversely anisotropic material similar to a tendon. By prestretching the scaffold prior to lamination, the degree of transverse anisotropy could be increased [3
]. Conversely, for repair of body wall, the UBM could be scraped circumferentially and laminated with random orientation of the sheets to provide an isotropic mechanical behavior. Additional study is necessary to verify these predictions.
It should be noted that the results from the present study are only applicable to the scaffolds prior to implantation. In the absence of chemical crosslinking, an ECM scaffold begins to be degraded immediately by the host, altering their mechanical properties during the process of remodeling. Previous studies have shown that ECM scaffolds are completely degraded within 60–90 days [25
], and the degradation products are small bioactive peptides that have chemotactic, bacteriostatic, and mitotic properties [27
]. The chemotactic peptides may be responsible for the recruitment of bone marrow derived progenitor cells that participate in the remodeling by differentiating into site specific cells in response to mechanical and biochemical cues [31
]. Since the mechanical environment is defined by the collagen fiber architecture and kinematics, the changes in these properties of the scaffold are an important topic of future study.
A limitation of the present study is that the specimens were only subjected to subfailure biaxial testing. Uniaxial testing to failure may have provided additional information about whether the increased collagen fiber alignment would result in changes in tensile properties of the tissue. Comparison of these results to previous work is confounded by the lack of control for orientation of the test articles relative to the anatomic orientation of the urinary bladder [34
]. The biaxial testing used in the present study was selected to provide a broad understanding of the material behavior within physiologically relevant ranges of load and strain. In addition, the data collected in this study will be useful in the development of structural constitutive models of the ECM of the urinary bladder [37
], which could be useful for predicting the remodeling response of a scaffold in vivo
or optimal mechanical loading regimens in vitro