Stress State and Strain Rate Dependence of the Human Placenta
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Maternal trauma (MT) in automotive collisions is a source of injury, morbidity, and mortality for both mothers and fetuses. The primary associated pathology is placental abruption in which the placenta detaches from the uterus leading to hemorrhaging and termination of pregnancy. In this study, we focused on the differences in placental tissue response to different stress states (tension, compression, and shear) and different strain rates. Human placentas were obtained (n = 11) for mechanical testing and microstructure analysis. Specimens (n = 4+) were tested in compression, tension, and shear, each at three strain rates (nine testing protocols). Microstructure analysis included scanning electron microscopy, histology, and interrupted mechanical tests to observe tissue response to various loading states. Our data showed the greatest stiffness in tension, followed by compression, and then by shear. The study concludes that mechanical behavior of human placenta tissue (i) has a strong stress state dependence and (ii) behaves in a rate dependent manner in all three stress states, which had previously only been shown in tension. Interrupted mechanical tests revealed differences in the morphological microstructure evolution that was driven by the kinematic constraints from the different loading states. Furthermore, these structure–property data can be used to develop high fidelity constitutive models for MT simulations.
KeywordsHuman placenta biomechanics Stress state dependence Strain rate dependence Maternal traumatic injury Placental abruption
Maternal trauma (MT) affects 5–8% of all pregnancies and is the leading nonobstetric cause of maternal death in the United States.7,10,31,33 It is also a source of serious injury and mortality for the unborn fetus with many life-long consequences caused by both explicit trauma and emergency preterm delivery.2,23 The most common cause of trauma is a motor vehicle accident (MVA) and the most commonly associated pathology is abruptio placentae (AP), detachment of the placenta from uterus.17 AP affects approximately 1% of all pregnancies, and leads to hemorrhaging and reduction of blood flow to the fetus, as well as maternal complications including disseminated intravascular coagulation and renal failure.3,4,17 MVAs during pregnancy are particularly troubling to the clinician, because it is difficult to predict the outcome for the mother and fetus, and therefore are difficult to effectively treat.10,18 MVAs can also cause additional less common pathologies not associated with AP such as amniotic fluid embolism, uterine rupture, and pelvic fractures.2,7,11
The placenta is a transient vascular organ that develops during pregnancy. It attaches by microvilli to the uterine decidua and acts, along with the umbilicus, as a nutritional and respiratory conduit between the mother and fetus. During pregnancy the uterus increases greatly in size and the placenta and membranes form to protect and nourish the fetus.14 The placenta is attached to the inner surface uterus and does not receive direct support from any boney or ligamentous tissues. This makes the pregnant mother and fetus particularly susceptible to trauma through MVA.28 MT is difficult to study since in vitro or in vivo testing of physical specimens carries many logistical and ethical issues. Despite these difficulties, it is important to have methods for understanding MT to allow manufacturers to create safer restraints for pregnant passengers.23
Computational simulations have been widely used in traumatic injury prediction.9,24,32,36 Recent research has shown promise in helping to understand the complex injury mechanisms involved in maternal injuries.12,29,30,34,39 These simulations require advanced constitutive relationships to describe the behavior of the tissues being studied. It has been shown in non-biological materials that the loading state can affect the material properties, but little has been done in the biological world to examine this phenomenon.13,19 Previous MT simulations have used material models based on the phenomenological response of the biomaterial29; furthermore, those pioneering efforts have not considered the stress state dependence of tissue components, which can be a limiting factor in the accuracy of these simulations since loading is usually complicated and multi-axial.
To fully understand and simulate AP from MT, the mechanical properties of the pregnant uterus, the placenta, and the interface between the two need to be known. Previous experimental biomechanics studies have investigated the material properties of human placentas.1,20,21,26,27 These studies have used tensile testing to evaluate and describe complex biomechanical properties of placentas including hyperelasticity, strain rate dependence, and viscoelasticity. To our knowledge, no study has directly investigated the effects of different loading states (tension, compression, and shear) on a human placenta although studies have shown that the mechanical behavior of biological materials might vary under different loading mode.16,38
In this study, we specifically investigated the mechanical behavior of human placentas under various stress states and strain rates. The stress state dependence of human placentas was evaluated by mechanical testing in the tensile, shear, and compressive loading states. Interrupted mechanical tests were also conducted and placenta microstructures were analyzed to reveal the intrinsic mechanisms of tissue behavior under different stress states. The data presented in this study will be used to develop stress-state dependent constitutive models for use in finite element simulations of pregnant females in MVA or other situations of interest (e.g., pregnant woman falling). A computational simulation built on thorough and accurate microstructure characterizations will yield better predictions, provide in-depth understanding of injury mechanisms, and assist in corresponding safety designs.
Materials and Methods
Human placentas from eleven donors were used in this study. All samples were obtained from uncomplicated singleton vaginal deliveries at Oktibbeha County Hospital in accordance with the Mississippi State University (MSU) Institutional Review Board (#08-275). Donating patients were screened for the presence of sexually transmitted diseases (including HIV, HBV, and HCV) and unclear perinatal history. Samples were placed in 4 °C Ringers lactate buffered saline (LBS) and transported immediately after delivery to MSU where all tests were performed in a BSL2 certified laboratory. All tests were performed within 24 h of delivery. Note that we used vaginally delivered placentas, because placentas obtained by caesarean delivery are pulled from the uterine wall and are usually already mechanically damaged. Due to the size limit of the placenta tissue, we were not able to perform tests for each strain-rate and stress-state condition within a single donor. However, donor placentas were randomly assigned to loading modalities (tension, compression, and shear). Within a loading modality, there were between three and four donors, each donor placenta contributed at least one test to each strain rate.
Dogbone shaped samples (Fig. 1b) were prepared with a grip-to-grip length of 40 mm, a width of 10 mm at the center of the dogbone, and a thickness of 5 mm. Samples were preloaded to 1 g, subjected to 10 cycles of 10% preconditioning, preloaded to 1 g, and then pulled to failure. The majority of the ends of the dogbone specimens were within the grips to minimize distortion of strain uniformity, and all tensile specimens failed in the middle region of the dogbone indicating that the specimen shape served its intended purpose. The tensile failure tests were conducted at rates of 40, 400, and 4000 μm/s (N = 4+ for each rate). In tensile testing, the displacement rates of 40, 400, and 4000 μm/s correspond to strain rates of 0.001, 0.01, and 0.1/s, respectively.
Unconfined Compression Testing
Cylindrical samples (Fig. 1c) were prepared with a grip-to-grip length of 16 mm and a radius of 19 mm. Specimens were mounted and secured with a small dot of PermaBond cyanoacrylate ester adhesive (PermaBond, Pottstown PA) to prevent slipping from the compression head. The very small amount of glue did not prevent the specimens from deforming as unconfined compression. Specimens were preloaded to 1 g, preconditioned for 10 cycles, re-preloaded to 1 g, and then loaded to 3000 g. Tests were conducted at rates of 40, 400, and 4000 μm/s (N = 5+ for each rate). In compression testing, the displacement rates of 40, 400, and 4000 μm/s correspond to strain rates of 0.0025, 0.025, and 0.25/s, respectively.
Rectangular samples (Fig. 1d) were prepared to have a width of 20 mm, length of 50 mm, and thickness of 10 mm. All samples were prepared such that the shear loading aligned parallel to the uterine wall. These samples were glued to the shear test setup (two parallel tissue mounting plates) using a minimal amount of PermaBond adhesive. Compressive load between the parallel shear loading surfaces was limited to the minimum necessary to secure the samples with glue. Samples were sheared to 100 g in the positive and negative directions for 10 cycles. The data from the final cycles were used for analysis, as that data represents the tissue behavior after preconditioning. Tests were conducted at rates of 40, 400, and 4000 μm/s (N = 5+ for each rate). In shear testing, the displacement rates of 40, 400, and 4000 μm/s correspond to strain rates of 0.004, 0.04, and 0.4/s, respectively. Data was recorded at sampling rate of 100/s. All data was processed for further comparison using a custom software tool.5,8
Mechanical Data Analyses
Similar to other types of soft tissues,6,37 the stress–strain curve of placenta tissues consists of a nonlinear region and a linear region. For each mechanical test, a linear fit was applied in the linear region of the stress–strain curve to determine the slope and x-intercept of the fitted line. The slope of this fitted line represents the tissue’s tensile, compressive, or shear modulus in the linear region (large-strain elastic modulus) and the x-intercept represents the tissue’s extensibility.6,15
Scanning Electron Microscopy
Scanning electron microscopy (SEM) was performed to visualize the microstructure of placentas. Samples were prepared by common SEM preparation methods. Briefly, specimens were fixed in half-strength Karnovsky’s fixative (2% paraformaldehyde, 2.5% glutaraldehyde, in 0.1 M phosphate buffer). Samples were further fixed in 1% osmium tetroxide and then dehydrated in a critical point dryer (Polaron E 3000 CPD). Dried samples were sputter coated with gold–palladium and observed using a Zeiss EVO 50 SEM (Zeiss, Thornwood, NY) equipped with a LaB6 electron gun and secondary electron detector.
Interrupted Mechanical Testing and Histology
Interrupted mechanical tests were performed to reveal the microstructure evolution of the placenta tissue as the applied load increased. Samples were prepared, as described earlier, for each stress state. After being mounted in the appropriate configuration for their stress state, each sample was deformed to a desired engineering strain value and held at that strain; the water bath was replaced with 10% neutral buffered formalin and the sample was allowed to fix for 24 h. The interrupted mechanical tests were performed at two strain levels for each stress state, the first near the transitional region (heel region) of the nonlinear stress–strain curve (22.3, 35.7, and 39% true strain for tension, compression, and shear, respectively), and the second in the linear region of the stress–strain curve (40.5, 91.6, and 79% true strain for tension, compression, and shear, respectively).
After fixation, samples were prepared for histology analysis. Samples were embedded in paraffin and cut into 5 μm sections. Samples were then stained with Haematoxylin & Eosin (H&E) and examined by light microscopy (Nikon EC600) to assess internal microstructure changes in response to the external loading, especially the alteration of blood vessel alignment and morphology.
All experimental data were presented as mean ± standard deviation. One way analysis of variances (ANOVA) was applied for statistical analysis (SigmaStat 3.0, SPSS Inc., Chicago, IL). Comparison among groups was considered significantly different at p < 0.05.
Strain rate comparisons for each stress-state reaffirmed the rate dependence of the placenta under tension but also introduced the notion of varied responses to compression and shear.
For compression the human placenta showed greater stresses and lesser extensibility at 0.25/s when compared to 0.025 or 0.0025/s; the trend continued when comparing the responses at 0.025–0.0025/s (Fig. 2b). Since the placenta comprises mainly small blood vessels, we believe compression to be highly affected by the fluid flowing out of these vessels. Note that the toe region of compressive stress–strain behavior is very long and shallow (Fig. 2b), which possibly implies fluid flowing from collapsing vessels with little resistance. The linear region is very steep and abrupt as the mechanical response shifts from vessel collapse to the compaction of collapsed vessels (Fig. 2b). At 0.25/s, the strain rate is high enough to experience greater resistance of flow from the collapsing vessels in both the transition region and linear region. Moreover, 0.025 and 0.0025/s are relatively slow allowing the fluid to flow with less resistance, hence lower displacement rates display a more abrupt increase in mechanical resistance at the transition region corresponding to the fully collapsed vessels.
The shear stress–strain behavior had a trend similar to compression in terms of the strain rate dependence (Fig. 2c). The shear curves for 0.04 and 0.004/s are very similar with long shallow toe region and an abrupt, steep linear region. 0.4/s in shear incurred higher stresses but smaller strains, with a less abrupt transition region.
The tension stress–strain behavior did not show a strong difference among the applied strain rates (Fig. 2a). This observation coincides well with previous tensile studies,26,27 which shows a rate dependence occurring between 7 and 0.7/s but no obvious rate dependence between 0.7 and 0.07/s. A strain-rate normalization of our data reduces our tension strain rates to 0.1, 0.01, and 0.001/s, which is not high enough to elicit a strong rate-dependence under tension.27 The tensile failure reported for true stress appears consistent with those reported in other studies; however, true strain values appears to be somewhat higher despite using a similar preload value.21,26,27 Possible contributions for the variation include that our strain rates were not as high as other studies, our testing configuration in which the specimen is fully submerged in saline throughout the test, or the use of preconditioning which was not clearly reported in the method sections of the previous studies.20,26,27
Stress-state comparisons at each strain rate domain show very clear and important results. Most apparent is the relatively low stress-state in shear when compared to tension and compression stress–strain behavior (Fig. 3). Because the placenta has a less significant shear stiffness as indicated by this data, shear loading could possibly be problematic. The tensile stiffness was very high as compared to shear. The stress-state dependence observed in this study highlights the importance of addressing how different stress states are related to the injury mechanisms of placenta abruption in the real world.
Histology of interrupted tension tests revealed that the tensile loading is dominated by blood vessel recruitment driven by kinematic texture and ultimately blood vessel failure, similar to the kinematically-driven mechanism of collagen fiber recruitment observed in other biological materials such as tendon and heart valve.22,25,35 Histology of the interrupted compression tests showed vessel collapse and compaction that dominated the compressive response of the placenta. In shear, blood vessel recruitment occurred diagonally from the kinematic rotational spin as the strain increased; later, blood vessel failure occurred as revealed by the histology results. The interrupted tests showed the deformation of blood vessels induced recruitment such that the morphological changes occurred. This gave rise to structural changes similar to textural softening as is often described in texture analysis (see Horstemeyer and co-workers8,19 for example). This is also important related to placental detachment, which is likely caused by a complex multiaxial loading event, involving the incremental failure of discrete microstructure elements that connect the placenta and uterus. As the placenta-uterus connections fail, the placental detachment increases, potentially leading to full abruption and termination of the pregnancy. Studying this interface from a mechanical standpoint remains a difficult research problem due to the ethical and logistical issues of procuring human samples, or appropriately similar animal specimens.
Future studies will include assessment of Poisson’s ratio of the human placenta to allow more accurate true stress–strain calculations than our current assumptions. Research is also being conducted to prepare structure-based constitutive relationships from our mechanical and structural characterizations. These relationships will provide more accurate description of the mechanical behavior in computational simulations of biological tissues. Further research should be conducted to evaluate failure in shear and compression, as well as the failure properties of the utero-placental junction.
The fact that displacement rate, as opposed to normalized strain rate, was held constant across stress states is a limitation of this study. Using the same normalized strain rates would more directly compare the different stress states. However, this limitation did not affect our conclusions on stress state dependence and strain rate dependence. Furthermore, our strain rates were not as high as those in other studies, which limits the degree to which the strain-rate sensitivity was elucidated. Strain and stress calculations are based on the grip-to-grip gauge length and the average tissue cross-section, respectively. For both sample dimension and strain measurement, our current method is less preferred than optical measurement techniques which better account for variation within sample. Optical tracking of local deformation provides a better indication of the strain field across the tissue, as compared with grip-to-grip measurement. Another limitation concerning dogbone shaped specimens for tensile testing involves consistency uniform middle region. Our specimens were prepared by hand dissection, assisted with cutting patterns. This has the advantage of allowing us to cut with minimal tissue deformation, but the disadvantage of being manually performed and therefore less reproducible. The natural tendency of human placenta to relax under stress may interfere with our interrupted mechanical testing. Since stress-relaxation begins immediately after loading, while formaldehyde fixation processes require hours to complete, the final fixed tissue sample may not exactly mirror the loading state being evaluated. Nevertheless, the significant microstructure alterations at different strain level (Figs. 6–8) demonstrate the effectiveness of tissue fixation in the interrupted mechanical testing to reveal internal structural evolution.
Our study reaffirmed previous observations of the strain rate dependence of the human placenta in tension and extended those observations to compressive and shear loading. We further demonstrated that the human placenta behaved quite differently under the different loading states with the placenta exhibiting greater stresses in tension, lesser stresses in compression, and the least in shear at the same strain levels. At 50% strain, the tensile stresses were approximately one order of magnitude greater indicating the greater resistance to the loading. Tissue extensibility exhibited an opposite trend: the highest extensibility in shear, less in compression, and least in tension. These results are important, because multi-axial loading conditions associated with trauma cannot be accurately simulated without constitutive relationships that account for this stress state difference. We further demonstrated that the human placenta undergoes a distinct microstructure evolution for each of the stress states observed. The reported data are important for creating structure-based constitutive models that capture complex tissue behaviors and consequently enable accurate computational simulations of MT in various boundary conditions.
This study is supported by the MAFES SRI (awarded to JL) and Health Resources and Services Administration (HRSA) (DHHS R1CRH10429-01-00). We thank Karen Tiffen, RNC, Chrissy Poole, RNC, Dana Brooks, RNC, Cindy Patton, RN, Heather McMillian, ST, Bella Oswalt, ST, Sonya Anderson, RN, Rene Guines, ST, and other staff members of the Labor & Delivery Unit at OCH Regional Medical Center for their assistance with patient eligibility and tissue procurement; we also appreciate help from Amanda Lawrence (MSU EM Center) for her assistance in SEM imaging. We would also like to thank the Center for Advanced Vehicular Systems (CAVS) at the Mississippi State University for helping to support this research effort.
- 2.Aboutanos, M. B. M. D. M. P. H., S. Z. M. D. Aboutanos, D. B. S. Dompkowski, T. M. M. D. Duane, A. K. M. D. Malhotra, and R. R. M. D. Ivatury. Significance of motor vehicle crashes and pelvic injury on fetal mortality: a five-year institutional review. J. Trauma-Inj. Infect. Crit. Care 65:616–620, 2008.CrossRefGoogle Scholar
- 14.Duma, S. M. Pregnant Occupants Biomechanics: Advances in Automobile Safety Research. Warrendale, PA: Society of Automotive Engineers, 2010.Google Scholar
- 15.Fung, Y. C. Biomechanics: Mechanical Properties of Living Tissues. New York: Springer, 1981.Google Scholar
- 22.Kastelic, J., and E. Baer. Deformation of tendon collagen. In: The Mechanical Properties of Biological Materials, edited by J. F. Vincient, and J. D. Currey. Cambridge: Society for Experimental Biology, 1980, pp. 397–433.Google Scholar
- 28.Mattox, K. L. M. D., and L. M. D. Goetzl. Trauma in pregnancy. Crit. Care Med. Crit. Illn. Pregnancy 33:S385–S389, 2005.Google Scholar
- 34.Rupp, J. D. K. K., S. Moss, J. Zhou, M. D. Pearlman, and L. W. Schneider. Development and testing of a prototype pregnant abdomen for the small-female hybrid III ATD. Stapp Car Crash J. 45:18, 2001.Google Scholar
- 38.Yamada, H. Strength of Biological Materials. Baltimore: Williams and Wilkinson, 1970.Google Scholar