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Experimental study on multi-step creep properties of rat skins
journal of the mechanical behavior of biomedical materials 46 (2015) 49 –58
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Research Paper
Experimental study on multi-step creep properties of rat skins Gang Chena, Shibo Cuia, Lin Youa, Yan Lib, Yun-Hui Meic,d,n, Xu Chena a
School of Chemical Engineering and Technology, Tianjin University, Tianjin, China Nursing College, Hebei Medical University, Hebei, China c Tianjin Key Laboratory of Advanced Joining Technology, Tianjin University, Tianjin, China d School of Materials Science and Engineering, Tianjin University, Tianjin, China b
art i cle i nfo
ab st rac t
Article history:
Tension, single-step creep, and multi-step creep of rat skins at room temperature were
Received 23 September 2014
experimentally studied. We studied the effects of loading histories of high stress creep, low
Received in revised form
stress creep, and stress relaxation on multi-step creep. Microstructure of rat skins after
31 January 2015
prescribed tests were observed microscopically with the help of standard hematoxylin and
Accepted 19 February 2015
eosin (H&E). The void ratios were also analyzed. The loading histories of high stress creep,
Available online 26 February 2015
low stress creep, and stress relaxation have significant influence on multi-step creep. We
Keywords:
found that the creep strain and its rate in the steady-state stage and the creep-fatigue life
Rat skin
of rat skins are sensitive to creep stress. Low stress creep after the loading history of high
Creep
stress creep is characterized as a recovery of strain and a zero strain rate. Both the loading
Multi-step
history of low stress creep and stress relaxation act as a recovery in multi-step creep, and
Microstructure
they are driven by a same mechanism in the creep strain and the void ratio of rat skins. The loading history, of which sequence is as followings successively: low stress creep, stress relaxation, and high stress creep, helps to obtain the largest creep strain at the lowest void ratio. & 2015 Elsevier Ltd. All rights reserved.
1.
Introduction
Skin, composed of epidermis, dermis, and hypodermis, is the largest organ of the body in vertebrates (Lamers et al., 2013). As the interface of our body with the outside world, skin provides a dynamic protective layer to the body. However, skin wounds, burn, and scars represent a major burden upon the world healthcare cost. As a result, autologous, allogeneic, or artificial skins are required in plastic and reconstructive surgery. Among
these, skin autograft is considered the current gold standard of care (Herndon et al., 1989; Herndon and Parks, 1986). Since skin behaves as a non-homogeneous, anisotropic, nonlinear, viscoelastic material subjected to stress (Fung, 1981), tissue expansion is a remarkable concept based on the skin's creep ability to obtain autograft. Mechanical creep (hereafter referred to as creep) shows the viscoelasticity of skin and is defined as the elongation of skin with a constant load over time beyond intrinsic extensibility (Johnson et al., 1993). It has been described
n Corresponding author at: Tianjin Key Laboratory of Advanced Joining Technology, Tianjin University, Tianjin, China. Tel.: 86 22 27408399. E-mail address: [email protected] (Y.-H. Mei).
http://dx.doi.org/10.1016/j.jmbbm.2015.02.020 1751-6161/& 2015 Elsevier Ltd. All rights reserved.
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journal of the mechanical behavior of biomedical materials 46 (2015) 49 –58
as the vehicle for wound closure with presuturing, rapid tissue expansion, skin-stretching devices, and skin retraction with undermining (Wilhelmi et al., 1998). A thorough understanding of the creep property plays an important role in clinical applications, especially in tissue expansion, expansion device, computer-assisted surgery and surgical robot design. Time-dependent behaviors of soft tissues, especially in response to creep/relaxation loading have been characterized in many studies (Hasan et al., 2014; Kang and Wu, 2011; Thornton et al., 2002; Wang and Ker, 1995; Wren et al., 2003). Among those tests, in-vitro test was still the most widely used method for studying the mechanical properties of soft tissues due to the advantages in clamping, loading, and strain measurement (Annaidh et al., 2012). Wang and Ker, (1995) found that creep rupture may occur even when the applied stress was far below the ultimate stress of a tensile test, and time to failure decreased exponentially with increasing stress for the wallaby tail tendons under creep loading. However, for the creep specimens of human Achilles tendon, there was no significant relationship between applied stress and time to failure, but time to failure decreased exponentially with increasing initial strain (strain when target stress is first reached) and decreasing failure strain (Wren et al., 2003). Zhang proposed that normal skin and expanded skin displayed a similar pattern during creep test (Zhang et al., 2006). Zhang's studies implied that creep behavior in artery wall became stable after 3 h (Zhang et al., 2008). Experimental studies showed creep response was negligible for decellularized aortic ECM and intact aortas (Zou and Zhang, 2012). Furthermore a reduction in relaxation was observed following decellularization, and might be caused by the reduction in glycosaminoglycan content (Converse et al., 2012). While the above studies have provided a fundamental understanding of the creep/relaxation responses of soft tissues, there are still some limitations remain. Most of those works were only focused on single-step creep/relaxation and lose sight of the previous loading history effect, which would occur in applications. For example, one standard surgical operation, named “skin expansion”, is making use of an expander to apply tension on the skin to obtain a larger area. Generally, skin is not expended to a preset stress directly but step by step (Zoellner et al., 2013). Such kind of scenario is called as multistep loading condition, which had received considerable attentions and studies in metals (Lin et al., 2013) and polymers (Shi et al., 2012), but not yet for soft tissues. Therefore, it is necessary to reveal the multi-step creep behavior of skin soft tissue to assess a proper clinical protocol in skin expansion surgery. In this study, rat skins, which have anatomical and biomechanical similarities to human skins (Crichton et al., 2011; Groves et al., 2013), are used to study the single-step and multistep creep. The effects of loading rate, loading level and loading sequence on tensile and creep behaviors are all investigated according to the experimental results. In order to make a thorough understanding on how the loading history affects multi-step creep, rupture and void ratio in collagen fiber of rat skin during monotonic and creep tests are observed by optical microscopy, and the micromechanism of multi-creep is discussed. The results will be useful to realize the effect of loading history on creep of skin tissue.
2.
Materials and methods
2.1.
Sample preparation
Rats, age from 2 to 3 months, with a weight of 20474 g were provided by Hebei Medical University, China. After these rats were anesthetized, skins were cut from the dorsal area with the length direction perpendicular to the direction of the spine (i.e. transverse specimens). The fat layer and hairs were removed from the specimens using a surgical scalpel. The specimens were cut into 15–25 mm long and 2–3 mm wide. All these specimens were preserved in 0.9% normal saline solution at room temperature. Samples were tested as soon as they were prepared. All the tests were finished within 11 h after death of the rats.
2.2.
Experimental setup
All the samples are tested on an in situ fatigue testing apparatus (IBTC-100, CARE Measure & Control Co., Ltd.) at room temperature. All the samples were mounted on the IBTC-100 by means of special flat clamps and immersed in a bath with 0.9% normal saline solution. To avoid the slipping or rupture of the clamped part, the clamps were covered with absorbent cotton. Fig. 1 shows a typical installed sample preparing for the further test by IBTC-100.
2.3.
Experimental design
In this work, the stress, σ, and the strain, ε, refer to the engineering stress and strain, respectively. They are calculated from the following equations: σ ¼ F=A0
ð1Þ
ε ¼ ðD D0 Þ=D0
ð2Þ
where F is the applied force, A0 is the initial area of the gauge cross section of the samples, and D and D0 are the current and initial gauge lengths, respectively. D and D0 are measured by the measurement of clampers displacement. This measure method had been proved equivalent to Digital Image Correlation (Annaidh et al., 2012). Moreover, local strain magnification does not occurs before rupture, and contraction of the cross section along the entire specimen ensures the strain uniform. The ultimate tensile strength, σmax, is defined as the maximum measured stress. The fracture strain, εmax, is defined as the strain corresponding with the maximum stress. Because precondition is considered as one way to ensure repeatability of results (Lanir and Fung, 1974a, b), all specimens
Fig. 1 – Stretching apparatus shown with rat skin in 0.9% normal saline (NS) solution.
journal of the mechanical behavior of biomedical materials 46 (2015) 49 –58
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Table 1 – Creep test conditions for rat skins. Specimen Num. ID
Step
C1 C2 C3 C4 C5 C6 C7 C8 MC1
Step Step Step Step Step Step Step Step Step Step Step Step Step Step Step Step Step Step Step Step
MC2
MCR1
MCR2
Stress/ MPa 1 1 1 1 1 1 1 1 1 2 3 1 2 3 1 2 3 1 2 3
Strain
2 3 4 5 6 7 8 9 2 4 2 4 2 4 2 Hold 4 4 Hold 4
Time/s
5000 5000 10,500 5000 5000 5000 5000 5000 3500 3500 3500 3500 3500 3500 3500 3500 3500 3500 3500 3500
Fig. 2 – Sketch of transverse section of rat skin.
were firstly preconditioned by ten loading–unloading cycles at a strain rate of 5%/s with a peak strain of 10%. Three types of tests were conducted: (1) uniaxial tensile tests at different stress rates to obtain the ultimate tensile strength and the fracture strain of rat skins, (2) single-step creep tests to illustrate the basic characteristics of creep-fatigue behaviors of rat skins, (3) multistep creep tests to study the effect of loading history on creepfatigue behaviors of rat skins. The prescribed loading conditions for all the creep tests are listed in Table 1.
2.4.
Histology
Firstly, the specimens of C3, MC1, MC2, MCR1, MCR2, as listed in Table 1, were embedded in paraffin. Then these specimens were sectioned perpendicular to the surface of the rat skin to obtain transverse histologic sections as sketched in Fig. 2. Finally, these histologic sections were stained with standard hematoxylin and eosin (H&E) and observed by optical microscopy to analyze the change of microstructures of rat skins.
3.
Results
3.1.
Tensile tests
Typical stress–strain curves of uniaxial tensile tests for rat skins at the stress rate of 0.01 MPa/s, 0.1 MPa/s, and 1 MPa/s are shown in Fig. 3(a). Three specimens were tested repeatedly for each stress rate. There is no significant dispersion in these curves especially when the stress rate is low. Furthermore, it can be seen that rat skin is highly nonlinear. The elastic module of rat skin seems to be independent of the tensile stress rate. Fig. 3(b) plots the relationship between stress rate and the ultimate tensile strength or the fracture strain. It is demonstrated that both the ultimate tensile strength and the fracture strain increase with increasing stress rate.
Fig. 3 – (a) Tensile stress vs. tensile strain curves of rat skins, and (b) ultimate tensile strength and fracture strain at different tensile stress rates.
3.2.
Single-step creep tests
The loading rate in single- and multi-step creep tests was set to 1 MPa/s. Fig. 4(a) illustrates a typical creep strain-time curve of rat skin at the stress of 7 MPa. The creep strain evolution can be divided into three stages: transient, steadystate, and tertiary (Li et al., 2013). Most of the creep strain accumulates in the transient creep stage. In this stage, a sharp decrease of the creep strain rate occurs. As a result, the steady-state stage is characterized as a low rate and has the longest duration. Finally, an accelerated increase of the creep strain in the tertiary stage leads to the ultimate failure of the rat skin. These characteristics of creep for rat skins in the present
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journal of the mechanical behavior of biomedical materials 46 (2015) 49 –58
stress affects the creep rupture of rat skin, and the creep life shortens with the increasing applied stress. It can also be observed in Fig. 4(b) that the influence of the creep stress on creep rupture is attributed to the strain rate in the steady-state stage. We take the minimum strain rate of the creep-time curves as the creep strain rate ε_ of the steady-state stage. The relationship between applied stress and creep strain rate in the steady-state stage is shown in Fig. 4(c). The strain rate in the steady-state stage nonlinearly rises with the applied stress. The repeatability of the mechanical behavior of biological soft tissue is usually not as good as metal, rubber, or plastic because the mechanical behavior of biological soft tissue should be dependent on age, size, weight, and grow environment of the rat. As a result, specimens from different rats show a visible deviation. For example, the median strain of 2 MPa creep is 33%, however, the largest one is 40% and the lowest one is 13%. Because of this deviation, multi-step creep tests were carried out. In one multistep creep test, only one specimen was used. The difference among the specimens is excluded as M.J. Munoz' work (Munoz et al., 2008).
3.3.
Fig. 4 – (a) Typical creep strain–time curve of rat skin at stress of 7 MPa. Creep behavior of rat skins obtained at different stresses including (b) creep strain, (c) steady-state creep strain rate.
study are consistent with other soft tissues which had been studied, including rat skin (Del Prete et al., 2004), pig skin (Kang and Wu, 2011), and bovine cornea (Boyce et al., 2007). Results of tensile creep tests for rat skins from the stress of 2 MPa to that of 9 MPa are shown in Fig. 4(b). It can be noted that the creep strain of rat skin is sensitive to the applied stress and increases with the increase of the creep stress. The applied
Multi-step creep tests
In order to ensure the multi-step creep with three loading steps, stresses of 2 MPa and 4 MPa were chosen because of their long duration in the steady-state stage. According to the characteristics in Fig. 4(b) that the applied stress has no influence on the creep strain in the initial part of the transient creep stage, the part that prior to 40 s in step 1 is hidden in order to analyze the effect of loading history. The following results of the multi-step tests are all presented in the same way. The loading history effects of the high stress creep and the low stress creep on multi-step creep are shown in Fig. 5(a) and (b), respectively. It can be seen that the creep strain in multi-step creep also mainly accumulates in the transient creep stage of step 1 like the single-step creep. For MC1, of which loading subsequence in creep stress is low-high-low, the creep strain rate in step 3 is close to zero. This means that there is almost no accumulation of creep strain in step 3. However, for MC2, of which subsequence of creep stress is high-low-high, the creep strain rate in step 3 exhibits in a similar way as that in step 1. Moreover, for MC2, a strain of 3% recovers when the stress turns from 4 MPa to 2 MPa, whereas the recovered strain rapidly regains when the sample is reloaded to 4 MPa. The influence of loading history of high-stress, i.e., 4 MPa, creep on low-stress, i.e., 2 MPa, creep behavior of rat skin is shown in Fig. 6(a). The creep strain under the applied stress of 2 MPa without any loading history increases greatly with time. However, a sharp drop of the creep strain is shown in step 2 of MC2 and step 3 of MC1 when the applied stress decreases from 4 MPa to 2 MPa. Furthermore, the creep strain rates in the step 2 of MC2 and the step 3 of MC1 become close to zero. The results are in accordance with those of bovine cornea (Boyce et al., 2007). Such low-stress creep after loading history of high-stress creep could be regarded as a recovery phase (Ruggles-Wrenn and Balaconis, 2008; Shi et al., 2012) for the skin. However, this recovery should be incomplete because the creep strain in step 3 of MC1 is larger than the ones in the step 2 of MC2 and the step 1 of MC1. Therefore,
journal of the mechanical behavior of biomedical materials 46 (2015) 49 –58
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Fig. 5 – Two types of multi-step creep: (a) sequence of stress is 2-4-2 MPa for MC1 and (b) sequence of stress is 4-2-4 MPa for MC2.
Fig. 6 – Effects of loading history of stress of (a) 4 MPa on creep strain of 2 MPa, and (b) 2 MPa on creep strain of 4 MPa.
the low-stress creep with the loading history of high-stress creep recovers the creep strain to some extents but shows almost no increase in creep strain. On the other hand, the influence of loading history of lowstress, i.e., 2 MPa, creep on high-stress, i.e., 4 MPa, creep behavior of rat skin is shown in Fig. 6(b). The creep strain under the applied stress of 4 MPa without any loading history increases significantly with time. As indicated by step 2 of MC1 and step 3 of MC2, the evolution of the creep strain with the loading history of low-stress creep also includes transient creep stage and steady-state creep stage as that without any loading history. Comparing the creep strain in step 3 of MC2 with that in step 1 of MC2, we find that the creep strain increases by 5% due to the recovery of the loading history of the low-stress, i.e., 2 MPa, creep in the step 2 of MC2. In addition, the duration of the transient stage in the step 3 of MC2 is shortened, and the creep strain rate of the steadystate stage reduces. The curve of step 3 MC2 coincides closely to step 1 of MC2. This is similar to Mullins effect in rubber like solids under cycle loading, which was first proposed by Mullins (Diani et al., 2009; Mullins, 1948). Therefore, it is concluded that the loading history of the low-stress creep could result in larger creep strain than single-step creep, but decrease the creep strain rate in the steady-state stage.
Stress relaxation could occur when tissue is loaded at a constant strain. The stress in tissue can even decrease to zero in rat brain tissue (Haslach et al., 2014). Because of this characteristic of stress recovery, the loading history of the stress relaxation is considered as another kind of recovery that corresponds to the loading history of the low-stress creep. Fig. 7(a) and (b) shows the results of the multi-step creep with the loading history of stress relaxation. The creep strain in step 3 evolves in a similar way as that in step 1. However, the duration of the transient creep stage with the stress relaxation history is much shorter than that with the creep history, such as the one in step 2 of MC1 or in step 3 of MC2. Although MCR1 presents a lower stress and a smaller strain in step 1 than MCR2, MCR1 shows much larger deformation in step 3. Furthermore, the influence of loading history of stress relaxation is shown in Fig. 8(a) and compared with that of the low-stress, i.e., 2 MPa, creep on the highstress, i.e., 4 MPa, creep behavior. Although the transient stage in step 3 of MCR2 is much shorter than that of MC2, the creep strain and its rate at the steady-state stage of MC2 approximately equal to those of MCR2. These results indicate that the two kinds of recovery, which are the loading history of the low stress creep and the stress relaxation, play the same role in creep strain and its rate. In the meantime, the
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journal of the mechanical behavior of biomedical materials 46 (2015) 49 –58
Fig. 7 – Multi-step creep with loading history of stress relaxation: (a) loading sequence is 2 MPa creep – stress relaxation – 4 MPa creep for MCR1, (b) loading sequence is 4 MPa creep – stress relaxation – 4 MPa creep for MCR2.
influence of creep stress in step 1 is further studied in the multi-step creep test with the loading history of stress relaxation. Fig. 8(b) shows that an interesting phenomenon can be observed though the stress in step 1 of MCR1 is smaller than that of MCR2, the strain in step 3 of the former is much larger than that of the latter. Furthermore, the strain rate in the steady-state stage for MCR1 is lower than that for MCR2, which implies the duration of the former may be longer than the latter. Therefore, the results demonstrate that low stress applied in the first step loading is much better to obtain a larger deformation. It can be concluded that the loading sequence of the low stress creep-the stress relaxation-the high stress creep contributes to produce larger strain and lower strain rate based on these macroscopic analyses.
3.4.
Optical microscopy observations
The transverse histologic sections of one as-received sample are shown in Fig. 9(a). The rat skin can be classified into three layers: epidermis (E), dermis (D), and hypodermis (H) (Subramanyan et al., 2007). Epidermis is shown in dark purple, and characterized as a water proof, protective, out layer (Zollner et al., 2013). The hypodermis connects skin to bone and muscle, consists primarily of muscle and adipocytes (Zollner et al., 2013). The
Fig. 8 – Effects of loading history of (a) low-stress creep and stress relaxation; (b) first step stress on creep behavior of rat skin.
dermal layer of skin is acknowledged to be a significant contributor to the mechanical behavior of the tissue (Agache et al., 1980). The fibers of the rat skin are believed to be the major determinant of the skin's biomechanical properties (Groves et al., 2013). Direct observation of the microstructures of fibers at various loading cases could provide anatomical data concerning the structural basis for the static mechanical properties of the skin. Fibers without loading are waved and crimped (Brown, 1973). When stretching the rat skin under the stress of 4 MPa over time, the fibers straighten and realign parallel to one another along the loading direction as shown in Fig. 9(b). A similar behavior was observed by Roy in White New Zealand rabbit arterial wall (Roy et al., 2010). However, ruptures of fibers are also observed. The ruptures may develop into unrecoverable damage, for example cell necrosis (Provenzano et al., 2002), or even failure for the rat skin (Pena, 2014). For the specimens with multi-step loadings, elongation and reorientation of their fibers along loading direction like single-step creep are also observed in Fig. 9(c)–(e). It can be seen that folds and waves of fibers diminish as the loading case changing from C3, MC2, MCR2 to MCR1. This implies that the elongation of fibers becomes greater with the sequence in MCR1. On the other hand, the fiber rupture from Fig. 9(b) to 9(e) also reduces, which denotes the damage
journal of the mechanical behavior of biomedical materials 46 (2015) 49 –58
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Fig. 9 – Microstructures of collagen fibers in rat skin in following loading case: (a) as-received sample, (b) C3, (c) MC2, (d) MCR2, (e) MCR1. E ¼epidermis, D¼ dermis, H¼hypodermis, LD¼ loading direction. 10.
due to the loading to the rat skin weakens in turn. According to the visual analysis of the microstructures, the specimen of loading sequence of the 2 MPa creep – stress relaxation – 4 MPa creep has the least damage compared with other three specimens at the same loading time.
Because porosity is usually related to the mechanical properties of materials and has been extensively used in bone (Seeman and Delmas, 2006; Zebaze et al., 2010) and polymer (Khatib et al., 2012), the gaps between fiber bundles in rat skin are concerned. Abundant gaps in the single-step creep are observed in Fig. 9(b),
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journal of the mechanical behavior of biomedical materials 46 (2015) 49 –58
Fig. 10 – Converted images of gaps between fibers of rat skins in following loading case: (a) C3, (b) MC2, (c) MCR2, (d) MCR1. and most of these gaps are along with the loading direction. However, the gaps diminish in multi-step creep tests, implying that the damage of the rat skin alleviates with a loading history of low stress creep or stress relaxation, as shown in Fig. 9(c)–(e). To quantitatively estimate the damage of the rat skin, void ratio of the microstructure of the dermis layer is utilized. High void ratio indicates more severe damage. Void ratio is measured by image analyze method. This method was detail introduced in (Rnjak-Kovacina et al., 2011). Void ratios are quantified from H&E
stained histologic sections. Voids and tissue show different colors after H&E staining. Voids are colorless and transparent while tissue is red. The images of the sections were converted to grayscale format firstly, and then to binary format. The converted images are shown as Fig. 10. The gaps between fiber bundles of the dermis for C3, MC2, MCR2, and MCR1 are shown as black pixels in Fig. 10. As a result, the void ratio is obtained by dividing the black area by the total section surface area in each image. Their void ratios are calculated and shown in Fig. 11. It
journal of the mechanical behavior of biomedical materials 46 (2015) 49 –58
4.
57
Conclusions
The present study gives an insight into the multi-step creep on both mechanical and microcosmic perspective. One important application of information regarding multi-step creep behavior is in assessing the appropriate clinical protocols, i.e., loading sequence, which should be accounted for during the surgery of skin expansion. First, the single-step creep tests were conducted to demonstrate the creep behavior of skin. Then the effects of loading histories of the high stress creep, the low stress creep, and the stress relaxation on multi-step creep tests were investigated. Finally, the microstructure of rat skins was observed by optical microscopy with the help of standard hematoxylin and eosin (H&E). The conclusions can be drawn as follows: Fig. 11 – Void ratios in different loading cases.
can also be seen that the damage of the single-step creep is the greatest with the highest void ratio, i.e., 16.67%. For MC2 and MCR2, the void ratios are 7.48% and 7.34%, respectively. This suggests that loading histories of low stress creep and stress relaxation helps to reduce the damage of rat skins. Moreover, their effects have no significant difference in lowering damage and both lessen half of the damage comparing with the singlestep creep. According to Figs. 5 and 7, both the loading history of the low stress creep and the stress relaxation enable to increase by 4% of the creep strain. Therefore, larger deformation and lower damage can be achieved in the multi-step creep with the loading histories of both the low stress creep and the stress relaxation. Moreover, the effects of both loading histories on the deformation and damage of rat skins are similar. Comparing MCR1 with MCR2, gaps in the former are far less and thinner than in the latter. Fig. 11 shows that the void ratio of the specimen with the loading sequence of 2 MPa creep – stress relaxation – 4 MPa creep is the lowest, which is 3.58%. The damage of MCR1 is less than half of that of MCR2. Hence, the low stress in step 1 helps to lessen the damage of fibers. On the other hand, the strain of MCR1 is also the largest among these loading cases as shown in Fig. 7. It can be concluded that the loading sequence of low stress creep – stress relaxation – high stress creep contributes to obtaining the largest creep strain with the lowest void ratio. It can be concluded that applying low stress creep and stress relaxation in advance could produce more skin in skin expansion operation than direct high stress creep, and less damage would be induced. From the abovementioned discussion on the aspects of mechanical properties and microstructures, the loading sequence of the low stress creep – stress relaxation – high stress creep produces the largest deformation but the lowest damage of the skin. However, there are visible differences between the diameters, strength, and densities of human and murine dermal fibers. In addition, other considerations include the degree of cross-linking within the fiber matrix, the degradation of the fibers and variations in the thickness of the dermal layer (Groves et al., 2013). These are likely to contribute to mechanical differences between human and animal skin. Therefore, further studies on human skin are necessary to determine the stresses in the first and the third steps and the relationship between the loading sequence and the damage of the skin.
(1) The creep strain and its rate of the steady-state stage, and creep-fatigue life are highly dependent on the applied stress. Creep strain and its rate of the steady-state stage increase with the applied stress, whereas the fatiguecreep life decreases. (2) Loading histories of the high-stress creep, the low-stress creep, and the stress relaxation have an important influence on multi-step creep. The low-stress creep after the loading history of the high-stress creep shows the strain recovery and no more creep strain accumulation. The history of stress relaxation also presents the strain recovery as that of high-stress creep. (3) Both the histories of the low-stress creep and the stress relaxation help to obtain larger strain than the single-step creep and decrease the void ratio in the rat skin. (4) The largest deformation and the lowest void ratio can be obtained in the multi-step with the loading sequence of the low stress creep – the stress relaxation – the high stress creep.
Acknowledgments The authors gratefully acknowledge the financial support from the National Natural Science Foundation of China (Nos. 11172202 and 61334010).
r e f e r e nc e s
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Effects of cryopreservation, decellularization and novel extracellular matrix conditioning on the quasi-static and timedependent properties of the pulmonary valve leaflet. Acta Biomater. 8, 2722–2729. Crichton, M.L., Donose, B.C., Chen, X.F., Raphael, A.P., Huang, H., Kendall, M.A.F., 2011. The viscoelastic, hyperelastic and scale dependent behaviour of freshly excised individual skin layers. Biomaterials 32, 4670–4681. Del Prete, Z., Antoniucci, S., Hoffman, A.H., Grigg, P., 2004. Viscoelastic properties of skin in Mov-13 and Tsk mice. J. Biomech. 37, 1491–1497. Diani, J., Fayolle, B., Gilormini, P., 2009. A review on the Mullins effect. Eur. Polym. J. 45, 601–612. Fung, Y.C., 1981. Biomechanics. Mechanical Properties of Living Tissues. Springer-Verlag, New York, NY, USA. Groves, R.B., Coulman, S.A., Birchall, J.C., Evans, S.L., 2013. An anisotropic, hyperelastic model for skin: experimental measurements, finite element modelling and identification of parameters for human and murine skin. J. Mech. Behav. Biomed. Mater. 18, 167–180. Hasan, A., Ragaert, K., Swieszkowski, W., Selimovic, S., Paul, A., Camci-Unal, G., Mofrad, M.R.K., Khademhosseini, A., 2014. Biomechanical properties of native and tissue engineered heart valve constructs. J. Biomech. 47, 1949–1963. Haslach, H.W., Leahy, L.N., Riley, P., Gullapalli, R., Xu, S., Hsieh, A.H., 2014. Solid-extracellular fluid interaction and damage in the mechanical response of rat brain tissue under confined compression. J. Mech. Behav. Biomed. Mater. 29, 138–150. Herndon, D.N., Barrow, R.E., Rutan, R.L., Rutan, T.C., Desai, M.H., Abston, S., 1989. A comparison of conservative versus early excision. Therapies in severely burned patients (discussion 552-543). Ann. Surg. 209, 547–552. Herndon, D.N., Parks, D.H., 1986. Comparison of serial debridement and autografting and early massive excision with cadaver skin overlay in the treatment of large burns in children. J. Trauma 26, 149–152. Johnson, T.M., Lowe, L., Brown, M.D., Sullivan, M.J., Nelson, B.R., 1993. Histology and physiology of tissue expansion. J. Dermatol. Surg. Oncol. 19, 1074–1078. Kang, G., Wu, X., 2011. Ratchetting of porcine skin under uniaxial cyclic loading. J. Mech. Behav. Biomed. Mater. 4, 498–506. Khatib, J.M., Wright, L., Mangat, P.S., Negim, E.M., 2012. Porosity and pore size distribution of well hydrated Cement-Fly AshGypsum pastes. Am.-Eurasian J. Sci. Res. 7, 142–145. Lamers, E., van Kempen, T.H.S., Baaijens, F.P.T., Peters, G.W.M., Oomens, C.W.J., 2013. Large amplitude oscillatory shear properties of human skin. J. Mech. Behav. Biomed. Mater. 28, 462–470. Lanir, Y., Fung, Y.C., 1974a. Two dimensional mechanical properties of rabbit skin. I. Experimental system. J. Biomech. 7, 29–34. Lanir, Y., Fung, Y.C., 1974b. Two dimensional mechanical properties of rabbit skin. II. Experimental results. J. Biomech. 7, 171–182. Li, X., Chen, G., Wang, L., Mei, Y.H., Chen, X., Lu, G.Q., 2013. Creep properties of low-temperature sintered nano-silver lap shear joints. Mater. Sci. Eng. A—Struct. Mater. Prop. Microstruct. Process. 579, 108–113. Lin, Y.C., Liu, Z.-H., Chen, X.-M., Chen, J., 2013. Stress-based fatigue life prediction models for AZ31B magnesium alloy under single-step and multi-step asymmetric stresscontrolled cyclic loadings. Comput. Mater. Sci. 73, 128–138. Munoz, M.J., Bea, J.A., Rodriguez, J.F., Ochoa, I., Grasa, J., del Palomar, A.P., Zaragoza, P., Osta, R., Doblare, M., 2008. An experimental study of the mouse skin behaviour: Damage and inelastic aspects. J. Biomech. 41, 93–99.
Mullins, L., 1948. Effect of stretching on the properties of rubber. Rubber Chem. Technol. 21, 281–300. Pena, E., 2014. Computational aspects of the numerical modelling of softening, damage and permanent set in soft biological tissues. Comput. Struct. 130, 57–72. Provenzano, P.P., Heisey, D., Hayashi, K., Lakes, R., Vanderby, R., 2002. Subfailure damage in ligament: a structural and cellular evaluation. J. Appl. Physiol. 92, 362–371. Rnjak-Kovacina, J., Wise, S.G., Li, Z., Maitz, P.K.M., Young, C.J., Wang, Y., Weiss, A.S., 2011. Tailoring the porosity and pore size of electrospun synthetic human elastin scaffolds for dermal tissue engineering. Biomaterials 32, 6729–6736. Roy, S., Boss, C., Rezakhaniha, R., Stergiopulos, N., 2010. Experimental characterization of the distribution of collagen fiber recruitment. J. Biorheol. 24, 84–93. Ruggles-Wrenn, M.B., Balaconis, J.G., 2008. Some aspects of the mechanical response of BMI 5250-4 neat resin at 191 degrees C: experiment and modeling. J. Appl. Polym. Sci. 107, 1378–1386. Seeman, E., Delmas, P.D., 2006. Bone quality – the material and structural basis of bone strength and fragility. New Engl. J. Med. 354, 2250–2261þ2212. Shi, S., Yu, D., Gao, L., Chen, G., Chen, J., Chen, X., 2012. Nonlinear viscoelastic-plastic constitutive description of proton exchange membrane under immersed condition. J. Power Sources 213, 40–46. Subramanyan, K., Misra, M., Mukherjee, S., Ananthapadmanabhan, K.P., 2007. Advances in the materials science of skin: a composite structure with multiple functions. MRS Bull. 32, 770–778. Thornton, G.M., Shrive, N.G., Frank, C.B., 2002. Ligament creep recruits fibres at low stresses and can lead to modulusreducing fibre damage at higher creep stresses: a study in rabbit medial collateral ligament model. J. Orthop. Res. 20, 967–974. Wang, X.T., Ker, R.F., 1995. Creep rupture of wallaby tail tendons. J. Exp. Biol. 198, 831–845. Wilhelmi, B.J., Blackwell, S.J., Mancoll, J.S., Phillips, L.G., 1998. Creep vs. stretch: a review of the viscoelastic properties of skin. Ann. Plast. Surg. 41, 215–219. Wren, T.A.L., Lindsey, D.P., Beaupre, G.S., Carter, D.R., 2003. Effects of creep and cyclic loading on the mechanical properties and failure of human achilles tendons. Ann. Biomed. Eng. 31, 710–717. Zebaze, R.M.D., Ghasem-Zadeh, A., Bohte, A., Iuliano-Burns, S., Mirams, M., Price, R.I., Mackie, E.J., Seeman, E., 2010. Intracortical remodelling and porosity in the distal radius and post-mortem femurs of women: a cross-sectional study. Lancet 375, 1729–1736. Zhang, E.-p., Liao, D.-h., Liu, A.-z., Wang, X.-b., Li, X.-y., Zeng, Y.-j., Wang, S.-j., 2006. Biomechanical characteristics investigation on long-term free graft with expanded porcine skin. Clin. Biomech. 21, 864–869. Zhang, W., Guo, X., Kassab, G.S., 2008. A generalized Maxwell Model for creep behavior of artery opening angle. J. Biomech. Eng. 130 054502-054502. Zoellner, A.M., Holland, M.A., Honda, K.S., Gosain, A.K., Kuhl, E., 2013. Growth on demand: reviewing the mechanobiology of stretched skin. J. Mech. Behav. Biomed. Mater. 28, 495–509. Zollner, A.M., Holland, M.A., Honda, K.S., Gosain, A.K., Kuhl, E., 2013. Growth on demand: reviewing the mechanobiology of stretched skin. J. Mech. Behav. Biomed. Mater. 28, 495–509. Zou, Y., Zhang, Y.H., 2012. Mechanical evaluation of decellularized porcine thoracic aorta. J. Surg. Res. 175, 359–368.
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Research Paper
Experimental study on multi-step creep properties of rat skins Gang Chena, Shibo Cuia, Lin Youa, Yan Lib, Yun-Hui Meic,d,n, Xu Chena a
School of Chemical Engineering and Technology, Tianjin University, Tianjin, China Nursing College, Hebei Medical University, Hebei, China c Tianjin Key Laboratory of Advanced Joining Technology, Tianjin University, Tianjin, China d School of Materials Science and Engineering, Tianjin University, Tianjin, China b
art i cle i nfo
ab st rac t
Article history:
Tension, single-step creep, and multi-step creep of rat skins at room temperature were
Received 23 September 2014
experimentally studied. We studied the effects of loading histories of high stress creep, low
Received in revised form
stress creep, and stress relaxation on multi-step creep. Microstructure of rat skins after
31 January 2015
prescribed tests were observed microscopically with the help of standard hematoxylin and
Accepted 19 February 2015
eosin (H&E). The void ratios were also analyzed. The loading histories of high stress creep,
Available online 26 February 2015
low stress creep, and stress relaxation have significant influence on multi-step creep. We
Keywords:
found that the creep strain and its rate in the steady-state stage and the creep-fatigue life
Rat skin
of rat skins are sensitive to creep stress. Low stress creep after the loading history of high
Creep
stress creep is characterized as a recovery of strain and a zero strain rate. Both the loading
Multi-step
history of low stress creep and stress relaxation act as a recovery in multi-step creep, and
Microstructure
they are driven by a same mechanism in the creep strain and the void ratio of rat skins. The loading history, of which sequence is as followings successively: low stress creep, stress relaxation, and high stress creep, helps to obtain the largest creep strain at the lowest void ratio. & 2015 Elsevier Ltd. All rights reserved.
1.
Introduction
Skin, composed of epidermis, dermis, and hypodermis, is the largest organ of the body in vertebrates (Lamers et al., 2013). As the interface of our body with the outside world, skin provides a dynamic protective layer to the body. However, skin wounds, burn, and scars represent a major burden upon the world healthcare cost. As a result, autologous, allogeneic, or artificial skins are required in plastic and reconstructive surgery. Among
these, skin autograft is considered the current gold standard of care (Herndon et al., 1989; Herndon and Parks, 1986). Since skin behaves as a non-homogeneous, anisotropic, nonlinear, viscoelastic material subjected to stress (Fung, 1981), tissue expansion is a remarkable concept based on the skin's creep ability to obtain autograft. Mechanical creep (hereafter referred to as creep) shows the viscoelasticity of skin and is defined as the elongation of skin with a constant load over time beyond intrinsic extensibility (Johnson et al., 1993). It has been described
n Corresponding author at: Tianjin Key Laboratory of Advanced Joining Technology, Tianjin University, Tianjin, China. Tel.: 86 22 27408399. E-mail address: [email protected] (Y.-H. Mei).
http://dx.doi.org/10.1016/j.jmbbm.2015.02.020 1751-6161/& 2015 Elsevier Ltd. All rights reserved.
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journal of the mechanical behavior of biomedical materials 46 (2015) 49 –58
as the vehicle for wound closure with presuturing, rapid tissue expansion, skin-stretching devices, and skin retraction with undermining (Wilhelmi et al., 1998). A thorough understanding of the creep property plays an important role in clinical applications, especially in tissue expansion, expansion device, computer-assisted surgery and surgical robot design. Time-dependent behaviors of soft tissues, especially in response to creep/relaxation loading have been characterized in many studies (Hasan et al., 2014; Kang and Wu, 2011; Thornton et al., 2002; Wang and Ker, 1995; Wren et al., 2003). Among those tests, in-vitro test was still the most widely used method for studying the mechanical properties of soft tissues due to the advantages in clamping, loading, and strain measurement (Annaidh et al., 2012). Wang and Ker, (1995) found that creep rupture may occur even when the applied stress was far below the ultimate stress of a tensile test, and time to failure decreased exponentially with increasing stress for the wallaby tail tendons under creep loading. However, for the creep specimens of human Achilles tendon, there was no significant relationship between applied stress and time to failure, but time to failure decreased exponentially with increasing initial strain (strain when target stress is first reached) and decreasing failure strain (Wren et al., 2003). Zhang proposed that normal skin and expanded skin displayed a similar pattern during creep test (Zhang et al., 2006). Zhang's studies implied that creep behavior in artery wall became stable after 3 h (Zhang et al., 2008). Experimental studies showed creep response was negligible for decellularized aortic ECM and intact aortas (Zou and Zhang, 2012). Furthermore a reduction in relaxation was observed following decellularization, and might be caused by the reduction in glycosaminoglycan content (Converse et al., 2012). While the above studies have provided a fundamental understanding of the creep/relaxation responses of soft tissues, there are still some limitations remain. Most of those works were only focused on single-step creep/relaxation and lose sight of the previous loading history effect, which would occur in applications. For example, one standard surgical operation, named “skin expansion”, is making use of an expander to apply tension on the skin to obtain a larger area. Generally, skin is not expended to a preset stress directly but step by step (Zoellner et al., 2013). Such kind of scenario is called as multistep loading condition, which had received considerable attentions and studies in metals (Lin et al., 2013) and polymers (Shi et al., 2012), but not yet for soft tissues. Therefore, it is necessary to reveal the multi-step creep behavior of skin soft tissue to assess a proper clinical protocol in skin expansion surgery. In this study, rat skins, which have anatomical and biomechanical similarities to human skins (Crichton et al., 2011; Groves et al., 2013), are used to study the single-step and multistep creep. The effects of loading rate, loading level and loading sequence on tensile and creep behaviors are all investigated according to the experimental results. In order to make a thorough understanding on how the loading history affects multi-step creep, rupture and void ratio in collagen fiber of rat skin during monotonic and creep tests are observed by optical microscopy, and the micromechanism of multi-creep is discussed. The results will be useful to realize the effect of loading history on creep of skin tissue.
2.
Materials and methods
2.1.
Sample preparation
Rats, age from 2 to 3 months, with a weight of 20474 g were provided by Hebei Medical University, China. After these rats were anesthetized, skins were cut from the dorsal area with the length direction perpendicular to the direction of the spine (i.e. transverse specimens). The fat layer and hairs were removed from the specimens using a surgical scalpel. The specimens were cut into 15–25 mm long and 2–3 mm wide. All these specimens were preserved in 0.9% normal saline solution at room temperature. Samples were tested as soon as they were prepared. All the tests were finished within 11 h after death of the rats.
2.2.
Experimental setup
All the samples are tested on an in situ fatigue testing apparatus (IBTC-100, CARE Measure & Control Co., Ltd.) at room temperature. All the samples were mounted on the IBTC-100 by means of special flat clamps and immersed in a bath with 0.9% normal saline solution. To avoid the slipping or rupture of the clamped part, the clamps were covered with absorbent cotton. Fig. 1 shows a typical installed sample preparing for the further test by IBTC-100.
2.3.
Experimental design
In this work, the stress, σ, and the strain, ε, refer to the engineering stress and strain, respectively. They are calculated from the following equations: σ ¼ F=A0
ð1Þ
ε ¼ ðD D0 Þ=D0
ð2Þ
where F is the applied force, A0 is the initial area of the gauge cross section of the samples, and D and D0 are the current and initial gauge lengths, respectively. D and D0 are measured by the measurement of clampers displacement. This measure method had been proved equivalent to Digital Image Correlation (Annaidh et al., 2012). Moreover, local strain magnification does not occurs before rupture, and contraction of the cross section along the entire specimen ensures the strain uniform. The ultimate tensile strength, σmax, is defined as the maximum measured stress. The fracture strain, εmax, is defined as the strain corresponding with the maximum stress. Because precondition is considered as one way to ensure repeatability of results (Lanir and Fung, 1974a, b), all specimens
Fig. 1 – Stretching apparatus shown with rat skin in 0.9% normal saline (NS) solution.
journal of the mechanical behavior of biomedical materials 46 (2015) 49 –58
51
Table 1 – Creep test conditions for rat skins. Specimen Num. ID
Step
C1 C2 C3 C4 C5 C6 C7 C8 MC1
Step Step Step Step Step Step Step Step Step Step Step Step Step Step Step Step Step Step Step Step
MC2
MCR1
MCR2
Stress/ MPa 1 1 1 1 1 1 1 1 1 2 3 1 2 3 1 2 3 1 2 3
Strain
2 3 4 5 6 7 8 9 2 4 2 4 2 4 2 Hold 4 4 Hold 4
Time/s
5000 5000 10,500 5000 5000 5000 5000 5000 3500 3500 3500 3500 3500 3500 3500 3500 3500 3500 3500 3500
Fig. 2 – Sketch of transverse section of rat skin.
were firstly preconditioned by ten loading–unloading cycles at a strain rate of 5%/s with a peak strain of 10%. Three types of tests were conducted: (1) uniaxial tensile tests at different stress rates to obtain the ultimate tensile strength and the fracture strain of rat skins, (2) single-step creep tests to illustrate the basic characteristics of creep-fatigue behaviors of rat skins, (3) multistep creep tests to study the effect of loading history on creepfatigue behaviors of rat skins. The prescribed loading conditions for all the creep tests are listed in Table 1.
2.4.
Histology
Firstly, the specimens of C3, MC1, MC2, MCR1, MCR2, as listed in Table 1, were embedded in paraffin. Then these specimens were sectioned perpendicular to the surface of the rat skin to obtain transverse histologic sections as sketched in Fig. 2. Finally, these histologic sections were stained with standard hematoxylin and eosin (H&E) and observed by optical microscopy to analyze the change of microstructures of rat skins.
3.
Results
3.1.
Tensile tests
Typical stress–strain curves of uniaxial tensile tests for rat skins at the stress rate of 0.01 MPa/s, 0.1 MPa/s, and 1 MPa/s are shown in Fig. 3(a). Three specimens were tested repeatedly for each stress rate. There is no significant dispersion in these curves especially when the stress rate is low. Furthermore, it can be seen that rat skin is highly nonlinear. The elastic module of rat skin seems to be independent of the tensile stress rate. Fig. 3(b) plots the relationship between stress rate and the ultimate tensile strength or the fracture strain. It is demonstrated that both the ultimate tensile strength and the fracture strain increase with increasing stress rate.
Fig. 3 – (a) Tensile stress vs. tensile strain curves of rat skins, and (b) ultimate tensile strength and fracture strain at different tensile stress rates.
3.2.
Single-step creep tests
The loading rate in single- and multi-step creep tests was set to 1 MPa/s. Fig. 4(a) illustrates a typical creep strain-time curve of rat skin at the stress of 7 MPa. The creep strain evolution can be divided into three stages: transient, steadystate, and tertiary (Li et al., 2013). Most of the creep strain accumulates in the transient creep stage. In this stage, a sharp decrease of the creep strain rate occurs. As a result, the steady-state stage is characterized as a low rate and has the longest duration. Finally, an accelerated increase of the creep strain in the tertiary stage leads to the ultimate failure of the rat skin. These characteristics of creep for rat skins in the present
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journal of the mechanical behavior of biomedical materials 46 (2015) 49 –58
stress affects the creep rupture of rat skin, and the creep life shortens with the increasing applied stress. It can also be observed in Fig. 4(b) that the influence of the creep stress on creep rupture is attributed to the strain rate in the steady-state stage. We take the minimum strain rate of the creep-time curves as the creep strain rate ε_ of the steady-state stage. The relationship between applied stress and creep strain rate in the steady-state stage is shown in Fig. 4(c). The strain rate in the steady-state stage nonlinearly rises with the applied stress. The repeatability of the mechanical behavior of biological soft tissue is usually not as good as metal, rubber, or plastic because the mechanical behavior of biological soft tissue should be dependent on age, size, weight, and grow environment of the rat. As a result, specimens from different rats show a visible deviation. For example, the median strain of 2 MPa creep is 33%, however, the largest one is 40% and the lowest one is 13%. Because of this deviation, multi-step creep tests were carried out. In one multistep creep test, only one specimen was used. The difference among the specimens is excluded as M.J. Munoz' work (Munoz et al., 2008).
3.3.
Fig. 4 – (a) Typical creep strain–time curve of rat skin at stress of 7 MPa. Creep behavior of rat skins obtained at different stresses including (b) creep strain, (c) steady-state creep strain rate.
study are consistent with other soft tissues which had been studied, including rat skin (Del Prete et al., 2004), pig skin (Kang and Wu, 2011), and bovine cornea (Boyce et al., 2007). Results of tensile creep tests for rat skins from the stress of 2 MPa to that of 9 MPa are shown in Fig. 4(b). It can be noted that the creep strain of rat skin is sensitive to the applied stress and increases with the increase of the creep stress. The applied
Multi-step creep tests
In order to ensure the multi-step creep with three loading steps, stresses of 2 MPa and 4 MPa were chosen because of their long duration in the steady-state stage. According to the characteristics in Fig. 4(b) that the applied stress has no influence on the creep strain in the initial part of the transient creep stage, the part that prior to 40 s in step 1 is hidden in order to analyze the effect of loading history. The following results of the multi-step tests are all presented in the same way. The loading history effects of the high stress creep and the low stress creep on multi-step creep are shown in Fig. 5(a) and (b), respectively. It can be seen that the creep strain in multi-step creep also mainly accumulates in the transient creep stage of step 1 like the single-step creep. For MC1, of which loading subsequence in creep stress is low-high-low, the creep strain rate in step 3 is close to zero. This means that there is almost no accumulation of creep strain in step 3. However, for MC2, of which subsequence of creep stress is high-low-high, the creep strain rate in step 3 exhibits in a similar way as that in step 1. Moreover, for MC2, a strain of 3% recovers when the stress turns from 4 MPa to 2 MPa, whereas the recovered strain rapidly regains when the sample is reloaded to 4 MPa. The influence of loading history of high-stress, i.e., 4 MPa, creep on low-stress, i.e., 2 MPa, creep behavior of rat skin is shown in Fig. 6(a). The creep strain under the applied stress of 2 MPa without any loading history increases greatly with time. However, a sharp drop of the creep strain is shown in step 2 of MC2 and step 3 of MC1 when the applied stress decreases from 4 MPa to 2 MPa. Furthermore, the creep strain rates in the step 2 of MC2 and the step 3 of MC1 become close to zero. The results are in accordance with those of bovine cornea (Boyce et al., 2007). Such low-stress creep after loading history of high-stress creep could be regarded as a recovery phase (Ruggles-Wrenn and Balaconis, 2008; Shi et al., 2012) for the skin. However, this recovery should be incomplete because the creep strain in step 3 of MC1 is larger than the ones in the step 2 of MC2 and the step 1 of MC1. Therefore,
journal of the mechanical behavior of biomedical materials 46 (2015) 49 –58
53
Fig. 5 – Two types of multi-step creep: (a) sequence of stress is 2-4-2 MPa for MC1 and (b) sequence of stress is 4-2-4 MPa for MC2.
Fig. 6 – Effects of loading history of stress of (a) 4 MPa on creep strain of 2 MPa, and (b) 2 MPa on creep strain of 4 MPa.
the low-stress creep with the loading history of high-stress creep recovers the creep strain to some extents but shows almost no increase in creep strain. On the other hand, the influence of loading history of lowstress, i.e., 2 MPa, creep on high-stress, i.e., 4 MPa, creep behavior of rat skin is shown in Fig. 6(b). The creep strain under the applied stress of 4 MPa without any loading history increases significantly with time. As indicated by step 2 of MC1 and step 3 of MC2, the evolution of the creep strain with the loading history of low-stress creep also includes transient creep stage and steady-state creep stage as that without any loading history. Comparing the creep strain in step 3 of MC2 with that in step 1 of MC2, we find that the creep strain increases by 5% due to the recovery of the loading history of the low-stress, i.e., 2 MPa, creep in the step 2 of MC2. In addition, the duration of the transient stage in the step 3 of MC2 is shortened, and the creep strain rate of the steadystate stage reduces. The curve of step 3 MC2 coincides closely to step 1 of MC2. This is similar to Mullins effect in rubber like solids under cycle loading, which was first proposed by Mullins (Diani et al., 2009; Mullins, 1948). Therefore, it is concluded that the loading history of the low-stress creep could result in larger creep strain than single-step creep, but decrease the creep strain rate in the steady-state stage.
Stress relaxation could occur when tissue is loaded at a constant strain. The stress in tissue can even decrease to zero in rat brain tissue (Haslach et al., 2014). Because of this characteristic of stress recovery, the loading history of the stress relaxation is considered as another kind of recovery that corresponds to the loading history of the low-stress creep. Fig. 7(a) and (b) shows the results of the multi-step creep with the loading history of stress relaxation. The creep strain in step 3 evolves in a similar way as that in step 1. However, the duration of the transient creep stage with the stress relaxation history is much shorter than that with the creep history, such as the one in step 2 of MC1 or in step 3 of MC2. Although MCR1 presents a lower stress and a smaller strain in step 1 than MCR2, MCR1 shows much larger deformation in step 3. Furthermore, the influence of loading history of stress relaxation is shown in Fig. 8(a) and compared with that of the low-stress, i.e., 2 MPa, creep on the highstress, i.e., 4 MPa, creep behavior. Although the transient stage in step 3 of MCR2 is much shorter than that of MC2, the creep strain and its rate at the steady-state stage of MC2 approximately equal to those of MCR2. These results indicate that the two kinds of recovery, which are the loading history of the low stress creep and the stress relaxation, play the same role in creep strain and its rate. In the meantime, the
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journal of the mechanical behavior of biomedical materials 46 (2015) 49 –58
Fig. 7 – Multi-step creep with loading history of stress relaxation: (a) loading sequence is 2 MPa creep – stress relaxation – 4 MPa creep for MCR1, (b) loading sequence is 4 MPa creep – stress relaxation – 4 MPa creep for MCR2.
influence of creep stress in step 1 is further studied in the multi-step creep test with the loading history of stress relaxation. Fig. 8(b) shows that an interesting phenomenon can be observed though the stress in step 1 of MCR1 is smaller than that of MCR2, the strain in step 3 of the former is much larger than that of the latter. Furthermore, the strain rate in the steady-state stage for MCR1 is lower than that for MCR2, which implies the duration of the former may be longer than the latter. Therefore, the results demonstrate that low stress applied in the first step loading is much better to obtain a larger deformation. It can be concluded that the loading sequence of the low stress creep-the stress relaxation-the high stress creep contributes to produce larger strain and lower strain rate based on these macroscopic analyses.
3.4.
Optical microscopy observations
The transverse histologic sections of one as-received sample are shown in Fig. 9(a). The rat skin can be classified into three layers: epidermis (E), dermis (D), and hypodermis (H) (Subramanyan et al., 2007). Epidermis is shown in dark purple, and characterized as a water proof, protective, out layer (Zollner et al., 2013). The hypodermis connects skin to bone and muscle, consists primarily of muscle and adipocytes (Zollner et al., 2013). The
Fig. 8 – Effects of loading history of (a) low-stress creep and stress relaxation; (b) first step stress on creep behavior of rat skin.
dermal layer of skin is acknowledged to be a significant contributor to the mechanical behavior of the tissue (Agache et al., 1980). The fibers of the rat skin are believed to be the major determinant of the skin's biomechanical properties (Groves et al., 2013). Direct observation of the microstructures of fibers at various loading cases could provide anatomical data concerning the structural basis for the static mechanical properties of the skin. Fibers without loading are waved and crimped (Brown, 1973). When stretching the rat skin under the stress of 4 MPa over time, the fibers straighten and realign parallel to one another along the loading direction as shown in Fig. 9(b). A similar behavior was observed by Roy in White New Zealand rabbit arterial wall (Roy et al., 2010). However, ruptures of fibers are also observed. The ruptures may develop into unrecoverable damage, for example cell necrosis (Provenzano et al., 2002), or even failure for the rat skin (Pena, 2014). For the specimens with multi-step loadings, elongation and reorientation of their fibers along loading direction like single-step creep are also observed in Fig. 9(c)–(e). It can be seen that folds and waves of fibers diminish as the loading case changing from C3, MC2, MCR2 to MCR1. This implies that the elongation of fibers becomes greater with the sequence in MCR1. On the other hand, the fiber rupture from Fig. 9(b) to 9(e) also reduces, which denotes the damage
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55
Fig. 9 – Microstructures of collagen fibers in rat skin in following loading case: (a) as-received sample, (b) C3, (c) MC2, (d) MCR2, (e) MCR1. E ¼epidermis, D¼ dermis, H¼hypodermis, LD¼ loading direction. 10.
due to the loading to the rat skin weakens in turn. According to the visual analysis of the microstructures, the specimen of loading sequence of the 2 MPa creep – stress relaxation – 4 MPa creep has the least damage compared with other three specimens at the same loading time.
Because porosity is usually related to the mechanical properties of materials and has been extensively used in bone (Seeman and Delmas, 2006; Zebaze et al., 2010) and polymer (Khatib et al., 2012), the gaps between fiber bundles in rat skin are concerned. Abundant gaps in the single-step creep are observed in Fig. 9(b),
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Fig. 10 – Converted images of gaps between fibers of rat skins in following loading case: (a) C3, (b) MC2, (c) MCR2, (d) MCR1. and most of these gaps are along with the loading direction. However, the gaps diminish in multi-step creep tests, implying that the damage of the rat skin alleviates with a loading history of low stress creep or stress relaxation, as shown in Fig. 9(c)–(e). To quantitatively estimate the damage of the rat skin, void ratio of the microstructure of the dermis layer is utilized. High void ratio indicates more severe damage. Void ratio is measured by image analyze method. This method was detail introduced in (Rnjak-Kovacina et al., 2011). Void ratios are quantified from H&E
stained histologic sections. Voids and tissue show different colors after H&E staining. Voids are colorless and transparent while tissue is red. The images of the sections were converted to grayscale format firstly, and then to binary format. The converted images are shown as Fig. 10. The gaps between fiber bundles of the dermis for C3, MC2, MCR2, and MCR1 are shown as black pixels in Fig. 10. As a result, the void ratio is obtained by dividing the black area by the total section surface area in each image. Their void ratios are calculated and shown in Fig. 11. It
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4.
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Conclusions
The present study gives an insight into the multi-step creep on both mechanical and microcosmic perspective. One important application of information regarding multi-step creep behavior is in assessing the appropriate clinical protocols, i.e., loading sequence, which should be accounted for during the surgery of skin expansion. First, the single-step creep tests were conducted to demonstrate the creep behavior of skin. Then the effects of loading histories of the high stress creep, the low stress creep, and the stress relaxation on multi-step creep tests were investigated. Finally, the microstructure of rat skins was observed by optical microscopy with the help of standard hematoxylin and eosin (H&E). The conclusions can be drawn as follows: Fig. 11 – Void ratios in different loading cases.
can also be seen that the damage of the single-step creep is the greatest with the highest void ratio, i.e., 16.67%. For MC2 and MCR2, the void ratios are 7.48% and 7.34%, respectively. This suggests that loading histories of low stress creep and stress relaxation helps to reduce the damage of rat skins. Moreover, their effects have no significant difference in lowering damage and both lessen half of the damage comparing with the singlestep creep. According to Figs. 5 and 7, both the loading history of the low stress creep and the stress relaxation enable to increase by 4% of the creep strain. Therefore, larger deformation and lower damage can be achieved in the multi-step creep with the loading histories of both the low stress creep and the stress relaxation. Moreover, the effects of both loading histories on the deformation and damage of rat skins are similar. Comparing MCR1 with MCR2, gaps in the former are far less and thinner than in the latter. Fig. 11 shows that the void ratio of the specimen with the loading sequence of 2 MPa creep – stress relaxation – 4 MPa creep is the lowest, which is 3.58%. The damage of MCR1 is less than half of that of MCR2. Hence, the low stress in step 1 helps to lessen the damage of fibers. On the other hand, the strain of MCR1 is also the largest among these loading cases as shown in Fig. 7. It can be concluded that the loading sequence of low stress creep – stress relaxation – high stress creep contributes to obtaining the largest creep strain with the lowest void ratio. It can be concluded that applying low stress creep and stress relaxation in advance could produce more skin in skin expansion operation than direct high stress creep, and less damage would be induced. From the abovementioned discussion on the aspects of mechanical properties and microstructures, the loading sequence of the low stress creep – stress relaxation – high stress creep produces the largest deformation but the lowest damage of the skin. However, there are visible differences between the diameters, strength, and densities of human and murine dermal fibers. In addition, other considerations include the degree of cross-linking within the fiber matrix, the degradation of the fibers and variations in the thickness of the dermal layer (Groves et al., 2013). These are likely to contribute to mechanical differences between human and animal skin. Therefore, further studies on human skin are necessary to determine the stresses in the first and the third steps and the relationship between the loading sequence and the damage of the skin.
(1) The creep strain and its rate of the steady-state stage, and creep-fatigue life are highly dependent on the applied stress. Creep strain and its rate of the steady-state stage increase with the applied stress, whereas the fatiguecreep life decreases. (2) Loading histories of the high-stress creep, the low-stress creep, and the stress relaxation have an important influence on multi-step creep. The low-stress creep after the loading history of the high-stress creep shows the strain recovery and no more creep strain accumulation. The history of stress relaxation also presents the strain recovery as that of high-stress creep. (3) Both the histories of the low-stress creep and the stress relaxation help to obtain larger strain than the single-step creep and decrease the void ratio in the rat skin. (4) The largest deformation and the lowest void ratio can be obtained in the multi-step with the loading sequence of the low stress creep – the stress relaxation – the high stress creep.
Acknowledgments The authors gratefully acknowledge the financial support from the National Natural Science Foundation of China (Nos. 11172202 and 61334010).
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