In vitro Response of the Bone Marrow-Derived Mesenchymal Stem Cells Seeded in a Type-I Collagen-Glycosaminoglycan Scaffold for Skin Wound Repair Under the Mechanical Loading Condition

In order to achieve successful wound repair by regenerative tissue engineering using mesenchymal stem cells (MSCs), it is important to understand the response of stem cells in the scaffold matrix to mechanical stress.
To investigate the clinical effects of mechanical stress on the behavior of cells in scaffolds, bone marrow-derived mesenchymal stem cells (MSCs) were grown on a type-I collagen-glycosaminoglycan (GAG) scaffold matrix for one week under cyclic stretching loading conditions.
The porous collagen-GAG scaffold matrix for skin wound repair was prepared, the harvested canine MSCs were seeded on the scaffold, and cultured under three kinds of cyclic stretching loading conditions ( 0%: control, 5% strain, 15% strain ). After 7 days incubation, MSCs were evaluated histologically and immunohistochemically regarding the proliferation and differentiation.
Cultured MSCs in the high strain (15% strain) group showed activea-smooth muscle actin (α-SMA) expression and poor differentiation into type-I collagen-positive cells, whereas enhanced differentiation into type-I collagen positive cells and a lack ofa-SMA expression where shown in the lower stress (5% strain) group. These results suggest that mechanical stress may affect the proliferation and differentiation of stem cells, and subsequently the wound healing process, through attachment interactions between the stem cells and scaffold matrix. Our findings provide an additional consideration for clinical treatment of wound repair using regenerative tissue engineering.     

Skin viscoelasticity studied in vitro by microprobe-based techniques

Time-dependent deformation of porcine skin was studied in vitro using specialized microprobe instruments. The deformation behavior of stratum corneum, dermis, and whole skin is examined in the context of results of creep strain, elastic stiffness, and viscoelastic constants obtained in terms of the hold time, loading/unloading rate, and maximum indentation depth (load). Skin time-dependent deformation is significantly influenced by dermis viscoelasticity up to a critical indentation depth (load) beyond which it is controlled by the outermost hard epidermis, particularly stratum corneum. Skin viscoelastic behavior under constant load (creep) and constant displacement (stress relaxation) is interpreted in the light of phenomenological observations and experimental trends.     

Unique behavior of dermal cells from regenerative mammal,the African Spiny Mouse,in response to substrate stiffness

The African Spiny Mouse (Acomys spp.) is a unique outbred mammal capable of full, scar-free skin regeneration. In vivo, we have observed rapid reepithelialization and deposition of normal dermis in Acomys after wounding. Acomys skin also has a lower modulus and lower elastic energy storage than normal lab mice, Mus musculus. To see if the different in vivo mechanical microenvironments retained an effect on dermal cells and contributed to regenerative behavior, we examined isolated keratinocytes in response to physical wounding and fibroblasts in response to varying substrate stiffness. Classic mechanobiology paradigms suggest stiffer substrates will promote myofibroblast activation, but we do not see this in Acomys dermal fibroblasts (DFs). Though Mus DFs increase organization of α-smooth muscle actin (αSMA)-positive stress fibers as substrate stiffness increases, Acomys DFs assemble very few αSMA-positive stress fibers upon changes in substrate stiffness. Acomys DFs generate lower traction forces than Mus DFs on pliable surfaces, and Acomys DFs produce and modify matrix proteins differently than Mus in 2D and 3D culture systems. In contrast to Acomys DFs “relaxed” behavior, we found that freshly isolated Acomys keratinocytes retain the ability to close wounds faster than Mus in an in vitro scratch assay. Taken together, these preliminary observations suggest that Acomys dermal cells retain unique biophysical properties in vitro that may reflect their altered in vivo mechanical microenvironment and may promote scar-free wound healing.     

Age-associated reduction of cell spreading induces mitochondrial DNA common deletion by oxidative stress in human skin dermal fibroblasts: implication for human skin connective tissue aging

Background

Reduced cell spreading is a prominent feature of aged dermal fibroblasts in human skin in vivo. Mitochondrial DNA (mtDNA) common deletion has been reported to play a role in the human aging process, however the relationship between age-related reduced cell spreading and mtDNA common deletion has not yet been reported.

Results

To examine mtDNA common deletion in the dermis of aged human skin, the epidermis was removed from full-thickness human skin samples using cryostat. mtDNA common deletion was significantly elevated in the dermis of both naturally aged and photoaged human skin in vivo. To examine the relationship between age-related reduced cell spreading and mtDNA common deletion, we modulated the shape of dermal fibroblasts by disrupting the actin cytoskeleton. Reduced cell spreading was associated with a higher level of mtDNA common deletion and was also accompanied by elevated levels of endogenous reactive oxygen species (ROS). Boosting cellular antioxidant capacity by using antioxidants was found to be protective against mtDNA common deletion associated with reduced cell spreading.

Conclusion

mtDNA common deletion is highly prevalent in the dermis of both naturally aged and photoaged human skin in vivo. mtDNA common deletion in response to reduced cell spreading is mediated, at least in part, by elevated oxidative stress in human dermal fibroblasts. These data extend current understanding of the mitochondrial theory of aging by identifying the connection between mtDNA common deletion and age-related reduction of cell spreading.     

Spatial patterning of cell proliferation and differentiation depends on mechanical stress magnitude

Mechanical stress has been proposed as a major regulator of tissue morphogenesis; however, it remains unclear what is the exact mechanical signal that leads to local tissue pattern formation. We explored this question by using a micropatterned cell aggregate model in which NIH 3T3 fibroblasts were cultured on micropatterned adhesive islands and formed cell aggregates (or “cell islands”) of triangular, square, and circular shapes. We found that the cell islands generated high levels of mechanical stresses at their perimeters compared to their inner regions. Regardless of the shape of cell islands, the mechanical stress patterns corresponded to both cell proliferation and differentiation patterns, meaning that high level of cell proliferation and differentiation occurred at the locations where mechanical stresses were also high. When mechanical stretching was applied to cell islands to elevate overall mechanical stress magnitudes, cell proliferation and differentiation generally increased with the relatively higher mechanical stresses, but neither cell proliferation nor differentiation patterns followed the new mechanical stress pattern. Thus, our findings indicate that a certain range of mechanical stress magnitudes, termed window stress threshold, drives formation of cell proliferation and differentiation patterns and hence possibly functions as a morphogenetic cue for local tissue pattern formation in vivo.     

The Three-Dimensional Human Skin Reconstruct Model: a Tool to Study Normal Skin and Melanoma Progression

Most in vitro studies in experimental skin biology have been done in 2-dimensional (2D) monocultures, while accumulating evidence suggests that cells behave differently when they are grown within a 3D extra-cellular matrix and also interact with other cells (1-5). Mouse models have been broadly utilized to study tissue morphogenesis in vivo. However mouse and human skin have significant differences in cellular architecture and physiology, which makes it difficult to extrapolate mouse studies to humans. Since melanocytes in mouse skin are mostly localized in hair follicles, they have distinct biological properties from those of humans, which locate primarily at the basal layer of the epidermis. The recent development of 3D human skin reconstruct models has enabled the field to investigate cell-matrix and cell-cell interactions between different cell types. The reconstructs consist of a "dermis" with fibroblasts embedded in a collagen I matrix, an "epidermis", which is comprised of stratified, differentiated keratinocytes and a functional basement membrane, which separates epidermis from dermis. Collagen provides scaffolding, nutrient delivery, and potential for cell-to-cell interaction. The 3D skin models incorporating melanocytic cells recapitulate natural features of melanocyte homeostasis and melanoma progression in human skin. As in vivo, melanocytes in reconstructed skin are localized at the basement membrane interspersed with basal layer keratinocytes. Melanoma cells exhibit the same characteristics reflecting the original tumor stage (RGP, VGP and metastatic melanoma cells) in vivo. Recently, dermal stem cells have been identified in the human dermis (6). These multi-potent stem cells can migrate to the epidermis and differentiate to melanocytes.     

The Effects of Mechanical Loading on Tendons - An In Vivo and In Vitro Model Study

Mechanical loading constantly acts on tendons, and a better understanding of its effects on the tendons is essential to gain more insights into tendon patho-physiology. This study aims to investigate tendon mechanobiological responses through the use of mouse treadmill running as an in vivo model and mechanical stretching of tendon cells as an in vitro model. In the in vivo study, mice underwent moderate treadmill running (MTR) and intensive treadmill running (ITR) regimens. Treadmill running elevated the expression of mechanical growth factors (MGF) and enhanced the proliferative potential of tendon stem cells (TSCs) in both patellar and Achilles tendons. In both tendons, MTR upregulated tenocyte-related genes: collagen type I (Coll. I ∼10 fold) and tenomodulin (∼3–4 fold), but did not affect non-tenocyte-related genes: LPL (adipocyte), Sox9 (chondrocyte), Runx2 and Osterix (both osteocyte). However, ITR upregulated both tenocyte (Coll. I ∼7–11 fold; tenomodulin ∼4–5 fold) and non-tenocyte-related genes (∼3–8 fold). In the in vitro study, TSCs and tenocytes were stretched to 4% and 8% using a custom made mechanical loading system. Low mechanical stretching (4%) of TSCs from both patellar and Achilles tendons increased the expression of only the tenocyte-related genes (Coll. I ∼5–6 fold; tenomodulin ∼6–13 fold), but high mechanical stretching (8%) increased the expression of both tenocyte (Coll. I ∼28–50 fold; tenomodulin ∼14–48 fold) and non-tenocyte-related genes (2–5-fold). However, in tenocytes, non-tenocyte related gene expression was not altered by the application of either low or high mechanical stretching. These findings indicate that appropriate mechanical loading could be beneficial to tendons because of their potential to induce anabolic changes in tendon cells. However, while excessive mechanical loading caused anabolic changes in tendons, it also induced differentiation of TSCs into non-tenocytes, which may lead to the development of degenerative tendinopathy frequently seen in clinical settings.     

A Coupled Experiment-finite Element Modeling Methodology for Assessing High Strain Rate Mechanical Response of Soft Biomaterials

This study offers a combined experimental and finite element (FE) simulation approach for examining the mechanical behavior of soft biomaterials (e.g. brain, liver, tendon, fat, etc.) when exposed to high strain rates. This study utilized a Split-Hopkinson Pressure Bar (SHPB) to generate strain rates of 100-1,500 sec^-1. The SHPB employed a striker bar consisting of a viscoelastic material (polycarbonate). A sample of the biomaterial was obtained shortly postmortem and prepared for SHPB testing. The specimen was interposed between the incident and transmitted bars, and the pneumatic components of the SHPB were activated to drive the striker bar toward the incident bar. The resulting impact generated a compressive stress wave (i.e. incident wave) that traveled through the incident bar. When the compressive stress wave reached the end of the incident bar, a portion continued forward through the sample and transmitted bar (i.e. transmitted wave) while another portion reversed through the incident bar as a tensile wave (i.e. reflected wave). These waves were measured using strain gages mounted on the incident and transmitted bars. The true stress-strain behavior of the sample was determined from equations based on wave propagation and dynamic force equilibrium. The experimental stress-strain response was three dimensional in nature because the specimen bulged. As such, the hydrostatic stress (first invariant) was used to generate the stress-strain response. In order to extract the uniaxial (one-dimensional) mechanical response of the tissue, an iterative coupled optimization was performed using experimental results and Finite Element Analysis (FEA), which contained an Internal State Variable (ISV) material model used for the tissue. The ISV material model used in the FE simulations of the experimental setup was iteratively calibrated (i.e. optimized) to the experimental data such that the experiment and FEA strain gage values and first invariant of stresses were in good agreement.     

Moderate Exercise Mitigates the Detrimental Effects of Aging on Tendon Stem Cells

Aging is known to cause tendon degeneration whereas moderate exercise imparts beneficial effects on tendons. Since stem cells play a vital role in maintaining tissue integrity, in this study we aimed to define the effects of aging and moderate exercise on tendon stem/progenitor cells (TSCs) using in vitro and in vivo models. TSCs derived from aging mice (9 and 24 months) proliferated significantly slower than TSCs obtained from young mice (2.5 and 5 months). In addition, expression of the stem cell markers Oct-4, nucleostemin (NS), Sca-1 and SSEA-1 in TSCs decreased in an age-dependent manner. Interestingly, moderate mechanical stretching (4%) of aging TSCs in vitro significantly increased the expression of the stem cell marker, NS, but 8% stretching decreased NS expression. Similarly, 4% mechanical stretching increased the expression of Nanog, another stem cell marker, and the tenocyte-related genes, collagen I and tenomodulin. However, 8% stretching increased expression of the non-tenocyte-related genes, LPL, Sox-9 and Runx-2, while 4% stretching had minimal effects on the expression of these genes. In the in vivo study, moderate treadmill running (MTR) of aging mice (9 months) resulted in the increased proliferation rate of aging TSCs in culture, decreased lipid deposition, proteoglycan accumulation and calcification, and increased the expression of NS in the patellar tendons. These findings indicate that while aging impairs the proliferative ability of TSCs and reduces their stemness, moderate exercise can mitigate the deleterious effects of aging on TSCs and therefore may be responsible for decreased aging-induced tendon degeneration.     

Onset of neonatal locomotor behavior and the mechanical development of Achilles and tail tendons

Tendon tissue engineering approaches are challenged by a limited understanding of the role mechanical loading plays in normal tendon development. We propose that the increased loading that developing postnatal tendons experience with the onset of locomotor behavior impacts tendon formation. The objective of this study was to assess the onset of spontaneous weight-bearing locomotion in postnatal day (P) 1, 5, and 10 rats, and characterize the relationship between locomotion and the mechanical development of weight-bearing and non-weight-bearing tendons. Movement was video recorded and scored to determine non-weight-bearing, partial weight-bearing, and full weight-bearing locomotor behavior at P1, P5, and P10. Achilles tendons, as weight-bearing tendons, and tail tendons, as non-weight-bearing tendons, were mechanically evaluated. We observed a significant increase in locomotor behavior in P10 rats, compared to P1 and P5. We also found corresponding significant differences in the maximum force, stiffness, displacement at maximum force, and cross-sectional area in Achilles tendons, as a function of postnatal age. However, the maximum stress, strain at maximum stress, and elastic modulus remained constant. Tail tendons of P10 rats had significantly higher maximum force, maximum stress, elastic modulus, and stiffness compared to P5. Our results suggest that the onset of locomotor behavior may be providing the mechanical cues regulating postnatal tendon growth, and their mechanical development may proceed differently in weight-bearing and non-weight-bearing tendons. Further analysis of how this loading affects developing tendons in vivo may inform future engineering approaches aiming to apply such mechanical cues to regulate engineered tendon formation in vitro.     

CDH1 is Identified as A Therapeutic Target for Skin Regeneration after Mechanical Loading

Rationale: Mechanical stimuli in the microenvironment are considered key regulators of cell function. Clinically, mechanical force (tissue expander) is widely used to regenerate skin for post-burn or trauma repair, implying that mechanical stretching can promote skin cell regeneration and proliferation. However, the underlying mechanism remains unknown.Methods: Microarray analysis was utilized to detect the hub gene. The expression of Cdh1 as examined in cells and tissues by western blot, q-PCR and immunohistochemistry staining respectively. Biological roles of Cdh1 was revealed by a series of functional in vitro and in vivo studies.Results: Microarray analysis identified Cdh1 as a hub gene related to skin regeneration during rat cutaneous mechanical loading. In vitro studies suggested that both mechanical loading and Cdh1 interference induced keratinocyte dedifferentiation and enhanced stemness, promoting cell proliferation and prevent apoptosis. Furthermore, the forkhead box O1/Krüppel-like factor 4 (FOXO1/KLF4) pathway was activated and contributed to the keratinocyte dedifferentiation. In vivo studies showed that mechanical loading and Cdh1 interference facilitated epidermal dedifferentiation and promoted dermal collagen deposition, and that Cdh1 overexpression could block such influence.Conclusions: In this study, we show that E-cadherin (CDH1), a well-known cell-cell adhesion molecule, plays a crucial role in mechanical stretch-induced skin cell regeneration and proliferation. We have shown for the first time the process by which mechanical stress is transmitted to the epidermis and induces a downstream signaling pathway to induce epidermal cells to differentiate. These findings demonstrate that Cdh1-induced keratinocyte dedifferentiation is a crucial event in mechanical stretch-mediated skin regeneration and that Cdh1 may serve as a potential therapeutic target for promoting skin regeneration.     

Dynamic mechanical properties of body-wall dermis in various mechanical states and their implications for the behavior of sea cucumbers

The dermis of the sea cucumber body wall is a typical catch connective tissue that rapidly changes its mechanical properties in response to various stimuli. Dynamic mechanical properties were measured in stiff, standard, and soft states of the sea cucumber Actinopyga mauritiana. Sinusoidal deformations were applied, either at a constant frequency of 0.1 Hz with varying maximum strain of 2%-20% or at a fixed maximum strain of 1.8% with varying frequency of 0.0005-50 Hz. The dermis showed viscoelasticity with both strain and strain-rate dependence. The dermis in the standard state showed a J-shaped stress-strain curve with a stiffness of 1 MPa and a dissipation ratio of 60%; the curve of the stiff dermis was linear with high stiffness (3 MPa) and a low dissipation ratio (30%). Soft dermis showed a J-shaped curve with low stiffness (0.3 MPa) and a high dissipation ratio (80%). The strain-induced softening was observed in the soft state. Stiff samples had a higher storage modulus and a lower tangent delta than soft ones, implying a larger contribution of the elastic component in the stiff state. A simple molecular model was proposed that accounted for the mechanical behavior of the dermis. The model suggested that stiffening stimulation increased inter-molecular bonds, whereas softening stimulation affected intra-molecular bonds. The adaptive significance of each mechanical state in the behavior of sea cucumbers is discussed.     

Myosin-II-Mediated Directional Migration of Dictyostelium Cells in Response to Cyclic Stretching of Substratum

Living cells are constantly subjected to various mechanical stimulations, such as shear flow, osmotic pressure, and hardness of substratum. They must sense the mechanical aspects of their environment and respond appropriately for proper cell function. Cells adhering to substrata must receive and respond to mechanical stimuli from the substrata to decide their shape and/or migrating direction. In response to cyclic stretching of the elastic substratum, intracellular stress fibers in fibroblasts and endothelial, osteosarcoma, and smooth muscle cells are rearranged perpendicular to the stretching direction, and the shape of those cells becomes extended in this new direction. In the case of migrating Dictyostelium cells, cyclic stretching regulates the direction of migration, and not the shape, of the cell. The cells migrate in a direction perpendicular to that of the stretching. However, the molecular mechanisms that induce the directional migration remain unknown. Here, using a microstretching device, we recorded green fluorescent protein (GFP)-myosin-II dynamics in Dictyostelium cells on an elastic substratum under cyclic stretching. Repeated stretching induced myosin II localization equally on both stretching sides in the cells. Although myosin-II-null cells migrated randomly, myosin-II-null cells expressing a variant of myosin II that cannot hydrolyze ATP migrated perpendicular to the stretching. These results indicate that Dictyostelium cells accumulate myosin II at the portion of the cell where a large strain is received and migrate in a direction other than that of the portion where myosin II accumulated. This polarity generation for migration does not require the contraction of actomyosin.     

A biphasic multilayer computational model of human skin

The present study investigates the layer-specific mechanical behavior of human skin. Motivated by skin’s histology, a biphasic model is proposed which differentiates between epidermis, papillary and reticular dermis, and hypodermis. Inverse analysis of ex vivo tensile and in vivo suction experiments yields mechanical parameters for each layer and predicts a stiff reticular dermis and successively softer papillary dermis, epidermis and hypodermis. Layer-specific analysis of simulations underlines the dominating role of the reticular dermis in tensile loading. Furthermore, it shows that the observed out-of-plane deflection in ex vivo tensile tests is a direct consequence of the layered structure of skin. In in vivo suction experiments, the softer upper layers strongly influence the mechanical response, whose dissipative part is determined by interstitial fluid redistribution within the tissue. Magnetic resonance imaging-based visualization of skin deformation in suction experiments confirms the deformation pattern predicted by the multilayer model, showing a consistent decrease in dermal thickness for large probe opening diameters.

     

RAGE mediates accelerated diabetic vein graft atherosclerosis induced by combined mechanical stress and AGEs via synergistic ERK activation

Aims/Hypothesis

Diabetes with hypertension rapidly accelerates vascular disease, but the underlying mechanism remains unclear. We evaluated the hypothesis that the receptor of advanced glycation end products (RAGE) might mediate combined signals initiated by diabetes-related AGEs and hypertension-induced mechanical stress as a common molecular sensor.

Methods

In vivo surgical vein grafts created by grafting vena cava segments from C57BL/6J mice into the common carotid arteries of streptozotocin (STZ)-treated and untreated isogenic mice for 4 and 8 weeks were analyzed using morphometric and immunohistochemical techniques. In vitro quiescent mouse vascular smooth muscle cells (VSMCs) with either knockdown or overexpression of RAGE were subjected to cyclic stretching with or without AGEs. Extracellular signal-regulated kinase (ERK) phosphorylation and Ki-67 expression were investigated.

Results

Significant increases in neointimal formation, AGE deposition, Ki-67 expression, and RAGE were observed in the vein grafts of STZ-induced diabetic mice. The highest levels of ERK phosphorylation and Ki-67 expression in VSMCs were induced by simultaneous stretch stress and AGE exposure. The synergistic activation of ERKs and Ki-67 in VSMCs was significantly inhibited by siRNA-RAGE treatment and enhanced by over-expression of RAGE.

Conclusion

RAGE may mediate synergistically increased ERK activation and VSMC proliferation induced by mechanical stretching with and without AGEs. It may serve as a common molecular bridge between the two, accelerating vascular remodeling. This study provides potential drug targets and novel therapeutic strategies for the treatment of vascular diseases resulting from diabetes with hypertension.     

Does subcutaneous adipose tissue behave as an (anti-)thixotropic material?

Although subcutaneous adipose tissue undergoes large deformations on a daily basis, there is no adequate mechanical model to describe the transfer of mechanical load from the skin throughout the tissue to deeper layers. In order to develop such a non-linear model, a set of experimental data is required. Accordingly, this study examines the long term behavior of adipose tissue under small strain and its response to various large strain profiles. The results show that the shear modulus dramatically increases to about an order of magnitude after a loading period between 250 and 1250 s, but returns to its initial value within 3 h of recovery from loading. In addition, it was observed that the stress–strain responses for various large strain history sequences are reproducible up to a strain of 0.15. For increasing strains, the stress decreases for subsequent loading cycles and, above 0.3 strain, tissue structure changes such that the stress becomes independent of the applied strain. From the results, it can be concluded that adipose tissue likely behaves as an (anti-) thixotropic material and that a Mooney–Rivlin model might be appropriate to simulate behavior at physiologically relevant high strains. However, before the model is developed more fully, further experimental research is needed to ratify that the material is (anti-)thixotropic.     

Rapid alignment of collagen fibers in the dermis of undermined and not undermined skin stretched with a skin-stretching device

A controlled, quantitative histochemical study was performed in five piglets to establish changes in undermined and not undermined stretched skin. The skin was stretched with a stretching device for 30 minutes to close a large skin defect. On each flank of the piglet, at a standard position, 9 x 9-cm wounds were created under general anesthesia. On one flank, the surrounding skin was undermined cranially and caudally over a 10-centimeter area. Sections of skin biopsies obtained during stretching were stained with picrosirius red and studied with routine light microscopy and polarized light microscopy in combination with image analysis. The length of collagen fibers was analyzed as a parameter of changes in the dermis resulting from skin stretching. This newly developed quantitative method appeared to be valid, specific, and reproducible, allowing for objective determination of changes in the length of the fibers in the plain of the sections. Changes in the orientation of collagen fibers in the dermis as a result of skin stretching were thereby determined. Epidermal thickness did not change significantly under the influence of stretching forces in both undermined and not undermined skin. However, the orientation of the collagen fibers changed significantly as a result of skin stretching. In undermined wounds, parallel alignment and elongation of the fibers in the plane of the sections was already observed after 15 minutes of stretching. The fibers became aligned in the direction of the stretching force, perpendicular to the wound margin. After 30 minutes of stretching, the mean major axes of the collagen fibers were longest in the plane of the sections (p < 0.001). This meant that elongation and parallel alignment of the collagen fibers had occurred. Stretching of not undermined skin for 15 minutes resulted in significantly stronger parallel alignment in the plane of the sections as compared with undermined skin. This was less well defined after 30 minutes of stretching in not undermined skin. It is concluded that skin stretching with a skin-stretching device for 30 minutes results in significant histomorphological changes of collagen fibers in the dermis of both undermined and not undermined skin. The fibers realign rapidly as a result of stretching forces and become aligned in the direction of the stretching force, perpendicular to the wound margin. These dynamic changes in collagen fibers explain the significantly decreased wound closing tension resulting from skin stretching and explain how skin stretches beyond its inherent extensibility.     

Viscoelastic response of human skin to low magnitude physiologically relevant shear

Percutaneous implants are a family of devices that penetrate the skin and all suffer from the same problems of infection because the skin seal around the device is not optimal. Contributing to this problem is the mechanical discontinuity of the skin/device interface leading to stress concentrations and micro-trauma that chronically breaks any seal that forms. In this paper, we have quantified the mechanical behavior of human skin under low-magnitude shear loads over physiological relevant frequencies. Using a stress-controlled rheometer, we have performed isothermal (37 degrees C) frequency response experiments between 0.628 and 75.39rad/s at 0.5% and 0.04% strain on whole skin and dermis-only, respectively. Step-stress experiments of 5 and 10Pa shear loads were also conducted as were strain sweep tests (6.28rad/s). Measurements were made of whole human skin and skin from which the epidermis was removed (dermis-only). At low frequencies (0.628-10rad/s), the moduli are only slightly frequency dependent, with approximate power-law scaling of the moduli, G' approximately G' approximately omega(beta), yielding beta=0.05 for whole skin and beta=0.16 for dermis-only samples. Step-stress experiments revealed three distinct phases. The intermediate phase included elastic "ringing" (damped oscillation) which provided new insights and could be fit to a mathematical model. Both the frequency and step-stress response data suggest that the epidermis provides an elastic rigidity and dermis provides viscoelasticity to the whole skin samples. Hence, whole skin exhibited strain hardening while the dermis-only demonstrated stress softening under step-stress conditions. The data obtained from the low-magnitude shear loads and frequencies that approximate the chronic mechanical environment of a percutaneous implant should aid in the design of a device with an improved skin seal.     

Mechanical Stretch on Human Skin Equivalents Increases the Epidermal Thickness and Develops the Basement Membrane

All previous reports concerning the effect of stretch on cultured skin cells dealt with experiments on epidermal keratinocytes or dermal fibroblasts alone. The aim of the present study was to develop a system that allows application of stretch stimuli to human skin equivalents (HSEs), prepared by coculturing of these two types of cells. In addition, this study aimed to analyze the effect of a stretch on keratinization of the epidermis and on the basement membrane. HSEs were prepared in a gutter-like structure created with a porous silicone sheet in a silicone chamber. After 5-day stimulation with stretching, HSEs were analyzed histologically and immunohistologically. Stretch-stimulated HSEs had a thicker epidermal layer and expressed significantly greater levels of laminin 5 and collagen IV/VII in the basal layer compared with HSEs not subjected to stretch stimulation. Transmission electron microscopy revealed that the structure of the basement membrane was more developed in HSEs subjected to stretching. Our model may be relevant for extrapolating the effect of a stretch on the skin in a state similar to an in vivo system. This experimental system may be useful for analysis of the effects of stretch stimuli on skin properties and wound healing and is also expected to be applicable to an in vitro model of a hypertrophic scar in the future.     

Mechanical effects of load speed on the human colon

The aim of this study was to examine the mechanical behavior of the colon using tensile tests under different loading speeds.Specimens were taken from different locations of the colonic frame from refrigerated cadavers. The specimens were submitted to uniaxial tensile tests after preconditioning using a dynamic load (1 m/s), intermediate load (10 cm/s), and quasi-static load (1 cm/s).A total of 336 specimens taken from 28 colons were tested. The stress-strain analysis for longitudinal specimens indicated a Young’s modulus of 3.17 ± 2.05 MPa under dynamic loading (1 m/s), 1.74 ± 1.15 MPa under intermediate loading (10 cm/s), and 1.76 ± 1.21 MPa under quasi-static loading (1 cm/s) with p < 0.001. For the circumferential specimen, the stress-strain curves indicated a Young’s modulus of 3.15 ± 1.73 MPa under dynamic loading (1 m/s), 2.14 ± 1.3 MPa under intermediate loading (10 cm/s), and 0.63 ± 1.25 MPa under quasi-static loading (1 cm/s) with p < 0.001. The curves reveal two types of behaviors of the colon: fast break behavior at high speed traction (1 m/s) and a lower break behavior for lower speeds (10 cm/s and 1 cm/s). The circumferential orientation required greater levels of stress and strain to obtain lesions than the longitudinal orientation. The presence of taeniae coli changed the mechanical response during low-speed loading.Colonic mechanical behavior varies with loading speeds with two different types of mechanical behavior: more fragile behavior under dynamic load and more elastic behavior for quasi-static load.