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How are collagen fibers in skin aligned with respect to skin surface?

How are collagen fibers in skin aligned with respect to skin surface?


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I read in one paper that collagen fibers in dermis are randomly oriented in direction with respect to the skin surface. I can't locate that paper now. However, I came across this paper that indicates that collagen is always aligned parallel to the skin surface (page 3). Which one is correct?


Collagen underneath the epidermis is not completely randomly oriented, in fact mostly they are directed along the muscle fibres beneath them.

This was first observed by Karl Langer and hence was given the name as langer's lines( of skin tension). This peculiar arrangement of collagen is responsible for maintaining integrity of skin at times of stretching force developed on them by the muscles. Had they perpendicular, then skin would tear easily.

This also has clinical relevance because during surgery if an incision is made along these lines the scar would be minimum.


Source: https://en.m.wikipedia.org/wiki/Langer%27s_lines


Fibrous protein finding may lead to improved bioprinting, tissue engineering

Fibrous proteins such as collagen and fibrinogen form a thin solid layer on the surface of an aqueous solution similar to the "skin" that forms on warm milk, according to a team of Penn State Researchers, who believe this finding could lead to more efficient bioprinting and tissue engineering.

In the human body, fibrous proteins provide structural support for cells and tissues and aid in biomechanics. Collagen makes up 80% of our skin and 10% of our muscles, while fibrinogen helps in blood clotting by forming the hydrogel fibrin.

"Collagen and fibrinogen protein solutions are widely used as precursors of collagen and fibrin hydrogels in tissue engineering applications," said Hemanth Gudapati, graduate student in engineering science and mechanics. "This is because collagen and fibrin, which are used as structural materials for tissue engineering similar to their role in the human body, are nontoxic, biodegradable and mimics the natural microenvironments of cells."

Gudapati and fellow researchers report, in Soft Matter, for the first time that fibrous proteins form a solid layer on the surface of water due to aggregation of proteins at the air/water interface. This solid layer interferes with accurate measurements of the solution's rheology, which is the study of fluid properties such as flow. Previously, it was only demonstrated that the other main type of protein, globular proteins, formed these solid layers at the air/water interface.

Accurate rheology measurements are vital for successful bioprinting. Measurement of viscosity is important for identifying what protein solutions are potentially printable, and for detecting inconsistencies in flow behavior among different batches of fibrous proteins.

"Collagen and fibrinogen are extracted from animals, and their flow behavior changes from batch to batch and with time," Gudapati said.

This in turn leads to a challenge for consistent bioprinting results.

"Accurate measurement of flow behavior helps in reliable or consistent delivery of the protein solutions during bioprinting," Gudapati said. "This helps in fabrication of things such as reliable organ-on-chip devices and disease models."

A potential solution for accurate measurement is to add a surfactant such as polysorbate 80 to prevent the formation of film at the air/water interface.

The research also identifies the concentrations of protein solutions which are potentially printable via inkjet bioprinting, along with identifying bioprinting operating parameters.

Gudapati said there were other findings in their research that will require further investigation. These included the possibility that the aggregated fibrous proteins at the air/water interface may get released from the interface and that these protein aggregates may cause further accumulation of the proteins in the solutions.

"The further bulk aggregation could be one of the reasons for poor alignment of collagen fibers or poor mechanical strength of fibrin outside the body, i.e., in vitro, which are the challenges facing tissue engineering applications at present," Gudapati said.

The work was done in the lab of Ibrahim Ozbolat, Hartz Family Career Development Associate Professor of Engineering Science and Mechanics, in collaboration with Ralph Colby, professor of materials science and engineering and chemical engineering.

"Dr. Colby's work with globular protein solutions influenced our work," Gudapati said. "For example, we realized that the fibrous proteins could be behaving similar to globular proteins at the air/water interface at the beginning of our research."

Along with Colby, Ozbolat and Gudapati, other authors of the Soft Matter paper include Daniele Parisi, graduate student in materials science and engineering.

The Osteology Foundation in Switzerland, the Hartz Family Career Development Professorship in Engineering and the Penn State Department of Engineering Science and Mechanics supported this research.


Producing Collagen Micro-stripes with Aligned Fibers for Cell Migration Assays

The orientation of collagen fibers in native tissues plays an important role in cell signaling and mediates the progression of tumor cells in breast cancer by a contact guidance mechanism. Understanding how migration of epithelial cells is directed by the alignment of collagen fibers requires in vitro assays with standardized orientations of collagen fibers.

Methods

To address this issue, we produced micro-stripes with aligned collagen fibers using an easy-to-use and versatile approach based on the aspiration of a collagen solution within a microchannel. Glass coverslips were functionalized with a (3-aminopropyl)triethoxysilane/glutaraldehyde linkage to covalently anchor micro-stripes of aligned collagen fibers, whereas microchannels were functionalized with a poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) (PEO-PPO-PEO) nonionic triblock polymer to prevent adhesion of the collagen micro-stripes.

Results

Using this strategy, microchannels can be peeled off to expose micro-stripes of aligned collagen fibers without affecting their mechanical integrity. We used time-lapse confocal reflection microscopy to characterize the polymerization kinetics of collagen networks for different concentrations and the orientation of collagen fibers as a function of the microchannel width. Our results indicate a non-linear concentration dependence of the area of fluorescence, suggesting that the architecture of collagen networks is sensitive to small changes in concentration. We show the possibility to influence the collagen fibril coverage by adjusting the concentration of the collagen solution.

Conclusion

We applied this novel approach to study the migration of epithelial cells, demonstrating that collagen micro-stripes with aligned fibers represent a valuable in-vitro assay for studying cell contact guidance mechanisms.


INTRODUCTION

Psoriasis is one of the most common immune-mediated chronic inflammatory skin disorders which affects approximately 1%–3% of the world's population. Active interactions between the immune system and the skin usually occur with systemic manifestation, especially arthritis (Weigle and McBane, 2013 ). Characteristic features of psoriasis include hyperproliferative keratinocytes, dilated blood vessels in the dermis and massive infiltration of leukocytes. Psoriasis causes cells to build up rapidly on the surface of the skin, forming itchy, dry patches, and thick silvery scales. The most common type of psoriasis, psoriasis vulgaris, accounts for 90% of the cases (Griffiths and Barker, 2007 ). Management of psoriasis involves either topical treatment with immunosuppressants, steroids and several other agents, or systemic treatment such as methotrexate administration, phototherapy, oral retinoids, and biological therapies, with topical treatment remaining the most widely used form of treatment. However, patients treated for psoriasis often present with relapse, adverse drug effects, and other reactions such as development of nonmelanoma skin cancer (Pouplard et al., 2013 ). Recommended treatment options for psoriasis in traditional medicine are change in lifestyle, preventive measures, as well as herbal therapy (Atyabi et al., 2016 ). Thus, a novel, more effective therapeutic strategy devoid of side effects is still desirable.

Dysregulation of the immune system is a major hallmark in the development of psoriasis. Regulatory T cells (Tregs) are considered to be inhibitors of autoimmune responses, but their role in the pathogenesis of psoriasis remains unclear. Psoriasis is considered a T-helper 1 (Th1) disease as evidenced by increased levels of cytokines belonging to the Th1 pathway (interferon gamma, interleukin (IL) 2 and 12) in psoriatic plaques (Griffiths and Barker, 2007 Traub and Marshall, 2007 ). Tregs suppress immune effectors including Th17 cells and maintain immune homeostasis (Sugiyama et al., 2005 Bovenschen et al., 2011 ). Therefore, to restore the dysregulated immune status in psoriasis, it is necessary to enhance Tregs and/or suppress immune effectors including Th17 cells.

However, other researchers have suggested involvement (Yu et al., 2007 ) or even a major role for keratinocytes (Kharaeva et al., 2009 ) in the development of psoriasis. Another feature of psoriatic skin is acanthosis, due to reduced epidermal apoptosis (Boehm, 2006 ). However, stimulation of apoptosis is associated with retrogression of psoriatic hyperplasia (Heenen and Simonart, 2008 ). Control of keratinocyte proliferation may constitute a valuable strategy in the management of psoriasis since reestablishment of the homeostatic regulation of keratinocyte proliferation and differentiation is fundamental for restoration of normal epidermis (Tse et al., 2006 ).

Black seed oil or nigella sativa oil is the oil extracted from black seeds (Nigella sativa), which are tiny, black colored seeds commonly called “black cumin.” Multiple in vivo and in vitro studies performed on human and laboratory animals have shown that N. sativa and its ingredients have a wide range of pharmacological actions including antinociceptive (Abdel-Fattah et al., 2000 ), anti-inflammatory, antihypertensive, antiasthmatic, hypoglycemic, antiparasitic, antimicrobial, antioxidant, and anticancer effects (Padhye et al., 2008 Randhawa and Alghamdi, 2011 Abel-Salam, 2012 ). In the study of El-Dakhakhny et al. ( 2002 ), it was stated that the anti-inflammatory effects of N. sativa oil and its active principle, thymoquinone, may be explained by their action as inhibitors of 5-Lipooxygenase products and the production of 5-hydroxyeicosatetraenoic acid in a concentration-dependent manner, which may be due to its antioxidative action. Moreover, the oil and its active ingredients showed beneficial properties in immunomodulation in that they augmented the immune responses of T cell- and natural killer cells (Salem, 2005 ).

Hence, the aim of this study was to investigate the effect of black seed oil on an Imiquimod (IMQ)-induced psoriasis-like rat model by light/electron microscopy as well as immunohistochemistry to assess the role of black seed oil in treating psoriasis.


Experimental and Modeling Study of Collagen Scaffolds with the Effects of Crosslinking and Fiber Alignment

Collagen type I scaffolds are commonly used due to its abundance, biocompatibility, and ubiquity. Most applications require the scaffolds to operate under mechanical stresses. Therefore understanding and being able to control the structural-functional integrity of collagen scaffolds becomes crucial. Using a combined experimental and modeling approach, we studied the structure and function of Type I collagen gel with the effects of spatial fiber alignment and crosslinking. Aligned collagen scaffolds were created through the flow of magnetic particles enmeshed in collagen fibrils to mimic the anisotropy seen in native tissue. Inter- and intra- molecular crosslinking was modified chemically with Genipin to further improve the stiffness of collagen scaffolds. The anisotropic mechanical properties of collagen scaffolds were characterized using a planar biaxial tensile tester and parallel plate rheometer. The tangent stiffness from biaxial tensile test is two to three orders of magnitude higher than the storage moduli from rheological measurements. The biphasic nature of collagen gel was discussed and used to explain the mechanical behavior of collagen scaffolds under different types of mechanical tests. An anisotropic hyperelastic constitutive model was used to capture the characteristics of the stress-strain behavior exhibited by collagen scaffolds.

1. Introduction

Collagen, one of the major extracellular (ECM) components, is critical to the mechanical properties of many types of biological tissues including tendons, ligaments, bones, blood vessels, and skin. Collagen scaffolds have been widely used in tissue engineering, drug delivery, wound healing, and neuroregeneration guide substrate [1–3] for its biocompatibility, low toxicity, and well-documented structural, physical, chemical, and immunological properties [4]. Most of these applications require the scaffolds to operate under mechanical stresses, and thus being able to control and tailor the structural-functional integrity becomes crucial. Type I collagen gel prepared from commercially available solutions has been used broadly in biomaterials research. However, they have extremely poor biomechanical properties compared to the native tissues that they are targeted to mimic or replace.

Collagen fibrils are strengthened by covalent crosslinks within and between the constituent collagen molecules. Aggregation of collagen fibrils forms a collagen fiber, which is the most abundant protein in the body. Collagen can self-assemble through an enzymatic formation of intermolecular crosslinks leading to a head to tail network within the fiber. The mechanical properties of collagen fibers primarily depend on the formation of intermolecular crosslinks within the fibers to prevent slippage under load [5]. However in the engineered collagen scaffolds, the density of this type of crosslinking is not large enough for practical applications. In addition to self-assembly, the overall mechanical strength of collagen fibers can be improved by increasing the density of inter- and intramolecular crosslink with various chemical reagents.

Glutaraldehyde (GA) is one of the most common chemical crosslinking reagents for collagen [6–11]. Collagen gels crosslinked with GA have already been studied for ocular surfaces [12], corneal tissue engineering scaffolds [4], and nanoscale collagen fibril scaffolds [8, 13]. GA helps to retain many of the viscoelastic properties of collagen fibrillar network, and it reacts relatively quickly. Addition of GA will induce covalent bonds between collagen fibrils from aldehyde-amino reactions as well as from aldol condensation [14]. This results in a more tightly crosslinked network. GA can also lead to intramolecular crosslinks formed between two α-chains by aldol condensation. Although widely used as a crosslinking reagent for collagen-based biomaterials, the cytotoxicity problem associated with GA is a recognized draw back and prevents its application from in vivo studies.

Recently genipin (GP), a compound extracted from the fruit of the Gardenia Jasminoides, has been shown to effectively crosslink cellular and acellular biological tissues as well as many biomaterials including hydrogels and hydrogel composites [7]. It was also found that GP is significantly less cytotoxic than GA [15, 16]. Such features make GP an alternative crosslinking agent for biomaterials with improved mechanical properties. Similar to GA, GP can also form intramolecular as well as intermolecular crosslinks in collagen [7]. GP spontaneously reacts with the primary amines, lysine and arginine residues, in collagen to form monomers that further crosslink the collagen [17]. However, the crosslinking procedure is a complex process, and little is known about the mechanical properties of collagen treated with GP.

Preferred collagen orientation along the dominant physiological loading direction has been observed in many previous studies [18, 19]. Mechanical anisotropy in native tissue is highly associated with fiber orientation. Assembly of collagen molecules in vitro remains a major challenge for fabricating the next generation of engineered tissues. There are several ways to achieve anisotropic-aligned collagen fibrils during assembly. Molecules can be aligned by flow, microfluidic channels [20] and the application of external anisotropic mechanical forces [21–23], electric currents [24], and magnetic fields. Constant magnetic fields are able to align collagen molecules because the collagen molecules have diamagnetic anisotropy. Barocas et al. [25] demonstrated the circumferential alignment of the collagen in a tubular mold. However, the small diamagnetism of collagen molecules requires Tesla-order strengths magnet [26]. Recently, Guo and Kaufman [27] utilized the flow of magnetic beads enmeshed in collagen fibrils to align collagen. The streptavidin-coated ferromagnetic beads (about 1.5 μm in diameter) were shown to facilitate collagen alignment under magnetic field as low as

. Collagen was aligned as the beads move towards the magnetic poles. The timescales of beads travel and gelation need to be comparable for the alignment to occur properly.

As a network with hierarchical structures, the relationship between the mechanical properties of collagen and its structures is obviously causal. The present study was designed to characterize the biaxial tensile and rheological mechanical properties of collagen scaffolds. Different concentrations of GP were used to modify the degree of crosslinking in the collagen scaffolds. The method of using the flow of magnetic beads enmeshed in collagen fibrils [27] was adapted to achieve the alignment of fibers in the scaffolds. The coupled effects of fiber alignment and crosslinking across hierarchies on the mechanical properties of collagen scaffolds were studied.

2. Materials and Methods

2.1. Sample Preparation

Genipin Crosslinked Collagen Scaffolds
Nutragen type I collagen solution (6 mg/mL) was purchased from Advanced BioMatirx. Collagen was dissolved in 0.01 N HCl with a pH value of approximately 2.0. Neutralized collagen solution was prepared by quickly mixing Nutragen collagen solution, 10x PBS (Fisher Scientific), and 0.1 M NaOH (Fisher Scientific) solution with a ratio of 8 : 1 : 1 at 4°C with a final collagen concentration of 4.8 mg/mL. The pH value of the solution was adjusted to be between 7.2

7.4. The neutralized solution was transferred into a custom-made square reservoir with a dimension of about 30 mm

30 mm that sits in a Petri dish. On each side of the reservoir, a 15 mm 1 mm notch was cut to fit the loading bars. Four porous polyethylene bars (18 mm 3 mm 1.5 mm) (Fisher Scientific) prethreaded with nylon sutures were placed by the sides of the reservoir. The dimension between polyethylene bars was about 15 mm 15 mm, and the thickness of the gel was about 1 mm. The solution was firstly kept in an incubator at 37°C for 12 hours for gelation [4, 6, 11]. During gelation, the polyethylene bars were polymerized into the collagen gels [28] (Figure 1). The collagen gels were then immersed in 0.03%, 0.1%, and 0.25% GP solutions for another 6 hours in the incubator for crosslinking [16].


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(b) Collagen scaffolds crosslinked with (a) 0.03% and (b) 0.1% GP for 6 hours. Four prethreaded polyethylene bars were polymerized into the collagen scaffolds sample for biaxial tensile testing.

Magnetically Aligned Collagen Gel
The aligned collagen scaffolds were obtained with the aid of a flow of magnetic particles embedded in collagen fibrils [27]. Briefly, neutralized collagen solutions were prepared as above. Streptavidin-coated iron oxide magnetic particles (Bangs Labs) of 1.5 μm in diameter were added into the neutralized collagen solution at a concentration of 0.1 mg/mL. The solution with beads was then transferred into the reservoir and incubated for 12 hours at 37°C, during which a magnetic bar was placed under the petri dish. The direction of the magnetic field was marked on the petri dish. After gelation, samples were immersed in 0.03%, 0.1%, and 0.25% GP solutions for further crosslinking.

2.2. Scanning Electron Microscopy (SEM)

The morphology of the collagen scaffolds was examined using a JOEL JSM-6100 SEM operated at 10 kV. To prepare the sample for SEM, crosslinked collagen gels were fixed with 4% paraformaldehyde in PBS for 1 hour at room temperature. The fixed samples were dehydrated in a graded distilled water/ethanol series: 30%, 50%, 70%, and 100% for 15 minutes each, followed by washes with a graded ethanol/HMDS series: 30%, 50%, 70%, and 100% for 15 minutes each, and finally allowed to dry overnight [29–31]. This drying process has been employed to avoid sample shrinkage. The dried hydrogels were sputter-coated with Pd/Au prior to SEM. SEM images were taken at multiple locations across the sample and used to qualitatively assess the alignment of collagen fibers.

2.3. Planar Biaxial Tensile Test

The tensile mechanical properties of collagen scaffolds were characterized using a planar biaxial tensile tester. In biaxial tensile testing, a roughly square-shaped specimen was mounted so that it could be stretched along both the

in-plane directions. Four carbon dots markers forming a 5 mm × 5 mm square were placed in the center of the testing specimen, and a CCD camera was used to track the position of markers from which the tissue strains in both directions can be determined throughout the deformation. Tensile tension was applied to the specimen and the load was measured using load cells during the loading and unloading processes. The square samples were loaded biaxially via sutures prethreaded to the polyethylene bars. A preload of 2 g was used to straighten the sutures. Samples were preconditioned equibiaxially for 8 cycles with a load of 10 g to achieve a repeatable material response. A half cycle time of 10 seconds was used. The preloaded state was used as the reference state for later strain calculation. The samples were then tested under load control method and subjected to a set of equibiaxial loads with the maximum loads varying from 70 g to 100 g. Cauchy stress and logarithm strain were calculated [32] and used for the description of the biaxial tensile mechanical behavior of collagen gel.

2.4. Rheometry Study

The mechanical properties of collagen gel were also assessed using a parallel plate rheometer (AR2000, TA Instrument). Neutralized collagen solution was prepared as stated above with a final collagen concentration of 4.8 mg/mL. The solution was transferred to P60 Petri dishes and incubated at 37°C for 12 hours. The collagen gels were then crosslinked with 0.03%, 0.1%, and 0.25% GP for 6 hours. Before rheometry tests, the gel samples of 60 mm in diameter and 2 mm in thickness were carefully removed from the Petri dish and transferred to the bottom plate of the rheometer. The temperature of the plate was set to 37°C. The top plate was lowered to a height of 0.9 mm. Frequency and strain sweep tests were performed. For frequency sweep test, the dynamic storage and loss moduli were evaluated at 1% shear strain amplitude at frequencies ranging from 0.1 to 10 Hz. For strain sweep test, the moduli were evaluated at shear strain ranging from 0 to about 10% at 5 Hz.

2.5. Constitutive Modeling

In order to capture the characteristics of the stress-strain behavior exhibited by collagen gels, an anisotropic hyperelastic constitutive model was used [33]. The strain energy function was originally introduced by Holzapfel et al. [34] for the description of the passive mechanical response of arterial tissue, in which each layer is treated as a fiber-reinforced material with the fibers corresponding to the collagenous component distributed helically around the arterial wall. The model was later generalized to include collagen fiber dispersion [33]. The structurally based constitutive model has material parameters possessing physical meanings that can be related to the structure and components of the material being studied. The constitutive model was chosen in this study as it has been successfully used in previous studies for collagenous cardiovascular tissue and elastin-degraded arteries [35–37]. The general form of the strain energy function is

. The response of the model is governed by two parts, the isotropic and anisotropic components. The first term in (1) captures the isotropic behavior of the matrix material.

is the first invariant of tensor

is the modified counterparts of right Cauchy-Green tensor [34]. is a stress-like material parameter associated with the isotropic part of the overall response of the tissue. The anisotropy of the tissue is captured by the second term in (1). An assumption in this model is that the collagen fibers are only active in extension and not in compression. The

stands for the Macauley bracket and imposes the condition that

. is another stress-like parameter, and is a dimensionless constant related to the collagen fibers. is the number of families of fibers, and κ is a measure of the dispersion of the fiber orientation around a mean direction, as shown in Figure 2(a). The mean direction of the fibers, γ, is defined as the angle between the circumference direction of the tissue and the mean direction of the fibers. is the orientation density function and gives the normalized number of fibers within a certain orientation with respect to the mean direction.

are squares of the stretches in the direction of the α family of fibers. Finally, is the inverse of the bulk modulus and is set to zero as the material is assumed to be incompressible.

is the elastic volume ratio. Note that for incompressible material. Interested readers are referred to Gasser et al. [33] for more detailed explanations of the model.


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Due to the symmetric loading conditions in biaxial tensile test, a quarter of the collagen sample was modeled in ABAQUS 6.8-4 with loading and boundary conditions shown in Figure 2(b). Shell edge loading

-symmetry boundary conditions were applied in order to simulate biaxial tensile testing experimental settings. General-purpose shell elements (S4R) with inherent plane stress assumption were used in the finite element model. Material parameters were adjusted to fit the simulation results to experimental stress-strain curves.

3. Results

The color appearance of collagen gels changes when crosslinked with crosslinking reagents. Thermally crosslinked collagen gels at 37°C without any crosslinking reagents have a whitish color and are semitransparent. These scaffolds are extremely fragile and cannot be tested mechanically. Collagen gels cross-linked with GP turn into bluish color and become opaque, as shown in Figure 1. The higher concentration of GP increases the intensity of blue.

Figure 3 shows the representative stress-strain responses of collagen scaffolds crosslinked with different concentration of GP. Samples were under equibiaxial tensile test with a maximum load of 70–100 g. The biaxial tensile test revealed that the stiffness of collagen gel increases with higher concentrations of GP. It is also noted that all collagen scaffolds exhibit isotropic mechanical behavior as seen from the equibiaxial testing results. To further compare the stiffness of crosslinked collagen gels, the tangent modulus

was obtained for each sample by differentiating the stress-strain curves and was estimated from

[38]. Averaged tangent moduli were then obtained for collagen scaffolds with 0.03%, 0.1%, and 0.25% GP. Figure 4 shows that the initial tangent moduli of collagen scaffold increase with the concentration of GP. The tangent moduli also increase with strain. For strain less than 2%, the tangent moduli of the 0.03% and 0.1% GP crosslinked collagen scaffolds increase faster than the 0.25% GP crosslinked one. However, the tangent modulus of the 0.25% GP crosslinked collagen gel remained the highest. When strain is higher than 2%, the tangent moduli of the 0.1% GP crosslinked scaffolds are about the same as the 0.25% GP crosslinked scaffolds. As the strain further increases, the tangent moduli increase at about the same rate for all the samples.


Representative stress-strain responses of collagen scaffolds crosslinked with 0.03%, 0.1%, and 0.25% GP subjected to equibiaxial tensile test. The maximum load is 70 g, 100 g, and 100 g for 0.03%, 0.1%, and 0.25% GP crosslinked samples, respectively.


Rheological testing with a parallel plate rheometer demonstrate that the storage

and loss moduli of GP-crosslinked collagen scaffolds both increase with GP concentration, as shown in Figures 5(a) and 5(b). The increases slightly with frequency, while the decreases with frequency initially followed by a slight increase. The of collagen gels is about 10 times greater than the which suggests that collagen gel is a predominantly elastic material with small viscosity. The variation of storage and loss modulus with shear strain amplitudes are presented in Figures 5(c) and 5(d). There is a slight decrease in the storage moduli and an increase in the loss moduli with increasing strain amplitude. Overall, the storage and shear moduli do not vary significantly within the shear strain range applied in this study.


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(d) (a, b) Dynamic storage and loss moduli from frequency sweep rheological tests (c, d) from strain sweep rheological tests of collagen scaffolds crosslinked with 0.03%, 0.1%, and 0.25% GP.

SEM was performed to examine the structure in the aligned collagen scaffolds. The nonaligned collagen gel was also examined for comparison. As shown in Figure 6(a), the fibers in the nonaligned collagen gel distribute randomly and there is no preferred fiber distribution. On the other hand, the fibers in the aligned collagen gel show an overall fiber alignment in a particular direction, as shown in Figure 6(b). The mechanical behavior of the aligned collagen scaffolds were tested using a biaxial tensile tester and compared with the results from the nonaligned ones. The samples were subjected to equibiaxial tensile test with one loading axis parallel to the fiber alignment direction while the other loading direction perpendicular to the fiber alignment direction. The nonaligned collagen scaffolds with magnetic beads imbedded were also tested to validate that the presence of beads would not affect the mechanical property of collagen scaffolds (results now shown). The stress-strain responses of the aligned collagen scaffolds are shown in Figure 7. Compared with the nonaligned scaffolds, the aligned ones demonstrate obvious anisotropic mechanical behavior as manifested by one direction being stiffer than the other. As expected, the scaffolds are stiffer in the direction parallel to fiber alignment than in the direction perpendicular to the fiber alignment. The nonaligned collagen scaffolds appear to be isotropic with the stress-strain curves falling in between those from the aligned scaffolds.


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(b) Scanning electron microscopy (SEM) images of (a) nonaligned and (b) aligned 0.03% GP crosslinked collagen scaffolds. All the scale bars represent 10 μm.


Representative stress-strain response of the aligned and non-aligned collagen scaffolds crosslinked with 0.03% GP. Equi-biaxial tensile test reveals obvious anisotropic mechanical behavior in the aligned collagen scaffold.

Figure 8 shows the simulation results. Experimental results are also plotted for comparison. For nonaligned collagen scaffolds, γ was set to 45° and κ was set to 0.333 to represent an isotropic material. The , , and values were then chosen in order to fit the model to experimental data. For the aligned scaffolds, was kept the same as the corresponding nonaligned one and appropriate choices of , , γ, and κ were made to fit the experimental data. The material parameters for the models are summarized in Table 1.


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(c) Simulation results of Cauchy stress versus logarithmic strain for isotropic and anisotropic collagen gels crosslinked with (a) 0.03% (b) 0.1% (c) 0.25% GP. Isotropic fits are shown with the dotted lines. Anisotropic fits are shown with solid lines. Experimental results are shown with the symbols.

4. Discussion

4.1. Effect of Crosslinking

The change of color in collagen scaffolds crosslinked with chemical reagents indicates microstructure changes during crosslinking. For GP crosslinked collagen, blue pigment is produced by the reaction between collagen and GP [39]. It was proposed that the reaction between GP and an amino acid in collagen molecule will form a monomer and further radical reaction will cause dimerization. The mixtures of polymers formed from these reactions are the cause of blue pigment [40]. Previous studies suggested that GP may form intramolecular and intermolecular crosslinks within collagen fibers in biological tissue [7].

Various methods have been developed to improve the mechanical properties of collagen scaffolds including physical crosslinking through exposure to a radiation sources such as UV light [41, 42] and chemical crosslinking by chemical reactions with various crosslinking reagents [6, 43]. Glutaraldehyde is commonly used for collagen-based biomaterials. It has been shown that the stiffness of collagen gel increases with higher GA concentration [11]. However, this trend stops when the GA concentration is higher than a threshold value. Previous study using dermal sheep collagen [6] showed that collagen network treated with 0.08% and 0.5% GA had a similar modulus. Sheu et al. [11] suggested that a complete crosslinking in collagen gel is reached when the GA concentration is 0.12%. After fully crosslinking, no more amino groups will be available to react with the aldehydes from GA. Lacking of amino groups in the collagen gel will lead to self-polymerization of GA, which may adversely affect the mechanical property of the entire collagen gel [9, 14]. Although GA crosslinking greatly improved the mechanical strength of collagen gel, the potential toxic effect has been a vital drawback of this commonly used chemical reagent for biological tissues. A few studies have demonstrated that GP has the potential to be used as a substitute crosslinking reagent [7, 15, 16, 44]. Among these studies, GP has been found to be significantly less cytotoxic than GA [15, 44]. From the mechanical perspective, previous studies found that GP can be used to stiffen collagen gels in a relatively short-time frame. It is also noted that the mechanical properties of collagen gel can be controlled by varying the concentration of GP. Sundararaghavan et al. [16] found that the degree of crosslinking and the storage modulus of GP-crosslinked collagen gel increased with higher GP concentration. Our results from both biaxial tensile testing (Figures 3 and 4) and rheometry measurements (Figure 5) show that the tangent modulus, the storage, and loss modulus of collagen scaffolds increase with GP concentration. Although genipin can be used to tune the overall stiffness of collagen scaffolds, higher concentrations of genipin can lead to significant cell death [16], which needs to be considered in applications of cell-populated collagen scaffolds.

4.2. Effect of Fiber Alignment

Mechanical properties of collagen scaffolds are not only affected by the degree of crosslinking, but also highly correlated with the orientation of collagen fibers in the scaffolds. Preferred fiber orientation along the dominant physiological loading direction has been observed in many previous studies on various tissues [45–47]. Collagen fiber orientation plays important roles in determining the mechanical functionalities of collagen-based native and engineered biological material. In the present study, the alignment of collagen fibers was achieved via the flow of streptavidin-coated magnetic beads bond with collagen fibrils proposed previously by Guo and Kaufman [27], to which interested readers are referred for greater detail. Briefly, the streptavidin coating contains an Arg-Tyr-Asp (RYD) amino acid sequence similar to the RGD receptor domain of fibronectin, which has a function of binding with collagen fibrils. Our results demonstrated that, with this simple technique, the resulted scaffolds possess obvious anisotropic mechanical properties as manifested by the stress-strain responses from equibiaxial tensile test (Figure 7). Such kind of preliminary results are usefully for future quantitative investigation of the structure-function relation of collagen scaffolds. The anisotropic scaffolds are stiffer in the direction that the fibers are aligned than in the direction perpendicular to the alignment.

Our results also demonstrated that crosslinking of the scaffolds can be used to tune the overall stiffness of the scaffolds without affecting the existence of anisotropy in the collagen gel. Previous study by Sung et al. [7] found that fixation of the aortic valves using GP and GA did not alter the mechanical anisotropy observed in fresh valve leaflets. They concluded that the intramolecular and intermolecular crosslinks introduced into the collagen fibrils during fixation are of secondary importance to the presence of structural and mechanical anisotropy in fresh leaflets. Paik et al. [48] studied the effect of nitrite on the mechanical properties of uniaxially and biaxially constrained collagen gels. They quantitatively demonstrated that the crosslinking agent did not alter the fiber distribution in collagen gels significantly. Their study also suggested that the stiffening of collagen fibers might be another source of the anisotropic mechanical behavior seen in the uniaxially constrained collagen gel, in addition to the reorientation of fibers in the constraint direction. In the present study, the reorientation of collagen fibers induced by magnetic flow plays a dominant role in controlling the anisotropic mechanical behavior of collagen scaffolds.

4.3. Comparison between Biaxial Tensile and Rheological Testing

Planar biaxial tensile testing was used in this study to characterize the tensile mechanical properties of collagen scaffolds. Planar biaxial tensile test with independent control of load in both perpendicular loading directions has been used broadly to study the mechanical behavior of various native and engineered biological tissues [28, 32, 49, 50]. Although it cannot replicate the physiological loading conditions, biaxial tensile test is sufficient on elucidating the anisotropic mechanical properties of biological tissues with plane stress assumptions. In the present study, equibiaxial tensile testing was performed to study the effects of crosslinking and fiber-alignment on the mechanical properties of collagen scaffolds. Rheological testing has been applied to study the mechanical properties of polymeric and biological materials [29, 51, 52]. Our results show that higher GP concentration will generate a collagen gel with higher dynamic storage and loss moduli, which is consistent with previous study by Sundararaghavan et al. [16]. Although biaxial tensile testing results show a similar trend, the tangent stiffness from biaxial tensile test is two to three orders of magnitude higher than the storage moduli from rheological measurements, which is much greater than 3 : 1 for ideal elastic networks. Higher ratio of tangent modulus to shear modulus has been observed in previous studies on biomaterials and hydrogel system. Using a three-point bending test, Spatz et al. [53] reported that the ratio of Young’s modulus to shear modulus of cortical bone is in the order of 20 : 1. They suggested that materials comprising stiff fillers embedded in a compliant matrix could have a rather low shear modulus. Such kind of properties allow hollow bones to react smoothly to local impacts, which otherwise may lead to failure. Study by Richter [54] on poly(vinyl methylether) hydrogels showed that the ratio of compressive to shear moduli varied from 4.8 to 10.9 [54], which was contributed to the inhomogeneity of the sample.

It is known that collagen gel is a biphasic system consisting of a fibrillar network structure filled with a large excess of interstitial fluid. Tensile tests on collagen gels probe the tensile behavior of the fibrillar network, and the interstitial flow resistance is negligible in extension tests [55]. Results from our biaxial tensile testing on collagen scaffolds give a nonlinear stress-strain response with a compliant “toe” region, followed by a stiff region. The initial compliant “toe” region is due to the network orientation changes toward the loading axis, while the stiff region corresponds to resistance from fibril extension [56]. In rheological test, the fluids or soft solids flow rather than deform elastically [57]. The top plate of the rheometer compresses the collagen gel, oscillates, and exerts a dynamic torsional force on the sample. During the unconfined compression fluid, escape may not be trivial anymore. Another reason which may contribute to the low-measured shear modulus is that, during rheological test, fluid running out of the collagen gel can act as a lubricant between the gel surface and top plate. Thus the gel might in fact experience smaller shear than that applied by the top plate. This lubricant effect may dominate the increase in storage and loss moduli associated with dehydration. Results from our study suggest that, in rheological test, the movement of the interstitial fluid play a significant role in determining the overall mechanical properties of a collagen gel. It is important to understand the different mechanical properties that a biphasic collagen gel can exhibit under specific loading conditions, as such understandings are necessary for mechanobiological studies in which ECM mechanics play critical roles.

4.4. Determination of Material Parameters in the Constitutive Model

The anisotropic constitutive model in (1) was proposed originally for simulating arterial tissue in which elastin is the primary means of support at low stress-loading regions and then collagen being the major load-bearing component at higher stresses [33, 34]. In an earlier study by Holzapfel et al. [58], individual layers of arteries were separately modeled with their own sets of parameters. Their study using the constitutive model by Holzapfel et al. [34] was successful in modeling the intimal and adventitial layers that lack two distinct structural components as the medial layer. Regarding the parameters in the model with respect to our collagen gels, there was no elastin or other ECM ground substance material that support the initial loading in this experiment. The parameter is likely more representative of some other phenomenon such as uncoiling of collagen fibers. Parameters γ and κ can vary the amount of anisotropy in the stress-strain curves, and κ has a greater effect on the amount of curvature in the stress-strain curve [33, 59]. Finally, the most significant effect of increasing/decreasing and is in translating the stress-strain curves to lower/higher strains, respectively. This inverse relationship in the material is logical as they describe the properties of the collagen fibers [58].

Examining the parameters for the aligned and nonaligned collagen gels, we initially attempted to take the parameters from the nonaligned simulations and only change γ and κ to create a best fit of the aligned collagen gel, since γ controls the orientation of the fibers, and κ determines the fiber dispersion (amount of fiber alignment). Fitting of γ and κ has generally been done phenomenologically instead of based on histology studies [33]. However, it is interesting to point out that our results indicate a correlation between the degree of anisotropy demonstrated by stress-strain curves and γ. Among the three aligned collagen scaffolds shown in Figure 8, the collagen scaffolds aligned with 0.03% GP possessing the highest amount of anisotropy has the smallest model parameter γ correspondingly. Unfortunately, a good fit of the aligned collagen gel was not possible without modification of the and as well. This seems to indicate that the alignment process is affecting the material properties of the collagen fibers and not just their orientation/dispersion.

In the nonaligned and aligned collagen scaffolds, values were increased with higher GP concentrations, which suggest higher values for stiffer tissues resulted from increased crosslinking. There was also an increase in the values of and with GP concentration. Previous studies have shown that the method of crosslinking can affect how the crosslinks are organized in the collagen fibers [60]. Genipin has been shown to not only cause collagen to become more crosslinked but also cause the fibers to merge together and form thicker structures [61]. Increase in and indicates that stronger fibers present in collagen gels with higher GP concentration. Takiuchi et al. [62] showed that crosslinking along fibers may happen more often than across fibers. They showed that during the process of fixing tendon samples in formalin, the stiffness along the fibers increased linearly with fixing time. However, this is not the case in the direction across the fibers. This was explained by a difference in the number and formation rate of crosslinks in the direction along the fibers and across the fibers.

4.5. Limitations

We would like to point out several limitations of the current study. The method for collagen fiber alignment is ideal for thin gels with thickness around 10

20 μm however, thicker gels were necessary for biaxial testing in this experiment. Thus, it might be possible that alignment of collagen fibers is not uniform in such relatively thick gel [27], which could have also caused the need to change the values of material parameters in the model. The fiber alignment method was adopted in this study for the easiness of experimental implementation. The overall research approach can be applied to other studies with more controllable alignment methods in the future. SEM was used in this study to qualitatively access the fiber alignment. Due to the higher concentration of collagen, our existing confocal microscopy capability was incapable of providing quantitative information on fiber orientation. More quantitative correlation between the structure and function of collagen scaffolds through the constitutive model might be possible by applying experimental techniques. For example, values for mean fiber orientation γ and dispersion around the main direction κ have been determined previously for collagen fibers in bladder tissue using small angle light scattering [63]. Using scanning electron microscopy techniques, fiber diameter can be quantified [61]. Fiber mechanical properties were investigated using nanoindentation [64] or micromanipulation/tensile testing of individual fibers [65].

5. Conclusions

Improving mechanical properties of collagen scaffolds via a controlled collagen assembly process will greatly broaden their application in numerous life science researches and will be a substantial step toward biomaterial research on tissue repair and replacement. In the present study, the mechanical properties of collagen scaffolds with the effects of crosslinking and fiber alignment were studied both experimentally and theoretically. Equibiaxial tensile tests were performed to probe the elastic properties of collagen fibrillar network within the collagen scaffolds. The stiffness of the collagen scaffolds increases with GP concentration. The anisotropic behavior of collagen scaffolds is correlated with the presence of preferred collagen fiber alignment. Our results demonstrated that crosslinking of the scaffolds can be used to tune the overall stiffness of the scaffolds without affecting the presence of anisotropy in the collagen matrix. The tangent stiffness from biaxial tensile tests is two to three orders of magnitude higher than the storage moduli from rheological measurements. This suggests that in rheological tests the movement of the interstitial fluid play a significant role in determining the overall mechanical properties of a collagen gel. Therefore, the highly hydrated collagen gel comprising stiff fibrillar network embedded in a very compliant matrix has rather low shear modulus. The stress-strain responses of both nonaligned and aligned collagen scaffolds were captured well with the anisotropic hyperelastic constitutive model. Results from simulations suggest possible changes of the mechanical property of collagen fiber in the aligned collagen scaffolds due to preferred crosslinking along the fibers. The structurally based constitutive model is promising in future studies on relating material parameters possessing physical meanings to the microstructure of collagen scaffolds.

Acknowledgments

The authors thank Xin Brown for assistance in rheology measurements. They also thank the National Science Foundation for funding through Grant CMMI-0954825.

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Copyright

Copyright © 2011 Bin Xu et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


Biochemical pathways that are triggered after UV irradiation activating cell surface cytokine and growth factor receptors

Human skin cells respond to UV radiation by activation of multiple cytokine and growth factor receptors. These include epidermal growth factors receptors (EGF-R), tumor necrosis factor (TNF)-α receptors, platelet activating factor (PAF) receptor, interleukin (IL)-1 receptor, insulin receptor and platelet derived growth factor. Amongst these, the EGF-R activation has been the most studied. It is a single chain 180 kDa transmembrane protein. The extracellular domain possesses high affinity binding for EGF and EGF-like ligands (transforming growth factor [TGF]-α, amphiregulin and heparin binding-EGF) ( Rittié and Fisher 2002 ). The intracellular domain possesses intrinsic tyrosine kinase activity. EGF-R also known as ErbB1 undergoes homo- or heterodimerization with either ErbB2 or ErbB3 resulting in the transphosphorylation of specific tyrosine residues. EGF-R tyrosine phosphorylation is a well-characterized marker for receptor activation and occurs within 10 minutes of UV irradiation. Notably, UV fails to induce EGF-R tyrosinase phosphorylation in cells expressing mutant EGF-R lacking tyrosine kinase activity. UV irradiation of EGF-R, like ligand activation, is dependent on EGF-R tyrosine kinase-catalysed trans-phosphorylation. Alternatively, it has been proposed that UV-induced EGF-R tyrosine phosphorylation results from inactivation of protein tyrosine phosphatases (PTPs) that function to maintain EGF-R in a dephosphorylated basal state. Inhibition by specific tyrosine kinase inhibitors results in a very rapid dephosphorylation of EGF-R. Treatment of the cells with UV irradiation substantially prolonged the life of the EGF-R phosphorylated tyrosinases, thus suggesting an inhibitory effect of UV on PTPs. This inhibitory activity by UV was sensitive to N-acetyl cysteine, a scavenger of reactive oxygen intermediates and could be mimicked by treating cells with H2O2. UV-induced inactivation of PTP activity is postulated to result from oxidation of a critical cysteine residue that is present in the catalytic active site of all PTP’s to sulfenic acid. This oxidation occurs by the exposure of the cysteine residue on the PTP to reactive oxygen species which are generated within the cells by UV irradiation ( Rittié and Fisher 2002 ). This inactivation of PTPs may result in the activation of other cell surface receptors and cytokine receptors which in turn leads to activation of small GTP-binding protein families such as the Rac, Ras, and Cdc42. These are either direct or indirect (via other GTP-binding proteins or ROS) upstream regulators of mitogen-activated protein kinases (MAPKs). The UV irradiation causes increased ROS production and simultaneous increase in ceramide levels which may also contribute to the activation of MAPK pathways. A major effector of the MAPK pathways is the transcription factor activator protein-1 (AP-1). AP-1 is constitutively composed of c-Fos and JunD proteins or the other Jun and Fos family proteins (c-Jun, Junb, FosB, Fra1, and Fra2) in the nonirradiated skin. The activation of MAPKs indirectly activates the transcription factors for AP-1 formation ie, transcription of the c-Fos and c-Jun genes. UV irradiation induces c-Jun mRNA and protein in human skin in vivo within 30 min and 1 hour, respectively, and protein levels remain elevated for at least 24 hours post UV irradiation. Increased levels of c-Jun compete with JunD for forming complexes with c-Fos resulting in c-Jun: c-Fos AP-1 complexes ( Rittié and Fisher 2002 ). Transcription of several MMP family members is regulated by this AP-1 complex formed throughout the epidermal and dermal cells. MMPs are a large family of zinc-requiring endoprotreases with a broad range of specificities that together have the capacity to degrade all the extracellular matrix proteins. Initially, MMPs are synthesized as zymogens (proenzymes) which undergo proteolytic degradation to be active. These are inhibited by tissue inhibitors of metalloproteinases (TIMPs). Several MMPs are upregulated by AP-1 including MMP-1 (interstitial collagenase or collagenase1), which intitiates the degradation of types I & III fibrillar collagens, MMP-9 (92 kDa gelatinase or gelatinase B) degrades the collagen fragments (gelatin) generated by collagenases and MMP-3 (stromelysin 1) further degrades collagen type IV of the basement membrane and activates proMMP-1. MMP-1, MMP-3, and MMP-9 transcripts are induced within 8 hours following UV irradiation. Thus, together MMPs have the capacity to completely degrade mature fibrillar collagen in the skin within 24 hours of UV exposure via the induction of transcription factor AP-1. In addition to causing collagen breakdown, UV radiation impairs new type I collagen synthesis and organization of collagen fibrils in skin in vivo. Down-regulation of type I collagen is mediated by down-regulation of the transcription of genes that encode for type I procollagen. Type I procollagen mRNA and protein expression levels are decreased within 8 hours following UV irradiation of the human skin in vivo and become essentially absent in the upper dermis within 24 hours after UV irradiation, consistent with the sustained induction of c-Jun and thus AP-1 activation.

The TGF-β is a major profibrotic cytokine, which regulates multiple cellular functions including differentiation, proliferation and induction of synthesis of major extracellular matrix (ECM) proteins – collagen and elastin ( Massague 1998 ). In human skin, TGF-β inhibits growth of epidermal keratinocytes and stimulates growth of dermal fibroblasts ( Massague 2000 ). TGF-β inhibits the expression of MMP-1 and MMP-3 by binding to a certain cell surface receptor complex (TGF-β receptor proteins: TβR I/II/III), thus preventing the breakdown of collagens. UV irradiation has been shown to impair the TGF-β signaling pathway by reducing TβRII expression and to a lesser extent the inhibitory Smad 7. Moreover, the connective tissue growth factor is down-regulated after UV irradiation ( Rittié and Fisher 2002 ).

In this article, we critically compare the clinical efficacy and safety of various retinoids that have been in the treatment and protection of skin aging.


Results

Structural Differences of Dermal Collagen Fibers Among Different Burns

We first performed depth-resolved SHG imaging for control, SDB, DDB, and DB samples. To visualize structural differences of the dermal collagen fibers at high image contrast, the power of the laser light incident on the sample was adjusted to 10 mW for the control, 15 mW for SDB, 25 mW for DDB, and 40 mW for DB. The resulting series of depth-resolved SHG images are shown in Fig. 3 ( imaged region = 600 × 600 μ m 2 , interval of scanning depth = 30 μ m ). Furthermore, a series of more precisely, depth-resolved SHG images was given in Video 1 ( imaged region = 600 × 600 μ m 2 , interval of scanning depth = 10 μ m ). In this experiment, we defined the skin surface as a depth of 0 µm. The skin surface location was determined by confocal microscopy, which is a function included in the present SHG microscopy system (not shown in Fig. 2). It is interesting that the depths that the SHG light started to appear are different among different degrees of burn: 140 µm for control, 120 µm for SDB, 90 µm for DDB, and 40 µm for DB. This result indicated that the thickness of the epidermis was decreased due to the thermal shrink caused by the skin burn. If the probing depth of SHG microscopy is determined based on the skin surface, the actual probing depth of the dermis was different among the control, SDB, DDB and DB. Therefore, it is important to investigate depth dependency of the dermal collagen fiber for each burn sample. In the control sample, we observed that fine collagen fibers in the papillary dermis changed to thick collagen fibers in the reticular dermis with increasing probing depth [see Fig. 3(a)]. On the other hand, depth-resolved SHG images of the SDB, DDB, and DB samples indicated that each burn spread uniformly over the whole depth of the dermis. In the SDB sample, the SHG vanishing patterns discussed later were overlaid on the fiber structure of the dermal collagen [see Fig. 3(b)]. In the DDB sample, the SHG vanishing patterns became finer than those in the SDB sample [see Fig. 3(c)]. Furthermore, the fibrous structures of the dermal collagen were almost entirely lost and changed into amorphous structures. In the DB sample, a little SHG light was observed only from aggregates of degenerated collagen [see Fig. 3(d)]. Thus, there was no depth dependency of the SHG vanishing pattern for each burn sample within the probing depth range used in the present system.


How are collagen fibers in skin aligned with respect to skin surface? - Biology

The functions of bird skin are the same as for other vertebrates &ndash to keep out pathogens and other potentially harmful substances, retain vital fluids and gases, and serve as a sensory organ. The continual renewal of the skin acts to repel parasitic microorganisms. The skin of birds also produces and supports feathers. With feathers, the skin also plays an important role in thermoregulation. Although largely covered by feathers, the integument is unfeathered on the beak, feet, and, in some species, other areas. In contrast to mammals, avian skin does not have sweat glands and sebaceous glands. Avian skin consists of two layers, the epidermis and dermis. The outer layer, the epidermis, is generally very thin and pliable. The dermis is thicker than the epidermis and contains blood vessels, fat deposits, nerves and free nerve endings, several types of neuroreceptors, and smooth muscles that move the feathers (Lucas and Stettenheim1972).

The epidermis, the most superficial layer of the skin, is thinner in birds than in mammals of comparable size, flexible, and smooth, and this is due, at least part, to selective pressures to minimize body weight for more efficient flight (Spearman 1966). The epidermis is thinnest in areas covered by feathers (both feather tracts and apteria Figure 1) and thickest in exposed, featherless areas, including the covering of the beak (rhamphotheca) and feet (podotheca). The epidermis has two main layers &ndash a superficial stratum corneum and a deeper strateus germinativium (Figure 2). The stratum corneum consists of flattened, keratinized cells. These cells are called keratinized because they contain a protein called keratin (and, specifically, beta keratin) that, along with extracellular lipids (fats) produced by epidermal cells, provide a tough, permeability barrier that prevents excessive evaporative water loss. The stratum corneum can be viewed as having a &lsquobrick-and-mortar&rsquo organization, with the keratin-enriched cells forming the &lsquobricks&rsquo and the extracellular lipids the &lsquomortar&rsquo (Elias and Menon 1991). However, compared to reptiles and even mammals, cells in the stratum corneum have less keratin and, as a result, this barrier is less stringent and can facilitate evaporative cooling while retaining the capacity for facultative waterproofing (Menon et al. 1996). The high body temperatures of birds, increased heat production during flight, insulation by plumage and the lack of sweat glands, require a higher rate of evaporative cooling through a relatively "leaky" epidermal permeability barrier. Importantly, however, the relatively permeability of the avian epidermis can be modified. For example, Menon et al. (1996) found that, within 16 hours of water deprivation, adult Zebra Finches can reduce water loss via the epidermis by 50% by the rapid secretion of epidermal lipids. A similar ability to influence water loss by regulating secretion of epidermal lipids has been reported in larks (Haugen et al. 2003), House Sparrows (Passer domesticus Muñoz-Garcia and Williams 2008), and the tropical Dusky Antbird (Cercomacra tyrannina Muñoz-Garcia and Williams 2007).


Figure 1. Feathered (feather tracts) and unfeathered (apteria) areas of the avian integument (From: Chuong et al. 2000).


Figure 2. Cross-section through the skin of a bird or mammal (From: Lillywhite 2006).

Besides forming a dynamic barrier that regulates water loss through the skin, epidermal lipids may also have antimicrobial properties and offer protection against ultraviolet light (Menon 1984). In addition, epidermal lipids are used for cosmetic coloration in the Japanese Crested Ibis (Nipponia nippon Wingfield et al. 2000). Before breeding, the skin of the neck and head starts secreting a black substance that the ibises apply to their white plumage (Figure 3). The extent of the secretory skin area and how much of the plumage is covered by the &lsquocosmetic&rsquo varies among individuals and this variation plays a role in mate choice. Young birds do not produce the black secretion at all.


Figure 3. Plumages of the Japanese Crested Ibis. (a) Typical non-breeding plumage. (b) Daubing behavior applying the pigment to plumage on the head, neck, and back. (c) &lsquoCosmetically colored&rsquo ibis. (d) After post-breeding molt (From: Wingfield et al. 2000).

Among many species of birds, the integument exhibits specialized modifications. For example, the skin on the head is unfeathered to varying degrees and distinctively colored in guineafowl, vultures, colies (Colius), and many storks, ibises, spoonbills, and cranes. The skin around the eyes is unfeathered and distinctively colored in other birds, such as cariamas, falcons, sheathbills (Chionis), parrots, cuckoos, broadbills, bare-eyes (Phlegopsis), lyrebirds (Menura), and helmet-shrikes (Prionops) (Stettenheim 2000). More generally, patches of bare skin, other than the bill and legs, can be found in birds belonging to at least 19 different orders and 62 families (Negro et al. 2006). Colored unfeathered areas on the head and necks of birds may be important in (1) intra- and intersexual communication, e.g., advertising status or quality, (2) thermoregulation, and (3) preventing the soiling of feathers for species that sometimes extend their heads into carcasses when feeding (e.g., vultures and condors).

Among birds with colorful skin (Figure 4), the coloration is due either to pigments, structural mechanisms in the epidermis, or to blood (and, specifically, hemoglogin in the red blood cells) in the superficial capillary network (Lucas 1970, Prum and Torres 2003).


Figure 4. Structurally colored ornaments of a sample of the piciform and passeriform birds examined: (A) Selenidera reinwardtii, (B) Ramphastos vitellinus, (C) Ramphastos toco, (D) Neodrepanis coruscans, (E) Philepitta castanea, (F) Myrmeciza ferruginea, (G) Gymnopithys leucapsis, (H) Procnias alba, (I) Perissocephalus tricolor, (J) Dyaphorophyia concreta, (K) Terpsiphone mutata and (L) Leucopsar rothschildi. A,F&ndashJ, reproduced with permission from VIREO B,C,L, reproduced with permission from Kenneth Fink H, reproduced with permission from Nate Rice D, reproduced with permission from Steve Zack K, reproduced with permission from Tom Schulenberg (From: Prum and Torres 2003).

Some species with bare skin on the head and neck alter skin color by changing blood flow to the area. For example, skin in the unfeathered areas of a Crested Caracaras (Polyborus plancus) head has a much denser supply of blood vessels than skin in feathered areas (Figures 5 and 6). Although they sometimes feed on carrion, the bare skin of caracaras is most important for thermoregulation. Caracaras are relatively large birds with generally dark plumage that are typically found in relatively hot areas. When their body temperature increases, blood flow to the areas of bare skin increases as vessels dilate. This increased blood flow causes the skin to become deeper red in color, but, most importantly, enhances the loss of heat across the bare skin. Bare skin on the head and neck likely serves a similar function for many species of birds because many such birds are relatively large, dark-plumaged birds that occur at low latitudes where heat dissipation may be of great importance (Negro et al. 2006).


Figure 5. A Crested Caracara showing unfeathered (a) and feathered (b) areas of the head (From: Negro et al. 2006).


Figure 6. Micrographs of cross-sections of skin of a Crested Caracara, a species with unfeathered areas on the head. (A) Unfeathered area (bare skin) on the face, and (B) feather-covered area on the head. Note the greater number of blood vessels in the unfeathered skin. Scale bars: 25 µm. e, epidermis c, collagen er, erythrocytes bv, blood vessels. (From: Negro et al. 2006).

For those species of birds with bare skin on the head and neck and where skin coloration is altered by changes in blood flow (&lsquoflushing&rsquo), thermoregulation was almost certainly the primary selective factor. However, in some species, flushing occurs in contexts unrelated to thermoregulation, such as during courtship or agonistic encounters. For example, the skin of turkeys becomes redder when courting females and when engaged in agonistic encounters with other males. This suggests that &lsquoflushing&rsquo can, for some species and in some contexts, also serve a signaling function. The ability to generate a deeper red coloration or maintain redder coloration for longer periods may be correlated with individual quality if doing so is energetically costly or potentially damaging to the body (Negro et al. 2006).

The colored skin of many birds is due to pigments, molecules that differentially absorb and emit wavelengths of visible light. Carotenoids are the pigments responsible for colorful skin (as well as feathers) in many birds, and typically generate a red, orange, or yellow hue. Birds cannot synthesize carotenoids so must acquire them in their diet. As a result, variation in carotenoid-based skin (or feather) coloration can provide conspecifics with information about individual quality and, specifically, the ability of different individuals to acquire a limited resource (Negro et al. 2002). For example, Red-legged Partridges (Alectoris rufa) have bills and eye rings (bare skin not feathers) that are reddish due to the presence of carotenoids and individuals with redder bills and eye rings are in better physical condition (Pérez-Rodríguez and Viñuela 2008). Similarly, the yellow-orange skin on the legs, feet, and ceres (skin at the base of the upper bill) of European Kestrels (Falco tinnunculus) is due to carotenoids and studies have revealed that male kestrels with more brightly colored skin are better hunters and have better quality territories (Casagrande et al. 2006).

For many birds, skin coloration is the result of optical interactions with biological nanostructures or, in other words, the microscopic structure of skin (Figure 7). Such structural colors occur in the skin, bill (ramphotheca), legs and feet (podotheca) in about 129 avian genera in 50 families from 16 avian orders. Structurally colored skin is present in more than 250 bird species, or about 2.5% of all bird species (Prum and Torres 2003). Examination of the color, anatomy, and nanostructure of structurally colored skin, ramphotheca, and podotheca from several different species of birds indicates that color, including ultraviolet, dark blue, light blue, green and yellow hues, is produced by coherent scattering (i.e., constructive interference) of light from arrays of parallel collagen fibers in the skin (dermis) (Figures 8 and 9). Scattering, in this case, simply means that light deviates from a straight path.

Visible light is, of course, composed of many colors of light with distinct wavelengths. Red light has a long wavelength (

700 nm), whereas violet and blue light has a much shorter wavelength (

400 nm). When visible light encounters particles with the same or larger diameter than its component wavelengths, those specific light photons are reflected. For example, particles that are 400 nm or slightly larger will selectively reflect blue light photons while allowing other light photons to pass. In the skin of birds light is reflected by collagen fibers (long, string-like protein molecules) that are arranged in a much more highly organized manner than in normal skin. For patches of skin that are a particular color (e.g., blue or green) all collagen fibers are the same thickness (Figure 8). As a result, each fiber scatters wavelengths of light that are in phase (Figure 10) and, therefore, are reinforced, producing very bright colors even more saturated than typical pigment-based colors. In addition to the reflection of light of certain wavelengths, skin structural coloration also requires a means to prevent the reflection or scattering of white light by deeper tissues below the color-producing nanostructures. In bird skin, this light-absorbing layer consists of a thick layer of melanin granules (melanosomes Figure 7).


Figure 7. Light micrographs of structurally colored, white and pigmented bird skin showing differences between structurally colored and non-structurally colored skin in the thickness of collagen layers : (A) Chaemapetes unicolor, dark blue (B) Numida meleagris, dark blue (C) Tragopan temminckii, light blue (D) Opisthocomus hoazin, dark blue (E) Ramphastos vitellinus, dark blue (F) Selenidera reinwardtii, green (G) Procnias nudicollis, white leg skin (H) Procnias nudicollis, structurally green throat skin and (I) Tragopan temminckii, red, lateral lappet patches. All specimens stained with Masson's trichrome, which stains collagen blue and cells red. All scale bars represent 100 µm, except in C, which represents 50 µm. Abbreviations: c, collagen macrofibrils cc, collagenocytes cp, capillaries e, epidermis m, melanosomes (From: Prum and Torres 2003).


Figure 8. Transmission electron micrographs showing the very regular nanostructured arrays of dermal collagen fibers from: (A) Oxyura jamaicensis, light blue (B) Numida meleagris, dark blue (C) Tragopan satyra, dark blue (D) Tragopan caboti, dark blue (E) Tragopan caboti, light blue (F) Tragopan caboti, orange (G) Syrigma sibilatrix, blue (H) Ramphastos toco, dark blue (I) Philepitta castanea, light blue (J) Gymnopithys leucapsis, light blue (K) Procnias nudicollis, green and (L) Terpsiphone mutata, dark blue. All images were taken at 30000 x. All scale bars represent 200 nm (From: Prum and Torres 2003).


Figure 9. Coherent scattering is differential interference or reinforcement of wavelengths scattered by multiple light-scattering objects (x, y). Coherent scattering of specific wavelengths is determined by the phase relationships among the scattered waves. Scattered wavelengths that are out of phase will cancel each other out, but scattered wavelengths that are in phase will be constructively reinforced and coherently scattered. Phase relationships of wavelengths scattered by two different objects (x and y) are given by the differences in the path lengths of light scattered by the first object (x: 1&ndash1') and a second object (y: 2&ndash2') as measured from planes perpendicular to the incident (a) and reflected (b) waves in the mean refractive index of the media (From: Prum and Torres 2003).

Figure 10. Left, In constructive interference (coherent scattering), two light waves (on bottom) are in phase so the combined waveform (top) is enhanced. Right, In destructive interference, two light waves (bottom) out of phase so they cancel out (top). (From: Wikipedia).

In some groups of birds (e.g., Phasianidae, Eurylaimidae, Cotingidae, Paradisaeidae, and Cnemophilidae), sexual selection is likely responsible for the structurally colored skin of males because most species are polygynous. In other groups of birds, including Ardeidae, Cariamidae, Bucerotidae, Ramphastidae, Meliphagidae, and Monarchidae, both sexes have integumentary structural colors, suggesting that such coloration may be important in both inter- and intrasexual communication (Prum and Torres 2003). Many birds with structurally colored skin are found in rainforests. For example, nearly all species with structurally colored skin in the orders Casuariiformes, Galliformes, Opisthocomiformes, Cuculiformes, Trogoniformes, Coraciiformes, Piciformes, and Passeriformes occur in tropical forests. The quality of ambient light in tropical forest habitats may favor the evolution of communication signals in the shorter wavelength portion of the visible spectrum (blue and green) and, if so, selection might favor structural colors because vertebrates, including birds, have no pigments that generate such colors (Prum and Torres 2003).


Surucua Trogon (Trogon surrucura)

Claws are found at the distal end of all toes of all birds and cover the bones of terminal phalanges. Some birds also have wing claws. The dorsal portion of claws is highly keratinized and calcified and is very hard. Claws are curved to varying degrees because the dorsal portion grows faster than the ventral portion. In addition to variation in curvature, claws also vary in relative length and pointedness (Stettenheim 2000). Among diving and swimming birds with webbed or partially webbed feet, such as gannets, waterfowl, and gulls, claws tend to be small, less curved, and flatter. At the extreme, grebes have very flat claws that increase foot surface area and aid in underwater propulsion. In contrast, birds that climb on vertical or nearly vertical surfaces, like woodpeckers and nuthatches, have sharply recurved and pointed claws to help grip the substrate (e.g., bark of a tree). Glen and Bennett (2007) examined claw morphology and placed terrestrial birds into six categories, with ground-dwelling birds having longer, less recurved claws and the amount of curvature increasing for birds that are increasingly arboreal in their foraging habits (Figure 11). Among raptorial birds that use their feet to capture and kill prey, claws (also called talons) are relatively long, highly recurved, and pointed.


Figure 11. Categories (GB, Gg, Ga, Ag, Aa and V) of avian toe claws based the degree of ground or tree foraging GB = &lsquoground-based&rsquo birds, limited to foraging on the ground Gg = &lsquodedicated ground foragers&rsquo Ga = &lsquopredominantly ground foragers&rsquo Ag = &lsquopredominantly arboreal foragers&rsquo Aa = &lsquodedicated arboreal foragers&rsquo V = &lsquovertical surface foragers&rsquo. Analysis of the toe claws of 249 species of birds revealed that claw curvature increases as tree foraging becomes more predominant (Glen and Bennett 2007).

Several species of birds representing 17 families of birds in eight orders Nightjars, and including herons, frigatebirds, and pratincoles, have pectinate middle claws with comb-like edges that are used for grooming and preening feathers (Figure 12 Stettenheim 2000, Moyer and Clayton 2003). For example, Fiery-necked Nightjars (Caprimulgus pectoralis) use their pectinated claw to groom their rather long rictal bristles (Jackson 2007), and nocturnally foraging nightjars, more generally, may use their pectinated claw to clean spider webs from their plumage (Masterson 1979).


Figure 12. A pectinate claw.

Wing claws can be found at the tip of the alular digit in several groups of birds, including loons, storks, owls, and some shorebirds, but are very small and non-functional. Young Hoatzins (Opisthocomus hoazin), however, have two, well-developed claws on each wing and use them for climbing shrubs and trees. These claws have an important function for Hoatzins, a Neotropical species, because they typically nest over water and nestlings sometimes jump from nests when threatened by a predator. Once in the water, they swim to nearby shrubs and trees and climbs upwards using their wing claws and feet. Young Hoatzins shed their wing claws when 70-100 days old (Thomas 1996).

Rhamphotheca

Bird bills consist of bones that form the cores of the upper and lower mandibles. However, the outer surface and part of the inner surface of these bones are covered with a modified integument called the rhamphotheca (Figure 13). The epidermis of the rhamphotheca is relatively thick, hard, and consists of heavily cornified cells (Lucas and Stetteheim 1972). These cells produce beta-keratin like that found in avian scales and claws and calcium deposited between the keratin proteins generally make the rhamphotheca hard and strong (Homberger and Brush 1986, Bonser 1996). The epidermis is tightly bound to bone by a thin dermal layer that contains numerous collagen fibers. The dermis also contains sensory receptors, including Herbst and Grandry corpuscles that are sensitive to touch and vibration (see "Sense organs" on the "Nervous System: Brain & Senses" page). The integument of the bill grows continually from the base and culmen, with growth directed rostrally so that there is continuous movement of the horny beak from base to tip. The rhamphotheca, particularly at the tomia, is worn away by abrasion from food and other materials and by friction where the upper and lower bills meet.


Figure 13. A sagittal section near midline of the upper beak of a 2-wk-old domestic chicken. The bill tip is to the right. The dorsal region shows the upper smooth portion of the bill covered by the rhamphotheca (Rh), whereas the ventral region shows the tomium or cutting edge of the upper bill. Beneath the rhamphotheca is the epidermis (Ep) that provides a constant supply of cellular material to form the outer, hardened covering of the beak. Internal to the epidermis is the dermis (Dr) layer that is the most heterogeneous of all tissue layers. It extends from the epidermal to bone layers (Bn). Prominent structures found in this region include mechanoreceptors (Herbst [Hb Cp]and Grandry [G Cp] corpuscles), blood vessels (BV), perineural sheaths, and free nerve endings, or nociceptors (pain) Scale bar, 400 µm. N = nerve Pr Sh = perineural sheaths (From: Kuenzel 2007).

The edges of the rhamphotheca (tomia) are relatively sharp in most species of birds. However, edges are finely serrated for straining small food particles in filter-feeding birds like flamingoes and some waterfowl. Species in a number of bird families, including Ardeidae, Cuculidae, Coraciidae, Picidae, and several others, have scopate tomia with very small (typically only 0.3-0.7 mm high and difficult to see without magnification) brush-like ridges that likely create friction and aid birds in capturing and holding food items (Figure 14 Gosner 1993). Tiny serrations along the tomia of one (just upper bill) or both tomia of some hummingbirds likely aid in capturing and holding insect prey and, in some species of nectar-robbers, piercing the base of flowers to gain access to nectar (Ornelas 1994 Figure 15). The tomia of fish-eating mergansers have numerous pointed projections that aid in capturing and holding their primary prey. Falcons and shrikes have single, sharp projections of the tomia on each side of their upper bill. Several authors have suggested that falcons use these &lsquotomial teeth&rsquo to break the neck of their prey, but there is little or no evidence to support this hypothesis. Rather, &lsquotomial teeth&rsquo are likely used to help firmly grip and pull flesh from prey (Csermely et al. 1998) and, for large falcons, to help grip and break long bones of wings and legs of smaller prey before swallowing (White et al. 2002).


Figure 14. Top, Scopate tomia of the fish-eating Ringed Kingfisher (Ceryle torquata). Bottom, scopate tomia of the insectivorous Laughing Kookaburra (Dacelo novaguineae). Tomia magnified 10X (From: Gosner 1993).


Figure 15. Upper mandibles of four species of hummingbirds showing a typical, smooth tomium (A, Violet Sabrewing, Campylopterus hemileucurus) plus the serrate tomia of the Sparkling Violetear (Colibri coruscans, B), Crowned Woodnymph (Thalurania ridgwayi, C), and Green-breasted Mango (Anthracothorax prevostii, D) (From: Ornelas 1994).

In some birds, including raptors (Falconiformes), owls (Strigiformes), parrots, cracids, and pigeons, the rhamphotheca at the base of the upper bill is called the cere (Figure 16 Stettenheim 1972). The cere is a thickened, often brightly colored portion of the integument that straddles the base of the nasal region. (Lucas 1979). Lucas and Stettenheim (1972) suggested that the cere may provide protection for the underlying elongated nasal fossa (cavities). However, because ceres are often brightly colored, individual variation in cere color may also convey information about individual quality. For example, the cere of male Montagu&rsquos Harriers (Circus pygargus) reflects light at wavelengths corresponding to yellow-orange (500-600 nm), but also reflects ultraviolet (UV 300-400 nm) wavelengths (Figure 17). Differences among males in the UV peak were found to be associated with differences in body mass and condition, suggesting UV reflectance conveys information about individual quality (Mougeot and Arroyo 2006).


Figure 16. The cere (C) of a Rock Pigeon located at the base of the upper bill. The arrow points to the external nares (O), operculum (a small disc of cartilage centered in the nostril that keeps foreign objects out of the nasal cavity) (From: Purton 1988).

Figure 17. A male Montagu's Harrier (a) showing the cere (skin area above the beak and between the eyes) ( b) Reflectance pattern of a typical male cere showing peaks in the yellow-orange wavelengths (500-600 nm) and in the ultraviolet (300&ndash400nm) (From: Mougeot and Arroyo 2006).

Bonser, B. H. C. 1996. Comparative mechanics of bill, claw and feather keratin in the Common Starling Sturnus vulgaris. Journal of Avian Biology 27: 175-177.

Casagrande , S., D. Csermely, E. Pini, V. Bertacche, and J. Tagliavini. 2006. Skin carotenoid concentration correlates with male hunting skill and territory quality in the kestrel Falco tinnunculus. Journal of Avian Biology 37: 190-196.

Chuong, C.-M., R. Chodankar, R. B. Widelitz, and T.-X. Jiang. 2000. Evo-Devo of feathers and scales: building complex epithelial appendages. Current Opinion in Genetics and Development 10: 449-456.

Csermely, D., L. Bertè, and R. Camoni. 1998. Prey killing by Eurasian Kestrels: the role of the foot and the significance of bill and talons. Journal of Avian Biology 29: 10-16.

Elias, P. M., and G. K. Menon. 1991. Structural and biochemical correlates of the epidermal permeability barrier. Advances in Lipid Research 24:1-26.

Glen, C. L., and M. B. Bennett. 2007. Foraging modes of Mesozoic birds and non-avian theropods. Current Biology 17: R911-R912.

Gosner, K. L. 1993. Scopate tomia: an adaptation for handling hard-shelled prey? Wilson Bulletin 105: 316-324.

Haugen, M., J. B. Williams, P. W. Wertz, and B. I. Tieleman. 2003. Lipids of the stratum corneum vary with cutaneous water loss among larks along a temperature-moisture gradient. Physiological and Biochemical Zoology 76: 907&ndash917.

Homberger, D. G., and A. H. Brush. 1986. Functional morphology and biochemical correlations of the keratinized structures of the African Gray Parrot, Psittacus erithacus (Aves). Zoomorphology 106: 103-114.

Jackson, H. D. 2007. Measurements and functions of the pectinated claws and rictal bristles of Fiery-necked Nightjars Caprimulgus pectoralis and some congeners. Ostrich 78: 641-643.

Kuenzel, W. J. 2007. Neurobiological basis of sensory perception: welfare implications of beak trimming. Poultry Science 86: 1273-1282.

Lillywhite, H. B. 2006. Water relations of tetrapod integument. Journal of Experimental Biology 209: 202-226.

Lucas, A. M. 1970. Avian functional anatomic problems. Fed. Proc. 29:1641-1648.

Lucas, A. M., and P. R. Stettenheim. 1972. Avian anatomy. Integument. Agriculture Handbook 362, U.S. Department of Agriculture, Washington, D.C.

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An aligned porous electrospun fibrous membrane with controlled drug delivery - An efficient strategy to accelerate diabetic wound healing with improved angiogenesis

A chronic wound in diabetic patients is usually characterized by poor angiogenesis and delayed wound closure. The exploration of efficient strategy to significantly improve angiogenesis in the diabetic wound bed and thereby accelerate wound healing is still a significant challenge. Herein, we reported a kind of aligned porous poly (l-lactic acid) (PlLA) electrospun fibrous membranes containing dimethyloxalylglycine (DMOG)-loaded mesoporous silica nanoparticles (DS) for diabetic wound healing. The PlLA electrospun fibers aligned in a single direction and there were ellipse-shaped nano-pores in situ generated onto the surface of fibers, while the DS were well distributed in the fibers and the DMOG as well as Si ion could be controlled released from the nanopores on the fibers. The in vitro results revealed that the aligned porous composite membranes (DS-PL) could stimulate the proliferation, migration and angiogenesis-related gene expression of human umbilical vein endothelial cells (HUVECs) compared with the pure PlLA membranes. The in vivo study further demonstrated that the prepared DS-PL membranes significantly improved neo-vascularization, re-epithelialization and collagen formation as well as inhibited inflammatory reaction in the diabetic wound bed, which eventually stimulated the healing of the diabetic wound. Collectively, these results suggest that the combination of hierarchical structures (nanopores on the aligned fibers) with the controllable released DMOG drugs as well as Si ions from the membranes, which could create a synergetic effect on the rapid stimulation of angiogenesis in the diabetic wound bed, is a potential novel therapeutic strategy for highly efficient diabetic wound healing.

Statement of significance: A chronic wound in diabetic patients is usually characterized by the poor angiogenesis and the delayed wound closure. The main innovation of this study is to design a new kind of skin tissue engineered scaffold, aligned porous poly (l-lactic acid) (PlLA) electrospun membranes containing dimethyloxalylglycine (DMOG)-loaded mesoporous silica nanoparticles (DS), which could significantly improve angiogenesis in the diabetic wound bed and thereby accelerate diabetic wound healing. The results revealed that the electrospun fibers with ellipse-shaped nano-pores on the surface were aligned in a single direction, while there were DS particles distributed in the fibers and the DMOG as well as Si ions could be controllably released from the nanopores on the fibers. The in vitro studies demonstrated that the hierarchical nanostructures (nanopores on the aligned fibers) and the controllable released chemical active agents (DMOG drugs and Si ions) from the DS-PL membranes could exert a synergistic effect on inducing the endothelial cell proliferation, migration and differentiation. Above all, the scaffolds distinctly induced the angiogenesis, collagen deposition and re-epithelialization as well as inhibited inflammation reaction in the wound sites, which eventually stimulated the healing of diabetic wounds in vivo. The significance of the current study is that the combination of the hierarchical aligned porous nanofibrous structure with DMOG-loaded MSNs incorporated in electrospun fibers may suggest a high-efficiency strategy for chronic wound healing.

Keywords: Aligned porous structure Angiogenesis Controlled drug delivery Diabetic wound healing Electrospun membranes.

Copyright © 2018 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.


Innovation

The closure of a full-thickness wound by wound contraction is a slow process and not commonly used. Skin grafting is fast and effective in closing a defect. However, grafting, a surgical procedure, creates a new wound that causes discomfort and poses cosmetic issues. New therapies that lead to faster wound contraction should reduce the need for grafting. The development of more efficient wound contraction, possibly by enhancing the rate of collagen organization that would hasten the rate of contraction and eliminate both a surgical procedure and a secondary defect and scar, would be welcome.