- Email: [email protected]
Exploring the collagen nanostructure of dermal tissues after injury
JBUR 5902 No. of Pages 6
burns xxx (2019) xxx –xxx
Available online at www.sciencedirect.com
ScienceDirect journal homepage: www.elsevier.com/locate/burns
Exploring the collagen nanostructure of dermal tissues after injury Feng Tian a,b, Yiwen Niu c , Yuzhi Jiang b, * a
Shanghai Synchrotron Radiation Facility, Zhangjiang Lab., Shanghai Advanced Research Institute, Chinese Academy of Sciences, Shanghai, 201204, China b Shanghai Burns Institute, Ruijin Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, 200025, China c The Department of Burns and Plastic Surgery, Ruijin Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, 200025, China
article info
abstract
Article history:
Scar often occurred during wound repair. It was known that there were differences in
Accepted 31 July 2019
collagen structure in dermal tissues at millimeter scale and micron scale, however, it was not
Available online xxx
known whether there were differences in collagen structure in dermal tissues at nanoscale during wound repair. In order to compare the difference at nanoscale, skin samples from
Keywords: Scar tissue Small angle X-ray scattering (SAXS) Wide angle X-ray scattering (WAXS) Azimuth angle Three dimensional (3D) structure Collagen Transmission electron microscopy (TEM)
1.
patients were selected, the control groups were the normal skin from the same patients. These samples were tested by the small angle X-ray scattering techniques (SAXS) and wide angle X-ray scattering (WAXS) techniques. The transmission electron microscopy (TEM) was used as a comparison. The results showed that there were not only significantly differences between the normal tissue and scar tissue, but also between the center and the margin of the scar tissue at nanoscale by SAXS and WAXS, which was not demonstrated by other studies. These findings demonstrated that the SAXS and WAXS were excellent tools to detect the collagen structure at nanoscale and the orientation of the collagen alignment, which was beneficial for skin tissue engineering and skin regenerative medicine. © 2019 Elsevier Ltd and ISBI. All rights reserved.
Introduction
Skin is the outlayer of the body. It is easy to be injured for various pathological reasons, such as abrasion, cut, burns. A scar often forms after wound healed, which results in aesthetic sequelae and even local dysfunction. These complications take greatly bad effect on human life and society. However, the problems cannot be resolved until now, the mechanism of scar formation also cannot be clarified; therefore, it is imperative to make research about it [1]. Skin is divided into three layers, the epithelium, the dermal tissues and the hypodermis [2]. The epithelium is the out layer
of the skin, which is composed of epithelium cells. The dermal tissues are mostly composed of the web of collagen fibers which weaved compactly, the collagen structure is the scaffold of the dermal tissues which provides the micro-environment for cells. The hypodermis is composed of fat tissues and other loose connective tissues. It was known that the scar tissues occurred when the total thickness or the deep thickness dermal tissue was injured [3,4]. When skin was injured, the process of wound repair started to initiate. The process of wound healing was divided into three stages: inflammation, proliferation and remodeling. During inflammation, the wound was covered by temporary matrix, which was formed by fibrin from the
* Corresponding author. E-mail address: [email protected] (Y. Jiang). https://doi.org/10.1016/j.burns.2019.07.040 0305-4179/© 2019 Elsevier Ltd and ISBI. All rights reserved.
Please cite this article in press as: F. Tian, et al., Exploring the collagen nanostructure of dermal tissues after injury, Burns (2019), https://doi.org/10.1016/j.burns.2019.07.040
JBUR 5902 No. of Pages 6
2
burns xxx (2019) xxx –xxx
broken blood vessel and included many inflammatory cells. The proliferation stage was followed after inflammation. During this stage, fibroblasts, recruited from neighbor tissues and remote tissues, started to secret collage fiber, which inter-weaved to form newly structure and replaced the temporary matrix. The new structure matured after remodeling stage and the scar tissue might occur [5]. Therefore, after skin injury, the integrity and continuity of the scaffold might be involved, taking effect on the fibroblasts behavior and resulting in scar formation. Collagen structure was composed of the collagen fibers, while collagen fibers were composed of the collagen molecules, suggesting that there was relationship between the structure at micro scale and the substructure at nanoscale. The change of substructure at nanoscale might take effect on the change of structure at micro scale. The scar tissues had different appearance from the normal tissues, indicating of the different collagen structure between the scar tissues and the normal tissues, which had been confirmed at millimeter scale and micron scale by many studies [5,13,14]. Histologically, the scar tissue was composed of roughly collagen fibers, while the normal tissue was composed of delicate collagen fibers. However, whether there were some differences between them at nanoscale had not been clearly resolved until now. Currently, there were many techniques to explore the structure at nanoscale, such as a transmission electron microscope (TEM), a small angle X-ray scattering (SAXS) and wide angle X-ray scattering (WAXS). TEM could not exhibit the differences between them [10], while the studies by SAXS and WAXS had not been executed. Thus, in order to resolve the problem, the small angle X-ray scattering (SAXS) and wide angle X-ray scattering (WAXS) were used to explore the difference between the normal tissue and scar tissue, and the difference between the center and the margin of the scar at nanoscale. TEM was also used as a comparison.
2.
Method and materials
2.1.
Subjects
Patients with hypertrophic scar were separately from the department of burn and plastic surgery, Ruijin Hospital, affiliated with Shanghai Jiaotong University, Shanghai, China. Informed consent was obtained from all patients and experimental protocols were separately approved by the ethic board of the Ruijin hospital(2013-CEB-No.150). Skin biopsies were obtained from 8 patients with hypertrophic scar for aesthetic surgery. The locations were from hand (2 patients), ankle (2 patients), abdomen (2 patients) and neck (2 patients) (average age 25.2 + 15.4 years). The median duration of hypertrophic scars was 4.1months (rang 3–5 months). The minimal area of scar was 2.5 1 cm2, the maximum area of scar was 3.5 2 cm2. The control samples were obtained close to the wound. The normal samples were trimmed into 0.2-1-cm strips, and at least of 2 samples were got from each patient. The samples were blinded for detected and analyzed.
2.2.
Skin preparation
The samples were cut into 0.2-1-cm strips and divided into two parts. One was treated for TEM; the other was treated for SAXS and WAXS.
2.3.
Small-angle X-ray scattering (SAXS) experiments
After the excess subcutaneous tissue were trimmed during operation, the skin samples were incubated in 0.5% dispase (Roche, Mannheim, Germany) at 4 C over night. The epithelium was stripped and then dehydrated by lyophilisation for 48 h before SAXS and WAXS. The SAXS measurements were carried out at BL16B1 beamline [6] at Shanghai Synchrotron Radiation Facility (SSRF), Shanghai, China. X-ray of 10 keV was used as the incident beam. The scattering intensity was recorded using a Rayonix SX-165 CCD detector (Rayonix, Evanston, IL, USA) with a resolution of 2048 2048 pixels. The distances between the sample and the detector were 1850 mm [7,8].
2.4.
Wide angle X-ray scattering (WAXS) experiments
The measurements were carried out at the same place. The scattering patterns were recorded with a two dimensional (2D) detector MAR CCD (MAR Charge Coupled Device, Marresearch GmbH, Norderstedt, Germany) with a 165 mm diameter, the single exposure times were about 100 s. The distances between the sample and the detector distances were 85 mm. The calibration was performed using silver behenate [9]. The same analysis method was used in both SAXS and WAXS. The scattering intensity was represented as a function of the modulus of the scattering vector: q = (4p/l)sinu, where l was the wavelength and 2u was the scattering angle. The 2D data were averaged to obtain the one dimensional (1D) profile by circular averaging with Fit2D software. The long period (L), which represented the average distance between the regions with equivalent electronic densities, was calculated using the equation: L = 2p/q, where q was the modulus of the scattering vector at the peak maximum.
2.5.
TEM sample preparation
For TEM observation, the skin was cut into 5 mm thick strips by scalpel. A primary fixation was performed by immersing the tissue sections in 2.5% paraformaldehyde, 2.5% glutaraldehyde in 0.1 M cadodylate buffer for 2 h and after fixation was done in 1% osmium tetroxide in 0.15 M cadodylate buffer for 12 h. The specimens were then stained in 1% uranyl acetate for 12 h and dehydrated by ascending ethanol series. The samples were then embeded and subsequently sectioned using a Leica Ultracut UCT ultramicrotome (Leica) and a Diatome diamonds knife. Ultramicrotome sections were then placed on copper grids for TEM observation and after stained with uranyl acetate for 1 h. Finally the samples were observed using a Jeol electron microscope (JEM 2000 FX, Jeol Japan) at 160 kv.
Please cite this article in press as: F. Tian, et al., Exploring the collagen nanostructure of dermal tissues after injury, Burns (2019), https://doi.org/10.1016/j.burns.2019.07.040
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2.6.
Statistical analysis
All data were expressed as mean + SD of at least three independent experiments. One-way analysis of variance was used for statistical difference. Statistical significance was set at P < 0.05.
3.
Results
3.1.
D SAXS and 2D WAXS human data comparison
There were significantly different between the normal tissues and the scar tissues in 2D WAXS, compared with 2D SAXS; there were also significantly different between the center of the scars and the margin of the scars in 2D WAXS, compared with 2D SAXS (Fig. 1). In order to clarify the differences, 2D data were transformed into the 1D data.
3.2.
1D data from SAXS and WAXS analysis
When transformed from the 2D data, the SAXS patterns showed that there were a series of Bragg reflections with the regular repeating distance which dominated them (Fig. 2), indicating that the skin samples were periodic structure at nanoscale. The relative peak intensity was higher in the scar tissue at the margin area, especially for the 6th peak, the peak intensities were higher after the 6th peak. The patterns on the other two samples were similar. The repeating distances were not significantly different, the distances were 64.0 2.1 nm (the center of scar tissue), 64.4 7.8 nm (the margin of scar
3
tissue) and 64.2 3.6 nm (normal tissue) [10], which was the D-period in TEM research [18]. When transformed from the 2D data, the WAXS patterns showed that there was a mountain which declined from left to right on the three samples (Fig. 3). On the normal tissues, there was higher mountain on whose left, there was an abrupt bottom, and on whose right, there was a smooth decline. On scar tissues, there was a lower mountain, on whose left, there was a nipple on the valley bottom at 1.5, on whose top, there was a nipple at 2.5; on whose right, and there was also a nipple at 3.75 on the course of the declination. Compared with the scars at the center location, there was a higher nipple on the bottom, and rugged hills on the scars at the margin location. Due to the differences among the three samples in 2D SAXS and WAXS and different relative intensities from integrated intensity curves, the azimuth angle u was obtained by integration (0 u 360 ). The 6th peak was selected because it had higher relative peak intensity. The analysis results found that there were significantly different at the annular analysis of the 6th reflection between the normal tissue and the scar tissue (Fig. 4). The reflection signals were stronger in scar tissues compared with the normal tissue. Two peaks were exhibited on both of the centers and the margins of the scars, the peaks were at 60 and 260 and the bottom between the peaks was at 150 on both the center and the margin of the scar tissue. On the contrary, there was a flat line on the normal tissue. In order to further analyze the differences between the different locations of the scars, the advanced version of the MATLAB-based SAXS utilities package (http://www.saxsutilities.eu) was used. The results showed that the SAXS pattern of the scar tissue at the marginal area was formed with two arcs
Fig. 1 – 2D SAXS and WAXS human data. (A) 2D SAXS pattern at the center area of the scar tissues; (B) 2D SAXS pattern at the marginal area of the scar tissues; (C) 2D SAXS pattern of the normal skin; (D) 2D WAXS pattern at the center area of the scar tissues; (E) 2D WAXS pattern at the marginal area of the scar tissues; (F) 2D WAXS pattern of the normal skin. Please cite this article in press as: F. Tian, et al., Exploring the collagen nanostructure of dermal tissues after injury, Burns (2019), https://doi.org/10.1016/j.burns.2019.07.040
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Fig. 2 – 1D SAXS. The peaks were divided according to horizontal ordinate. The results showed that the skin samples were periodic structures at nanoscale. The range of q was 2.18 nm 1–0.17 nm 1, which equaled to 2.88– 36.63 nm. The three samples were similar.
Fig. 3 – 1D WAXS. The range of q was 4.69 nm 1–0.87 nm 1, which equaled to 1.36–7.8 nm. There were significantly different among the three samples.
with significant preferential scattering intensity distribution (Fig. 5). The SAXS pattern of the skin at the central scar area was formed with the four arc patterns, but the intensity increased at broader azimuthally angles, which suggested that the mechanical stress, which exerting on the collagen fibers, on the center area was more dispersed compared with on the margin area of the scar tissues. There was not significantly difference among the different anatomical locations by SAXS and WAXS.
3.3.
TEM
There was not significantly difference between the normal skin and scar tissue by TEM. There were also no statistically difference between the margin area and the center area of the
Fig. 4 – The azimuth angle of the 6th peak from three different samples from 1D SAXS, treated by Fit 2D software. There were significantly different between the normal and scar samples. There were similar between different locations of the scar samples.
Fig. 5 – The azimuth angle of the 6th peak from the scar tissues from 1D SAXS, treated by its advanced version. There were significantly different between the center area and the margin area of the scar samples.
scar tissues by TEM (Fig. 6). There was not significantly difference among the different anatomical locations.
4.
Discussion
Skin is the outlayer of the human body and provides protection from the environment. Therefore, it is easy to be injured. When the wound repaired, the scar tissues often form which have ugly appearances and uncomfortable feelings, such as itching and pain, even result in local dysfunction. These imperfections take greatly negative effect on the human life and society. Therefore, it is the great impetus to treat them [1]. However,
Please cite this article in press as: F. Tian, et al., Exploring the collagen nanostructure of dermal tissues after injury, Burns (2019), https://doi.org/10.1016/j.burns.2019.07.040
JBUR 5902 No. of Pages 6
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Fig. 6 – TEM of the center area of the scar tissues, the margin area of the scar tissues and the normal tissues from hand (the same scar tissue, the same patient). (A) The center area of the scar tissues; (B) The margin area of the scar tissues; (C) the normal tissues.
the mechanism of the scar formation still cannot be clarified and the treatments have not been satisfied until now [11]. Skin is divided into three layers: epithelium, dermal tissue and hypodermal tissue [2]. The epithelium is composed of cells; the dermal tissues are mostly composed of collagen fibers which weaved together into collagen structure as a scaffold. When the deep thickness dermal tissues and the full thickness of the dermal tissues were injured [3,4], the scaffold of the dermal tissues, which was composed by collagen structures, was mostly injured, this would affect the repair process and result in scar formation, indicating of the important role of the integration and continuity of the scaffold. Currently, there were obvious differences between the normal tissue and scar tissues by appearance and immunohistology. In scar tissues, HE staining section showed that the epidermal ridges were obviously lacked and the dermis was thicker, hyperemic and more cells infiltrated heavily, which represented more contribution to the greater deposition of extracellular matrix (ECM) in the dermis [12]. Compared with the normal skin, the collagen fibers were less structured, tougher, less clearly demarcated [13], which was consistent with our previous findings [14]. Our previous findings showed that the normal tissue had the feature of ovalshaped webs, which was not found in scar tissues, suggesting that the compromised ability of weaving of collagen fibers occurred after skin injury. Due to the structure at micro scale was composed of the substructure at nanoscale, there was a series of questions based on these findings: Was there difference at substructure at nanoscale between the scar tissues and the normal tissues? At which scale was the ability of weaving of collagen fiber injured? At which scale did the scar formation initiate? In order to resolve these questions, the exploration of the substructure at nanoscale should be made. TEM was the golden standard for presenting the structure of collagen fibers at nanoscale, however, it could not make differentiate between the normal skin and the scar tissue [10], which confirmed by our research. Apart from TEM, SXAS and WXAS were also excellent tools to explore the substructure at nanoscale. Here the SXAS and WXAS technique were applied in order to resolve these problems.
SAXS was a small-angle scattering technique. The elastic scattering of X-rays which passing through a sample was recorded at very low angles (typically 0.1–10 ). It was capable of delivering structural information of macromolecules between 5 and 25 nm, of repeat distances in partially ordered systems up to 150 nm [15,16]. WAXS was a wide-angle scattering technique; it could be operated at the same platform as SAXS. The distance from sample to the detector was shorter and the diffraction angles were larger. The analysis protocol of the two techniques was the same. In this paper, SAXS and WAXS showed there were differences among the normal tissue, the center area and the margin area of the scar tissues. However, the differences were more obvious by WAXS. From 2D data, the samples were composed of several concentric circles. The color of the rim of the circles was different and the arcs of the circle were different in different degrees, which meant the different intensity distribution, suggesting that the samples had the periodic structure; the samples had the feature of anisotropy because there were different mechanical properties at different directions. The results were consistent of Rosado C’s studies [17]. After transformation from the 2D data, the tissues were further presented as a periodic structure, which was marked by different peaks (Fig. 2). The periodic distance were measured, which was the characteristic signal from the stacking of human skin collagen fibers [7,18], and confirmed by our TEM research. The 6th peak was more obviously different at the 1D data and thus was selected and analyzed by the azimuth angle u, which meant the different intensity reflected in 360 direction and could demonstrate the distribution of the mechanical stress and the orientation of collagen fiber alignment. There were significantly different types of curves and the relative intensity between the normal tissue and scar tissue if treated by Fit 2D software, suggesting that there were more tensions in scar tissues. In order to explore the substructure between the different locations of the same tissues, the 6th peaks at 1D data from SAXS were further treated by its advanced version of the MATLABbased SAXS utilities package (http://www.saxsutilities.eu), the
Please cite this article in press as: F. Tian, et al., Exploring the collagen nanostructure of dermal tissues after injury, Burns (2019), https://doi.org/10.1016/j.burns.2019.07.040
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results showed that there were more preferential scattering intensity distribution at the margin area of the scar tissue, while there were more extensive intensity distribution which increased at broader azimuthally angles at the center area of the scar tissue, suggesting that there were more mechanical stress at the margin area, while there were more evenly distributed orientation of collagen structures at the center area. In conclusion, compared with the normal tissues, the scar tissues had more tensions; while there were more tensions at the margin area of the scar tissue, compared with at the center of the scar tissues. Although there were many other proteins in the dermal tissues, the quantity of them were small, compared with collagen protein. They could not affect periodic locations of the collagen structure by SAXS and WAXS. Furthermore, it was difficult to make differentiate among the other proteins. Currently, the SAXS and WAXS techniques could only be used to detect the collagen structure of the skin tissues, the other protein, such as the structure of the keratin or the structure of the elastin, could not be detected. In addition, the research could not find any structural differences in different locations, which was confirmed by our clinic. Clinically, a skin defect from any regions, such as the back of the hand, could be theoretically transplanted by the full thickness skin grafts from many other different donor locations, such as the abdomen, the thigh, and the forearm. Because the samples used in TEM was dehydrated, the samples used in SAXS and WAXS were also dehydrated. The lyophilisation was selected because it was widely used in the preparation of biological product and in the biological molecular experiments without changing their biological activity.
5.
Conclusion
The orientation of the collagen alignment was different among the normal tissue and the different locations of the scar tissue at nanoscale. The scar tissue had more tension compared with the normal tissues. The margin area of the scar tissues had more tensions compared with the center of the scar tissues. In addition, compared with other measurement, the SAXS and WAXS could provide the 3D information about the tissues. They were convenient tools in exploring the difference of scar and normal tissue.
Competing interests The authors declare that they have no conflict of interest.
Acknowledgement
REFERENCES
[1] Gurtner GC, Werner S, Barrandon Y, Longaker MT. Wound repair and regeneration. Nature 2008;453(7193):314–21. [2] Martin Paul. Wound healing-aiming for perfect skin regeneration. Science 1997;276(5309):75–81. [3] Dunkin CS, Pleat JM, Gillespie PH, Tyler MP, Roberts AH, McGrouther DA. Scarring occurs at a critical depth of skin injury: precise measurement in a graduated dermal scratch in human volunteers. Plast Reconstr Surg 2007;119:1722–32. [4] Kwan PO, Tredget EE. Biological priciples of scar and contracture. Hand Clin 2017;33(2):277–92. [5] Ogawa Rei. Recent advances in scar biology. Int J Mol Sci 2018;19(6):1749. [6] Zeng Jianrong, Bian Fenggang, Wang Jie, Li Xiuhong, Wang Yuzhu, Tian Feng, et al. Performance on absolute scattering intensity calibration and protein molecular weight determination at BL16B1, a dedicated SAXS beamline at SSRF. J Synchrotron Radiat 2017;24:509–20. [7] Cocera Mercedes, Rodriguez Gelen, Rubio Laia, Barbosa-Barros Lucyanna, Benseny-Cases Nuria, Cladera Josep, et al. Characterisation of skin states by non-crystalline diffraction. Soft Matter 2011;7:8605–11. [8] Tang Zhongfeng, Wang Mouhua, Tian Feng, Xu Lu, Guozhong Wu. Crystal size shrinking in radiation-induced crosslinking of polytetrafluoroethylene: synchrotron small angle X-ray scattering and scanning electron microscopy analysis. Eur Polym J 2014;59:156–60. [9] Podorov SG, Faleev NN, Pavlov KM, Pagain DM, Stepanov SA, Forster E. A new approach to wide-angle dynamical X-ray diffraction by deformed crystals. J Appl Crystallogr 2006;39:652–5. [10] Jiang Yuzhi, Tian Feng, Wang Zhiyong, Niu Yiwen, Yang Jianfei, Song Fei, et al. Exploring nanoscale structure change of dermal tissues suffering injury by small angle X-ray scattering and transmission electron microscopy. Mol Biol Rep 2019; 46:67–76. [11] Shukla Vijay K, Raj M, Luc T, Satyajit P. Management of wound healing, New Delhi. India: Jaypee Brothers Med Pub. Ltd.; 2007 [12] Meenakshiv J, Jayaramank M, Babu Ramkrishnan M. Ultrastructural differentiation of abnormal scars. Ann Burns Fire Disasters 2005;18(2):83–8. [13] Knapp TR, Daniels RJ, Kaplan EN. Pathologic scar formation, morphologic and biochemical correlates. Am J Pathol 1977;86 (1):47–69. [14] Jiang Yuzhi, Shuliang Lu. Three-dimensional insights into dermal tissue as a cue for cellular behavior. Burns 2014;40 (2):191–9. [15] Glatter O, Kratky O, editors. Small angle X-ray scattering.. London: Academic Press; 1982. [16] Yang Wen, Sherman Vincent R, Gludovatz Bernd, Schaible Eric, Stewart Polite, Robert O, et al. On the tear resistance of skin. Nat Commun 2015;6:6649. [17] Rosado C, Antunes F, Barbosa R, Fernando R, Estudante M, Silva HN, et al. About the in vivo quantitation of skin anisotropy. Skin Res Technol 2017;23(3):429–36. [18] http://www.esrf.eu/UsersAndScience/Experiments/CRG/ BM26/SaxsWaxs/Rattail..
This research was supported by the National Natural Science Foundation of China (Grant No. 81272110 and 30872685).
Please cite this article in press as: F. Tian, et al., Exploring the collagen nanostructure of dermal tissues after injury, Burns (2019), https://doi.org/10.1016/j.burns.2019.07.040
burns xxx (2019) xxx –xxx
Available online at www.sciencedirect.com
ScienceDirect journal homepage: www.elsevier.com/locate/burns
Exploring the collagen nanostructure of dermal tissues after injury Feng Tian a,b, Yiwen Niu c , Yuzhi Jiang b, * a
Shanghai Synchrotron Radiation Facility, Zhangjiang Lab., Shanghai Advanced Research Institute, Chinese Academy of Sciences, Shanghai, 201204, China b Shanghai Burns Institute, Ruijin Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, 200025, China c The Department of Burns and Plastic Surgery, Ruijin Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, 200025, China
article info
abstract
Article history:
Scar often occurred during wound repair. It was known that there were differences in
Accepted 31 July 2019
collagen structure in dermal tissues at millimeter scale and micron scale, however, it was not
Available online xxx
known whether there were differences in collagen structure in dermal tissues at nanoscale during wound repair. In order to compare the difference at nanoscale, skin samples from
Keywords: Scar tissue Small angle X-ray scattering (SAXS) Wide angle X-ray scattering (WAXS) Azimuth angle Three dimensional (3D) structure Collagen Transmission electron microscopy (TEM)
1.
patients were selected, the control groups were the normal skin from the same patients. These samples were tested by the small angle X-ray scattering techniques (SAXS) and wide angle X-ray scattering (WAXS) techniques. The transmission electron microscopy (TEM) was used as a comparison. The results showed that there were not only significantly differences between the normal tissue and scar tissue, but also between the center and the margin of the scar tissue at nanoscale by SAXS and WAXS, which was not demonstrated by other studies. These findings demonstrated that the SAXS and WAXS were excellent tools to detect the collagen structure at nanoscale and the orientation of the collagen alignment, which was beneficial for skin tissue engineering and skin regenerative medicine. © 2019 Elsevier Ltd and ISBI. All rights reserved.
Introduction
Skin is the outlayer of the body. It is easy to be injured for various pathological reasons, such as abrasion, cut, burns. A scar often forms after wound healed, which results in aesthetic sequelae and even local dysfunction. These complications take greatly bad effect on human life and society. However, the problems cannot be resolved until now, the mechanism of scar formation also cannot be clarified; therefore, it is imperative to make research about it [1]. Skin is divided into three layers, the epithelium, the dermal tissues and the hypodermis [2]. The epithelium is the out layer
of the skin, which is composed of epithelium cells. The dermal tissues are mostly composed of the web of collagen fibers which weaved compactly, the collagen structure is the scaffold of the dermal tissues which provides the micro-environment for cells. The hypodermis is composed of fat tissues and other loose connective tissues. It was known that the scar tissues occurred when the total thickness or the deep thickness dermal tissue was injured [3,4]. When skin was injured, the process of wound repair started to initiate. The process of wound healing was divided into three stages: inflammation, proliferation and remodeling. During inflammation, the wound was covered by temporary matrix, which was formed by fibrin from the
* Corresponding author. E-mail address: [email protected] (Y. Jiang). https://doi.org/10.1016/j.burns.2019.07.040 0305-4179/© 2019 Elsevier Ltd and ISBI. All rights reserved.
Please cite this article in press as: F. Tian, et al., Exploring the collagen nanostructure of dermal tissues after injury, Burns (2019), https://doi.org/10.1016/j.burns.2019.07.040
JBUR 5902 No. of Pages 6
2
burns xxx (2019) xxx –xxx
broken blood vessel and included many inflammatory cells. The proliferation stage was followed after inflammation. During this stage, fibroblasts, recruited from neighbor tissues and remote tissues, started to secret collage fiber, which inter-weaved to form newly structure and replaced the temporary matrix. The new structure matured after remodeling stage and the scar tissue might occur [5]. Therefore, after skin injury, the integrity and continuity of the scaffold might be involved, taking effect on the fibroblasts behavior and resulting in scar formation. Collagen structure was composed of the collagen fibers, while collagen fibers were composed of the collagen molecules, suggesting that there was relationship between the structure at micro scale and the substructure at nanoscale. The change of substructure at nanoscale might take effect on the change of structure at micro scale. The scar tissues had different appearance from the normal tissues, indicating of the different collagen structure between the scar tissues and the normal tissues, which had been confirmed at millimeter scale and micron scale by many studies [5,13,14]. Histologically, the scar tissue was composed of roughly collagen fibers, while the normal tissue was composed of delicate collagen fibers. However, whether there were some differences between them at nanoscale had not been clearly resolved until now. Currently, there were many techniques to explore the structure at nanoscale, such as a transmission electron microscope (TEM), a small angle X-ray scattering (SAXS) and wide angle X-ray scattering (WAXS). TEM could not exhibit the differences between them [10], while the studies by SAXS and WAXS had not been executed. Thus, in order to resolve the problem, the small angle X-ray scattering (SAXS) and wide angle X-ray scattering (WAXS) were used to explore the difference between the normal tissue and scar tissue, and the difference between the center and the margin of the scar at nanoscale. TEM was also used as a comparison.
2.
Method and materials
2.1.
Subjects
Patients with hypertrophic scar were separately from the department of burn and plastic surgery, Ruijin Hospital, affiliated with Shanghai Jiaotong University, Shanghai, China. Informed consent was obtained from all patients and experimental protocols were separately approved by the ethic board of the Ruijin hospital(2013-CEB-No.150). Skin biopsies were obtained from 8 patients with hypertrophic scar for aesthetic surgery. The locations were from hand (2 patients), ankle (2 patients), abdomen (2 patients) and neck (2 patients) (average age 25.2 + 15.4 years). The median duration of hypertrophic scars was 4.1months (rang 3–5 months). The minimal area of scar was 2.5 1 cm2, the maximum area of scar was 3.5 2 cm2. The control samples were obtained close to the wound. The normal samples were trimmed into 0.2-1-cm strips, and at least of 2 samples were got from each patient. The samples were blinded for detected and analyzed.
2.2.
Skin preparation
The samples were cut into 0.2-1-cm strips and divided into two parts. One was treated for TEM; the other was treated for SAXS and WAXS.
2.3.
Small-angle X-ray scattering (SAXS) experiments
After the excess subcutaneous tissue were trimmed during operation, the skin samples were incubated in 0.5% dispase (Roche, Mannheim, Germany) at 4 C over night. The epithelium was stripped and then dehydrated by lyophilisation for 48 h before SAXS and WAXS. The SAXS measurements were carried out at BL16B1 beamline [6] at Shanghai Synchrotron Radiation Facility (SSRF), Shanghai, China. X-ray of 10 keV was used as the incident beam. The scattering intensity was recorded using a Rayonix SX-165 CCD detector (Rayonix, Evanston, IL, USA) with a resolution of 2048 2048 pixels. The distances between the sample and the detector were 1850 mm [7,8].
2.4.
Wide angle X-ray scattering (WAXS) experiments
The measurements were carried out at the same place. The scattering patterns were recorded with a two dimensional (2D) detector MAR CCD (MAR Charge Coupled Device, Marresearch GmbH, Norderstedt, Germany) with a 165 mm diameter, the single exposure times were about 100 s. The distances between the sample and the detector distances were 85 mm. The calibration was performed using silver behenate [9]. The same analysis method was used in both SAXS and WAXS. The scattering intensity was represented as a function of the modulus of the scattering vector: q = (4p/l)sinu, where l was the wavelength and 2u was the scattering angle. The 2D data were averaged to obtain the one dimensional (1D) profile by circular averaging with Fit2D software. The long period (L), which represented the average distance between the regions with equivalent electronic densities, was calculated using the equation: L = 2p/q, where q was the modulus of the scattering vector at the peak maximum.
2.5.
TEM sample preparation
For TEM observation, the skin was cut into 5 mm thick strips by scalpel. A primary fixation was performed by immersing the tissue sections in 2.5% paraformaldehyde, 2.5% glutaraldehyde in 0.1 M cadodylate buffer for 2 h and after fixation was done in 1% osmium tetroxide in 0.15 M cadodylate buffer for 12 h. The specimens were then stained in 1% uranyl acetate for 12 h and dehydrated by ascending ethanol series. The samples were then embeded and subsequently sectioned using a Leica Ultracut UCT ultramicrotome (Leica) and a Diatome diamonds knife. Ultramicrotome sections were then placed on copper grids for TEM observation and after stained with uranyl acetate for 1 h. Finally the samples were observed using a Jeol electron microscope (JEM 2000 FX, Jeol Japan) at 160 kv.
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2.6.
Statistical analysis
All data were expressed as mean + SD of at least three independent experiments. One-way analysis of variance was used for statistical difference. Statistical significance was set at P < 0.05.
3.
Results
3.1.
D SAXS and 2D WAXS human data comparison
There were significantly different between the normal tissues and the scar tissues in 2D WAXS, compared with 2D SAXS; there were also significantly different between the center of the scars and the margin of the scars in 2D WAXS, compared with 2D SAXS (Fig. 1). In order to clarify the differences, 2D data were transformed into the 1D data.
3.2.
1D data from SAXS and WAXS analysis
When transformed from the 2D data, the SAXS patterns showed that there were a series of Bragg reflections with the regular repeating distance which dominated them (Fig. 2), indicating that the skin samples were periodic structure at nanoscale. The relative peak intensity was higher in the scar tissue at the margin area, especially for the 6th peak, the peak intensities were higher after the 6th peak. The patterns on the other two samples were similar. The repeating distances were not significantly different, the distances were 64.0 2.1 nm (the center of scar tissue), 64.4 7.8 nm (the margin of scar
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tissue) and 64.2 3.6 nm (normal tissue) [10], which was the D-period in TEM research [18]. When transformed from the 2D data, the WAXS patterns showed that there was a mountain which declined from left to right on the three samples (Fig. 3). On the normal tissues, there was higher mountain on whose left, there was an abrupt bottom, and on whose right, there was a smooth decline. On scar tissues, there was a lower mountain, on whose left, there was a nipple on the valley bottom at 1.5, on whose top, there was a nipple at 2.5; on whose right, and there was also a nipple at 3.75 on the course of the declination. Compared with the scars at the center location, there was a higher nipple on the bottom, and rugged hills on the scars at the margin location. Due to the differences among the three samples in 2D SAXS and WAXS and different relative intensities from integrated intensity curves, the azimuth angle u was obtained by integration (0 u 360 ). The 6th peak was selected because it had higher relative peak intensity. The analysis results found that there were significantly different at the annular analysis of the 6th reflection between the normal tissue and the scar tissue (Fig. 4). The reflection signals were stronger in scar tissues compared with the normal tissue. Two peaks were exhibited on both of the centers and the margins of the scars, the peaks were at 60 and 260 and the bottom between the peaks was at 150 on both the center and the margin of the scar tissue. On the contrary, there was a flat line on the normal tissue. In order to further analyze the differences between the different locations of the scars, the advanced version of the MATLAB-based SAXS utilities package (http://www.saxsutilities.eu) was used. The results showed that the SAXS pattern of the scar tissue at the marginal area was formed with two arcs
Fig. 1 – 2D SAXS and WAXS human data. (A) 2D SAXS pattern at the center area of the scar tissues; (B) 2D SAXS pattern at the marginal area of the scar tissues; (C) 2D SAXS pattern of the normal skin; (D) 2D WAXS pattern at the center area of the scar tissues; (E) 2D WAXS pattern at the marginal area of the scar tissues; (F) 2D WAXS pattern of the normal skin. Please cite this article in press as: F. Tian, et al., Exploring the collagen nanostructure of dermal tissues after injury, Burns (2019), https://doi.org/10.1016/j.burns.2019.07.040
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Fig. 2 – 1D SAXS. The peaks were divided according to horizontal ordinate. The results showed that the skin samples were periodic structures at nanoscale. The range of q was 2.18 nm 1–0.17 nm 1, which equaled to 2.88– 36.63 nm. The three samples were similar.
Fig. 3 – 1D WAXS. The range of q was 4.69 nm 1–0.87 nm 1, which equaled to 1.36–7.8 nm. There were significantly different among the three samples.
with significant preferential scattering intensity distribution (Fig. 5). The SAXS pattern of the skin at the central scar area was formed with the four arc patterns, but the intensity increased at broader azimuthally angles, which suggested that the mechanical stress, which exerting on the collagen fibers, on the center area was more dispersed compared with on the margin area of the scar tissues. There was not significantly difference among the different anatomical locations by SAXS and WAXS.
3.3.
TEM
There was not significantly difference between the normal skin and scar tissue by TEM. There were also no statistically difference between the margin area and the center area of the
Fig. 4 – The azimuth angle of the 6th peak from three different samples from 1D SAXS, treated by Fit 2D software. There were significantly different between the normal and scar samples. There were similar between different locations of the scar samples.
Fig. 5 – The azimuth angle of the 6th peak from the scar tissues from 1D SAXS, treated by its advanced version. There were significantly different between the center area and the margin area of the scar samples.
scar tissues by TEM (Fig. 6). There was not significantly difference among the different anatomical locations.
4.
Discussion
Skin is the outlayer of the human body and provides protection from the environment. Therefore, it is easy to be injured. When the wound repaired, the scar tissues often form which have ugly appearances and uncomfortable feelings, such as itching and pain, even result in local dysfunction. These imperfections take greatly negative effect on the human life and society. Therefore, it is the great impetus to treat them [1]. However,
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Fig. 6 – TEM of the center area of the scar tissues, the margin area of the scar tissues and the normal tissues from hand (the same scar tissue, the same patient). (A) The center area of the scar tissues; (B) The margin area of the scar tissues; (C) the normal tissues.
the mechanism of the scar formation still cannot be clarified and the treatments have not been satisfied until now [11]. Skin is divided into three layers: epithelium, dermal tissue and hypodermal tissue [2]. The epithelium is composed of cells; the dermal tissues are mostly composed of collagen fibers which weaved together into collagen structure as a scaffold. When the deep thickness dermal tissues and the full thickness of the dermal tissues were injured [3,4], the scaffold of the dermal tissues, which was composed by collagen structures, was mostly injured, this would affect the repair process and result in scar formation, indicating of the important role of the integration and continuity of the scaffold. Currently, there were obvious differences between the normal tissue and scar tissues by appearance and immunohistology. In scar tissues, HE staining section showed that the epidermal ridges were obviously lacked and the dermis was thicker, hyperemic and more cells infiltrated heavily, which represented more contribution to the greater deposition of extracellular matrix (ECM) in the dermis [12]. Compared with the normal skin, the collagen fibers were less structured, tougher, less clearly demarcated [13], which was consistent with our previous findings [14]. Our previous findings showed that the normal tissue had the feature of ovalshaped webs, which was not found in scar tissues, suggesting that the compromised ability of weaving of collagen fibers occurred after skin injury. Due to the structure at micro scale was composed of the substructure at nanoscale, there was a series of questions based on these findings: Was there difference at substructure at nanoscale between the scar tissues and the normal tissues? At which scale was the ability of weaving of collagen fiber injured? At which scale did the scar formation initiate? In order to resolve these questions, the exploration of the substructure at nanoscale should be made. TEM was the golden standard for presenting the structure of collagen fibers at nanoscale, however, it could not make differentiate between the normal skin and the scar tissue [10], which confirmed by our research. Apart from TEM, SXAS and WXAS were also excellent tools to explore the substructure at nanoscale. Here the SXAS and WXAS technique were applied in order to resolve these problems.
SAXS was a small-angle scattering technique. The elastic scattering of X-rays which passing through a sample was recorded at very low angles (typically 0.1–10 ). It was capable of delivering structural information of macromolecules between 5 and 25 nm, of repeat distances in partially ordered systems up to 150 nm [15,16]. WAXS was a wide-angle scattering technique; it could be operated at the same platform as SAXS. The distance from sample to the detector was shorter and the diffraction angles were larger. The analysis protocol of the two techniques was the same. In this paper, SAXS and WAXS showed there were differences among the normal tissue, the center area and the margin area of the scar tissues. However, the differences were more obvious by WAXS. From 2D data, the samples were composed of several concentric circles. The color of the rim of the circles was different and the arcs of the circle were different in different degrees, which meant the different intensity distribution, suggesting that the samples had the periodic structure; the samples had the feature of anisotropy because there were different mechanical properties at different directions. The results were consistent of Rosado C’s studies [17]. After transformation from the 2D data, the tissues were further presented as a periodic structure, which was marked by different peaks (Fig. 2). The periodic distance were measured, which was the characteristic signal from the stacking of human skin collagen fibers [7,18], and confirmed by our TEM research. The 6th peak was more obviously different at the 1D data and thus was selected and analyzed by the azimuth angle u, which meant the different intensity reflected in 360 direction and could demonstrate the distribution of the mechanical stress and the orientation of collagen fiber alignment. There were significantly different types of curves and the relative intensity between the normal tissue and scar tissue if treated by Fit 2D software, suggesting that there were more tensions in scar tissues. In order to explore the substructure between the different locations of the same tissues, the 6th peaks at 1D data from SAXS were further treated by its advanced version of the MATLABbased SAXS utilities package (http://www.saxsutilities.eu), the
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results showed that there were more preferential scattering intensity distribution at the margin area of the scar tissue, while there were more extensive intensity distribution which increased at broader azimuthally angles at the center area of the scar tissue, suggesting that there were more mechanical stress at the margin area, while there were more evenly distributed orientation of collagen structures at the center area. In conclusion, compared with the normal tissues, the scar tissues had more tensions; while there were more tensions at the margin area of the scar tissue, compared with at the center of the scar tissues. Although there were many other proteins in the dermal tissues, the quantity of them were small, compared with collagen protein. They could not affect periodic locations of the collagen structure by SAXS and WAXS. Furthermore, it was difficult to make differentiate among the other proteins. Currently, the SAXS and WAXS techniques could only be used to detect the collagen structure of the skin tissues, the other protein, such as the structure of the keratin or the structure of the elastin, could not be detected. In addition, the research could not find any structural differences in different locations, which was confirmed by our clinic. Clinically, a skin defect from any regions, such as the back of the hand, could be theoretically transplanted by the full thickness skin grafts from many other different donor locations, such as the abdomen, the thigh, and the forearm. Because the samples used in TEM was dehydrated, the samples used in SAXS and WAXS were also dehydrated. The lyophilisation was selected because it was widely used in the preparation of biological product and in the biological molecular experiments without changing their biological activity.
5.
Conclusion
The orientation of the collagen alignment was different among the normal tissue and the different locations of the scar tissue at nanoscale. The scar tissue had more tension compared with the normal tissues. The margin area of the scar tissues had more tensions compared with the center of the scar tissues. In addition, compared with other measurement, the SAXS and WAXS could provide the 3D information about the tissues. They were convenient tools in exploring the difference of scar and normal tissue.
Competing interests The authors declare that they have no conflict of interest.
Acknowledgement
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This research was supported by the National Natural Science Foundation of China (Grant No. 81272110 and 30872685).
Please cite this article in press as: F. Tian, et al., Exploring the collagen nanostructure of dermal tissues after injury, Burns (2019), https://doi.org/10.1016/j.burns.2019.07.040