Development of a novel collagenous matrix based on tissue

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Development of a novel collagenous matrix based on tissuemimicking advanced collagen aggregate synthetically crosslinked with biological crosslinkers, OCS and #-ODAP for wound healing Liu Xinhua, Zhuan Yan, Xuechuan Wang, Luo Xiaomin, Taotao Qiang, and Weihua Dan ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/ acssuschemeng.8b04529 • Publication Date (Web): 01 Nov 2018 Downloaded from http://pubs.acs.org on November 5, 2018

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ACS Sustainable Chemistry & Engineering

Development of a novel collagenous matrix based on tissue-mimicking advanced collagen aggregate synthetically crosslinked with biological crosslinkers, OCS and β-ODAP for wound healing Xinhua Liu a, b,* , Zhuan Yana, b, Xuechuan Wanga, b,*, Xiaoming Luoa, b, Taotao Qianga, b, Weihua Dan c* aCollege

of Bioresources Chemical and Materials Engineering, Shaanxi University of Science & Technology, WeiYang District, Xi’an 710021, Shaanxi, China

bNational

Demonstration Center for Experimental Light Chemistry Engineering Education,

Shaanxi University of Science & Technology, WeiYang District, Xi’an 710021, Shaanxi,China cResearch

Center of Biomedical Engineering, Sichuan University, No.24 South Section 1, Yihuan Road, Chengdu 610065, China

*Corresponding author: [email protected]; [email protected]; [email protected]

Abstract In this paper, the novel tissue-mimicking advanced collagen aggregate (TA-CA) consisting of collagen fibers and collagen fiber bundles functionally crosslinked with dialdehyde chitosan (OCS) and dencichine (β-ODAP) was developed as an efficacious wound healing device with hemostatic property. The synergistic cross-linking effects of β-ODAP and OCS on TA-CA was systematically studied by taking OCS, glutaraldehyde (GA), genipine (GP) and β-ODAP as control groups. The results showed that the thermal stability, mechanical properties, hydrophilicity, enzyme resistance and hemostatic properties of TA-CA modified by β-ODAP and OCS (OCS/β-ODAP-TA-CA) were significantly improved. Meanwhile, the MTT assay and CLSM observation showed the OCS/β-ODAP-TA-CA scaffold presented no cytotoxicity, better cell proliferation, and enhanced viability of primary fibroblast. OCS/β-ODAP-TA-CA scaffold was implanted into Sprague-Dawley rats to evaluate

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the ability to repair full-thickness skin wounds regarding collagen crosslinked with OCS/β-ODAP

(OCS/β-ODAP-Col)

as

control.

Results

revealed

that

OCS/β-ODAP-TA-CA scaffold could better accelerate the wound healing process and more effectively promote bFGF, VEGF and PFDF expression compared with OCS/β-ODAP-Col. Overall, this work contributed a new insight into the development of TA-CA scaffold modified with OCS and β-ODAP with excellent biocompatibility and hemostatic properties, and further confirmed TA-CA could serve as a better alternative source of collagens for biological applications. Keywords: collagen aggregate, dialdehyde chitosan, pseudo-ginseng, synergetic crosslinking modification, hemostatic materials

Introduction Collagen is the most fundamental constituents in extracellular matrices of the connective tissues in all multicellular organisms. With the advantages of high biocompatibility, appropriate biodegradability, sustainability, and only weakly antigenicity, collagen is widely used in the field of biomedical materials.1-5 At present, collagen raw materials available in the market is usually the collagen molecules produced by the degradation of collagen aggregates through biological enzymes. So they present disadvantages like poor mechanical properties, discomfort of biodegradability, inferior stability (thermostability and structural-stability), etc. Namely, most of the properties are not comparable to natural collagen, which greatly limits their biomedical applications.6-8 From the perspective of collagen’s biosynthesis in vivo, tissue-mimicking advanced collagen aggregate (TA-CA) has a complete three helix structure and they are the elementary building blocks in the collagen-rich tissues easily recognized by a 65–67 nm axial periodicity , so they showed many advantages, such as the glory of biology, strong enzyme resistance and so on. Therefore, it is assumed that if TA-CA can replace collagen as a raw material for collagen based medical materials, it will make up the deficiencies of the physical and chemical


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properties of collagen materials,9-10 thus providing new ideas for the construction of collagen based medical materials. In recent years, to signally enhance the natural physicochemical performances, collagen-based biomaterials are necessary to be crosslinked chemically or physically. Thereinto, synthetic chemical modifiers, including histone deacetylase (HDACS), formaldehyde, glutaraldehyde are most commonly utilized. However, these chemical modifiers still have the problems of poor cytocompatibility and unsatisfactory physical and chemical properties.11 Therefore, some biocompatible crosslinkers including proanthocyanidin and aldehyde alginate are brcoming more and more popular among researchers.12-14 It is particularly important to note whether the biocompatibility of collagen after modification can reach the medical standard. Therefore, developing a kind of ideal biotype cross-linking agents suitable for protein modification is expected. This kind of cross-linking agent itself should have excellent biocompatibility and will not introduce extra toxicity to collagen. Besides, it can also produce multi molecular crosslinking effects among collagen molecules. What’s more, it can obviously improve the physical and chemical properties of collagen noumenon. Chitosan (CS) is derived from chitin which is the second natural organic polymer material. Owing to its efficacy of antibiosis, anti-inflammatory and hemostasis as well as good biocompatibility and biodegradability, chitosan has been widely used in wound dressings, tissue engineering scaffolds and absorbable hemostatic.15-17 The chemically active aldehydes in dialdehyde chitosan (OCS) derived from chitosan (CS) can provide binding sites for free amino groups of proteins to form the stable Schiff’s base, as demonstrated in our previuos study. In addition, the flexibility of the molecular chain could be obviously improved after the oxidation of chitosan, which could slow down the steric hindrance effects of the molecular chain and provided more favorable structural conditions for the coupling of proteins. Furthermore, our previous report showed that the formed stable Schiff’s base between OCS and collagen has no significative effect on the structural integrity and bioactivity of collagen. Based on this, the feasibility of utilizing OCS as a novel chemical crosslinker in the modification of TA-CA is necessary to be evaluated.



Pseudo-ginseng has been widely used in the practice of traditional Chinese hemostatic medicine for thousands of years. It has remarkable curative effect in treating traumatic injuries, stomach and duodenal ulcers and other internal bleeding.18 In 1981, hemostatic component of pseudo-ginseng first isolated and identified, i.e. “dencichine” (β-ODAP).19 Studies have revealed that dencichine is responsible for the medicinal herb’s main haemostatic and platelet-increasing properties in vivo.20-22 Our group have conducted collagen-dencichine composite sponge and demonstrated that the hemostatic properties and physicochemical properties of collagen had been significantly improved by β-ODAP. However, the role of β-ODAP in playing in the modification of TA-CA’s structure and properties remains unknown, and it could effectively promote the development of novel functionalized hemostatic biomaterials. In this article, TA-CA consisting of collagen fibers and collagen fiber bundles was derived from porcine acellular dermal matrix (pADM). Dialdehyde chitosan (OCS) with appropriate oxidation degree was prepared by selective oxidation of chitosan with sodium periodate. The dencichine (β-ODAP) was extracted from traditional Chinese hemostatic medicine, pseudo-ginseng. Functional modification of TA-CA with β-ODAP and OCS was carried out through synergistic action. The synergistic cross-linking effects of β-ODAP and OCS on TA-CA were systematically studied by taking OCS, glutaraldehyde (GA) , conventional crosslinker genipine (GP) and β-ODAP as control groups. This work will lay a solid theoretical and experimental foundation for building functional hemostasis materials based on tissue-mimicking advanced collagen aggregates. In this paper, we described a novel and facile strategy to fabricate TA-CA scaffold functionally modified with OCS and β-ODAP (OCS/β-ODAP-TA-CA). The thermal stability, mechanical properties, hydrophilicity, enzyme resistance and hemostatic properties in vitro and in vivo of OCS/β-ODAP-TA-CA scaffold were systematically characterized, and the actual bioactivity of final OCS/β-ODAP-TA-CA scaffold was determined by detecting the in vitro cell adhesion, distribution and proliferation and measuring the in vivo wound healing effects, particularly assessing the expression of in vivo basic fibroblast growth factor (bFGF), vascular endothelial


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growth

factor

(VEGF)

and

platelet

derived

growth

factor

(PDGF)

of

OCS/β-ODAP-TA-CA scaffold by immunohistochemistry to evaluate its efficacy. This work will lay a solid theoretical and experimental foundation for building functional hemostasis materials based on tissue-mimicking advanced collagen aggregates.

Experimental Chemicals and materials TC-CA was extracted and purified by the combination of acid and enzyme from porcine skin according to our previous methods and in turn lyophilized in a freeze dryer.23 pADM, OCS and β-ODAP were prepared in reference to our previous reports.24-25 Unless noted otherwise, all chemicals and reagents were purchased from Sigma–Aldrich (St. Louis, MO, USA). Preparation

of

TA-CA,

β-ODAP/OCS

synergistic

crosslinked

TA-CA

membrane(β-ODAP/OCS-TA-CA), β-ODAP/OCS synergistic crosslinked collagen membrane(β-ODAP/OCS-Col), GA crosslinked TA-CA membrane and GP crosslinked TA-CA membrane The preparation method of TA-CA according to our previous work.26 The perparation process of β-ODAP/OCS-TA-CA is as follows. The concentration of TA-CA swelling solution was diluted to 5mg/mL by 0.5M acetic acid sodium acetate buffer solution whose pH is 4.5. It was followed by the slow addition of 30% OCS and 15% β-ODAP to the TA-CA swelling solution and the solution was magnetically agitated at low temperature for 48h. After the mixed solution was supersonic in ultrasonic cleaner (40 kHz, 120W) for 1min, several drops of ethanol were added to remove the bubble thoroughly. Finally, the β-ODAP/OCS-TA-CA was prepared by drying at room temperature for a week.23-24 The preparation process of β-ODAP/OCS-Col changed the TA-CA mentioned in the perparation process of β-ODAP/OCS-TA-CA into the collagen available in the market, and the rest of the operations are the same. In addition, the preparation method of GA crosslinked



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TA-CA membrane and GP crosslinked TA-CA membrane refer to Macaya, D. J.27 Cross-linking index The crosslinking index of Col was determined by ninhydrin method and the crosslinking

index

was

Crosslink density (%) =

calculated.

Specific

(NH2before −NH2after ) NH2before

formula

is

as

follows.28

× 100

(1)

In the formula (1) , NH2before is the free amino content of Col before crosslinking, and NH2after is the content of free amino group after crosslinking. Besides, for β-ODAP, OCS, β-ODAP/OCS groups, NH2before is the total free amino content of Col and β-ODAP or OCS or β-ODAP/OCS. Atomic force microscopy (AFM) The mica tablets carrying samples were directly placed on the AFM(SPM-9600, SHIMADZU Co., Ltd.) sample table and the observation was set in non-contact tapping mode. The length of the AFM probe cantilever is 180 m, the tip radius of curvature is 10 nm, and the force constant is 2.8 N/m. During the observation, each sample was randomly selected in 6 different areas for scanning. Differential scanning calorimetry (DSC) The 3-5 mg samples before and after modification were sealed in the DSC (DSC-200PC PHOX, Netzsch Co., Ltd.) aluminum crucible respectively. The temperature range was set from 20℃ ~120℃ and the heating rate was 10 ℃ /min. Nitrogen was used as the protection atmosphere (the flow rate was 60mL/min). Mechanical properties The maximum elongation, tensile strength and elongation at break of membrane material were synthetically measured according to the previous reported method.29 Each sample was tested for three times and the average value was calculated. Surface contact angle The surface hydrophobicity of the membrane materials was measured by the video contact angle measuring instrument (OCA-H200, Dataphysics Co., Ltd.) with droplet method. The deionized water after boiling was carefully dripped onto the surface of films to collect the instant images of the contact interface between the


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droplets and the surface of the films. The samples were tested in six different surface areas and the average value was calculated. In vitro biodegradability study The initial mass of vacuum freeze-dried membrane materials was recorded as W0. The membrane samples were soaked in the degradation solution and the samples were degraded in a constant temperature biochemical incubator at 37 ℃. After a period of time, the sample quality was weighted and recorded as Wt after sampling and vacuum freeze-drying. The degradation rate of membrane material is as follows. Degradation rate (%) = (W0- Wt)/W0×100%

(2)

Evaluation of hemostatic performance in vitro According to the practice of Esmedere ES,30 the activated partial thromboplastin time(APTT), prothrombin time (PT), thrombin time(TT) and total coagulation time(CT) of TA-CA before and after crosslinked were evaluated. In vitro biocompatibility study According to the international standard31-32 and the method of Streifel. BC,33 the cytotoxicity of TA-CA was evaluated by MTT method with L929 as the model cell.34-35 The growth and proliferation of cells on a sample composite sponge were observed by confocal laser scanning microscopy (CLSM).36 In vivo study Fifteen Sprague-Dawley rats, weighing 300-350 g, were randomly assigned to three groups (the control (GA), β-ODAP/OCS-Col and β-ODAP/OCS-TA-CA groups). All experimental animals were handled according to the guidelines formulated by the National Institutes of Health on human use and care of laboratory animals. All procedures performed on animals were approved by the Animal Care and Use Committee of Sichuan University. A rat model of wound healing was constructed on the basis of our previous study.37 All rats were euthanized at post-operation 1, 2 and 3 weeks and each specimen with surrounding tissues were collected. Paraffin-embedded tissue samples were sectioned to a thickness of 5 μm and stained with hematoxylin and eosin (H&E) according to standard protocols.38 Furthermore, the expression level of basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF)



and PDGF have been tested by ELISA kit according to previous study .39-41。

Results and discussion Cross-linking index analysis The cross-linking index between various modifiers and collagen differs with their molecular interaction mode and binding sites. The cross-linking index between various modifiers and collagen is shown in Fig.1. It could be found that the cross-linking index between β-ODAP and TA-CA presents the lowest. This is probably because the hydrogen bonds and ionic bonds with smaller bond energy fail to make them bind firmly, as a consequence, the cross-linking index could not be further promoted with the dosage of β-ODAP increased. Our previous study has proved that the complex interaction force of Schiff’s base, hydrogen bonds, ion bonds and hydrophobic bond could be formed among TA-CA, β-ODAP and OCS.21-23 Therefore, the cross-linking index between TA-CA and β-ODAP/OCS exhibits the highest. It is also confirmed that the positive synergistic crosslinking effect of β-ODAP and OCS can sostenuto enhance the cross-linking efficiency with TA-CA. Meanwhile, compared with OCS, although the mechanism of molecular interaction with TA-CA is similar, because of the relatively short molecular chain length of GP and GA, these two kinds of modifiers can not form a strong intermolecular crosslinking effects with TA-CA according to the so-called "Zero-length cross-linking mode" ,42-43 which seriously limits the cross-linking index with TA-CA.

Figure 1. The effect of GP, GA, β-ODAP, OCS and β-ODAP/OCS on the crosslink density of TA-CA.


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AFM analysis AFM could be used to analyze the effects of different cross-linking modification methods on the microstructure of TA-CA, especially its typical alternating gap and overlap regions along the collagen fibril, i.e. the D-periodic structure. Fig.2 shows the AFM images observed from the air-dried samples of TA-CA under different crosslinking methods. It is found that after crosslinking, TA-CA still presents the typical three-dimensional netlike structure. Fig.2 exhibits compact inter-fiber entanglement and tightly woven collagen fiber bundles or collagen fibrils with uneven diameters and non-orientated arrangement, and the typical D-periodic cross-striated patterns about 65nm along the longitudinal direction of TA-CA were clearly observed, indicating that the microstructure integrity of crosslinked TA-CA could be still maintained after the chemical modification of GP, GA, β-ODAP, OCS and β-ODAP/OCS. Even more notably, as shown in Fig.2, the degree of entanglement of collagen aggregates become much more obvious after crosslinked by OCS or β-ODAP/OCS, which might prove that OCS with the nature of sufficient molecular chain length could form multi-scale intermolecular crosslinking with TA-CA. Therefore, its cross-linking index is higher and many multitudinous interlaced network microstructure of collagen aggregates takes shape, which are significative to improve the mechanical properties and structural stability of TA-CA. Further, the D-periodicity of a small amount of blocky fibers could not discern clearly after crosslinked by GP, GA, OCS and β-ODAP/OCS, which indicates that the integrated forces between GP, GA, OCS, β-ODAP/OCS and TA-CA including Schiff’s base, hydrogen bond and ion bond, has negative effects on the natural short N- and C-terminal overlap area within the staggered array of collagen molecules in TA-CA.



Figure 2. The AFM images of TA-CA after crosslinked by β-ODAP（A）, OCS（B）, β-ODAP/OCS（C）, GP（D）and GA（E）.

DSC analysis The primary purpose of chemical cross-linking of collagen is to improve its thermal stability and make it more resistant to phase transformation under external heat. Obviously, the thermal denaturation temperature (Td) of TA-CA has been improved to varying degrees after crosslinked by β-ODAP, OCS, β-ODAP/OCS, GP and GA. Fig.3 exhibits that the increase in Td of crosslinked TA-CA presents not apparent. The reason may be that the side chain polar groups (-NH2, -OH, -COOH, etc.) of TA-CA are relatively less compared to collagen molecules, leading to a inadquatesource of efficient crosslinking effects. Coupled with its more complex crisscross interlacing network proved by AFM analysis, a steric hindered chemical microenvironment could be formed, therefore, it becomes more detrimental to the penetration and combination of the crosslinkers. The Td of TA-CA could reach to 87 ℃ after crosslinked by β-ODAP/OCS, which shows higher that of TC-CA crosslinked by other crosslinkers, and the thermal stability is improved significantly, which is in agreement with the results of cross-linking index. It indicates that OCS with long enough molecular segments can form intermolecular schiff’s base covalent


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bond with the primary amino groups of TA-CA so that collagen fibers in TC-CA could be concatenated in the form of a five carbon bridge. In addition, the -NH2 and -OH polar groups in OCS and β-ODAP molecules can further produce physical crosslinking with TA-CA and the synergistic croslinking effects has greatly enhanced the thermal stability of TA-CA.

Figure 3. DSC curves of uncrosslined TA-CA(73.7℃) and TA-CA after crosslinked by β-ODAP(74.0℃), OCS(90.5℃）, β-ODAP/OCS（96.0℃）, GP（77.8℃） and GA(76.7℃).

Mechanical properties As is known, the mechanical properties of collagen could be influnced greatly by different cross-linking methods. Fig.4 exhibits the effect of different crosslinking methods on the mechanical properties of TA-CA. As shown in Fig.4, β-ODAP/OCS, OCS, GA, GP and β-ODAP could enhance the mechanical properties of TA-CA to different degrees. Generally, the higher crosslinking index, the greater influences of crosslinking agents on the mechanical properties of collagen. Actually, β-ODAP/OCS can significantly improve the mechanical properties of TA-CA. The tensile strength and elongation at break of TA-CA crosslinked by β-ODAP/OCS are 5.8MPa and 45.7%, respectively, which are significantly better than those of TA-CA crosslinked by OCS, GA, GP or β-ODAP. It might further confirm that OCS can bond the free amido of adjacent or relatively distant collagen fibers in the form of "five carbon bridge" Schiff’s base to form crisscross interlaced interlacing network through its long "arm spread". At the same time, OCS and β-ODAP can also crosslinked with TA-CA



by physical binding mode with relatively weak hydrogen bonds and ionic bonds. Therefore, the multiscale synergistic cross-linking effects among the intramolecular and intermolecular collagen fibers can promote the basic three-dimensional micro-network structure of TA-CA to form a more regular and ordered "rigid supramolecular aggregate structure", which might make the strength of TA-CA significantly improved. In addition, the more stable supramolecular 3D interleaving network makes TA-CA more resistant to deformation when it is subjected to external stretching, which is more likely to lead to the phenomenon of "brittle fracture".

Figure 4. The effect of different crosslinking methods on the mechanical properties of TA-CA ((A) No crosslinking group as the control, (B) β-ODAP group, (C) GP group, (D) GA group, (E) OCS group and (F) β-ODAP/OCS group).

Surface contact angle analysis Studies have shown that the surface of biomaterials with proper hydrophilicity is conducive to the adhesion and ingrowth of subsequent inoculated cells.44 Therefore, it is of great significance to improve the surface hydrophilicity of TA-CA through proper crosslinking. Fig.5 presents the effects of different crosslinking methods on the surface contact angle of TA-CA. As shown in Fig.5, except for GA, the Water contact angle (WCA) values of TA-CA under different crosslinking methods decrease to varying degrees, which is attributed to the existence of more polar functional groups, including -NH2, -OH, -C=O and glycosidic bonds in the molecular chain of β-ODAP and OCS. Although the -NH2 content in TA-CA decreases after crosslinking, more polar groups could be introduced, which could effectively enhance the hydrophilicity of TA-CA. Meanwhile, β-ODAP and TA-CA are only combined with hydrogen bonds


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and ionic bonds, which could not reduce the polar functional groups of TA-CA. Therefore, it can be concluded that β-ODAP has greatest influences on the hydrophilicity of TA-CA. The WCA value of TA-CA can be reduced to ~60° by the synergistic crosslinking effects of β-ODAP/OCS. The hydrophilicity of TA-CA could be meaningfully improved, and the results accord with the expected architecture design.

Figure 5. The effect of different crosslinking methods on the Water contact angle(WCA) of TA-CA ((A) No crosslinking group as the control, (B) GA group, (C) GP group, (D) OCS group, (E) β-ODAP group and (F) β-ODAP/OCS group).

Resistance to enzyme degradation in vitro Designing biomaterials which can adapt the rate of biodegradation to the rate of tissue growth and repair has always a research focus. The commonly used methods to regulate the biodegradability of collagen matrix biomedical materials are crosslinking or specific structure design. Fig.6 shows the degradation of TA-CA after different cross-linking methods in type I bacterial collagenase physiological degradation fluid. As shown in Fig.6, β-ODAP, OCS, β-ODAP/OCS, GP and GA could enhance the enzyme resistance of TA-CA in varying degrees. The results of resistance to enzyme degradation in vitro are in agreement with that of DSC analysis. Under the positive synergistic cross-linking interactions, including the Schiff’s base covalent bonds, hydrogen bonds, ion bonds, etc., the TA-CA only degrades by 10% after 7d. After 29d, there is still 60% of TA-CA remained undegraded, which suggests that the enzyme



resistance of TA-CA cross-linked by β-ODAP/OCS is significantly improved. However, β-ODAP whose cross-linking effect is only a physical compound to TA-CA, therefore it has little effects on the enzyme resistance of TA-CA. It is worth noting that the basic tissue morphology and microstructurecollagen matrix scaffolds need to be maintained in a specific time frame in the tissue repair process in order to preserve the corresponding physical and chemical properties and biological functions. Therefore, it is of great significance to improve the enzyme resistance performance of TA-CA in the physiological environment to its subsequent biomedical applications.

Figure 6. The effect of different crosslinking methods on the resistance to degradation of TA-CA ((A) No crosslinking group as the control, (B) β-ODAP group, (C) GP group, (D) GA group, (E) OCS group and (F) β-ODAP/OCS group).

Hemostatic performance in vitro The TT and APTT could mainly reflect the endogenous coagulation efficiency of the hemostatic, while PT is an important indicator of the exogenous coagulation system. The APTT, PT and TT of TA-CA under different cross-linking methods are shown in Figure 7. Evidently, β-ODAP can significantly reduce the APTT and TT of TA-CA but has no significant effects on PT. OCS, GP and GA have no prominent effects on APTT, TT and PT of TA-CA, suggesting that β-ODAP can promote the expression of coagulation factor (VIII, IX, XI) of TA-CA. Therefore, the endogenous coagulation process of TA-CA could be accelerated by β-ODAP. However, β-ODAP, OCS, β-ODAP/OCS, GP and GA could not dramatically activate the expression of


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coagulation factors V, VII and X in the exogenous coagulation system. Moreover, OCS, GP and GA had no distinct effects on the endogenous coagulation process of TA-CA. To a certain extent, it may be inferred that OCS, GP and GA have indirect coagulation effects. Further, the CT of TA-CA under different cross-linking methods is presented in Fig.8. It can be seen from Fig.8 that CT of TA-CA has different degrees of descending after crosslinked by β-ODAP, OCS, β-ODAP/OCS, GP and GA. Moreover, its CT decreases significantly after crosslinked by β-ODAP/OCS. Combined with the conclusions from Fig.7, the coagulation mechanism of TA-CA after crosslinked by the composite synergistic crosslinker, β-ODAP/OCS, could be summarized as follows: (1) the TA-CA noumenon obviously activates the endogenous coagulation system and then forms physiological clotting; (2) β-ODAP can promote the expression of endogenous coagulation factor (VIII, IX, XI) and increase fibrin level, and then induce the first and second phase aggregation of platelet; (3) the indirect hemostasis effects of OCS can also improve the hemostatic performances of TA-CA. Furthermore, the CT of TA-CA after crosslinked by GA and GP has also declined but it is not obvious, which may be related to the more compact 3D network structure.

Figure 7. The effect of different crosslinking methods on the APTT, PT and TT of TA-CA.



Figure 8. The effect of different crosslinking methods on the CT of TA-CA. Uncrosslinking group (A), β-ODAP (B), OCS (C), β-ODAP/OCS (D), GA（E）and GP (F).

In vitro cytotoxicity Up to date, the high or relatively high cytotoxicity of chemical cross-linkers has been found in the literature, which has confined the biomedical applications of crosslinked collagen matrix biomaterials accordingly. The effects of different crosslinking methods on the growth activity of L929 fibroblasts on TA-CA are shown in Fig.9 and Fig.10. It can be found that compared with the blank control group, the GA group shows an obvious inhibitory effect on cell growth. Meanwhile, as shown in Fig.10, the number of cells seems to be relatively few. Therefore, we can infer that GA completely destroys the superior cytocompatibility of TA-CA and shows great cytotoxicity. After crosslinked by β-ODAP, OCS, β-ODAP/OCS and GP, TA-CA could obviously promote cell growth and proliferation. Furthermore, the cytocompatibility of TA-CA crosslinked by 30%β-ODAP/15%OCS is even better than that of genipin, an acknowledged nontoxic biologic cross-linking agent for collagen. It has been confirmed repeatedly that the diffusion of the excessive free aldehyde groups could react with amino group of proteins or polysaccharides on and inside the fibroblasts, leading to the cytotoxicity nature. Therefore, a slight excess of aldehyde groups of OCS could be be completely eliminated by the amino group of β-ODAP though the schiff’s base reaction. Moreover, the TA-CA backbone can also cover some of the free aldehyde groups, hence, the synergistic effects lead to the more


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superior biocompatibility of 30%β-ODAP/15%OCS crosslinked TA-CA.

Figure 9. The effect of different crosslinking methods on the proliferation of fibroblasts on TA-CA at different time intervals (1d, 3d and 5d).

Figure 10. CLSM images of fibroblasts on TA-CA after crosslinked by different methods at day 3. GA（A）,β-ODAP（B）,OCS（C）,β-ODAP/OCS（D）and GP（E）.

In vivo evaluation of wound healing Histological study Representative histological changes of wound sections stained with H&E photographs of wound healing for the control group (GA), β-ODAP/OCS-Col and



β-ODAP/OCS-TA-CA group at time point of 1, 2 and 3 weeks are shown in Fig.11. Different degrees of inflammation were found in the wound one week later. The control group and β-ODAP/OCS-Col group had relatively strong inflammatory reaction and a large number of inflammatory cells were gathered at the wound healing area, compared to β-ODAP/OCS-TA-CA group. Noted that there were no statistically significant differences in the number of inflammatory cells in β-ODAP/OCS-TA-CA group. Moreover, the reborn collagen fibers in control group and β-ODAP/OCS-Col group seemed to be non-uniformly distributed and appeared looser and less well-ordered. After 2 weeks of post-surgery, it was found that inflammatory cells in each group decreased significantly, especially in β-ODAP/OCS-TA-CA group. Meanwhile, the formation of epithelium was evident with dense collagen fibers in β-ODAP/OCS-Col and β-ODAP/OCS-TA-CA groups. However, there was a greater degree of granulation tissue in β-ODAP/OCS-TA-CA group. Besides, the dermal layer in both groups was basically repaired, and the basal cells were neatly arranged and small vascular tissue could be observed. After 3 weeks, the natural frame-structure of the skin in β-ODAP/OCS-TA-CA group was completely reconstructed. And the basal cells were arranged in a single layer and arranged closely, and there were more fibroblasts in the dermis. Noted that the skin appendages including the hair follicle and the sebaceous gland, could be also observed obviously. Further, compared with β-ODAP/OCS-Col group, the epidermis of the newborn skin seemed to be thicker, and the collagen fibers of the dermis were arranged in a transverse order, and the knitting was more compact in β-ODAP/OCS-TA-CA group, suggesting a better restorative efficiency of wound healing.


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Figure 11. HE staining diagram of three kinds of scaffolds (a: control group GA, b: col, c: β-ODAP/OCS-TA-CA) postoperatively (a1,b1,c1: 1 week; a2,b2,c2: 2 weeks; a3,b3,c3: 3 weeks) ; (the black arrows point at epithelial cell group and the blue arrows point at regenerated epithelial tissue).

Immunohistological study The growth factors can stimulate the formation of granulation tissue and the matrix, promote the epithelialization and the healing process of the skin wounds. Therefore, these factors play a very important role in the process of skin wound healing.45 Thereinto, bFGF, VEGF and PDGF are particularly important for the formation of healing matrix. The specific expression in the process of skin wound repair can induct the formation of capillaries and reflect the ability of inducing angiogenesis and regeneration.46-47 Figure 12-14 illustrated the immunohistochemical results of bFGF, VEGF and PDGF expression in the control group (GA), β-ODAP/OCS-Col and β-ODAP/OCS-TA-CA groups, respectively. From the analysis of H&E stained sections of wound healing, the wound healing ability of β-ODAP/OCS-TA-CA group presented significantly better than that of β-ODAP/OCS-Col and control groups. Likewise, Figs. 12-14 presented the same



would healing effect as that of the H&E staining images (β-ODAP/OCS-TA-CA > β-ODAP/OCS-Col > GA group). Specifically, at the same time point after post-surgery, β-ODAP/OCS-TA-CA group exhibited higher positive expression than that of β-ODAP/OCS-Col and control groups on the expression level of bFGF, VEGF and PDGF, which mainly attributed to the initial preferable biocompatibility of β-ODAP/OCS-TA-CA, promoting the growth and proliferation of fibroblasts, and thus effectively enhancing the cell secretion of the corresponding growth factors. Noted that these growth factors can form positive feedback regulation with cell growth and proliferation, thereby accelerating the wound healing process. In summary, β-ODAP/OCS-TA-CA presented better bioactive nature in promoting the expression level of bFGF, VEGF and PDGF, which was beneficial for wound healing. The results further confirms the obvious advantages of TA-CA in biological function, and has the potential to replace traditional collagen raw material (Col) as a new generation of collagous matrix for biomedical materials.

Figure 12. bFGF diagram of three kinds of scaffolds (a: control group GA, b: col, c: β-ODAP/OCS-TA-CA) postoperatively (a1,b1,c1: 1 week; a2,b2,c2: 2 weeks; a3,b3,c3: 3 weeks) .


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Figure 13. PDGF diagram of three kinds of scaffolds (a: control group GA, b: col, c: β-ODAP/OCS-TA-CA) postoperatively (a1,b1,c1: 1 week; a2,b2,c2: 2 weeks; a3,b3,c3: 3 weeks) .

Figure 14. VEGF diagram of three kinds of scaffolds (a: control group GA, b: col, c: β-ODAP/OCS-TA-CA) postoperatively (a1,b1,c1: 1 week; a2,b2,c2: 2 weeks; a3,b3,c3: 3 weeks) .

Conclusions ACS Paragon Plus Environment


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In this report, a new type of skin wound repair collagenous material based on the novel tissue-mimicking advanced collagen aggregate (TA-CA) was successfully fabricated after the functionally crosslinked with dialdehyde chitosan (OCS) and dencichine (β-ODAP). The thermal stability, mechanical properties, hydrophilicity, enzyme resistance and hemostatic properties of TA-CA were significantly improved by the comprehensive crosslinking effects of OCS and β-ODAP through the complex interaction force of Schiff’s base, hydrogen bonds, ion bonds and hydrophobic bond. Furthermore, after crosslinked, TA-CA still maintained a special 3D hierarchical structure and its typical D-periodic cross-striated patterns about 65nm were clearly discerned. Moreover, OCS/β-ODAP-TA-CA scaffold presented no cytotoxicity nature and could accelerate the proliferation and adhesion of fibroblasts. In the in vivo animal experiment, the results indicated that COL-PDA-PADM could effectively promote the expression of bFGF, VEGF and PFDF, possibly leading to an enhanced wound healing process of skin. In conclusion, OCS/β-ODAP-TA-CA with excellent biocompatibility and hemostatic properties is expected to become a new generation of collagenous hemostatic materials and TA-CA could serve as a better alternative source of collagens for biological applications.

Acknowledgements We sincerely acknowledge the funding and generous support of National Key R&D Program of China (2017YFB0308500); National Natural Science Foundation of China(21808133).

Author information Corresponding

author:

Xinhua

Liu

[[email protected]];

Xuechuan

Wang[ [email protected]]; Weihua Dan[[email protected]]. The authors declare no financial interest.

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Table of contents

Synopsis Sustainable tissue-mimicking collagen aggregate crosslinked with OCS and β-ODAP was developed as an efficacious wound healing device with hemostatic property.


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Development of a novel collagenous matrix based on tissue

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