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oxidized konjac composite hydrogel
Journal Pre-proof Preparation and characterization of carboxymethyl chitosan/ collagen peptide/oxidized konjac composite hydrogel
Chang Zhang, Xiaoshuang Yang, Wanqing Hu, Xianshun Han, Lihong Fan, Shengxiang Tao PII:
S0141-8130(19)37235-6
DOI:
https://doi.org/10.1016/j.ijbiomac.2020.01.127
Reference:
BIOMAC 14431
To appear in:
International Journal of Biological Macromolecules
Received date:
7 September 2019
Revised date:
7 January 2020
Accepted date:
13 January 2020
Please cite this article as: C. Zhang, X. Yang, W. Hu, et al., Preparation and characterization of carboxymethyl chitosan/collagen peptide/oxidized konjac composite hydrogel, International Journal of Biological Macromolecules(2020), https://doi.org/ 10.1016/j.ijbiomac.2020.01.127
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© 2020 Published by Elsevier.
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Preparation and characterization of Carboxymethyl Chitosan/Collagen Peptide/Oxidized Konjac Composite Hydrogel Chang Zhanga, Xiaoshuang Yanga, Wanqing Hua, Xianshun Hana,Lihong Fana*， Shengxiang Taob* School of Chemistry, Chemical Engineering and Life Sciences, Wuhan
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Zhongnan Hospital of Wuhan University，Wuhan 430071, China
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University of Technology, Wuhan 430070, China
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*Corresponding author. Tel.: +86 27 87859019; fax: +86 27 87859019
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Email addresses: [email protected]. (L. Fan)
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Abstract：
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[email protected]（S.Tao）
Medical dressings are used to protect damaged skin from external factors and provide a good healing environment.Hydrogels are aggregates of hydrophilic polymers and water that have a three-dimensional space and can absorb large amounts of water. It has been widely studied in the field of biomedical materials.In this study,we prepared a novel composite hydrogel combined carboxymethyl chitosan， collagen peptide and oxidized konjac，all three materials have been shown to be biocompatible.Then,we set up different hydrogels and tested hydrogels with different proportions.The structures of CMCS（carboxymethyl chitosan）/COP（collagen
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peptide）/OKGM（oxidized konjac） hydrogels were characterized by IR, NMR, X-ray diffraction and SEM.The effects of hydrogels on the growth of NS-FB and HS-FB cells were studied in vitro.The results of these tests show that the composite hydrogel has excellent mechanical properties and biological activity, and has potential application in wound dressing field.
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Keywords：Oxidized konjac; Carboxymethyl chitosan;Collagen Peptide；Hydrogels.
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1. Introduction
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Hydrogels are aggregates of hydrophilic polymers and water that have a
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three-dimensional space which can absorb large amounts of water. Typical hydrogels
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contain more than 90% water[1,2].The material state of the hydrogel is between liquid and solid, and has both the viscoelastic properties of the solid and the fluidity of the
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liquid.The hydrogel material has a soft and moist surface, it will not irritating
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response when in contact with human tissue, and its morphology is similar to the extracellular matrix of living tissue, and it can be used as an ideal material in the field of biomedicine[3,4].Hydrogel medical dressing is a kind of new dressing with soft surface and certain mechanical strength, and similar to human tissue. It can provide a good moisturizing healing environment for wounds, and can prevent external microorganisms and bacteria from invading the body.
Carborylmethy Chitosan (CMCS) is one of the most common derivatives of CS （Chitosan）. It is obtained by the substitution reaction of reactive amino groups and hydroxyl groups on the CS molecular chain with chloroacetic acid under specific
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conditions[5].According to the substitution position of carboxymethyl group, it can be divided into O-position, N-position and N, O-position CMCS.Scheme 1 is a schematic diagram of the molecular structure of three kinds of CMCS.CMCS overcomes the huge defect that CS is insoluble in water. Due to the introduction of hydrophilic group carboxymethyl group, the regularity of CS crystal is destroyed and
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its crystallinity is reduced[6], which significantly improves its water solubility and
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greatly expands CS applications.CMCS has antibacterial activity which is similar to
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CS because its amino cations can interfere with the synthesis of macromolecular
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substances on the surface of bacteria and change the permeability of cell walls.CMCS
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also has anti-tumor properties. The surface of tumor cells has a certain negative charge, which can attract the positive charge on CMCS through electrostatic
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interaction, thus inhibiting the growth of tumor cells.In addition, CMCS has the effect
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of promoting fibroblast growth and reducing scar formation[7].As a safe biological material, CMCS can promote the healing of wounds and reduce the formation of scars. Therefore, it has a good application prospect in the fields of wound repair and plastic surgery.
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Scheme.1. Molecular structure of three CMCS
Konjac Gulcomannnan (KGM) is a renewable natural high molecular polysaccharide which is extracted from the bulb of Anorphophallus Konjac.KGM has a molecular weight of 200-2000 kDa, which is composed of D-glucose and D-mannose in a molar ratio of 1:1.6[8], and its main chain is linked by β-1,4
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glycosidic bonds, at the C3 position of the main chain. There is a branch consisting of
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β-1,3 glycosidic bonds, about one branch per 68 sugar residues, and a branch length
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of 3 or 4 sugar residues[9], present on D-mannose Acetyl group[10,11].There are a
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large number of active hydroxyl groups in the KGM molecule, which facilitates
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various modifications to improve their performance and expand their applications.
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Scheme 2 is a schematic diagram of the molecular structure of KGM.
Scheme.2. Molecular structure of KGM
KGM has water solubility, which can absorb water about 100 times its own weight. During the dissolution process, since the diffusion rate of water molecules is much larger than the diffusion rate of KGM molecules, KGM particles swell and
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eventually form a liquid with a large viscosity. KGM has excellent gel properties. When acetyl groups are lost, KGM molecules form network structures through hydrogen bonding. For example, gelatin and agarose gels melt with increasing temperature.KGM can form a thermoreversible gel with unstable 3d network structure through strong synergistic effect with xanthan gum, carrageenan and other hydrophilic
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gums[12,13].In addition, KGM is a soluble dietary fiber with high fiber, low fat and
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low calorie, which has the effect of losing weight[14], lowering blood sugar, lowering
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blood fat and anti-tumor.KGM is widely used in medical, food, chemical industry[15]
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and other fields due to its unique physical and chemical properties and excellent
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physiological activity.KGM has good biocompatibility, biodegradability and renewability. It can be prepared into medical wound dressings to provide a moist
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healing environment for wounds, as well as hemostasis and healing.KGM is a kind of
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high quality dietary fiber with various physiological functions, which has been processed into a natural health food and has the functions of losing weight, lowering blood sugar, lowering blood fat, smoothing bowel and laxative, and improving human immunity.KGM can be used as food additive, such as emulsifier, thickener, meat binder, gelling enhancer, etc.
Collagen is a bioactive polymer synthesized by animal cells and widely found in skin, bones, tendons, ligaments and other connective tissues, accounting for about 30% of the total protein in the body[16]. It is an important part of the extracellular matrix.Collagen molecule is a rod-shaped spiral structure formed by the winding of
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three peptide chains. This stable structure enables collagen which has ability to support, protect and form a boundary between organs and the body in animals.Collagen has the advantages of good biological activity, tissue adhesion, low immunogenicity and hydrophilicity, etc. It can also interact with cells, participate in cell signal transduction, regulate cell migration, proliferation, differentiation and other
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physiological activities[17].Collagen peptide (COP) is a type of small molecule
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product produced by degradation of Collagen by enzyme treatment.In addition to
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typical characteristics such as high biological activity, small molecular weight, easy
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absorption and non-toxic and harmless, COP has many excellent biological functions
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because its content of proline and hydroxyproline is significantly higher than other
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active peptides.For example, antioxidant properties and the ability to promote healing.
It has been reported that COP can promote cartilage repair [18] and alveolar bone
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regeneration [19], and can also promote multi-differentiation of various stem cells [20]..Liu et al.[21] prepared a compound hydrogel containing COP. The experiment showed that the compound hydrogel had excellent physicochemical properties and good biocompatibility, and could promote cell proliferation.
In this study, KGM was used as raw material to prepare konjac oxide (OKGM) under the oxidation of sodium periodate. With OKGM as crosslinking agent and carboxymethyl chitosan/collagen peptide (CMCS/COP) through schiff base reaction, carboxymethyl
chitosan/collagen
peptide/konjac
oxide
(CMCS/COP/OKGM)
composite hydrogel was prepared.The materials used are biocompatible, and
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considering the characteristics of each material, a new compound hydrogel was designed for the practical application of wound dressing. The composite hydrogel was characterized by FTIR , NMR, XRD and SEM, and its gel time, swelling rate, water evaporation rate and hemolysis rate were measured.The effect of compound hydrogel on the growth of NS-FB and HS-FB cells was studied in vitro.
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2.Materials and methods
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2.1.Materials
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Sodium iodate (NaIO4), ethylene glycol (C2H6O2), potassium iodide (KI),
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sodium bicarbonate (NaHCO3), sodium thiosulfate (Na2S2O3), sodium chloride
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(NaCl), sodium dihydrogen phosphate (NaH2PO4), and disodium hydrogen phosphate (Na2HPO4) are all AR（Analytical reagent） and come from sinopharm chemical reagent
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co.,LTD.Konjac (KGM) is purchased from hubei Johnson konjac co., LTD.The purity
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of KGM is 95% and relative molecular mass is about 800,000. The degree of substitution is not explained.CMCS/COP composite material is self-made material; fresh anti-coagulation mouse blood comes from Wuhan Third Hospital.Collagen is derived from fish collagen and purchased through sinopharm chemical reagent co., LTD.
Chitosan purchased from sinopac group, deacetylation degree 80.0 ~ 95.0； burning residue (in terms of sulfate), % ≤1.5；drying loss, % ≤10.0；heavy metal (in terms of Pb), % ≤0.0015；arsenic (As), % ≤0.00006；viscosity, mPa·s 50 ~ 800；deacetylation degree, % 80.0 ~ 95.0；acetic acid insoluble substance, % ≤1.0. 2.2.Methods
Journal Pre-proof 2.2.1.preparation of OKGM The experimental method is to weigh an appropriate amount of KGM and add it to a three-necked flask, then add 500 mL of distilled water, and mechanically stir at room temperature until the KGM is completely dissolved.An appropriate amount of NaIO4 was dissolved in 30 mL distilled water to form a uniform solution, and was
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added to KGM solution drop by drop. The temperature was controlled at 30℃, and
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the reaction was conducted for 12 h.Add 10 mL C2H6O2 to react with excessive NaIO4,
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stop after 1 hour, pour the reaction solution into the dialysis bag, dialysis for 72 hours,
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change water twice a day, freeze dry and reserve, and the product is OKGM，The
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reaction equation is shown in Scheme 3.
Scheme.3. preparation of OKGM
2.2.2.Determination of oxidation degree of OKGM The oxidation degree of OKGM (DO, %) was determined by iodimetry.The oxidation degree of OKGM refers to the percentage of the total number of urylation units in the KGM molecule.This experiment indirectly calculated the oxidation consumption of NaIO4 by measuring the excessive content of NaIO4 in the preparation process of OKGM, and finally calculated the oxidation degree of
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OKGM.Specific operations are as follows:Take 5 mL of the mixed solution after the oxidation reaction, put it in the dark condition, add 10 mL of sodium bicarbonate solution (10 wt%) and 2 mL of potassium iodide solution (20 wt%), and let it sit for 20 minutes.Sodium thiosulfate standard solution was prepared. Starch solution was used as indicator to titrate the mixed solution and determine the iodine content.
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Formula1
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M 148 2 n 214 100 % DO M1
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According to formula 1, the oxidation degree of OKGM could be calculated.
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Where, M1 is the mass of konjac, with unit of g;M2 is the mass of sodium
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periodate added, in g;148 is the molar mass of konjac unit in g/mol;214 is the molar
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mass of sodium periodate in grams per mole;N is the amount of excess sodium periodate in moles.According to formula 1, the oxidation degree of OKGM was
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calculated to be 50%.
2.2.3.Preparation of CMCS/COP 2.2.3.1.The purification of MTGase（microbial trans glutaminase ）
(1) Preparation of buffer solution: A certain amount of Na2HPO4 and NaH2PO4 were weighed and dissolved in distilled water, and 1000 mL volumetric flask was added to prepare a 0.2 mol/L standard solution. The two standard solutions were mixed and adjusted to a pH of 6, as a buffer solution, and used.
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(2) Purification of MTGase: The purchased MTGase was added to the above-prepared buffer solution, fully dissolved by mechanical stirring, and the centrifuge speed was set at 3500 rpm/min, and centrifuged for 15 min. The supernatant was taken, vacuum-filtered three times, dialyzed in distilled water for three days, twice changed with water, and then dried using a freeze dryer to obtain
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purified MTGase powder, which was stored in a 4 ℃ refrigerator and set aside.
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2.2.3.2.The preparation of CMCS/COP
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The CMCS solution, the COP solution and the purified MTGase solution each
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having a mass fraction of 1 wt% were separately prepared using the above buffer
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solution. After the preparation, they were poured into a three-necked flask, placed in
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an oil bath, set at a temperature of 50 ℃, mechanically stirred for 4 h, and then the temperature was adjusted to 90 ℃ for 10 min to deactivate the high temperature of
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the MTGase. The reaction solution was cooled to room temperature, circulated and filtered three times, placed in distilled water for three days, periodically changed, and then dried using a freeze dryer to obtain a CMCS/COP composite. 2.2.4.Preparation of CMCS/COP/OKGM hydrogels A certain amount of CMCS/COP samples were weighed and added to distilled water to prepare 5 wt% CMCS/COP solution, which was mechanically stirred until completely dissolved and stored in the refrigerator for later use.Similarly, appropriate amount of OKGM was added to distilled water to prepare 10 wt% OKGM solution,
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which was mechanically stirred until completely dissolved.Add 0.4,0.8,1.2,1.6 and 2.0mL of OKGM solution to 2.0mL of CMCS/COP solution, and prepare hydrogels in different proportions after fully stirring.According to the different ratios of OKGM and CMCS/COP (0.2:1, 0.4:1, 0.6:1, 0.8:1, 1:1), the Numbers were CCGM-2, CCGM-4, CCGM-6, CCGM-8, and CCGM-10, respectively.
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Scheme 4 shows the reaction equation of preparing CMCS/COP/OKGM
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composite hydrogel. The reaction mechanism is the schiff base reaction between the
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residual amino group on CMCS/COP and the aldehyde group on OKGM.As shown in
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Scheme 5, CCGM-2, CCGM-4, CCGM-6, CCGM-8 and CCGM-10 hydrogels were
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prepared on a 12-well plate. All the samples were smooth, clear and transparent, with
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a yellowish tint, and became darker as the OKGM ratio increased.
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Scheme.4. Reaction equation of CMCS/COP/OKGM hydrogel preparation
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Scheme.5. CCGM-2（a）、CCGM-4（b）、CCGM-6（c）、CCGM-8（d）、
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CCGM-10（e） hydrogel sample
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2.2.5.Structural characterization of materials (1)Infrared spectrum characterization: KGM, OKGM and CMCS/COP/OKGM samples were ground into powder, followed by potassium bromide tablets and infrared characterization by Nicoet170SX type Fourier transform infrared spectrometer of Nicoet, USA, and the wavelength range was set at 500-4000 cm-1.
(2) NMR characterization: KGM and OKGM samples were ground into powder and fully dissolved in 0.5 mL D2O. 1H-NMR characterization was performed by amx-500 nuclear magnetic resonance instrument of Bruker company, Germany, and the temperature was set at 25 ℃.
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(3) X-ray diffraction characterization: KGM and OKGM samples were ground into powder and XRD analysis was conducted by D8 Advance X-ray diffractometer of Bruker company, Germany.
The parameters set in the experiment are: copper target, nickel filter plate, pipe flow 40 mA, pipe pressure 40 KV, scanning range 5-45°, step length 0.02°.
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(4) Sem characterization: freeze-dried, incised and gold-sprayed sections of
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CCGM-2, CCGM-6 and CCGM-10 hydrogel samples were used to observe the
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cross-section morphology of hydrogel samples by VEGA3 scanning electron
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microscope from Tescan company of the Czech republic.
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2.2.6.1.Gel time determination
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2.2.6.Study on the properties of CMCS/COP/OKGM hydrogels
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CMCS/COP and OKGM were dissolved in water respectively to prepare 5 wt% and 10 wt% solutions.CMCS/COP aqueous solution was injected into the flat bottom tube with a syringe.Add the teflon magnetic agitator into the flat bottom tube and set the rotation speed to 100 RPM /min. After the rotation is stabilized, add the OKGM aqueous solution and start timing until the magnetic agitator stops rotating. The total rotation time is the gel time. 2.2.6.2.Determination of swelling rate The samples of CCGM-2, CCGM-4, CCGM-6, CCGM-8 and CCGM-10 hydrogels were placed in a freeze-dryer, taken out 48 hours later, weighed and
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recorded as the initial weight M1. The Na2HPO4/NaH2PO4 buffer solution was prepared with a pH of 7.4.Place the freeze-dried sample in the buffer solution for water absorption and swelling, and weigh it regularly until the sample weight no longer increases, that is, the swelling balance is reached, and the residual water on the sample surface is erased. Record the weight at this time as the swelling balance
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weight M2.The swelling rate (SR) of the hydrogel sample was calculated according to
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M1 M 2 100% M2
Formula 2
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SR
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the formula 2, and the experiment was repeated three times.
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2.2.6.3.Determination of water evaporation rate
Samples of CCGM-2, CCGM-4, CCGM-6, CCGM-8 and CCGM-10 hydrogels
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were taken from freeze-dried samples at room temperature.After immersion in
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distilled water and reaching swelling equilibrium, residual water on the sample surface was wiped off, weighed and recorded as swelling equilibrium mass Ma.The oven temperature was set at 50 ℃ and the relative humidity was set at 50%. The samples after swelling and equilibrium were placed in the oven. The weight of the samples was measured periodically and recorded as Mb.Until the weight of the hydrogel sample is no longer reduced, weigh it and record it as Mc.Calculated by the formula 3 hydrogel samples of Water evaporation rate (Water evaporation rate, WER), the experiment repeated three times.
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WER
Ma Mb 100% Ma Mc
Formula 3
2.2.6.4.Determination of hemolysis rate The fresh anticoagulated mouse blood was diluted with an equal amount of physiological saline and stored in a refrigerator for use.The freeze-dried hydrogel
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samples of CCGM-2, CCGM-4, CCGM-6, CCGM-8 and CCGM-10 were ground into
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powder, then 25 mg was evenly dispersed in 10 mL normal saline, 0.1 mL diluted anticoagulant mouse blood was added, and placed in a constant temperature tank at
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37.5 ℃ for 1 h.Subsequently, the centrifuge was centrifuged for 10 min (rpm 1500
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rpm/min), the wavelength of the ultraviolet-visible spectrophotometer was set to 545
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nm, and the absorbance of the supernatant was measured.The Hemolysis rate (HR) of
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three times.
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hydrogel samples was determined by formula 4, and the experiment was repeated
HR
A1 A3 100% A 2 A3
Formula 4
In the formula 4, HR represents the hemolysis rate, A1 represents the absorbance of the hydrogel sample group, and A2 is the absorbance of the positive control group (10 mL distilled water, 0.1 mL diluted anticoagulated mouse blood, non-aqueous gel sample material), A3 is Absorbance of the negative control group (10 mL normal saline, 0.1 mL diluted anticoagulated mouse blood, non-aqueous gel sample material).
2.3.Determination of in vitro cell compatibility
Journal Pre-proof 2.3.1.Experimental main materials and instruments Experimental cells: Human skin fibroblasts (NS-FB) and human hypertrophic scar fibroblasts (HS-FB) were purchased at Beijing North Natron Biotechnology Research Institute.
The main materials of the experiment: DMEM medium, fetal bovine serum,
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streptomycin mixture, trypsin, CCK-8, cell culture flask, 96-well cell culture plate, all
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purchased from Wuhan Huashun Biotechnology Company. OKGM and CMCS/COP
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composites are self-made materials.
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Main instruments: super clean table, CO2 constant temperature incubator,
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inverted optical microscope, enzyme marker.
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2.3.2.Preparation of cell suspension and seed plate
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(1) preparation of cell culture medium: 50 mL fetal bovine serum was taken with pipette and put into 500 mL DMEM culture medium, 5 mL penicillins mixture was added, shaken well and set aside.
(2) cell culture: NS-FB and HS-FB cells were inoculated in T25 cell culture bottles respectively, 5 mL of the cell culture medium prepared above was added, and placed in CO2 incubator at a constant temperature of 37.5℃.The growth state of the cells was observed by inverted optical microscope on a regular basis every day, and the medium was changed according to the growth condition of the cells.When the cell density reached 80%, trypsin was used for digestion, and the logarithmic growth cells
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were obtained by passage three times at the ratio of 1 to 3.
(3) cell inoculation: the logarithmic growth cells were digested with trypsin and dispersed evenly in the cell culture medium.A cell suspension with a concentration of 1×104 cells /mL was prepared by plate counting method, which was inoculated into a 96-well cell culture plate with an addition amount of 100 L per hole.After
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inoculation, the cells were transferred to a CO2 constant temperature incubator and
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2.3.3.Preparation of hydrogel extract
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cultured for 24 hours to make the cells stick to the wall.
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CMCS/COP freeze-dried samples and OKGM freeze-dried samples were ground
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into powder, and after uv irradiation for 2 h, CCGM-2, CCGM-4, CCGM-6, CCGM-8
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and CCGM-10 hydrogel samples were prepared with aseptic distilled water in ultra-clean table.The sample was extracted with cell culture medium at a mass ratio of
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1:10, and after 24 h, it was sterilized by filtration using a 0.22 μm filter to obtain a leach solution of the hydrogel sample, which was sealed and stored in a 4℃ refrigerator for use.
2.3.4.Determination of absorbance by CCK-8 method
The adherent cells were removed from the CO2 constant temperature incubator, the medium was sucked out, and 100 L of the hydrogel sample extract was added respectively.Then placed in a cell culture incubator, removed one day later, aspirate the medium, and replace with 100 L of fresh medium.10 L CCK-8 stain was added to
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each hole with pipette gun, and the culture was continued for 3 h after vibration defoamed.The wavelength of the microplate reader was set at 450 nm to determine the absorbance of each hole.
According to the formula for calculating the Relative cell survival rate (4-5) (Relative cell viability, RCV).
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OD1 OD2 100% OD3 OD2
Formula 5
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RCV=
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Where, RCV represents the relative survival rate of cells, OD1 is the absorbance
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of sample group, OD2 is the absorbance of blank group (only medium and cck-8, no
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cells), and OD3 is the absorbance of positive control group (fresh medium replaces
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sample extract). 3.Results and discussion
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3.1.Determination of in vitro cell compatibility
Journal Pre-proof Fig.1. infrared spectra of KGM (a), OKGM (b) and CMCS/COP/OKGM (c) hydrogels Figure 1 is the infrared spectrum diagram of KGM, OKGM and CMCS/COP/OKGM hydrogel. The infrared spectrum curve of KGM was observed, and the analysis of its characteristic absorption peak was shown in table 1.
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Table 1. Each peak of KGM infrared spectrum belongs to Position of absorption peak Serial number
Peak on the ownership of the
3420
2
2925
O-H stretching vibration
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1
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(cm-1)
1734
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3
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The stretching vibration of C-H of methyl or hypomethyl groups
C=O on the acetyl group
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The absorption peaks in table 1 are consistent with the KGM standard infrared
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spectrum[22，23]. Compared with the infrared spectrum of KGM, the analysis of the new characteristic absorption peaks in the infrared spectrum of OKGM is shown in table 2.
Table 2. Attribution of new peaks in OKGM infrared spectrum Position of absorption peak Serial number
Peak on the ownership of the
(cm-1)
1
1720
Symmetric vibrations of the aldehyde group
2
895
The hemiacetal structure contracts in vibration
As it can be seen from table 2, two new characteristic absorption peaks [24] appeared in the infrared spectrum of OKGM, which fully reflected the appearance of
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the aldehyde group, indicating that the hydroxyl group in the KGM molecule was oxidized to the aldehyde group, and OKGM was successfully prepared.By observing figure 1 and comparing the infrared spectrum curve of OKGM, it can be found that in the Fourier infrared spectrum of CMCS/COP/OKGM hydrogel, the two strong vibration absorption peaks (1720 and 895 cm-1) originally belonging to OKGM
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basically disappear completely.It Indicated that the aldehyde group was consumed by
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the reaction during the preparation of hydrogels.At the same time, the stretching
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vibration absorption peak (1640 cm-1) of C=N was observed, which indicated that the
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aldehyde group on OKGM crosslinked with the amino group on CMCS/COP, and
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finally formed the composite hydrogel.
3.2.1H Nuclear Magnetic Resonance Spectrum
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Figure 2 shows 1H-NMR spectra of KGM and OKGM.In the 1H-NMR spectrum
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of KGM, the peak with a chemical shift of 2.18 PPM represents the hydrogen of methyl on the ester bonded acetyl group on the molecular chain sugar residues.The chemical displacement peak in the range of 3.37-4.14 PPM represents the displacement peak of other hydroxyl hydrogen on the sugar ring, and the chemical displacement peak of 4.45 PPM represents the displacement peak of hydroxyl hydrogen on the C6 position of the sugar residue molecule.These conform to KGM standard nuclear magnetic hydrogen spectra [25].By observing 1H-NMR spectrum of OKGM, it can be found that OKGM possesses all characteristic absorption peaks of KGM, in addition to which two new absorption peaks are generated.The chemical
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displacement peaks of 6.49 and 9.24 PPM represent the active hydrogen on the enol carbon.In general, under alkaline conditions, the aldehyde group of OKGM will appear as enol, and in acidic conditions, as free aldehyde groups.As the system was neutral, the peaks of the two sites were weak, and the peaks of 6.49 PPM and 9.24 PPM were not observed in the 1H-NMR spectra of KGM, indicating that KGM was
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successfully oxidized and the aldehyde group was formed.
Fig.2. 1H-NMR spectra of KGM (a) and OKGM (b) 3.3.X ray diffraction analysis Figure 3 shows the X-ray diffraction patterns of KGM and OKGM. It can be seen that the two samples have similar patterns, with no obvious sharp crystal peak, and the overall shape is a steamed bread shaped dispersion ring, which is an
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amorphous structure.
KGM OKGM
(a)
10
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Intensity(a.u.)
(b)
30
20
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2/()
40
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Fig.3. XRD spectra of KGM (a) and OKGM (b)
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3.4.Sem analysis
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FIG. 4 is the sem image of the cross section of CCGM-2, CCGM-6 and CCGM-10 composite hydrogel samples, with magnification of 200 and 1000
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times.Overall observation of the three kinds of composite hydrogels shows that the three kinds of hydrogels have uniform pore structure with good continuity. When magnified to 1000 times, it can be seen that the pore structure is smooth and flat with a certain thickness.This excellent three-dimensional space structure is conducive to the free diffusion of water molecules, and its solid skeleton structure enables the hydrogel to absorb a lot of water without breaking.
Longitudinal comparison of the three kinds of composite hydrogels showed that the pore structure of CCGM-2,CCGM-6 and CCGM-10 showed a weak trend of
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decreasing successively.The reason may be that with the increase of the content of OKGM in the compound hydrogel, the concentration of aldehyde group in the reaction process increases, and more aldehyde group binds to the amino group in CMCS/COP, leading to a higher degree of reaction between OKGM and CMCS/COP, resulting in a denser skeleton structure.Therefore, the pore diameter will decrease.The
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water absorption properties of hydrogels are mainly demonstrated in three aspects,
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including hydrogel materials, structures, and pore sizes.This composite hydrogel is
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mainly composed of oxidized konjac, which has been proven to have excellent water
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absorption properties. From the XRD results, the composite hydrogel has an
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amorphous structure, and the results of scanning electron microscopy also prove the composite Hydrogels have excellent pore structure. These experiments have proved
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their excellent water absorption properties. In the actual use of hydrogels, this
properties.
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conclusion is also proved. This composite hydrogel has excellent water absorption
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（a1）（×200）
（b2）（×1000）
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（b1）（×200）
（c1）（×200）
（c2）（×1000）
FIG. 4.Hydrogel profiles of CCGM-2（a）,CCGM-6（b）,CCGM-10（c） Sem image
3.5.Gel time analysis Figure 5 shows the gel time of CCGM-2, CCGM-4, CCGM-6, CCGM-8 and CCGM-10 hydrogels. It can be seen that all hydrogels have shorter gel time, which is less than 30 s, and the shorter gel time has higher requirements for clinical operation speed.The gel time of each composite hydrogel sample was compared longitudinally,
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and it was found that CCGM-2, CCGM-4, CCGM-6, CCGM-8, and CCGM-10 had a tendency to decrease first and then increase.As the compound hydrogels have the same composition, only the ratio of CMCS/COP to OKGM is different. As the content of OKGM increases, the concentration of aldehyde group in the reaction process increases, so that more aldehyde groups bind to the amino group in CMCS/COP,
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accelerating the reaction speed.Therefore, when the ratio of OKGM increased from
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0.2 to 0.6, the gelation time decreased successively, and CCGM-6 had the shortest
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gelation time (17 s).When the OKGM ratio continues to increase from 0.6 to 1, the
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excess aldehyde group has no time to bind with the amino group, but leads to a
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decrease in the overall concentration of the components in the system, slowing down the reaction rate and increasing the gel time. The longest gel time of CCGM-10 is 28
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s.Compared with other wound dressings, the gel time of this composite hydrogel is
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very short, which puts forward higher requirements for clinical operation. However, due to its rapid gel time, this material can quickly close the wound, rapidly shape the wound, reduce the risk of wound exposure during operation and shorten the operation time.
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30
Gelation Time (s)
25 20 15 10 5
CCGM-4
CCGM-6
CCGM-8 CCGM-10
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CCGM-2
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FIG. 5.Gel time of CCGM-2, CCGM-4, CCGM-6, CCGM-8, CCGM-10 hydrogel
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3.6.Swelling rate analysis
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Swelling rate is one of the important indexes for evaluating hydrogel dressings. When hydrogel reaches equilibrium in water absorption and swelling, the more water
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it can retain, the stronger its ability to provide a moist treatment environment for
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wounds, which plays an important role in patients' recovery as soon as possible. Therefore, it is of great significance to investigate the swelling performance of hydrogel.Figure 6 shows the swelling rate of CCGM-2, CCGM-4, CCGM-6, CCGM-8 and CCGM-10 hydrogels. It can be seen that the swelling rate is about 500%, indicating that the hydrogels have good swelling performance.Longitudinal comparison of each composite hydrogel showed that the swelling ratios of CCGM-2, CCGM-4, CCGM-6, CCGM-8, and CCGM-10 hydrogels decreased in turn.The swelling rate of CCGM-2 was 581%.The reason may be that with the increase of OKGM content in the compound hydrogel, the concentration of aldehyde group in the
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reaction process increases, and more aldehyde groups bind to the amino group in CMCS/COP.As a result, the degree of cross-linking between OKGM and CMCS/COP is higher, forming a denser skeleton structure, which affects the entry of free water.At the same time, the volume of water molecules can be reduced, which results in decreased swelling performance, which is the same as the result of SEM.
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600
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400
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300
200
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Swelling Rate (%)
500
100
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0
CCGM-2
CCGM-4
CCGM-6
CCGM-8 CCGM-10
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FIG. 6. Swelling rate of CCGM-2, CCGM-4, CCGM-6, CCGM-8, CCGM-10 hydrogels 3.7.Water evaporation rate analysis Figure 7 is the water evaporation rate of CCGM-2, CCGM-4, CCGM-6, CCGM-8, CCGM-10 hydrogel samples.It can be seen from the curve that the change trend of water evaporation rate of each compound hydrogel with time is basically similar, fast before and slow after 24 h, and the hydrogel basically reaches a stable after 24 h, and the hydrogel still retains a certain amount of water.Longitudinal comparison of each composite hydrogel, it can be found that with the increase of
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OKGM content in the composite hydrogel, the water evaporation rate has a tendency to decrease, probably because more aldehyde groups are introduced due to the increase of OKGM content, and there are more The combination of multiple aldehyde groups with the amino group in CMCS/COP makes the crosslinking of OKGM and CMCS/COP become higher, resulting in a denser pore structure, which affects the
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convection with air and slows the evaporation of free water. This results in a decrease
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in water evaporation rate, which is the same as the SEM results. 110
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80 70 60
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Water loss(%)
90
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CCGM-10 CCGM-8 CCGM-6 CCGM-4 CCGM-2
100
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50 40 30
4
6
8
10
12
14
16
18
20
22
24
26
Time (h)
FIG. 7. Water evaporation rate of CCGM-2, CCGM-4, CCGM-6, CCGM-8, CCGM-10 hydrogels 3.8.Hemolysis rate analysis The hemolysis rate of biological materials is an important indicator for judging the blood compatibility of the material. The hemolysis phenomenon refers to the phenomenon that hemoglobin escapes after the contact of foreign matter with blood
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causes the red blood cells to rupture[26].As wound dressing, hydrogel can directly contact the blood, so its hemolysis rate must be controlled within a certain range. The international standard for the hemolysis rate of biological materials is less than 5%.Therefore, it is of great significance to determine the hemolysis rate of the compound hydrogel for its clinical application.Figure 8 shows the hemolysis in the
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gel sample group, the positive control group and the negative control group after 1 h
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of contact with the blood and centrifugation for 10 min. It can be observed that the
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blood cells in the positive control group have ruptured and the solution is uniform and
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red.In the negative control group, blood cells were deposited at the bottom of the tube,
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and the solution was clear and transparent.The hemolysis phenomenon in the gel sample group was similar to that in the negative control group. The solution was
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basically clear and transparent, and the blood cells and gel material were deposited at
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the bottom, indicating that the hydrogel material could maintain the integrity of the blood cell structure without causing hemolysis reaction.
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（2）
（3）
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（1）
FIG. 8. The figure shows the negative sample group（1）, gel control group（2） and
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positive control group（3）.
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Table 3 shows the hemolysis rate of CCGM-2, CCGM-4, CCGM-6, CCGM-8,
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and CCGM-10 hydrogel samples after 1 h of contact with blood.It can be seen from the table that the hemolysis rate of all samples is between 0.81% and 2.67%, which conforms to the standard of less than 5% of biological materials and has good blood compatibility.
Table 3. Hemolysis rate of CCGM-2, CCGM-4, CCGM-6, CCGM-8, CCGM-10 hydrogels Sample
Hemolysis rate（%）
CCGM-2
0.81
CCGM-4
2.67
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0.89
CCGM-8
1.78
CCGM-10
1.62
3.9.In vitro compatibility analysis of CMCS/COP/OKGM hydrogels Figure 9 shows the relative survival rates of NS-FB and HS-FB cells cultured in
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CCGM-2, CCGM-4, CCGM-6, CCGM-8, and CCGM-10 hydrogel extracts.By
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observing the survival rate of NS-FB cells, it was found that there was no significant
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difference in the relative survival rate of cultured ns-fb cells between different sample
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extracts.Ccgm-2 hydrogel extract had the highest survival rate, up to 105%.The cell
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survival rate decreased slightly with the increase of OKGM ratio in various ratios of hydrogels,It may be that uncrosslinked OKGM leads to the high concentration of
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OKGM in the hydrogel extract, and excessive aldehyde group is introduced, thus
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disrupting the nutrient balance of the medium.It may also lead to changes in cell osmotic pressure, which affects cell proliferation.However, the survival rate of ccgm-10 hydrogel extract was the lowest, but reached 93%, indicating that the hydrogel had good compatibility with ns-fb cells and could promote the proliferation of NS-FB cells.
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CCGM-2 CCGM-4 CCGM-6 CCGM-8 CCGM-10
Relative Cell Viability (%)
120 100 80 60 40 20 0 HS-FB
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NS-FB
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FIG. 9. Relative survival rate of NS-FB and HS-FB cells cultured in CCGM-2,
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CCGM-4, CCGM-6, CCGM-8 and CCGM-10 hydrogel extracts
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For HS-FB cells, the relative survival rates of all hydrogel sample extracts were
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greater than 88%.There was no significant difference in cell survival rate between
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different hydrogel samples,The cell survival rate decreased slightly with the increase of OKGM ratio in all hydrogels.It is also because too much aldehyde group is
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introduced, which damages the nutrient balance of the culture medium and even changes in the osmotic pressure of cells, thus affecting the proliferation of cells.In conclusion, this composite hydrogel had little effect on HS-FB cell proliferation.In this composite hydrogel, collagen peptide is an important component, and its cell experiments have also proved the biocompatibility of the composite hydrogel. Long-term laboratory studies have found that this bio-derived collagen peptide, With good biological activity, we also carried out cell experiments, confirming our view that collagen peptides still have good biological activity in composite hydrogels. 4. Conclusion
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In this paper, Regarding the innovation of this paper, three natural sources of raw materials were used, which were modified according to their physical and chemical properties, and the three materials were cross-linked to form a composite new hydrogel, using a series of tests. It has proved its physical and chemical properties, and has carried out cell experiments to confirm its prospects for clinical use, making it
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have the potential for industrialization.KGM was used as raw material to prepare
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OKGM under the oxidation of NaIO4. Using OKGM as crosslinking agent, schiff base
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reaction was conducted with CMCS/COP prepared in chapterⅡto obtain CMCS/COP
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complex hydrogels, which were numbered CCGM-2, CCGM-4, CCGM-6, CCGM-8
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and CCGM-10 hydrogels according to the different contents of OKGM and CMCS/COP.The structure was characterized by infrared, nuclear magnetic, X-ray
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diffraction and scanning electron microscopy. The gel time, swelling ratio, water
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evaporation rate and hemolysis rate of the composite hydrogel were tested. The cells were in vitro with NS-FB and HS-FB cells. Experiments were conducted to investigate the effects of composite hydrogels on the growth of these two cells.
Infrared, nuclear magnetic and XRD results showed successful oxidation of OKGM and successful preparation of CMCS/COP/OKGM composite hydrogel.Sem test showed that all hydrogels had continuous and uniform pore structure, and the pore diameter decreased with the increase of OKGM content in hydrogels.The gel time test showed that the CMCS/COP/OKGM hydrogel had a shorter gel time, less than 30 s, and the gel time decreased first and then increased with the increase of OKGM
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content. CCGM-6 hydrogel has the shortest gel time is 17 s.The swelling ratio test showed that the CMCS/COP/OKGM hydrogel had good swelling performance, the swelling ratio was about 500%, and the CCGM-2 swelling rate was the highest, which was 581%.The water evaporation rate test showed that the CMCS/COP/OKGM hydrogel had good water retention ability, and the water evaporation rate of the
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hydrogel decreased with the increase of the content of OKGM, and the CCGM-10
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hydrogel could retain 20% water after 24 h.The hemolysis rate test showed that the
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hemolysis rate of CMCS/COP/OKGM hydrogel was between 0.81-2.67%, all of
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which were lower than 5%, showing excellent blood compatibility.The cell
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experiments showed that the relative survival rate of NS-FB cells cultured by CMCS/COP/OKGM hydrogel extract was more than 93%, which had good
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cytocompatibility and could promote the proliferation of NS-FB cells.The relative
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survival rate of HS-FB cells cultured by CMCS/COP/OKGM hydrogel was more than 88%, and there was no obvious side effect on HS-FB cells.
In summary, this paper prepared a chitosan-based natural polysaccharide composite hydrogel, and carried out a series of structural characterization and performance tests. The results show that the hydrogel has good swelling performance and long time. Its water retention capacity, excellent blood compatibility and cytocompatibility, as well as the function of promoting wound healing.It will have a broad application prospects in the field of wound dressings. Acknowledgements
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The work was supported by the National Natural Science Foundation of China (Foundation No. 51773161), The Special Funds Project of Major New Products of Hubei Province (Foundation No. 20132h0040), University-industry Cooperation Projects of The Ministry of Education of Guangdong province (Foundation No. 2012B091100437), The innovation fund project of the Ministry of Science and of
Small
and
Medium-sized
Enterprises
(Foundation
No.
of
Technology
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11C26214202642, No. 11C26214212743), Zhuhai Science and Technology Plan
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Projects (Foundation No. 2011B050102003), Wuhan Science and Technology
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Development (Foundation No. 201060623262), The Fundamental Research Funds for
Reference
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the Central Universities(Foundation No. 2014-zy-220).
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Chang Zhang, Xiaoshuang Yang, Wanqing Hu, Xianshun Han, Lihong Fan, Shengxiang Tao PII:
S0141-8130(19)37235-6
DOI:
https://doi.org/10.1016/j.ijbiomac.2020.01.127
Reference:
BIOMAC 14431
To appear in:
International Journal of Biological Macromolecules
Received date:
7 September 2019
Revised date:
7 January 2020
Accepted date:
13 January 2020
Please cite this article as: C. Zhang, X. Yang, W. Hu, et al., Preparation and characterization of carboxymethyl chitosan/collagen peptide/oxidized konjac composite hydrogel, International Journal of Biological Macromolecules(2020), https://doi.org/ 10.1016/j.ijbiomac.2020.01.127
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© 2020 Published by Elsevier.
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Preparation and characterization of Carboxymethyl Chitosan/Collagen Peptide/Oxidized Konjac Composite Hydrogel Chang Zhanga, Xiaoshuang Yanga, Wanqing Hua, Xianshun Hana,Lihong Fana*， Shengxiang Taob* School of Chemistry, Chemical Engineering and Life Sciences, Wuhan
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a
Zhongnan Hospital of Wuhan University，Wuhan 430071, China
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b
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University of Technology, Wuhan 430070, China
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*Corresponding author. Tel.: +86 27 87859019; fax: +86 27 87859019
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Email addresses: [email protected]. (L. Fan)
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Abstract：
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[email protected]（S.Tao）
Medical dressings are used to protect damaged skin from external factors and provide a good healing environment.Hydrogels are aggregates of hydrophilic polymers and water that have a three-dimensional space and can absorb large amounts of water. It has been widely studied in the field of biomedical materials.In this study,we prepared a novel composite hydrogel combined carboxymethyl chitosan， collagen peptide and oxidized konjac，all three materials have been shown to be biocompatible.Then,we set up different hydrogels and tested hydrogels with different proportions.The structures of CMCS（carboxymethyl chitosan）/COP（collagen
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peptide）/OKGM（oxidized konjac） hydrogels were characterized by IR, NMR, X-ray diffraction and SEM.The effects of hydrogels on the growth of NS-FB and HS-FB cells were studied in vitro.The results of these tests show that the composite hydrogel has excellent mechanical properties and biological activity, and has potential application in wound dressing field.
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Keywords：Oxidized konjac; Carboxymethyl chitosan;Collagen Peptide；Hydrogels.
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1. Introduction
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Hydrogels are aggregates of hydrophilic polymers and water that have a
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three-dimensional space which can absorb large amounts of water. Typical hydrogels
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contain more than 90% water[1,2].The material state of the hydrogel is between liquid and solid, and has both the viscoelastic properties of the solid and the fluidity of the
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liquid.The hydrogel material has a soft and moist surface, it will not irritating
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response when in contact with human tissue, and its morphology is similar to the extracellular matrix of living tissue, and it can be used as an ideal material in the field of biomedicine[3,4].Hydrogel medical dressing is a kind of new dressing with soft surface and certain mechanical strength, and similar to human tissue. It can provide a good moisturizing healing environment for wounds, and can prevent external microorganisms and bacteria from invading the body.
Carborylmethy Chitosan (CMCS) is one of the most common derivatives of CS （Chitosan）. It is obtained by the substitution reaction of reactive amino groups and hydroxyl groups on the CS molecular chain with chloroacetic acid under specific
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conditions[5].According to the substitution position of carboxymethyl group, it can be divided into O-position, N-position and N, O-position CMCS.Scheme 1 is a schematic diagram of the molecular structure of three kinds of CMCS.CMCS overcomes the huge defect that CS is insoluble in water. Due to the introduction of hydrophilic group carboxymethyl group, the regularity of CS crystal is destroyed and
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its crystallinity is reduced[6], which significantly improves its water solubility and
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greatly expands CS applications.CMCS has antibacterial activity which is similar to
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CS because its amino cations can interfere with the synthesis of macromolecular
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substances on the surface of bacteria and change the permeability of cell walls.CMCS
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also has anti-tumor properties. The surface of tumor cells has a certain negative charge, which can attract the positive charge on CMCS through electrostatic
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interaction, thus inhibiting the growth of tumor cells.In addition, CMCS has the effect
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of promoting fibroblast growth and reducing scar formation[7].As a safe biological material, CMCS can promote the healing of wounds and reduce the formation of scars. Therefore, it has a good application prospect in the fields of wound repair and plastic surgery.
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Scheme.1. Molecular structure of three CMCS
Konjac Gulcomannnan (KGM) is a renewable natural high molecular polysaccharide which is extracted from the bulb of Anorphophallus Konjac.KGM has a molecular weight of 200-2000 kDa, which is composed of D-glucose and D-mannose in a molar ratio of 1:1.6[8], and its main chain is linked by β-1,4
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glycosidic bonds, at the C3 position of the main chain. There is a branch consisting of
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β-1,3 glycosidic bonds, about one branch per 68 sugar residues, and a branch length
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of 3 or 4 sugar residues[9], present on D-mannose Acetyl group[10,11].There are a
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large number of active hydroxyl groups in the KGM molecule, which facilitates
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various modifications to improve their performance and expand their applications.
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Scheme 2 is a schematic diagram of the molecular structure of KGM.
Scheme.2. Molecular structure of KGM
KGM has water solubility, which can absorb water about 100 times its own weight. During the dissolution process, since the diffusion rate of water molecules is much larger than the diffusion rate of KGM molecules, KGM particles swell and
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eventually form a liquid with a large viscosity. KGM has excellent gel properties. When acetyl groups are lost, KGM molecules form network structures through hydrogen bonding. For example, gelatin and agarose gels melt with increasing temperature.KGM can form a thermoreversible gel with unstable 3d network structure through strong synergistic effect with xanthan gum, carrageenan and other hydrophilic
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gums[12,13].In addition, KGM is a soluble dietary fiber with high fiber, low fat and
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low calorie, which has the effect of losing weight[14], lowering blood sugar, lowering
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blood fat and anti-tumor.KGM is widely used in medical, food, chemical industry[15]
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and other fields due to its unique physical and chemical properties and excellent
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physiological activity.KGM has good biocompatibility, biodegradability and renewability. It can be prepared into medical wound dressings to provide a moist
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healing environment for wounds, as well as hemostasis and healing.KGM is a kind of
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high quality dietary fiber with various physiological functions, which has been processed into a natural health food and has the functions of losing weight, lowering blood sugar, lowering blood fat, smoothing bowel and laxative, and improving human immunity.KGM can be used as food additive, such as emulsifier, thickener, meat binder, gelling enhancer, etc.
Collagen is a bioactive polymer synthesized by animal cells and widely found in skin, bones, tendons, ligaments and other connective tissues, accounting for about 30% of the total protein in the body[16]. It is an important part of the extracellular matrix.Collagen molecule is a rod-shaped spiral structure formed by the winding of
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three peptide chains. This stable structure enables collagen which has ability to support, protect and form a boundary between organs and the body in animals.Collagen has the advantages of good biological activity, tissue adhesion, low immunogenicity and hydrophilicity, etc. It can also interact with cells, participate in cell signal transduction, regulate cell migration, proliferation, differentiation and other
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physiological activities[17].Collagen peptide (COP) is a type of small molecule
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product produced by degradation of Collagen by enzyme treatment.In addition to
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typical characteristics such as high biological activity, small molecular weight, easy
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absorption and non-toxic and harmless, COP has many excellent biological functions
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because its content of proline and hydroxyproline is significantly higher than other
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active peptides.For example, antioxidant properties and the ability to promote healing.
It has been reported that COP can promote cartilage repair [18] and alveolar bone
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regeneration [19], and can also promote multi-differentiation of various stem cells [20]..Liu et al.[21] prepared a compound hydrogel containing COP. The experiment showed that the compound hydrogel had excellent physicochemical properties and good biocompatibility, and could promote cell proliferation.
In this study, KGM was used as raw material to prepare konjac oxide (OKGM) under the oxidation of sodium periodate. With OKGM as crosslinking agent and carboxymethyl chitosan/collagen peptide (CMCS/COP) through schiff base reaction, carboxymethyl
chitosan/collagen
peptide/konjac
oxide
(CMCS/COP/OKGM)
composite hydrogel was prepared.The materials used are biocompatible, and
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considering the characteristics of each material, a new compound hydrogel was designed for the practical application of wound dressing. The composite hydrogel was characterized by FTIR , NMR, XRD and SEM, and its gel time, swelling rate, water evaporation rate and hemolysis rate were measured.The effect of compound hydrogel on the growth of NS-FB and HS-FB cells was studied in vitro.
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2.Materials and methods
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2.1.Materials
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Sodium iodate (NaIO4), ethylene glycol (C2H6O2), potassium iodide (KI),
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sodium bicarbonate (NaHCO3), sodium thiosulfate (Na2S2O3), sodium chloride
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(NaCl), sodium dihydrogen phosphate (NaH2PO4), and disodium hydrogen phosphate (Na2HPO4) are all AR（Analytical reagent） and come from sinopharm chemical reagent
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co.,LTD.Konjac (KGM) is purchased from hubei Johnson konjac co., LTD.The purity
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of KGM is 95% and relative molecular mass is about 800,000. The degree of substitution is not explained.CMCS/COP composite material is self-made material; fresh anti-coagulation mouse blood comes from Wuhan Third Hospital.Collagen is derived from fish collagen and purchased through sinopharm chemical reagent co., LTD.
Chitosan purchased from sinopac group, deacetylation degree 80.0 ~ 95.0； burning residue (in terms of sulfate), % ≤1.5；drying loss, % ≤10.0；heavy metal (in terms of Pb), % ≤0.0015；arsenic (As), % ≤0.00006；viscosity, mPa·s 50 ~ 800；deacetylation degree, % 80.0 ~ 95.0；acetic acid insoluble substance, % ≤1.0. 2.2.Methods
Journal Pre-proof 2.2.1.preparation of OKGM The experimental method is to weigh an appropriate amount of KGM and add it to a three-necked flask, then add 500 mL of distilled water, and mechanically stir at room temperature until the KGM is completely dissolved.An appropriate amount of NaIO4 was dissolved in 30 mL distilled water to form a uniform solution, and was
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added to KGM solution drop by drop. The temperature was controlled at 30℃, and
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the reaction was conducted for 12 h.Add 10 mL C2H6O2 to react with excessive NaIO4,
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stop after 1 hour, pour the reaction solution into the dialysis bag, dialysis for 72 hours,
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change water twice a day, freeze dry and reserve, and the product is OKGM，The
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reaction equation is shown in Scheme 3.
Scheme.3. preparation of OKGM
2.2.2.Determination of oxidation degree of OKGM The oxidation degree of OKGM (DO, %) was determined by iodimetry.The oxidation degree of OKGM refers to the percentage of the total number of urylation units in the KGM molecule.This experiment indirectly calculated the oxidation consumption of NaIO4 by measuring the excessive content of NaIO4 in the preparation process of OKGM, and finally calculated the oxidation degree of
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OKGM.Specific operations are as follows:Take 5 mL of the mixed solution after the oxidation reaction, put it in the dark condition, add 10 mL of sodium bicarbonate solution (10 wt%) and 2 mL of potassium iodide solution (20 wt%), and let it sit for 20 minutes.Sodium thiosulfate standard solution was prepared. Starch solution was used as indicator to titrate the mixed solution and determine the iodine content.
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Formula1
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M 148 2 n 214 100 % DO M1
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According to formula 1, the oxidation degree of OKGM could be calculated.
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Where, M1 is the mass of konjac, with unit of g;M2 is the mass of sodium
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periodate added, in g;148 is the molar mass of konjac unit in g/mol;214 is the molar
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mass of sodium periodate in grams per mole;N is the amount of excess sodium periodate in moles.According to formula 1, the oxidation degree of OKGM was
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calculated to be 50%.
2.2.3.Preparation of CMCS/COP 2.2.3.1.The purification of MTGase（microbial trans glutaminase ）
(1) Preparation of buffer solution: A certain amount of Na2HPO4 and NaH2PO4 were weighed and dissolved in distilled water, and 1000 mL volumetric flask was added to prepare a 0.2 mol/L standard solution. The two standard solutions were mixed and adjusted to a pH of 6, as a buffer solution, and used.
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(2) Purification of MTGase: The purchased MTGase was added to the above-prepared buffer solution, fully dissolved by mechanical stirring, and the centrifuge speed was set at 3500 rpm/min, and centrifuged for 15 min. The supernatant was taken, vacuum-filtered three times, dialyzed in distilled water for three days, twice changed with water, and then dried using a freeze dryer to obtain
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purified MTGase powder, which was stored in a 4 ℃ refrigerator and set aside.
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2.2.3.2.The preparation of CMCS/COP
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The CMCS solution, the COP solution and the purified MTGase solution each
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having a mass fraction of 1 wt% were separately prepared using the above buffer
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solution. After the preparation, they were poured into a three-necked flask, placed in
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an oil bath, set at a temperature of 50 ℃, mechanically stirred for 4 h, and then the temperature was adjusted to 90 ℃ for 10 min to deactivate the high temperature of
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the MTGase. The reaction solution was cooled to room temperature, circulated and filtered three times, placed in distilled water for three days, periodically changed, and then dried using a freeze dryer to obtain a CMCS/COP composite. 2.2.4.Preparation of CMCS/COP/OKGM hydrogels A certain amount of CMCS/COP samples were weighed and added to distilled water to prepare 5 wt% CMCS/COP solution, which was mechanically stirred until completely dissolved and stored in the refrigerator for later use.Similarly, appropriate amount of OKGM was added to distilled water to prepare 10 wt% OKGM solution,
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which was mechanically stirred until completely dissolved.Add 0.4,0.8,1.2,1.6 and 2.0mL of OKGM solution to 2.0mL of CMCS/COP solution, and prepare hydrogels in different proportions after fully stirring.According to the different ratios of OKGM and CMCS/COP (0.2:1, 0.4:1, 0.6:1, 0.8:1, 1:1), the Numbers were CCGM-2, CCGM-4, CCGM-6, CCGM-8, and CCGM-10, respectively.
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Scheme 4 shows the reaction equation of preparing CMCS/COP/OKGM
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composite hydrogel. The reaction mechanism is the schiff base reaction between the
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residual amino group on CMCS/COP and the aldehyde group on OKGM.As shown in
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Scheme 5, CCGM-2, CCGM-4, CCGM-6, CCGM-8 and CCGM-10 hydrogels were
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prepared on a 12-well plate. All the samples were smooth, clear and transparent, with
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a yellowish tint, and became darker as the OKGM ratio increased.
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Scheme.4. Reaction equation of CMCS/COP/OKGM hydrogel preparation
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Scheme.5. CCGM-2（a）、CCGM-4（b）、CCGM-6（c）、CCGM-8（d）、
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CCGM-10（e） hydrogel sample
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2.2.5.Structural characterization of materials (1)Infrared spectrum characterization: KGM, OKGM and CMCS/COP/OKGM samples were ground into powder, followed by potassium bromide tablets and infrared characterization by Nicoet170SX type Fourier transform infrared spectrometer of Nicoet, USA, and the wavelength range was set at 500-4000 cm-1.
(2) NMR characterization: KGM and OKGM samples were ground into powder and fully dissolved in 0.5 mL D2O. 1H-NMR characterization was performed by amx-500 nuclear magnetic resonance instrument of Bruker company, Germany, and the temperature was set at 25 ℃.
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(3) X-ray diffraction characterization: KGM and OKGM samples were ground into powder and XRD analysis was conducted by D8 Advance X-ray diffractometer of Bruker company, Germany.
The parameters set in the experiment are: copper target, nickel filter plate, pipe flow 40 mA, pipe pressure 40 KV, scanning range 5-45°, step length 0.02°.
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(4) Sem characterization: freeze-dried, incised and gold-sprayed sections of
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CCGM-2, CCGM-6 and CCGM-10 hydrogel samples were used to observe the
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cross-section morphology of hydrogel samples by VEGA3 scanning electron
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microscope from Tescan company of the Czech republic.
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2.2.6.1.Gel time determination
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2.2.6.Study on the properties of CMCS/COP/OKGM hydrogels
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CMCS/COP and OKGM were dissolved in water respectively to prepare 5 wt% and 10 wt% solutions.CMCS/COP aqueous solution was injected into the flat bottom tube with a syringe.Add the teflon magnetic agitator into the flat bottom tube and set the rotation speed to 100 RPM /min. After the rotation is stabilized, add the OKGM aqueous solution and start timing until the magnetic agitator stops rotating. The total rotation time is the gel time. 2.2.6.2.Determination of swelling rate The samples of CCGM-2, CCGM-4, CCGM-6, CCGM-8 and CCGM-10 hydrogels were placed in a freeze-dryer, taken out 48 hours later, weighed and
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recorded as the initial weight M1. The Na2HPO4/NaH2PO4 buffer solution was prepared with a pH of 7.4.Place the freeze-dried sample in the buffer solution for water absorption and swelling, and weigh it regularly until the sample weight no longer increases, that is, the swelling balance is reached, and the residual water on the sample surface is erased. Record the weight at this time as the swelling balance
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weight M2.The swelling rate (SR) of the hydrogel sample was calculated according to
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M1 M 2 100% M2
Formula 2
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SR
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the formula 2, and the experiment was repeated three times.
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2.2.6.3.Determination of water evaporation rate
Samples of CCGM-2, CCGM-4, CCGM-6, CCGM-8 and CCGM-10 hydrogels
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were taken from freeze-dried samples at room temperature.After immersion in
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distilled water and reaching swelling equilibrium, residual water on the sample surface was wiped off, weighed and recorded as swelling equilibrium mass Ma.The oven temperature was set at 50 ℃ and the relative humidity was set at 50%. The samples after swelling and equilibrium were placed in the oven. The weight of the samples was measured periodically and recorded as Mb.Until the weight of the hydrogel sample is no longer reduced, weigh it and record it as Mc.Calculated by the formula 3 hydrogel samples of Water evaporation rate (Water evaporation rate, WER), the experiment repeated three times.
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WER
Ma Mb 100% Ma Mc
Formula 3
2.2.6.4.Determination of hemolysis rate The fresh anticoagulated mouse blood was diluted with an equal amount of physiological saline and stored in a refrigerator for use.The freeze-dried hydrogel
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samples of CCGM-2, CCGM-4, CCGM-6, CCGM-8 and CCGM-10 were ground into
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powder, then 25 mg was evenly dispersed in 10 mL normal saline, 0.1 mL diluted anticoagulant mouse blood was added, and placed in a constant temperature tank at
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37.5 ℃ for 1 h.Subsequently, the centrifuge was centrifuged for 10 min (rpm 1500
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rpm/min), the wavelength of the ultraviolet-visible spectrophotometer was set to 545
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nm, and the absorbance of the supernatant was measured.The Hemolysis rate (HR) of
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three times.
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hydrogel samples was determined by formula 4, and the experiment was repeated
HR
A1 A3 100% A 2 A3
Formula 4
In the formula 4, HR represents the hemolysis rate, A1 represents the absorbance of the hydrogel sample group, and A2 is the absorbance of the positive control group (10 mL distilled water, 0.1 mL diluted anticoagulated mouse blood, non-aqueous gel sample material), A3 is Absorbance of the negative control group (10 mL normal saline, 0.1 mL diluted anticoagulated mouse blood, non-aqueous gel sample material).
2.3.Determination of in vitro cell compatibility
Journal Pre-proof 2.3.1.Experimental main materials and instruments Experimental cells: Human skin fibroblasts (NS-FB) and human hypertrophic scar fibroblasts (HS-FB) were purchased at Beijing North Natron Biotechnology Research Institute.
The main materials of the experiment: DMEM medium, fetal bovine serum,
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streptomycin mixture, trypsin, CCK-8, cell culture flask, 96-well cell culture plate, all
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purchased from Wuhan Huashun Biotechnology Company. OKGM and CMCS/COP
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composites are self-made materials.
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Main instruments: super clean table, CO2 constant temperature incubator,
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inverted optical microscope, enzyme marker.
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2.3.2.Preparation of cell suspension and seed plate
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(1) preparation of cell culture medium: 50 mL fetal bovine serum was taken with pipette and put into 500 mL DMEM culture medium, 5 mL penicillins mixture was added, shaken well and set aside.
(2) cell culture: NS-FB and HS-FB cells were inoculated in T25 cell culture bottles respectively, 5 mL of the cell culture medium prepared above was added, and placed in CO2 incubator at a constant temperature of 37.5℃.The growth state of the cells was observed by inverted optical microscope on a regular basis every day, and the medium was changed according to the growth condition of the cells.When the cell density reached 80%, trypsin was used for digestion, and the logarithmic growth cells
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were obtained by passage three times at the ratio of 1 to 3.
(3) cell inoculation: the logarithmic growth cells were digested with trypsin and dispersed evenly in the cell culture medium.A cell suspension with a concentration of 1×104 cells /mL was prepared by plate counting method, which was inoculated into a 96-well cell culture plate with an addition amount of 100 L per hole.After
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inoculation, the cells were transferred to a CO2 constant temperature incubator and
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2.3.3.Preparation of hydrogel extract
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cultured for 24 hours to make the cells stick to the wall.
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CMCS/COP freeze-dried samples and OKGM freeze-dried samples were ground
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into powder, and after uv irradiation for 2 h, CCGM-2, CCGM-4, CCGM-6, CCGM-8
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and CCGM-10 hydrogel samples were prepared with aseptic distilled water in ultra-clean table.The sample was extracted with cell culture medium at a mass ratio of
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1:10, and after 24 h, it was sterilized by filtration using a 0.22 μm filter to obtain a leach solution of the hydrogel sample, which was sealed and stored in a 4℃ refrigerator for use.
2.3.4.Determination of absorbance by CCK-8 method
The adherent cells were removed from the CO2 constant temperature incubator, the medium was sucked out, and 100 L of the hydrogel sample extract was added respectively.Then placed in a cell culture incubator, removed one day later, aspirate the medium, and replace with 100 L of fresh medium.10 L CCK-8 stain was added to
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each hole with pipette gun, and the culture was continued for 3 h after vibration defoamed.The wavelength of the microplate reader was set at 450 nm to determine the absorbance of each hole.
According to the formula for calculating the Relative cell survival rate (4-5) (Relative cell viability, RCV).
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OD1 OD2 100% OD3 OD2
Formula 5
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RCV=
-p
Where, RCV represents the relative survival rate of cells, OD1 is the absorbance
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of sample group, OD2 is the absorbance of blank group (only medium and cck-8, no
lP
cells), and OD3 is the absorbance of positive control group (fresh medium replaces
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sample extract). 3.Results and discussion
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3.1.Determination of in vitro cell compatibility
Journal Pre-proof Fig.1. infrared spectra of KGM (a), OKGM (b) and CMCS/COP/OKGM (c) hydrogels Figure 1 is the infrared spectrum diagram of KGM, OKGM and CMCS/COP/OKGM hydrogel. The infrared spectrum curve of KGM was observed, and the analysis of its characteristic absorption peak was shown in table 1.
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Table 1. Each peak of KGM infrared spectrum belongs to Position of absorption peak Serial number
Peak on the ownership of the
3420
2
2925
O-H stretching vibration
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1
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(cm-1)
1734
lP
3
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The stretching vibration of C-H of methyl or hypomethyl groups
C=O on the acetyl group
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The absorption peaks in table 1 are consistent with the KGM standard infrared
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spectrum[22，23]. Compared with the infrared spectrum of KGM, the analysis of the new characteristic absorption peaks in the infrared spectrum of OKGM is shown in table 2.
Table 2. Attribution of new peaks in OKGM infrared spectrum Position of absorption peak Serial number
Peak on the ownership of the
(cm-1)
1
1720
Symmetric vibrations of the aldehyde group
2
895
The hemiacetal structure contracts in vibration
As it can be seen from table 2, two new characteristic absorption peaks [24] appeared in the infrared spectrum of OKGM, which fully reflected the appearance of
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the aldehyde group, indicating that the hydroxyl group in the KGM molecule was oxidized to the aldehyde group, and OKGM was successfully prepared.By observing figure 1 and comparing the infrared spectrum curve of OKGM, it can be found that in the Fourier infrared spectrum of CMCS/COP/OKGM hydrogel, the two strong vibration absorption peaks (1720 and 895 cm-1) originally belonging to OKGM
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basically disappear completely.It Indicated that the aldehyde group was consumed by
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the reaction during the preparation of hydrogels.At the same time, the stretching
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vibration absorption peak (1640 cm-1) of C=N was observed, which indicated that the
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aldehyde group on OKGM crosslinked with the amino group on CMCS/COP, and
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finally formed the composite hydrogel.
3.2.1H Nuclear Magnetic Resonance Spectrum
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Figure 2 shows 1H-NMR spectra of KGM and OKGM.In the 1H-NMR spectrum
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of KGM, the peak with a chemical shift of 2.18 PPM represents the hydrogen of methyl on the ester bonded acetyl group on the molecular chain sugar residues.The chemical displacement peak in the range of 3.37-4.14 PPM represents the displacement peak of other hydroxyl hydrogen on the sugar ring, and the chemical displacement peak of 4.45 PPM represents the displacement peak of hydroxyl hydrogen on the C6 position of the sugar residue molecule.These conform to KGM standard nuclear magnetic hydrogen spectra [25].By observing 1H-NMR spectrum of OKGM, it can be found that OKGM possesses all characteristic absorption peaks of KGM, in addition to which two new absorption peaks are generated.The chemical
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displacement peaks of 6.49 and 9.24 PPM represent the active hydrogen on the enol carbon.In general, under alkaline conditions, the aldehyde group of OKGM will appear as enol, and in acidic conditions, as free aldehyde groups.As the system was neutral, the peaks of the two sites were weak, and the peaks of 6.49 PPM and 9.24 PPM were not observed in the 1H-NMR spectra of KGM, indicating that KGM was
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successfully oxidized and the aldehyde group was formed.
Fig.2. 1H-NMR spectra of KGM (a) and OKGM (b) 3.3.X ray diffraction analysis Figure 3 shows the X-ray diffraction patterns of KGM and OKGM. It can be seen that the two samples have similar patterns, with no obvious sharp crystal peak, and the overall shape is a steamed bread shaped dispersion ring, which is an
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amorphous structure.
KGM OKGM
(a)
10
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Intensity(a.u.)
(b)
30
20
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2/()
40
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Fig.3. XRD spectra of KGM (a) and OKGM (b)
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3.4.Sem analysis
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FIG. 4 is the sem image of the cross section of CCGM-2, CCGM-6 and CCGM-10 composite hydrogel samples, with magnification of 200 and 1000
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times.Overall observation of the three kinds of composite hydrogels shows that the three kinds of hydrogels have uniform pore structure with good continuity. When magnified to 1000 times, it can be seen that the pore structure is smooth and flat with a certain thickness.This excellent three-dimensional space structure is conducive to the free diffusion of water molecules, and its solid skeleton structure enables the hydrogel to absorb a lot of water without breaking.
Longitudinal comparison of the three kinds of composite hydrogels showed that the pore structure of CCGM-2,CCGM-6 and CCGM-10 showed a weak trend of
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decreasing successively.The reason may be that with the increase of the content of OKGM in the compound hydrogel, the concentration of aldehyde group in the reaction process increases, and more aldehyde group binds to the amino group in CMCS/COP, leading to a higher degree of reaction between OKGM and CMCS/COP, resulting in a denser skeleton structure.Therefore, the pore diameter will decrease.The
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water absorption properties of hydrogels are mainly demonstrated in three aspects,
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including hydrogel materials, structures, and pore sizes.This composite hydrogel is
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mainly composed of oxidized konjac, which has been proven to have excellent water
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absorption properties. From the XRD results, the composite hydrogel has an
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amorphous structure, and the results of scanning electron microscopy also prove the composite Hydrogels have excellent pore structure. These experiments have proved
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their excellent water absorption properties. In the actual use of hydrogels, this
properties.
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conclusion is also proved. This composite hydrogel has excellent water absorption
Journal Pre-proof （a2）（×1000）
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（a1）（×200）
（b2）（×1000）
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（b1）（×200）
（c1）（×200）
（c2）（×1000）
FIG. 4.Hydrogel profiles of CCGM-2（a）,CCGM-6（b）,CCGM-10（c） Sem image
3.5.Gel time analysis Figure 5 shows the gel time of CCGM-2, CCGM-4, CCGM-6, CCGM-8 and CCGM-10 hydrogels. It can be seen that all hydrogels have shorter gel time, which is less than 30 s, and the shorter gel time has higher requirements for clinical operation speed.The gel time of each composite hydrogel sample was compared longitudinally,
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and it was found that CCGM-2, CCGM-4, CCGM-6, CCGM-8, and CCGM-10 had a tendency to decrease first and then increase.As the compound hydrogels have the same composition, only the ratio of CMCS/COP to OKGM is different. As the content of OKGM increases, the concentration of aldehyde group in the reaction process increases, so that more aldehyde groups bind to the amino group in CMCS/COP,
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accelerating the reaction speed.Therefore, when the ratio of OKGM increased from
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0.2 to 0.6, the gelation time decreased successively, and CCGM-6 had the shortest
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gelation time (17 s).When the OKGM ratio continues to increase from 0.6 to 1, the
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excess aldehyde group has no time to bind with the amino group, but leads to a
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decrease in the overall concentration of the components in the system, slowing down the reaction rate and increasing the gel time. The longest gel time of CCGM-10 is 28
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s.Compared with other wound dressings, the gel time of this composite hydrogel is
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very short, which puts forward higher requirements for clinical operation. However, due to its rapid gel time, this material can quickly close the wound, rapidly shape the wound, reduce the risk of wound exposure during operation and shorten the operation time.
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30
Gelation Time (s)
25 20 15 10 5
CCGM-4
CCGM-6
CCGM-8 CCGM-10
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CCGM-2
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0
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FIG. 5.Gel time of CCGM-2, CCGM-4, CCGM-6, CCGM-8, CCGM-10 hydrogel
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3.6.Swelling rate analysis
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Swelling rate is one of the important indexes for evaluating hydrogel dressings. When hydrogel reaches equilibrium in water absorption and swelling, the more water
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it can retain, the stronger its ability to provide a moist treatment environment for
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wounds, which plays an important role in patients' recovery as soon as possible. Therefore, it is of great significance to investigate the swelling performance of hydrogel.Figure 6 shows the swelling rate of CCGM-2, CCGM-4, CCGM-6, CCGM-8 and CCGM-10 hydrogels. It can be seen that the swelling rate is about 500%, indicating that the hydrogels have good swelling performance.Longitudinal comparison of each composite hydrogel showed that the swelling ratios of CCGM-2, CCGM-4, CCGM-6, CCGM-8, and CCGM-10 hydrogels decreased in turn.The swelling rate of CCGM-2 was 581%.The reason may be that with the increase of OKGM content in the compound hydrogel, the concentration of aldehyde group in the
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reaction process increases, and more aldehyde groups bind to the amino group in CMCS/COP.As a result, the degree of cross-linking between OKGM and CMCS/COP is higher, forming a denser skeleton structure, which affects the entry of free water.At the same time, the volume of water molecules can be reduced, which results in decreased swelling performance, which is the same as the result of SEM.
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600
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400
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300
200
lP
Swelling Rate (%)
500
100
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0
CCGM-2
CCGM-4
CCGM-6
CCGM-8 CCGM-10
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FIG. 6. Swelling rate of CCGM-2, CCGM-4, CCGM-6, CCGM-8, CCGM-10 hydrogels 3.7.Water evaporation rate analysis Figure 7 is the water evaporation rate of CCGM-2, CCGM-4, CCGM-6, CCGM-8, CCGM-10 hydrogel samples.It can be seen from the curve that the change trend of water evaporation rate of each compound hydrogel with time is basically similar, fast before and slow after 24 h, and the hydrogel basically reaches a stable after 24 h, and the hydrogel still retains a certain amount of water.Longitudinal comparison of each composite hydrogel, it can be found that with the increase of
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OKGM content in the composite hydrogel, the water evaporation rate has a tendency to decrease, probably because more aldehyde groups are introduced due to the increase of OKGM content, and there are more The combination of multiple aldehyde groups with the amino group in CMCS/COP makes the crosslinking of OKGM and CMCS/COP become higher, resulting in a denser pore structure, which affects the
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convection with air and slows the evaporation of free water. This results in a decrease
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in water evaporation rate, which is the same as the SEM results. 110
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80 70 60
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Water loss(%)
90
-p
CCGM-10 CCGM-8 CCGM-6 CCGM-4 CCGM-2
100
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50 40 30
4
6
8
10
12
14
16
18
20
22
24
26
Time (h)
FIG. 7. Water evaporation rate of CCGM-2, CCGM-4, CCGM-6, CCGM-8, CCGM-10 hydrogels 3.8.Hemolysis rate analysis The hemolysis rate of biological materials is an important indicator for judging the blood compatibility of the material. The hemolysis phenomenon refers to the phenomenon that hemoglobin escapes after the contact of foreign matter with blood
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causes the red blood cells to rupture[26].As wound dressing, hydrogel can directly contact the blood, so its hemolysis rate must be controlled within a certain range. The international standard for the hemolysis rate of biological materials is less than 5%.Therefore, it is of great significance to determine the hemolysis rate of the compound hydrogel for its clinical application.Figure 8 shows the hemolysis in the
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gel sample group, the positive control group and the negative control group after 1 h
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of contact with the blood and centrifugation for 10 min. It can be observed that the
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blood cells in the positive control group have ruptured and the solution is uniform and
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red.In the negative control group, blood cells were deposited at the bottom of the tube,
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and the solution was clear and transparent.The hemolysis phenomenon in the gel sample group was similar to that in the negative control group. The solution was
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basically clear and transparent, and the blood cells and gel material were deposited at
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the bottom, indicating that the hydrogel material could maintain the integrity of the blood cell structure without causing hemolysis reaction.
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（2）
（3）
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（1）
FIG. 8. The figure shows the negative sample group（1）, gel control group（2） and
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positive control group（3）.
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Table 3 shows the hemolysis rate of CCGM-2, CCGM-4, CCGM-6, CCGM-8,
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and CCGM-10 hydrogel samples after 1 h of contact with blood.It can be seen from the table that the hemolysis rate of all samples is between 0.81% and 2.67%, which conforms to the standard of less than 5% of biological materials and has good blood compatibility.
Table 3. Hemolysis rate of CCGM-2, CCGM-4, CCGM-6, CCGM-8, CCGM-10 hydrogels Sample
Hemolysis rate（%）
CCGM-2
0.81
CCGM-4
2.67
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0.89
CCGM-8
1.78
CCGM-10
1.62
3.9.In vitro compatibility analysis of CMCS/COP/OKGM hydrogels Figure 9 shows the relative survival rates of NS-FB and HS-FB cells cultured in
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CCGM-2, CCGM-4, CCGM-6, CCGM-8, and CCGM-10 hydrogel extracts.By
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observing the survival rate of NS-FB cells, it was found that there was no significant
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difference in the relative survival rate of cultured ns-fb cells between different sample
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extracts.Ccgm-2 hydrogel extract had the highest survival rate, up to 105%.The cell
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survival rate decreased slightly with the increase of OKGM ratio in various ratios of hydrogels,It may be that uncrosslinked OKGM leads to the high concentration of
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OKGM in the hydrogel extract, and excessive aldehyde group is introduced, thus
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disrupting the nutrient balance of the medium.It may also lead to changes in cell osmotic pressure, which affects cell proliferation.However, the survival rate of ccgm-10 hydrogel extract was the lowest, but reached 93%, indicating that the hydrogel had good compatibility with ns-fb cells and could promote the proliferation of NS-FB cells.
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CCGM-2 CCGM-4 CCGM-6 CCGM-8 CCGM-10
Relative Cell Viability (%)
120 100 80 60 40 20 0 HS-FB
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NS-FB
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FIG. 9. Relative survival rate of NS-FB and HS-FB cells cultured in CCGM-2,
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CCGM-4, CCGM-6, CCGM-8 and CCGM-10 hydrogel extracts
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For HS-FB cells, the relative survival rates of all hydrogel sample extracts were
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greater than 88%.There was no significant difference in cell survival rate between
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different hydrogel samples,The cell survival rate decreased slightly with the increase of OKGM ratio in all hydrogels.It is also because too much aldehyde group is
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introduced, which damages the nutrient balance of the culture medium and even changes in the osmotic pressure of cells, thus affecting the proliferation of cells.In conclusion, this composite hydrogel had little effect on HS-FB cell proliferation.In this composite hydrogel, collagen peptide is an important component, and its cell experiments have also proved the biocompatibility of the composite hydrogel. Long-term laboratory studies have found that this bio-derived collagen peptide, With good biological activity, we also carried out cell experiments, confirming our view that collagen peptides still have good biological activity in composite hydrogels. 4. Conclusion
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In this paper, Regarding the innovation of this paper, three natural sources of raw materials were used, which were modified according to their physical and chemical properties, and the three materials were cross-linked to form a composite new hydrogel, using a series of tests. It has proved its physical and chemical properties, and has carried out cell experiments to confirm its prospects for clinical use, making it
of
have the potential for industrialization.KGM was used as raw material to prepare
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OKGM under the oxidation of NaIO4. Using OKGM as crosslinking agent, schiff base
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reaction was conducted with CMCS/COP prepared in chapterⅡto obtain CMCS/COP
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complex hydrogels, which were numbered CCGM-2, CCGM-4, CCGM-6, CCGM-8
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and CCGM-10 hydrogels according to the different contents of OKGM and CMCS/COP.The structure was characterized by infrared, nuclear magnetic, X-ray
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diffraction and scanning electron microscopy. The gel time, swelling ratio, water
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evaporation rate and hemolysis rate of the composite hydrogel were tested. The cells were in vitro with NS-FB and HS-FB cells. Experiments were conducted to investigate the effects of composite hydrogels on the growth of these two cells.
Infrared, nuclear magnetic and XRD results showed successful oxidation of OKGM and successful preparation of CMCS/COP/OKGM composite hydrogel.Sem test showed that all hydrogels had continuous and uniform pore structure, and the pore diameter decreased with the increase of OKGM content in hydrogels.The gel time test showed that the CMCS/COP/OKGM hydrogel had a shorter gel time, less than 30 s, and the gel time decreased first and then increased with the increase of OKGM
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content. CCGM-6 hydrogel has the shortest gel time is 17 s.The swelling ratio test showed that the CMCS/COP/OKGM hydrogel had good swelling performance, the swelling ratio was about 500%, and the CCGM-2 swelling rate was the highest, which was 581%.The water evaporation rate test showed that the CMCS/COP/OKGM hydrogel had good water retention ability, and the water evaporation rate of the
of
hydrogel decreased with the increase of the content of OKGM, and the CCGM-10
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hydrogel could retain 20% water after 24 h.The hemolysis rate test showed that the
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hemolysis rate of CMCS/COP/OKGM hydrogel was between 0.81-2.67%, all of
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which were lower than 5%, showing excellent blood compatibility.The cell
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experiments showed that the relative survival rate of NS-FB cells cultured by CMCS/COP/OKGM hydrogel extract was more than 93%, which had good
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cytocompatibility and could promote the proliferation of NS-FB cells.The relative
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survival rate of HS-FB cells cultured by CMCS/COP/OKGM hydrogel was more than 88%, and there was no obvious side effect on HS-FB cells.
In summary, this paper prepared a chitosan-based natural polysaccharide composite hydrogel, and carried out a series of structural characterization and performance tests. The results show that the hydrogel has good swelling performance and long time. Its water retention capacity, excellent blood compatibility and cytocompatibility, as well as the function of promoting wound healing.It will have a broad application prospects in the field of wound dressings. Acknowledgements
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The work was supported by the National Natural Science Foundation of China (Foundation No. 51773161), The Special Funds Project of Major New Products of Hubei Province (Foundation No. 20132h0040), University-industry Cooperation Projects of The Ministry of Education of Guangdong province (Foundation No. 2012B091100437), The innovation fund project of the Ministry of Science and of
Small
and
Medium-sized
Enterprises
(Foundation
No.
of
Technology
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11C26214202642, No. 11C26214212743), Zhuhai Science and Technology Plan
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Projects (Foundation No. 2011B050102003), Wuhan Science and Technology
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Development (Foundation No. 201060623262), The Fundamental Research Funds for
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