Design of biostable scaffold based on collagen crosslinked by dialdehyde chitosan with presence of gallic acid

International Journal of Biological Macromolecules 130 (2019) 836–844

Contents lists available at ScienceDirect

International Journal of Biological Macromolecules journal homepage: http://www.elsevier.com/locate/ijbiomac

Design of biostable scaffold based on collagen crosslinked by dialdehyde chitosan with presence of gallic acid Pemo Bam, Anindita Bhatta, Ganesan Krishnamoorthy ⁎ Natural Products Chemistry Groups, Chemical Sciences and Technology Division, CSIR-North East Institute of Science & Technology, Jorhat 785 006, Assam, India

a r t i c l e

i n f o

Article history: Received 29 October 2018 Received in revised form 18 February 2019 Accepted 2 March 2019 Available online 4 March 2019 Keywords: Collagen Dialdehyde chitosan Gallic acid Scaffold Biostable

a b s t r a c t In this study, we have prepared the biostable collagen scaffold which is crosslinked by dialdehyde chitosan (DAC) with presence of Gallic acid (GA) and characterized its physico-chemical, biostable and biocompatible properties. The digital photographic and scanning electron microscopic (SEM) images of the prepared collagen scaffold is exposed well with properly oriented interconnected porous natured structure. The appearance of diffraction peaks showed slightly crystalline characteristic when compared to others. The differential scanning calorimetric (DSC) and thermogravimetric analysis (TGA) measurements indicates well significantly increased denaturation temperature (TD) and decreased decomposition rate. FT-IR result suggests the structural integrity of collagen which favours the molecular stability. The dialdehyde groups from DAC crosslinked with collagen functional groups that increase the molecular crosslinking owing to the large number of amino groups in its molecular chain. This scaffold exhibited 87% resistance against collagenolytic degradation by collagenase. The results showed that the improved biostability which prevents the free access of the collagenase to binds with the collagen triple helical chains. This scaffold confirm high biocompatibilities; enhanced cell proliferation and adhesions properties. This results gains new insight into the collagen scaffold to improves the biostability. This could be suitable method to preparation of collagenous biomaterials for tissue engineering applications. © 2019 Elsevier B.V. All rights reserved.

1. Introduction In recent years, natural polymer of the collagen along with the dialdehyde polysaccharides (DAPs) have been contributing to the development of wide variety of biomaterials which can be used as for design of drug delivery systems and tissue engineering materials with huge enhancements. The DAPs; dialdehyde chitosan (DAC), dialdehyde alginate (DAA), dialdehyde starch (DAS) and dialdehyde cellulose (DACe) are polymeric dialdehydes which derived from naturally occurring polysaccharides. These DAPs can be used as additive and crosslinking agents to the collagen matrix in an effort to improve the physico-chemical and biological properties [1]. Both the biopolymers, collagen and DAPs, based hybrid biomaterials exhibit excellent biodegradability, good bioresorbable, superior biocompatibilities; cell proliferation and adhesion properties, and weak antigenicity when compared with combination of other natural polymers [2–5]. The DAPs crosslinked collagen improves the thermal and mechanical properties [6]. There are many researchers reported on DAPs substituted collagen based hybrid biomaterials; 3-D collagen scaffold, film, sponge and nanocomposite have been successfully fabricated [5–10]. The DAPs can ⁎ Corresponding author. E-mail address: [email protected] (G. Krishnamoorthy).

https://doi.org/10.1016/j.ijbiomac.2019.03.017 0141-8130/© 2019 Elsevier B.V. All rights reserved.

be used for decellularized tissue preparation and biological tissue fixation [11–14]. Collagen is a coiled-coil fibrous protein present in extracellular matrix which contains both, acidic and basic amino acids, and may bear either a positive or negative charge depending on the pH condition. Various studies reported that applications of collagen as drug delivery system are burn and wound dressing materials, shields in ophthalmology and as tissue engineering are porous scaffolds, filaments, thinfilms for tissue implant that promote the cell and tissue attachment and growth [15–18]. The DAPs; DAC, DAA, DAS and DACe are valuable crosslinkers which has been received enormous attentions in the varied biomedical fields [19–22]. These polymeric dialdehydes are considered as a safer additives and green cross-linking agent for various biomaterials and nanomaterials, and also can use as a biological tissue fixation and tanning agents [1]. Compared with other crosslinking agent, DAPs exhibited satisfactory penetrability and crosslinking reactivity in collagen fiber network, and thus performed more favorable stability in terms of higher shrinkage temperature [1]. These are easily reacting with amine groups of proteins in mild conditions, at physiological pH, with high efficiency. Additionally, these have been reported that as a component for achieving the biocompatible matrix, there into the haemocompatibility is exceptional. These are biodegradable, biocompatible

P. Bam et al. / International Journal of Biological Macromolecules 130 (2019) 836–844

837

and toxically acceptable which could provide desired biological strength. The DAC can modify the collagen materials with desirable physicochemical and biological properties are considered [5]. The DAC could be promoted the thermostability and fibrillogenesis of collagen and formed stable Schiff's. These materials could target of high attention for the design of scaffolds as membrane and as implant for wellcontrolled delivery of therapeutic drugs. The combination of both the biopolymers collagen and DAC could renovate the existing state of drug delivery and tissue engineering [5]. Hence, the DAC might have an opportunity to react with the free amino groups within collagen molecules. However, DAC into the collagen scaffolds, which reduces its versatility to make different kinds of end products and gives the hardness and toughness but could not give desirable structural integrity. To enhance the structural integrity and biostability of collagen scaffold, we have introduced the gallic acid (GA) during the cross-linking process with DAC. The GA is 3, 4, 5-trihydroxybenzoic acid which has been considered as a suitable crosslinking option to replace other crosslinking agents to construct the biocompatible biomaterials [23]. Similar to other plant polyphenolics, GA gives considerable stability to form multiple H-bonds with collagen based products. This GA improves the collagen stability as confirmation by an increased resistance to biodegradation against collagenase activity [23]. This polymeric GA such as tannic acid has been suggested as a potential cross-linking agent and reported to improve the stability of the collagen in cardiovascular implants [24]. Based on the polyphenols binding to collagen, four mechanisms for interaction between GA and collagen have been postulated, viz., non-covalent, covalent, ionic and H-bonding or hydrophobic interactions. The GA derivatives have sufficient –OH and –COOH groups to effectively form strong multiple complexes with collagen and other macromolecules [25,26]. In order to improve the structural stability of collagen scaffold, DAC along with GA are added during the collagen crosslinking process. Combination of these crosslinking agents could improve the structural integrity and desired properties thereby expect to yield a wide variety of biostable collagen scaffolds. In this study, an attempt has been made to prepare a DAC conjugated biostable collagen scaffold with presence of GA. The report seeks to overcome the disadvantages of the existing collagen scaffold. The above crosslinking process by using prepared DAC along with GA is as an effective to accelerate the collagen fibrous network and uniform surface morphologies. The biodegradation rate, morphological characteristics, structural and thermal stability, and biocompatibility have compared with collagen alone, DAC, GA and DAC-GA crosslinked.

solution. Subsequently, the product was purified by dialysis against 0.2 M NaCl for 2 days and thereafter against water for another two days. Finally, the obtained product was freeze-dried to get a fine powder.

2. Materials and methods

2.3. Characterization

2.1. Materials

2.3.1. Physico-chemical properties The following physico-chemical techniques were used for the characterizations of collagen scaffold. The SEM (Carl Zeiss Eigma-FESEM, Xmax Oxford Instruments) was used for analysis of scaffold surface morphological properties. The specimen was cut from the official sampling position with uniform thickness without any pre-treatment. The crystalline and amorphous state of the scaffold was evaluated by XRD (Rigaku X-ray diffractrometer). The X-ray generator was operated at 40 KV tube voltages and 40 mA of tube current, using the Kα lines of copper as the radiation source. The scanning angle ranged from 5 to 100 °C for 45 min in step scan mode (step width 3°/min). The FT-IR spectra (PerkinElmer 1640 FT-IR Spectrometer) were recorded as an average of 50 scans. The spectra represented the average of 50 scans. All spectra were recorded from 400 to 4000 cm−1 with a resolution of 2 cm−1. The endothermic peak temperature was recorded as the denaturation temperature (TD) of the scaffold which analyzed by using differential scanning calorimetric (DSC) technique. The thermal behaviour was studied by heating 4 ± 0.5 mg of each sample in a

All reagents and chemicals were used of analytical grade. Chitosan (Low MW, 75–85% deacetylated), Gallic acid and Clostridium histolyticum collagenase (Type IA) (ChC) were procured from Sigma Chemicals Co., USA. All other reagents and chemicals were obtained from SRL Ltd., India. All other, fibroblast, culture media and antibiotics, chemicals for these studies were purchased from Sigma-Aldrich and Fluka Chemicals. 2.2. Design of DAC-GA crosslinked collagen scaffold 2.2.1. Preparation of DAC The DAC was prepared according to the previously reported method [5]. In brief, DAC was prepared by adding 15 mg/ml sodium periodate in 20 ml water (as oxidant) was added drop-wise into the 1 g chitosan solution at pH 4.5 under stirring. The reaction was continuously stirred in dark at room temperature. After 48 h to terminate the reaction, 10 ml ethylene glycol was added into solution for 2 h. To get the precipitated crude product, 5 g NaCl and 800 ml pure ethanol were added into

2.2.2. Isolation of type I collagen Acid soluble bovine Achilles collagen type I was extracted, purified and determined here according to the previously described method [23]. The bovine Achilles tendons, collected fresh from local slaughter house, were manually dissected out from surrounding fascia, followed by washing in distilled water. They were cut into small bits of 3–4 mm each with a sharp knife and were solubilized. In brief, the bits were minced in a Mincer and washed using nonionic surfactant. The washed tissues were suspended in sodium peroxide solution (0.3%) for swelling. The tissue was washed with distilled water (qs) after the coagulation of the swollen mass and then suspended in phosphate buffer solution of pH 8.5 and treated overnight with trypsin (0.5% w/w). The tissue was again washed in distilled water to deactivate the enzyme and the dissolved salts were removed. The coagulated tissue was swollen again in distilled water after adjusting the pH of water to 2.5 with HCl, and treated with Pepsin (0.3% w/w) overnight. After the 2nd enzyme treatment tissues were washed repeatedly in water to deactivate the enzyme. The coagulated collagen was dissolved in Millipore water (0.06 μs purity) acidified to pH 3.5 using HCl to get pure collagen solution. The undissolved proteins were removed by centrifugation at 10000 rpm for 30 min. All the above operations were performed at a temperature of 15 ± 2 °C. Quantity of collagen was determined by Hydroxyproline (Hyp) assay method [23]. 2.2.3. Preparation of collagen scaffold Collagen was dissolved in 0.5 M acetic acid to prepare 1% (w/v) solution. After deaeration under reduced pressure to remove entrapped air bubbles, the collagen solution was injected into disposable plastic petri dishes, frozen at −20 °C then −80 °C for 4 h, and then lyophilized for 24 h to obtain a porous collagen scaffold. 2.2.4. Crosslinking of collagen scaffold The collagen scaffold was dehydrated under vacuum for 24 h before any additional chemical crosslinking. In all the cross-linking experiments, the scaffold was incubated with or without 0.5% of prepared DAC (pH 5.5) and 0.1% GA (pH 5.5) solution. After incubation for 24 h at room temperature, the scaffold was neutralized, washed with double distilled water and lyophilized then used for further characterizations.

838

P. Bam et al. / International Journal of Biological Macromolecules 130 (2019) 836–844

covered in pan under nitrogen gas flow at 20 ml/min. The investigation was carried out over the temperature range of 25–300 °C with a heating rate of 10 °C min−1 with an empty aluminum pan as the reference. The % weight loss of the scaffold was studied using thermogravimetric analysis (TGA). The thermal behavior was studied by heating 4 ± 0.5 mg of each individual scaffold in a sample pan under nitrogen gas flow at 20 ml/min. The above investigation was carried out over the temperature range 25–800 °C with a heating rate of 10 °C min−1. The water-uptake and swelling ratios were assessed by using reported method [30]. The water-uptake and swelling ratios were obtained by incubation of the collagen, collagen-GA, collagen-DAC and collagen-DAC-GA scaffolds in water at room temperature for 2 h. Water uptake ratio of scaffolds were assessed as follows. The scaffold was weighted and then equilibrated for 2 h in PBS (pH 7.2) at room temperature. After removal of additional water, it was weighted again. The water-uptake ratio was calculated by following formula. The wateruptake ratio is defined as (Wt-W0)/(W0).

gentamycin (160 mg/mL) and amphotericin B (3 mg/mL) at 37 °C humidified with 5% of CO2. After 72 h, the percentages of viable cells were assessed using MTT assay. 2.4.2. Cell adhesion NIH3T3 cells were incubated with the crosslinked scaffold which placed at the bottom of a 96 well tissue culture plates for 72 h. At each time interval, scaffold was removed then rinsed with PBS. The average number of cells was counted from PBS. 2.5. Statistical analysis All the experiments are repeated thrice and expressed as the mean ± standard deviation (SD). The significant level was set as * p b 0.0001, ** p b 0.0002 and *** p b 0.0005. 3. Results 3.1. Synthesis and characterization

Water uptake ratio ¼ ðWt −W0 Þ=ðW0 Þ  100 Swelling ratios of scaffolds were assessed as follows. After hydrating in water for 1 h, the sample was equilibrated for 2 h in PBS (pH 7.2) at room temperature, blotted with filter paper to remove excess surface water and then weighted. The scaffold was then placed in deionized water to remove the buffer salts and air-dried to constant weight. The swelling ratio is defined as (St-S0)/(S0). Swelling ratio ¼ ðSt −S0 Þ=ðS0 Þ  100 In addition to assessment of weight and calculation of the swelling ratio, the volume changes of the different cylindrical formed collagen scaffolds before contact with fluid (dry samples) and after removal of fluid (dried samples) were determined. 2.3.2. Biodegradation rate The percentage (%) of the crosslinked collagen scaffolds degradation against collagenase activity was calculated by the method described earlier [23]. The degradation of scaffold was monitored by using Hyp assay method by release of soluble form of Hyp from insoluble collagen. The method of determining Hyp involves the oxidation of Hyp to pyrrole-2-carboxylic acid, which complexes with pdimethylaminobenzaldehyde exhibiting maximum absorbance at 557 nm.

During the cross-linking process, DAC with addition of GA is irreversibly bound with the –NH2 groups of collagen by intra-versus inter molecular cross linking and through coordinate covalent linkage. The stable Schiff's base between DAC and collagen with GA has significant effects to improve the structural integrity of collagen. The studies supporting the Schiff bases formation between DAC and bovine serum albumin (BSA) gave the resulting material a higher thermal stability and DACe crosslinked chitosan have high protein adsorption capacity [27,28]. GA is composed of a –OH and – COOH groups with one galloyl residue. This galloyl unit improves the collagen stability which evidenced by an increased resistance to degradation against collagenase activity [23] and has been suggested as a potential crosslinking agent. These functional groups can react with functional groups located in collagen, such as -NH 2 or –OH groups. The galloyl group is able to form ionic bonds with positively charged amino acids and that can also form ion dipole interactions with water. These interactions are very important and improve the stabilities of collagen scaffold. In the study collagen-DAC in partially anionic in charge is treated with acetic acid in order to convert the cationic charge, prior to crosslinking. The GA crosslinked collagen is negatively charged and then neutralized. This is achieved by using mild alkalis such as sodium hydroxide. During neutralization, the pH is raised to 7 and further lyophilized. Schematic representations of proposed structure of collagen side chain crosslinking with DAC and GA are shown (Scheme 1). The aldehyde groups in

%Soluble collagen ¼ %Hyp  7:4 Based on the soluble (solubilized due to enzymatic hydrolysis) collagen content in the supernatant solution of the ChC treated scaffold. The % degradation of collagen for collagen, collagen-GA, collagen-DAC, collagen-DAC-GA scaffolds are calculated as %collagen degradiation    Initial collagen‐Soluble collagen ¼ 100−  100 Initial collagen

2.4. Biocompatibility analyses 2.4.1. Cell viability assay Monolayers of fibroblast cell lines, NIH3T3 [National Centre for Cell Science (NCCS), Pune, India] were grown on crosslinked scaffold in a 96 well culture plate (Corning, NY) and maintained in Dulbecco's Modified Eagles Medium (DMEM) with 10% Fetal Calf Serum (FCS) supplemented with penicillin (120 units/mL), streptomycin (75 mg/mL),

Scheme 1. Schematic of DAC and GA residues complexation with collagen molecules. DAC with addition of GA is irreversibly bind with the collagen scaffold by intra-versus inter molecular cross linking with collagen –NH2 groups through coordinate covalent linkage. The stable Schiff's base between DAC and collagen has significantly improved the structural integrity of collagen.

P. Bam et al. / International Journal of Biological Macromolecules 130 (2019) 836–844

DAC can react with amino groups, hydroxyl groups, the phenolic ring of tyrosine, and the imidazole ring of histidine, which could result in the formation of inter- and intra-molecular cross-links in collagens. The DAC and GA are mediated to form aldehyde groups and Hbonding interactions; lysine and hydroxylysine, a divalent amine, and then cross-links glucose units by reacting with these aldehyde groups (Scheme 1). The DAC addition does not affect the secondary structure of the collagen but forming intermolecular cross-linking. The intra- and inter-molecular interactions and the cross-linking should improve the properties of the collagen scaffold. As results, the biostability and structural integrity, textural, thermal stability, water uptake and swelling ability, and as well biocompatibility increased.

839

3.2. SEM analysis The native, GA and DAC and DAC-GA crosslinked collagen scaffolds are shown at a magnification of × 500 which depicted in the Fig. 1 and well interconnected porous structure is indicated in DAC-GA crosslinked one. The porous network of this scaffold seems to be less dispersed when compared to others. In principle, increased density of the DAC-GA crosslinked scaffold exhibit the pore diameters ranging from 75 to 150 μm and slightly dispersed porous structure when compared to native, GA and DAC. At the same time, we could see that DAC-GA crosslinked collagen scaffold had higher microporosity, and the porosity is better than that of native, GA and DAC crosslinked. An ideal scaffold to use in tissue engineering application should possess

Fig. 1. A) Digital photographic and B) SEM images of the crosslinked collagen scaffolds are shown at a magnification of ×500 at pH 7. DAC-GA crosslinked collagen scaffold exhibit slightly dispersed porous structure when compared to the collagen alone, collagen-GA and collagen-DAC.

840

P. Bam et al. / International Journal of Biological Macromolecules 130 (2019) 836–844

the attribute of a homogenous microporosity. Since the addition of DACGA improves the collagen structural stability.

A)

-40 -35 -30

3.3. XRD analysis

Collagen Alone Collagen-GA Collagen-DAC Collagen-DAC-GA

The X-ray diffraction patterns for DAC-GA crosslinked collagen scaffold is shown in the Fig. 2. The diffraction patterns for native, GA, DAC and DAC-GA crosslinked collagen scaffolds are show broaden in the same region of the spectrum indicating the amorphous and crystalline characteristics. The appearance of DAC-GA crosslinked collagen diffraction peaks shows slightly crystalline characteristic when compared to others.

Heat Flow (mW/mg)

-25 -20 -15 -10 -5 0 5 10

3.4. DSC analysis

15 20 20

40

60

80

100

120 140

160

180 200 220

240

Temperature (°C)

B)

100 90 80

% Weight loss

The DAC-GA crosslinked collagen scaffold exhibit an increased in the TD values when compared to native one. The TD of DAC-GA crosslinked collagen was computed from DSC data (Fig. 3A) and also presented in Table 1. The prepared pure collagen TD is 69. DSC curve for DAC-GA crosslinked collagen showed characteristic endothermic transitions of structural changes of the collagen triple helix i.e., the protein denaturation. This may be due to net increase in the number of inter and intra molecular crosslinks and interactions between the collagen with DAC and GA. It is known that TD of collagen matrix is paralleled by destruction of H-bonds and hydrophobic interactions between the protein subunits and helix-coil transformation. The hydrophobic interactions determined predominantly by glycine residues and H-bonds formed between Hyp residues play an important role in stabilization of collagen molecules. Presumably, the combination of high hydrophobicity and capacity to form peptide bonds permits these molecules incorporate in certain areas of collagen fibrils and promote stabilization of the collagen scaffold structure. This could be possibly due to the decrease in the availability of the active sites in the scaffold. The TD of scaffold depends on stable intra and intermolecular cross-links formed through the DACGA.

Collagen Alone Collagen-GA Collagen-DAC Collagen-DAC-GA

70 60 50 40 30 0

100

200

300

400

500

600

700

Temperature ( C )

3.5. TGA analysis The TGA analysis of crosslinked collagen scaffold is shown in Fig. 3B. The % weight loss is presented in Table 1. The TGA profile of native, GA and DAC and DAC-GA are shown an initial loss on drying below 100 °C

Fig. 3. DSC and TGA thermograms of the crosslinked scaffolds. A) The collagen-DAC-GA scaffold is exhibited an increased TD value when compared to collagen alone. B) Around 68.98, 52.45, 40.97 and 29.56% weight losses were observed for collagen alone, collagenGA and collagen-DAC and DAC-GA samples, respectively in the temperature range of 200–400 °C.

5000

Intensity (a.u)

4000

(about 4%) and a second weight loss between 200 and 400 °C which is due to the slight water bound in samples. Around 68.98, 52.45, 40.97 and 29.56% weight loss were observed for native, GA and DAC and DAC-GA samples, respectively in the temperature range 200–400 °C (See Table 1). This stage is associated with slow decomposition of scaffold over a temperature range of 150–500 °C. The collagen-DAC-GA decreases the weight loss of collagen scaffold. The corresponding weight losses for all collagen scaffolds are not significantly different. The results are showed with the decreased weight loss of DAC-GA compared to others. In the above cases, DAC-GA crosslinked collagen scaffold lead to a different thermal profile when compared to others.

Collagen Alone Collagen-GA Collagen-DAC Collagen-DAC-GA

3000

2000

1000

3.6. FT-IR spectral analysis 0 0

10

20

30

40

50

60

70

2 (degree) Fig. 2. XRD spectra of the crosslinked scaffolds. The diffraction patterns for collagen alone, collagen-GA, collagen-DAC and collagen-DAC-GA are shown broaden in the same region of the spectrum indicating the amorphous and crystalline characteristics. The appearance of collagen-DAC-GA diffraction peaks are shown slightly crystalline characteristic when compared to others.

The FT-IR spectrum of DAC shows the absorption bands at 1632 and 1560 cm−1 are assigned to the symmetric and asymmetric stretching vibration of C3-C2 bond which shown in the Fig. 4A. The bands around 1118 and 1031 cm−1 are corresponding to the C_O stretching vibration from C3-OH and C6-OH, respectively. The relatively intense peaks of chitosan situated at 1595 cm−1, 1078 cm−1 and 1028 cm−1 disappear totally after oxidization on DAC, which might indicate that the unit cyclic structure of chitosan is pyranosidic ring-opened and parts of –

P. Bam et al. / International Journal of Biological Macromolecules 130 (2019) 836–844

841

Table 1 % weight loss, hydrothermal stability (TD), % water-uptake and swelling properties of DAC-GA crosslinked collagen scaffold. Process

% Weight loss 200–400 °C

TD(°C)

% Water uptake

% Swelling ratio

Collagen alone Collagen-GA Collagen-DAC Collagen-DAC-GA

68.98 52.45 40.97 29.56

293 ± 3 291 ± 3 284 ± 3 283 ± 3

77 ± 3 78 ± 3 71 ± 3 65 ± 4

25 ± 2 15 ± 3 13 ± 2 13 ± 4

OH and -NH are oxidized into aldehyde groups can clearly confirm that from DAC. The band at 1735 cm−1, a characteristic absorption peak of aldehyde groups, can clearly confirm that from DAC. The structural changes of DAC-GA crosslinked collagen amide I, amide II and amide III bands which shown in the Fig. 4B and Table 2. The spectra were scaled to equal absorption at 3268 cm−1, assigned to the –CH2 group, which remains unchanged during the crosslinking process. The crosslinking of collagen scaffold by DAC with presence of GA

A)

40 35

% Transmittance

30 25 20

3.7. Water-uptake and swelling properties

15 Chitosan DAC

10 5 500

1000

1500

2000

2500

3000

3500

4000

-1

Wavelength (cm )

B) 105 100

% Transmittance

95 90 85 Collagen Alone Collagen-GA Collagen-DAC Collagen-DAC-GA

80 75

65

1000

1500

2000

2500

3000

Water-uptake and swelling ratios of crosslinked collagen scaffold is presented in the Table 1. The water-uptake of native, GA and DAC and DAC-GA scaffolds were 77 ± 3, 78 ± 3, 71 ± 3 and 65 ± 4% at pH 7.2 respectively. The swelling ratio of native, GA and DAC and DAC-GA scaffolds were 25 ± 2, 15 ± 3, 13 ± 2 and 13 ± 4% at pH 7.2 respectively. The formation of DAC crosslinked collagen scaffold can decrease more or less the amount of hydrophilic groups and also presented lower swelling ratio, this decrease should be mainly attributed to the partial diminishing of the hydrophilic groups of the scaffold after crosslinking when compared to GA treated. The initially existing hydrophilic groups, such as amino and carboxyl groups were partially transformed into hydrophobic amide groups under the DAC-GA treatment. The water uptake and swelling ratios of the DAC-GA crosslinked scaffolds are still huge enough to meet the demand of the scaffolds used in tissue engineering, consequently both the properties can be controlled by characteristics of DAC-GA. 3.8. Biodegradation analysis

70

60 500

results in an increased absorption at 1232 and 1047 cm−1 when compared to the collagen alone, GA and DAC scaffold. The spectra showed the retention of all the major three amide bands viz. I, II, and III in the region of 1600–1670 cm−1, 1510–1560 cm−1 and 1220–1450 cm−1, symmetric stretching of carboxylate salts region 1403 cm−1 and ester bond 1082 cm−1, which validated the structural integrity in the conformation of the collagen molecule. The absorbance at the region of collagen bend (1640–1690 cm−1) decreased, characteristic region (1620–1630 cm−1) increased, as well as the increased ester bond (1080 cm−1), while the symmetric stretching of carboxylate salts (1403 cm−1) was decreased compared to C\\H bond (2958 cm−1), indicating that the amidation reaction has occurred between carboxyl groups and available amino groups of Lys and Hyl residues. The possible reason may be that the Schiff's base might have a negligibly negative influence on the structure of collagen. Nevertheless, electrostatic interactions between DAC and collagen occupy primary role, which facilitate the process of the self-aggregation of collagen molecules. Therefore, we may draw a conclusion that the structural integrity and stability of collagen is improved after crosslinking.

3500

4000

-1

Wavelength (cm ) Fig. 4. FT-IR spectra of crosslinked collagen scaffold. A) The relatively intense peaks of chitosan situated at 1595 cm−1, 1078 cm−1 and 1028 cm−1 disappear totally after oxidization, which might indicate the formation of dialdehyde groups. B) The absorbance at the region of collagen bend (1640–1660 cm−1) decreased, characteristic region (1620–1630 cm−1) was increased, as well as the increase of ester bond (1080 cm−1), while the symmetric stretching of carboxylate salts (1403 cm−1) was decreased compared to C\ \H bond (2958 cm−1) in collagen-DAC-GA when compared to collagen alone, collagen-GA and collagen-DAC which is indicating that the amidation reaction has occurred between carboxyl groups and available amino groups of Lysine and Hydroxylysine residues.

Percentage of biodegradation (based on Hyp released) of crosslinked collagen scaffold by treatment of collagenase have been determined (Fig. 5). Significant reduction in the biodegradation is observed for GA, DAC and DAC-GA as against 98% degradation in the case of native one at 72 h period of incubation. DAC-GA crosslinked scaffold reduced biodegradation rate when compared to collagen alone, GA and DCA crosslinked. More recently, tannic acid and gallic acid (GA) molecules have been shown to stabilize collagen not only through H-bonding but also by hydrophobic interactions [29,30]. The previous studies also supporting for this work to stabilization of collagen by hydrolysable and condensed tannin against collagenase activity [31]. The hydrophobic core of the GA molecule, likely incorporates itself into hydrophobic areas, while –OH groups of the GA may establish multiple H-bonds with neighboring collagen-DAC molecules, resulting in improved stabilization of collagen that prevent the free access of collagenase to reactive

842

P. Bam et al. / International Journal of Biological Macromolecules 130 (2019) 836–844

Table 2 FT-IR analysis of the DAC-GA crosslinked collagen scaffold. Region

Collagen alone

Collagen-GA

Collagen-DAC

Collagen-DAC-GA

3301 2891 1629 1562 1383 1023

3219 2898 1629 1533 1356 1023

Peak wave number (cm−1) Amide A Amide B Amide I Amide II Amide III

3281 2864 1629 1549 1376 1023

Assignment

3308 2851 1629 1562 1411 1023

sites on the collagen chains. The dialdehyde groups can crosslink between the collagen functional groups and increase the intra and intermolecular cross-linking owing to the large number of amino groups in its molecular triple helical chain. A schematic representation of interaction of collagen with DAC and galloyl unit of GA is leading to polymerization and complex formation which shown in Scheme 1. This is also possible that GA (either free or bound to collagen) might well bind the collagenase rendering it inactive. Hence, this could be inferred that the inhibition of collagenase activity on collagen scaffold which could be most likely due to the blocking of the reactive sites in collagen by GA, thereby collagenase unable to bind and degrade the DAC crosslinked collagen.

N-H stretching with H-bond CH2 asymmetrical stretching C=O stretching N-H bending coupled with C\ \N bending and CH2 bending C-N stretching coupled with N\ \H bending and CH2 wagging C-O stretching Skeletal stretching

cell-adhesion ratio on crosslinked scaffold significantly increased (Fig. 6B). DAC-GA exhibited more fibroblast adhesion than GA and DAC alone. This is interesting to note that fibroblast adhesion to crosslinked scaffolds demonstrated similar trend to native scaffold. It suggests that DAC-GA may induce the adhesion and spreading of fibroblast than native, GA and DAC. In this study, we confirmed that DAC

A) 110

105

In this scaffold, cells exhibited better proliferation than those on the native, GA and DAC crosslinked one (Fig. 6A). The fibroblast in direct contact with DAC-GA crosslinked collagen scaffold is typical morphology. There is no abnormal morphology or cellular lysis was detected. The structural appearance of cultured fibroblast on this scaffold is linear patterns of arrangement with the cells oriented towards the edges of the membrane. Further, the cultured fibroblast on this scaffold is a regular arrangement with a tightly packed striated and spindle shape appearance. This indicates that DAC assisted GA crosslinked collagen scaffold has potential for tissue engineering applications. The DAC could improve the cell adhesion to the surface of scaffold. Introducing DAC into collagen favorable for the cell's adhesion, growth and proliferation are studied [5]. After GA is introduced into the DAC-collagen, the

100

100

*

*

*

Collagen

Collagen-GA

*

*

95

90

85

80 Collagen-DAC Collagen-DAC-GA

Scaffolds

24 hrs 48 hrs 72 hrs

80

60

**

*

80

*

*

**

40 30

**

20

** ***

*

100

% Cell Adhesion

*

70

50

*

B)

*

90

% Biodegradation

% Cell viability

3.9. Biocompatibility analysis

*

* *

60

40

20

10 Collagen

Collagen-GA

Collagen-DAC Collagen-DAC-GA

Scaffolds

0

Collagen

Collagen-GA

Collagen-DAC Collagen-DAC-GA

Scaffolds Fig. 5. Percentage of biodegradation of the DAC-GA crosslinked scaffolds at pH 7.2. The significant reduction of biodegradation is observed for GA, DAC and DAC-GA crosslinked scaffolds as against 98% degradation in the case of native one at 72 h period of incubation. DAC-GA crosslinked scaffold reduced biodegradation rate when compared to collagen alone, collagen-GA and collagen-DCA.

Fig. 6. A) Percentage cell viability and B) Cell adhesion properties of the DAC-GA crosslinked scaffold. DAC-GA crosslinked collagen scaffold exhibited better cell proliferation than collagen alone, collagen-GA and collagen-DAC. After GA is introduced into the collagen-DAC, the cell-adhesion ratio on scaffold increased significantly.

P. Bam et al. / International Journal of Biological Macromolecules 130 (2019) 836–844

along with GA is an effective biological crosslinker which is more suitable for the preparation of collagen based scaffolds. 4. Discussion In this study, we investigated the DAC initiated GA assisted crosslinked collagen scaffold. This crosslinking stabilize the collagen through H-bonding interaction and covalent crosslinking. The DAC with presence of GA on collagen was found to enhance the biostability by using this effective crosslinking process. SEM was used for revealing the influence of DAC-GA treatment on the microstructure. The results indicate that the scaffold could preserve the porous structure during the cross-linking treatment in the presence of DAC-GA. It is a crucial aspect to evaluate a cross-linking method to modify a tissue engineering scaffold. Cell culture results demonstrate that the ability to induce cell infiltration and growth of the original scaffold have been preserved after cross-linking treatment in the presence of DAC-GA. As expected, enhanced TD can be surely gained when the crosslinking is conducted in the presence of DAC-GA. The DAC-GA can diminish the biodegradation ultimately improve biostability. Recent work, the increased in thermostability, mechanical properties and low susceptibility towards biodegradation of DACe crosslinked collagen structure are reported [4]. Another study reported the enhanced intensity and elasticity by addition of DAA into the collagen solution [32]. The addition of DAA into chitosan/silk fibroin blending membrane to promote the cell attachment and proliferation were reported [33]. Various study reported that DAPs crosslinked collagen scaffold for tissue fixation to enhancing the structural integrity intensity and elasticity [3,34]. The general dialdehyde click chemistry for amine bioconjugation provides convincing evidence to synthesis stable molecules [35]. The DAS could be an efficient additive to the collagen matrix in improving its physical and biological properties [2]. This crosslinking study suggests that free amino groups of collagen are initially reacting with DAC and then bridging by the GA which producing more extensive intra and inter molecular crosslinking. The DAC crosslinked collagen can interact with GA which is mediated by –OH group through available –COOH group of GA and amide group of collagen molecule as well as the effect of both ionic and covalent bonds. The gallolyl group is known to provide extensive H-bonding capacity with collagen which stabilizes the collagen triple helical chains [25]. Recent studies, GA and tannic acid have been shown to stabilize the collagen which is not only through H-bonding but also by hydrophobic interactions [23,29]. These results suggested that the steric structure of GA containing galloyl unit is on important unit for the stabilization of EDC/NHS crosslinked collagen against collagenase activity. This GA is induced the cross linking of DAC-collagen chains and also protected potential cleavage sites from collagenase attack. The strong binding of galloyl unit arises from both H-bonding and hydrophobic interactions with the collagen fibrils. Our results showed that efficacy of GA on blocking crosslinked collagen degradation by indirect inhibition of collagenase activity. All the results positively demonstrated that DAC-GA possesses the capacity to protect the collagen scaffold. We found that DAC-GA with collagen converted about 92% to stable form. Furthermore, the good biocompatibility of DAC-GA, accompanied by the improved TD and biostability, a better preservation of the original biocompatibility of scaffold can be expected. Attachment of DAC-GA to scaffold can increase the polar groups on the surface. So the cell adhesion rate on collagen-DAC-GA scaffold is higher than on native, DAC and GA treated collagen scaffold. And also DAC-GA improves the hydrophobicity of the scaffold. The addition of GA into the DAC crosslinked scaffold which could not decrease the cytocompatibility. DAC possesses the positive and negative charges which could be widely used to enhance cell attachment and adhesion, growth and differentiation of many cell types. This DAC-GA crosslinked is resistance to enzymatic degradation and could be used to culture a wide variety of cell types. Overall, it can be concluded that DAC-GA crosslinked collagen not only having

843

biostability and structural integrity and also superior biocompatibility; cell proliferation and adhesion.

5. Conclusion In conclusion, we have prepared the improved biostable and highly biocompatible collagen scaffold which cross-linked by DAC with presence of GA. This work has provided the convincing evidence to improve the biostability and structural integrity of scaffold. Therefore, combination of DAC with GA provides a selective crosslinking seems to be influenced strongly by structure and nature. These cross-linkers could comprise an important aid to prevention of collagen degradation. The formed stable Schiff's base between the collagen and DAC with GA has significant effects on improvement of the micro-structural integrity. This cross-linking improves the texture, thermal, structural and biostability, as well as swelling and water uptake properties. This scaffold is more stable than the native ones; native, GA and DAC. Furthermore, introducing DAC-GA into the collagen scaffold may favor the cell's adhesion, growth and proliferation. Hence, this cross-linking may find the potential use in the preparation of collagenous biomaterials for tissue engineering applications. Acknowledgements One of the authors Dr G. Krishnamoorthy acknowledges Department of Science and Technology (DST) - Science and Engineering Research Board (SERB), New Delhi, Government of India for his awarded Young Scientist grant support. The author GK is thankful to the Dr Archanamoni Das, Senior Scientist, Natural Products Chemistry, CSTD, CSIR-NEIST, Jorhat for her valuable support. The author GK is thankful to the Director, CSIR-NEIST, Jorhat for providing laboratory facilities. Conflict of interest The authors declare that they have no conflict of interest. Research involving human participants and/or animals This chapter does not contain any studies with human participants or animals performed by any of the authors. Informed consent Informed consent was obtained from all individual participants included in the study. References [1] W. Ding, Y.N. Wang, J. Zhou, B. Shi, Effect of structure features of polysaccharides on properties of dialdehyde polysaccharide tanning agent, Carbohydr. Polym. 201 (2018) 549–556. [2] Y. Liu, G. Acharya, C.H. Lee, Effects of dialdehyde starch on calcification of collagen matrix, J. Biomed. Mater. Res. A 99 (2011) 485–492. [3] Y. Hu, L. Liu, Z. Gu, W. Dan, N. Dan, X. Yu, Modification of collagen with a natural derived cross-linker, alginate dialdehyde, Carbohyd. Polym. 102 (2014) 324–332. [4] K. Pietrucha, M. Safandowska, Dialdehyde cellulose-crosslinked collagen and its physicochemical properties, Process Biochem. 50 (2015) 2105–2111. [5] X. Liu, N. Dan, W. Dan, J. Gong, Feasibility study of the natural derived chitosan dialdehyde for chemical modification of collagen, Int. J. Biol. Macromol. 82 (2016) 989–997. [6] K. Pietrucha, E. Marzec, M. Kudzin, Pore structure and dielectric behaviour of the 3D collagen-DAC scaffolds designed for nerve tissue repair, Int. J. Biol. Macromol. 92 (2016) 1298–1306. [7] T. Du, Z. Chen, H. Li, X. Tang, Z. Li, J. Guan, C. Liu, Z. Du, J. Wu, Modification of collagen-chitosan matrix by the natural crosslinker alginate dialdehyde, Int. J. Biol. Macromol. 82 (2016) 580–588. [8] T. Liu, L. Shi, Z. Gu, W. Dan, N. Dan, A novel combined polyphenol-aldehyde crosslinking of collagen film - applications in biomedical materials, Int. J. Biol. Macromol. 101 (2017) 889–895. [9] B. Kaczmarek, A. Sionkowska, A.M. Osyczka, The application of chitosan/collagen/ hyaluronic acid sponge cross-linked by dialdehyde starch addition as a matrix for

844

[10]

[11]

[12]

[13]

[14]

[15] [16]

[17] [18] [19]

[20] [21]

[22]

P. Bam et al. / International Journal of Biological Macromolecules 130 (2019) 836–844 calcium phosphate in situ precipitation, Int. J. Biol. Macromol. Part A. 107 (2018) 470–477. J. Skopinska-Wisniewska, K. Wegrzynowska-Drzymalska, A. Bajek, M. Maj, A. Sionkowska, Is dialdehyde starch a valuable cross-linking agent for collagen/elastin based materials? J. Mater. Sci. Mater. Med. 27 (2016) 67. Y. Xu, L. Li, X. Yu, Z. Gu, X. Zhang, Feasibility study of a novel crosslinking reagent (alginate dialdehyde) for biological tissue fixation, Carbohyd. Polym. 87 (2012) 1589–1595. Y. Xu, C. Huang, L. Li, X. Yu, X. Wang, H. Peng, Z. Gu, Y. Wang, In vitro enzymatic degradation of a biological tissue fixed by alginate dialdehyde, Carbohyd. Polym. 95 (2013) 148–154. X. Wang, Y. Wang, L. Li, Z. Gu, X. Yu, Feasibility study of the naturally occurring dialdehyde carboxymethyl cellulose for biological tissue fixation, Carbohyd. Polym. 115 (2015) 54–61. X. Wang, Z. Gu, H. Qin, L. Li, X. Yang, X. Yu, Crosslinking effect of dialdehyde starch (DAS) on decellularized porcine aortas for tissue engineering, Int. J. Biol. Macromol. 79 (2015) 813–821. L. Cen, W. Liu, L. Cui, W. Zhang, Y. Cao, Collagen tissue engineering: development of novel biomaterials and applications, Pediatric Res. 63 (2008) 492–496. R. Berisio, A.D. Simone, A. Ruggiero, R. Improta, L. Vitagliano, Role of side chains in collagen triple helix stabilization and partner recognition, J. Pept. Sci. 15 (2009) 131–140. P.T. Ashwin, P.J. McDonnell, Collagen cross-linkage: a comprehensive review and directions for future research-review, Bri. J. Ophthalmol. 94 (2010) 965–970. H.P. Bachinger, K. Mizuno, J.A. Vranka, S.P. Boudko, Collagen formation and structure, Comprehensive Natural Products II. Chapter 5 (16) (2010) 469–530. B.E. Christensen, I.M.N. Vold, K.M. Varum, Chain stiffness and extension of chitosans and periodate oxidized chitosans studied by size-exclusion chromatography combined with light scattering and viscosity detectors, Carbohyd. Polym. 74 (2008) 559–565. Y. Feng, L. Yang, F. Li, A novel sensing platform based on periodate-oxidized chitosan, Anaytical Methods 2 (2010) 2011–2016. K.A. Kristiansen, A. Potthast, B.E. Christensen, Periodate oxidation of polysaccharides for modification of chemical and physical properties, Carbohydr. Res. 345 (2010) 1264–1271. X. He, R. Tao, T. Zhou, C. Wang, K. Xie, Structure and properties of cotton fabrics treated with functionalized dialdehyde chitosan, Carbohyd. Polym. 103 (2014) 558–565.

[23] G. Krishnamoorthy, R. Selvakumar, T.P. Sastry, S. Sadulla, A.B. Mandal, M. Doble, Experimental and theoretical studies on gallic acid assisted EDC/NHS initiated crosslinked collagen scaffolds, Mater. Sci. Eng. C Mater. Biol. Appl. 43 (2014) 164–171. [24] J.C. Isenburg, D.T. Simionescu, N.R. Vyavahare, Tannic acid treatment enhances biostability and reduces calcification of glutaraldehyde fixed aortic wall, Biomaterials. 26 (2005) 1237–1245. [25] A. Sionkowska, B. Kaczmarek, K. Lewandowska, Modification of collagen and chitosan mixtures by the addition of tannic acid, J. Mol. Liq. 199 (2014) 318–323. [26] M.E. Tedder, J. Liao, B. Weed, C. Stabler, H. Zhang, A. Simionescu, D.T. Simionescu, Stabilized collagen scaffolds for heart valve tissue engineering, Tissue Eng. Part A. 15 (2009) 1257–1268. [27] U.J. Kim, Y.R. Lee, T.H. Kang, J.W. Choi, S. Kimura, M. Wadaa, Protein adsorption of dialdehyde cellulose-crosslinked chitosan with high amino group contents, Carbohyd. Polym. 163 (2017) 34–42. [28] L. Zhang, Q. Zhang, Y. Zheng, Z. He, Y. Da, Study of Schiff base formation between dialdehyde cellulose and proteins, and its application for the deproteinization of crude polysaccharides extracts, Ind. Crop. Prod. 112 (2018) 532–540. [29] G. Krishnamoorthy, P.K. Sehgal, A.B. Mandal, S. Sadulla, Studies on collagen-tannic acid collagenase ternary system; inhibition of collagenase against collagenolytic degradation of extracellular matrix component of collagen, J. Enzyme Inhibit. Med. Chemi. 27 (2012) 451–457. [30] G. Krishnamoorthy, P.K. Sehgal, A.B. Mandal, S. Sadulla, Development of D-lysine assisted EDC/NHS initiated crosslinking of collagen matrix for design of scaffold, J. Biomed. Mater. Res. Part-A. 101A (2013) 1173–1183. [31] G. Krishnamoorthy, B. Madhan, S. Sadulla, J. Raghava Rao, W. Mathulatha, Stabilization of collagen by plant polyphenolics Acacia mollissima and Terminalia Chebula, J. Appl. Polym. Sci. 108 (2008) 199–205. [32] S. Zhu, X. Yu, S. Xiong, R. Liu, Z. Gu, J. You, T. Yin, Y. Hu, Insights into the rheological behaviors evolution of alginate dialdehyde crosslinked collagen solutions evaluated by numerical models, Mater. Sci. Eng. C Mater. Biol. Appl. 78 (2017) 727–737. [33] Z. Gu, H. Xie, C. Huang, L. Li, X. Yu, Preparation of chitosan/silk fibroin blending membrane fixed with alginate dialdehyde for wound dressing, Int. J. Biol. Macromol. 58 (2013) 121–126. [34] L. Ge, Y. Xu, X. Li, L. Yuan, H. Tan, D. Li, C. Mu, Fabrication of antibacterial collagen based composite wound dressing, ACS Sustain. Chem. Eng. 6 (2018) 9153–9166. [35] S. Elahipanah, P.J. O'Brien, D. Rogozhnikov, M.N. Yousaf, General dialdehyde click chemistry for amine bioconjugation, Bioconjug. Chem. 28 (2017) 1422–1433.