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In vitro biocompatibility determination of acellular aortic matrix of buffalo origin.

Introduction

Abdominal wall defects pose a great challenge to the reconstructive surgeons even today. The principal concept in management of abdominal wall defects is "tension-free" closure. Previously several synthetic materials were used to achieve this objective. However due to their suboptimal performance in clinical settings, many investigators have shown interest in the utilization of extracellular matrix scaffolds for abdominal wall reconstruction. Biological scaffolds composed of naturally occurring extracellular matrix (ECM) are being widely used in biomaterial research now a days because of their superiority in biocompatibility when compared to other materials. The ECM provide a supportive medium or conduit for blood vessels, nerves and lymphatics along with the diffusion of nutrients from the blood to the surrounding cells. For these reasons, the ECM has been developed as a biological scaffold for tissue engineering applications in virtually every body system.

However, the presently available biological materials have significant limitations. Complications associated with these costly materials, such as rapid break down and loss of the graft material, especially in infected fields, and undesirable host foreign body reaction have been reported in animal and human studies when used to repair abdominal wall defects (1). As a consequence the search for the optimal source of reconstruction material for clinical use continues.

The decellularized vascular matrices retain their natural biological composition and three- dimensional architecture suitable for cell adhesion and proliferation. Therefore, these are used as scaffolds in various urological and vascular applications. The blood vessel matrix derived from porcine aorta served as a viable option in the repair of abdominal wall tissue defects (2). Unmodified natural materials, upon implantation are subjected to chemical and enzymatic degradation, thereby seriously decreasing the life of the prosthesis. The use of these natural biomaterials has typically required chemical or physical pre-treatment aimed at preserving the tissue by enhancing the resistance of the material to enzymatic or chemical degradation, reducing the immunogenicity of the material and sterilizing the tissue. Fixing interspecies extracellular matrices with a crosslinking reagent may reduce their immunogenicity. In addition, cross-linking is used to improve mechanical properties and enhance resistance to degradation.

In the present study efficacy of cross-linking agents like GA, BDDGE and EDC on aortic acellular matrix graft was evaluated by in vitro biocompatibility determination.

Materials and Methods

Collection and processing of aorta

Fresh posterior aorta of buffalo origin was collected from the local abattoir and immediately preserved in ice-cold sterile phosphate buffered saline (pH 7.4) containing antibiotic (Amikacin--1mg/ml) and 0.02% EDTA. The maximum time period between tissue procurement and processing was less than 4 h. It was then thoroughly washed with sterile phosphate buffered saline to remove all the adherent blood. The blood vessel was cut into 2x2 [cm.sup.2] pieces to carry out decellularization protocol.

Decellularization

Non ionic biological detergent (1%) (Name of the detergent is not disclosed and nomenclature used as XY) was used for decellularization. Aortic samples were immersed in 20 ml of 1% XY solution and then were subjected to continuous shaking for 48 h in an orbital shaker at the rate of 180 rpm and at 37[degrees]C to provide better contact of tissue with chemicals. After completion of protocol, the matrices were preserved in 10% formal saline solution for histopathological examination to check the acellularity of matrix.

Crosslinking

The acellular aortic matrix obtained after decellularization procedure were cross-linked by the following reagents.

a) Glutaraldehyde (GA) as a standard / control (0.6% in phosphate buffered saline)

b) 1,4-butanediol diglycidylether (BDDGE) (1% in phosphate buffered saline)

c) 1-ethyl-3-(3-dimethyl aminopropyl carbodiimide (EDC) (1% in phosphate buffered saline)

The amount of solution used to crosslink each sample was 20 ml. Tissues of each study group were kept for 12, 24, 48 and 72h in different chemical agents for crosslinking. The solution was changed at 24 h time interval. The cross-linking was done at room temperature (37[degrees]C) with continuous agitation (180 rpm in orbital shaker) for better penetration of cross-linkers.

In vitro biocompatibility determination

In-vitro biocompatibility determination was done on the basis of the following parameters:

Enzymatic degradation

In vitro collagenase enzymatic degradation

In-vitro collagenase enzymatic degradation was performed as per the method described by Connolly et al. (3).

In vitro elastase enzymatic degradation

Elastase enzymatic degradation was performed as per the method described by Leach et al. (4).

Moisture content analysis/Swelling ratio

The moisture content was analyzed as per the method of Sung et al. (5).

Free amino group contents

Ninhydrin assay was used to determine the free amino group content of each test sample as per the procedure of Sung et al. (5).

Fixation Index

The fixation index was determined by ninhydrin assay as per the procedure of Sung et al. (6).

Free protein content

The free protein contents were estimated by the methods of Lowery et al. (7).

Molecular weight

Molecular weight analysis of the biomaterials cross-linked with various reagents was done by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) as per the method of Lastowka et al. (8).

Statistical analysis

The data was analysed by ANOVA and Student's t-test as per Snedecor and Cochran (9).

Results and Discussion

Decellularization

The native aortic tissue treated with 1% XY for 48 h was softer and whiter when compared to native aortic tissue. Microscopically, There was no evidence of cellularity of tissue (fig. 1). All the layers of aorta were intact. The collagen fibres were compactly arranged than the native tissue (fig.2). Ionic detergents have been reported for vascular tissue decellularization because of its ease and ability to lyse cells uniformly within all layers of the tissue (10). Furthermore, its mechanism of action may make it less damaging than other enzymatic, mechanical, or tissue fixation methods. In the present study, the cell extraction using ionic detergent was effectively achieved without significant disturbances in extracellular matrix morphology and strength.

Crosslinking

The acellular aortic tissues were exposed to standardized concentration of different chemicals for cross-linking. The aortic tissue treated with 0.6% GA showed yellowish coloration as compared to other tissues cross-linked with cross-linking agents. These tissues were slightly swollen and hard. One percent BDDGE treated tissues were more swollen, pliable and slightly stiffer when compared to EDC treated tissues. One percent EDC treated tissues were white in colour and soft in consistency.

In vitro biocompatibility determination

In vitro biocompatibility determination of acellular aortic matrix was based on the observations recorded in following parameters:

In vitro enzymatic degradation

In vitro enzymatic degradation studies of native, acellular and cross-linked acellular aortic matrix were conducted using different concentrations of collagenase, and elastase enzymes at different time intervals.

In vitro collagenase degradation

Among all the cross-linked tissues, weight loss was minimal in aortic tissues cross-linked with GA for 72 h followed by EDC treated tissues for 24 h and maximum in BDDGE treated tissues for 72 h (Fig.3a, 3b, 3c and 3d). GA reacts with '- amino group of lysyl residues in proteins which induces the formation of interchain crosslinks and stabilizes tissues against chemical and enzymatic degradation depending upon the extent of cross-linking (11). Therefore prolonged crosslinking periods resulted in increased resistance to enzymatic degradation. BDDGE cross-linked tissues showed more weight loss when compared to EDC and GA cross-linked tissues. Crosslinking with 1,4-butanediol diglycidyl ether can be achieved under acidic (pH 4.5) or basic (pH 9.0-10.0) conditions, resulting in completely different materials concerning mechanical properties and in-vitro stability towards enzymes. Zeeman et al. (12) reported that crosslinking of dermal sheep collagen with BDDGE at a pH of 4.5 afforded materials which degrade even after crosslinking for 10 days. Dewangan et al. (15) also reported a linear increase in the rate of weight loss (percent0 of tissue samples as with digestion time intervals.

In vitro elastase degradation

At all digestion intervals, weight loss was minimal in aortic tissues cross-linked with GA followed by BDDGE treated tissues for 48 h and maximum in aortic tissues cross-linked with EDC for 48 h (Fig.4a,4b,4c and 4d). Partial distortion of the triple-helix caused by acylation is more important than shielding or blocking of specific sites for enzymes in determining the degradation rate of this material. A method to determine the influence of the distortion of the helix on the resistance against enzymatic degradation is the use of elastase as a degrading enzyme. The elastase is capable of cleaving peptide bonds of denatured collagen. On the contrary, native collagen is not degraded. In the present study, degradation was less in all GA treated tissues suggesting that these materials were either not denatured or that the denatured segments were tightly cross-linked. However, degradation was more in BDDGE and EDC cross-linked tissues which implies that some distortion of the helical structure was present.

Moisture content

Among the cross-linked tissues, moisture content was lowest in tissues cross-linked with GA for 24 h and 48 h and was highest in tissues cross-linked with EDC for 48 h (Fig. 5a, 5b, 5c and 5d). The denaturation of collagen (cross-linked) led to increased swelling by conversion of the collagen triple helix structure to a random coil conformation, because of the increased accessibility of collagen peptide chains to hydration (13). In the present study the cross-linked samples revealed lower moisture percentage as compared to control as also reported by Dewangan et al. (15) when acellular urinary bladder crosslinkd by GA, BDDGE, HMD and EDC. This may be attributed to the shrinkage of tissue during fixation which reduces the free volume in tissue and thus expelled some water molecules out of the fixed tissue. GA crosslinked acellular dermis revealed lower moisture contents, which indicate that the fixation with aldehydes cause more shrinkage of tissue as compare to other groups, expelling more number of water molecules out of the fixed tissue.

Free amino group

Among the cross-linked tissues, free amino group concentration was minimal in tissues cross-linked with EDC for 24 h and maximum in tissues cross-linked with EDC for 72 h (Fig. 6a, 6b, 6c and 6d). The analysis of free amino group contents indirectly indicates the degree of the cross-linking. The amount of free amino group is inversely proportional to the degree of the cross-linking. All the cross-linking agents had bound the free amino acids as compared to the uncross-linked tissues. The cross linking initiated by GA occurs by reaction of the GA aldehyde groups with two collagen a-amine groups of either lysine or hydroxylysine residues. In the crosslinking, two amine (NH3) groups were used in every GA induced primary amine cross-linking (14). Crosslinking involves the reaction of amine groups of (hydroxy) lysine residues with epoxide groups of the BDDGE molecules, resulting in formation of secondary amines. Only single amino group was used in each cross-linking so the free amino acids were available more in number in comparison to the GA, but less than other groups. The cross-linking between the amino acids without incorporating itself by EDC revealed that the free amino acid concentration was slightly more than GA (15).

Fixation index

The values of fixation index in GA, BDDGE and EDC cross-linked tissues were significantly different within a group at various cross-linking intervals. The fixation index was lowest in tissues cross-linked with GA for 48 h and highest in tissues cross-linked with EDC for 24 h (Fig. 8). Fixation index is used to estimate the percentage of amino groups within the tissue reacted with the crosslinking agent. A higher fixation index implies a lower level of free amino groups left in the fixed tissue. Sung et al. (6) reported significant increase in fixation index of epoxy-fixed and glutaraldehyde fixed porcine arteries. In the present study higher fixation index was observed in aortic tissues cross-linked with EDC for 24 h indicating less free amino groups in the cross-linked tissue.

Free protein

Among cross-linked tissues, free protein concentration was highest in tissues cross-linked with BDDGE and EDC for 48 h whereas, it was lowest in tissues cross-linked with EDC for 24 h (Fig. 7a, 7b, 7c and 7d). In the present study, the significant decrease in the free protein of crosslinking samples was observed as compared to the uncrosslinked acellular and native samples. The cross-linking binds the peptide and form large molecule of protein that was also evident in SDS-PAGE, in which the large molecule was unable to pass through the gel and therefore a particular band remained absent. Dewangan et al. (15) reported minimun reduction in free protein contents in HMD crosslinked and maximum reduction in GA and EDC crosslinked bladder acellular matrix.

Molecular weight

SDS-PAGE was performed to determine the cross-linking ability of different chemicals. Cross-linking resulted in the formation of high molecular weight protein which was determined by the expression of protein bands (Fig. 9). The typical aortic collagen pattern is represented in the native aorta (Lane 2). In the SDS resolving gel, the collagen bands showed molecular weight of about 25, 30, 45, 50, 60, 66 and 200 kDa. Native collagen molecules remained in the stacking gel. After decellularization process, the soluble protein decreased as revealed in SDS-PAGE of acellular aorta (Lane 3). The protein band pattern of collagen in acellular aorta in SDS-PAGE did not show any high protein band. The GA treated acellular aortic matrix graft did not show any higher protein band pattern in SDS-PAGE gel. The GA cross-linked collagen resulted in the formation of large covalently cross-linked complex. This complex did not disassociate by chemical treatment with sample buffer and was too large to enter the stacking gel. Therefore, all the GA treated acellular aortic matrix graft did not show any band pattern in SDS-PAGE gel. The EDC and BDDGE treated tissues produced a characteristic cross-linking of the proteins which was observed in all cross-linked samples, suggesting that the chemical treatment had effectively cross-linked the different chains of collagen proteins resulting into formation of high mass which did not find entry even in the stacking gel (15). Therefore, all the EDC and BDDGE treated acellular aortic matrix grafts did not show any band pattern in SDS-PAGE gel. Dewangan et al. (15) also reported the same type of results when acelluar bladder matrix was crosslinked with GA and EDC.

Acknowledgement

The authors acknowledge the financial assistance received from the Department of Biotechnology (DBT), Ministry of Science and Technology, New Delhi, India to carry out this work.

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[15.] Rukmani Dewangan, A. K. Sharma, Naveen Kumar, S. K. Maiti, Himani Singh, A. K. Gangwar, Sameer Shrivastava, Sonal and Amit Kumar, In-vitro biocompatibility determination of bladder acellular matrix graft. Trends in Biomaterials andArtificial Organs, 25, 161-171(2011).

J. Devarathnam (1), A. K. Sharma (1), R.B.Rai (1), S. K. Maiti (1), Sameer Shrivastava (2), Sonal (2), Naveen Kumar (1) *

(1) Division of Surgery, (2) Divission of Animal Biotechnology, Indian Veterinary Research Institute, Izatnagar 243122, Uttar Pradesh, India

* Corresponding Author Email: naveen.ivri1961@gmail.com

Received 6 January 2014; Accepted 11 June 2014; Available online 1 July 2014
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Title Annotation:Original Article
Author:Devarathnam, J.; Sharma, A.K.; Rai, R.B.; Maiti, S.K.; Shrivastava, Sameer; Sonal; Kumar, Naveen
Publication:Trends in Biomaterials and Artificial Organs
Article Type:Report
Date:Jul 1, 2014
Words:2927
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