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In vitro evaluation of cross-linked native and acellular caprine pericardium.

Introduction

Collagen is the major structural proteins of connective tissues. It constitute more than one-third of the total body proteins in mammals (1). The chemical, structural, and biological properties of collagen make this material very suitable for biomedical applications. Nowadays, collagen based biomaterials such as aorta (2-4), dermis (5-7), diaphragm (8), and urinary bladder (9) were used for soft tissue repair.

In order to render collagen suitable for tissue engineering applications, the mechanical strength of collagen must be enhanced. Cross-linking is an effective method to control resorption rate of collagen based biomaterials and to prevent a rapid elution of the material into wound fluids. The efficiency and extent of cross-linking reactions depend upon the thickness of the layers of the collagenous tissue and defines the magnitude of the penetration. The other parameters like concentration of the cross-linker, cross-linking time and the temperature of crosslinking solutions affects the cross-linking. A balance must be achieved for attaining enough reliable cross-links for the biomaterials to last lifetime of the recipients yet, permit the biomaterial to perform as it would be in its natural state. The methods that have been developed do not and probably cannot satisfy the dual requirements. In the present study in-vitro evaluation of cellular and acellular caprine pericardium following crosslinking with glutaraldehyde (GA), glyoxal (GO) diphenyl phosphoryl azide (DPPA), and ethylene glycol diglycidyl ether (EGDGE) was carried out.

Materials and Methods

Harvest and preparation of acellular pericardium

Fresh caprine pericardium was procured from the local abattoir and immediately preserved in cold phosphate buffer saline (PBS) solution. The pericardial tissue was washed throughly with PBS to remove the adhered blood. Excess fatty layer was carefully trimmed and pericardial tissue was equally divided into two portions. One portion was cross-linked with 0.6% glutaraldehyde (GA), 1% glyoxal (GO), 1% diphenylphosphorylazide (DPPA) and 1% ethylene glycol diglycidyl ether (EGDGE), and other portion was made acellular using anionic biological detergent and then cross-linked with similar crosslinking agents. To make acellular, pericardial tissue was cut into 2 x 2 [cm.sup.2] in size and placed in 4% sodium deoxycholate solution for 24 h under constant agitation and temperature. Finally decellurized tissue was thoroughly washed in PBS and stored in PBS solution containing 1% amikacin at 4[degrees]C. Fresh pericardium preserved in PBS was used as control.

Crosslinking process

The native as well as acellular pericardium was crosslinked with 0.6% GA, 1% GO, 1% DPPA and 1% EGDGE. The amount of solutions used to crosslink each sample was 20 ml and were changed at every 24 h. Tissues of each study group were kept for 12, 24, 48 and 72 h in crosslinking solution under constant agitation and temperature. Samples of each group were taken out at various elapsed fixation periods. The physical properties of crosslinked samples were compared, in vitro, to those of the acellular counterparts. The physical properties were determined on the basis of following parameters:

Gross observations

A gross observation of tissue were made after cross-linking with GA, GO, DPPA and EGDGE at given concentration and duration. It included change in colour, consistency, swelling and stiffness.

In-vitro non-enzymatic degradation

Native and acellular pericardium before and after cross-linking were subjected to in-vitro non-enzymatic degradation tests as per the procedure described earlier [10]. Pre-weighed specimens were immersed for 0, 1, 3, 5 and 7 days at 37[degrees]C in isotonic saline solution containing 1% sodium azide. The values of weight loss were expressed in percentage.

In-vitro enzymatic degradation

In-vitro enzymatic degradation was performed using 1% cyanogen bromide, papain and pepsin enzymes solution. Samples were degraded for 12, 24, 48 and 72 h time intervals. The values of weight loss were expressed in percentage.

Estimation of free protein contents

Free protein contents of the specimens were determined after cross-linking with GA, GO, DPPA and EGDGE by the method described earlier [11] using bovine serum albumin (BSA) as a standard. The values of protein contents were expressed in mg/ ml.

Estimation of Free amino group concentration

Ninhydrin assay was used to determine the free amino group concentration of each test sample as per the procedure of Sung et al. [12].

Fixation Index

The fixation index was determined by ninhydrin assay as per the procedure of Sung et al. [13]. The values of fixation index were expressed in percentage.

Moisture content

The moisture content was analyzed as per the method of Sung et al. [12], and expressed as percentage (%).

Molecular weight analysis

It was performed by 10% sodium dodecyl sulphate polyacrylamide gel electrophorosis (SDS-PAGE). Briefly, 100 mg of each test sample was triturated with 1 ml of 10% SDS and supernatant was obtained after centrifugation at 10,000 rpm for 10 minutes. 1 x Sample buffer was added to the supernatant in the ratio 1:1 and heated for 10 minutes. The solution was allowed to cool at room temperature. The sample was loaded onto 7 slot applicator. The finished gel was stained with Comassie blue and de-stained with 20% methanol and 10% acetic acid. Known molecular weight marker was used to calibrate the gel. The values of high molecular weight protein bands were expressed in kDa.

Statistical analysis

The parameters were evaluated by ANOVA and Student's paired t-test as per Snedecor and Cochran [14].

Results

Microscopic observation of acellular pericardium revealed that the protocol for making them acellular was effective. It resulted in complete removal of cells. No nuclear bodies were seen. The tissue was primarily composed of extracellular matrix.

Gross observations

The gross observations of tissues were made after cross-linking the biomaterials at fixed concentration of different cross-linking agents and duration. Pericardium was exposed to appropriate concentration of different cross-linking solutions. Pericardium treated with 0.6% GA showed yellowish coloration as compared to other cross-linking agents. Pericardium treated with 1% glyoxal changed to milky white colour. DPPA treated pericardium showed comparatively more swelling and softness than natural tissue. EGDGE treated pericardium was stiffer as compared to GO, DPPA treated and uncross-linked pericardium. Crosslinking with different chemicals for 12 h did not show any change in colour, except in GA treated tissue that showed mild yellow colouration. Pericardium on treatment for 24, 48 and 72 h with different chemicals became stiff and hard in comparison to those treated for 12 h. The treatment with GA, GO and DPPA for 72 h showed some degree of tissue degradation, with white flakes on the surface, loss of elasticity and increased hardness which was not found fit to act as scaffold. Pericardium cross-linked for 24 h with all cross-linking agents at room temperature were found best.

In-vitro non-enzymatic degradation

The rate of weight loss (percent) of native as well as acellular pericardium cross-linked with different chemicals followed by non-enzymatic degradation is presented in table1.

Native pericardium: The rate of weight loss of tissue crosslinked with GA, GO, DPPA and EGDGE increased with the increase in time intervals of non-enzymatic digestion. Therefore, the tissues digested for 7 days exhibited maximum weight loss. Similar pattern in the rate of weight loss was recorded in all the tissues cross-linked for four different time intervals (12, 24, 48 and 72 h) with all cross-linking agents. The minimum weight loss was seen in the tissue cross-linked for 12 and 24 h with GO and digested for 1 day. Whereas, the maximum weight loss was seen in the tissue cross-linked for 12 h with EGDGE and then digested for 5 and 7 days. The weight loss in uncross-linked sample after digestion was slow in comparison to the crosslinked tissue samples on different test intervals observed during the period of studied.

Acellular pericardium: The weight loss in acellular pericardium treated with GA was 31.65[+ or -]0.002 % at day 1 and it was maximum when compared with the tissues treated for 12 h with GO, DPPA and EGDGE. Similarly, the greatest reduction in weight loss was observed at day 3 in the tissue treated with GA for 12 h in comparison to tissue treated with other 3 agents for 12 h. At day 5, the weight loss in the tissues treated for 72 h with DPPA was maximum (40.54 [+ or -] 0.006%). At day 7, the treatment with DPPA and EGDGE for 72h exhibited the maximum weight loss.

In-vitro enzymatic degradation

The degradation of the samples was studied by exposing the materials to cyanogen bromide, pepsin and papain solution.

In-vitro cyanogen bromide degradation: The rates of weight loss (percent) of native and acellular pericardium due to cyanogen bromide degradation are presented in table2.

The weight loss of caprine native pericardium cross-linked with GA and DPPA for 12 h and treated with cyanogen bromide for 12 h exhibited lesser degree of weight loss in comparison to that observed in 72 h cross-linked tissues. In contrast, the tissue cross-linked with GO and EGDGE for 12 h showed maximum weight loss and degree of weight loss reduced as the cross linking time increased. Therefore, the weight loss in 72 h crosslinked sample was least. The weight loss observed in DPPA and EGDGE cross-linked samples differed significantly (P<0.05) from uncross-linked samples.

The weight loss of acellular pericardium cross-linked with GA for 12 h exhibited maximum weight loss on 12 h digestion, whereas, the weight loss was minimum in 72 h cross-linked samples. In contrast, the samples cross-linked with GO, DPPA and EGDGE, on 12 h digestion showed lesser degree of weight loss than that of 72 h cross-linked samples. In 24, 48 and 72 h digested samples the pattern of weight loss in all the four treatment groups was similar to that observed in 12 h digested samples. The values recorded in 72 h cross-linked samples with GA differed significantly (P<0.05) to those cross-linked for 12, 24 and 48 h in all the samples digested for four different time intervals.

In-vitro pepsin degradation: The rates of weight loss (percent) of native and acellular pericardium due to pepsin degradation are presented in table3. At 12 h of pepsin digestion cross-link native pericardium with GA and DPPA for four different periods exhibited the increasing trend in weight loss with the increase in cross-linking time. In GO treated tissue sample the reduction in weight was seen with the increase in cross-link time. In EGDGE treated samples the weight loss was more or less similar in the tissue cross-linked for four different periods of time.

In 12 digested acellular pericardium, which was cross-linked with GA and EGDGE exhibited increasing trend in the weight loss with the increase in cross-linking time. In contrast, a decreasing order in weight loss with the increase in cross-linking time was recorded in the tissues cross-linked with GO and DPAA. The values recorded in 12 and 24 h GA treated samples differed significantly (P<0.05) from 48 and 72 h treated samples on 12 h digestion. At 24, 48 and 72 h digested samples no definite trend in the weight loss in different groups was observed.

In-vitro papain degradation: The rates of weight loss (percent) of native and acellular pericardium due to papain degradation are presented in table 4. The weight loss in the tissue crosslinked with GA and EGDGE exhibited progressive reduction with the increase in cross-linking time in all the tissues digested for 12, 24, 48 and 72 h of digestion with papain. In GO treated samples the reduction in weight loss was also seen at 12 and 24 h of digestion. In DPPA treated samples no systematic pattern in the weight loss was seen after 48 and 72 h of enzymatic digestion. The weight loss at 12 h digestion in 12 h cross-linked sample with GA differed significantly (P<0.05) to that of recorded in 24, 48 and 72 h cross-linked samples.

Protein contents

The free protein contents of native and acellular pericardium is presented in table 5. Mean[+ or -]SE values of free protein (mg/ml) after cross-linking with GA, GO, DPPA and EGDGE is presented in table 4.The free protein content in tissues significantly reduced (P<0.05) after cross-linking for 12 h with all the chemicals in each group in comparison to native pericardium (control). The GA and EGDGE produced significant fall (P<0.05) in protein content at subsequent treatment intervals. In contrast, a significant (P<0.05) rise in protein contents was recorded after treatment with GO and DPPA for 24, 48 and 72 h. The crosslinking of acellular pericardium with GA, GO, DPPA and EGDGE for 12 h exhibited significant (P<0.05) fall in the protein content in comparison to untreated acellular pericardium. Therefore, the treatment of pericardium with GA, GO, DPPA and EGDGE did not show any definite change in the values of protein between 24-72 h, however, a significant (P<0.05) difference was observed between the values recorded at these time intervals.

Free amino group contents determination

The free amino group contents of native and acellular pericardium is presented in table 5.The free amino group concentration in native pericardium after cross-linking with GA, GO, DPPA and EGDGE had significantly (P<0.05) reduced in the samples for 12 h. The concentration of free amino group progressively reduced as the contact time of tissue with crosslinking agents had increased. The free amino group concentration of acellular pericardium in 12 h treated tissue with GA, GO, DPPA and EGDGE were significantly (P<0.05) lower than that of the uncross-linked samples. The free amino group concentration progressively reduced as the cross-linking time increased in each treatment group. The GA and DPPA treatment had produced greater reduction in the free amino group concentration in comparison to GO and EGDGE treatment in both tissues.

Fixation index

Fixation index of native and acellular pericardium are presented in table 5.The values of fixation index after cross-linking the tissues for 12 h were minimum and increased with increase in cross-linking time in all the four groups. A progressive increase in fixation index with the increase in cross-linking time was recorded with all the four agents.

Moisture content analysis/ swelling ratio

Moisture percentages of native and acellular pericardium are presented in table 5. The treatment of tissue with GA, GO, DPPA and EGDGE for 12 h reduced the moisture contents of the tissue, which was significant (P<0.05) in GA, GO and DPPA treated tissue sample. A progressive reduction in the moisture content was recorded with the increased cross-linking time and therefore the minimum moisture was recorded in the tissue treated for 72 h with each cross-linking agents.

Molecular weight analysis

Cross-linking resulted in the formation of high molecular weight protein which determines the expression of protein bands. SDSPAGE of native and acellular pericardium crosslinked for 12 h are presented in figure 1. Protein bands of 14.4, 25, 45, 66.2 and 116 kDa were seen in uncross-linked native pericardium. In DPPA and EGDGE treated pericardium 66.2 kDa and 116 kDa bands were seen and in GO treated pericardium only 116 kDa band was evident. Lower molecular weight protein bands (14.4 kDa and 25 kDa) were noticed in GA treated pericardium. Acellular cross-linked pericardium showed 25, 35, 45 and 116 kDa protein bands. In GO treated tissue the protein bands of 30 kDa and 116 kDa were observed. However, GA, DPPA and EGDGE treated acellular cross-linked pericardium did not show any protein band.

Discussion

Decellularization process leads to removal of nucleus and cytoplasmic cellular components, lipids and its membranes along with soluble proteins and basement membrane components of cellular material, while preserving the components of extracellular matrix (ECM) which consist of primarily of elastin, insoluble collagen and tightly bound glycosaminoglycans [15]. Acellular tissue matrices are biocompatible, slowly degraded upon implantation and are replaced and remodeled by the extracellular matrix proteins synthesized and secreted by ingrowing host cells, which reduce the inflammatory response [16]. After the removal of cells and cell debris the intact ECM of the acellular tissue itself may elicit an immune response [17].

[FIGURE 1 OMITTED]

The tissues cross-linked with EGDGE were more pliable than GA cross-linked tissue. Pericardiums treated with GA were stiffer as compared to other cross-linking agents. In GA there are only one carbon-carbon bonds, which are known to be relatively inflexible, therefore, the GA fixed tissue is usually comparatively stiffer than the EGDGE-fixed tissues [18,19]. Cheung et al. [20] proposed that lower concentrations have been found to be better in bulk tissue cross-linking compared to higher concentration. 0.6% concentration of GA and 1% concentration of GO, DPPA and EGDGE were more suitable and ideal for surgical implantation. High concentration of GA promoted rapid surface cross-linking of tissue, generating a barrier that impeded or prevented the further diffusion of glutaraldehyde into the tissue bulk. Similarly, Santillan-Doherty et al. [21] recommended the use of lower concentration of GA for the preservation of biological tissue. At six hour duration cross-linking the pericardium did not differ much from the native pericardium. Prolonged contact time of 24 h was suggested for biological fixation by GA and genipin [22].

The cross-links that are derived non-enzymatically occur more adventitiously and are important to patho-biological processes [23]. In present study native and acellular pericardium crosslinked by GO showed maximum resistant toward non-enzymatic degradation. The glyoxal treatment caused the cross-linking between the free amine groups of soy and the aldehyde groups of GO [10]. The resulting cross-links are mainly of intramolecular nature. Thus, the degradation behaviour of the glyoxal treated materials should be mainly related with their intramolecular cross-linking density. Vaz et al. [10] also reported that after 24 h of immersion, non-cross-linked soy retained over 92% of its original weight, whereas a 1% and 0.7% decrease in weight were observed for glyoxal cross-linked soy and heat treated soy, respectively.

In present study the native and acellular pericardium crosslinked with GO showed greater resistance to cyanogen bromide, pepsin and papain degradation. Cyanogen bromide cleaves proteins at methionine residues of collagen whereas, elastin lacks methionine, therefore, it is resistant to cyanogen bromide digestion. Pepsin cleaved the carboxylic groups of aromatic amino acids such as phenylalanine and tyrosine. It did not cleave at bonds with valine, alanine or glycine, which allows the maintenance for the triple helix. Therefore, the selective cleavage of pepsin occur at the N and C telopeptide nonhelical regions breaking the peptide bonds and forming an atelopeptide collagen molecule capable of dissolving in aqueous solution [24].

In general, papain is known to cleave peptide bonds of basic amino acids, such as leucine or glycine, and also to hydrolyze esters and amides. Papain is also a non-specific enzyme that is active to chitosan depolymerization [25] as well as cellulose, pectinase, pepsine, and wheat germ lipase [26]. According to the results, the papain activity varied depending on the degree of deacetylation, which was observed in the case of lysozyme. Therefore, it was suggested that papain recognizes N-acetylglucosamine residue as well as lysozyme.

The biomaterials cross-linked with EGDGE and DPPA did not show any appreciable resistance to cyanogen bromide, pepsin and papain degradation. Polyglycidyl ethers have gained much attention as cross-linking reagents for collagen based biomaterials [27] and fixation using PEG resulted in materials having good mechanical properties [28] a decreased calcification and lower cytotoxicity [29]. Use of EGDGE might have also resulted in an undefined cross-linked structure because of the polyfunctionality [30].

GA and EGDGE cross-linked samples produced significant fall in protein contents at subsequent treatment intervals. The decrease in free protein contents indicated the efficacy of the cross-linking of the biomaterials. Reduction in the free protein contents was least in GO cross-linked samples, while it was highest in GA treated samples. It indicated that the GA crosslinked the biomaterials effectively and efficiently and the free protein was not available in these tissues. 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. Kumar et al. (31, 32) also reported significant decrease in free protein contents with increased cross-linking time intervals with different cross-linking agents.

The free amino group analysis indicated that GA and DPPA have the greatest ability to cross-link insoluble collagen fibrils. It was observed that the GA induced cross-linking resulted in the least number of free amines. Similar findings were also reported by Lastowka et al. [33]. Kumar et al. [31, 32] also reported significant decrease in the free amino group concentration with increased cross-linking time intervals in acellular bubaline acellular intestinal matrix and fish swim bladder respectively.

The rate of tissue fixation can be determined by monitoring the change in free amino group contents, denaturation temperature and moisture content of fixed tissues [12]. A fixation index is used to estimate the percentage of amino groups within the tissue. A higher fixation index often implies a lower level of free amino groups left in the fixed tissues. The fixation index in present study was in order of GO>EGDGE>GA>DPPA in cross-linked tissue samples at 72 h. The rate of GA fixation was faster than that of epoxy fixation. Sung et al. [13] reported that the epoxy-fixed and GA-fixed porcine arteries showed significant increase in fixation index as compared to the fresh one. The resistance of porcine arteries against bacterial collagenase in-vitro degradation increased significantly after cross-linking with the epoxy compound or GA. The reduction of the tensile strength of the epoxy-fixed artery after bacterial collagenase degradation was more severe than its -GA-fixed counterpart. Generally, with increasing pH, temperature or fixative concentration, the fixation indices and denaturation temperatures of the epoxy-fixed porcine arteries had increased. Similarly in present study the fixation index increased with the increase in cross-linking time. Sung et al. [13] developed an improved pericardial substitute. An epoxy-fixed biological patch with ionically bound heparin was developed and also evaluated the cross-linking characteristics of the newly developed biological patch using the glutaraldehyde-fixed counterpart as a control. The epoxy-fixed biological patch appeared more similar to the native pericardium in colour and was more pliable than its glutaraldehyde-fixed counterpart. Both the epoxy- and glutaraldehyde-fixed biological patches had significant increase in fixation index and denaturation temperature as compared to the fresh one. Similar finding were observed in present study. The EGDGE fixed biomaterials were similar to native pericardium in colour and were more pliable than its glutaraldehyde-fixed counterpart. Both, the epoxy- and glutaraldehyde-fixed biological patches had significant increase in fixation index.

The moisture contents of the cross-linked samples showed significant reduction in moisture percentage (except EGDGE) as compared to control samples. It is evident from the moisture percentage studies that cross-linking reduced the equilibrium moisture percent of collagenous matrices [22]. The swelling ratio of the single step neutral cross-linking with EGDGE was greater than the swelling ratio of either the single step alkaline or the double step alkaline procedures [34]. The cross-linking pH has effect on the modulation of the swelling ratio. After fixation it was noted that the epoxy fixed tissues were more pliable than the glutaraldehyde fixed tissue. This may be due to the relatively higher moisture contents of the epoxy fixed tissues than the glutaraldehyde-fixed counterpart [13]. Similar finding were also reported in the present study. Sung et al. [12] reported that the moisture content of glutaraldehyde and genipin crosslinked tissues was significantly lower than the fresh tissue. Similarly, in the present study the cross-linked samples revealed lower moisture percentage as compared to control. This may be attributed to the shrinkage of tissue during fixation which reduces the free volume in tissue and thus expels some water molecules out of the tissue during fixation. GA and GO cross-linked samples of pericardium and diaphragm (native as well as acellular samples) revealed significant reduction in moisture contents, which indicated that the fixation with aldehydes caused more shrinkage of tissue as compared to other groups. Expulsion of more number of water molecules out of the fixed tissue will cause more shrinkage. Choi et al. [35] reported that the highly cross-linked sponges with EDC showed lower water uptake. Similarly in present study in 72 h cross-linked samples minimal moisture content was recorded. Perme et al. [36] used GA, formaldehyde (FA), polyethylene glycol, hexamethylene diisocyanate (HMDC), 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) to cross-link bovine pericardium. GA and FA treated native and acellular bovine pericardium showed significant decrease in moisture percentage at 12, 48 and 72 h interval as compared to NSS treated control samples. HMDC treated biomaterials did not show any significant difference at different time intervals. Purohit [37] reported that the moisture percent significantly decreased with the increased cross-linking time intervals within the same cross-linking agents. At 6 h of crosslinking the over all sequence of the moisture per cent was HMD>BDE>GA>EDC in Skin. The GA cross-linked group showed the significant reduction in moisture percentage at subsequent time intervals. Similar finding were also observed in present study, where GA, GO and DPPA cross-linked biomaterials showed the significant reduction in moisture percentage at subsequent time intervals.

Crosslinking resulted in the formation of high molecular weight protein which determines the expression of protein bands. Native pericardium treated with GA showed less amount of low molecular weight proteins, which indicated that GA had the greatest ability to cross-link the biomaterials. In GO exposed pericardium high molecular weight protein (116 kDa) was expressed, which indicated the lesser ability of GO to crosslink the biomaterials in comparison to GA. DPPA and EGDGE expressed 66.2 kDa and 116 kDa bands therefore indicated the minimum ability to cross-link the biomaterials. Similar to native pericardium, acellular pericardium treated with GA, DPPA and EGDGE exhibited greater ability to cross-link the biomaterials in comparison to GO. Lastowka et al. [33] reported the formation of higher molecular weight protein following the GA treatment. Further, these higher molecular weight proteins were found maximum in tissues exposed to GA as compared to dehydrothermal drying and microbial transglutaminase method of cross-linking. Vaz et al. [10] reported that the glyoxal crosslinked soy samples showed a very weak intensity, partly reflecting the low solubility of this protein in the electrophoresis.

Conclusions

In this study, the technique for making acellular scaffold from caprine pericardium was found successful as complete acellularity of pericardium was obtained. The GA and DPPA treatment had produced greater reduction in the free amino group concentration in comparison to GO and EGDGE treatment. GO cross-linked pericardium showed greater resistance to enzymatic as well as non-enzymatic degradation. GO treatment showed high molecular weight protein bands than GA, DPPA and EGDGE on the SDS-PAGE. Pericardium cross-linked for 24 h with all the four cross-linking agents at room temperature were found best for in-vivo studies.

Acknowledgements

The authors are thankful to the Department of Biotechnology, Government of India, New Delhi, for the financial support to undertake this project work.

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[30.] R. Zeeman, P. J. Dijkstra, P. B. van Wachem, M. J. van Luyn, M. Hendriks, P. T. Cahalan, and J. Feijen, Successive epoxy and carbodiimide cross-linking of dermal sheep collagen. Biomaterials, 20, 921-931 (1999).

[31.] V. Kumar, N. Kumar, H. Singh, A. K. Gangwar, R. Dewangan, A. Kumar, R. B. Rai In vitro evaluation of bubaline acellular small intestinal matrix. International Journal of Bioassays, 2, 581-587 (2013).

[32.] V. Kumar, N. Kumar, H. Singh, A. K. Gangwar, R. Dewangan, A. Kumar, R. B. Rai, Effects of crosslinking treatments on the physical properties of acellular fish swim bladder. Trends in Biomaterials & Artificial Organs, 27, 93-101 (2013).

[33.] A. Lastowka, G. J. Maffia and E. M. Brown, A comparison of chemical, physical and enzymatic cross-linking of bovine type I collagen fibrils. JALCA, 100, 196-202 (2004).

[34.] J. B. Leach, J. B. Wolinsky, P. J. Stone and A. Wong, Crosslinked a-elastin biomaterials: towards a processable elastin mimetic scaffold. Acta Biomaterialia 1: 155-164 (2005).

[35.] H. Choi, M. Lee, M. Kim and C. Kim, Effect of additives on the physicochemical properties of liquid suppository bases. International Journal of Pharmaceutics, 190, 13-19 (1999).

[36.] H. Perme, A. K. Sharma, N. Kumar, H. Singh, R. Dewangan and S. K. Maiti, In-vitro biocompatibility evaluation of crosslinked cellular and acellular bovine pericardium. Trends in Biomaterials & Artificial Organs, 23, 66-75 (2009).

[37.] S. Purohit, Biocompatibility testing of acellular dermal grafts in a rabbit model: an in-vitro and in-vivo study. PhD thesis submitted to deemed university. Indian Veterinary research Institute, Izatnagar (Utter Pradesh) (2008).

Amit Kumar, A. K. Sharma, Naveen Kumar *, S. K. Maiti, Rukmani Dewangan, Vineet Kumar, Himani Singh

Division of Surgery, Indian Veterinary Research Institute, Izatnagar 243122, Uttar Pradesh, India

Received 14 August 2014; Accepted 8 December 2014; Published online 13 April 2015

# Coresponding author: Dr. Naveen Kumar; E-mail: naveen@ivri.res.in
Table 1: Mean [+ or -] SE of rate of weight loss (percent) after
non-enzymatic degradation (in isotonic saline solution containing 1%
sodium azide) of native as well as acellular pericardium of caprine
cross-linked with GA, GO, DPPA and EGDGE

                          Cross     Isotonic Saline Solution
                Cross     Linking   digestion
Biomaterials    linking   Time
                Agents    (hr)      1st day

                          12        18.33 [+ or -] 0.002 (aA)
                GA        24        18.98 [+ or -] 0.001 (bA)
                          48        19.43 [+ or -] 0.001 (cA)
                          72        19.54 [+ or -] 0.001 (dA)
                          12        02.97 [+ or -] 0.001 (aA)
                          24        02.92 [+ or -] 0.001 (aA)
                GO        48        00.77 [+ or -] 0.000 (bA)
                          72        00.24 [+ or -] 0.000 (cA)
Native                    12        06.21 [+ or -] 0.004 (aA)
  pericardium   DPPA      24        08.33 [+ or -] 0.006 (bA)
                          48        11.05 [+ or -] 0.000 (cA)
                          72        17.36 [+ or -] 0.006 (dA)
                          12        13.52 [+ or -] 0.002 (aA)
                EGDGE     24        15.81 [+ or -] 0.003 (bA)
                          48        16.52 [+ or -] 0.003 (cA)
                          72        19.11 [+ or -] 0.001 (dA)
Control                   -         03.77 [+ or -] 0.001 (A)
                          12        31.65 [+ or -] 0.002 (aA)
                GA        24        28.63 [+ or -] 0.003 (bA)
                          48        25.18 [+ or -] 0.005 (cA)
                          72        22.51 [+ or -] 0.003 (dA)
                          12        15.03 [+ or -] 0.004 (aA)
                GO        24        23.36 [+ or -] 0.005 (aA)
                          48        25.25 [+ or -] 0.012 (bA)
                          72        27.97 [+ or -] 0.012 (cA)
                          12        11.04 [+ or -] 0.003 (aA)
Acellular       DPPA      24        14.36 [+ or -] 0.011 (aA)
  pericardium             48        20.51 [+ or -] 0.002 (aA)
                          72        29.72 [+ or -] 0.008 (bA)
                          12        11.54 [+ or -] 0.002 (aA)
                EGDGE     24        21.44 [+ or -] 0.001 (bA)
                          48        24.95 [+ or -] 0.002 (cA)
                          72        26.11 [+ or -] 0.001d (A)
Control                   --        17.53 [+ or -] 0.001 (A)

                          Cross     Isotonic Saline Solution
                Cross     Linking   digestion
Biomaterials    linking   Time
                Agents    (hr)      3rd day

                          12        19.98 [+ or -] 0.002 (aB)
                GA        24        20.51 [+ or -] 0.001 (bB)
                          48        20.74 [+ or -] 0.001 (cB)
                          72        20.55 [+ or -] 0.001 (dB)
                          12        06.66 [+ or -] 0.001 (aB)
                          24        19.22 [+ or -] 0.001 (bB)
                GO        48        08.87 [+ or -] 0.000 (cB)
                          72        05.31 [+ or -] 0.003 (dB)
Native                    12        06.44 [+ or -] 0.004 (aB)
  pericardium   DPPA      24        11.01 [+ or -] 0.005 (bB)
                          48        11.81 [+ or -] 0.001 (cB)
                          72        20.73 [+ or -] 0.006 (dB)
                          12        33.01 [+ or -] 0.007 (aB)
                EGDGE     24        24.51 [+ or -] 0.003 (bB)
                          48        32.32 [+ or -] 0.004 (cB)
                          72        24.0 [+ or -] 30.007 (dB)
Control                   -         03.91 [+ or -] 0.001 (B)
                          12        32.21 [+ or -] 0.002 (aB)
                GA        24        34.21 [+ or -] 0.001 (bB)
                          48        26.06 [+ or -] 0.005 (cB)
                          72        24.51 [+ or -] 0.006 (cB)
                          12        15.51 [+ or -] 0.003 (B)
                GO        24        26.57 [+ or -] 0.005 (B)
                          48        26.21 [+ or -] 0.012 (B)
                          72        28.22 [+ or -] 0.002 (B)
                          12        21.72 [+ or -] 0.011 (aB)
Acellular       DPPA      24        21.74 [+ or -] 0.007 (aB)
  pericardium             48        30.21 [+ or -] 0.008 (aB)
                          72        35.86 [+ or -] 0.006 (bB)
                          12        25.05 [+ or -] 0.005 (aB)
                EGDGE     24        25.65 [+ or -] 0.001 (aB)
                          48        33.96 [+ or -] 0.009 (bB)
                          72        31.81 [+ or -] 0.001 (cB)
Control                   --        23.77 [+ or -] 0.011 (B)

                          Cross     Isotonic Saline Solution
                Cross     Linking   digestion
Biomaterials    linking   Time
                Agents    (hr)      5th day

                          12        20.48 [+ or -] 0.002 (aC)
                GA        24        21.66 [+ or -] 0.001 (bC)
                          48        22.77 [+ or -] 0.001 (cC)
                          72        22.41 [+ or -] 0.001 (dC)
                          12        09.55 [+ or -] 0.001 (aC)
                          24        19.61 [+ or -] 0.001 (bC)
                GO        48        09.25 [+ or -] 0.000 (aC)
                          72        06.25 [+ or -] 0.003 (bC)
Native                    12        09.33 [+ or -] 0.004 (aC)
  pericardium   DPPA      24        13.37 [+ or -] 0.006 (bC)
                          48        15.21 [+ or -] 0.001 (cC)
                          72        24.44 [+ or -] 0.005 (dC)
                          12        43.18 [+ or -] 0.002 (aC)
                EGDGE     24        30.54 [+ or -] 0.003 (bC)
                          48        38.08 [+ or -] 0.004 (cC)
                          72        29.86 [+ or -] 0.001 (dC)
Control                   -         04.11 [+ or -] 0.001 (C)
                          12        32.71 [+ or -] 0.002 (aC)
                GA        24        36.37 [+ or -] 0.001 (bC)
                          48        26.27 [+ or -] 0.005 (cC)
                          72        25.01 [+ or -] 0.006 (cC)
                          12        15.83 [+ or -] 0.003 (aC)
                GO        24        28.23 [+ or -] 0.004 (bC)
                          48        26.56 [+ or -] 0.012 (bC)
                          72        30.15 [+ or -] 0.003 (bC)
                          12        23.73 [+ or -] 0.010 (aC)
Acellular       DPPA      24        23.34 [+ or -] 0.007 (aC)
  pericardium             48        31.86 [+ or -] 0.009 (aC)
                          72        40.54 [+ or -] 0.006 (bC)
                          12        36.43 [+ or -] 0.000 (aC)
                EGDGE     24        35.14 [+ or -] 0.001 (bC)
                          48        35.01 [+ or -] 0.009 (cC)
                          72        37.64 [+ or -] 0.003 (dC)
Control                   --        24.91 [+ or -] 0.011 (C)

                          Cross     Isotonic Saline Solution
                Cross     Linking   digestion
Biomaterials    linking   Time
                Agents    (hr)      7th day

                          12        22.76 [+ or -] 0.001 (aD)
                GA        24        23.54 [+ or -] 0.000 (bD)
                          48        23.27 [+ or -] 0.001 (cD)
                          72        22.91 [+ or -] 0.001 (dD)
                          12        09.88 [+ or -] 0.001 (aD)
                          24        19.87 [+ or -] 0.001 (bD)
                GO        48        09.72 [+ or -] 0.000 (aD)
                          72        06.48 [+ or -] 0.003 (cD)
Native                    12        11.81 [+ or -] 0.003 (aD)
  pericardium   DPPA      24        15.88 [+ or -] 0.009 (bD)
                          48        18.66 [+ or -] 0.002 (cD)
                          72        30.91 [+ or -] 0.005 (dD)
                          12        43.58 [+ or -] 0.002 (aD)
                EGDGE     24        31.06 [+ or -] 0.003 (bD)
                          48        38.51 [+ or -] 0.004 (cD)
                          72        30.71 [+ or -] 0.001 (dD)
Control                   -         06.63 [+ or -] 0.000 (D)
                          12        32.88 [+ or -] 0.002 (aD)
                GA        24        37.93 [+ or -] 0.001 (bD)
                          48        26.47 [+ or -] 0.005 (cD)
                          72        25.41 [+ or -] 0.006 (cD)
                          12        16.06 [+ or -] 0.004 (D)
                GO        24        28.65 [+ or -] 0.004 (D)
                          48        26.91 [+ or -] 0.012 (D)
                          72        31.66 [+ or -] 0.002 (D)
                          12        26.08 [+ or -] 0.011 (aD)
Acellular       DPPA      24        25.28 [+ or -] 0.007 (aD)
  pericardium             48        35.08 [+ or -] 0.008 (aD)
                          72        45.88 [+ or -] 0.006 (bD)
                          12        38.11 [+ or -] 0.001 (aD)
                EGDGE     24        37.07 [+ or -] 0.001 (bD)
                          48        36.91 [+ or -] 0.011 (cD)
                          72        45.87 [+ or -] 0.008 (dD)
Control                   --        25.21 [+ or -] 0.011 (D)

Table2: Mean [+ or -] SE of rate of weight loss (percent) after
cyanogen bromide digestion of native as well as acellular pericardium
of caprine cross-linked with GA, GO, DPPA and EGDGE

                          Cross     Cyanogen Bromide
Biomaterials    Cross     Linking   digestion
                linking   Time
                Agents    (hr)      12hr

                          12        01.88 [+ or -] 0.000 (A)
                GA        24        04.15 [+ or -] 0.001 (A)
                          48        06.27 [+ or -] 0.002 (A)
                          72        06.73 [+ or -] 0.002 (A)
                          12        04.19 [+ or -] 0.002 (A)
                GO        24        02.86 [+ or -] 0.001 (A)
                          48        01.86 [+ or -] 0.001 (A)
                          72        01.69 [+ or -] 0.000 (A)
Native                    12        06.37 [+ or -] 0.001 (aA)
  pericardium   DPPA      24        07.31 [+ or -] 0.000 (aA)
                          48        08.32 [+ or -] 0.001 (aA)
                          72        13.01 [+ or -] 0.003 (bA)
                          12        10.41 [+ or -] 0.002 (aA)
                EGDGE     24        06.07 [+ or -] 0.001 (bA)
                          48        05.08 [+ or -] 0.002 (bA)
                          72        02.94 [+ or -] 0.002 (cA)
Control                   --        06.88 [+ or -] 0.003 (A)
                          12        10.94 [+ or -] 0.002 (aA)
                GA        24        06.19 [+ or -] 0.000 (aA)
                          48        04.03 [+ or -] 0.001 (aA)
                          72        01.81 [+ or -] 0.000 (bA)
                          12        03.74 [+ or -] 0.001 (A)
                GO        24        04.56 [+ or -] 0.000 (A)
                          48        05.81 [+ or -] 0.003 (A)
                          72        05.97 [+ or -] 0.000 (A)
                          12        03.74 [+ or -] 0.001 (A)
Acellular       DPPA      24        04.56 [+ or -] 0.000 (A)
  pericardium             48        05.81 [+ or -] 0.003 (A)
                          72        05.97 [+ or -] 0.000 (A)
                          12        07.18 [+ or -] 0.004 (A)
                EGDGE     24        09.69 [+ or -] 0.003 (A)
                          48        10.36 [+ or -] 0.004 (A)
                          72        11.34 [+ or -] 0.003 (A)
Control                   --        15.31 [+ or -] 0.000 (A)

                          Cross     Cyanogen Bromide
Biomaterials    Cross     Linking   digestion
                linking   Time
                Agents    (hr)      24hr

                          12        04.02 [+ or -] 0.000 (A)
                GA        24        14.94 [+ or -] 0.006 (B)
                          48        13.47 [+ or -] 0.005 (B)
                          72        12.58 [+ or -] 0.004 (B)
                          12        10.92 [+ or -] 0.003 (B)
                GO        24        07.57 [+ or -] 0.003 (B)
                          48        02.74 [+ or -] 0.001 (B)
                          72        04.96 [+ or -] 0.001 (B)
Native                    12        14.17 [+ or -] 0.003 (aB)
  pericardium   DPPA      24        14.81 [+ or -] 0.002 (aB)
                          48        18.62 [+ or -] 0.001 (aB)
                          72        23.93 [+ or -] 0.006 (bB)
                          12        19.13 [+ or -] 0.001 (aB)
                EGDGE     24        10.81 [+ or -] 0.001 (bB)
                          48        09.74 [+ or -] 0.003 (bB)
                          72        03.48 [+ or -] 0.002 (cB)
Control                   --        13.46 [+ or -] 0.002 (B)
                          12        29.48 [+ or -] 0.002 (aB)
                GA        24        11.81 [+ or -] 0.000 (aB)
                          48        07.71 [+ or -] 0.001 (aB)
                          72        03.02 [+ or -] 0.000 (bB)
                          12        07.03 [+ or -] 0.002 (B)
                GO        24        08.68 [+ or -] 0.000 (B)
                          48        06.52 [+ or -] 0.003 (B)
                          72        07.95 [+ or -] 0.000 (B)
                          12        07.03 [+ or -] 0.002 (B)
Acellular       DPPA      24        08.68 [+ or -] 0.000 (B)
  pericardium             48        06.52 [+ or -] 0.003 (B)
                          72        07.95 [+ or -] 0.000 (B)
                          12        11.22 [+ or -] 0.004 (B)
                EGDGE     24        16.71 [+ or -] 0.003 (B)
                          48        15.74 [+ or -] 0.006 (B)
                          72        13.91 [+ or -] 0.003 (B)
Control                   --        28.39 [+ or -] 0.004 (B)

                          Cross     Cyanogen Bromide
Biomaterials    Cross     Linking   digestion
                linking   Time
                Agents    (hr)      48hr

                          12        06.98 [+ or -] 0.001 (B)
                GA        24        16.83 [+ or -] 0.006 (C)
                          48        14.52 [+ or -] 0.005 (C)
                          72        14.62 [+ or -] 0.005 (C)
                          12        11.59 [+ or -] 0.003 (C)
                GO        24        08.19 [+ or -] 0.004 (C)
                          48        04.08 [+ or -] 0.001 (C)
                          72        08.11 [+ or -] 0.001 (C)
Native                    12        15.52 [+ or -] 0.003 (aC)
  pericardium   DPPA      24        18.91 [+ or -] 0.004 (aC)
                          48        19.74 [+ or -] 0.001 (aC)
                          72        27.67 [+ or -] 0.007 (bC)
                          12        23.85 [+ or -] 0.001 (aC)
                EGDGE     24        16.81 [+ or -] 0.005 (bC)
                          48        13.57 [+ or -] 0.004 (bC)
                          72        11.98 [+ or -] 0.001 (cC)
Control                   --        14.23 [+ or -] 0.002 (C)
                          12        35.05 [+ or -] 0.001 (aC)
                GA        24        24.66 [+ or -] 0.006 (aC)
                          48        09.44 [+ or -] 0.001 (aC)
                          72        05.78 [+ or -] 0.000 (bC)
                          12        07.73 [+ or -] 0.002 (C)
                GO        24        09.94 [+ or -] 0.001 (C)
                          48        07.23 [+ or -] 0.003 (C)
                          72        08.01 [+ or -] 0.000 (C)
                          12        07.73 [+ or -] 0.002 (C)
Acellular       DPPA      24        09.94 [+ or -] 0.001 (C)
  pericardium             48        07.23 [+ or -] 0.003 (C)
                          72        08.01 [+ or -] 0.000 (C)
                          12        18.62 [+ or -] 0.003 (C)
                EGDGE     24        20.72 [+ or -] 0.004 (C)
                          48        21.78 [+ or -] 0.010 (C)
                          72        15.91 [+ or -] 0.003 (C)
Control                   --        34.44 [+ or -] 0.006 (C)

                          Cross     Cyanogen Bromide
Biomaterials    Cross     Linking   digestion
                linking   Time
                Agents    (hr)      72hr

                          12        07.67i0.00 1c
                GA        24        18.55 [+ or -] 0.006 (D)
                          48        15.64 [+ or -] 0.005 (D)
                          72        17.81 [+ or -] 0.005 (D)
                          12        12.15 [+ or -] 0.003 (D)
                GO        24        09.28 [+ or -] 0.004 (D)
                          48        12.79 [+ or -] 0.005 (D)
                          72        10.72 [+ or -] 0.000 (D)
Native                    12        17.36 [+ or -] 0.003 (aD)
  pericardium   DPPA      24        20.66 [+ or -] 0.004 (aD)
                          48        20.17 [+ or -] 0.001 (aD)
                          72        28.05 [+ or -] 0.007 (bD)
                          12        26.96 [+ or -] 0.001 (aD)
                EGDGE     24        18.01 [+ or -] 0.005 (bD)
                          48        14.72 [+ or -] 0.004 (bD)
                          72        15.61 [+ or -] 0.001 (cD)
Control                   --        15.24 [+ or -] 0.002 (D)
                          12        36.58 [+ or -] 0.001 (aD)
                GA        24        27.78 [+ or -] 0.005 (aD)
                          48        17.77 [+ or -] 0.001 (aD)
                          72        08.49 [+ or -] 0.000 (bD)
                          12        08.73 [+ or -] 0.002 (D)
                GO        24        10.92 [+ or -] 0.001 (D)
                          48        07.71 [+ or -] 0.003 (D)
                          72        08.08 [+ or -] 0.000 (D)
                          12        08.73 [+ or -] 0.002 (D)
Acellular       DPPA      24        10.92 [+ or -] 0.001 (D)
  pericardium             48        07.71 [+ or -] 0.003 (D)
                          72        08.08 [+ or -] 0.000 (D)
                          12        20.49 [+ or -] 0.003 (D)
                EGDGE     24        22.12 [+ or -] 0.004 (D)
                          48        22.50 [+ or -] 0.010 (D)
                          72        16.97 [+ or -] 0.002 (D)
Control                   --        36.54 [+ or -] 0.007 (D)

Table3: Mean [+ or -] SE of rate of weight loss (percent) after pepsin
digestion of native as well as acellular pericardium of caprine
cross-linked with GA, GO, DPPA and EGDGE

                          Cross     Pepsin digestion
                Cross     Linking
Biomaterials    linking   Time
                Agents    (hr)      12hr

                          12        00.35 [+ or -] 0.000 (aA)
                GA        24        02.41 [+ or -] 0.000 (bA)
                          48        02.51 [+ or -] 0.001 (bA)
                          72        04.02 [+ or -] 0.000 (cA)
                          12        03.28 [+ or -] 0.001 (A)
                GO        24        00.37 [+ or -] 0.000 (A)
                          48        00.59 [+ or -] 0.000 (A)
                          72        00.17 [+ or -] 0.000 (A)
Native                    12        03.28 [+ or -] 0.001 (A)
  pericardium   DPPA      24        00.37 [+ or -] 0.000 (A)
                          48        00.59 [+ or -] 0.000 (A)
                          72        00.17 [+ or -] 0.000 (A)
                          12        07.47 [+ or -] 0.000 (aA)
                EGDGE     24        08.11 [+ or -] 0.001 (bA)
                          48        09.57 [+ or -] 0.001 (cA)
                          72        09.75 [+ or -] 0.002 (cA)
Control                   --        06.69 [+ or -] 0.003 (A)
                          12        04.43 [+ or -] 0.001 (aA)
                GA        24        04.54 [+ or -] 0.000 (aA)
                          48        05.23 [+ or -] 0.001 (bA)
                          72        05.36 [+ or -] 0.007 (cA)
                          12        03.08 [+ or -] 0.002 (aA)
                GO        24        02.36 [+ or -] 0.000 (bA)
                          48        01.99 [+ or -] 0.001 (cA)
                          72        01.53 [+ or -] 0.000 (cA)
                          12        09.91 [+ or -] 0.000 (A)
Acellular       DPPA      24        09.44 [+ or -] 0.000 (A)
  pericardium             48        08.66 [+ or -] 0.000 (A)
                          72        03.35 [+ or -] 0.002 (A)
                          12        05.63 [+ or -] 0.001 (A)
                EGDGE     24        10.24 [+ or -] 0.002 (A)
                          48        16.08 [+ or -] 0.002 (A)
                          72        16.26 [+ or -] 0.002 (A)
Control                   --        06.38 [+ or -] 0.001 (A)

                          Cross     Pepsin digestion
                Cross     Linking
Biomaterials    linking   Time
                Agents    (hr)      24hr

                          12        00.71 [+ or -] 0.000 (A)
                GA        24        03.77 [+ or -] 0.000 (B)
                          48        05.71 [+ or -] 0.001 (B)
                          72        09.31 [+ or -] 0.006 (B)
                          12        05.41 [+ or -] 0.001 (B)
                GO        24        00.69 [+ or -] 0.000 (B)
                          48        01.21 [+ or -] 0.001 (B)
                          72        00.53 [+ or -] 0.000 (B)
Native                    12        05.41 [+ or -] 0.001 (B)
  pericardium   DPPA      24        00.69 [+ or -] 0.000 (B)
                          48        01.21 [+ or -] 0.001 (B)
                          72        00.53 [+ or -] 0.000 (B)
                          12        16.31 [+ or -] 0.006 (B)
                EGDGE     24        17.58 [+ or -] 0.000 (B)
                          48        27.52 [+ or -] 0.004 (B)
                          72        13.96 [+ or -] 0.002 (B)
Control                   --        11.99 [+ or -] 0.005 (B)
                          12        12.52 [+ or -] 0.002 (aB)
                GA        24        10.09 [+ or -] 0.001 (bB)
                          48        05.95 [+ or -] 0.001 (aB)
                          72        17.23 [+ or -] 0.012 (aB)
                          12        05.43 [+ or -] 0.003 (aB)
                GO        24        04.59 [+ or -] 0.001 (bB)
                          48        04.15 [+ or -] 0.002 (bB)
                          72        01.84 [+ or -] 0.000 (cB)
                          12        15.93 [+ or -] 0.001 (A)
Acellular       DPPA      24        18.81 [+ or -] 0.001 (B)
  pericardium             48        18.05 [+ or -] 0.001 (A)
                          72        05.19 [+ or -] 0.001 (B)
                          12        11.02 [+ or -] 0.003 (B)
                EGDGE     24        21.02 [+ or -] 0.004 (B)
                          48        29.79 [+ or -] 0.003 (B)
                          72        24.98 [+ or -] 0.001 (B)
Control                   --        12.86 [+ or -] 0.001 (B)

                          Cross     Pepsin digestion
                Cross     Linking
Biomaterials    linking   Time
                Agents    (hr)      48hr

                          12        03.11 [+ or -] 0.001 (B)
                GA        24        07.03 [+ or -] 0.001 (C)
                          48        09.88 [+ or -] 0.003 (C)
                          72        10.71 [+ or -] 0.006 (C)
                          12        08.71 [+ or -] 0.001 (C)
                GO        24        01.14 [+ or -] 0.000 (C)
                          48        07.72 [+ or -] 0.004 (C)
                          72        05.44 [+ or -] 0.002 (C)
Native                    12        08.71 [+ or -] 0.001 (C)
  pericardium   DPPA      24        01.14 [+ or -] 0.000 (C)
                          48        07.72 [+ or -] 0.004 (C)
                          72        05.44 [+ or -] 0.002 (C)
                          12        21.79 [+ or -] 0.005 (C)
                EGDGE     24        21.26 [+ or -] 0.003 (C)
                          48        30.61 [+ or -] 0.006 (C)
                          72        15.67 [+ or -] 0.001 (C)
Control                   --        16.43 [+ or -] 0.004 (C)
                          12        25.38 [+ or -] 0.001 (aC)
                GA        24        12.89 [+ or -] 0.001 (bC)
                          48        27.93 [+ or -] 0.005 (cC)
                          72        26.78 [+ or -] 0.012 (aC)
                          12        19.71 [+ or -] 0.017 (aC)
                GO        24        06.25 [+ or -] 0.001 (bC)
                          48        11.87 [+ or -] 0.006 (cC)
                          72        04.24 [+ or -] 0.000 (bC)
                          12        22.11 [+ or -] 0.001 (B)
Acellular       DPPA      24        26.39 [+ or -] 0.001 (C)
  pericardium             48        25.31 [+ or -] 0.002 (B)
                          72        11.21 [+ or -] 0.003 (C)
                          12        14.81 [+ or -] 0.003 (C)
                EGDGE     24        33.34 [+ or -] 0.004 (C)
                          48        37.01 [+ or -] 0.003 (C)
                          72        35.58 [+ or -] 0.005 (C)
Control                   --        22.21 [+ or -] 0.003 (C)

                          Cross     Pepsin digestion
                Cross     Linking
Biomaterials    linking   Time
                Agents    (hr)      72hr

                          12        05.75 [+ or -] 0.002 (C)
                GA        24        16.36 [+ or -] 0.003 (D)
                          48        18.34 [+ or -] 0.004 (D)
                          72        14.88 [+ or -] 0.007 (D)
                          12        09.43 [+ or -] 0.001 (D)
                GO        24        01.46 [+ or -] 0.000 (D)
                          48        08.34 [+ or -] 0.004 (D)
                          72        05.98 [+ or -] 0.002 (D)
Native                    12        09.43 [+ or -] 0.001 (D)
  pericardium   DPPA      24        01.46 [+ or -] 0.000 (D)
                          48        08.34 [+ or -] 0.004 (D)
                          72        05.98 [+ or -] 0.002 (D)
                          12        28.71 [+ or -] 0.007 (D)
                EGDGE     24        28.54 [+ or -] 0.004 (D)
                          48        34.26 [+ or -] 0.005 (D)
                          72        24.36 [+ or -] 0.009 (D)
Control                   --        22.03 [+ or -] 0.009 (D)
                          12        46.67 [+ or -] 0.003 (D)
                GA        24        15.99 [+ or -] 0.001 (D)
                          48        29.74 [+ or -] 0.005 (D)
                          72        27.80 [+ or -] 0.012 (D)
                          12        22.79 [+ or -] 0.018 (aD)
                GO        24        09.52 [+ or -] 0.001 (bD)
                          48        23.74 [+ or -] 0.008 (aD)
                          72        05.11 [+ or -] 0.000 (cD)
                          12        22.61 [+ or -] 0.001 (C)
Acellular       DPPA      24        29.06 [+ or -] 0.000 (D)
  pericardium             48        30.94 [+ or -] 0.002 (C)
                          72        20.44 [+ or -] 0.004 (D)
                          12        17.31 [+ or -] 0.004 (D)
                EGDGE     24        35.35 [+ or -] 0.005 (D)
                          48        43.93 [+ or -] 0.001 (D)
                          72        37.84 [+ or -] 0.005 (D)
Control                   --        29.83 [+ or -] 0.009 (D)

Table4: Mean [+ or -] SE of rate of weight loss (percent) after papain
digestion of native as well as acellular pericardium of caprine
cross-linked with GA, GO, DPPA and EGDGE

                          Cross
                Cross     Linking      Papain digestion
Biomaterials    linking   Time
                Agents    (hr)      12hr

                          12        03.93 [+ or -] 0.002 (aA)
                GA        24        01.71 [+ or -] 0.000 (bA)
                          48        01.61 [+ or -] 0.001 (bA)
                          72        01.16 [+ or -] 0.000 (bA)
                          12        04.06 [+ or -] 0.000 (A)
                GO        24        03.91 [+ or -] 0.000 (A)
                          48        01.53 [+ or -] 0.001 (A)
                          72        00.41 [+ or -] 0.000 (A)
Native                    12        01.51 [+ or -] 0.000 (A)
  pericardium   DPPA      24        02.11 [+ or -] 0.000 (A)
                          48        05.67 [+ or -] 0.001 (A)
                          72        05.97 [+ or -] 0.001 (A)
                          12        22.97 [+ or -] 0.003 (A)
                EGDGE     24        22.98 [+ or -] 0.003 (A)
                          48        19.95 [+ or -] 0.007 (A)
                          72        17.66 [+ or -] 0.007 (A)
Control                   --        08.16 [+ or -] 0.001 (A)
                          12        03.31 [+ or -] 0.000 (A)
                GA        24        03.41 [+ or -] 0.001 (A)
                          48        03.43 [+ or -] 0.000 (A)
                          72        03.44 [+ or -] 0.001 (A)
                          12        04.65 [+ or -] 0.000 (A)
                GO        24        01.62 [+ or -] 0.001 (A)
                          48        01.09 [+ or -] 0.000 (A)
                          72        01.04 [+ or -] 0.001 (A)
                          12        08.32 [+ or -] 0.003 (A)
                          24        05.94 [+ or -] 0.002 (A)
                DPPA      48        05.59 [+ or -] 0.000 (A)
                          72        04.61 [+ or -] 0.001 (A)
                          12        26.68 [+ or -] 0.006 (aA)
                EGDGE     24        26.16 [+ or -] 0.000 (aA)
                          48        24.71 [+ or -] 0.005 (bA)
                          72        23.23 [+ or -] 0.005 (cA)
Control                   --        08.35 [+ or -] 0.001 (A)

                          Cross     Papain digestion
                Cross     Linking
Biomaterials    linking   Time
                Agents    (hr)      24hr

                          12        07.68 [+ or -] 0.004 (aB)
                GA        24        05.03 [+ or -] 0.001 (bB)
                          48        02.72 [+ or -] 0.001 (cB)
                          72        02.45 [+ or -] 0.012 (cA)
                          12        06.42 [+ or -] 0.001 (B)
                GO        24        07.35 [+ or -] 0.000 (B)
                          48        03.48 [+ or -] 0.002 (B)
                          72        01.26 [+ or -] 0.000 (B)
Native                    12        03.32 [+ or -] 0.000 (B)
  pericardium   DPPA      24        06.01 [+ or -] 0.001 (B)
                          48        13.09 [+ or -] 0.002 (B)
                          72        11.85 [+ or -] 0.005 (B)
                          12        23.49 [+ or -] 0.003 (B)
                EGDGE     24        23.67 [+ or -] 0.003 (B)
                          48        21.66 [+ or -] 0.006 (B)
                          72        19.09 [+ or -] 0.007 (B)
Control                   --        21.94 [+ or -] 0.000 (B)
                          12        09.06 [+ or -] 0.000 (B)
                GA        24        08.41 [+ or -] 0.003 (B)
                          48        04.31 [+ or -] 0.000 (B)
                          72        07.77 [+ or -] 0.001 (B)
                          12        09.86 [+ or -] 0.000 (B)
                GO        24        03.98 [+ or -] 0.001 (B)
                          48        03.01 [+ or -] 0.001 (B)
                          72        01.91 [+ or -] 0.001 (B)
                          12        20.96 [+ or -] 0.003 (B)
                          24        13.23 [+ or -] 0.004 (B)
                DPPA      48        18.01 [+ or -] 0.001 (B)
                          72        09.21 [+ or -] 0.001 (B)
                          12        29.13 [+ or -] 0.006 (aB)
                EGDGE     24        29.37 [+ or -] 0.001 (aB)
                          48        29.23 [+ or -] 0.007 (aB)
                          72        25.69 [+ or -] 0.005 (bB)
Control                   --        14.69 [+ or -] 0.001 (B)

                          Cross     Papain digestion
                Cross     Linking
Biomaterials    linking   Time
                Agents    (hr)      48hr

                          12        08.77 [+ or -] 0.005 (aC)
                GA        24        05.61 [+ or -] 0.001 (bC)
                          48        04.67 [+ or -] 0.001 (cC)
                          72        04.22 [+ or -] 0.000 (cB)
                          12        09.11 [+ or -] 0.001 (C)
                GO        24        12.41 [+ or -] 0.002 (C)
                          48        04.35 [+ or -] 0.002 (C)
                          72        02.12 [+ or -] 0.000 (C)
Native                    12        09.21 [+ or -] 0.000 (C)
  pericardium   DPPA      24        10.76 [+ or -] 0.001 (C)
                          48        17.18 [+ or -] 0.002 (C)
                          72        14.62 [+ or -] 0.004 (C)
                          12        30.24 [+ or -] 0.007 (C)
                EGDGE     24        29.43 [+ or -] 0.001 (C)
                          48        25.05 [+ or -] 0.005 (C)
                          72        23.06 [+ or -] 0.009 (C)
Control                   --        31.62 [+ or -] 0.006 (C)
                          12        31.86 [+ or -] 0.004 (C)
                GA        24        08.98 [+ or -] 0.004 (C)
                          48        07.11 [+ or -] 0.001 (C)
                          72        08.53 [+ or -] 0.001 (C)
                          12        17.74 [+ or -] 0.002 (C)
                GO        24        11.98 [+ or -] 0.004 (C)
                          48        12.73 [+ or -] 0.001 (C)
                          72        10.83 [+ or -] 0.001 (C)
                          12        30.18 [+ or -] 0.002 (C)
                          24        18.52 [+ or -] 0.008 (C)
                DPPA      48        26.42 [+ or -] 0.003 (C)
                          72        14.81 [+ or -] 0.002 (C)
                          12        32.91 [+ or -] 0.005 (aC)
                EGDGE     24        34.04 [+ or -] 0.000 (bC)
                          48        35.72 [+ or -] 0.010 (cC)
                          72        28.21 [+ or -] 0.006 (dC)
Control                   --        30.41 [+ or -] 0.003 (C)

                          Cross     Papain digestion
                Cross     Linking
Biomaterials    linking   Time
                Agents    (hr)      72hr

                          12        12.41 [+ or -] 0.005 (aD)
                GA        24        07.93 [+ or -] 0.001 (bD)
                          48        06.51 [+ or -] 0.001 (cD)
                          72        04.22 [+ or -] 0.012 (cB)
                          12        11.49 [+ or -] 0.001 (D)
                GO        24        12.81 [+ or -] 0.002 (D)
                          48        05.85 [+ or -] 0.002 (D)
                          72        02.78 [+ or -] 0.000 (D)
Native                    12        16.82 [+ or -] 0.003 (D)
  pericardium   DPPA      24        15.62 [+ or -] 0.001 (D)
                          48        21.31 [+ or -] 0.002 (D)
                          72        19.01 [+ or -] 0.003 (D)
                          12        34.56 [+ or -] 0.008 (D)
                EGDGE     24        30.13 [+ or -] 0.001 (D)
                          48        30.12 [+ or -] 0.007 (D)
                          72        24.65 [+ or -] 0.008 (D)
Control                   --        33.22 [+ or -] 0.007 (D)
                          12        34.89 [+ or -] 0.004 (D)
                GA        24        14.92 [+ or -] 0.006 (D)
                          48        08.08 [+ or -] 0.001 (D)
                          72        09.25 [+ or -] 0.001 (D)
                          12        19.07 [+ or -] 0.003 (D)
                GO        24        16.53 [+ or -] 0.001 (D)
                          48        13.94 [+ or -] 0.001 (D)
                          72        24.09 [+ or -] 0.000 (D)
                          12        32.52 [+ or -] 0.002 (D)
                          24        22.98 [+ or -] 0.008 (D)
                DPPA      48        29.31 [+ or -] 0.003 (D)
                          72        15.37 [+ or -] 0.002 (D)
                          12        41.85 [+ or -] 0.010 (aD)
                EGDGE     24        39.21 [+ or -] 0.001 (bD)
                          48        39.66 [+ or -] 0.012 (bD)
                          72        31.24 [+ or -] 0.006 (cD)
Control                   --        34.93 [+ or -] 0.006 (D)

Table5: Mean [+ or -] SE values of free protein ([micro] g/ml),
free amino group concentration (pg/ml), fixation-index (percent)
and moisture contents (percent) after cross-linking with GA, GO,
DPPA and EGDGE

                          Cross
                Cross     Linking   Protein Free
Biomaterials    linking   Time
                Agents    (hr)

                          12        0.233 [+ or -] 0.001 (a)
                GA        24        0.182 [+ or -] 0.002 (b)
                          48        0.176 [+ or -] 0.001 (c)
                          72        0.194 [+ or -] 0.001 (d)
                          12        0.185 [+ or -] 0.002 (a)
                GO        24        0.233 [+ or -] 0.011 (b)
                          48        0.218 [+ or -] 0.002 (c)
                          72        0.208 [+ or -] 0.001 (d)
Native                    12        0.264 [+ or -] 0.001 (a)
  pericardium   DPPA      24        0.267 [+ or -] 0.002 (b)
                          48        0.252 [+ or -] 0.001 (c)
                          72        0.274 [+ or -] 0.001 (d)
                          12        0.300 [+ or -] 0.001 (a)
                EGDGE     24        0.217 [+ or -] 0.001 (b)
                          48        0.199 [+ or -] 0.002 (c)
                          72        0.197 [+ or -] 0.004 (d)
Control                   --        0.352 [+ or -] 0.001 (C)
                          12        0.202 [+ or -] 0.002 (a)
                GA        24        0.237 [+ or -] 0.001 (b)
                          48        0.202 [+ or -] 0.001 (c)
                          72        0.240 [+ or -] 0.001 (d)
                          12        0.182 [+ or -] 0.003 (a)
                GO        24        0.228 [+ or -] 0.001 (b)
                          48        0.246 [+ or -] 0.002 (c)
                          72        0.189 [+ or -] 0.003 (d)
                          12        0.255 [+ or -] 0.001 (a)
Acellular       DPPA      24        0.242 [+ or -] 0.001 (b)
  pericardium             48        0.238 [+ or -] 0.003 (c)
                          72        0.255 [+ or -] 0.001 (d)
                          12        0.325 [+ or -] 0.001 (a)
                EGDGE     24        0.300 [+ or -] 0.000 (b)
                          48        0.288 [+ or -] 0.002 (c)
                          72        0.258 [+ or -] 0.003 (d)
Control                   --        0.290 [+ or -] 0.000 (C)

                          Cross
                Cross     Linking   Amino Group
Biomaterials    linking   Time
                Agents    (hr)

                          12        15.26 [+ or -] 0.003 (a)
                GA        24        08.86 [+ or -] 0.002 (b)
                          48        07.46 [+ or -] 0.010 (c)
                          72        05.52 [+ or -] 0.004 (d)
                          12        31.72 [+ or -] 0.001 (a)
                GO        24        26.60 [+ or -] 0.003 (b)
                          48        22.40 [+ or -] 0.001 (c)
                          72        19.72 [+ or -] 0.001 (d)
Native                    12        08.00 [+ or -] 0.002 (a)
  pericardium   DPPA      24        06.60 [+ or -] 0.001 (b)
                          48        05.66 [+ or -] 0.006 (c)
                          72        03.40 [+ or -] 0.001 (d)
                          12        34.40 [+ or -] 0.001 (a)
                EGDGE     24        28.15 [+ or -] 0.002 (b)
                          48        16.86 [+ or -] 0.001 (c)
                          72        08.26 [+ or -] 0.001 (d)
Control                   --        40.12 [+ or -] 0.001 (C)
                          12        14.20 [+ or -] 0.002 (a)
                GA        24        13.13 [+ or -] 0.005 (b)
                          48        09.86 [+ or -] 0.002 (c)
                          72        05.93 [+ or -] 0.002 (d)
                          12        27.86 [+ or -] 0.001 (a)
                GO        24        21.26 [+ or -] 0.001 (b)
                          48        18.73 [+ or -] 0.001 (c)
                          72        16.86 [+ or -] 0.002 (d)
                          12        07.40 [+ or -] 0.004 (a)
Acellular       DPPA      24        06.40 [+ or -] 0.001 (b)
  pericardium             48        05.60 [+ or -] 0.004 (c)
                          72        02.80 [+ or -] 0.002 (d)
                          12        07.40 [+ or -] 0.004 (a)
                EGDGE     24        06.40 [+ or -] 0.001 (b)
                          48        05.60 [+ or -] 0.004 (c)
                          72        02.80 [+ or -] 0.002 (d)
Control                   --        31.46 [+ or -] 0.002 (C)

                          Cross
                Cross     Linking   Fixation Index
Biomaterials    linking   Time
                Agents    (hr)

                          12        61.83 [+ or -] 1.20 (a)
                GA        24        77.83 [+ or -] 1.20 (b)
                          48        83.50 [+ or -] 0.86 (c)
                          72        86.16 [+ or -] 2.35 (d)
                          12        20.66 [+ or -] 0.60 (a)
                GO        24        30.16 [+ or -] 2.60 (b)
                          48        41.50 [+ or -] 0.57 (c)
                          72        50.66 [+ or -] 0.60 (d)
Native                    12        80.00 [+ or -] 1.44 (a)
  pericardium   DPPA      24        83.50 [+ or -] 0.60 (b)
                          48        85.83 [+ or -] 3.05 (c)
                          72        91.16 [+ or -] 0.72 (d)
                          12        13.83 [+ or -] 0.66 (a)
                EGDGE     24        26.16 [+ or -] 1.20 (b)
                          48        57.83 [+ or -] 0.72 (c)
                          72        79.33 [+ or -] 0.72 (d)
Control                   --        --
                          12        55.90 [+ or -] 0.72 (a)
                GA        24        59.20 [+ or -] 3.63 (a)
                          48        64.16 [+ or -] 3.94 (a)
                          72        81.53 [+ or -] 1.80 (b)
                          12        13.40 [+ or -] 0.52 (a)
                GO        24        33.90 [+ or -] 1.11 (b)
                          48        41.76 [+ or -] 1.08 (c)
                          72        47.56 [+ or -] 1.47 (d)
                          12        76.96 [+ or -] 2.92 (a)
Acellular       DPPA      24        80.13 [+ or -] 0.63 (b)
  pericardium             48        82.56 [+ or -] 2.95 (c)
                          72        91.30 [+ or -] 1.30 (d)
                          12        22.93 [+ or -] 1.31 (a)
                EGDGE     24        42.60 [+ or -] 0.52 (b)
                          48        54.20 [+ or -] 0.20 (c)
                          72        56.50 [+ or -] 0.34 (d)
Control                   --        --

                          Cross
                Cross     Linking   Moisture Content
Biomaterials    linking   Time
                Agents    (hr)

                          12        48.66 [+ or -] 00.08 (a)
                GA        24        46.46 [+ or -] 00.14 (b)
                          48        42.53 [+ or -] 00.26 (c)
                          72        35.33 [+ or -] 00.08 (d)
                          12        66.86 [+ or -] 00.20 (a)
                GO        24        59.86 [+ or -] 00.23 (b)
                          48        56.33 [+ or -] 00.16 (c)
                          72        56.03 [+ or -] 00.26 (d)
Native                    12        66.93 [+ or -] 01.79 (a)
  pericardium   DPPA      24        65.06 [+ or -] 01.41 (b)
                          48        64.90 [+ or -] 00.76 (c)
                          72        61.63 [+ or -] 01.05 (d)
                          12        70.10 [+ or -] 04.12
                EGDGE     24        67.36 [+ or -] 02.27
                          48        62.40 [+ or -] 05.06
                          72        62.23 [+ or -] 05.57
Control                   --        70.76 [+ or -] 00.14 (C)
                          12        59.16 [+ or -] 00.12 (a)
                GA        24        56.76 [+ or -] 00.18 (b)
                          48        49.26 [+ or -] 00.08 (c)
                          72        38.26 [+ or -] 00.14 (d)
                          12        66.60 [+ or -] 00.15 (a)
                GO        24        52.90 [+ or -] 00.05 (b)
                          48        40.86 [+ or -] 00.08 (c)
                          72        28.26 [+ or -] 00.12 (d)
                          12        71.33 [+ or -] 00.63 (a)
Acellular       DPPA      24        65.56 [+ or -] 00.43 (b)
  pericardium             48        56.10 [+ or -] 04.80 (c)
                          72        50.26 [+ or -] 01.37 (d)
                          12        63.73 [+ or -] 03.25 (a)
                EGDGE     24        61.90 [+ or -] 01.52 (b)
                          48        53.40 [+ or -] 02.51 (c)
                          72        52.83 [+ or -] 00.54 (d)
Control                   --        79.86 [+ or -] 00.06 (C)
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Title Annotation:Original Article
Author:Kumar, Amit; Sharma, A.K.; Kumar, Naveen; Maiti, S.K.; Dewangan, Rukmani; Kumar, Vineet; Singh, Hima
Publication:Trends in Biomaterials and Artificial Organs
Article Type:Report
Geographic Code:1USA
Date:Apr 1, 2015
Words:11058
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