In-vitro biocompatibility determination of bladder acellular matrix graft.
Abstract: In-vitro biocompatibility testing of bladder acellular matrix graft (BAMG) was carried out after cross-linking with 0.6% Glutaraldehyde (GA), 1% Hexamethylene diisocyanate (HMD), 1% 1,4-butanediol diglycidyl ether (BDDGE) and 1% 1-ethyl3- (3-dimethyl aminopropyl) carbodiimide (EDC) for 12, 24, 48 and 72 h duration at room temperature. The uncross-linked acellular bladder was used as control. In-vitro studies included enzymatic degradation (collagenase, elastase and trypsin), free amino group determination, moisture content percentage, molecular weight analysis using SDS-PAGE and free protein content estimation. Enzymatic degradation was carried out by treating BAMG with collagenase (20 U/ml), elastase (0.1 U/ml) and trypsin (0.004 Anson U/ml). HMD treated biomaterials were found highly resistant among the cross-linked samples to enzymatic degradation. The GA and HMD treated bladder acellular matrix had greatest reduction in free amino group contents in comparison to EDC, BDDEGE and control samples. The maximum dehydration (least moisture content) was recorded in EDC treated BAMG, followed by HMD, BDDGE and GA treated samples at respective time intervals. In SDS-PAGE the GA and EDC treated samples did not show any band pattern whereas, HMD and BDDGE showed protein bands almost similar to that of acellular bladder (control), but were of reduced density and less intensity. Reduction in the free protein contents was minimum in HMD cross-linked samples, while it was maximum in GA and EDC treated samples.
Article Type: Report
Subject: Bladder (Physiological aspects)
Artificial organs (Usage)
Artificial organs (Health aspects)
Biocompatibility (Measurement)
Authors: Dewangan, Rukmani
Sharma, A.K.
Kumar, Naveen
Maiti, S.K.
Singh, Himani
Gangwar, A.K.
Shrivastava, Sameer
Sonal
Kumar, Amit
Pub Date: 10/01/2011
Publication: Name: Trends in Biomaterials and Artificial Organs Publisher: Society for Biomaterials and Artificial Organs Audience: Academic Format: Magazine/Journal Subject: Health Copyright: COPYRIGHT 2011 Society for Biomaterials and Artificial Organs ISSN: 0971-1198
Issue: Date: Oct, 2011 Source Volume: 25 Source Issue: 4
Product: Product Code: 3842132 Artificial Organs NAICS Code: 339113 Surgical Appliance and Supplies Manufacturing
Geographic: Geographic Scope: India Geographic Code: 9INDI India
Accession Number: 304842732
Full Text: Introduction

The lack of tissue or organ for transplantation is a serious issue, the most formidable barrier to making transplantation a routine medical treatment is the immune system. The immune system has evolved elaborate and effective mechanisms to protect the organism from attack by foreign agents, and these same mechanisms cause rejection of graft from anyone who is not genetically identical to the recipient. Most tissue engineering approaches to the restoration and repair of damage tissues require a scaffold material upon which cells can attach, proliferate, and differentiate, hopefully into a functionally and structurally appropriate tissue for the body location into which it is placed. Collagen-based biomaterials derived from natural tissue have been used extensively as implants to help alleviate the chronic shortage of autologous grafti materials. Collagen is generally treated as "self tissue by recipients into which it is implanted and begins to degrade immediately. It is broken down in the tissues by the catabolic processes, including degradation by specific collagenase enzyme and phagocytosis. Therefore, in the exploitation of tissue as clinical material, this deterioration must be arrested and deferred, preferably beyond the recipient's natural life. These materials require a degree of processing to prepare the collagen for implantation. The first step in this approach is to identify a suitable allogenic or xenogenic tissue and modify the structure to give a material that will be immunologically inert, mechanically robust, and will support cell attachment and proliferation (1). It was hypothesized that cell extraction from biological tissue may remove their cellular antigens as a means for reducing the antigenic response to xenograft materials; extraction removes lipid membranes and membrane-associated antigens as well as soluble proteins (2, 3). However, even with complete extraction of cellular proteins, it would still be anticipated a cross-species response directed towards the structural proteins if acellular tissues were used as a xenograft (4). This cross-species response due to the structural proteins may be further reduced by modifying acellular tissues with cross linking agents (5). Because of immediate degradation of biological tissues obtained from the abattoir, cadaver or patient and the presence of antigenicity in allogenic or xenogenic tissues, the fresh biological tissues can not directly be preserved and applied. The aim is to prolong the original structural and mechanical integrity and remove or at least neutralize the antigenic properties attributed to these materials. The advantages of cross-linking with different chemicals included: (a) preserving the tissue by enhancing the resistance of the material to enzymatic or chemical degradation (b) reducing the immunogenicity of the material and (c) sterilizing the tissue matrix to be implanted and maintaining their mechanical properties (6). The rate of their biodegradation can be reduced by treating them with different cross-linking agents. Cross-linking reinforces the collagen structure by introducing intra- and inter-molecular cross-links between collagen molecules. 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, the time and the temperature of exposure affects the cross-linking (7). So there is need to search for an ideal chemical reagent for cross-linking of collagen material. There are numerous cross-linking agents, but till now the ideal cross-linking agent without the disadvantages of immunogenicity, cytotoxicity and mineralization is yet to be discovered. Therefore, the objective of this study was to determine in-vitro biocompatibility of different cross-linked bladder acellular matrix graft.

Materials and Methods

Preparation of bladder acellular matrix graft

The fresh urinary bladder of pig after collection from abattoir was rinsed with normal saline to remove the adhered blood. The maximum time period between the retrieval and initiation of protocols was less than 4 h. The tissues were cut into 2cm x 2 cm pieces and were placed in 0.5 % anionic biological detergent for 24 h with continuously agitated. Then, the tissue was thoroughly washed in phosphate buffer saline (PBS) solution. Acellularity was confirmed by histological examination. The prepared BAMG were stored in PBS solution containing 1% amikacin at 4[degrees]C until use. The optimized bladder acellular matrix grafts (BAMG) of porcine were cross-linked using following four reagents. The detail outline of cross-linking in different groups is presented in table. The amount of solution used to cross-link each sample was 20 ml. Tissues of each study group were kept for 12, 24, 48 and 72 h in chemicals for cross-linking at room temperature. The solution was changed at 12 h time interval.

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

i) Gross observations

A gross observation of tissue after cross-linking was examined for the change in colour, consistency, swelling and stiffness.

ii) In-vitro enzymatic degradation

In-vitro enzymatic degradation was performed using collagenase, elastase and trypsin enzymes.

a) In-vitro collagenase enzymatic degradation

In-vitro collagenase enzymatic degradation was performed as per the method described by Connolly et al. (8). The samples were transferred to 2 ml microcentrifuge tube containing 1.75 ml of 20 U/ml Collagenase Type-IV-S (Sigma Aldrich Co., St Louis, MO, USA, C1889) obtained from Clostridium histolyticum in phosphate buffered saline with 0.2 mg/ ml sodium azide was added to each tube and incubated for 6, 12 and 24 h at 37[degrees]C. The tissues were blotted and the mass was carefully determined. Weight loss of biomaterial was calculated that of the original tissue.

b) In-vitro elastase enzymatic degradation

Elastase enzymatic degradation was performed as per the method described by Leach et al. (9). The samples were transferred to 2 ml micro-centrifuge tube containing 1.75 ml of 0.1 U/ml elastase (pancreatic solution, type I: from porcine pancreas, Sigma Aldrich Co., St Louis, MO, USA, E1250) in PBS with 0.2 mg/ml sodium azide and incubated for 6, 12 and 24 h at 37[degrees]C. The tissues were blotted and the mass was recorded. Weight loss of biomaterial was calculated that of the original tissue.

c) In-vitro trypsin enzymatic degradation

The procedure of trypsin enzymatic degradation was similar to elastase enzymatic degradation. The samples were transferred to 2 ml microcentrifuge tube containing 1.75 ml of 0.004 Anson U/ml trypsin (Sigma Aldrich Co., St. Louis, MO, USA, 15117-3) in PBS with 0.2 mg/ml sodium azide and incubated for 6, 12 and 24 h at 37[degrees]C. Weight loss of biomaterial was then calculated that of the original tissue.

iii) Free amino group contents determination

Ninhydrin assay was used to determine the free amino group content of each test sample as per the procedure of Sung et al. (10). The sample was lyophilized for 24 h and weighed. Subsequently, the lyophilized tissue was heated with 2.67% ninhydrin solution in water bath for 20 minutes. After heating, the optical absorbance of the solution was recorded with spectrophotometer at a wavelength of 570 hm.

iv) Moisture content analysis / swelling ratio

The moisture content was analyzed as per the method of Sung et al. (10). The values of moisture content were expressed in percentage. The moisture content of the test tissue was calculated as follows:

Moisture content (%) = (Wet tissue weight - Dry tissue weight) x 100 / Wet tissue weight

v) Free protein content estimation

The free protein contents were estimated by the methods of Lowry et al. (11) using bovine serum albumin (BSA) as a standard. The absorbance was measured at 750 hm. The increase in the absorbance against the blank was used for calculations. The values of protein contents were expressed in mg/ml.

Concentration of test protein = (Absorbance of test protein / Absorbance of standard protein) x Concentration of standard protein

vi) Molecular weight analysis

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. (12). It was performed by 10% sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). Samples were prepared by triturating 100 mg of cross-linked and native biomaterials with 10% sodium dodecyl sulphate (1 ml) and supernatant was obtained after centrifugation at 10,000 rpm for 10 minutes. 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. Then the sample was loaded onto 14 slot applicator. The finished gel was stained with Coomassie blue and destained 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.

vii) Statistical analysis

The data was analysed by ANOVA and Student's t-test as per Snedecor and Cochran (13).

Results

Preparation of bladder acellular matrix graft

In the present study, the decellularization process successfully removed the nucleus and cytoplasmic cellular components of the graft and the resulting into full-thickness bladder acellular matrix graft (BAMG) of porcine origin consisting of primarily collagen and elastin having good tensile strength. The protocol for conversion of porcine urinary bladder into bladder acellular matrix graft was effective and acellularity was confirmed histologically. The graft showed complete acellularity with normal collagen fibres arrangement. The process had been shown to effectively remove nucleus and cytoplasmic cellular components and preserving the original structural arrangement of extracellular matrix components. Masson's trichrome stain showed that the matrix was collagen rich membrane. Hematoxylin and Eosin staining and electronic microscopy did not reveal any cellular elements. These grafts were used for cross-linking with different cross-linkers.

i) Gross observations

The gross observations of tissue were made after cross-linking at given concentration and duration.

ii) In-vitro enzymatic degradation

In-vitro enzymatic degradation studies of BAMG was done using different concentrations of collagenase, elastase and trypsin enzymes at different time intervals.

a) In-vitro collagenase degradation

The results of in-vitro collagenase degradation at different time intervals are presented in Fig. 1. A linear increase in the rate of weight loss (percent) of tissue samples was recorded with the increase in collagenase (20U/ml) digestion time intervals. In contrast, linear decrease in weight loss was observed with the increase in cross-linking time intervals. The treatment of the collagenase caused progressive reduction in the weight of cross-linked acellular bladder matrix prepared with the increasing cross-linking time. The reduction in the weight of acellular uncross-linked bladder (control) was 27.47 [+ or -] 1.07 percent. The digestion with collagenase for 12 h and 24 h produced a significant (P<0.05) reduction in the weight of acellular bladder matrix in GA treated samples for all the four different cross-linking time intervals. The values recorded in all these samples were significantly (P<0.05) lower than the control group. Progressive reduction in the weight loss of samples treated with HMD, BDDGE and EDC was also seen with the increase in cross-linking time in each group. The values of weight recorded for cross-linked samples remained lower in comparison to the values of uncross-linked samples (control).

b) In-vitro elastase degradation

The results of in-vitro elastase degradation at different time intervals are presented in Fig. 2. There was increase in the weight loss (percent) of the cross-linked bladder acellular matrix with the increase in the elastase (0.1U/ ml) digestion time. Therefore, the minimum weight loss of samples was recorded at 6 h digestion with elastase and maximum weight loss of tissue samples was recorded at 24 h digestion with elastase in all the samples cross-linked with different cross-linkers for four different periods of time. A progressive reduction of variable degree in the weight of bladder acellular matrix was observed with the increase in cross-linking time in all the four groups with different cross-linkers. In GA treated tissue samples for four different time intervals a significant (P<0.05) reduction in the weight loss of bladder acellular matrix was recorded after elastase digestion for 6, 12 and 24 h. The values recorded in HMD-12 and HMD-72 samples at 6, 12 and 24 h were significantly (P<0.05) different. The samples cross-linked with BDDGE-48 and BDDGE-72 exhibited significant (P<0.05) reduction in weight loss in comparison to control values after digestion for 6 and 12 h with elastase, whereas, in 24 h digested samples the non-significant (P>0.05) reduction in the weight of tissue was observed. The samples cross-linked with EDC for four different periods exhibited significant (P<0.05) reduction in the weight of samples in comparison to control group in 6, 12 and 24 h digested samples. The values recorded in 6 h and 12 h elastase digested samples remained significantly (P<0.05) different in each sample cross-linked for a particular time interval.

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c) In-vitro trypsin degradation

The results of in-vitro trypsin degradation at different time intervals are presented in Fig. 3. Increased rate of weight loss (percent) was observed with the increase in time intervals of trypsin (0.004 Anson U/ml) digestion. However, decreased rate of weight loss was observed with the increase in cross-linking time intervals. The GA and HMD cross-linked samples exhibited significant (P<0.05) reduction in the weight of bladder acellular matrix after 6, 12 and 24 h of trypsin digestion in comparison to control values. In BDDGE and EDC cross-linked samples, the values recorded after trypsin digestion for all the three time intervals were non-significant (P>0.05). However, a progressive reduction in the weight loss with increase in the cross-linking time intervals in both groups was recorded. Further, the samples cross-linked for a particular time exhibited significant (P<0.05) difference in the values with the increase in the trypsin digestion time. The weight loss in acellular uncross-linked bladder matrix (control) was greater in comparison to cross-linked samples. Whereas, the weight loss of tissue was maximum in the native bladder samples.

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iii) Free amino group concentrations determination

The free amino group concentrations at different time intervals are presented in Fig. 4. The standard curve was plotted using different concentrations (20, 40, 60, 80, 100 and 150 [micro]g/ml) of glycine to calculate the free amino group contents in cross-linked samples. The free amino acid group concentration decreased significantly (P<0.05) with the increase in cross-linking time intervals within the same treatment group. The free amino acid group concentrations of acellular bladder (control) and native bladder are 77.00 [+ or -] 0.002 [micro]g/ml and 73.33 [+ or -] 0.002 [micro]g/ml respectively. These values are significantly (P<0.05) higher than those recorded in the cross-linked samples in each group. The BDDGE and EDC cross-linked groups showed non-significant (P>0.05) reduction in free amino group concentration at 24 h and later at 48 h the reduction in the values was significant (P<0.05). In GA and HMD treated groups the free amino acid group concentration had significantly (P<0.05) reduced with increase in cross-linking time intervals.

iv) Moisture content percentage

The moisture content percentages at different time intervals are presented in Fig. 5. The moisture content percentage had decreased significantly (P<0.05) with the increase in cross-linking time intervals within the group for each cross-linking agent. The GA, HMD and BDDGE cross-linked groups showed the significant (P<0.05) reduction in moisture percentage with increase in cross-linking time intervals. Whereas, in EDC treated tissue samples, non-significant (P>0.05) reduction in moisture percentage was recorded in all cross-linked samples. The moisture content in 12 h cross-linked samples was maximum which reduced significantly (P<0.05) with the increase in cross-linking time in GA, HMD and BDDGE treated samples. The moisture content of native bladder was almost similar to the moisture content of 12 h samples cross-linked with four different cross-linkers. However, the maximum values (81.87 [+ or -] 3.58%) of moisture were recorded in acellular uncross-linked bladder matrix. The over all sequence of the moisture content percent was acellular >GA >Native bladder > BDDGE > HMD > EDC in 12 h cross-linked group.

[FIGURE 7 OMITTED]

v) Free protein content estimation

The free content at different time intervals is presented in Fig. 6. The free protein concentrations significantly (P<0.05) decreased with the increase in cross-linking time intervals with each cross-linker. These values were significantly (P<0.05) different from control values recorded for each cross-linked sample. The free protein was higher (0.513 [+ or -] 0.002 mg/ml) in acellular uncross-linked bladder samples (control) in comparison to cross-linked samples. However, the free protein content was maximum (0.579 [+ or -] 0.003 mg/ml) in native tissue samples.

vi) Molecular weight analysis

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. The results are presented in Figure 7. The typical bladder collagen pattern is represented in the native bladder (Lane 6). In the SDS resolving gel, the collagen bands showed molecular weight of about 30, 49, 88, 130, 140 and 150 kDa. A band corresponding to a protein of about 25 kDa was also visible in the gel, which may be due to presence of some non-specific protein in porcine bladder. Native collagen molecules remained in the stacking gel. After decellularization process, the soluble protein decreased as revealed in SDS-PAGE of acellular bladder (Lane 5). The protein band pattern of collagen in acellular bladder in SDS-PAGE did not show any high protein band, whereas, only lower protein bands of about 88, 49, 30 and 25 kDa molecular weight were seen. The GA treated bladder acellular matrix graft did not show any higher protein band pattern in SDS-PAGE gel. All the GA treated bladder acellular matrix graft did not show any band pattern in SDS-PAGE gel. The protein bands corresponding to about 88, 30 and 25 kDa were visible in the SDS gel with variable density for HMD cross-linked samples for different time periods. The density of protein bands had reduced in the SDS-PAGE for the cross-linked samples. The protein band of 49 kDa molecular weight disappeared from all the HMD cross-linked samples as seen in the SDS gel. The density of bands was more in 12 h treated samples. The density of bands had reduced in 48 h treated samples which indicated that the cross-linking had increased with increase in cross-linking time interval. The protein band patterns of collagen produced by protein of cross-linked samples were similar to that observed in acellular tissue (control) but with less intensity. The protein band pattern of collagen in the tissues treated with 1% BDDGE prepared in sterile PBS and cross-linked for different time intervals are corresponding to about 88, 49, 30 and 25 kDa were visible in the SDS gel for the samples cross-linked for all the four time periods. The protein band patterns of collagen were almost similar to that of acellular tissue (control) but were of less intensity. In the other group, where bladder acellular matrix graft was treated with 1% EDC (prepared in sterile PBS), 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. Therefore, all the EDC treated bladder acellular matrix graft did not show any band pattern in SDS-PAGE gel.

Discussion

Before biomaterials can be applied for its clinical use, the tissue response to these biomaterials had to be evaluated in-vitro. This approach is to identify a suitable allogenic or xenogenic tissue and modify the structure to give a material that will be immunologically inert, mechanically robust, and will support cell attachment and proliferation (1). The preparation of natural matrices commonly involves a combination of physical methods to delaminate layers of tissue, followed by chemical and enzymatic methods to remove cell bodies from the remaining ECM (14) and such decellularisation strategies, designed to limit the immunogenicity of the matrix. Decellularization process may attenuate severe xenogenic immune response (15), but the removal of cellular components may not be sufficient to eliminate inflammation, and fixation techniques may still be necessary to prevent degradation (16). It was also important to keep in mind that, even after the removal of cells and debris from the biomaterials the extracellular matrix of the acellular tissue itself may elicit some amount of immune response (17). Chemical cross-linking of collagen had been used for several years to improve scaffold stability (18, 19). Control resorption of biological biomaterials is essential where it is to be used for tissue regeneration. Cross-linking is an effective method to control resorption rate of collagen based biomaterials and to prevent a rapid elution of the materials into the wound fluid (20). The process of cross-linking involves the chemical agents initiating ideally, irreversible and stable intra- and inter-molecular chemical bonds between collagen molecules. Intermolecular cross-linking of collagen in-vitro by physical treatment or by chemical agents modified the properties of biomaterials (21, 22, 23). It reduces the solubility, water absorption and biodegradability of the collagen biomaterials and increases its mechanical properties (24). The antigenicity of a collagen biomaterial can be reduced by the process of cross-linking (25). The inflammatory reaction depended both on the site of implantation and the species in which the collagen biomaterial was implanted (26).

In the present study, the decellularization process successfully removed the nucleus and cytoplasmic cellular components of the graft and the resulting into full-thickness bladder acellular matrix graft (BAMG) of porcine origin consisting of primarily collagen and elastin having good tensile strength. The extraction protocol was designed to reduce the antigenic response to a xenograft material. The process had been shown to effectively remove nucleus and cytoplasmic cellular components, lipid membranes and membrane-associated antigens as well as soluble proteins, while preserving the original structural arrangement of extracellular matrix components which consist of primarily of insoluble collagen and elastin fibres which are embedded in a ground substance of glycosaminoglycans (3). On the basis of the histological observations showed an intact mesh with no evidence of cells, nuclei or other cell fragments. The resultant biomaterial had shown that underlying bladder histoarchitecture was retained. Similarly, Geng et al. (27) prepared xenogenic bladder submucosa acellular matrix (BSAM) without using any enzymatic treatment and only biological detergent 0.5% SDS and dH2O were used. The BSAM was white, semi-translucent and approximately 0.1 to 0.2 mm thick. Masson's trichrome stain showed that the matrix was collagen rich membrane. Hematoxylin and Eosin staining and electronic microscopy did not reveal any cellular elements.

GA treated (0.6%) tissues were hard/stiff as compared to other cross-linking agents. In glutaraldehyde, there is only one carbon-carbon bond (C-C), which is known to be relatively inflexible; therefore, the glutaraldehyde fixed tissue is usually comparatively stiff (28). However, lower concentrations have been found to be better in bulk tissue cross-linking compared to higher concentration (29). HMD treated tissue were also stiff similar to GA treated tissue. BDDGE and EDC treated tissues were found to be soft in consistency, which may be attributed to their more absorption of moisture as compared to GA treated tissues which showed less swelling or low moisture percentage. However, GA cross-linked biomaterials have been shown to release toxic monomeric GA upon hydrolyzation of the material (30, 31). HMD treated tissues showed formation of white flakes. On rinsing tissue with PBS the solution became cloudy. However, Loke et al. (32) recommended the use of 2-propanol as solvent for HMD, as the isocyanate group is highly reactive to water, destroying the isocyanate functionality before it can react with collagen. Similarly, in present study, propanol was used to prepare HMD solution for cross-linking. The cross-linking of the collagen based tissue with glycidyl ether based reagents resulted in good stability towards enzymatic degradation with excellent mechanical property (33), decreased calcification and a lower cytotoxicity (34) as compared to GA cross-linked tissue. However, use of these reagents resulted in an undefined cross-linked structure because of the polyfunctionality. The EDC offers the method for generating cross-links between corresponding reaction sites, without itself being incorporated (7). To overcome problems associated with reagent toxicity of the GA, carbodiimides have been used to cross-link collagen. Carbodiimides activate the carboxylic acid groups of glutamic or aspartic acid residues to react with amine groups of another chain, forming amide bonds (35). Collagen scaffolds cross-linked with EDC have been shown to possess decreased enzymatic degradation rates, but improved the mechanical properties (36).

The cross-linking with different chemicals retards resorption of collagen based biomaterials in tissues. Therefore, the study of enzymatic degradation can be a good model for evaluation of the resorption rate in tissue. In-vitro degradation studies (degradation with collagenase, elastase and trypsin) revealed that uncross-linked tissues were more prone to enzymatic degradation than the cross-linked tissue. Fixing of samples with cross-linking agents resulted in increased resistance against enzymatic degradation due to increase in cross-linking degree. The increased resistance against enzymatic degradation may be due to the inhibition of enzyme-substrate interaction via the hidden or altered the cleavage sites of collagen by the cross-linking agents (5). Collagen based biomaterials are biocompatible and non-toxic to tissues and have well-documented structural, physical, chemical, biological and immunological properties. Additionally, mechanical and to some extent immunologic properties of collagen scaffolds can be influenced by modification of matrix properties (porosity, density) or by different chemical treatment affecting its degradation rate (37,38). Cross-linking of tissue was done by carboxyl-amine group intrahelical (EDC/NHS), carboxyl-carboxyl group interhelical (BDDGE), and amine-amine group intermolecular (GA) cross-linking between the polymer and the collagen helix, which was a component of the native tissue. The intrahelically cross-linked tissue showed weaker stability against heat and degradation caused by collagenase compared to the interhelically cross-linked tissue. The tissue intermolecularly cross-linked with polymer showed the highest stability against heat and degradation caused by collagenase (39). The bladder acellular matrix graft samples of the present study revealed that the increase in cross-linking time intervals decreased the enzymatic degradation. The results of the present study revealed that the cross-linked bladder acellular matrix graft of porcine origin had shown significantly (P<0.05) increased resistant to various concentration of collagenase, elastase and trypsin degradation at different time intervals as compared to acellular uncross-linked bladder matrix (control). The GA and HMD treated tissue have shown significantly (P<0.05) increased resistance to collagenase and elastase degradation at different time intervals. However, uncross-linked tissue samples whether native or acellular, degraded earlier as compared to cross-linked tissue samples. The HMD and GA treated samples showed increased resistance to trypsin degradation as compared to other groups. All the cross-linked bladder acellular matrix graft also showed increased resistance to trypsin degradation than the control group. BDDGE treated tissue samples did not show any significant (P>0.05) difference in resistance to enzymatic degradation when compared with control. The results obtained from in-vitro collagenase degradation experiments demonstrated the increased resistance of cross-linked samples to enzymatic degradation. Bacterial collagenase from Clostridium histolyticum is capable of cleaving peptide bonds within the triple helical structure and has a specificity for the Pro-X-Gly-Pro-Y region, splitting between X and Gly. This region is found about 40 times in the a-chain. Olde-Damink et al. (35) observed the increased loss of weight and tensile strength after exposing the dermal sheep collagen to the bacterial collagenase with increased time intervals and decreased loss of weight and tensile strength with increased degree of cross-linking. Similarly in the present study, the cross-linked bladder acellular matrix graft showed the similar trend on enzymatic degradation. The decrease in the rate of weight loss of cross-linked versus uncross-linked samples during enzymatic degradation is most probably owing to interference of the penetration of the enzyme into the collagen fibres (40). This reduced penetration will substantially decrease the surface area available for adsorption of the enzyme in the cross-linked collagen network and finally decreases the surface degradation rate. Ester bonds can be more easily cleaved by enzymes than the secondary amine bonds (41). Cross-linking also increases the mechanical properties (42). In contrast, to our finding Bakos and Koniarova (20) observed that 5% solution of HMD strongly inhibited the enzymatic degradation of biomaterials, which could be due to the higher concentration of the HMD. Woodroof (43) reported that higher concentration of GA increased the stiffness of the biomaterials due to intra- and inter-molecular cross-links. In the present study, it was observed that 0.6% GA solution increased the stiffness of the material. Similar observations on chemical concentration for cross-linking of biomaterials have been reported by Santillan-Doherty et al. (44). Aldehyde (GA) reacted with the amino groups of lysyl residues in protein (collagen) and induced the formation of interchain cross-links (45), which stabilized the tissue against chemical and enzymatic degradation depending upon the extent of cross-linking (46, 47).

The free amino group contents analysis indirectly indicates the degree of the cross-linking. The amount of free amino group is inversely proportional to the degree of the cross-linking. In the present study, the free amino group analysis indicated that GA and HMD have the greatest ability to cross-link insoluble collagen fibrils. Lastowka et al. (12) reported that the GA induced cross-linking resulted in the least number of free amines. 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 cross-linking, two amine (-NH3) groups were used in every GA induced primary amine cross-linking (48). Thus, the free amino groups were less in number. In HMD, the isocyanate groups reacted with amino, amide and guanidine groups of polypeptide chain to form stable bonds in water environment (49). Thus, the various functional groups were used in cross-linking; therefore, the free amino acids were available more in quantity. Cross-linking involves the reaction, of amine groups of (hydroxy) lysine residues with epoxide groups of the BDDGE molecules, resulting in the formation of secondary amines. The only single amino group was used in each cross-linking; hence the free amino acids were available more in number in comparison to the GA. The cross-linking between the amino acids without incorporating itself by EDC revealed that the free amino acid concentration was more than GA. Sung et al. (10) reported that higher temperature resulted in lower free amino group contents. It is also known that the reduction of free amino groups in biological tissue diminishes its antigenicity (50). Bovine pericardium cross-linked by GA showed significant decrease in the free amino group content of the cross-linked samples (51). Zeeman et al. (36) reported a decrease in free amino group contents of GA treated dermal collagen. By observing the free amino group contents in different cross-linking agents a general trend was observed in this study. All the cross-linking agents had bound the free amino acids as compared to the acellular uncross-linked bladder matrix (control). Among all the cross-linked samples, GA bound the maximum of free amino acids. Similar findings were also reported by Jorge-Herrero et al. (52). The GA treatment of bladder acellular matrix graft was found more protective than HMD, EDC and BDDGE treatment, which had bound the minimum number of free amino groups.

All cross-linked samples showed reduction in moisture percentage at different cross-linking time intervals as compared to the acellular uncross-linked bladder matrix (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 fixed tissue. The EDC cross-linked biomaterials showed least moisture percentage than all other cross-linked biomaterials. The over all sequence of the moisture percent was acellular>GA>Native bladder>BDDGE>HMD>EDC in 12 h cross-linked group. The 72 h cross-linked samples were found to have minimum moisture content in all cross-linked samples. It is evident from the moisture percentage studies that cross-linking reduced the moisture percent of collagenous matrices. Similar finding have also been reported by Kato and Silver (53). The degree of moisture percentage decreases non-linearly with increasing cross-linking density. Choi et al. (54) reported that the highly cross-linked sponges with EDC showed lower water uptake. Courtman et al. (3) and Chang et al. (55) observed the most notable changes in the acellular tissue. The tissue was swollen and the thickness had increased by about 25%, attributable to an increase in water content, probably due to removal of non-polar tissue lipids. Similarly, in this study, the acellular uncross-linked bladder matrix (control) showed higher moisture content than native bladder. Sung et al. (10) reported that the moisture contents of glutaraldehyde cross-linked tissues were significantly lower than the fresh tissue. GA cross-linked BAMG revealed significant (P<0.05) reduction in moisture content, which indicated that the fixation with aldehydes caused more shrinkage of tissue as compared to control group (acellular uncross-linked bladder matrix). Expulsion of more number of water molecules out of the fixed tissue will cause more shrinkage.

In the cross-linked protein there is masking of immunogenic epitopes and therefore, there is either delayed or altogether no immune response in host body resulting into successful tissue regeneration. Once the protein is cross-linked in the acellular graft, it will delay the degradation of transplanted tissue, thereby, providing sufficient line for the host body to replace the damaged tissue. Bladder acellular matrix graft treated with GA and EDC showed less amount of low molecular weight proteins, which indicated that GA and EDC had the greatest ability to cross-link the biomaterials. Both GA and EDC were found to cross-link bladder protein very efficiently. This was evidenced by absence of specified bands in the SDS-PAGE gel. Where as, HMD treated tissue showed three characteristic protein bands and BDDGE treated tissue showed four protein band patterns in the SDS-PAGE gel. However, in both groups, the density and intensity of bands in SDS-PAGE gel had decreased with the increase in cross-linking time intervals. This indicates that both had minimum ability to cross-link the biomaterials due to expression of more protein bands. Therefore, the maximum ability to cross-link bladder acellular matrix graft was seen with GA, followed by EDC. The results of SDS-PAGE revealed that BDDGE possesses poorest cross-linking ability among all the chemical agents used, as four protein bands were visible in SDS-PAGE gel. GA at concentration of 0.6% V/V as well as 1% EDC gave satisfactorily similar pattern of cross-linked proteins. However, in a report by Lastowka et al. (12) it was shown that 0.5% GA was sufficient to cause cross-linking of protein.

All the cross-linked bladder acellular matrix graft produced significant (P<0.05) decrease in protein contents with the increase in cross-linking time intervals as compared to the control. The decrease in free protein contents indicated the efficacy of the cross-linking of the biomaterials. The free protein contents release was minimum in GA cross-linked samples while it was maximum in HMD treated samples. It was indicated that the GA cross-linked the biomaterials effectively 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. Purohit (56) also reported significant (P<0.05) decrease in free protein contents with the increase in cross-linking time intervals with different cross-linking agents viz. GA, HMD, BDDGE and EDC as compared to the uncross-linked native skin of the rabbit.

The sequence obtained from in-vitro evaluation of cross-linked bladder acellular matrix graft with different cross-linking agents was HMD>GA>EDC>BDDGE. The HMD treated bladder acellular matrix grafts showed least degradation with enzymatic treatment. However, grossly graft was sticky with white flakes on the surface of the graft which made the biomaterial unfit for suturing. SDS-PAGE also showed little cross-linking of this graft. The GA treated tissue came next after HMD treated tissue. GA had the greatest ability to cross-link the biomaterial as visualized in the SDS gel, but they became harder in consistency after cross-linking. EDC had maximum ability to cross-link bladder graft as evidenced by absence of protein band pattern in SDS gel. Therefore, EDC treated grafts were selected for further in-vivo biocompatibility study.

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 research work.

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Rukmani Dewangan, A.K. Sharma, Naveen Kumar, S.K. Maiti, Himani Singh, A.K. Gangwar, Sameer Shrivastava*, Sonal* and Amit Kumar

Division of Surgery, 'Division of Animal Biotechnology Indian Veterinary Research Institute, Izatnagar 243122, U.P.

Received 4 May 2011; Accepted 8 September 2011; Available online 8 September 2011
Groups      Observations

GA          Light yellowish colouration, slightly swollen
            and hard

HMD         Milky white and flakes on surface, stiff

BDDGE       No change in colour, more swollen, pliable an
            soft in consistency

EDC         Light brownish colour and soft in consistency


Table 1: Concentration of cross-linking agents

Groups    Reagents                              Concentration

GA        Glutaraldehyde                        0.6 percent in PBS

HMD       Hexamethylene diisocyanate            1 percent in propanol

BDDGE     1,4-butanediol diglycidyl ether       1 percent in PBS

EDC       1-ethyl-3-(3-dimethyl aminopropyl)    1 percent in PBS
          carbodiimide
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