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Effects of crosslinking treatments on the physical properties of acellular fish swim bladder.

Introduction

Biological materials from both allogenic and xenogenic tissue sources, have been used to construct extracellular matrices for in vivo tissue regeneration [1-5]. However, the outbreak of bovine spongiform encephalopathy and the foot-and-mouth disease have caused restrictions on use of extracellular matrix (ECM) from bovine or porcine origin. Therefore, the ECM from fish swim bladder may be good substitutes for the repair of tissue defects. It was hypothesized in the literature that cell extraction from biological tissues may remove their cellular antigens [6]. As a means for reducing the antigenic response to xenograft materials, extraction removes lipid membranes and membrane-associated antigens as well as soluble proteins [7]. However, even with complete extraction of cellular proteins, it would still be anticipated a crossspecies response directed toward the structural proteins if acellular tissues were used as a xenograft [7]. This cross-species response due to the structural proteins may be further reduced by crosslinking [8]. Due to the high enzymatic turnover rate of ECM proteins in the body, the stabilization of protein based materials, by crosslinking, is required to assure the respective integrity and the desired mechanical properties during an implantation period [9]. Glutaraldehyde (GA) is the most extensively used crosslinking reagent [10]. However, GA is known to elicit cytotoxicity, and calcification of GA treated ECM on implantation have caused great concern [10]. 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. The aim of the present study is to assess the effect of various crosslinking agents on the physical properties of acellular swim bladder of fish origin. The crosslinking agents used included GA, 1, 4-butanediol diglycidyl ether (BDDGE), and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) at their respective optimal concentrations.

Materials and Methods

Standard reagents were obtained from Sigma-Aldrich (St. Louis, Missouri, USA) unless otherwise noted.

Harvest and acellularization of swim bladder

The swim bladder of fresh water fish (Labeo rohita) was collected from the Krishi Vigyan Kendra, Indian Veterinary Research Institute, Izatnagar, Uttar Pradesh, India, and immediately preserved in chilled (4[degrees]C) sterile phosphate buffered saline (PBS) (pH 7.4) containing 0.1% amikacin and 0.02% EDTA. It was washed thoroughly with sterile PBS to remove all adherent blood and mucus. Swim bladders were cut into 2 x 2 cm pieces, and were placed in different concentrations of an ionic biological detergent to carry out acellurization protocols. They were subjected to microscopic examination at 12, 24, 48, and 72 hours intervals to optimize the acellularization protocols. The prepared acellular swim bladder (ASB) was stored in PBS solution containing 0.1% amikacin solution at -20[degrees]C until use.

Crosslinking of the ASB

The ASB was crosslinked with 0.6% GA, 1% BDDGE, and 1% EDC. The amount of solutions used to crosslink each sample was 20 ml and were changed at every 24 hours. Tissues of each study group were kept for 12, 24, 48, and 72 hours in chemicals for crosslinking under constant agitation and temperature (37[degrees]C).

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:

i) Gross observations

Gross observations of test samples were made after treating with GA, BDDGE, and EDC. It included change in colour, consistency, swelling and stiffness of test samples.

ii) Degradation tests

a) Non-enzymatic degradation test

In-vitro non-enzymatic degradation of test samples was performed as per the procedure of Vaz et al. [11]. Preweighed dry specimens were immersed for 0, 1, 3, 5, and 7 days at 37[degrees]C in an isotonic saline solution containing 0.1% sodium azide (Na[N.sub.3]). The values of weight loss were expressed in percentage.

b) Enzymatic degradation test

In-vitro collagenase enzymatic degradation of test samples was performed as per the method described by Connolly et al. [12]. The samples were equilibrated overnight in PBS with 0.2 mg/ml sodium azide (NaN3) as preservative. The samples were then removed from the solution, excess moisture was blotted from the surface and the initial mass was recorded. The samples were transferred to 2 ml microcentrifuge tube and 1.75 ml of 20 U/ml Collagenase Type-I from Clostridium histolyticum in PBS with 0.2mg/ml sodium azide was added to each tube and incubated for 12, 24, 48, and 72 hours intervals at 37[degrees]C. The tissues were blotted and the mass was determined. Weight loss of biomaterial was then calculated that of the original tissue. The values of weight loss were expressed in percentage.

iii) Determination of free protein

The free protein contents of test samples were estimated as per the methods of Lowry et al. [13], using bovine serum albumin (BSA) as a standard. The absorbance was measured at 750 nm. The increase in the

absorbance against the blank was used for calculation. The values of protein contents were expressed in mg/ml.

iv) Determination of free amino group contents

Free amino group contents of test samples were determined by ninhydrin assay as per the procedure of Sung et al. [14], using glycine as a standard. The samples were lyophilized for 24 hours and weighed. Subsequently, the lyophilized tissues were heated with 2.67% ninhydrin solution in water bath for 20 minutes. The optical absorbances of the solutions were recorded at 570 nm with spectrophotometer, and linear standard graph was obtained. Sample concentrations were determined from the standard linear graph. The values of free amino groups were expressed in mg/ml.

v) Determination of fixation index

The fixation index of test samples were determined by ninhydrin assay as per the procedure of Sung et al. [15] and were expressed in percentage.

vi) Determination of free hydroxyproline contents

The free hydroxyproline contents of test samples were estimated as per method of Neuman and Logan [16], using hydroxyproline stock solution as a standard. The optical absorbances of the solutions were recorded at of 550 nm with spectrophotometer. Sample concentrations were determined from a linear hydroxyproline standard curve with regression coefficient > 0.98.

vii) Moisture content analysis

The moisture contents of test samples were analyzed as per the method of Sung et al. [14], and expressed as percentage (%). The wet tissue was sandwiched between the two paper towels and a 40 grams weight was applied on top for 10 sec. The weight of the tissue was recorded as wet tissue weight. Subsequently, the tissue was dried for 24 hours and weighed again (dry tissue weight). Finally, the moisture contents of the test tissue were calculated as follows:

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

viii) SDS-PAGE analysis

Molecular weight analyses of test samples were performed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) as per the method described by Lastowka et al. [17]. Briefly, 10 mg of each sample were triturated with tissue homogenizer buffer (50 mM Tris, 150 mM NaCl, 0.25% Triton X-100 and 0.5% SDS, pH 7.4) (1ml) and supernatant was obtained after centrifugation at 10,000 rpm for 10 minutes. The supernatant were mixed with an equal volume of sample buffer (1xsample buffer: 62.5 mM Tris, 2% SDS, 10% glycerol, 0.0125% bromophenol blue, pH 6.8). Samples were heat-denatured for 5 minutes at 90[degrees]C and were subjected to SDS-PAGE analysis as described by Laemmli [18], using 4% stacking gels and 10% resolving gels in a Mini Protean II unit (Bio-Rad Laboratories, Hercules, CA, USA) at 50 mA/gel. After fractionation, the gel was stained in a staining solution (50% methanol, 10% glacial acetic acid, 0.25% Coomassie Brilliant Blue R-250) for 10 minutes. Next, the gel was destained in a solution containing 250 mL methanol, 100 mL acetic acid, and 650 mL distilled water until protein fractions appeared clear. Pre-stained markers from Bio-Rad were used for molecular weight determination.

Statistical analysis

Results were expressed as mean [+ or -] SE. The means of parametric observations were compared by analysis of variance (ANOVA) as described by Snedecor and Cochran (19). For each comparison, differences between groups were considered significant at P < 0.05.

Results

Acellularization of swim bladder

Figure 1 and 2 showed the microscopic image of native as well as ASB (H&E, X40). The native swim bladder treated with 0.5% ionic biological detergent for 24 hours under constant agitation showed complete loss of cellularity. The tunica externa and interna were completely acellular. Contrary to native tissue the collagen fibres were loosely arranged.

i) Gross observations

GA treated ASB was yellowish in colour, slightly swollen and hard, which further increased with the increasing crosslinking intervals as compared to control. BDDGE treated ASB was pliable, soft in consistency, and more swollen as compared to control. EDC treated ASB was white in colour and soft in consistency as compared to control.

ii) Degradation tests

a) Nonenzymatic degradation test

Rate of weight loss (percent) of test samples following non-enzymatic degradation are presented in figure 3. The weight loss was significantly (P<0.05) decreased in crosslinked ASB at different degradation intervals as compared to control. Additionally, it was noted that the rate of weight loss of the GAtreated ASB was significantly (P<0.05) lowered than BDDGE, and EDC treated ASB during the entire period of observations.

b) Enzymatic degradation test

Rate of weight loss (percent) of test samples after collagenase enzymatic degradation at various degradation intervals are presented in figure 4. The weight loss was significantly (P<0.05) decreased in crosslinked ASB at various degradation intervals as compared to control. Among all the crosslinked tissues, weight loss was minimal in ASB cross-linked with GA for 72 hours followed by BDDGE treated tissues for 72 hours, and maximum in EDC treated tissues for 12 hours. The values of uncrosslinked ASB were significantly (P<0.05) different from different cross-linked groups.

iii) Determination of free protein

Mean [+ or -] SE of free protein concentration (mg/ml) of crosslinked ASB and control are presented in figure 5. The free protein concentration of ASB (control) was 1.54 [+ or -] 0.038 mg/ml, which was significantly (P<0.05) higher then cross-linked ASB at different cross-linking intervals. In GA, BDDGE, and EDC crosslinked ASB, free protein concentration vary significantly (P<0.05) within the group at various crosslinking intervals. Among crosslinked tissues, free protein concentration was highest in tissues cross-linked with EDC for 12 hours whereas, it was lowest in tissues crosslinked with GA for 72 hours.

iv) Determination of free amino group contents

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 crosslinked samples. The free amino group concentrations of crosslinked ASB and control are presented in figure 6. The free amino acid group concentrations of ASB was 97.793 [+ or -] 0.034 [micro]/ml which was significantly (P<0.05) higher when compared to crosslinked ASB. In GA, BDDGE and EDC crosslinked ASB, free amino group concentration at 48, and 72 hours crosslinking intervals was significantly (P<0.05) lower than the free amino group concentration at 12, and 24 hours crosslinking intervals. Among the crosslinked tissues, free amino group concentration was minimal in tissues crosslinked with GA for 72 hours and maximum in tissues crosslinked with EDC for 12 hours.

v) Determination of fixation index

Mean [+ or -] SE of fixation index (percent) of crosslinked ASB is presented in figure 7.0. In GA, BDDGE, and EDC crosslinked tissues, fixation index at 12, and 24 hours crosslinking intervals was significantly (P<0.05) lower than the free amino group concentration at 48, and 72 hours cross-linking intervals. The fixation index was lowest in tissues crosslinked with EDC for 12 hours and highest in tissues crosslinked with GA for 72 hours.

vi) Determination of free hydroxyproline contents

The standard curve was plotted using different concentrations (5, 10, 15, and 20 [micro]g/ml) of standard cis-4-hydroxy-L-proline to calculate the free hydroxyproline contents in crosslinked samples. The free hydroxyproline contents of native swim bladder, and ASB were 4.581 [+ or -] 0.001 [micro]g/ml and 1.098 [+ or -] 0.020 [micro]g/ml respectively which were significantly (P<0.05) higher compared to crosslinked ASB.

vii) Moisture contents analysis

Mean [+ or -] SE of moisture contents (percentage) of samples are presented in figure 8.0. Moisture contents were significantly (P<0.05) higher in ASB when compared to crosslinked ASB. In GA fixed samples, moisture content at 72 hours was significantly (P<0.05) lower than at 12, and 24 hours. In BDDGE and EDC cross-linked samples, moisture contents at 24, 48, and 72 hours was significantly (P<0.05) lower compared to at 12 hours.

ix) 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 protein bands of native, acellular and cross-linked ASB were visualized in figure 9.0 and 10. The typical native swim bladder collagen pattern is visualized in Lane 1. In the SDS resolving gel, the collagen bands showed molecular weight of about 50 and 85 kDa. Native collagen molecules remained in the stacking gel. After acellularization, the soluble protein decreased as revealed in SDS-PAGE of ASB matrix (Lane 2). All the GA fixed ASB did not show any band pattern in SDS-PAGE gel (Lane 3-6). BDDGE-12 treated ASB showed lower molecular protein band (Lane 7). BDDGE-24, BDDGE 48 and BDDGE-72 treated ASB did not show any band pattern in SDS-PAGE gel. The EDC fixed ASB did not show any band pattern in SDS-PAGE gel.

Discussion

The goal of an acellularization is to efficiently remove all cellular and nuclear material while minimizing any adverse effect on the composition, biological activity, and mechanical integrity of the ECM [20]. The present investigation analyzed the effects of an ionic biological detergent on swim bladder of fresh water fish (Labeo rohita). Ionic biological detergents are effective for solubilizing both cytoplasmic and nuclear cellular membranes [21]. Ionic biological detergents are used extensively in acellularization protocols due to their mild effects on tissue structure. These surfactants disrupt lipid--protein and lipid--lipid interactions, but generally leave protein--protein reactions intact with the result of maintaining their functional conformations [20]. Ionic detergents are very effective for removing cellular remnants of native tissues [20]. The cell extraction was effectively achieved without significant disturbances in ECM morphology and strength. Results of this study also supports that ionic detergent is viable option for acellularization of the fish swim bladder.

GA crosslinking of collagenous tissues significantly reduced the biodegradation, made them biocompatible and preserved anatomic integrity and flexibility [22]. GA treated tissues were stiff in consistency when compared to the control. In GA, there are only carbon bonds (C-C), which are known to be relatively inflexible. Therefore, the GA treated tissue is comparatively stiffer [15,23]. As the crosslinking time increased the tissue became stiffer and yellowish in colour. Upon fixation, GA degrades to glutaric acid, which is light yellowish in colour. This might be responsible for yellow colour of the GA fixed tissues. After proper fixation of tissue the colour and the tensile strength of the biomaterial had changed. The tensile strength had increased and the graft was found ideal to hold and replace the damaged tissue. The contact time of 24 hours was suggested for biological fixation by GA [15]. In this study, ASB was crosslinked for four different time intervals ranging between 12 and 72 hours. The ASB on treatment for 24, 48, and 72 hours with different chemicals became stiff and hard in comparison to those treated for 12 hours. GA treated ASB was swollen and hard, which further increased with the increase in contact time with chemical. As the cross-linking time interval increased, the GA treated ASB became more yellowish as compared to 12 hours treated ASB. BDDGE treated ASB did not show any change in colouration but were more swollen, pliable and soft in consistency than acellular uncrosslinked tissue. The EDC treated ASB did not show any change in colouration but were soft in consistency than that of control. Apparently significant difference was observed in physical nature of ASB after treatment with different chemicals at 24, 48, and 72 hours time intervals. Crosslinking time of 48 hours has been found ideal for crosslinking the biomaterials [24,25].

In-vitro biodegradation and mechanical testing demonstrated that collagen treated with GA for 4 hours had significantly increased both resistant to collagenase and mechanical strength as compared to untreated control [26]. Increase in the crosslinking time caused the surface erosion of the graft, attributable to the inability of penetration of degrading solution in the matrix, which would be able to retain the strength for a longer period during degradation [11]. In this study, in-vitro nonenzymatic degradation test showed that the rate of weight loss of crosslinked tissues were significantly (P<0.05) lower than control. The ASB treated with GA showed maximum resistant towards non-enzymatic degradation. Silva et al. [27] assessed the in-vitro non-enzymatic degradation of chitosan membranes after crosslinking by GA in 30 ml of isotonic saline solution for 7, 14, 30, and 60 day tests period. The mechanical resistance to stretching decreased sharply when swollen in an aqueous environment, mainly due to the high hydration equilibrium degree of chitosan materials. However, neutralized chitosan membranes and membrane crosslinked with small amounts of GA became more flexible.

In vitro collagenase enzymatic degradation study revealed that crosslinked tissues were less prone to enzymatic degradation as compared to control. This resistance may be due to the inhibition of enzyme substrate interaction via the hidden or altered cleavage sites of the collagen by crosslinking agents [28]. In the present study, weight loss of both crosslinked and uncrosslinked tissues increased after exposing to bacterial collagenase with increased time intervals. Similar observations were made by Olde Daminik et al. [29] with sheep dermal collagen and Dewangan et al. [30] with porcine bladder matrix. Among the crosslinked tissues, the ASB crosslinked with GA showed less weight loss when compared to BDDGE and EDC crosslinked tissues. GA reacts with the e-amino group of lysyl residues in proteins (e.g., collagen), which induces formation of interchain crosslinks and stabilizes tissues against chemical and enzymatic degradation depending on the extent of crosslinking [23,31]. BDDGE crosslinking involves the reaction of amine groups of lysine or hydroxylysine residues present in the collagen with epoxide groups of BDDGE molecules, resulting in formation of secondary amines. In this study, BDDGE crosslinked ASB showed less weight loss when compared to EDC crosslinked tissues and more weight loss when compared to GA crosslinked tissues. The reaction of collagen with EDC will generally lead to zero-length crosslinks between or inside the helices, while GA and BDDGE can also form intermicrofibrillar crosslinks. Intra- and interhelical crosslinks will enhance the resistance towards collagenase degradation. EDC crosslinked ASB showed less resistance to enzymatic degradation when compared to GA and BDDGE crosslinked tissues.

The ASB treated with GA, BDDGE, and EDC showed significantly (P<0.05) reduced free protein concentrations as compared to control, at different crosslinking intervals. The crosslinking binds the peptides and forms 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. Similar observations were made by Kumar et al. [24] after treating bubaline acellular small intestinal matrix with GA, BDDGE, and EDC.

The analysis of free amino group contents indirectly indicates the degree of the crosslinking. The amount of free amino group is inversely proportional to the degree of the crosslinking. The free amino group analysis indicated that GA has the greatest ability to cross-link insoluble collagen fibrils. Significantly (P<0.05) lower free amino group concentrations were observed in ASB treated with GA, BDDGE, and EDC as compared to control, at different crosslinking intervals. Similar observations were made by Zeeman et al. [32] after crosslinking of sheep dermal collagen with BDDGE. Lastowka et al. [17] reported that the GA induced crosslinking resulted in the least number of free amines. Perme et al. [25] also reported significant decrease in the free amino group concentrations in bubaline pericardium with increasing crosslinking intervals. Dewangan et al. [30] reported a decrease in the free amino group contents of BDDGE, EDC and GA treated porcine bladder matrix.

The rate of tissue fixation can be determined by monitoring the change in free amino group contents, denaturation temperature and moisture contents of fixed tissues [14]. Fixation index is used to estimate the percentage of amino groups within the tissue reacted with the crosslinking agent. A higher fixation index implies a lower level of free amino groups left in the fixed tissue. The fixation index in present study was in order of GA>BDDGE>EDC in crosslinked tissue samples at 72 hours. The rate of GA fixation was faster than that of epoxy fixation. Sung et al. [15] reported significant increase in fixation index of epoxy-fixed and GA fixed porcine arteries. Higher fixation index was observed in ASB crosslinked with GA for 72 hours indicating less free amino groups in the crosslinked tissue.

The analysis of free hydroxyproline contents indirectly indicates the degree of the crosslinking. Free hydroxyproline contents of native swim bladder were significantly higher when compared to tissues crosslinked with GA, BDDGE, and EDC at all crosslinking intervals. Walter et al. [33] reported significant (P<0.05) decrease in free hydroxyproline contents in human root dentin matrix treated with GA.

The moisture contents of GA, BDDGE, and EDC treated acellular swim bladder were significantly (P<0.05) lower as compared to control. The degree of moisture percentage decreases non-linearly with increasing crosslinking density. Kato and Silver [34] conducted studies on moisture percentage of tissue and observed that crosslinking reduces the equilibrium moisture percent of collagenous matrices. Choi et al. [35] reported that the highly crosslinked sponges with EDC showed lower water uptake. Leach et al. [36] observed that the swelling ratio of the single step neutral cross-linking with ethylene glycol diglycidyl ether was greater than either the single step alkaline or the double step alkaline procedures. Sung et al. [14] reported that the moisture contents of glutaraldehyde crosslinked tissues were significantly lower than the fresh tissue. The crosslinked samples also 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 expelled some water molecules out of the fixed tissue. GA crosslinked ASB revealed lower moisture contents, which indicate that the fixation with aldehydes cause more shrinkage of tissue as compare to other groups, expelling more number of water molecules out of the fixed tissue. In the present study; however, the increase in the cross-linking duration had significantly (P<0.05) decrease the free amino group concentrations, fixation index, moisture contents and free protein contents.

Once the protein is crosslinked in the ASB, it will delay the degradation of transplanted tissue, thereby, providing sufficient line for the host body to replace the damaged tissue. The ASB treated with GA, EDC and BDDGE showed fewer amounts of low molecular weight proteins, which indicated efficient crosslinking. This was evidenced by absence of specified bands in the SDS-PAGE gel. Therefore, the maximum ability to crosslink the tissues was seen with GA. Lastowka et al. [17] reported that 0.5% GA was sufficient to cause crosslinking of protein. BDDGE and EDC have almost equal ability to crosslink the tissues.

Conclusion

In the present study the technique for making ASB was a success as complete acellularity of fish swim bladder was obtained. GA had the greatest ability to crosslink the swim bladder but, they are potentially carcinogenic. In-vitro biodegradation tests demonstrated that ASB crosslinked with BDDGE for 48 hours had significantly increased resistant to bacterial collagenase as compared to EDC treated and untreated control tissues. Therefore, ASB fixed with 1% BDDGE for 48 hours can be used for repair of soft tissue defects.

Acknowledgements

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

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Vineet Kumar (a,b) *, Naveen Kumar (b), Himani Singh (b), A.K. Gangwar (b), Rukmani Dewangan (b), Amit Kumar (b) and R.B. Rai (c)

(a) Department of Veterinary Surgery & Radiology, College of Veterinary Science & Animal Husbandry, Junagadh 362001, Gujarat.

(b) Division of Surgery, (c) Division of Pathology, Indian Veterinary Research Institute, Izatnagar-243122, Uttar Pradesh, India

* Corresponding author: Vineet Kumar, bharadwaj374@gmail.com

Received 8 August 2012; Accepted 16 June 2013; Available online 21 July 2013
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Title Annotation:Original Article
Author:Kumar, Vineet; Kumar, Naveen; Singh, Himani; Gangwar, A.K.; Dewangan, Rukmani; Kumar, Amit; Rai, R.B
Publication:Trends in Biomaterials and Artificial Organs
Article Type:Report
Date:Jul 1, 2013
Words:5361
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