In vivo determination of biocompatibility of bladder acellular matrix in a rabbit model.
Abstract: The present study was carried out for in-vivo biocompatibility testing of cross-linked bladder acellular matrix graft (BAMG) with of 0.6% Glutaraldehyde(GA), 1% Hexamethylene diisocyanate (HMD), 1% 1, 4-butanediol diglycidyl ether (BDDGE) and 1% 1-ethyl-3-(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-vivo biocompatibility testing of above cross-linked bladder acellular matrix grafts was determined by subcutaneous implantation of graft on either side of spine of rabbits. The native bladder, acellular and cross-linked BAMG were implanted in separate rabbit under general anesthesia. These grafts were retrieved back at 15, 30, 60 and 90 post-operative implantation days and were evaluated using parameters like macroscopic and microscopic examination, immunoblotting, lymphocyte proliferation assay and ELISA. Macroscopically, all the retrieved biomaterials were found covered with fibrous connective tissue. However, it was thin at 15 days and later on its density increased with maximum at day 90. By day 90, EDC treated grafts for 24 h and 72 h were completely resorbed while EDC treated 12 and 48 h grafts were partially resorbed; whereas GA, HMD and BDDGE cross-linked biomaterials were partially resorbed. The histopathological examination revealed that the bladder acellular matrix graft treated with GA underwent minimum degradation at day 90 post-implantation, which indicated that degradation was least as compared to other cross-linking agents. The native bladder was found to induce more host inflammatory reaction as compared to its acellular graft. All the cross-linked bladder acellular matrix graft showed similar type of inflammatory reaction as compared to acellular uncross-linked bladder matrix (control), which was confined to the periphery of the graft without infiltration within the graft. The reaction became more intense at day 90 in GA and BDDGE treated BAMG samples. The HMD-12 h and HMD-48 h treated BAMG showed good healing of tissue appeared within the graft which was having less inflammatory exudates by day 90. The HMD-24 h and HMD-72 h treated graft showed heavy infiltration of cellular debris and disintegrated collagen scaffold in time dependent manner. The EDC-24 h and EDC-72 h treated BAMG showed host reaction, confined to the periphery around graft upto 60 days post-implantation, but at day 90 the graft was completely reabsorbed. At different cross-linking time intervals the host graft reaction of BDDGE treated graft was similar. The immunoblotting revealed that HMD and BDDGE cross-linked graft showed humoral response. In lymphocyte proliferation assay, the minimum SI was recorded in the tissues cross-linked with GA-48 h which indicated that GA cross-linked tissue had least ability to trigger CMI response in host and the maximum SI was seen in HMD-48 h treated samples. The GA and EDC cross-linked BAMG showed minimum SI value as compared to other cross-linked samples. ELISA revealed less immune response towards the EDC cross-linked grafts.
Article Type: Report
Subject: Biocompatibility (Research)
Homografts (Research)
Authors: Dewangan, Rukmani
Sharma, A.K.
Kumar, Naveen
Maiti, S.K.
Singh, Himani
Kumar, Amit
Shrivastava, Sameer
Sonal
Singh, Rajendra
Pub Date: 01/01/2012
Publication: Name: Trends in Biomaterials and Artificial Organs Publisher: Society for Biomaterials and Artificial Organs Audience: Academic Format: Magazine/Journal Subject: Health Copyright: COPYRIGHT 2012 Society for Biomaterials and Artificial Organs ISSN: 0971-1198
Issue: Date: Jan, 2012 Source Volume: 26 Source Issue: 1
Topic: Event Code: 310 Science & research
Geographic: Geographic Scope: India Geographic Code: 9INDI India
Accession Number: 304842703
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. A variety of scaffold materials are available, each with different physical properties and each associated with a specific and unique host response when implanted in mammalian hosts. Collagen-based biomaterials derived from natural tissue have been used extensively as implants to help alleviate the chronic shortage of autologous grafting 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. It was hypothesized that cell extraction from biological tissue may remove their cellular antigens (1,2) as a means for reducing the antigenic response to xenograft materials, extraction removes lipid membranes and membrane-associated antigens as well as soluble proteins. 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 (3). 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.

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. The prepared BAMG were stored in PBS solution containing 1% amikacin at 4[degrees]C until use. The BAMG were cross-linked with of 0.6% Glutaraldehyde(GA), 1% Hexamethylene diisocyanate (HMD), 1% 1,4-butanediol diglycidyl ether (BDDGE) and 1% 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide (EDC) for 12, 24, 48 and 72 h duration at room temperature. In-vivo determination of biocompatibility was done on the basis of the following parameters:

[FIGURE 1a OMITTED]

Subcutaneous implantation of cross-linked grafts

Eighteen (18), clinically healthy adult New Zealand white rabbits of either sex were used for the experiment. Sixteen animals were randomly divided into four groups. These groups comprised of four animals were further divided into four subgroups for 12, 24, 48 and 72 h cross-linking time intervals of tissue samples and in remaining two rabbits, one rabbit was used for control study in which acellular uncross-linked bladder matrix was implanted while in second rabbit native bladder was implanted subcutaneously. Animals were maintained under uniform conditions of feeding, management and environment throughout experimental period. The either side of spine of all the animals was properly clipped, shaved and scrubbed with 5% cetrimide and chlorhexidine solution and painted with povidone iodine solution. Four subcutaneous pouches (2 on either side of the spine) were prepared. The four same cross-linked biomaterials of 10 mm x 20 mm in size were taken and implanted in each pouch. The native bladder, acellular and cross-linked bladder acellular matrix grafts were implanted in separate rabbit. These grafts were retrieved back at 15, 30, 60 and 90 post-operative implantation days. The specimen for histopathological examination was preserved in 10% formalin saline solution. The tissues were processed by routine paraffin embedding technique and the sections were cut at 5 micron thickness. The sections were stained with hematoxylin and eosin by using the method described by Tyrell et al. (4) to evaluate the tissue reaction. The sections were examined for inflammatory reaction around the implant material, degenerative changes of the graft, neovascularization, lymphocytes infiltration and fibroblastic proliferation. Special staining for collagen fibres (Masson's trichrome stain) and elastin fibres (Verhoeff's stain) was also done.

[FIGURE 1b OMITTED]

[FIGURE 1c OMITTED]

[FIGURE 1d OMITTED]

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Immunological studies

a) Immunoblotting

Humoral immune response toward xenogenic bladder acellular matrix graft was studied performing immunoblotting using sera from 15 days post-implanted rabbit and immunoblotting was done as per method described by Talbot et al. (5).

b) Lymphocyte proliferation assay

The cell mediated immune response toward xenogenic bladder acellular matrix graft was studied by lymphocyte proliferation assay as per the method of Kruisbeek and Shevach (6) with some modifications. The OD 570 nm of each well was recorded in a plate reader keeping a reference wave length at 690 nm. The stimulation index (SI) was calculated using the following formula. SI MTT = (OD of stimulated cultures/OD of unstimulated cultures)

Where, SI MTT is the stimulation index determined by MTT assay.

c) ELISA

To evaluate the immunocompatibility of the cross-linked biomaterials ELISA was performed. The test was done as per standard protocol. The values of antibodies titer (absorbance) were expressed in ng/ml.

Statistical analysis

The data was analysed by ANOVA and Student's t-test as per Snedecor and Cochran (7). (1973).

Results

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.

[FIGURE 3 OMITTED]

Subcutaneous implantation of the cross-linked grafts

a) Gross observations

At day 15: All the biomaterials could easily be retrieved from the surrounding host tissue (except HMD cross-linked graft). The grafts were covered with thin white fibrous connective tissue. Infection or pus formation in the vicinity of the implanted biomaterials was not seen in any of the rabbits. The implanted biomaterials at day 15 did not show any significant loss of mass (resorption) when compared with pre-implanted graft.

At day 30 and day 60: The implanted biomaterials were more deeply seated within the fibrous connective tissue. The grafted tissue was difficult to retrieve as compared to day 15 tissue samples. There was slight decrease in the mass of the implanted biomaterials than that of pre-implanted graft.

At day 90: The implanted tissues were quite deeply seated within the host tissue and were difficult to separate from the host tissue. The GA, HMD and BDDGE treated grafts were present deep beneath the fibrous connective tissue and had significantly reduced in size due to resorption. By day 90 EDC treated grafts for 12 h and 48 h were partially resorbed where as 24 h and 72 h treated grafts had completely resorbed.

b) Histopathological observations

The histopathological evaluation of the subcutaneous implanted graft materials was done and a score card was prepared (Fig. 1, a-d). The histopathological picture of the retrieved tissue samples cross-linked with GA, HMD, BDDGE and EDC at 15, 30, 60 and 90 days are depicted in Fig. 2 to Fig.5. Masson's Trichrome and Verhoeff's staining was done for the visualization of collagen and elastin fibres, respectively.

GA treated bladder acellular matrix graft

The histopathological picture of the retrieved tissue samples cross-linked with GA at 15, 30, 60 and 90 days are depicted in Fig 2. On day 15, in GA-12 h graft there was a zone of heavy necrotic cellular debris (lymphocytes, macrophages, few neutrophils) both at the interface and to some extent within the grafted matrix. By day 30, the cellular necrotic mass was slightly reduced at both the places. There was no evidence of invasion of granulation tissue inside the matrix. By day 60, the changes were almost similar to that in day 30. By day 90, the enwrapped healing tissue was found pushing the necrotic debris at the interface and to some extent within the grafted matrix, undergoing moderate resolution. On day 15, the GA-24 h graft showed moderate necrotic debris at the interface and within the graft compared to GA-12 h graft. On day 30, the changes were almost similar to that in day 15. On days 60 and 90, the necrotic cellular debris persisted which resulted into degradation/disruption of scaffold in time depended fashion with scanty presence of healing reaction at the interface. The GA-48 h treated scaffold showed heavy deposition and invasion of necrotic cellular debris with moderate disruption/disintegration of the collagen bundles on day 15. On day 30 to day 90, the changes remained severe, both at the interface and within the graft. The scaffold was more disrupted and disintegrated in time dependent fashion with calcification of collagen bundles. On day 15 and 30, the GA-72 h graft showed severe necrotic debris both at the interface was covered with moderate granulation tissue which invaded the graft at the interface. On day 60 the inflammatory exudate was mild; the scaffold was moderately disrupted and invaded by the fibroblast. On day 90, the spaces within the collagen bundles were occupied with fibroblast sheets and few immune cells. The similar healing tissue was seen at the interface.

[FIGURE 4 OMITTED]

HMD treated bladder acellular matrix graft

The histopathological picture of the retrieved tissue samples cross-linked with HMD at 15, 30, 60 and 90 days are depicted in Fig 3. On day 15, the HMD-12 h graft showed mild to moderate presence of necrotic cellular debris both at the interface and there was moderate presence of fibroblasts/fibrocytes strip having infiltrated MNCs and eosinophils. By day 30, the inflammatory reaction almost resolved and replaced by more amount of healing tissue (fibroblasts, few fibrocytes and neovessels). By day 60, the granulation tissue became more pronounced with mild to moderate number of MNCs (lymphocytes, plasma cells, few mast cells) and few eosinophils. By day 90, the granulation tissue became more organized and compact around the matrix as well as inside the mildly degraded matrix. On day 15 and 30, HMD-24 h graft showed heavy necrotic cellular debris outside as well as within the graft with moderate amount of healing connective tissue. However, on day 60 and 90, the granulation tissue moderately increased with increased number of fibroblast/neovessels and MNCs. On day 15, HMD-48 h graft showed moderate necrotic inflammatory exudates over and within the mildly disrupted scaffold. By days 30 and 60, the necrotic debris had reduced and replaced with moderate amount of healing tissue at the interface and at places within the graft. By day 90, good healing tissue appeared within the graft which was having less inflammatory exudates. The HMD-72 h graft showed heavy infiltration of cellular debris and disintegrated collagen scaffold in time dependent manner.

[FIGURE 5 OMITTED]

BDDGE treated bladder acellular matrix graft

The histopathological picture of the retrieved tissue samples cross-linked with BDDGE at 15, 30, 60 and 90 days are depicted in Fig 4. The changes of BDDGE-12 h graft, on day 15 and day 30 showed the presence of mild granulation tissue visible focally over the necrosed debris at the interface. However, on day 60, there was heavy necrotic cellular debris (lymphocytes, macrophages) seen at both the places. The necrosed mass at the interface was covered with thick granulation tissue (fibroplasias and neo-vascularization) infiltrated with moderate number of MNCs and few eosinophils. By day 90, the necrosed masses mildly reduced. On day 15 the BDDGE-24 h scaffold mildly disintegrated within the necrotic debris at the interface which was covered with thin layer of connective tissue. On subsequent days the inflammatory exudate became more intense with disruption of scaffold in time dependent fashion. The BDDGE-48 h graft showed severe inflammatory exudate both at the interface and inside the mildly degraded graft on days 15 and 30 with no evidence of healing connective tissue. However, on days 60 and 90, the scaffold was found adhered with thick band of fibrous connective tissue having infiltrated MNCs, which in turn supported by host connective tissue. The necrotic debris was reduced in time dependent manner. The changes in BDDGE-72 h graft on days 15 and 30 were less compared to BDDGE-48 h on the same days. On days 60 and 90, the heavy necrotic debris was covered with thick band of fibrous connective tissue at the interface.

[FIGURE 6 OMITTED]

EDC treated bladder acellular matrix graft

The histopathological picture of the retrieved tissue samples cross-linked with EDC at 15, 30, 60 and 90 days are depicted in Fig 5. On day 15, in EDC-12 h graft there was severe cellular reaction at the interface and between the moderately degraded collagen bundles of the graft. By day 30, the graft was attached with moderate amount of fibrous tissue, having infiltrated polymorphs and MNCs. By day 60, the changes were almost similar with little increase in healing tissue. By day 90, good amount of healing tissue with increase number of fibroblasts/ fibrocytes was seen merged with moderately degraded scaffold. The EDC-24 h graft, on day 15 there was moderate exudate both at interface and within the mild to moderately hyalinized collagen of the scaffold. On day 30 and 60 the scaffold further reduced in mass with more amount of necrotic inflammatory exudate at the interface and the graft with little connective tissue on the interface. By day 90, the graft completely resorbed and could not be collected for histopathological evaluation. On day 15, there was extensive necrotic exudate at both the places with moderate disintegration of EDC-48 h scaffold. By day 30 and 60, the necrotic debris further reduced and the moderately disintegrated graft was covered at places with thick lumps of fibrous connective tissue. Further, by day 90, the graft almost resorbed. The EDC-72 h graft on days 15 and 30; thick necrotic inflammatory exudate was seen at the interface and within the periphery of the graft causing its disintegration. On day 60, the graft was heavily covered with necrotic exudate resulting into disintegration of the graft at the periphery. On day 90, the graft was completely resorbed at the implanted site.

Acellular uncross-linked bladder matrix graft

Acellular uncrossed-linked porcine bladder graft on day 15 showed moderate cellular reaction at the interface and within the mildly degraded graft. The granular tissue infiltrated with MNCs and eosinophils was present as a thick lump at the interface attached with the periphery of the graft. On day 30, the graft was moderately disintegrated and the necrotic mass at the interface was encased in fibrous cellular tissue. On day 60, the graft degraded and covered with thin fibrous connective tissue. On day 90 the inflammation was subsided and degraded graft was surrounded by thick fibrocellular tissue.

Uncross-linked native bladder

On day 15, native porcine bladder showed extensive necrotic reaction at the interface and the periphery of the graft. However, by day 30 the inflammatory exudate was remarkably reduced and found replaced with more thick and mature connective tissue in close association with fascia of the host. On day 60, the moderately disintegrated collagen showed calcification. On day 90, there was moderate fibrinocellular reaction at the interface and the graft was moderately degraded along with calcification.

Immunological studies

a) Immunoblotting

Humoral immune response of the rabbit towards xenogenic bladder acellular matrix graft implant was assessed by performing immunoblotting using sera from 15 days post-implanted rabbit. The SDS-PAGE gel of HMD and BDDGE treated group, which depicted characteristic bands of collagen, was used as a model to assess the antibody binding in the immunoblot. In all the four subgroups viz. 12, 24, 48 and 72 h treated with HMD and BDDGE, sera obtained from rabbits implanted with corresponding bladder acellular matrix grafts was utilized for western blot. The HMD treated graft showed collagen bands corresponding to about 88, 30 and 25 kDa present in immunoblotting, signify that antibodies against these proteins were generated in the host rabbit. These findings for the groups HMD-12, HMD-24, HMD-48 and HMD-72 and acellular (control) group are presented in the fig. Similarly, blot of BDDGE treated graft revealed collagen bands corresponding to about 88, 49, 30 and 25 kDa which signify that antibodies against these proteins were generated in the host rabbit. These findings for the graft cross-linked with BDDGE for different time intervals along with acellular group (control) are presented in the fig.

b) Lymphocyte proliferation assay

The cell mediated immune response towards xenogenic cross-linked bladder acellular matrix graft and acellular uncross-linked bladder as control with and without Con A, cultured in rabbit lymphocytes was analyzed. When rabbit lymphocytes were stimulated in-vitro using acellular uncross-linked bladder antigen (control), it was observed that the stimulation index (SI) was 3.127 [+ or -] 0.005, which was found to highest in comparison to either native tissue samples or different cross-linked BAMG samples under study (Fig. 6). The SI of lymphocytes in response to GA cross-linked BAMG was analyzed. The SI was minimum (0.939 [+ or -] 0.000) in GA-48 samples and maximum (1.740 [+ or -] 0.003) in GA-12 samples. The SI of tissue treated with GA for both 12 h and 72 h showed a higher SI, indicating that treatment upto 48 h is sufficient to cross-link the tissue, thus generating the least CMI in the host. The SI of HMD-12 samples was significant (P<0.05) lower in comparison to other HMD cross-linked samples, whereas SI of HMD-48 was significantly (P<0.05) higher than control group. A significantly (P<0.05) lower SI was recorded in the acellular bladder matrix cross-linked with BDDGE for 72 h and maximum SI was seen in BDDGE-24 samples. The SI of EDC-72 samples was significantly (P<0.05) lower than all other EDC cross-linked samples. The SI recorded in all the EDC cross-linked samples was significantly (P<0.05) lower than control group. These SI values were significantly (P<0.05) different from control values recorded for each cross-linked sample. The values of SI recorded for all the cross-linked samples were significantly (P<0.05) lower in comparison to the values of uncross-linked samples (control) except for HMD-48 samples. Therefore, the minimum SI was recorded in the tissues cross-linked with GA-48 h and the maximum SI was seen in HMD-48 h treated samples at all the cross-linking time intervals.

c) ELISA

The immunoreactivity of the graft material was assessed by ELISA. The serum samples were collected before implantation and on day 15, 30, 60 and 90 post-implantation. These samples were examined for the extent of antibody generated towards the graft component. A fixed dilution of antibody of 1:100 was used throughout the experiment. The absorbance was increased on day 15 when compared to pre-implanted values and thereafter significantly (P<0.05) decreased with increased time intervals from day 15 to day 90 post-implantation. Pre-implanted serum was taken as basal values (0.403 [+ or -] 0.003 ng/ml) to compare the magnitude of response. In serum of rabbits received the GA cross-linked bladder acellular matrix graft at day 15, the value of absorbance were significantly (P<0.05) higher as compared to pre-implanted values. The values showed significant reduction (P<0.05) on day 30. These values further reduced significantly (P<0.05) at day 60 in all the tissues cross-linked with GA for different time intervals. At day 90, values remained significantly (P<0.05) lower as compared to day 60 in GA-12, GA-48 and GA-72 except in GA-24. At day 15 post-implantation, the values of antibody titer of HMD, BDDGE and EDC cross-linked bladder acellular matrix graft samples showed significant (P<0.05) elevation as compared to pre-implanted values. At days 30, 60 and 90 post-implantation, the values of antibody titer significantly (P<0.05) decreased in all the BDDGE cross-linked samples for all cross-linking time intervals. The values at day 90 were comparable to pre-implanted values. The values of all the cross-linked groups were significantly (P<0.05) higher than control (acellular) group at different time interval at day 15, 30 and 90 but remained non-significantly (P>0.05) higher at day 60.

Discussion

Before biomaterials can be applied for its clinical use, the tissue response to these biomaterials had to be evaluated in-vitro and in-vivo. 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 (8). 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 (9) and such decellularisation strategies, designed to limit the immunogenicity of the matrix. Decellularization process may attenuate severe xenogenic immune response (10), but the removal of cellular components may not be sufficient to eliminate inflammation, and fixation techniques may still be necessary to prevent degradation (11). 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 (12). Chemical cross-linking of collagen had been used for several years to improve scaffold stability (13,14). 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 (15). 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 (16, 17, 18). It reduces the solubility, water absorption and biodegradability of the collagen biomaterials and increases its mechanical properties (19). The antigenicity of a collagen biomaterial can be reduced by the process of cross-linking (20). The inflammatory reaction depended both on the site of implantation and the species in which the collagen biomaterial was implanted (21).

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 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 (2). 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. (22) 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.

Subcutaneous implantation of the cross-linked grafts

a) Gross observations

In this study, a uniform layer of white connective tissue was found covering all the implanted biomaterials at day 15 and 30 post-implantation. However, it was dense at day 30. Shoukry et al. (23) also observed similar observations where commercial polyester fabric was used to repair the abdominal hernias and defects in horse. At day 90 the implanted biomaterials were present beneath the fibrous connective tissue and revealed significant loss of graft due resorption. Similarly, Kanade et al. (24) found uniform layer of connective tissue covering the graft material when diaphragm was used as prosthetic materials for the repair of ventral abdominal wall defects in bovines. Deokiouliyar et al. (25) also reported similar findings when glycerol treated pericardium was used for hernioplasty in bovines.

In the present study, EDC treated 12 and 48 h grafts were partially resorbed while 24 and 72 h treated samples showed complete resorption at day 90, whereas GA, HMD and BDDGE cross-linked biomaterials were partially resorbed. The scaffold used for tissue regeneration must provide the necessary support until the new tissue achieved its biological function (26). During tissue regeneration the host cells begin to secrete their own extracellular matrix, the scaffold degrades and is eventually eliminated from the body (27). From this study, it was indicated that cross-linking with certain chemicals resulted into slower degradation of the graft. The size of the subcutaneously implanted biomaterial decreased with the increase in post implantation period, due to degradation. Benghuzzi (28) reported that the rate of degradation of an implant depends heavily on the site chosen for implantation in the organism. Liang et al. (29) observed that fresh and 30% cross-linked acellular tissue with genipin degraded significantly at 1 month postoperatively and completely disappeared by 90th day. In contrast, the 60% and 90% cross-linked acellular tissues were still present at 1 year post-operatively.

b) Microscopic observations

The biocompatibility of cross-linked biomaterials was assessed from the induction of a transitional inflammatory response and the acceptance of these implanted biomaterials. The native bladder was found to induce more host inflammatory reaction as compared to its acellular counterpart which was characterized by infiltration of mononuclear cells consisted of macrophage, lymphocytes, eosinophils and fibroblast which persisted upto 90 days post-implantation. Whereas, all the cross-linked bladder acellular matrix graft showed similar type of inflammatory reaction as compared to acellular uncross-linked bladder matrix (control) which was confined to the periphery of the graft without infiltration of mononuclear cells within the graft, however, the reaction became more intense at day 90 in GA and BDDGE treated BAMG. Removal of cell from graft decreased the antigenicity. It was suggested that an ideal removal method should not compromise graft structure and mechanical properties (30). Acellular bladder showed less host inflammatory reaction as compared to its native counterpart after implantation in rabbit, suggesting the high degree of biocompatibility of the acellular collagen: elastin matrices. Similar results were obtained using the acellular bovine pericardium by Gilberto and Pereira (31). The GA-24 h and GA-48 h treated BAMG showed heavy deposition and invasion of necrotic cellular debris by lymphocytes, macrophages, few neutrophils with moderate disruption/disintegration of the collagen bundles upto day 90. However, the HMD-12 h and HMD-48 h treated BAMG showed mild to moderate presence of necrotic cellular debris both at the interface upto day 60. By day 90, good healing of tissue appeared within the graft which was having less inflammatory exudates. Whereas, HMD-24 h and HMD-72 h treated graft showed heavy infiltration of cellular debris and disintegrated collagen scaffold in time dependent manner. There was only mild to moderate granulation tissue seen at the interface of the graft till day 90. Remnant cell components in xenografts may contribute to, calcification and/or immunogenic reaction (2). In practical, lipids and DNA fragments are likely to play a role in calcification. Calcification of biological tissue had been described as one of the major causes of failure of bioprosthetic heart valves derived from glutaraldehyde treated bovine pericardium or porcine aortic valves (32,33,34). The GA-48 h and native samples showed necrosis with calcification of the graft at 90 and 60 days post-implantation respectively, while no calcification was observed with other cross-linked samples and acellular uncross-linked bladder matrix (control). van Luyn et al. (35) reported that in the implanted collagenous matrices cross-linked with glutaraldehyde, the pathologic calcification may seriously compromise their long-term functionality. Calcification limited the durability of implanted biomaterials as it decreased the function and resulted in early degradation of implants. Similar finding were also observed by Purohit (36), where GA treated rabbit dermis was implanted subcutaneously and the histological examination showed calcification at day 7 post-implantation. The xenograft after implantation was infiltrated with inflammatory cells. It was because of the graft reacted as foreign material and the host immune response occurred. However, native bladder revealed more cellular and immune response due to provoke of the immune response due to presence of the cellular antigen. There are no multinucleated muscle giant cells observed in all the histological cross-linked BAMG samples except the GA-24 h at 15 day intervals. Similar finding were also observed by Liang et al. (29), where fresh pericardia were severely degraded and filled with inflammatory cells. Courtman et al. (2) hypothesized that cell extraction process decreased the antigenic load within the tissue due to the elimination of cellular antigens. However, the EDC-24 h and EDC-72 h treated BAMG showed host reaction, confined to the periphery with formation of densely thick sheet of fibroblast/neo-vessels and inflammatory cells around graft upto 60 days post-implantation and at day 90 the graft was completely resorbed but in other cross-linked BAMG and acellular uncross-linked bladder matrix (control) complete resorption was not seen at day 90. While the EDC-12 h and EDC-48 h treated BAMG revealed severe cellular reaction at host graft junction attached with moderate amount of fibrous tissue, having infiltrated polymorphs and MNCs. By day 60, the necrotic debris further reduced and moderately disintegrated graft was covered at places with thick lumps of fibrous connective tissue. Further, by day 90, the graft was almost resorbed. The host graft reaction of BDDGE treated graft was almost similar with all the grafts cross-linked for different time intervals. The necrotic debris was reduced in time dependent manner and on subsequent days the scaffold was found adhered with thick band of fibrous connective tissue having infiltrated MNCs, which in turn supported by host connective tissue. Similar observations were found by Pieper et al. (27) at week 4, where the implanted matrix was covered by cellular rim composed of fibroblast, macrophages and lymphocytes while native diaphragm showed heavy infiltration of inflammatory cells with necrosis at host graft junction. A cellular rim composed of fibroblasts, macrophages and some lymphocytes and giant cells was present at the outer margins of the matrix. Cellular infiltration had clearly decreased for the cross-linking matrices, with and without attached glycosaminoglycans. Lymphocytes were observed at the implantation site of the collagenous matrices. These cells were able to secrete various mediators which were involved in immunological and inflammatory responses (37).

Immunological studies

a) Immunoblotting

The sera obtained from HMD and BDDGE treated tissue transplanted rabbit reacted with protein bands of collagen showed that antibodies to these polypeptides were generated in the host body in response to graft transplanted rabbit. There was positive reaction in the western blot showing that in case of xenogenic graft, humoral immune responses were generated in host animals. Hudson et al. (38) reported that the acellularity of the nerve graft reduced the antigenicity level nearer to the isograft. Purohit (36) reported that immunoblotting showed humoral response against HMD cross-linked allogenic acellular dermal graft.

b) Lymphocyte proliferation assay

Lymphocyte proliferation assay (LPA) measures the ability of lymphocytes placed in short-term tissue culture to undergo a clonal proliferation when stimulated in-vitro by a foreign molecule (antigen/mitogen). An immune response against non-self and self-antigens is initiated by presentation of the antigen in a suitable form to T cells. Antigen can only be presented to T cells in the context of molecules of MHC (39). Lymphocytes consist of various subpopulations with distinctive functions, which play important roles in immune responses (40). Activation and proliferation of these subpopulations can be achieved by treating them with antigens. In the present study, for decellularization of porcine urinary bladder tissue 0.5% anionic biological detergent for 24 h was used and the antigen prepared from this acelluar tissue (control) showed highest SI in MTT assay. The SI recorded for cross-linked samples was lower in comparison to the values of uncross-linked samples (control). The greater ability of this antigen to stimulate the lymphocytes in-vitro may be attributed to the fact that on treatment with biological detergent, the bonds between protein molecules are broken and results into a change from quaternary and tertiary structure to primary and secondary structures. Whereas, when BAMG was treated with different cross-linkers, there was formation of bonds between protein molecules due to cross-linking which results in less number of bands as evidenced in SDS-PAGE. Therefore, the acellular antigen had greater ability to trigger CMI response in host because of presence of shorter peptide fragments which can be presented to the immune systern by MHC class II pathway and stimulate the CD4 lymphocytes. Whereas, the cross-linked BAMG is not processed in the body to form shorter immunogenic fragments which can elicit the CMI in host. This may also be because of the fact that on cross-linking tissues with different chemicals, the site where biological enzymes act in-vivo, are masked and the cross-linked tissue is no longer broken down into smaller peptide fragments to elicit immune response. The minimum SI was recorded in the tissues cross-linked with GA-48 and the maximum SI was seen in HMD-48 samples at all the cross-linking time intervals. This indicates that GA cross-linked tissue had least ability to trigger CMI response in host. The GA and EDC cross-linked BAMG showed minimum SI, which indicated that GA and EDC had the greatest ability to cross-link the biomaterials as was also evident by absence of specified protein bands in the SDSPAGE gel. Whereas, HMD and BDDGE cross-linked tissues showed higher SI as compared to other cross-linked tissue. In the SDS-PAGE also more number of protein bands were visible and hence indicated less degree of cross-linking of bladder acellular matrix graft in comparison to GA and EDC. Cross-linking of the proteins on treatment with GA and EDC might have masked immunogenic epitopes and therefore, there is either delayed or altogether no cellular immune response (CMI) in host body.

c) ELISA

The immune response to xenogenic transplantation included both natural and induced humoral components. The presence of antibodies to xenogenic collagen was an epiphenomenon and not an indicator for rejection of the implant (41). In the present study, a general trend was observed in the absorbance. The absorbance increased significantly (P<0.05) on day 15 and then decreased on day 30 which followed upto day 90. The pre-implanted serum values were taken as basal values (0.403 [+ or -] 0.003 ng/ml) to compare the magnitude of response and the values of cross-linked BAMG were found to be significantly (P<0.05) higher. The response was slightly higher in native tissue as compared to acellular uncross linked bladder matrix (control). Similar results were also observed with glutaraldehyde treatment study using bovine pericardial tissue, when implanted in rat, exhibited T-cell cytotoxicity and antibody response (42). Singh (43) (2004) reported significant (P<0.05) elevation of absorbance upto day 90 in glutaraldehyde treated diaphragm and pericardium used for abdominal wall reconstruction in rabbits. Wu et al. (2002) also found that xenogenic acellular dermal matrix produced immunogenic and inflammatory reaction at early stage of implantation which decreased gradually and the increased expression of Th2 cytokine might be related to humoral immune response. A significant (P<0.05) decrease in absorbance with the increase in cross-linking time intervals in all the cross-linked groups has been reported. The GA cross-linked skin graft showed more immune response than all other cross-linked groups. However, in the uncross-linked native skin at day 15 and day 60 post-implantation, significant (P<0.05) elevation in the values of antibody titer of the all other cross-linked acellular dermis groups was recorded (36). In present study, EDC-24 h and EDC-72 h cross-linked biomaterials were completely resorbed at 90 days. Due to this, the minimal immune response was observed in EDC cross-linked BAMG biomaterials. The minimal immunological reaction was observed in all the cross-linked BAMG in comparison to the acellular uncross-linked bladder matrix (control) and native bladder. EDC cross-linked grafts showed minimum SI values and ELISA revealed less immune response and were more compatible with better handling qualities than other cross-linked grafts.

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, Amit Kumar, Sameer Shrivastava *, Sonal,* Rajendra Singh **

Division of Surgery, * Division of Animal Biotechnology, ** CADRAD Indian Veterinary Research Institute, Izatnagar 243 122, U.P., India

Received 28 May 2011; Accepted 9 October 2011; Available online 8 February 2012
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