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Efficacy of bladder acellular matrix graft with and without cell seeding for cystoplasty in rabbits.

Introduction

Severe bladder dysfunction induced by disease or surgical intervention can result in chronic urinary incontinence and increased upper urinary tract pressure leading to irreversible kidney damage. Currently, the treatment of choice in these patients is enterocystoplasty with the aim to increase bladder capacity and lower the storage pressure (1). Unfortunately, this procedure fails to restore the emptying function and is associated with many complications such as metabolic disturbance, increased mucus production, urolithiasis, infections and even malignant diseases (2). In view of these disadvantages, alternative substrates have been investigated with only limited success. Tissue engineering or cell culture techniques may offer new treatment options for patients with severe bladder dysfunction (1). Several studies have confirmed feasibility of bladder reconstruction using engineered segments which were formed using biomaterials seeded with autologous cells in vitro (3,4). The choice of a suitable scaffold for cell delivery and /or the ingrowths of bladder wall components are recognized as one of the key factors that determine regenerative capacities and graft function in augmentation cystoplasty. The engineered scaffold must provide optimal structural integrity to withstand in-vivo requirements and appropriate regulation of cell behaviour (5). The homologous and heterologous bladder acellular matrix graft (BAMG) has been demonstrated to serve as a scaffold for the ingrowth of all bladder wall components in rats (6-8) and pigs (9, 10). BAMG is collagen-based non-immunogenic membranes derived from homologous or heterologous tissues. BAMG permits the regeneration of native epithelium acting as a scaffold, allowing cross-healing of the edges and also promoting angiogenesis and growth of smooth muscle bundles (11). Encouraged by the promising results, present study was conducted to test the efficacy of bladder acellular matrix graft with and without cell seeding for cystoplasty in rabbits and to evaluate the process of bladder regeneration.

Materials and Methods

Primary rabbit urinary bladder cell culture (smooth muscle cells)

Rabbit urinary bladder smooth muscle cells were cultured as per the modified method of Atala et al. (12) and Geng et al. (13). Bladder was excised and rinsed in sterile Hank's balanced salt solution (HBSS) (Hi Media Laboratories Pvt. Ltd., India). Under sterile condition, it was dissected and minced into approximately 1 [mm.sup.3] fragments with scissor. Bladder fragments were placed in a 0.25 % solution of collagenase Type-IV-S (Lifecare Pvt. Ltd., India) obtained from Clostridium histolyticum and incubated at 37[degrees]C for 30 min. The digested cell suspension was filtered with muslin cloth in a beaker, and centrifuged at 2,000 rpm. Supernatant was discarded and the cell pellets were washed twice with HBSS. The cell pellets were resuspended in 4:1 mixture of Dulbecco's modified eagle's medium (DMEM) (Hi Media Laboratories Pvt. Ltd., India, AT 067 01) and Ham's F12 nutrient mixture with L-glutamine (Gibco, 11765) containing 100 mg/ml penicillin and 100 mg/ml streptomycin with 10% fetal bovine serum (Sigma Aldrich Co., St Louis). Verification of cell pellets was done by examining aliquots of the final cell suspension microscopically on a hemocytometer. Efficiency of cell recovery and cell viability was determined using the trypan blue exclusion method and cells were counted and then transferred in tissue culture flask. The cells were incubated in a humidified C[O.sub.2] incubator containing 5% C[O.sub.2] and maintained at 37[degrees]C. The cultures were left undisturbed for 1 week to promote its expansion and the media was changed twice a week. At the same time the muscle cell cultures were also examined until 80% confluence was achieved. The expanded smooth muscle cells were trypsinized, washed and collected as a pellet. The cells were then seeded on the BAMG.

Preparation of bladder acellular matrix graft (BAMG)

The urinary bladder of pig was collected from local abattoir in ice cold sterile phosphate buffered saline (PBS, pH 7.4) solution. It was rinsed with PBS 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 2 x 2 [cm.sup.2] pieces and were placed in 0.5% sodium deoxycholate for 24 h under constant agitation. Then, the tissue was washed thoroughly in PBS solution. The prepared BAMG was cross-linked with 1% 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide and stored in PBS solution containing 0.1% amikacin at -20[degrees]C until use.

Seeding of bladder smooth muscle cells on BAMG

Seeding on BAMG was done as per the method of Brown et al. (14). BAMG was cut to fit the wells of 24 well culture plates and soaked in series of 70% ethanol rinse. These grafts were re-hydrated in sterile PBS containing 1% penicillin-streptomycin for 24 h and then equilibrated in DMEM for 48 h prior to seeding at 37[degrees]C in a humidified atmosphere of 5% C[O.sub.2] On the day of seeding, the expanded smooth muscle cells were trypsinized and washed. The cells were seeded on the BAMG at 1x[10.sup.5] to 1x[10.sup.7] cells per [cm.sup.2] and thereafter, 500 [micro]l of DMEM with 10% fetal bovine serum containing 100 mg/ml penicillin and 100 mg/ml streptomycin was carefully pipetted down to the cell-matrix complexes. All culture were incubated at 37[degrees]C in a humidified atmosphere of 5% C[O.sub.2] and allowed to attach for upto 7 days. Then cell-seeded BAMG was used for cystoplasty in rabbit.

Experimental Design

Eighteen clinically healthy adult New Zealand white rabbits of either sex were used in this study. The necessary permission for conducting the experiment had been taken from the Institute Ethical Committee for Animal Experimentation, IVRI. The animals were randomly divided into three groups. Group A (n=6; 1% EDC cross-linked BAMG), Group B (n=6; cell-seeded BAMG) and Group C (n=6; control). General anesthesia was induced with xylazine (5 mg/kg intramuscularly) and ketamine (44 mg/kg intramuscularly). The ventral abdomen was prepared for aseptic surgery. To expose the urinary bladder, ventral midline incision was made. A defect of 10x10 [mm.sup.2] was created in urinary bladder and immediately repaired with or without cell seeded BAMG. The defect in control group was repaired with autograft. In all animals, the grafts were implanted with continuous suturing pattern using 4-0 PGA absorbable suture. The efficacy of BAMG was tested on the basis of the following parameters.

Clinical observations

Feeding pattern and general behavioral changes in all animals were observed daily during the observation period. Rectal temperature was recorded before operation and on days 1, 2, 3, 5, 7 and 15 after the implantation.

Serum urea nitrogen and creatinine assessment

The serum samples were collected before the operation and on days 7, 15, 30 and 90 after the implantation. The level of serum urea nitrogen and serum creatinine was estimated using biochemistry autoanalyzer (Vitros DT chemistry autoanalyzer).

Radiological observations

Positive contrast radiography of urinary bladder was performed using contrast agent (diatrizoate meglumine and diatrizoate sodium-76%) on days 30, 60 and 90 post-implantation to evaluate the healing status. 5 ml contrast agent was infused into bladder using infant feeding tube of size 5 inch and dorso-ventral view radiograph was taken at 47 kvp and 10 mAs.

Ultrasonography

Ultrasonography was performed on days 10 and 45 post-implantation on commercially available scanner with linear/convex 5-MHz transducer. The shape of the reconstructed bladder, wall thickness, repair or grafted site and the presence of calculi or casts, if any were evaluated.

Laparoscopy

Laparoscopic observations were done on days 15 and 60 post-implantation, for the presence of adhesions, shape of the reconstructed bladder, status of the graft (contracture of BAMG) and repair site.

Gross examinations

Three animals from each group were euthanized at day 30 and 90 post-implantation. The reconstructed urinary bladder was grossly examined for the presence of adhesions, status of reconstructed site, fate of BAMG and for the presence of any calculi or any other abnormalities.

Histopathological observations

Representative tissue from the host bladder together with graft was collected at post-implantation day 30 and 90. The tissue was preserved in neutral buffered formalin and processed by routine paraffin embedding technique. The tissue sections of 5 micron thickness were cut and stained with hematoxylin-eosin (H&E). The healing at the cellular level was evaluated as per the method of Ayyildiz et al. (15).

Histochemical observations

Representative tissue from the host bladder together with the graft were obtained at post-implantation day 30 and 90 after euthanizing 3 animals from each group at each time interval. The development of collagen and elastin fibres of the tissue was confirmed by Masson's trichrome and Verhoeff's stain respectively.

Statistical analysis

All the data are presented as mean [+ or -] standard error of mean. The data were analysed by ANOVA and Student's t-test as per Snedecor and Cochran (16). Data were considered statistically significant different if P<0.05.

Results

Primary rabbit bladder cell culture (Smooth muscle cells)

After 4 to 5 days of seeding, the cell reached 60 % confluency and exhibited different morphological pattern like fusiform, triangular and stellate shapes, characteristic of smooth muscle cells (Fig. 1,a). On 7th day post seeding, nuclear fission was evident and cell population achieved about 90 % confluency. In this state, smooth muscle cells displayed an elongated, spindle shaped morphology with a centrally located nucleus and cell borders appeared to be straight and well defined (Fig.1,b-c). The cells became fusiform and cell nodules formed a typical structure of multiple layers with a "hills and valleys" appearance (Fig. 1, d). When viewed in phase contrast and differential interference contrast (DIC) microscopy, 3 dimensional spindle shaped morphology of smooth muscle cells were evident (Fig. 1,e-f). This type of morphology of cells was characteristically seen at about 7 day post seeding.

Microscopic observation of bladder acellular matrix graft

The acellularity of prepared BAMG was confirmed histologically which showed complete acellularity with normal collagen fibres arrangement. The nucleus and cytoplasmic cellular components were successfully removed with no evidence of remaining cells, nuclei or other cell fragments.

Bladder smooth muscle cells seeding on bladder acellular matrix

The pH of the culture medium decreased after 1 to 2 days of seeding, indicative of vigorous cell metabolism and progressive proliferation of the seeded cells. In 4-5 days, growths of smooth muscle cells with spindle shaped morphology were seen at the surface of the BAMG. After this cell seeded BAMG was used for cystoplasty in rabbit.

Clinical observations

Animals of different groups started taking feed and water partially within 24 h of operation, but remained slightly dull for 3 days. Post-operatively, animals of all groups passed blood tinged urine for 2 days. During urination all animals assumed a haunched back posture. All animals survived the surgery without any complications; there were no urinary leakage or extravasation of urine. None of the animals died of causes related to immunogenic reactions or clinical rejection.

The rectal temperature increased significantly (P<0.05) in all groups upto day 3 post-implantation. The peak value was recorded at day 3. Thereafter, significant (P<0.05) decrease was observed from day 5 to day 15 post-implantation. At day 15 values were nearly similar to that of the preoperative values.

Serum urea nitrogen and creatinine assessment

The peak value of SUN was recorded at day 7 in all groups. Thereafter, the values showed a declining trend and returned to physiological range at day 90. The comparison among the different groups showed that SUN values recorded at particular time interval were significantly (P<0.05) different from each other.

The peak value of serum creatinine was recorded at day 7 and the values were significantly (P<0.05) higher up to 15th post-operatively in all groups. Thereafter, the creatinine values did not show any significant difference (P>0.05) from their corresponding pre-operative values.

Radiological observations

The radiological observations of reconstructed urinary bladder of all groups at different time intervals are presented in Fig. 2. On day 30, in groups A and B, the luminal surface was smooth along the entire periphery of the bladder except at the reconstructed site where the wall appeared thickened and hazy. However, in group C (control), the reconstructed site appeared smooth and did not differ from the adjacent bladder wall. On day 60, in all the three groups, the site of anastomosis was no longer distinguishable from the adjacent bladder wall. On day 90, in group A, the reconstructed site of bladder wall visualized as hazy, relatively rough, thickened and uneven surface which had a flat or bulging appearance. Where as, in group B, the reconstructed site was smooth but slightly thicker than the adjacent normal urinary bladder wall. There was no evidence of detachment of the graft in both groups A and B. However, in group C, cystogram showed almost normal shape of urinary bladder.

Ultrasonographic observations

The ultrasonographic observations of reconstructed urinary bladder of all groups at different time intervals are presented in Fig. 3. Ultrasonographic examinations performed at day 10 and 45 post-implantation showed no pathological changes. On day 10, ultasonographic examination revealed uniformly dilated urinary bladder with no evidence of leakage and no evidence of bladder calculus in any of the group. On day 45, in groups A and B, the bladder showed thickened, hyperechoic wall with hyperechoic intraluminal materials without any acoustic shadow i.e. pseudomasses. Whereas, in control group (group C), the bladder wall had similar morphological appearance to its pre-operative state.

Laparoscopic observations

The laparoscopic observations of reconstructed urinary bladder of all groups at different time intervals are presented in Fig. 4. On day 15 and day 60, only minor adhesions to the surrounding tissues were observed in all the animals of different groups. There were no signs of hyperplasia, tumor or scarring at the graft margins in any animal. The bladder displayed normal reservoir in size and configuration. There was no evidence of diverticulum formation or urine leakage.

Gross observations

On day 30, there were fine adhesions on the peritoneal side of the graft between the matrix and to the overlying peritoneum and tissues in all the animals of different groups. The normal saline distended bladders were uniformly dilated with no evidence of leakage. In all the groups, no calculus was observed within the lumen of the urinary bladder. On day 90, in groups A and B, the region of the graft had a slight white colour than normal host tissue. Grossly, the bladders were uniformly dilated with no evidence of diverticular formation; however, there was scarring of the graft centrally.

Histopathological observations

The histopathological observations of reconstructed urinary bladder of all groups at different time intervals are presented in Fig. 5. In group A, at day 30, the cross-linked BAMG was highly necrosed and disintegrated. Adjacent to it, the host bladder at the margins showed denuded lining epithelium, sub-mucosal engorgement and infiltration of mono nuclear cells/lymphoid follicles. In addition, the muscle bundles at the margins showed giant cell formation. At day 90, the graft was covered with uroepithelium from the margins of the host tissue and the sub-mucosa was thinly populated with granulation tissue. The muscle fascicles were arranged irregularly with many giant cells.

In group B, at day 30, the cell-seeded BAMG treated bladder showed re-epithelization at the margins of the host bladder. The underlying sub-mucosa showed hypervascular granulation tissue, where as the fibrocytes were arranged parallel to the serosa. There were focal areas of necrosis with mild cellular infiltration around the regenerating muscle bundles. At day 90, there was complete re-epithelization and laying down of the submucosa and muscle fibers similar to the native bladder.

In group C on day 30, the epithelium lining of urinary bladder was intact and at places hypercellular. Around sutured material there was fibrocellular reaction with presence of lymphoid aggregates. The muscle bundles in the inflamed area were mildly disorganized and surrounded by fibrovascular tissue. By day 90, the bladder lining appeared vesiculated and the sub-mucosal cells were loosely arranged. The muscle bundles were arranged normally as in case of native bladder.

Histochemical observations

The histochemical observations of reconstructed urinary bladder of different groups at different time intervals were presented in Fig. 6. Section stained with Masson's trichrome stain at day 30 showed that the collagen fibres mass and its compactness was more at healing site in group B as compared with groups A and C. By day 90, the thickness of collagen matrix was further increased in group B than group A, whereas in group C the collagen content was less at this stage. Section stained with Verhoeff's stain at day 30 showed more density of elastin fibres in group B than groups A and C which was further increased at day 90.

Discussion

Tissue engineering had been proposed as an alternative approach to generate bladder tissues for reconstruction. Presently, both cell-seeded and unseeded types of tissue engineering production are being tried for reconstruction of the urinary tract (17). Cell-unseeded techniques involve direct in-vivo placement of a cell unseeded biodegradable material that will function as a scaffold to allow the cells that constitute the tissue being replaced to grow inside and form the tissue structure. Cell-seeded techniques need to seed biodegradable materials in-vitro the culturally expanded cells that are harvested from the host tissue and to form the complex patch (18). In the present study, invitro growth of rabbit bladder smooth muscle cells seeded on xenogenic BAMG in order to demonstrate the reaction between bladder smooth muscle cells and BAMG, and the possible use of BAMG as a cell deliverer was evaluated. The smooth muscle cells were applied as the seeding cells on the BAMG. In this study, smooth muscle cells were harvested by collagenase digestion of rabbit urinary bladder. The isolated cells were cultured in-vitro to expand into sufficient number for seeding on xenogenic BAMG (12, 13). Smooth muscle cells were cultured and passaged in routine manner. After 4 to 5 days of post seeding, cells exhibited different morphological pattern like fusiform, triangular and stellate shapes, characteristic of smooth muscle cells. In the confluent state, smooth muscle cells displayed an elongated, spindle shaped morphology with a centrally located nucleus (14). The cells became fusiform and cell nodules formed a typical structure of multiple layers with a "hills and valleys" appearance (13,19). The bladder smooth muscle cells cultured in-vitro were seeded onto BAMG (14) and after 4-5 days, growth of smooth muscle cells with spindle shaped morphology were seen at the surface of the BAMG. The cell seeded graft was used for cystoplasty in rabbit.

The animal started taking feed and water from next day of operation but dullness, depression and partial anorexia observed for first 3 post-operative days were probably due to surgical trauma and stress. Similar findings have also been reported after bladder augumentation in goats (20). The passing of blood tinged urine for the first 2 days could be attributed to surgical trauma inflicted during resection and reconstruction of the urinary bladder. The urine was clear after second day that indicates the satisfactory healing at the host graft junction. During urination almost all the animals of different groups assumed a haunched back posture, which was not an abnormal manifestation during the first 10 days following cystoplasty. The probable reason of such manifestation might be attributed to the diminished tone of bladder in the early post-operative period. BAMG would not have been stretched fully and become functional which resulted in the diminished capacity of the bladder in the initial phase of implantation. In addition, the graft and suture material might have also acted as irritant. No animal died of causes related to immunogenic reactions or clinical rejection. In this study, the decreased antigenicity made the heterologous BAMG transplants feasible without immunosuppression. Similar findings were reported by Piechota et al. (6,8) in rats, Merguerian et al. (9) in pigs and Probst et al. (21) in dogs after cystoplasty using BAMG.

Serum urea nitrogen significantly (P<0.05) increased during the first 15 days in all groups following bladder reconstruction and showed its peak value on day 7 postoperatively, which decreased gradually and returned to pre-operative values by day 30. The probable reason of elevated serum urea nitrogen following cystoplasty might be diffusion of urine from anastomotic site and grafts. The reconstructed bladder might have resulted to incomplete emptying of urinary bladder and initial diffusion of urine along the suture line and its subsequent absorption. This diffusion possibly was more in immediate post-operative day which resulted in peak serum urea nitrogen on day 7. The acellular matrix by virtue of its more intracellular and intercellular space might have resulted in more diffusion of urine into peritoneal cavity and subsequently increased the serum urea nitrogen level. It was further supported by the fact that, as the urinary bladder regained the normal function with the regeneration of host tissue in the grafted areas, the serum urea nitrogen values also came down to the base line level in the animals of all the groups irrespective of grafts used. Increased BUN in goats following cystoplasty with allogenic acellular bladder has been reported (20).

A gradual increase in serum creatinine in the early postoperative days with peaks at day 7 might be the result of impaired bladder function. As the surface area of grafts reduced due to regeneration of uroepithelium and the character of grafts changed due to deposition of fibrin, the serum creatinine levels also decreased and became almost normal by day 30 in all groups. Similar findings have also been reported by earlier workers (20). In the groups A and B, the increase in the serum creatinine values were more and persisted longer than group C which might be attributed to the histological makeup of the acellular graft. The acellular matrix might have resulted in more diffusion of urinary solutes and subsequent higher serum creatinine for longer duration. Creatinine levels have also been recorded within the reference range (4-18 mg/dl) in all the experimental dogs repaired with homologous bladder acellular matrix graft during the study periods (21).

No radiological evidence of such complication was recorded in any animal of different groups. The reconstructed urinary bladders maintained their normal shape and location in all the animals irrespective of the grafts used. On day 30, the reconstructed sites of bladder wall were visualized as hazy, relatively rough, thickened and uneven surface in all the animals of groups A and B. This was probably due to the presence of graft which was at different stages of replacement by the regenerated bladder epithelium. Whereas, in group C, the reconstructed site appeared smooth and not recognized from adjacent bladder wall and indicated good regeneration. Cystography performed at 8 weeks after surgery in rabbit repaired with homologous bladder extracellular matrix graft (BECM) showed bladder in normal size and configuration, without notable difference from native bladder (18). In the present study, radiographic cystogram recorded on day 60 in all the three groups, the anastomotic site was no longer distinguishable from adjacent bladder wall, indicated smooth integration of the grafted site into whole bladder body without diverticular or scarred stricture formation. Bladder was smooth and oval, closely resembling to the normal shape of bladder. Similarly, cystographic appearances were observed by Piechota et al. (22) after 4 months postcystoplasty in rats and Probst et al. (21) at 7 months after BAMG-cystoplasty in dog. On day 90, in group A, the reconstructed site of bladder wall appeared relatively thicker and flat in cystogram suggested that newly formed granulation tissue was not organized with relatively less stretchability. At the same stage of observation, in the animals of group B acellular bladder graft was completely resorbed from the site and subsequently complete epithelialization of the regenerated tissue imparted smooth surface of bladder lumen in the radiograph. But the area appeared thicker than the adjacent bladder wall in radiograph which might be attributed to incomplete organization of the regenerated tissue. There was no evidence of detachment of the graft in both group A and B. However, in group C, there was no marked change in the shape of reconstructed bladder. Cystogram showed uniformity with the normal bladder wall, which was indicative of regeneration of host tissue and its subsequent organization. These findings also suggested the complete detachment/resorption of the graft from the reconstructed site. Similar cystographic finding was observed by Ghosh (20) using autogenous and acellular allogenic urinary bladder and seromuscular layer of rumen for cystoplasty in goats.

In this study, ultrasonographic examinations did not reveal any pathological change. On day 10, ultasonographic examination revealed uniformly dilated bladder with no evidence of leakage in all the three groups. This confirmed the promising outcome of bladder augmentation with xenogenic BAMG with and without cell seeding. On day 45, in group C, the bladder wall had similar morphological appearance to its pre-operative state and bladder wall thickness had increased. There were no signs of calculi formation. Probst et al. (21) reported similar finding after 7 months of urinary bladder augmentation in dogs repaired with homologous BAMG. In this study, at same stage of observation in groups A and B, the bladder showed thickened, hyperechoic wall with hyperechoic intraluminal materials without any acoustic shadow i.e. pseudomasses. This finding suggested irregular shape of reconstructed bladder is related to the contour at the anastomosis of graft to bladder, as well as to the shape of the graft used in the surgery. Pseudomasses when attached to the bladder produced sonographic patterns that differed significantly from that of the adjacent wall. Free-floating collections are usually attributed to mucus. The shape of reconstructed bladder was variable and slightly different from that of normal urinary bladder. Similar findings were also reported by Hertzberg et al. (23).

Minor adhesions to the surrounding tissues were observed in all groups irrespective of observation time intervals. Post-operative adhesion is normal response occurs after surgery. This adhesion is first step in healing process after surgery and is caused by an inflammatory response to tissue damage. All groups showed normal bladder size and configuration at different time interval of observation. There was no urine leakage and no signs of hyperplasia, tumor or scarring at the graft margins. No difference could be detected macroscopically between the regenerated and the normal bladder epithelium by cystoscopy in porcine, where bladder augmentation was done using allogenic acellular dermis (24). Similar findings were reported by Probst et al. (21) at 7 months of cystoscopy in dog where bladder was augmented with homologous bladder acellular matrix graft.

At different stage of gross observation, the reconstructed bladders with different grafts showed mild to moderate adhesions on the peritoneal side of the graft between the matrix and to the overlying peritoneum and tissues in all groups. Post-operative intraperitoneal adhesions following abdominal surgery are a matter of common occurrence. These adhesions are perpetuated by host of associated factors, the important one being the denudation of serosal surface following abdominal surgery. Since the cystectomy and subsequent cystoplasty involved extensive surgical manipulation inside the abdominal cavity, intraperitoneal adhesions were expected. Formation of adhesions between reconstructed bladder and intraperitoneal organs in cystoplasties with different grafts have been reported (6,7,20,21). On day 30, no evidence of urine leakage and no calculus were observed within the lumen of the urinary bladder in all the animals of three groups. Similar findings were reported by Ayyildiz et al. (15) where bladder augmentation was done using porcine acellular collagen matrix in rabbit. Merguerian et al. (9) reported that none of the BAM-augmented bladders had stones within their lumen. In groups A and B, the graft appeared slightly white in colour than normal host tissue. On day 90, grossly the bladders were uniformly dilated with no evidence of diverticular formation; however, there was scarring of the graft centrally. Similar observations were reported by Yang et al. (18) in rabbit bladder repaired with homologous bladder extracellular matrix graft (BECM) and by Merguerian et al. (9) and Brown et al. (10) in pig bladder repaired with bladder acellular matrix allograft.

The key measures of successful bladder reconstruction are determined firstly by collagen content and amount of functional detrusor smooth muscle within the augmented or regenerated bladder (6). The composition and texture of the graft material may be key factors for the extent and quality of regeneration (6,22). Many investigators have demonstrated the regeneration of epithelial cells and smooth muscles of host tissue after cystoplasty with BAMG (6,9,11,14,18). Similarly, the grafted animals in this study showed regeneration of all bladder wall components with BAMG serving as a scaffold in all the animals of different groups.

The excised urinary bladder epithelium regenerates at faster pace which start from adjacent edge of the host bladder epithelium (11). By day 30, in the group A, the cross-linked BAMG was highly necrosed and disintegrated. Adjacent to it, the host bladder at the margins showed denuded lining epithelium, sub-mucosal engorgement and infiltration of MNCs/lymphoid follicles. At the same time of observation in group B, grafted bladder showed re-epithelization at the margins of the host bladder. The underlying sub-mucosa showed hypervascular granulation tissue, where as the fibrocytes were arranged parallel to the serosa. There were focal areas of necrosis with mild cellular infiltration around the regenerating muscle bundles. The presence of inflammatory cells underneath the epithelium suggested the interaction of epithelium and inflammatory cells in order to initiate the process of stimulating bladder wall regeneration (11). However, in group C, the epithelium lining of urinary bladder was intact. The early presence of uro-epithelium and its well organization is considered to be an index of quick regeneration of the host tissue as it also influences differentiation of regeneration smooth muscle (11). A possible explanation for smooth muscle regeneration in the BAMG includes (i) migration of neighbouring mature cells; (ii) de-differentiation, migration and redifferentiation of mature cells; and (iii) infiltration of myofibroblasts with subsequent differentiation into smooth muscle cells (21). In the present study, the central hypercollagenous area in the graft showed incomplete integration of the BAMG segments. These zones were also seen in the earlier states and vanished during completion of the regeneration process.

At day 90, in group A, the graft was covered with uroepithelium and the sub-mucosa was thinly populated with granulation tissue. The muscle fascicles were arranged irregularly with many giant cells. BAMG-augmentation cystoplasty can lead to morphologic and functional regeneration of the bladder, preserving its low-pressure reservoir function. Because BAMG-regenerated bladders exhibit functional innervation that is similar to normal, they can work in coordination with the host bladder components, thus generating adequate intravesical pressure to produce sustained voiding (22). The histologic studies with homologous (rat) and heterologous (hamster, rabbit, dog and pig) BAMG material for augmentation cystoplasty in rats, suggested that bladder regeneration is facilitated by the closest possible structural match of the matrix graft and the host bladder wall interstitium (6,8,22). Porcine acellular collagen matrix was grafted in rabbit bladder and micro calcification of the material, irregular detrusor regeneration and disarrangement of the smooth muscle fibres that migrated in to the material were observed through the bladder wall (15). In this study, the remnant of graft showed foci of calcification. This might be due EDC cross-linked BAMG used for cystoplasty. In groups B and C, there was complete re-epithelization and sub-mucosa and muscle fibers were arranged similar to the native bladder. These all are suggestive of normal and usual progressive healing process of grafted bladder. Yoo et al. (25) using homologous bladder submucosa as graft for bladder augmentation, demonstrated that bladder augmented with graft seeded with urothelial cell showed an increased bladder capacity compared with bladder augmented with cell-free graft. However, Geng et al. (13) in experimental studies observed that acellular matrices with no cells implanted in the bladder contract with time and lead to diminished bladder capacity. Oberpenning et al. (3) reported that beagle dog's bladder was replaced by tissue engineered bladder, in which synthetic material PGA was used as a cell deliverer. Bladder autoaugmentation in rabbit using cell-seeded PGA with autologous smooth muscle cells, 3 months after surgery regenerated smooth muscle bundles were detected. Thus, cell-seeded PGA polymer facilitated smooth muscle regeneration, offered sufficient bladder wall backup (19). The grafted bladder showed normal histological structure consisted of urothelium, submucosa and muscle (3,9,10,17 18). In the present study, the grafted bladder acellular matrix showed evidence of decreased antigenicity, complete luminal epithelialization, angiogenesis and reconstruction of smooth muscle cells. In the literatures, several authors consider that the state of the art graft should be completely eliminated after acting as a backbone to the bladder tissue regeneration. Thus, the present results support the hypothesis that BAMG allow the functional regeneration of bladder substitute and prove to be a viable surgical alternative to bladder augmentation. Although a complete understanding of the mechanisms involved in the normal development and regeneration of the urinary bladder remained to be elucidated, the findings of the present study suggest its important clinical potential in bladder reconstruction.

Abundant deposition of collagen fibres were observed in the animals at day 30 postoperatively which gradually diminished in density due to differentiation and further maturation of regenerated tissue on day 90. The deposition of collagen was more prominent at the junctional site, indicating the extension of collagen fibres from the host towards the graft, which was considered to be normal healing (26). In the present study, the more deposition of collagen fibres mass at healing site in the animals of group B was suggestive of early granulation tissue formation than that of groups A and C. However, workers have observed deposition of new elastin fibres in the repaired arteries (27) and reconstructed urinary bladders with different graft materials (20,26). In the present study, the regenerating elastic fibres were seen in the newly formed regenerated bladder tissue in all animals of the different groups in varying degree on 30th post-operative day. With the passage of time, the elastic fibres were found more matured/dense and placed in a orderly manner in differentiating layers. Bladder reconstructed with different grafts in all the animals showed normal distribution of elastic fibres in host graft tissue which might be attributed to the optimal regeneration of the host tissue over the grafted site.

Conclusion

In conclusion, the present study showed successful invitro seeding of rabbit bladder smooth muscle cells on bladder acellular matrix graft of porcine origin and cell-seeded BAMG was successfully used for cystoplasty in rabbits. The decreased antigenicity made the heterologous BAMG transplants feasible without immunosuppression. No animal died of causes related to immunogenic reactions or clinical rejection. Regeneration of urinary bladder was found faster in cell-seeded BAMG over cross linked BAMG. zzz Acknowledgement

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

Division of Surgery, * Division of Veterinary Biotechnology, ** Centre for Animal Disease Research and Diagnosis, Indian Veterinary Research Institute, Izatnagar 243122, Uttar Pradesh, India

Received 27 July 2012; Accepted 22 January 2013; Available online 8 June 2013
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Title Annotation:Original Article
Author:Dewangan, Rukmani; Sharma, A.K.; Kumar, Naveen; Kumar, Amit; Singh, Himani; Kumar, Vineet; Maiti, S.
Publication:Trends in Biomaterials and Artificial Organs
Article Type:Report
Date:Apr 1, 2013
Words:6688
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