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Development of bioengineered porcine small intestinal submucosal scaffolds for reconstructive surgery.


Small intestinal submucosaL matrix is derived from small intestine of swine. It consist of stratum compactum layer of tunica mucosa, the tunica muscularis mucosa and the tunica submucosa (Badylak, 1993). This biomaterial is mainly composed of collagen, proteoglycan, glycoprotein and glycosaminoglycans (Badylak et al., 1999). It also contain some natural growth factors such as basic fibroblast growth factor (basic FGF) and transforming growth factor beta (TGF-beta). As a biomaterial SIS provides a scaffold to reconstruct and repair the host tissue and it signals surrounding host cells to grow, to form new vessels and to foster cellular differentiation which initiates site specific tissue remodeling (Hodde et al., 2001; Zhang et al, 2003; Campodonico et al, 2004). SIS has good tensile strength (Whitson et al., 1998), non immunogenic (Badylak, 1993), resistant to infection (Badylak et al., 1994) and when used as a xenograft material promotes wound healing by providing a scaffold for tissue ingrowth (Kropp and Cheng, 2000). Early clinical studies showed that SIS is devoid of any significant side effects, including infection or inflammatory complications (Rutner et al., 2003; Jones et al., 2005). But recently it has been reported that some porcine cells remained in the SIS could trigger a potential adverse immune response elicited by cell membrane epitopes, allogenic or xenogenic DNA and damage associated molecular pattern molecules (Gilbert et al, 2006; Lozte et al, 2007; Bianchi et al, 2007).

Adult stem cells are unspecialized cells, which show self-renewal and can selfmaintain for a long time with the potential to commit to a cell lineage with specialized functions. The most investigated adult stem cells are mesenchymal stem cells (MSCs). This cell type holds significant promise for the engineering of musculoskeletal structures. Bone marrow stroma represents the major source of MSCs. These MSCs are characterized by a high proliferation potential and the capability to differentiate into progenitor cells for distinct mesenchymal tissues (Caplan, 1991). MSCs develop into distinct terminal differentiated cells and tissues including bone, cartilage, fat, muscle, tendon, and neural tissue (Ringe et al., 2002). However, even with an increasing number of cell passages, MSCs do not spontaneously differentiate (Pittenger et al.,1999). MSC-based therapies can be autologous (from self) and thus, eliminate the issues of immunorejection and pathogen transmission, or allogeneic for potentially off-the-shelf availability. Regardless of cell source (autologous vs allogeneic), MSCs will likely generate better tissue grafts than artificial materials. In the present study we evaluated the efficacy of decellularization of small intestinal submucosa with an ionic biological detergent followed by in-vitro seeding with rabbit mesenchymal cells (rMSCs) to develop bioengineered matrix which can further used in reconstructive surgery.

Materials and Methods

Collection, isolation and in-vitro culturing of rabbit bone marrow mesenchymal stem cells (r-MSC)

Collection of bone marrow: The area of iliac crest on either side in New Zealand White rabbit was prepared in aseptic manner (Fig.3). The bone marrow aspirate was collected with the help of an 18 G bone marrow biopsy needle from the posterior aspect of iliac crest under ketamine xylazine anesthesia. The biopsy needle was advanced by rotating it slowly until the bony cortex was penetrated.aspect of iliac crest. The stylet of the biopsy needle was removed and 2.5 ml of bone marrow was aspirated into a hypodermic syringe containing 1000 IU units of heparin under negative pressure. The same procedure was followed in the contra lateral bone to collect another 2.5 ml of bone marrow aspirate in the same syringe. Thus a total quantity of 5ml of bone marrow aspirate was collected from a single animal.

Isolation and in-vitro culture of bone marrow The bone marrow sample was diluted in equal amount of Dulbecco's phosphate buffered saline (DPBS) and was layered on density gradient medium (Ficoll-Hypaque) at 2:1 ratio of BM sample and density gradient medium. The sample was subjected to gradient centrifuge at 2000 rpm for 30 minutes and the buffy coat containing mononuclear cells was collected from the interface. The buffy coat was taken in the test tube along with the Modified Eagle's Medium--low glucose (DMEM-LG) containing antibiotic and subjected for centrifugation at 2000 rpm for 10 minutes. Now, the cell pellet was taken and diluted with same media containing antibiotics was again subjected for centrifugation at 2000 rpm for 10 minutes. The cell pellets were re-suspended, counted and plated at 2 x [10.sup.5] cells/[cm.sup.2] in 25[cm.sup.2] culture flasks. The cells were maintained in DMEM-LG containing 10% fetal bovine serum (FBS) and antibiotics (mixture of 100 units/ml of penicillin and 100 units/ml of streptomycin) in an incubator with atmosphere of 5% C[O.sub.2], 21% [O.sub.2], 95% humidity at 37[degrees]C. After 4-5 days of primary culture, the non adherent cells were removed by changing the medium. The media was changed at every 3 days thereafter till it attained 80-90% confluency.

Expansion of r-MSC

Once the cells attained 80-90% confluence, the cells (BMSCs) were passaged several times to increase cell population. For that, old culture media was removed and cells were washed two times with DPBS (without Ca and Mg). Trypsin (0.25%) was added into the flask and was kept in C[O.sub.2] incubator for 5-10 min for detachment of cells. Equal volume of growth media with FBS was added to the flask and was mixed gently with 5 strokes and transferred into a 15ml sterile tube and centrifuged at 2000 rpm for 4 minutes at room temperature, supernatant was completely removed. Then the cell pellet was re-suspended in culture media and subjected for washing and cell suspension were counted. Cells were counted using a hemocytometer and reseeded in flasks with pre-warmed growth medium at approximately 100,000 cells per 75[cm.sup.2] flasks (1500 cells per [cm.sup.2]). Cells were examined regularly for its viability, morphological features and confluence under high power microscope. After adjusting the cell count to 2 x [10.sup.4]/ml, the cells were used for transplantation/seeding with decellularized intestine and cornea.

Preparation of decellularized porcine small intestinal submucosa (SIS)

Porcine small intestine was collected from local abattoir and were cleaned thoroughly by sterile physiological normal saline. The small intestine was cleaned thoroughly. The serosa and muscular layer was removed by scrapping. The small intestine was cut into 1 x 1 [cm.sup.2] pieces and placed in sterile phosphate buffer saline (PBS) containing cocktail of antibiotics (penicillin, streptomycin and amphotericin). The porcine SIS was made acellular by treating with 1% SDS in an orbital shaker for 12 h. The prepared acellular scaffolds were thoroughly washed with PBS solution and placed in 70% ethanol for 12 h. It was again washed thoroughly in PBS and stored at -20[degrees]C in PBS solution containing mixture of antibiotics. The prepared acellular matrix of SIS was subjected for histological and scanning electron microscopic (SEM) examination. For histological examination the matrix was placed in 10% formaldehyde solution and for SEM examination the sample was kept in 2% glutaraldehyde. The decellularized matrix was subjected to histological and scanning electron microscpical examination.

Histological examination

Prepared decellularized matrix was scaffolds were cut into small pieces and fixed in 10% formalin for 48-72 h. The matrices were processed in routine manner and 4 micrometer thick sections were cut and stained with Haematoxylin and Eosin as per the standard procedure. The stained sections were examined for degree of decellularization and arrangement of collagen fibers.

Scanning electron microscopic examination

The tissue matrices were fixed in 2% gluteraldehyde for 24 h. The samples were washed three times with 0.1M cacodylate buffer in 1 M hydrogen chloride (pH 7.4) and then dehydrated in a series of ethanol solutions. The tissues were first kept in 30, 50 and 70% ethanol for 10 minutes each, then in 90 and 100% ethanol for 15 min each. The tissue samples were then dried in a Critical Point Dryer using carbondioxide as the transitional fluid and mounted on aluminium stubs using adhesive silicone tape. A thin layer of gold ion sputtering was done at 7-10 mA and 1-2 kV for 15 min. The specimens were observed at Jeol ion sputter Model JFC 1600 under appropriate acceleration voltage and magnification range of the unit calibrated using colloidal latex particles of 1[micro] for the study.

Seeding of mesenchymal stem cells on decellularized porcine small intestinal submucosa

The decellularized small intestinal submucosal matrices were washed 4-5 times with antibiotic containing Dulbecco's Modified Eagle Medium (DMEM) and were placed in 6 well culture plate. The cells were trypsinized so as to detach the monolayer of cells from the flask. Growth medium i.e. DMEM containing 10% fetal bovine serum was added and mixed properly to get single cell suspension. The cells were seeded on decellularized matrices at the rate of 2 x [10.sup.4] cells/[cm.sup.2]. It was maintained at 37[degrees]C in a humidified atmosphere of 5% C[O.sub.2] in a C[O.sub.2] incubator. The growth media was changed every 48 hours for upto 14 days. The seeded biomatrices were observed and processed for morphological assessment on day 14. The seeded biomaterial was preserved in 10% formalin for histopathological and in 2% gluteraldehyde for scanning electron microscopic examination.


Collection and in-vitro culturing of rabbit mesenchymal stem cells (r-MSC)

Bone marrow was obtained from the iliac crest of adult NZW rabbits and the separated buffy coat was plated in T 25 flask containing media. The cells were maintained in a humidified atmosphere of 5% CO2 and observed daily under phase contrast microscope to assess the viability and proliferation of cells. The non adherent cells were removed by changing the media. Once the cells attained 90% confluence they were passaged on to T75 flask after detaching the monolayer with 0.25% trypsin. The characteristic spindle shape and plastic adherent property showed by the cultured cells indicated the presence of rabbit mesenchymal stem cells (Fig.1).







Macroscopic, histological and SEM examination of scaffold

Macroscopic observation after decellularization revealed soft, spongy and glistening small intestinal submucosa (Fig. 2). It clearly shows transparent thin soft membranous structure (Fig. 3). Histologically native SIS showed the absence of lamina propria, tunica muscularis and loose connective tissue with blood vessels and lymphatics (Fig. 4). SEM examination of scaffold shows smooth surface (Fig. 5). Histologically, after decellularization the SIS lost cellularity and no nuclei were visible only acellular tissue matrix was seen (Fig. 6). SEM examination of scaffold also shows acellularitry and porosity (Fig. 7).

Seeding of r-MSC on decellularized porcine small intestinal submucosa

The confluent P3 monolayer cells were detached using 0.25% trypsin and seeded at the rate of 2 x [10.sup.4] cells/[cm.sup.2] on 1 x 1 [cm.sup.2] sized decellularized scaffold kept in each well of 6 well culture plate. The scaffolds were kept in C[O.sub.2] incubator and media was changed on every 48 hours upto 14 days. Acceptable growth and attachment of cells were observed on 14th day of seeding on the scaffolds by scanning electron microscopy. Histological examination revealed mesenchymal stem cell nest in the decellularized scaffolds (Fig.8) which was clearly visible in SEM examination (Fig.9).


Decellularized tissues and organs have been successfully used as scaffolds in a variety of tissue engineering/regenerative medicine applications and the decellularization methods varied a widely as the tissues and organs of interest. Biologic scaffolds derived from decellularized tissues and organs have been successfully used in both pre-clinical animal studies and in human clinical applications (Wainwright, 1995; Chen et al., 1999; Dellegren et al., 1999; Harper., 2001; Metcalf et al., 2002; Lee, 2002; Badylak, 2004; Kolker et al., 2005). Removal of cells from a tissue or an organ leaves the complex mixture of structural and functional proteins that constitute the extracellular matrix (ECM). Several methods can be used to facilitate decellularization of tissue, included freezing and thaw, direct pressure and agitation and use of biological detergents

Histologically, loss of cellular details and nuclear remnants indicated an effective decellularization of the small intestinal submucosa. The fundamental ultrastructure of small intestinal submucosa appeared to be preserved in scanning electron microscopy. SDS is very effective for removal of cellular components from tissue. Compared to other detergents, SDS yield more complete removal of nuclear remnants and cytoplasmic protein such as vimentin (Woods and Gratzer, 2005). SDS tends to disrupt the native tissue structure and cause a decrease in the glycosaminoglycan (GAG) concentration and a loss of collagen integrity(Gilbert et al., 2006). The mesenchymal stem cell seeded scaffold showed cell nests in histology and spindle shaped cell studded submucosal surface in scanning electron microscopy. Tan et al., (2006) also successfully seeded mesenchymal stem cells on the small intestinal submucosa for use as a vascular graft in rabbit model.





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P. Sangeetha, S.K Maiti, Naveen Kumar *, Kiranjeet Singh, Aswathy Gopinathan, A.R. Ninu, Rashmi, V. Remya, T.B. Sivanarayanan, A Mohsina, P. Tamil Mahan, Divya Mohan

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

Received 26 March 2015; Accepted 7 April 2015; Published online 13 April 2015

# Coresponding author: Dr. Naveen Kumar; E-mail:
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Title Annotation:Original Article
Author:Sangeetha, P.; Maiti, S.K.; Kumar, Naveen; Singh, Kiranjeet; Gopinathan, Aswathy; Ninu, A.R.; Rashmi
Publication:Trends in Biomaterials and Artificial Organs
Article Type:Report
Geographic Code:1USA
Date:Apr 1, 2015
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