Process Development to Prepare an Acellular Matrix from Bovine Omasum Using Biological Detergents and Enzymes.
Wound healing is a fundamental response to tissue injury that ultimately results in restoration of tissue integrity. This response is achieved mainly by the synthesis of the connective tissue matrix . Most wounds can heal naturally, but full-thickness skin wounds greater than 1 cm in diameter in human being needs a skin graft to prevent scar formation, morbidity and cosmetic deformities .
The tissue engineering approach for skin substitutes has relied upon the creation of three-dimensional scaffolds as extracellular matrix (ECM) analogue to guide cell adhesion, growth, and differentiation to form skin- functional and structural tissue . The three-dimensional scaffolds can not only cover wound and provide a physical barrier against external infection as wound dressing, but also can provide support both for dermal fibroblasts and the overlying keratinocytes for skin tissue engineering. A successful tissue scaffold should exhibit appropriate physical and mechanical characteristics and provide an appropriate surface chemistry and nano and microstructures to facilitate cellular attachment, proliferation, and differentiation.
The use of acellular scaffolds of xenogenic origin, ECM resulting after removal of cells, as skin substitute is an acceptable modality for treating dermal wounds. Biological scaffold derived from decellularized tissues are in use as surgical implants and scaffolds for regenerative medicine because extracellular matrix secreted from resident cells of each tissue and organ can provide favourable micro-environment that affects cell migration, proliferation and differentiation [4,6].
"Forestomach matrix" (FM) provides a number of advantages over other scaffolds and is useful in a variety of clinical and therapeutic applications, including wound repair and tissue regeneration . Bovine omasum matrix is selected for this study as histology shows that the lamina propria is unusually dense, whereas the abluminal side of the FM scaffold is structured as an open omasum matrix. The dense layer of ECM from the lamina propria contributed to the increased thickness and strength of FM scaffolds compared to those derived from other organs. This structure makes the FM well suited for encouraging epithelial regeneration on the dense luminal side of the matrix and fibroblast invasion on the less dense abluminal side of the matrix, when used as a medical device for tissue regeneration. These differences serve an important role in epithelial regeneration, as the dense side acts as a barrier to cell migration, while the less dense side does not present a barrier and therefore allows cell invasion. The forestomach matrix scaffolds can be used to promote, stimulate, or increases proliferation of cells near by the scaffold attachment site as well as increases vascularization of a tissue or organ . The relative size and thickness of ECM in tissue offers a solution to generate relatively large format ECM based biomaterial with good performance characteristics .
Materials and Methods
Standard reagents were obtained from Sigma-Aldrich (St. Louis, Missouri, USA) unless otherwise noted.
Preparation of acellular matrix from bovine omasum
Omasum of buffalo was procured from the local abattoir. Immediately, after collection the omasum was be kept in cold physiological saline solution containing 0.02% EDTA and antibiotic (Amikacin @ 1 mg/ml). The tissue was thoroughly rinsed with normal saline before the start of protocol. The maximum time period between retrieval and the initiation of protocol was less than 4 h. Tissue was cut from sides to obtain a flat sheet and was cut into 4x4 [cm.sup.2] size pieces and kept in PBS for 4 h at 4[degrees]C.
Protocols for de-epithelialization and delamination of bovine omasum
After thorough cleaning of the bovine omasum, deepithelialization was done in hypertonic solution 2M NaCl for 6 h. The keratinised epithelium of omasum was easily scrubbed off by blunt surface of BP-handle. The serosal layer was separated mechanically with forceps. Microscopically, native bovine omasum showed keratinised epithelium on mucosal surface. Lamina propria is the luminal portion of the propriasubmucosa, which includes a dense layer of extracellular matrix and serosal layer.
Protocols for decellularization of bovine omasum
The omasum after de-epithelialization and delamination was cut into 4x4[cm.sup.2] size pieces and placed in biological detergent and enzymes to carry out decellurization protocols. They were subjected to microscopic examination at 12, 24, 48, and 72 h intervals to optimize the decellularization protocols. The prepared acellular omasum matrix was stored in PBS solution containing 1mg/ml amikacin at 4[degrees]C until use. Ionic detergent (SDS) , Non-ionic detergents , Zwitterionic detergent  and enzyme (Trypsin)  was used in 0.5% concentration for decellularization of bovine omasum. Treatment with chemical was done upto 72 h and tissues were subjected to continuous agitation at 370C on orbital shaker at the speed of 200 rpm. The solutions were changed at every 12h intervals. Finally the tissues were thoroughly rinsed thrice (2 h each) with sterile PBS on orbital shaker and samples were collected at 12, 24, 48 and 72 h time intervals for evaluation of parameters. Macroscopic and microscopic examination was done at different time intervals.
The parameters for biological evaluation included:
a. Histopathological evaluation
The tissue samples collected at different time intervals during standardization of decellularization protocols were subjected for histological examination. The samples were fixed in 10% formal saline solution, dehydrated in ethanol, cleared in xylene and embedded in paraffin to get 5micron thin sections. The sections were stained with hematoxylin and eosin staining. Masson's trichrome staining was done for assessing collagen fiber arrangement.
b. DNA quantification
The DNA contents analysis before the start of protocols and after completion of protocolswas done as per method described by .
c. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE)
The expression of protein bands was determined by the sodium dodecyl sulphate polyacrylamide gel electrophoresis as per Laemmli .
Gross and Histopathological Observations
Gro ss examination at 6h time interval revealed that the keratinised epithelium was easily scrubbed off by blunt edge of BP handle while the serosal layer was delaminated by applying mechanical assistance and results in complete de-epithelialisation and delamination. The de-epithelialized omasum matrices were subjected to histological examination. The tissue section of 5 micron thickness were cut and stained with haematoxylin and eosin staining. Microscopic examination also revealed complete removal of keratinised epithelium and serosal layer.
Histopathological evaluation showed that the delaminated omasum treated with ionic detergent (0.5% SDS) for 12 h under constant agitation showed 85% to 90% loss of cellularity along with presence of cellular debris are presented in (Fig 1a). Treatment for 24 h showed slight increase in loss of cellularity i.e. more than 90% and cellular debris is presented in (Fig 1b). At 48 h time interval sub mucosal layer was completely acellular. The collagen fibers were compact with moderate porosity than the native tissue. No debris was observed between the spaces of thick collagen fibers are presented in (Fig 1c). At 72 h time interval ECM showed thin, loosely arranged collagen fibres with very high porosity. No cellular debris was evident are presented in (Fig 1d).
The treatment with non-ionic detergent (0.5% Triton X-100) for 12 h under constant agitation showed 30% to 40% loss of cellularity with high cellular content, thick and compact cellular fiber arrangement with least porosity (F ig 2a). At 24 h no further loss of cellularity was observed. High cellular content, thick and compact collagen fiber arrangement with least porosity was observed (Fig 2b). At 48 h time interval loss of cellularity increased up to 60% with moderate cellular debris and porosity (Fig 2c). At 72 h time interval, there was no further decrease in cellularity and porosity was observed. Mild cellular debris and damage in collagen fiber arrangement was observed at this stage (Fig 2d).
The treatment with zwitter-ionic detergent (0.5% Trin-BP) for 12 and 24 h showed no effect on loss of cellularity (Fig 3a & b).
The high cellular contents were noted at these time intervals. At 48 h and 72 h, nearly 80% decellularization with loss of cellular debris and damage in collagen fiber arrangement was observed (Fig 3c & d).
Treatment enzyme with (Trypsin) was inefficient in decellularization during various time intervals. No loss in cellular content with any effect on collagen fibre arrangement was observed at different time intervals (Fig 4a-d).
The DNA from the samples was isolated and concentration was measured using Nanodrop to measure the effectiveness of decellularization method. The quantification was a direct measure to confirm the effectiveness of the processes, since the DNA is present in active nucleus of cell. In the native omasum, abundant cell components and nucleic acids were present. However, after the decellularization, cells and nucleic acids were hardly observed in ECM. The DNA contents (ng/[micro]l) before and after decellularization with ionic detergents and enzyme are presented in table 1. The DNA concentration in native bovine omasum was 82.40 ng/[micro]l. After decellularization the DNA concentration significantly decreased in SDS treated group but no difference was observed in other detergents treated as well as enzyme treated group at 24 and 48 h time intervals.
SDS-PAGE analysis in 10% polyacrylamide gel showed the expression of the proteins of the tissues namely native bovine omasum, decellularised bovine omasum with 0.5% SDS at 48 h interval and its comparison with different biological detergents at same percentage and at 24 h interval. The decellularized bovine omasal laminae showed two protein bands (thick), one of 31 KDa and another of 116 KDa at 24 h and one protein band (thin) of 31 KDa at 48 h are presented in Fig 5a & b. At 48 h, majority of the proteins removed and leaves only the collagen.
The objective of the study was to access the effectiveness of decellularization protocols and to determine an appropriate application time for the treatments. The native bovine omasum was subjected to treatment with hypertonic solution 2M NaCl for 6 h. At 6h time interval the keratinised mucosal layer was easily scrubbed off and serosal layer was separated with slight mechanical assistance. The isolated delaminated bovine omasum was kept in 70% ethanol for sterilization for 4 h and later on washed thoroughly with distilled water for 24-48 h. The goal of decellularization was to efficiently remove all cellular and nuclear materials while minimizing any adverse effect on the composition, biological activity, and mechanical integrity of the native extra cellular matrix . Decellularization can be brought by physical, chemical, and enzymatic methods which leave a material composed of extra cellular matrix (ECM) components. In the present study chemical methods were used to prepare acellular matrix. These acellular matrices retained their natural mechanical properties and promote remodeling by neovascularization and recellularization by the host .
The acellular tissue matrices are biocompatible, slowly degraded upon implantation and are replaced and remodelled by the extracellular matrix proteins synthesized and secreted by in growing host cells, which reduce the inflammatory response . The acellular matrices support the regeneration of tissues with no evidence of immunogenic reaction . However, even after the removal of cells and cell debris the intact extracellular matrix of the acellular tissue may itself elicit an immune response  .
In the present study four different protocols were used to obtain the acellular ECM from the delaminated bovine omasum. The delaminated bovine omasum was subjected to ionic (SDS, 0.5%), non-ionic (0.5%), zwitterionic detergents (0.5%) and enzyme (Trypsin 0.5%) treatment. The time of reaction (12, 24, 48 and 72 h) was optimised to obtain the acellular ECM. The delaminated bovine omasum was subjected to SDS (0.5%) treatment for 24 h became 90% acellular with mildly thick collagen fibers. The cellular debris was seen in between the void spaces of collagen fibers in the samples. Therefore, time intervals were increased to remove this cellular debris. At 48 h, complete acellularity with no cellular debris was observed. No nuclear bodies were seen and the tissue was primarily composed of extracellular matrix. Further increase in time interval to 72 h resulted in distributed and damaged collagen fibers. In the present study desired results were achieved after 48 h of treatment with 0.5% SDS detergent. The propria-submucosa layer was completely acellular. The collagen fibres were thick and arranged in longitudinal and transverse manner as compared to the native tissue. SDS is very effective for removal of cellular components from tissue. Compared to other detergents, SDS yields complete removal of nuclear remnants and cytoplasmic proteins, such as vimentin . Ionic detergents are effective for solubilizing both cytoplasmic and nuclear cellular membranes, but tend to denature proteins by disrupting protein-protein interactions . These surfactants disrupt lipid-protein and lipid-lipid interactions, but generally leave protein-protein reactions intact with the result of maintaining their functional conformations . The cell extraction was effectively achieved without significant disturbances in extracellular matrix morphology and strength. The extraction protocol for decellularization of delaminated bovine reticulum by 0.5% SDS treatment for 48 h has been documented by Hasan et al. . Non-ionic biological detergents (Triton X-100) do not usually denature proteins and interaction between peptides remains intact, therefore considered most suitable to investigate the subunits structure of membranes proteins. SDS differs very much from non-ionic detergent Triton-X 100. It is relatively non-denaturing. It inhibits protein-protein and protein-lipid binding . Zwitterionic detergents exhibits properties of non-ionic and ionic detergents are efficient cell removal with ECM disruption similar to that of Triton-X 100. Treatment with 0.5% Trin-BP for 48 h and 72 h showed damage in collagen fiber arrangement and up to 80% loss of cellularity was observed. The enzymatic decellularization using trypsin cleaved peptide bonds on the C side of Arg and Lys  but not completely decellularized the tissue.
To measure the effectiveness of decellularization method, the DNA from cells of the samples was isolated and the DNA concentration was measured using Nanodrop. This quantification was an indirect measure to confirm the effectiveness of the processes, since the DNA is present in active nucleus of cell. The concentration of native bovine omasum DNA (P<0.01) was 82.40 [+ or -] 1.41 ng/[micro]l. Treatment with SDS (0.5% concentration) showed lowest values 4.30 [+ or -] 0.14 ng/ml of DNA content in bovine omasum. Significant decrease (P<0.01) in DNA contents showed the effectiveness of treatment for decellularization. Significant decrease (P<0.05) in DNA contents was also observed by Poonam (2014)  while preparing acellular cholecyst matrices from bovine and porcine origin. Treatment with SDS showed effective removal of cells. Quantification of residual DNA in animal- derived biological scaffold materials is one of technical specifications for evaluating decellularization process and immunotoxicity risk (Xu et al., 2012) .
SDS-PAGE analysis for native bovine omasum laminae revealed presence of different proteins and after decellularization of it, the majority of the proteins were removed and only two protein bands (thick), one of 31 kDa for 24 h and another of 116 kDa and one protein band (thin) of 31 kDa at 48 h were present. This reveals that the decellularization procedure removed most of the proteins and leaves only the collagen which was the main component of the extracellular matrix. SDS- PAGE analysis of native rat and rabbit skin for tissue proteins revealed two protein bands (one thin and one thick) above 66 kDa .
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Priya Singh (1), A.K. Sharma (1), Naveen Kumar (1) *, P. Tamil Mahan (1), P. Sangeetha (1), Ajit Kumar Singh (1), Deepti Sharma (1), S.K. Maiti (1), Sonal Saxena (2), Sameer Shrivastava (2), K.P. Singh (3)
(1) Division of Surgery, (2) Division of Veterinary Biotechnology, (3) Centre for Animal Disease Research and Diagnosis, Indian Veterinary Research Institute, Izatnagar 243122, Uttarpradesh, India
Received 15 November 2016; Accepted 11 January 2017; Published online 31 December 2017
* Coresponding author: Dr. Naveen Kumar; naveen. ivri 1961 @gmail. com
Caption: Figure 1: a-d: Microphotograph of bovine omasum after treatment with 0.5% of SDS at 12, 24, 48 and 72h (H&E Stain, 100X)
Caption: Figure 2: a-d: Micrograph of bovine omasum after treatment with 0.5% of Triton-X100 at 12, 24, 48 and 72h (H&E Stain, 100X)
Caption: Figure 3: a-d: Micrograph of bovine omasum after treatment with 0.5% of TnB at 12, 24, 48 and 72h (H&E Stain, 100X)
Caption: Figure 4: a-d: Micrograph of bovine omasum after treatment with 0.5% of trypsin at 12, 24, 48 and 72h (H&E Stain, 100X)
Caption: Figure 5: a,b: SDS-PAGE of bovine omasum laminae at 24h and 48h post treatment
Table 1: Mean [+ or -] SD values of DNA content (ng/il) after decellularization Time intervals (h) Groups 12 Native SDS 15.80 [+ or -] 1.98 * 82.40 [+ or -] 1.41 Tritron-X 100 73.40 [+ or -] 1.98 TnB 74.20 [+ or -] 0.99 Trypsin 79.60 [+ or -] 1.13 Time intervals (h) 24 48 Native 12.30 [+ or -] 1.84 * 4.30 [+ or -] 0.14 ** 82.40 [+ or -] 1.41 63.10 [+ or -] 1.56 58.30 [+ or -] 1.70 * 72.10 [+ or -] 0.28 44.90 [+ or -] 2.12 * 77.50 [+ or -] 1.70 77.20 [+ or -] 3.25 * differ significantly (P<0.05) ** differ significantly (P<0.01)
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|Title Annotation:||Original Article|
|Author:||Singh, Priya; Sharma, A.K.; Kumar, Naveen; Mahan, P. Tamil; Sangeetha, P.; Singh, Ajit Kumar; Sharma|
|Publication:||Trends in Biomaterials and Artificial Organs|
|Date:||Jan 1, 2017|
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