Extraction techniques for the decellularization of rat dermal constructs.
The skin losses can occur due to acute trauma, resection of cutaneous malignancies, donor site harvesting, chronic wounds or even surgical interventions. The full thickness injuries are characterized by the complete destruction of epithelial regenerative tissues. Currently, autologous skin grafting is the treatment of choice for full thickness skin injuries. In this process donor site also heal with some scarring and may be very painful. The large wound that can not be corrected by conventional surgical procedures requires substitute of missing tissue to keep the wound free of infection, to reduce pain and ensure early wound healing . In the cases of extensive injuries, donor sites are extremely limited and in such cases alternative life saving approaches are required. This may include the use of bioengineered skin substitutes. The dermal substitutes effectively close the wound and help to guide cells during granulation tissue formation, fibroblast proliferation, angiogenesis and epithelialization . Tissue engineering is a promising strategy for tissue regeneration. The concept of tissue engineering allows in-vitro expansion of isolated cells using cell culture techniques and their transplantations for organ regeneration. A tissue engineered construct is produced by culturing the required cell types on a biocompatible scaffold or extracellular matrix (ECM) . The scaffolds have been developed from synthetic biodegradable polymers, natural polymers and natural matrices derived from decellularised tissue . The natural bio-scaffold have advantages over synthetic material that it mimic natural ECM structure and composition, simulate natural stimulatory effects of ECM on cells and allows the incorporation of growth factors and other matrix proteins to further enhance cell functions. The scaffold allows cell invasion, their proliferation and secretion of own extra cellular matrix for longer duration leading to a complete and natural tissue replacement .
In skin tissue engineering the ideal matrix/scaffold should be able to provide the right biological and physiological environment to ensure homologous distribution of cells and ECM. It should also provide the right size and morphology of the neo-tissue required. It must not induce a toxic or immune response or result in extensive inflammation. It should have low level of disease risk, be slowly biodegradable, support the reconstruction of normal tissue and have similar mechanical and physical properties to the skin that it replaces. The matrix also supplies growth factors and cell signaling molecules that are required by the cells to self organize in to a 3-D functional tissue . Dermal substitute scaffolds promote fibroblast adhesion, growth and infiltration which accelerate and enhance dermal and epidermal regeneration . To accommodate large number of cells the scaffold need to be highly porous with large surface to volume ratio . The rationale for decellularization of xenogenic scaffolds is to remove antigenicity that might trigger a destructive immune response. A decellularized xenogenic tissue may be a viable option as a replacement tissue, as the antigenic cellular material will be removed while preserving the relatively non-immunogenic ECM . Therefore, the present study was undertaken to assess the effect of different decellularization treatments of rat dermis.
Materials and Methods
The present study was conducted in the Biomaterials and Bioengineering Laboratory, Division of Surgery, Indian Veterinary Research Institute, Izatnagar-243 122 (UP).
Preparation of acellular dermal matrix from rat skin
The fresh skin was procured by euthanizing rats. Just after collection, the skin was preserved in cold phosphate buffer saline (PBS) containing antibiotic (0.1% Amikacin). Thereafter, skin was rinsed with PBS to remove the adhered blood. The maximum time period between collection of skin and initiation of tissue processing was less than 4h. After initial washing, the tissue samples were cut into 20 x 20 mm2 pieces and one piece was subjected to normal histological examination.
Protocols for de-epithelialization of rat skin
Epidermis of the skin was removed by placing skin pieces in hypertonic solution. The composition of solution includes 605 mg of tris base, 4 gram of sodium chloride, 202.5 mg of EDTA in 100 ml phosphate buffer saline. The skin pieces were subjected to continuous agitation at 37[degrees]C for 8 h on orbital shaker at the speed of 150 rpm. The solution was changed at every 4h. Macroscopic and microscopic examination was done at 4 and 8h time intervals.
Protocols for decellularization of rat skin after deepithelialization
The cellular dermis was further subjected to different protocols for making the dermis acellular. The decellularization treatment included: 1. Hypertonic solution (605 mg tris base, 4 gram sodium chloride, 202.5 mg EDTA in 100 ml phosphate buffer saline), 2. Triton X 100 (1%) with Tri n-butyl phosphate (0.25%), 3. Sodium dodecyl sulphate (SDS) (0.5%) with Tri n-butyl phosphate (0.25%), 4. Triton X 100 (1%) with Tri n-butyl phosphate (0.25%), 5. Sodium deoxycholate (SD) (1%) and 6. Sodium deoxycholate (SD) (2%).
The dermal tissues were subjected to continuous agitation at 37[degrees]C for 24 and 48h on orbital shaker at the speed of 150 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. Macroscopic and microscopic examination was done at different time intervals.
Normal skin architecture is depicted in figure 1. The rat skin consisted of thick epidermis followed by highly cellular dermal matrix. Masson trichome staining also revealed highly cellular epidermis with abundance of collagen fibres (Fig.2).
De-epithelialization of rat skin
At 4h time interval the epidermis could not be separated from the dermis. At 6h interval the epidermis was separated with more mechanical assistance. However, at 8h the epidermis was separated spontaneously or with minimal mechanical assistance (Fig.3) and resulted in completely de-epithelialized dermis matrix (Fig. 4).
The de-epithelialized dermal 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 epidermis from the skin.
Decellularization of rat skin after de-epithelialization
In the present study five different protocols were used to obtain the acellular dermis from the deepithelialized skin. Treatment with hypertonic saline solution for 24h showed acellularity with moderate thicker collagen fibers. The cellular debris was observed between void spaces (Fig.5). The de-epithelialized skin subjected to Triton X-100 (nonionic detergent) treatment showed acellularity with thick collagen fibers (Fig.6). There was some cellular debris in between the void spaces of collagen fibres. At 48h complete acellularity and thin collagen fibres with increased porosity was observed (Fig.7). Treatment with SDS (ionic detergent) for 24h showed acellularity with thicker collagen fibers (Fig.8). At 48h the collagen fibres were thinner with increased spaces between them (Fig.9). Treatment with 1% SD, an ionic detergent showed effective removal of cellular remnants at 48h (Fig 10). Increase in concentration from 1% to 2% resulted in thinning of collagen fibres with increased porosity (Fig.11). No nuclear bodies were seen and the tissue was primarily composed of extracellular matrix. At 48h all the samples treated with different protocols showed complete acellularity with removal of cellular debris from the tissue.
The objective of the study was to acessed the effectiveness of multiple decellularization protocols and to determine an appropriate application time for the treatments.
Optimization of protocols for de-epithelialization of rat skin
The rat skin was subjected to hypertonic saline for 8h. At 6h interval the epidermis was separated with more mechanical assistance. At 8h, the epidermis was separated spontaneously or with minimal mechanical assistance. Osmotic shock with hypertonic solution was used to lyse the cells within tissues . The chelating agent, EDTA, forms a ring shaped molecular complex that firmly binds and isolates a central metal ion. It has been shown that divalent [Ca.sup.++] and [Mg.sup.++] are necessary for cell attachment to collagen and fibronectin at the Arg-Gly-Asp receptor. By binding the divalent cations that are present at the cell adhesions to the ECM, the EDTA facilitates removal of cellular material from the tissue. Trypsin (0.5%) and 2M sodium chloride have been successfully used for deepithelialization of rabbit skin . Rakhorst et al.  developed a method for de-epithelialization and decellularization of human skin. The epidermis was peeled off from the dermis after overnight incubation in 10X phosphate buffer saline with antibiotic cocktail and EDTA.
Optimization of protocols for de-cellularization of rat skin after de-epithelialization
In the present study five different protocols were used to obtain the acellular dermis from the de-epithelized skin. The de-epithelialized skin was subjected to hypertonic solution, SDS (0.5%), Triton X-100 (1%) and SD (1% and 2%). The de-epithelialized skin subjected to different chemical treatments for 24h became 100% acellular with thick collagen fibers. The cellular debris was seen in between the void spaces of collagen fibers in all the samples. Therefore, time interval was increased to remove this cellular debris. At 48h, samples showed complete acellularity with removal of cellular debris from the tissue. No nuclear bodies were seen and the tissue was primarily composed of extracellular matrix.
The rationale for decellularization of xenogenic scaffolds is to remove antigenicity that might trigger a destructive immune response. Before implantation of xenogenic biological scaffold the tissue antigenicity must be abolished by de-cellularization treatment in order to avoid destructive immune response. Decellularization of donor skin is one method to retain the native collagen configuration and fiber orientation while removing cell components . A decellularization protocol generally begins with lysis of the cell membrane using physical treatments or ionic solutions followed by separation of cellular components from the ECM using enzymatic treatments, solubilization of cytoplasmic and nuclear cellular components using detergents and finally removal of cellular debris from the tissue. These steps can be coupled with mechanical agitation to increase their effectiveness. Decellularization of the skin has been done by various chemical and enzymatic techniques to remove the cellular components.
Following decellularization, allresidual chemicals must be removed to avoid an adverse host tissue response to the chemical . Removal of cells from the tissue leaves the complex mixture of structural and functional proteins that constitute the ECM. Decellularization of tissues can be done by physical methods (agitation, sonication, freezing and thawing), chemical methods (alkaline or acids, ionic, non-ionic and zwitter ionic detergents, Tri (n-butyl) phosphate, hypotonic and hypertonic treatments and chelating agents), enzymatic methods (trypsin) and protease inhibitors . The tissue from which ECM harvested, species of origin, decellularization protocol and method of sterilization affects the composition and ultarstructure of ECM and accordingly affects the host tissue response to the ECM scaffold following implantation. Assessment of de-cellularization of biological scaffolds for tissue engineering has been relied primarily on histological cellularity .
Immersion and agitation method of decellularization for dermis have been described . Crapo et al.  suggested that the denser tissues such as dermis, tendon and trachea required prolonged agitation protocols lasting from days to months. However, in the present study desired results were achieved after 48h of treatment with different biological detergents. Badylak and Gilbert  suggested that the cells and cell products cannot be completely removed from dense tissues like dermis even with the most rigorous processing methods. However, in the present study complete acellularity was observed after 48h of treatment.
Hypotonic/hypertonic treatment has been reported as an effective decellularization agent [9,17]. The Hypertonic saline solution lyses the cells within the tissues by the mechanism of osmotic shock and dissociation of DNA from proteins . It causes cell lysis, but does not generally remove the resultant cellular remnants from the tissues . Tri (n-butyl) phosphate (TBP) is an organic solvent that is used for decellularization of tendon and ligament grafts. Treatment with TBP yielded complete removal of nuclear remnants. It does not affect the tensile strength of the collagen fibers but led to decrease in collagen content . TBP has minimal effect on the mechanical behavior of the ECM.
Triton-X100 is a non ionic detergent and has been used extensively for decellularization of tissues because of their relatively mild effect upon tissue structure. Non-ionic detergents disrupt lipid-lipid and lipid-protein interactions but leave protein-protein interactions intact . Exposure of tissues to Triton-X100 for periods ranging from several hours to 14 days led to complete loss of GAGs and decrease in laminin and fibronectin content of the tissue . Triton-X100 can effectively remove cell residues from thicker tissues.
SDS is an ionic detergent and it solubilize cell membranes and dissociates DNA from proteins. Therefore, it is effective in removing cellular material from the tissues. Sodium dodecyl sulphate is typically more effective for removing cell residues and cytoplasmic protein such as vimectin from tissue compared to other detergents but is also more disruptive to ECM . The removal of ECM proteins and DNA by detergents increases with exposure time . Sodium dodecyl sulphate is more effective than triton-X100 for removing nuclei from the dense tissue. Sodium dodecyl sulphate tends to disrupt the native tissue structure and causes a decrease in the GAG concentration and a loss of collagen integrity . Sodium deoxycholate sulphate (SD) is also an ionic detergent which is very effective for removing cellular remnants. SD was shown not to alter the structural properties of ECM structure  as also observed in the present study, but tends to cause greater disruption to the native tissue architecture so it should be used in mild concentration.
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 work.
[1.] Z. Ruszczak, Effect of collagen matrices on dermal wound healing. Adv. Drug Del. Rev., 55, 1595-1611 (2003).
[2.] H. Shin, S. Jo and A.G. Mikos, Biomimetic materials for tissue engineering. Biomaterials, 24, 4353-4364 (2003).
[3.] B.E. Uygun, A. Soto-Gutierrez, H. Yagi, M. Izamis, M.A. Guzzardi, C. Shulman, Organ reengineering through development of a transplantable recellularized liver graft using decellularized liver matrix. Nat. Med., 16, 814-820 (2010).
[4.] M. Smith, P. Mcfetridge, T. Bodamyali, J.B. Chaudhuri, J.A. Howell, C.R. Stevens and M. Horrocks, Porcine derived collagen as a scaffold for tissue engineering. Trans I. Chem. E., 78(C), 19-24 (2000).
[5.] B.S. Kim and D.J. Mooney, Development of biocompatible synthetic extracellular matrices for tissue engineering. Trends in Biotech., 16, 224 230 (1998).
[6.] V.L. Lamme, R.T. Van Leeuwen, K. Brandsma, J. Van Marie and E. Middlekoop, Higher number of autologous fibroblasts in an artificial dermal substitute improve tissue regeneration and modulate scar tissue formation. J. Path., 190 (5), 595-603 (2000).
[7.] K. Whang, C.K. Thomas, K.E. Nuber and A. Heely, A novel method to fabricate bio-reabsorable scaffolds, Polymer, 36, 837 (1995).
[8.] T.W. Gilbert, T.L. Sellaro and S.F. Badylak, Decellularization of tissues and organs. Biomaterials, 27, 3675-3683 (2006).
[9.] S.L. Dahl, J. Koh, V. Prabhakar and L.E. Niklason, Decellularized native and engineered arterial scaffolds for transplantation. Cell transplant, 12, 659-666 (2003).
[10.] S. Purohit, Biocompatibility testing of acellular dermal grafts in a rabbit model: An in-vitro and In-vivo study. Ph.D. Thesis submitted to Deemed University, I.V.R.I., Izatnagar, Bareilly (UP)-243 122 (2008).
[11.] H.A. Rakhorst, S.J.P. Sluijs, W.M.W. Tra, J.W. Van Neck, G.J.V.M. Van Osch, S.E.R. Hovins, A.W. El Ghalbzouri and S.O.P. Hofer, Fibroblasts accelerate culturing on mucosal substitutes. Tissue Engineering, 12, 2321-2331 (2006).
[12.] D.Y. Lee, H.T. Ahn and K.H. Cho, A new skin equivalent model: dermal substrate that combines deepidermized dermis with fibroblasts populated collagen matrix. J. Dermatol. Sci., 23, 132-137 (2000).
[13.] A. C. Goncalves, L. G. Griffiths, R. V. Anthony and E. C. Orton, Decellularization of bovine pericardium for tissue engineering by targeted removal of xenoantigens. The J. Heart Valve Diseases, 14, 212-217 (2005).
[14.] S.R. Meyer, B. Chiu, T. A. Churchill, L. Zhu, J.R. Lakey and D.B. Ross, Comparison of aortic valve allograft decellularization technique in rats. J. Biomed. Mater. Res. A, 79(2), 254-262 (2006).
[15.] P.M. Crapo, T.W. Gilbert and S.F. Badylak, An overview of tissue and whole organ decellularization processes. Biomaterials, 32, 3233-3243 (2011).
[16.] S.F. Badylak and T.W. Gilbert, Immune response to biologic scaffold materials. Semin Immunol., 20(2), 109-116 (2008).
[17.] B. D. Elder, S. V. Eleswarapu and K. A. Athanasiou, Extraction techniques for the decellularization of tissue engineered articular cartilage constructs. Biomaterials, 30(22), 3749-3756 (2009).
[18.] B. Cox and A. Emili, Tissue subcellular fractionation and protein extraction for use in mass-spectrometry based proteomics, Nat. Protoc., 1(4), 1872-1878 (2006).
[19.] T. Woods and P.F. Gratzer, Effectiveness of three extraction techniques in the development of a decellularized bone-anterior cruciate ligament bone graft. Biomaterials, 26, 7339-7349 (2005).
[20.] A.M. Seddon, P. Curnow and P.J. Booth, Membrane proteins, lipids and detergents: not just a soap opera. Biochem. Biophys. Acta., 1666, 105-117 (2004).
[21.] R.W. Grauss, M.G. Hazekamp, F. Oppenhuizen, C.J. Van Munsteren, A.C. Giltenberger-de Groot and M.C. De Ruiter, Histological evaluation of decellularized porcine aortic valves: matrix changes due to different decellularization methods. Eur. J. cardiothorac. Surg., 27, 566-571 (2005).
[22.] S. Funamoto, K. Nam, T. Kimura, A. Murakoshi, Y. Hashimoto and K. Nibaya, The use of high hydrostatic pressure treatment to decellularize blood vessels. Biomaterials, 31(13), 3590-3595 (2010).
[23.] M. T. Kasimir, E. Rieder, G. Seebacher, G. Silberhumer, E. Wolner and G. Wiegel, Comparison of different decellularization procedures of porcine heart valves. Int. J. Artif. Organs, 26(5), 421-427 (2003).
Naveen Kumar, A.K. Gangwar, A.K. Sharma, Mamta Negi, Sameer Shrivastava *, Dayamon D. Mathew, V. Remya, Sonal*, S.K. Maiti, Kh. Sangeeta Devi, Vineet Kumar, P.W. Ramteke, D.T. Kaarthick, N.P. Kurade **
Division of Surgery, * Division of Animal Biotechnology, ** Division of Pathology, Indian Veterinary Research Institute, Izatnagar 243122, Uttar Pradesh, India
Received 30 January 2013; Accepted 16 June 2013; Available online 21 July 2013
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|Title Annotation:||Original Article|
|Author:||Kumar, Naveen; Gangwar, A.K.; Sharma, A.K.; Negi, Mamta; Shrivastava, Sameer; Mathew, Dayamon D.; Re|
|Publication:||Trends in Biomaterials and Artificial Organs|
|Date:||Jul 1, 2013|
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