sheep colon acellular matrix: immunohistologic, biomechanical, scanning electron microscopic...

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Journal of Bioscience and BioengineeringVOL. xx No. xx, 1e6, 2013

Sheep colon acellular matrix: Immunohistologic, biomechanical, scanningelectron microscopic evaluation and collagen quantification

Abdol-Mohammad Kajbafzadeh,* Ahmad Masoumi, Mohammad Hosseini, Mohammad Amin Borjian,Aram Akbarzadeh, and Mohammad Javad Mohseni

Pediatric Urology Research Center, Section of Tissue Engineering and Stem Cell Therapy, Department of Pediatric Urology, Children’s Hospital Medical Center,Pediatric Center of Excellence, 62 Qarib St, Keshavarz Blvd, Tehran 1419733151, Iran

Received 11 November 2012; accepted 12 July 2013Available online xxx

* CorrespondE-mail add

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Colon decellularization provides three-dimensional biologic scaffold without any cell elements with preservation ofextracellular matrix in order to enable autologous cell seeding for tissue augmentation without any immunologicalresponse. This study was performed to investigate the safety and feasibility of sheep colon decellularization as a firststep of colon tissue engineering. The process of sheep colon decellularization was done in four stages which includedscaffold preparation, histologic examination and microscopic investigations to reveal the remaining cellular deposits,biomechanical evaluation and collagen quantification studies by measurement of hydroxyproline content of normal anddecellularized sheep colon. Decellularized colon scaffold revealed complete cell removal under a light microscope while40,6-diamidino-2-phenylindole, di-hydrochloride (DAPI) staining confirmed no deoxyribonucleic acid (DNA) residues.Decellularized colon displayed preserved ultrastructure, comparable biophysical properties (resistance to unidirectionalstretch forces) and higher hydroxyproline content. The results of biomechanical tests proved that the decellularizedmatrix did not bear any unexpected damages or structural changes which would make it unable to tolerate in vivo forcesand stretches. The microscopic images captured after staining the tissue with Picro-sirius red also showed that thecollagen in extracellular matrix is well preserved; this was confirmed by scanning electron microscopy. This implies thatthe scaffold prepared by this method is suitable for tissue augmentation or transplantation.

� 2013, The Society for Biotechnology, Japan. All rights reserved.

[Key words: Decellularization; Tissue engineering; Sheep; Colon; Extracellular matrix (ECM)]

Tissue engineering employs combination of cell grafting sci-ences, material sciences and engineering (1). Natural extracellularmatrix (ECM) provides a unique compatibility feature which makesit an excellent tissue substitute for surgical operations. It alsoprovides a proper environment for cell renewal partly due tocellecell interaction and also by providing a structural support andextracellular proteins which are needed for cell growth andregeneration. Decellularized ECM contains growth factors whichenhances graft substitution (2).

A vast variety of measures have been undertaken in order toremove cell deposits from extracellular matrix. Predictably, eachmeasure affects ECM structure and the recipient response toengrafted tissue, in its own way. Most decellularization mea-sures include both mechanical and chemical processes. Me-chanical measures mainly aim at cell membrane, while chemicalmeasures focus both on cell membrane integrity and celleECMinteractions (3e6). In order to provide a biomechanically suit-able scaffold and foster efficient reseeding the tissue and organ,the decellularization processes must preserve the structural andultrastructural components, biophysical properties, and somebiochemical components of ECM. The ECM is composed of

ing author. Tel./fax: þ 98 21 66565400.ress: [email protected] (A.-M. Kajbafzadeh).

e see front matter � 2013, The Society for Biotechnology, Japan..org/10.1016/j.jbiosc.2013.07.006

this article in press as: Kajbafzadeh, A.-M., et al., Sheep colon ac evaluation and collagen quantification, J. Biosci. Bioeng., (20

various types of collagen (7) and thus hydroxyproline is a usefulindex of collagen content considering that it makes up to 13.2%of the total collagen material (8).

Decellularization processes have been tried for a vast variety oftissues, Including: heart valves (9e12), blood vessels (13e16), skin(17), nerves (18,19), skeletal muscle (20), tendons (21), ligaments(22), small intestine sub-mucosa (7,23,24), bladder (25,26), andliver (27). There is limited published research on colon decellula-rization which is the focus of this study.

In the present study, sheep colon tissues were undergone achemical process to remove the cell deposits from the extracellularmatrix and the outcome of the process was evaluated using lightand electron microscopy, as well as biomechanical tests. We alsoindirectly determined the amount of tissue collagen by measuringthe amount of hydroxyproline content of tissues through acidhydralization technique (28).

MATERIALS AND METHODS

This studywas performed in four stages including: decellularization and scaffoldpreparation, histologic examination and microscopic trials for detection ofremaining cellular deposits, biomechanical evaluation and collagen quantificationstudies by measurement of hydroxyproline content of normal and decellularizedsheep colon.

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FIG. 1. (A) Normal colon prior to decellularization, (B) initial decellularization process, (C) complete decellularized colon.

FIG. 2. Hematoxylin and Eosin light microscope slide; showing complete acellularizedcolon tissue scaffold with no remaining cell nuclei or debris.

2 KAJBAFZADEH ET AL. J. BIOSCI. BIOENG.,

Scaffold preparation Ten colons from healthy mature sheep of the samegender were chosen, eight colons were used for decellularization and twowere usedfor the control group. Colon tissues were put in a solution of PBS (phosphate-buff-ered saline), penicillin, gentamicin and amphotericin B with pH of 7.4 and trans-ported within 2 h to the lab (Fig. 1).

After removal of visceral fat from the colon, all tissues were put into a mixture ofdistilled water and gentamicin for 24 h, so that the blood remained in the tissuewould be washed out. Solution was maintained at 4�C and was renewed once after12 h. The colons were then considered ready for decellularization process. Fordecellularization, each colon sample was sequentially undergone the following fivetreatments: 4 h in HBSS (Hank’s Buffered Salt Solution) while shaking at (70 rpm);8 h in SDS (sodium dodecyl sulfate) (2%) solution while shaking at (70 rpm); 10 h in1% Triton-X Sigma solution while shaking at (70 rpm); 30 min in tripsin solution(0.5%) at the temperature of 38�C in a tissue incubator. Finally, to wash out theremaining materials and liquids used in the decellularization process from tissues,all colons were soaked in PBS solution for 10min. This final stepwas repeated for tentimes.

Histopathologic studies Biopsy specimens were fixed for 24 h in 10%neutral buffered formalin solution in PBS (pH 7.4). The multiple biopsies fromacellular colon were then washed in distilled water (dH2O), dehydrated in gradedalcohol, embedded in paraffin and sectioned at 5 mm thickness. Tissue slides werestained with Hematoxylin and Eosin (H&E) (Leica, Wetzlar, Germany) and Picro-Sirius Red (Sigma, Poole, UK). From paraffin embeded tissue specimens, 4 mm/5 mmslices were prepared and evaluated by the histopathologic laboratory of CancerInstitute of Tehran University of Medical Sciences. Control normal colon biopsieswere also prepared by the same technique.

Biomechanical tests As shown in the previous studies, mechanicalresistance of a tissue will be modified after decellularization and scaffold prep-aration processes, the extent of which is related to the experimental protocol.Such modifications are influenced by different factors such as local micro-envi-ronment of tissue, decellularization process and mechanical forces on the tissue.A reliable method for estimation of modifications in the mechanical features oftissue is biomechanical testing. So to estimate these modifications followingdecellularization five pieces of eight decellularized colon tissues and five piecesof two control colons were selected and underwent a unidirectional stretch testusing a standard machine (model: Hct 400/25, Zwick/Roell, Ulm, Germany) inthe biomechanical testing center of medical engineering laboratory of AmirkabirUniversity. The resistance of acellular and normal cellular colons under unidi-rectional stretch (before tearing off) was calculated at room temperature(20 � 1�C) and the related force-displacement diagrams were illustrated by thecomputer.

Scanning electron-microscopy To prepare a suitable scaffold, collagen fil-aments need to be kept intact after all the decellularization and scaffold prepara-tion procedures. It is believed that no experimental protocols can keep theextracellular matrices intact. In order to check this prediction; a scanning electronmicroscopy was performed on acellular and normal colons as control group. Biopsyspecimens were fixed in 2% glutaraldehyde in 0.1 M phosphate buffer and left for24 h at 3�C. Following washing with 0.1 M phosphate buffer, they were cut intosegments of approximately 1 cm length. After washing process using PBS, everytissue sample was treated by ethanol gradient in order to be dehydrated. For thispurpose each tissue sample was put in eight ethanol beakers with increasing purityfrom 30% to 100%, each for 1 h. After dehydration process using ethanol gradient,the tissue biopsies were kept under the drying hood for 12 h. Then each sample(including one sample from the control group) was transported to SEM laboratory.In the laboratory acellular and normal specimens were covered with a gold micro-layer and observed by scanning electron-microscope to obtain images withdifferent magnifications. In order to reduce bias rate, all experiments includinghistopathologic examinations, biomechanical tests and SEMs were performed byblinded specialists.

Fluorescence microscope For assessing the nuclear remnants in the tissueswe used fluorescence microscopy by staining the tissue with DAPI. DAPI specificallystains the dsDNA by attaching the A-T rich regions of DNA, with little or no cyto-plasmic labeling (29). After fixation in formalin theywere equilibratedwith PBS. The

Please cite this article in press as: Kajbafzadeh, A.-M., et al., Sheep colon amicroscopic evaluation and collagen quantification, J. Biosci. Bioeng., (20

DAPI staining solution was then added to the coverslip preparations, ensuring thatthe tissues are completely covered by the solution. After incubation for 5 min inDAPI solution, colon specimens were rinsed in PBS several times. Finally, all colonspecimens were viewed by a fluorescence microscope with appropriate filters.

Collagen quantification Hydroxyproline concentration of the native anddecellular colon as an index of collagen content was determined according to apreviously describedmethod (30). Tissue slices of approximatelywere hydrolyzed in5 ml of 6 N HCl at 110�C for 14e16 h, 500 ml ChloramineeT solution,0.14 gChloramineeT (Sigma), 2 ml of distilled water and 8 ml citrate/acetate bufferwere added to each sample and the samples were incubated at room temperaturefor 20 min. One milliliter of Ehrlich’s reagent (Sigma) was then added, and theresulting mixture was incubated at 65�C for 20 min. Absorbance at 546 nm wasmeasured by spectrophotometer (Synergy HT1, Bio-Tek, VT, USA). Results wereexpressed as milligram of hydroxyproline per gram of wet tissue (mg/g wettissue). Statistical analysis of the results was performed using the analysis ofvariance and Duncan tests. Results were expressed as mean � standard deviation.A significant result was assumed if p was less than 0.05.

RESULTS

Histopathologic studies Evaluation of tissue specimens bylight microscopy showed complete cell removal. Light microscopicH&E images depicted that there were no remaining cell nucleifollowing scaffold preparation (Fig. 2). These findings confirmedthat the present protocol can be used for future colondecellularization purposes ensuring that no cell debris willremain after this protocol. Sirius red staining also confirmed that


FIG. 3. Picro-Sirius Red staining (A) before trypsin treatment and (B) after using trypsin.

VOL. xx, 2013 SHEEP COLON ACELLULAR MATRIX 3

the collagenous structure of the acellular scaffold was wellpreserved (Fig. 3).

Biomechanical tests Statistical analysis revealed that thenormal colon tissues went under a mean displacement of 9.70 mmwith a mean force of 6.81 N (Newtons), while the acellular colontissues tolerated a mean force of 7.77 N with a mean displacementof 9.31mm. Thismeans that the acellular matrix can tolerate higherforces before rupture compared with the native tissue. This is anexpectable finding regarding the higher content of collagen inacellular scaffolds as shown by collagen quantification studies. Theforceedisplacement diagram of one of the acellular colons and oneof the normal colons obtained by this method is shown in Fig. 4.

Scanning electron-microscopy Scanning electron-micro-scopic images captured from a decellularized colon tissue samplealso showed complete cell removal with non-damaged matrixscaffold (Figs. 5 and 6). Complete cell removal reduces the rate ofgraft rejection. Intact matrix structure ensures that scaffoldprovides an almost normal resistance to the in-vivo forces;similar to that of a non-manipulated graft. Interestingly, theacellular matrix showed a collagen fiber network more tightlycompacted compared to that of control tissue.

FIG. 4. (A) Force-displacement diagram of the normal cellular colon tissue sample. (B) F


Collagen quantification Native (four replicates) and decellu-larized colon (four replicates) were analyzed for collagen content(Fig. 7). Hydroxyproline content of decellularized tissue(9.31 � 1.09 mg/g) was significantly higher (p < 0.05) compared tothe native tissue (4.83� 0.69 mg/g).

Fluorescence microscope Visualization by fluorescence mi-croscopy proved that decellularized colon tissuewas almost clear ofDNA residues. Images captured by fluorescence microscope areshown in Fig. 8.

DISCUSSION

Tissue transplantation still encounters several undesiredsequelae such as inflammation, tissue distortion, scar formation,calcification and sclerosis, obstruction and graft rejection. All thesereactions seem to be related to foreign antigens of the donor graft.Cellular antigens of allogenic or exogenic tissue grafts are recog-nized as intruders by recipient immune system and an antibodymediated response results in graft rejection. However, the use ofimmune-suppressive drugs and modern surgical methods hasminimized untoward reactions. Another strategy is using sterile

orce-displacement diagram of acellularized colon tissue sample. KN, kilo Newton.


FIG. 5. SEM images captured from control (cellular) colon sample.

FIG. 6. SEM images captured from decellularized colon sample showing complete cell removal and intact scaffold structure.


Please cite this article in press as: Kajbafzadeh, A.-M., et al., Sheep colon acellular matrix: Immunohistologic, biomechanical, scanning electronmicroscopic evaluation and collagen quantification, J. Biosci. Bioeng., (2013), http://dx.doi.org/10.1016/j.jbiosc.2013.07.006

FIG. 7. Hydroxyproline contents of native and decellularized colon tissues. Error barsindicate standard deviation. Data were statistically different (p < 0.05) according to 1-way ANOVA.

VOL. xx, 2013 SHEEP COLON ACELLULAR MATRIX 5

ECM that does not trigger an immune reaction and is mostlypreserved across different species. ECM grafts are usually signifi-cantly tolerated, even by exogenic recipients (27,31e33). ECM hasseveral favorable features that make it suitable for regenerativesurgical operations. ECM also provides mechanical support for cellrenewal through structural support, cellecell interactions andextracellular proteins. Remaining of some growth factors even insterile engrafted ECM provides additional stimuli for cell substi-tution (2).

Protocols which are currently used to prepare an acellularscaffold usually use physical processes, ion solutions and enzymesto remove cell deposits from ECM. Multiple tests have been pro-posed to evaluate the efficacy of decellularization process such asH&E, DAPI (3).

In 2003, a collaborative project between Friedrich-SchillerUniversity in Germany and Industrial University of Swinburne,Melbourne investigated the effect of decellularized matrices inheart valve xenograft transplantation. Main goal of this project wasto examine different decellularization protocols regarding theireffect on transplanted tissue integrity. Impact of decellularizationtime was clearly shown in this project, i.e., by increasing thedecellularization time; graft’s mechanical resistance may decreaseproportionately (34).

In a similar study, inUniversityof Pittsburgh,differentmechanicaland chemical (acid-base solutions, non-ionic detergents, Zwitter-ionic detergents, Tributyl phosphate, enzymes, hypo and hypertonicsolutions and chelating agents) methods were used in tissue decel-lularization protocols and their effect on the decellularized ECMintegrity was compared. They also studied the impact of chemicaldeposit removal (3).

FIG. 8. Fluorescence microscopic images after DAPI staini


In a study from Rice University in 2009 the methods of decellu-larization of bladder, cardiovascular structures, knee joint meniscusand temporo-mandibular joint (TMJ) were reviewed. They empha-sized the limitations in joint cartilage decellularization. In this studya biphasic method using SDS (1%), SDS (2%), Tributyl-phosphate(2%), Triton X-100 (2%) and hypo/hypertonic solutions were tested.Results showed incredible compatibility and non-immunogenicityof grafted matrices (5).

Du et al. evaluated the relation of decellularization and tissueresistance in porcine corneal scaffold. They showed that mere useof SDS (0.5 or 1%) for 24 h removes all stromal cell deposits off thecornea and provides a great resistance and integrity for the tissueafter transplantation (35).

In the present study we used a new protocol to remove thecellular components from extracellular matrix to provide a suit-able biologic scaffold from ship colon. Scanning electron-micro-scope (SEM) images and mechanical tests proved that acellularmatrix did not bear any unexpected damage or change whichwould make it inappropriate for in-vivo forces and stretches. Onthe other hand, microscopic images proved that with applicationof this protocol, no cell deposits can be found after decellulariza-tion process. Histopathologic evaluation proved the absence ofcellular deposits within acellular matrix in all the decellularizedscaffolds. The tensile strength of acellular matrix was highercompared with the native tissue. This might be due to the relativeincrease of collagen in the tissue and the loss of cellular compo-nents during decellularization. The collagen content of an acellulartissue is variable depending on the protocol used to prepare thescaffold. For instance, in five different liver decellularization pro-tocols, the collagen content was decreased in four techniques andincreased in just one protocol (36). The best protocol is the onethat best preserves the biochemical and biophysical properties ofthe tissue.

To the best of our knowledge this is the first protocol describedfor decellularizing colon with complete removal of cell DNA indecellularized colon and preserved biomechanical properties asshown by biomechanical tests, SEM images and collagen quantifi-cation tests.

In conclusion, the natural ECM scaffolds produced by thismethod may represent an innovative platform for large bowelbioengineering. Nevertheless, more studies are needed to sub-stantiate our findings, by grafting acellularized colon matrices tothe experimental animals in order to evaluate recellularization bythe autologous stem cells. However, these preliminary data showedthat the chance of graft rejection would be low, even in the case ofxenografts.

ng of (A) normal colon and (B) decellularized tissue.



References

1. Koh, C. J. and Atala, A.: Tissue engineering, stem cells, and cloning: opportu-nities for regenerative medicine, J. Am. Soc. Nephrol., 15, 1113e1125 (2004).

2. Santucci, R. A. and Barber, T. D.: Resorbable extracellular matrix grafts inurologic reconstruction, Int. Braz. J. Urol., 31, 192e203 (2005).

3. Gilbert, T. W., Sellaro, T. L., and Badylak, S. F.: Decellularization of tissues andorgans, Biomaterials, 27, 3675e3683 (2006).

4. Nakase, Y., Nakamura, T., Kin, S., Nakashima, S., Yoshikawa, T., Kuriu, Y.,Sakakura, C., Yamagishi, H., Hamuro, J., Ikada, Y., Otsuji, E., andHagiwara, A.: Intrathoracic esophageal replacement by in situ tissue-engi-neered esophagus, J. Thorac. Cardiovasc. Surg., 136, 850e859 (2008).

5. Elder, B. D., Eleswarapu, S. V., and Athanasiou, K. A.: Extraction techniquesfor the decellularization of tissue engineered articular cartilage constructs,Biomaterials, 30, 3749e3756 (2009).

6. Piterina, A. V., Callanan, A., Davis, L., Meaney, C., Walsh, M., andMcGloughlin, T. M.: Extracellular matrices as advanced scaffolds for vasculartissue engineering, Biomed. Mater. Eng., 19, 333e348 (2009).

7. Badylak, S. F., Tullius, R., Kokini, K., Shelbourne, K. D., Klootwyk, T.,Voytik, S. L., Kraine, M. R., and Simmons, C.: The use of xenogeneic smallintestinal submucosa as a biomaterial for Achilles tendon repair in a dog model,J. Biomed. Mater. Res., 29, 977e985 (1995).

8. Stegemann, H. and Stalder, K.: Determination of hydroxyproline, Clin. Chim.Acta, 18, 267e273 (1967).

9. Bader, A., Schilling, T., Teebken, O. E., Brandes, G., Herden, T., Steinhoff, G.,and Haverich, A.: Tissue engineering of heart valvesehuman endothelial cellseeding of detergent acellularized porcine valves, Eur. J. Cardiothorac. Surg., 14,279e284 (1998).

10. Booth, C., Korossis, S. A., Wilcox, H. E., Watterson, K. G., Kearney, J. N.,Fisher, J., and Ingham, E.: Tissue engineering of cardiac valve prostheses I:development and histological characterization of an acellular porcine scaffold,J. Heart Valve Dis., 11, 457e462 (2002).

11. Grauss, R. W., Hazekamp, M. G., Oppenhuizen, F., van Munsteren, C. J., Git-tenberger-de Groot, A. C., and DeRuiter, M. C.: Histological evaluation ofdecellularised porcine aortic valves: matrix changes due to different decellu-larisation methods, Eur. J. Cardiothorac. Surg., 27, 566e571 (2005).

12. Kasimir, M. T., Rieder, E., Seebacher, G., Silberhumer, G., Wolner, E.,Weigel, G., and Simon, P.: Comparison of different decellularization pro-cedures of porcine heart valves, Int. J. Artif. Organs, 26, 421e427 (2003).

13. Conklin, B. S., Richter, E. R., Kreutziger, K. L., Zhong, D. S., and Chen, C.:Development and evaluation of a novel decellularized vascular xenograft, Med.Eng. Phys., 24, 173e183 (2002).

14. Dahl, S. L., Koh, J., Prabhakar, V., and Niklason, L.,E.: Decellularized nativeand engineered arterial scaffolds for transplantation, Cell Transplant, 12,659e666 (2003).

15. Schmidt, C. E. and Baier, J. M.: Acellular vascular tissues: natural biomaterialsfor tissue repair and tissue engineering, Biomaterials, 21, 2215e2231 (2000).

16. Uchimura, E., Sawa, Y., Taketani, S., Yamanaka, Y., Hara, M., Matsuda, H.,and Miyake, J.: Novel method of preparing acellular cardiovascular grafts bydecellularization with poly(ethylene glycol), J. Biomed. Mater. Res. A., 67,834e837 (2003).

17. Chen, R. N., Ho, H. O., Tsai, Y. T., and Sheu, M. T.: Process development of anacellular dermal matrix (ADM) for biomedical applications, Biomaterials, 25,2679e2686 (2004).

18. Hudson, T. W., Liu, S. Y., and Schmidt, C. E.: Engineering an improved acellularnerve graft via optimized chemical processing, TissueEng., 10, 1346e1358 (2004).

19. Kim, B. S., Yoo, J. J., and Atala, A.: Peripheral nerve regeneration using acel-lular nerve grafts, J. Biomed. Mater. Res. A, 68, 201e209 (2004).


20. Borschel, G. H., Dennis, R. G., and Kuzon, W. M., Jr.: Contractile skeletalmuscle tissue-engineered on an acellular scaffold, Plast. Reconstr. Surg., 113,595e602 (2004).

21. Cartmell, J. S. and Dunn, M. G.: Effect of chemical treatments on tendoncellularity and mechanical properties, J. Biomed. Mater. Res., 49, 134e140(2000).

22. Woods, T. and Gratzer, P. F.: Effectiveness of three extraction techniques inthe development of a decellularized bone-anterior cruciate ligament-bonegraft, Biomaterials, 26, 7339e7349 (2005).

23. Badylak, S. F., Lantz, G. C., Coffey, A., and Geddes, L. A.: Small intestinalsubmucosa as a large diameter vascular graft in the dog, J. Surg. Res., 47, 74e80(1989).

24. Kropp, B. P., Eppley, B. L., Prevel, C. D., Rippy, M. K., Harruff, R. C.,Badylak, S. F., Adams, M. C., Rink, R. C., and Keating, M. A.: Experimentalassessment of small intestinal submucosa as a bladder wall substitute, Urology,46, 396e400 (1995).

25. Freytes, D. O., Badylak, S. F., Webster, T. J., Geddes, L. A., and Rundell, A. E.:Biaxial strength of multilaminated extracellular matrix scaffolds, Biomaterials,25, 2353e2361 (2004).

26. Gilbert, T. W., Stolz, D. B., Biancaniello, F., Simmons-Byrd, A., andBadylak, S. F.: Production and characterization of ECM powder: implicationsfor tissue engineering applications, Biomaterials, 26, 1431e1435 (2005).

27. Lin, P., Chan, W. C., Badylak, S. F., and Bhatia, S. N.: Assessing porcine liver-derived biomatrix for hepatic tissue engineering, Tissue Eng., 10, 1046e1053(2004).

28. Neuman, R. E. and Logan, M. A.: The determination of hydroxyproline, J. Biol.Chem., 184, 299e306 (1950).

29. Kapuscinski, J.: DAPI: a DNA-specific fluorescent probe, Biotech. Histochem.,70, 220e233 (1995).

30. Woessner, J. F., Jr.: The determination of hydroxyproline in tissue and proteinsamples containing small proportions of this imino acid, Arch. Biochem. Bio-phys., 93, 440e447 (1961).

31. Bernard, M. P., Chu, M. L., Myers, J. C., Ramirez, F., Eikenberry, E. F., andProckop, D. J.: Nucleotide sequences of complementary deoxyribonucleic acidsfor the pro alpha 1 chain of human type I procollagen. Statistical evaluation ofstructures that are conserved during evolution, Biochemistry, 22, 5213e5223(1983).

32. Bernard, M. P., Myers, J. C., Chu, M. L., Ramirez, F., Eikenberry, E. F., andProckop, D. J.: Structure of a cDNA for the pro alpha 2 chain of human type Iprocollagen. Comparison with chick cDNA for pro alpha 2(I) identifies struc-turally conserved features of the protein and the gene, Biochemistry, 22,1139e1145 (1983).

33. Constantinou, C. D. and Jimenez, S. A.: Structure of cDNAs encoding the triple-helical domain of murine alpha 2 (VI) collagen chain and comparison to humanand chick homologues. Use of polymerase chain reaction and partiallydegenerate oligonucleotide for generation of novel cDNA clones, Matrix, 11,1e9 (1991).

34. Schenke-Layland, K., Vasilevski, O., Opitz, F., Konig, K., Riemann, I.,Halbhuber, K. J., Wahlers, T., and Stock, U. A.: Impact of decellularization ofxenogeneic tissue on extracellular matrix integrity for tissue engineering ofheart valves, J. Struct. Biol., 143, 201e208 (2003).

35. Du, L., Wu, X., Pang, K., and Yang, Y.: Histological evaluation and biome-chanical characterisation of an acellular porcine cornea scaffold, Br. J. Oph-thalmol., 95, 410e414 (2011).

36. Kajbafzadeh, A. M., Javan-Farazmand, N., Monajemzadeh, M., andBaghayee, A.: Determining the optimal decellularization and sterilizationprotocol for preparing a tissue scaffold of a human-sized liver tissue,Tissue Eng. Part C Methods, 19, 642e651 (2013).


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