Acta Biomaterialia 9 (2013) 52515261

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Acta Biomaterialia
journal homepage: www.elsevier.com/locate/actabiomat

Biochemical changes caused by decellularization may compromise mechanical integrity of tracheal scaffolds
L. Partington a,b, N.J. Mordan c, C. Mason a, J.C. Knowles c,d, H-W. Kim d,e, M.W. Lowdell b, M.A. Birchall f,g, I.B. Wall a,d,
a

Department of Biochemical Engineering, University College London, Torrington Place, London WC1E 7JE, UK Department of Haematology, University College London, Medical School, Rowland Hill St., London NW3 2PF, UK Division of Biomaterials and Tissue Engineering, UCL Eastman Dental Institute, 256 Grays Inn Road, London WC1X 8LD, UK d Department of Nanobiomedical Science and WCU Research Centre, Dankook University, Cheonan 330-714, South Korea e Institute of Tissue Regeneration Engineering (ITREN), Dankook University, Cheonan 330-714, South Korea f UCL Ear Institute, The Royal National Throat Nose and Ear Hospital, 330 Grays Inn Road, London WC1X 8DA, UK g Department of Otolaryngology, University of California Davis Medical Center, 2521 Stockton Blvd., Suite 7200, Sacramento, CA 95817, USA
b c

a r t i c l e

i n f o

a b s t r a c t
Tissue-engineered airways have achieved clinical success, but concerns remain about short-term loss of biomechanical properties, necessitating a stent. This study investigated the effect of chemicalenzymatic decellularization on biochemical properties of trachea important for cell attachment and vascularization (bronectin and laminin) and cartilage matrix homeostasis (type II collagen and glycosaminoglycans (GAG)), as well as biomechanical status. Native trachea was used as a control, and NDC trachea stored in phosphate buffered saline (PBS) in parallel to decellularization was used as a time-matched control. Decellularization removed most cells, but chondrocytes and DNA remained after 25 cycles. Fibronectin was retained throughout the lamina propria and laminin at basement membranes. DNA accumulation along ECM bres was seen. A decline in soluble collagen was observed in decellularized tissue. GAG content of cartilage rings was reduced, even in PBS control tissue from 20 cycles onwards (p < 0.05), but decellularization caused the greatest loss (p < 0.01). Tensile strength declined throughout the process, but was signicant only at later time points. The data demonstrate that the substantial reduction in GAG might contribute to loss of mechanical integrity of biotracheas. Overcoming structural changes that cause an imbalance in cartilage matrix equilibrium will be necessary to optimize clinical benet, enabling widespread use of biotracheas. 2012 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Article history: Received 17 June 2012 Received in revised form 29 September 2012 Accepted 3 October 2012 Available online 8 October 2012 Keywords: Tracheal prosthesis Mechanical properties Glycosaminoglycans Scaffold

1. Introduction Whole organ tissue engineering has become a signicant focus for biomedicine in recent years. Patients have already beneted from transplantation of tissue-engineered hollow organs and tissues such as bladder [3], urethra [31] and airway [27], and the knowledge gained in these areas will facilitate clinical translation for other hollow tube tissues such as oesophagus and bowel. A number of different biomaterial scaffolds have been considered for production of tissue-engineered airways including both natural and synthetic materials [6]. The use of decellularization methods to obtain natural scaffolds derived from tissue originally formed via in vivo organogenesis has been seen as an exciting milestone, as the intrinsic scaffold properties should be closely
Corresponding author at: Department of Biochemical Engineering, University College London, Torrington Place, London WC1E 7JE, UK. Tel.: +44 (0) 20 7679 3918. E-mail address: i.wall@ucl.ac.uk (I.B. Wall).

matched to the biological requirement in the engineered tissue product. The rst transplantation of tissue-engineered airway tissue into human was conducted in 2004, using decellularized pig intestinal tissue and the patients own cells [29]. The following year, a previously described detergentenzymatic decellularization method was adapted to be permissive to cellular repopulation of the scaffold [13]. The transplantation of bioengineered tracheas using decellularized human cadaveric trachea has met with great reported success [17,25,27]. However, both recipients have needed stents to maintain an open airway. Furthermore, in a subsequent cohort of nine patients requiring airway replacement, treated on a compassionate basis, three of the patients required a stent in the transplanted airway [5]. Tracheal collapse is caused by a biomechanical failure in the tissue, although the nature of the pathology leading to biomechanical weakness was unknown in those cases. Understanding the impact of decellularization on extracellular matrix (ECM) integrity and biomechanical properties in hollow

1742-7061/$ - see front matter 2012 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.actbio.2012.10.004

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tube tissues will be critical if the methods are to be employed in routine clinical practice, not just for airways, but other types of hollow organ too. There are several critical factors to consider when producing decellularized scaffolds for tissue-engineered grafts. First, many immunologic cells are present in the respiratory mucosa, and it is necessary to completely remove them and immunologically active protein that could trigger an adverse host immune responses [9,10,36]. The decellularization process meets this requirement [23,27]. Second, the scaffold needs to support revascularization, and therefore retention of basement membrane structures in their correct anatomical locations is important for facilitating vascularization, either by vasculogenesis or angiogenic ingrowth of vessels, so that it can occur rapidly following transplantation. Indeed, orthotopic revascularization of bioengineered trachea takes place readily over the space of a few weeks [2,14,19,27,28]. This time period is tolerated owing to the relatively low oxygen requirement of chondrocytes and the continued air contact of the epithelial cells that make up the biotrachea [26]. Finally, production of decellularized scaffolds that are biomechanically similar to native tissue depends on retention of structural proteins that are appropriately anchored. The tracheal cartilage plays a crucial role in maintaining a rigid open airway structure during breathing [33]. Collagen brils have an important role in conferring tensile strength to the tissue. To create a rigid cartilage matrix, glycosaminoglycan (GAG)-rich proteoglycans occupy the space between the brils, creating a highly hydrated matrix. Functional coupling between the brillar collagen and hydrated GAG-rich proteoglycans is essential for maintaining biomechanically sound cartilage [39], and loss of either or both components could disrupt the biochemical equilibrium. Indeed, both collagen and proteoglycan are lost at sites of tracheal collapse [30]. The current study aimed to investigate how chemical enzymatic decellularization already used for clinical production of tissue-engineered airways affects the structural and biomechanical properties of the resulting scaffold. It considered basement membrane proteins that facilitate cellular repopulation of the scaffold, along with type II collagen and non-collagenous GAG components that cooperate to confer tensile and compressive properties to the tissue. The study also investigated whether biochemical changes had any impact on the biomechanical properties of the scaffold.

solution at 4 C overnight and, the following day, the next cycle was initiated. Non-decellularized (NDC) tracheas stored in PBS for time periods corresponding to the different decellularization cycles were used as a time control for the decellularization process. 2.2. Histological processing of tracheal tissue Tracheal tissue from native and decellularized trachea was processed for resin-embedded histology in preparation for haematoxylin and eosin staining. Native and decellularized tracheal samples from different cycle numbers were pre-xed in 3% glutaraldehyde (Agar Scientic, Stansted, UK) in 0.1 M sodium cacodylate (Agar Scientic) (pH 7.4) at room temperature for 30 min, trimmed to 12 mm thick, then xed in 3% glutaraldehyde solution at 4 C for 3 h. The samples were dehydrated in a graded series of alcohols from 20% ethanol to 90% ethanol, then inltrated with LR white resin hard grade (Agar Scientic) at a ratio of 1:1 LR white to 90% ethanol for 30 min, then LR white for 30 min, both at room temperature, fresh LR white at 4 C overnight, then fresh LR white for 30 min at room temperature. Samples were then embedded into the LR white resin containing LR white accelerator (Agar Scientic) and incubated at 20 C for 1 h followed by 24 h at 4 C to polymerize the LR white, followed by nal curing at room temperature for 2 days. The resin embedded samples were trimmed from the resin and 2 lm sections cut using a microtome (Ultracut E, Reichert-Jung, Depew, USA) and semi-xed to a microscope slide on the heating block for at least 24 h. For cryosectioning, decellularized and PBS control tracheal samples from a series of cycle numbers were immersed in 20% sucrose solution (Sigma) diluted in distilled water at 4 C for 24 h, rinsed briey in PBS, embedded in O.C.T. compound (CTL Scientic Supply Corp, Deer Park, USA) and slowly frozen in liquid nitrogen cooled isopentane. Sections of 6 lm were prepared on a Cryotome FSE Cryostat (Thermo Scientic, Loughborough, UK) and stored on microscope slides at 20 C until required. 2.3. Haematoxylin and eosin staining of tracheal tissue sections Slides containing resin-embedded sections were immersed in Mayers haematoxylin (Sigma) for 1 h, blued in gently running tap water for 20 min, then immersed in 1% eosin Y (Sigma) for 1 h then rinsed very gently in static tap water, all at room temperature. The sections were dried on the heating block for a few minutes then mounted in DPX mountant. The sections were then observed under an optical microscope (BX50, Olympus, UK) and imaged (Coolsnap-Pro cf color, Media Cybernetics, Bethesda, USA). The total numbers of cells or nuclei were visualized at 100 by light microscopy. Five sections were counted per sample. The mean standard deviation (SD) was determined for each analysis. One-way ANOVA was used to compare decellularized matrices ndings with native trachea ndings. 2.4. Alcian blue staining for GAG analysis For Alcian blue staining, frozen sections were brought to room temperature, xed for 1 min in 10% formalin (Sigma) and, following a brief wash in distilled water, immersed in Alcian blue pH 2.5 in 3% acetic acid (Sigma) for 5 min, briey rinsed in distilled water, then washed well in running tap water for 5 min then counterstained with neutral red for 1 min. The slides were then rinsed in 100% IMS, dehydrated through alcohol, cleared in xylene (Sigma) and then mounted with DPX mountant (Sigma). Imaging was performed on the Nikon Eclipse TE2000-U with NIS-Elements BR software (Nikon UK Ltd, Kingston upon Thames, UK). ImageJ (NIH, USA) was used to annotate the images.

2. Methods 2.1. Preparation of bioengineered porcine tracheal matrices Whole cadaveric tracheas (>10 cm) were freshly harvested from 10 male pigs (>25 weeks, 80100 kg). Five of the tracheas were subjected to decellularization, while the remaining ve were stored in phosphate buffered saline (PBS) for the corresponding length of time. Tracheas were decellularized using a previously described detergentenzymatic method [8,27]. Briey, the tracheas were washed three times with PBS (Sigma, Poole, UK) containing 1% v/v antibiotic and antimycotic (Sigma), and then incubated in sterile ltered water for 72 h at 4 C. Next, 25 cycles of detergentenzyme decellularization were used. In the detergent step, tracheas were incubated with 4% w/v sodium deoxycholate (Sigma) diluted in distilled water and rotated continuously for 4 h at room temperature. Detergent was then removed and, in the enzyme step, DNase-I (2 kU ml1; Sigma) in 1 M NaCl (Sigma) was used, with continuous rotation for 3 h at room temperature. After three 10-min wash steps with sterile ltered water, trachea samples were stored in PBS containing 1% antibiotic and antimycotic

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2.5. Immunouorescence labelling for analysis of ECM components Immunohistochemical (IHC) analysis was performed on frozen sections. The sections were brought to room temperature, xed in acetone then washed three times in Tris-buffered saline (TBS) with 0.025% Triton X-100 for 5 min. Sections were then blocked in 10% normal serum with 1% BSA in TBS for 2 h at room temperature. Sections were then incubated overnight at 4 C with the primary antibody (all from Abcam, Cambridge, UK), which was type II collagen (ab3092 and ab300), bronectin (ab23516) or laminin (ab11575), all at 1:100 dilution in 1% BSA in TBS. Following 2 5 min washes in TBS 0.025% Triton, the sections were incubated for 1 h at room temperature with the Alexauor 488 or 555 conjugated secondary antibodies (A-11001, A-11008 or A21436; Invitrogen, Paisley, UK) and then, following three washes in TBS, counterstained with 40 ,6-diamidino-2-phenylindole (DAPI). For negative controls, the primary antibody was omitted. IHC imaging was performed on the Nikon Eclipse TE2000-U with NIS-Elements BR software (Nikon). ImageJ was used to annotate the images. 2.6. Analysis of DNA, protein and GAG content DNA was extracted from tissue using the DNeasy Blood & Tissue Kit (Qiagen, Crawley, UK) according to the manufacturers instructions. DNA was quantied using the Nanodrop (ND1000; NanoDrop Products, Wilmington, USA). Protein content was evaluated in the tissue, following papain (Sigma) digestion using the Pierce BCA Assay (Thermo Fisher Scientic, Cramlington, UK), according to the manufacturers instructions, and quantied at 562 nm on the Tecan Sare2 (Tecan, Reading, UK). GAG content was evaluated, following papain (Sigma) digestion, using the BlyscansGAG Assay (Biocolor Ltd, Carrickfergus, UK), according to the manufacturers instructions, and quantied at 656 nm on the Tecan Sare2 (Tecan). Soluble collagen content in the tissue was evaluated, following digestion in 3% v/v acetic acid (Sigma) and 0.01% w/v pepsin (Sigma) overnight at 4 C, using the Blyscan Sircol Assay (Biocolor, UK) according to the manufacturers instructions, and quantied at 555 nm on the Tecan Sunrise (Tecan). Total collagen content in the tissue was evaluated following hydrolysis with 6 M HCl overnight at 95 C, using the Quickzyme Total Collagen Assay (2BScientic, Upper Heyford, UK) according to the manufacturers instructions, and quantied at 570 nm on the Tecan Sunrise (Tecan). For each DNA, protein, GAG and collagen quantitative test, the concentration was normalized to wet weight, and then the mean SD was determined. Two-way ANOVA was used to nd any statistically signicant differences between native and decellularized trachea, as well as NDC PBS control tissue. 2.7. Sectioning for transmission electron microscopy Ultrathin sections (90100 nm) were cut on an ultramicrotome (Reichert UltracutE, Cambridge Instruments, Cambridge, UK) with a diamond knife (Diatome AG, Biel, Switzerland). These sections were mounted onto Formvar/Carbon coated 200 mesh gold grids (Agar Scientic), stained with 0.1% (w/v) uranyl acetate in absolute alcohol for 5 min, followed by 5 min in Reynolds lead citrate, then examined by transmission electron microscopy (TEM) using a JEOL 100CX II transmission electron microscope (JEOL UK, UK) operating at 80 kV. The images were recorded on Kodak EM 4638 lm (TAAB, UK) and scanned to provide a digital format.

2.8. Mechanical testing The mechanical properties of the cartilage rings for native, DC and NDC PBS control tracheas from various cycle numbers were evaluated in the circumferential axis using tensile and compression testing. The Instron 5565 (Instron, High Wycombe, UK) was used to assess and record in real time the tensile properties of each sample in the circumferential axis. Tissue was prepared as 10 2 mm dogbone samples and clamped into the specimen grips. The uniaxial tension was increased at a constant rate of 100 mm min1 until rupture of the cartilage, which was conrmed through observation of tears in the cartilage tissue and assessment of the stressstrain curves. Three parameters were determined from the tensile stressstrain curves: the Youngs (tensile) modulus, the ultimate tensile strength (stress at failure) and the strain at failure. All were plotted as the mean SD for each analysis. Two-way ANOVA was used to compare DC matrices ndings with native and NDC trachea ndings over the decellularization treatment. The PerkinElmer DMA 7E (PerkinElmer, Seer Green, UK) was used to assess and record in real time the compressive properties of each sample. Trachea specimens 5 5 n mm (W D H) were prepared with the adventitia and lamina propria removed. The exact measurements were inputted into the software, and the specimen positioned in the instrument. A force was then applied at 200 mN min1 up to 6000 mN and the Youngs (compressive) modulus (MPa) SD was determined for each analysis. Two-way ANOVA was used to compare data from DC matrices with those of native and NDC PBS control trachea over the decellularization treatment. 3. Results 3.1. Preparation of bioengineered porcine tracheal matrices Macroscopic and microscopic inspection of the trachea revealed a loss of cellular material in the DC tracheal matrices (Fig. 1B and D) when compared with the freshly harvested samples (Fig. 1A and C). However, DC trachea retained the gross anatomical structure typical of native trachea even after 25 cycles of decellularization (Fig. 1B). 3.2. Impact of decellularization on tracheal tissue and chondrocyte survival Tracheal scaffolds appeared completely acellular in the submucosal region after 25 cycles of decellularization compared with native trachea (Fig. 2A and B). In the cartilage, chondrocytes were still present (Fig. 2C and D). Quantitative analysis of histologic sections revealed that mucosal cells were all absent following the initial water incubation stage. This was evident throughout the mucosa (Fig. 2E) in DC compared with native trachea (0 cells vs. 8910 1280; P < 0.005), as well as sub-mucosa (0 cells vs. 7639 2418, P < 0.005) and adventitia layers (0 cells vs. 1298 346, P < 0.005). Residual nuclear material was still seen in the DC scaffolds, predominantly in the mucosa, after 25 cycles of decellularization (Fig. 2B). Complete clearance of cells from the cartilage was not fully achieved, with some chondrocytes remaining after decellularization (649 192 cells vs. 1257 412, P < 0.01; Fig. 2F). Morphological observations, based on the descriptions by Roach and colleagues [32] suggested that chondrocytes did undergo apoptosis, which was evident from cycle 10 onwards (Fig. 2E). 3.3. Protein and DNA quantication in DC tracheas DNA concentration underwent a substantial decrease from 742.2 236.5 ng mg1 in native trachea to 264.7 69.9 ng mg1

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Fig. 1. (A and C) Macroscopic and microscopic view of native trachea and (B and D) DC tracheal scaffold after 25 cycles of detergentenzymatic decellularization. Scale bar in (C and D) = 100 lm.

after 25 cycles of decellularization (P < 0.005; Fig. 3A). However, even in corresponding PBS controls, time-matched to 25 cycles, a similar reduction in DNA concentration was seen (284.1 37.7 ng mg1; P < 0.005) despite lack of treatment with DNase. Analysis of total protein concentration revealed no statistically signicant difference between native trachea (97.8 6.9 lg mg1) and either DC tissue (95.7 20.8 lg mg1; P > 0.1) or timematched NDC PBS control (95.3 8.6 lg mg1; P > 0.1; Fig. 3B). 3.4. Impact of decellularization on bronectin and laminin localization Laminin and bronectin are both important for cell attachment, migration and revascularization [34,21,12,22,16]. Laminin was localized to basement membrane structures in both native and DC trachea (Fig. 4AC). Fibronectin was present throughout the whole lamina propria, and its expression pattern remained largely unaffected by the decellularization process (Fig. 4DF). DAPI counterstaining indicated that nuclear material accumulated abundantly in the mucosal glands and along ECM bres (Fig. 4C and F). 3.5. Collagen content and ultrastructure of DC tracheal cartilage To investigate potential collagen loss, antibodies specic to type II collagen were used to immunolabel collagen in the cartilage. Using the monoclonal antibody ab3092 (Fig. 5AC), type II collagen appeared less intensely labelled after decellularization compared with that of native trachea, but this was not the case for timematched NDC PBS controls (Fig. 5AC). Immunouoroscent labelling with the polyclonal ab300 also revealed a reduction in uorescence after decellularization compared with both native

and time-matched NDC PBS controls (Fig. 5DF). Quantication of collagen content revealed an overall decline in soluble collagen content in the DC scaffolds when compared with the time-matched NDC PBS controls at cycles 10 (0.19 0.17 lg mg1 vs. 0.76 0.84 lg mg1), 20 (0.13 0.08 lg mg1 vs. 0.56 0.26 lg mg1) and 25 (0.06 0.04 lg mg1 vs. 0.49 0.49 lg mg1) (P > 0.01) (Fig. 5G). Subsequent quantitation of total collagen revealed no overall statistical differences between the native tissue (63.6 10.1 lg mg1), the decellularized scaffolds (53.2 9.8 lg mg1) and the time-matched NDC PBS controls (53.1 15 lg mg1) at cycle 25. However, a clear non-signicant trend of gradually declining collagen was observed (Fig. 5H). TEM images of the tracheal cartilage matrix were obtained to assess the ultrastructural arrangement and appearance of the collagen brils (Fig. 5I and J). Uniform orientation of bres was noted, with no observable differences between DC and native trachea. 3.6. Impact of decellularization on GAG content of tracheal tissue GAG were assessed, as they contribute to the function of proteoglycans in creating a highly hydrated matrix, and a ne balance between these and the collagen bres is needed to maintain matrix homeostasis. Alcian blue staining revealed that GAG were substantially removed from tracheal cartilage in both DC and timematched NDC PBS control tissue compared with native cartilage, but was more prominent with decellularization (Fig. 6AF). This was conrmed by quantitative analysis of GAG over the course of decellularization, with a concentration of 3.38 0.27 lg mg1 in native trachea, reducing to 2.21 0.73 lg mg1 in DC (P < 0.01 vs. native) and 2.45 0.42 lg mg1 in PBS control tissues (P < 0.01 vs. native; Fig. 6G).

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Fig. 2. H&E staining of (A and B) tracheal mucosal tissue and(C and D) cartilage. Mucosal tissue of native tracheas is highly cellular (A; white arrows indicate examples of cell nuclei), whereas 25 decellularization cycles completely removes cells (B), leaving only weak residual nuclear material (B; black arrows). In the cartilage compartment, large cell nuclei were visible in the chondrocytes of native tracheas (C; black arrows), the chondrocytes visibly lling their lacunae (white arrows). Cartilage of the resulting scaffold after 25 cycles of decellularization (D) displayed evidence of nuclear shrinkage (black arrow) and blebbing of the cell remnants into the lacuna (white arrow). Quantitative analysis revealed that a majority of the cells are cleared from tracheal mucosal regions during the initial water treatment (E). However, this does not account for cellular debris that requires repeated decellularization cycles to be cleared. A decrease in the total number of chondrocytes was observed throughout the decellularization process (F), which correlates with an increase in the number of chondrocytes undergoing apoptosis. Values are mean SD; N = 5; P < 0.05, P < 0.01, P < 0.005. Scale bar in (AD) = 10 lm.

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(32.2 4%) compared with native tissue (35.6 7.6%) or the corresponding NDC PBS control tissue (31.3 6.1%). Similarly, the compression testing (Fig. 7D) revealed that the application of compressive forces up to 6000 mN also resulted in no difference in the observed static compressive modulus between the DC tissue at cycle 25 (1.45 0.12 MPa) and the NDC PBS control tissue at cycle 25 (1.46 0.14 MPa), as well as no overall difference in compressive modulus with increasing number of decellularization cycles when compared with native tissue (1.4 0.12 MPa) (Fig. 7D). Strangely, a single signicant increase was noted in the NDC PBS control at cycle 10 vs. native trachea, but a subsequent Grubbs test revealed there to be a single outlying observation contributing to this.

4. Discussion A previously described decellularization protocol was employed to prepare tracheal scaffolds and assessed multiple parameters post-decellularization. It was found that the decellularization protocol effectively removed cells from most regions of the trachea; however, the presence of chondrocytes was still detectable after 25 cycles, as previously reported [27]. Additionally, DNA was still detected after 25 decellularization cycles. The mass removal of supercial donor cell populations occurred very efciently during the rst stage of the decellularization protocol. Cells in all the exposed regions of the trachea, such as mucosa, sub-mucosa and adventitia were all lysed during the 72 h water treatment. However, hyaline cartilage is highly hydrated by nature, and the water treatment did not disrupt the ECM or have a substantial impact on the removal of chondrocytes. Complete removal of chondrocytes was not fully achieved even after 25 cycles of decellularization, although chondrocyte apoptosis did take place, and therefore it is likely that the chondrocytes were ultimately removed via a combination of apoptosis in conjunction with clearance via the decellularization treatment. This would explain previous observations where loss of chondrocytes in the cartilage was evident after 22 cycles (minimum of 66 days) when the chemicalenzymatic method incorporated a water step into each cycle [13], yet when using the chemicalenzymatic method lacking the water step, chondrocytes were still visible after 25 cycles (minimum of 25 days) [8,27]. However, cartilage is an immunologically privileged tissue, and therefore the presence of chondrocytes could be tolerated during the matrix remodelling and cellular replacement of donor chondrocytes with the patients own [4,15]. While cartilage might pose only a limited immunological threat, nucleic acids can also cause concern. Intracellular material (including nucleic acids) released from lysed cells can bind to surrounding ECM components, and the subsequent decellularization cycles were required to remove these components. Nuclear material is known to be immunogenic, even triggering autoimmune responses due to sequence and epigenetic changes that can occur with increasing age [1]. It can also potentially trigger inammatory responses in the recipient if any residual donor DNA remains in the scaffold [24,40]. The present data indicate that it was not fully removed from the tissue even after 25 decellularization cycles. Highly sensitive DAPI staining indicated that DNA was still bound to ECM bres in the lamina propria, where it was seen as brilar deposits. In addition, DNA was also detected within mucosal glands that were surrounded by intact bronectin and laminin membrane structures, even though H&E staining indicated that cells had been removed. Therefore, studies that have employed only H&E staining to demonstrate removal of cellular material must be viewed with caution, owing to the risk of incomplete DNA removal. However, many commercially available scaffolds for regenerative medicine interventions in other tissue compart-

Fig. 3. DNA and protein content in trachea following decellularization. A decrease in the residual DNA concentration was observed throughout the decellularization process in DC trachea and in NDC control trachea stored in PBS in parallel (A). However, a higher degree of clearance was observed in DC vs. NDC PBS control trachea. Total protein concentration remained constant and did not decrease throughout the decellularization process (B). Values are mean SD; N = 5; P < 0.01, P < 0.005.

3.7. Impact of decellularization on biomechanical properties of tracheal tissue The mean value of the tensile modulus (Fig. 7A) of the DC trachea decreased in a non-signicant fashion with increasing decellularization cycles. However, at cycle 25 (tensile modulus 39.86 6.54), a statistically signicant difference was seen compared with native tissue, which had a tensile modulus of 54.07 9.41 MPa (P < 0.05), a reduction of 26% in stiffness. The DC tissue also had a lower stiffness than NDC PBS-stored tissue at cycles 10, 20 and 25 (39.86 6.54 MPa vs. 53.36 7.13 MPa at cycle 25 respectively; P < 0.005). A decrease was also an observed in the ultimate tensile strength (Fig. 7B) of the DC trachea, which correlated well with the observed tensile modulus data, with the DC tissue breaking at lower mean ultimate tensile strength values compared with the NDC PBS-stored tissue at cycles 10, 20 and 25 (9.1 1.7 vs. 12 2.3 MPa at cycle 25; P < 0.005). Again, this was the equivalent of 24% decrease in mean strength of DC tissue compared with PBS-stored controls. Compared with native tissue, which had a breaking strength of 12.1 2.7 MPa, only DC tissue after 25 cycles had undergone a signicant reduction in ultimate tensile strength (P < 0.05), again a decrease of 25%. There was no difference in strain at failure (Fig. 7C) between DC tissue

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Fig. 4. Immunouorescentlabelling of ECM components laminin and bronectin demonstrates that they are conserved over the course of the decellularization. Laminin staining in native trachea (A, B) was localized to the lamina propria (lp) and connective tissue (ct) but not in the cartilage (ca). In the lamina propria, the mucosal glands (mg) and basement membrane (bm) are clearly stained. In the DC trachea, the same structures are positively stained (C). Fibronectin staining of native (D, E) and DC (F) trachea revealed similar patterns of expression between the two and included the adventitia (ad) as well as at a low level in the cartilage. Co-staining with DAPI indicated that genetic material remained after decellularization, much of which was accumulated in mucosal glands (C00 ) or bound to matrix brils in the lamina propria (F00 ). Scale bar: (A and D) = 200 lm; (B, C, E, F) = 50 lm.

ments have DNA fragments remaining after decellularization [18]. This is because the DNA is particularly sticky and can bind to bres in the ECM, as seen in this study and elsewhere [24]. If remnants exist as fragments of <300 bp, they are considered much less likely to induce inammatory and tissue remodelling events [18], and the DNase step was used to degrade DNA to small fragments, if not completely remove it. The process uses 1 M NaCl as the DNase buffer, but this may not be optimal. Both Ca2+ and Mg2+ facilitate the activity of DNase I, and therefore a buffer containing these ions could be more optimal for DNA removal during decellularization, and future efforts might focus on exploring such optimization methods. Adequate removal of residual cellular and genetic material is required to reduce the risk of inammation mediated via the Th1 pathway leading to eventual immune rejection of the graft upon transplantation [4]. While adequate removal of potentially immunogenic material is essential, a careful balance is required to ensure important components of the ECM are retained. Critically, these are components such as bronectin, collagen and laminin, which aid recellularization by promoting cell adhesion, migration and differentiation. Laminin is particularly important as a major basement membrane component that supports re-epithelialization of the lumen and neovascularization [37]. It was therefore promising to see that bronectin and laminin had expression patterns in DC tissue similar to those in native trachea, as cell attachment in these regions should not be impeded. Also, the combined structural integrity of basement membrane components observed, along with retention of angiogenic factors such as bFGF that has been previously reported [8], suggests that neovascularization should be well supported upon transplantation of the graft. Indeed, revascularization of transplanted biotracheas occurs readily over several weeks in humans [27,17], and the intact basement membrane structures reported in this study will inevitably support vascular responses. Retention of matrix molecules that impart mechanical strength on the tissue is also required to support a functional airway. However, type II collagen, soluble collagen and GAG were found to undergo decline throughout the decellularization process. It

has recently been reported that GAG loss accompanies decellularization [20]. In the current paper, loss of GAG was also observed in the NDC PBS control tissue over time, and therefore decellularization may only accelerate the decline in GAG that occurs over time anyway. The decellularization process took 6 weeks to complete, a substantial length of time, in which tissue degradation can occur. Therefore, signicantly reducing the decellularization time could result in better retention of GAG. Changes in the cartilage and collagen structure were not evident at the ultrastructural level. However, decreased collagen content was seen in DC cartilage using IHC analysis compared with the NDC PBS control matrices. The present results indicate that the decellularization solution diffused though the cartilage over time, resulting in removal of type II collagen deep within the cartilage tissue. Again, the effects seemed to increase gradually over time, and reducing the length of the DC process could moderate the loss of type II collagen. In spite of the collagen and GAG loss, there was still an abundance of each after 25 cycles, and total collagen was certainly not signicantly altered, even though a non-signicant trend of decline was seen. Also, the gross anatomical structure of the cartilage rings was retained after the decellularization process. The hyaline cartilage of the trachea has an important structural role in that the tracheal rings must maintain an open airway, even in the presence of intrathoracic pressure changes that are created during breathing. This structural property is the result of functional coupling between high osmotic pressure provided by the GAG-rich proteoglycans and tensile strength conferred by the brillar collagen network at the microscopic level. Altering the matrix constituents in terms of quantities, ratios and arrangements of ECM molecules could consequently alter the functional properties, particularly at the biomechanical level. For example, age-related decline in proteoglycan content of cartilage leads to an increase in elastic modulus [33], making the tissue stiffer. Tracheal cartilage is relatively complicated, as its biomechanical properties are nonlinear [38] but, to take a holistic view, the circumferential strength of the cartilage rings is determined by their structural composition. If any adverse structural changes in terms of content or

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Fig. 5. Immunolabelling of type II collagen within cartilage matrix using two different antibodies (AF) suggested that, compared with native trachea (A and D), DC trachea (C and F) but not PBS-stored trachea (B and E) experienced a decline in collagen content. Quantitative analysis of collagen content indicated that decellularization reduced the amount of soluble collagen compared with time-matched PBS stored tissue (G), but did not signicantly affect total collagen content (H). TEM images showed that the ultrastructure of collagen was similar in native (I) and DC (J) trachea, with parallel bundles of collagen bres in both. Scale bar for (AF) = 100 lm; (I and J) = 40,000 magnication. Values are mean SD; N = 5; P < 0.01.

arrangement of specic ECM constituents takes place due to decellularization, there is a risk of accelerated structural weakening and increased potential for collapse in the grafted trachea. Previously, tracheal stenosis was found to be associated with signicant loss of the GAG-rich proteoglycan Aggrecan and type I collagen, but not type II collagen [30]. This also parallels the functional decline in articular cartilage caused by osteoarthritis, where early indicators include loss of GAG-rich proteoglycans and then, later, signicant collagen loss [35]. Indeed, in tracheal cartilage, it was found that substantial decreases in GAG content were evident throughout the decellularization process and in PBS-stored tissue,

but total collagen content appeared to be largely unaffected, at least in terms of concentration. Even so, the GAG loss could reect a change in tissue structure that may manifest biomechanical instability in vivo. Maintaining circumferential strength in DC matrices is a key requirement for scaffolds that will have a structural role. It is particularly important in the case of trachea because, in case of mechanical failure, using a stent can lead to complications such as infection. If stenting led to the problems that necessitated a tracheal transplant in the rst instance, then, rather than being cured, the patient is potentially starting the cycle all over again. As

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Fig. 6. Histochemical analysis of GAG content in tracheal cartilage. Alcian blue staining revealed that native trachea was rich in GAG, evidenced by the bright blue staining throughout the cartilage (ca; A and D). DC trachea displayed a substantial loss of GAG, evidenced by a decrease in staining intensity (C and F). Even in tracheal tissue stored in PBS as a time-matched control for DC tissue underwent a visible decline in staining intensity (B and E), although not to the same extent as the DC tissue. Mucosal glands (mg) of the lamina propria (lp) showed weak GAG staining. Quantitative analysis of GAG content (G) revealed that a signicant decrease is observed in DC trachea from 20 decellularization cycles onwards. Furthermore, NDC tissue stored for the same period of time in PBS also underwent a signicant reduction in GAG content. Scale bar: (A C) = 500 lm; (DF) = 200 lm. Values are mean SD; N = 5; P < 0.05, P < 0.01.

biomechanical strength is a function of the hyaline cartilage rings, the biomechanical strength in DC cartilage rings was assessed. A reduction in Youngs modulus and ultimate tensile strength was observed in DC trachea from cycle 10 onwards compared with PBS-stored trachea and could be related to the decline in GAG, type II collagen and in soluble collagen. The biomechanical weakness could potentially lead to tracheal collapse in vivo under negative air pressure during breathing. This echoes the ndings of signicant loss of GAG-rich proteoglycan and type I collagen at sites of fracture in tracheal collapse in vivo [30]. Loss of type II collagen and GAG along with the corresponding matrix strength was seen to occur later in the decellularization process and therefore an optimized process using fewer cycles or reduced time could better preserve type II collagen, GAG and cartilage strength. Previous studies using a similar decellularization protocol found that biomechanical strength became signicantly reduced only after 27 cycles [8], which is later than the time at which reduced biomechanical integrity was observed. It has also been reported that long-term (12 months) storage of DC trachea in PBS at 4 C led to signicant alteration in matrix properties and a reduction in mechanical strength [7]. Interestingly, it was observed that GAG content was reduced in NDC trachea stored in PBS for relatively short periods of time, cycle-matched to the DC tissue from 20 cy-

cles onwards. It is not surprising, however, as a study assessing the effect of storage on articular cartilage indicated that, after just 12 days of refrigerated storage, the biomechanical properties of the tissue become affected [11] and observations of altered GAG levels might explain the biomechanical compromise. One also cannot preclude the possibility that the potential failure of engineered airways in vivo is due not just to ECM destruction during decellularization, but also to a failure of host chondrocyte repopulation, migration and chondrogenesis after seeding. Functional chondrocytes are crucial for the re-establishment of cartilage homeostasis post-transplant and the failure of stem cell-derived chondrocytes to undergo appropriate maturation and integration in the donor scaffold could itself be a substantial cause of failure in airway grafts. 5. Conclusions The current chemicalenzymatic decellularization protocol potentially weakens the structure of the trachea, resulting in loss of circumferential strength, which could result in collapse of the trachea in vivo. In addition to this, aspects of the protocol require optimization to ensure effective removal of all immunogenic material within the shortest time period with minimal cycles, which

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could be benecial to maintaining strength, as increasing decellularization cycles leads to a reduction in GAG and potentially type II collagen and a reduction in tensile strength of the tissue. The development of a novel method for decellularizing tracheas rapidly with optimized decellularization solutions and storage conditions would hopefully address the issues currently faced, and move the research from hospital-exempt procedures into clinical practice. Acknowledgements L.P. acknowledges support from the Engineering and Physical Science Research Council (EPSRC) Industrial Doctoral Training Centre in Bioprocess Engineering Leadership (EP/G034656/1) and the UK Stem Cell Foundation I.W., J.K. and H.W.K. are members of the WCU (World Class University) program in Nanobiomedical Science (National Research Foundation of Korea Grant Number R31-10069 funded by the Ministry of Education, Science and Technology). References
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Fig. 7. Tensile testing of cartilage demonstrated that DC tissue had lower stiffness (A) and lower ultimate tensile strength (B) than the corresponding time-matched PBS controls before failure. Tensile strain values did not change with increasing number of decellularization cycles compared with the time-matched PBS controls (C). DMA compression testing also demonstrated that decellularization did not affect compressive modulus values compared with the time-matched PBS controls (D). Values are mean SD; N = 5; P < 0.05, P < 0.005.

L. Partington et al. / Acta Biomaterialia 9 (2013) 52515261 [21] Herard AL, Pierrot D, Hinnrasky J, Kaplan H, Sheppard D, Puchelle E, et al. Fibronectin and its alpha 5 beta 1-integrin receptor are involved in the wound-repair process of airway epithelium. Am J Physiol 1996;271: L72633. [22] Hocking DC, Chang CH. Fibronectin matrix polymerization regulates small airway epithelial cell migration. Am J Physiol Lung Cell Mol Physiol 2003;285:L16979. [23] Jungebluth P, Go T, Asnaghi A, Bellini S, Martorell J, Calore C, et al. Structural and morphologic evaluation of a novel detergentenzymatic tissueengineered tracheal tubular matrix. J Thorac Cardiovasc Surg 2009;138:58693. discussion 5923. [24] Keane TJ, Londono R, Turner NJ, Badylak SF. Consequences of ineffective decellularization of biologic scaffolds on the host response. Biomaterials 2012;33:177181. [25] Laurance J. British boy receives trachea transplant built with his own stem cells. BMJ 2010;340:c1633. [26] Macchiarini P, Birchall M. Stem cell Hype in tracheal transplantation? A response. Transplantation 2010;90:9289. [27] Macchiarini P, Jungebluth P, Go T, Asnaghi MA, Rees LE, Cogan TA, et al. Clinical transplantation of a tissue-engineered airway. Lancet 2008;372:202330. [28] Macchiarini P, Mazmanian GM, de Montpreville V, Dulmet E, Fattal M, Lenot B, et al. Experimental tracheal and tracheoesophageal allotransplantation. ParisSud University Lung Transplantation Group. J Thorac Cardiovasc Surgery 1995;110:103746. [29] Macchiarini P, Walles T, Biancosino C, Mertsching H. First human transplantation of a bioengineered airway tissue. J Thorac Cardiovasc Surg 2004;128:63841. [30] Mankarious LA, Adams AB, Pires VL. Patterns of cartilage structural protein loss in human tracheal stenosis. Laryngoscope 2002;112:102530.

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