Advances in Medical Sciences 59 (2014) 299–307
Contents lists available at ScienceDirect
Advances in Medical Sciences journal homepage: www.elsevier.com/locate/advms
Original Research Article
Effect of three decellularisation protocols on the mechanical behaviour and structural properties of sheep aortic valve conduits Reza Khorramirouz a, Shabnam Sabetkish a, Aram Akbarzadeh a, Ahad Muhammadnejad b, Reza Heidari a, Abdol-Mohammad Kajbafzadeh a,* a
Pediatric Urology Research Center, Section of Tissue Engineering and Stem Cells Therapy, Children’s Hospital Medical Center, Tehran University of Medical Sciences, Tehran, Iran Cancer Research Center, Iranian Cancer Institute, Department of Pathology, Tehran University of Medical Sciences, Tehran, Iran
b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 28 February 2014 Accepted 13 August 2014 Available online 26 August 2014
Purpose: To determine the best method for decellularisation of aortic valve conduits (AVCs) that efficiently removes the cells while preserving the extracellular matrix (ECM) by examining the valvular and conduit sections separately. Material/methods: Sheep AVCs were decellularised by using three different protocols: detergent-based (1% SDS + 1% SDC), detergent and enzyme-based (Triton + EDTA + RNase and DNase), and enzyme-based (Trypsin + RNase and DNase) methods. The efficacy of the decellularisation methods to completely remove the cells while preserving the ECM was evaluated by histological evaluation, scanning electron microscopy (SEM), hydroxyproline analysis, tensile test, and DAPI staining. Results: The detergent-based method completely removed the cells and left the ECM and collagen content in the valve and conduit sections relatively well preserved. The detergent and enzyme-based protocol did not completely remove the cells, but left the collagen content in both sections well preserved. ECM deterioration was observed in the aortic valves (AVs), but the ultrastructure of the conduits was well preserved, with no media distortion. The enzyme-based protocol removed the cells relatively well; however, mild structural distortion and poor collagen content was observed in the AVs. Incomplete cell removal (better than that observed with the detergent and enzyme-based protocol), poor collagen preservation, and mild structural distortion were observed in conduits treated with the enzyme-based method. Conclusions: The results suggested that the detergent-based methods are the most effective protocols for cell removal and ECM preservation of AVCs. The AVCs treated with this detergent-based method may be excellent scaffolds for recellularisation. ß 2014 Medical University of Bialystok. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.
Keywords: Aorta Heart valve Aortic conduit Acellular Sheep
1. Introduction Valvular heart diseases are important causes of mortality. Congenital and acquired valvular dysfunction is frequently treated by heart valve replacement, which is a common procedure worldwide [1]. Aneurysmal rupture is one of the leading causes of death, with an incidence of 3–4% [2]. However, the current treatments for aortic valve defects in children are not satisfactory,
* Corresponding author at: Pediatric Urology Research Center, Section of Tissue Engineering and Stem Cells Therapy, Children’s Hospital Medical Center, Tehran University of Medical Sciences, No. 62, Dr. Qarib’s Street, Keshavarz Boulevard, 1419433151 Tehran, Iran. Tel.: +98 21 66565400; fax: +98 21 66565400. E-mail address: [email protected] (A.-M. Kajbafzadeh).
and the incidence of regurgitation and stenosis are more common in children than in older patients. Children and neonates treated with mechanical valve prostheses can be confronted with significant postoperative complications due to the lack of appropriately sized prostheses. Therefore, to minimise probable postoperative complications, ideally, in terms of cell removal and mechanical properties, a decellularised aortic valve conduit (AVC) should be used as a substitute for juvenile patients. Surgical treatment of ascending aortic aneurysms accompanied with AV regurgitation is still challenging, and the management is a debatable matter. Therefore, alternative treatments are needed to reduce the incidence of this life-threatening event. Mechanical and bio-prosthetic valves are the two conventional substitutes for dysfunctional heart valves. However, the application
http://dx.doi.org/10.1016/j.advms.2014.08.004 1896-1126/ß 2014 Medical University of Bialystok. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.
300
R. Khorramirouz et al. / Advances in Medical Sciences 59 (2014) 299–307
of these replacement valves is associated with several disadvantages, including their inability to grow, which is a particular problem in patients that are still growing, structural valve deterioration, the need for long-term anticoagulation therapy, immunological responses, and the absence of remodelling [3]. To overcome these limitations, decellularised heart valve conduits can be used as alternatives with physiological functions that are similar to those of natural valves, with the least immunogenic potential [4]. The process of engineering AVCs includes an effective method for decellularisation of both the valve and conduit sections for further in vitro or in vivo cell seeding. A critical point of the decellularisation process is the preservation of the extracellular matrix (ECM) in both sections. The goal of decellularisation is maximum cell and nuclei removal to minimise the cellular debris remaining in the AVCs and greatly lessen the adverse immune responses, inflammation, fibrous scarring, and calcification [4–7]. A practical scaffold should be capable of self-repair and remodelling, be durable, and not induce rejection. Therefore, it is necessary to determine the best decellularisation protocol for complete cell removal and preservation of the tissue microstructural features of the AVCs. In this study, we compared three different protocols for the decellularisation of AVCs, and we introduced an ideal method that strikes a balance between maximum cell removal and preservation of tissue microstructure, with optimal biochemical properties to avoid possible complications encountered after transplantation. 2. Material and methods 2.1. Tissue preparation The ethical committee of Tehran University of Medical Sciences approved this study. Twelve sheep, with a mean body weight of 47 5 kg, were sedated by an injection of xylazine (0.15 mL per 10 kg body weight; IM). The animals were anesthetised by administration of atropine sulphate (11 mg per 10 kg body weight), propofol (5 mg per kg body weight), and diazepam (0.27 mg per kg body weight). Hearts were harvested under sterile conditions and placed in Hank’s balanced salt solution (HBSS) containing antibiotics (penicillin, gentamycin, streptomycin, and amphotericin). AVCs were precisely dissected and decellularised. Harvested AVCs had a thin rim of subvalvular muscle tissue and a short arterial segment. A total of 12 AVCs were included in the study, and they were randomly divided into 3 decellularisation groups and a control group. 2.2. Decellularisation process Nine AVCs were treated with 3 different decellularisation protocols (detergent-, enzyme-, and detergent and enzyme-based), with 3 AVCs per group. Before starting each treatment, all valves and conduits were placed in PBS for 90 min, and the solution was changed every 30 min to rinse the tissues. Then, each group was treated as described below. All materials were purchased from Sigma–Aldrich (Belgium) unless stated otherwise. 2.2.1. Protocol 1 (1% SDS + 1% SDC) In this detergent-based decellularisation method, AVCs were treated with 1% (w/v) sodium dodecyl sulphate (SDS) and 1% (w/v) sodium deoxycholate (SDC) dissolved in distilled water for 48 h at room temperature. Then, they were washed with distilled water 2 times at 4 8C for 12 h each. In the final stage of the decellularisation procedure, the AVCs were washed in phosphate buffered saline (PBS) (137 mmol/L NaCl, 2.7 mmol/L KCl, 1.78 mmol Na2HPO42H2O, and 2 mmol/L KH2PO4, pH 7.4) for 24 h by changing the solution every 12 h. This step was performed to eliminate the applied detergents.
2.2.2. Protocol 2 (1% Triton + EDTA + RNase and DNase) In this detergent and enzyme-based method, the AVCs were placed in 1% (w/v) Triton X-100 with a combination of 1 mmol/L ethylenediaminetetraacetic acid (EDTA), 20 mg/mL RNase, and 200 mg/mL DNase (dissolved in PBS) at 37 8C for 24 h. In the second step, samples were incubated in HBSS (137 mmol/L NaCl, 5.4 mmol/L KCl, 0.5 mmol/L KH2PO4, 4.1 mmol/L NaHCO3, and 0.33 mmol/L Na2HPO4 without Ca, Mg, and phenol red) at 4 8C for 72 h. The solution was changed every 12 h (6 times). 2.2.3. Protocol 3 (0.1% Trypsin + RNase and DNase) All the steps in this protocol were similar to those in protocol 2. The only difference was the use of 0.1% (w/v) Trypsin (TS) instead of Triton X-100. All the above-mentioned steps were conducted under continuous shaking on a mechanical shaker at 50 revolutions per minute (rpm). 2.3. Histological examination and DAPI staining To evaluate and compare the effectiveness of our decellularisation protocols in removing the cellular components of both the valvular and conduit sections, native and decellularised AVCs were cut into 3 mm 4 mm sections and fixed in 10% neutral buffered formalin (Merck, Darmstadt, Germany) at pH 7.4 for 48 h at room temperature. Then, they were washed in distilled water, dehydrated in a graded series of ethanol, embedded in paraffin, and cut into 5–8-mm thick sections. To visualise remnant deoxyribonucleic acids (DNA), the sections were stained with 40 ,6-diamidino-2-phenylindole (DAPI; Biotium, Inc.). To enhance fluorescence, the DAPI solution was diluted to 30 nM in PBS, and 300 mL was pipetted directly onto all samples. After incubation in the dark for 30 min and rinsing with distilled water, the slides were evaluated with a fluorescent microscope. Images were taken with a Nikon digital camera (DXM 1200). Picrosirius red and Russell–Movat pentachrome staining were also used for collagen typing and histological evaluation of the ECM. For Picrosirius red staining, paraffin-embedded blocks were cut into 5-mm slices. The sections were deparaffinised and hydrated in graded series of alcohol. The slides were stained for 1 h in Picrosirius red solution (0.1% Sirius red in saturated picric acid), followed by two washes of 0.5% acetic acid without counterstaining. Under polarised microscopy, type I collagen fibres are seen as thick, strongly birefringent, yellow-orange fibres, and type III fibres are seen as thin, weakly birefringence, greenish fibres. Different connective tissue constituents are highlighted by pentachrome staining. Using this technique, collagen, elastin, muscle, mucin, and fibrin can be differentiated. For Movat pentachrome staining, paraffin-embedded blocks were cut into 5-mm slices, deparaffinised, and hydrated in a graded series of alcohol. Briefly, mucin was stained with Alcian blue 8GS and converted to insoluble blue pigment by treatment with alkaline alcohol; nuclei and elastic fibres were stained black with Verhoeff’s haematoxylin; fibrinoid, fibrin, and muscle were stained red with Crocein scarlet-acid fuchsin solution; and collagen and reticular fibres were stained yellow with Saffron. 2.4. Scanning electron microscopy (SEM) Valvular and conduit sections of AVCs treated with the 3 decellularisation protocols as well as fresh control specimens were fixed with 2.5% glutaraldehyde (Merck, Darmstadt, Germany). Then, they were washed in PBS 3 times for 60 min each to remove the glutaraldehyde. Both native and decellularised samples were fixed with 1% osmium tetroxide, 0.8% potassium
R. Khorramirouz et al. / Advances in Medical Sciences 59 (2014) 299–307
301
ferrocyanide, and 1% tannic acid. Then, the samples were completely dehydrated using a graded series of ethanol (30%, 50%, 70%, 90%, and 100%; Merck) for 10 min each. Finally, the samples were sputter coated with gold to improve the conductivity of electrons and to visualise the surfaces via SEM (Hitachi-54160, Japan). Finally, all the samples were examined in a Jeol JSM-6340F Field Emission Scanning Electron Microscope. The acceleration voltage was adjusted to 10 kV, and a working distance of 8 mm was used. Secondary and backscattered electrons were used to obtain the images.
endothelial layer in the intima layer with interstitial cells and smooth muscle cells in the media layer. The collagen fibres in the media layer were stained by Movat pentachrome staining. Elastin fibres were also present in the media layer. Picrosirius staining showed the presence of collagen type I in the media layer (Figs. 1A and 2A). DAPI staining showed cellularity in all 3 layers (Fig. 3A and B), and SEM examination showed an intact ultrastructure within the endothelial layer (Figs. 4A and 5A).
2.5. Mechanical properties A tensile-test device (Hct 400/25; Zwick/Roell, Germany) was used to compare the tensile strength of the native and decellularised conduit sections. Both longitudinal and transitional tensile strength was measured in 2 separate tests. To calculate the physical properties of the longitudinal (maximum strength and Young’s modulus) and transitional fibres (force/displacement), samples were cut into 4 mm 4 mm strips. The specimens were clamped in sample holders with a constant elongation rate of 0.1 mm/s (6 mm/min) at room temperature. Each sample was subjected to mounting uniaxial tensile testing until the tissue tore and the load disappeared, as demonstrated by the device. The maximal point indicated by the device was considered the maximum pressure tolerance. Four samples from each group were tested, and the results were averaged.
H&E and DAPI staining confirmed that the detergent-based method was the most effective protocol for removing cells and intracellular debris from all parts of the AVCs. The samples were well decellularised in the intima, media, and adventitious layers, with relative structural integrity. The ECM in both the valvular and conduit sections was satisfactorily preserved. Pentachrome staining of the AVCs showed preservation of elastin and collagen fibres, especially in the media layer. Pentachrome staining of the conduits demonstrated mild a decrease in elastin content compared to the control samples. Picrosirius red staining of valvular and conduit sections showed collagen preservation in the media layer (Figs. 1B, 2B, 3C and D). Under SEM, no cellularity or structural distortion were detectable in either the inflow or outflow sides of the valves and conduits in the decellularised constructs. Therefore, the ultrastructure was preserved after the decellularisation procedure (Figs. 4B and 5B).
2.6. Hydroxyproline content
3.3. Protocol 2: (1% Triton + EDTA + RNase and DNase)
The collagen content of the valvular and conduit portions of the native and decellularised AVCs was quantitatively measured by hydroxyproline evaluation. The collagen content of 4 replicates of the native and decellularised heart valve samples was analysed. A 10-mg/mL 4-hydroxy-L-proline stock solution was diluted to make hydroxyproline standards (0.75–25 mg/mL). The collagen content per wet weight of the native and decellularised samples was assessed by measuring the total amount of hydroxyproline according to a previously described method [8]. Tissue samples were hydrolysed in 5 mL of 6 N HCl at 110 8C for 14–16 h. In the next step, the samples were incubated in 500 mL of chloramine-T solution (0.14 g chloramine-T, 2 mL of distilled water, and 8 mL of citrate/acetate buffer) at room temperature for 20 min. One millilitre of Ehrlich’s reagent (10.13 g of p-dimethylaminobenzaldehyde, 41.85 mL of 1-propanol, and 17.55 mL of 70% perchloric acid; Fluka Chemicals) was then added to the specimens. The resulting mixture was incubated at 65 8C for 20 min, and then the absorbance at 546 nm was measured with a spectrophotometer (BioTek Synergy HT1 microplate reader). The hydroxyproline concentration was calculated as the mg of hydroxyproline per g of wet tissue (mg/g wet tissue). All measurements were expressed as mean standard error of the mean (SEM). Data were analysed using SPSS version 16 (SPSS Inc., Chicago, IL, USA). Differences between groups were analysed by one-way ANOVA and Duncan’s tests. A p value less than 0.05 was considered statistically significant.
In the conduits and in each layer of the AVs treated with this protocol, cells and nuclei were observed, especially in the media layer by both H&E and DAPI staining. Pentachrome staining of the AVs showed good collagen preservation in all 3 layers. In the conduit portion, the collagen and elastin fibres were well preserved in the media layer (Figs. 1C, 2C, 3E and F). SEM examination of the AVs confirmed the distortion of the ultrastructure, with multiple fenestrations as well as a patchy cell distribution. However, cells remained in the conduits and the ultrastructure was well preserved with no media distortion (Figs. 4C and 5C).
3.2. Protocol 1: (1% SDS + 1% SDC)
3.4. Protocol 3: (0.1% Trypsin + RNase and DNase) The worst results were observed in the AVCs treated with this protocol. Picrosirius staining revealed poor collagen type I staining in both the valves and conduits. The results of the Pentachrome staining were in agreement with the Picrosirius staining regarding the collagen content in all 3 layers. Furthermore, the elastin fibres were completely eliminated (Figs. 1D and 2D). Using this enzymatic decellularisation method, the AV samples were well decellularised in all 3 layers as confirmed by DAPI staining (Fig. 3G and H). However, the conduits were not well decellularised in all 3 layers, although the remaining cellularity was less than that observed with the Triton-based protocol. Mild structural distortion and pore formation in the valvular and conduit sections was confirmed by SEM evaluation (Figs. 4D and 5D).
3. Results 3.5. Mechanical properties 3.1. Control cellular AVCs A normal meshwork of structural proteins was observed under light microscopy. Ultrastructure examination by SEM also showed a confluent monolayer of endothelial cells covering both the inflow and outflow tracts. This pattern is referred to as cobblestone morphology. H&E staining of the cellular AVCs showed a confluent
All decellularised aortic conduits underwent tensile testing to evaluate the resistance to biomechanical forces. Protocol 1 showed the following mechanical properties: maximal longitudinal strength = 0.39 MPa, Young’s modulus = 0.7 MPa, and transitional strength = 42 N/mm. However, the biophysical properties of the native conduits were as follow: maximal longitudinal
302
R. Khorramirouz et al. / Advances in Medical Sciences 59 (2014) 299–307
Fig. 1. H&E, Red Sirius (light microscope), Red Sirius (fluorescence microscope), and Pentachrome staining of AVs. (A) Control. (B) Decellularised valve with SDS 1% and SDC 1%. (C) Decellularised valve with Triton–EDTA–RNase and DNase. (D) Decellularised valve with Trypsin and RNase and DNase.
strength = 0.47 MPa, Young’s modulus = 0.81 MPa, and transitional strength = 43 N/mm). Conduits treated by using protocol 2 exhibited the following biophysical properties: maximal longitudinal strength = 0.3 MPa, Young’s modulus = 0.6 MPa, and transitional strength = 33 N/mm. The conduits treated by using the third protocol had the following biophysical properties: maximal longitudinal strength = 0.19 MPa, Young’s modulus = 0.39 MPa, and transitional strength = 20 N/mm. Overall, at strain smaller than 0.5, protocol 3 is closest to the native tissue, but its final strength is much lower than the native tissue. The final strength of protocol 1 is closest to the native tissue, but is much stiffer at small strains and getting softer when strain increases. Moreover, at small displacement, protocol 2 is the closest to the native tissue (Fig. 6). However, regarding the fact that the modulus is the slop of each stress-strain curve and the slop of each curve is changing with strain; the overall averaged modulus is not a good value to compare. 3.6. Hydroxyproline evaluation The hydroxyproline content of the conduit sections treated by using protocol 1 (0.66 0.01 mg/mg), protocol 2 (0.69 0.01 mg/ mg), and protocol 3 (0.65 0.02 mg/mg) was significantly higher (p < 0.05) than that of the native tissue (0.54 0.02 mg/mg).
The collagen content of the AVs decellularised with protocol 1 (0.24 0.1 mg/g) and protocol 2 (0.18 0.008 mg/g) was significantly higher (p < 0.05) than that of the native AV (0.16 0.01 mg/g) (Fig. 7). 4. Discussion In the current study, the characteristics of AVCs treated by using distinct decellularisation methods were compared to determine the optimal approach for cell removal and ECM preservation. The comparative approach of this study, in which we separately examined the valvular and conduit sections of decellularised AVCs obtained from the same decellularisation solutions, is a concept that has not been previously described. In addition, in the current study, we aimed to determine the best AVC decellularisation protocol for complete cell removal and to provide greater insight into the initial functional state of the decellularised AVC. Our results demonstrated that a detergent-based method has gained credibility because it completely removed the cells while preserving the ECM in both the valve and conduit sections relatively well which may reduce the probable postoperative complications. Cardiovascular disease is still the leading cause of death worldwide. In 2006, over 300,000 heart valve surgeries were performed, and this number is expected to triple by 2050 [9]. However, their effects on public health are significantly
R. Khorramirouz et al. / Advances in Medical Sciences 59 (2014) 299–307
303
Fig. 2. H&E, Red Sirius (light microscope), Red Sirius (fluorescence microscope), and Pentachrome staining of conduit sections. (A) Control. (B) Decellularised conduit with SDS 1% and SDC 1%. (C) Decellularised conduit with Triton–EDTA–RNase and DNase. (D) Decellularised conduit with Trypsin and RNase and DNase.
understated. Owing to these alarming statistics, an urgent call to action is necessary. Several decellularised tissues and organs treated by using different decellularisation protocols have been applied in tissue engineering and regenerative medicine. The efficacies reported in the literature for the methods that are most commonly used to generate decellularised heart valves differ widely. In addition, concomitant decellularisation of valvular and conduit segments of sheep AVCs has not been previously reported. Abnormalities of the aortic media can result in dilation of the proximal ascending aorta. This dilation is the most common congenital defect of the bicuspid aortic valve [10]. Decellularised heart valves can play a crucial role in the treatment of congenital valvular heart diseases. They are capable of growth, repair, and remodelling which makes them a practical substitute for valvular transplantation, especially in the paediatric population [11,12]. In early studies, attempts were made to construct engineered valves from polyglycolic acid fibre matrix with myofibroblasts and endothelial cells, the results of which were satisfactory in developing leaflets with similar functional and morphological properties compared with native structures [13,14]. Later studies introduced tissue-engineered heart valves treated with decellularisation processes, and investigated the fate of seeded cells in these manipulated tissues, with promising results both in vitro and in vivo [5,15,16]. Despite increasing interest among researchers to develop bioengineered heart valves, there are still many challenges in this field, and greater insight into the behaviour of AVCs after
decellularisation is needed. Various approaches have been used to develop valves with satisfactory properties, including construction of a 3D valvular structure using resorbable synthetic polymers, the development of decellularised xenogeneic tissues followed by direct implantation or in vitro cell seeding, and fabrication of hybrid heart valves from decellularised xenogeneic tissues enhanced by bioresorbable polymers [17,18]. These decellularised scaffolds can replace mechanical valves in patients confronted with several complications, such as systemic embolisation, bleeding, and valve obstruction due to thrombosis or pannus formation [19]. An effective decellularisation method for heart valves must remove the cellular and nuclear components that are responsible for evoking an immune response and causing subsequent damage to the valvular structure. It is worth noting that the damage induced by the decellularisation techniques is much less than the damage incurred by the recipient’s immune response to the transplanted tissue-engineered grafts [20]. In the study by Steven et al., 3 different decellularisation protocols were used for AVs, including detergent, enzymatic, and osmotic lysis methodologies. In vitro assays demonstrated that the detergent-based methodology was the most effective decellularisation protocol in terms of cell removal and preservation of the ECM [21]. In another study, porcine aortic valve leaflets were treated with 2 different decellularisation methods, including a Triton X-100/SDC and a detergent–enzyme digestion method. The results demonstrated that the Triton X-100/SDC method was better for cell removal and collagen fibre preservation [22]. In the study by Somers et al.,
304
R. Khorramirouz et al. / Advances in Medical Sciences 59 (2014) 299–307
Fig. 3. DAPI staining. (A) Control group of AVs. (B) Control group of conduit. (C) Decellularised valve treated with protocol 1. (D) Decellularised conduit treated with protocol 1. (E) Decellularised valve treated with protocol 2. (F) Decellularised conduit treated with protocol 2. (G) Decellularised valve treated with protocol 3. (H) Decellularised conduit treated with protocol 3.
4 different decellularisation methods were applied to porcine AVs by using Trypsin, osmotic, Trypsin-osmotic, or detergent-osmotic solutions. The results showed that the Trypsin-osmotic method was the most effective for nearly complete cell removal [7]. In one study in 2008 [23], 3 different decellularisation methods were used for the AV leaflets: SDS, Trypsin, and Triton X-100. The results demonstrated that the tri-layered structure of the leaflet decellularised with SDS was more similar to the tri-layered structure of the native AV, and the fibrosa layer was better stained compared to the other treated AVs. A certain amount of damage to the ECM will inevitably occur when the cells detach from the surrounding matrix in each of the decellularisation procedures. Some authors have reported that Trypsin-based decellularisation protocols [24] lead to extensive destruction of the elastin elements in the ECM of the heart valves [21]. Other studies showed satisfactory decellularisation of heart valves with ECM preservation after treatment with 0.1% Trypsin [7,25–27] or DNase and RNase [24]. This may be due to the differences in the origin of the semilunar valves (rat, porcine, and sheep) or the concentration of the consumed enzymes and the duration of enzymatic digestion [27,28]. In one recent study, an AVC decellularisation protocol with SDS in the presence of protease inhibitors was used. Histological analysis demonstrated that the major structural components were maintained [29]. In the current study, treatment with 1% Triton X-100 was the least effective method, in terms of cell removal, compared with the other decellularisation protocols. One of the goals of this study was to compare the effect of different decellularisation solutions on cell removal and ECM preservation in both the valvular and conduit sections. The durability of the AVC scaffolds, with respect to the increased mechanical stress on the conduit section, was also discussed in the current study. In the current study, hydroxyproline evaluation of the AVCs showed increased collagen content in all the treated samples compared to the native samples. Aortic samples from AVCs treated with each of the protocols had higher collagen content that the conduit samples, which may be due to their
Fig. 4. Scanning electron microscopy of AVs. (A) Control. (B) Decellularised valve with protocol 1. (C) Decellularised valve with protocol 2. (D) Decellularised valve with protocol 3.
R. Khorramirouz et al. / Advances in Medical Sciences 59 (2014) 299–307
305
Fig. 5. Scanning electron microscopy of conduit segments. (A) Control. (B) Decellularised valve with protocol 1. (C) Decellularised valve with protocol 2. (D) Decellularised valve with protocol 3.
naturally higher collagen content. We also demonstrated that among all the samples, those treated with the enzyme-based protocol had the lowest collagen content in the conduit and valvular segments. Our findings suggested that detergent-based methods using 1% SDS and 1% SDC (w/v) were the most effective decellularisation protocols for removing all cell types in the 3 layers of the AV leaflets and conduits. This method may be considered a practicable procedure to obtain an empty matrix for further in vivo evaluations. Accordingly, the ECM structure of both parts of the conduits was well preserved, with no loss or disruption of the major structural components. This scaffold can be considered as an acceptable and reliable substitute for transplantation of AVCs, especially in patients suffering from ascending aortic aneurysms and dissection. In contrast, treatment of conduits with the enzymebased protocol led to a notable deterioration of the ECM and incomplete decellularisation of the treated AVs. Although some limitations seem to exist, more studies are currently in progress to evaluate the efficacy of the present method of AVC decellularisation for surgical treatment of annuloaortic ectasia, dissections, and ascending aortic aneurysms.
A biocompatible decellularised AVC could be useful in patients with ascending aortic aneurysm, elongation, and severe AV incompetence. However, more studies should be performed to fully confirm this method of decellularisation as the gold standard for the treatment of AVC diseases. It should also be mentioned that the lack of degenerative traits makes these AVCs a promising alternative for further AV transplantation. This study demonstrated the state of the art in tissue engineering techniques for the development of decellularised AVCs from an experimental point of view. The results of the current study estimated the availability and feasibility of using AVC scaffolds for the clinical surgery. Additionally, growing and adaptively decellularised AVCs can be manufactured, and they will be able to prevent reoperations, especially in children. Although many issues regarding decellularised heart valve tissues still need to be resolved, our results provide new information on the safety of decellularised AVCs for transplantation. The findings of the current study will have a significant impact on future investigations in the field of aortic heart valve tissue engineering. Nevertheless, to ensure that a completely
Fig. 6. Comparison of biomechanical properties. (A) Transitional tensile strengths in native conduit, decellularised conduit with protocol 1, decellularised conduit with protocol 2, and decellularised conduit with protocol 3. (B) Longitudinal tensile strengths in native conduit, decellularised conduit with protocol 1, decellularised conduit with protocol 2, and decellularised conduit with protocol 3.
306
R. Khorramirouz et al. / Advances in Medical Sciences 59 (2014) 299–307
Fig. 7. Biochemical assays. (A) Hydroxyproline contents of native and decellularised AVs. (B) Hydroxyproline contents of native and decellularised conduits. Result of collagen analysis. Each column represents the mean value Standard Error.
cell-free matrix has been achieved that could be used for in vivo implantation; a DNA-assay should be performed, which is one of the limitations of the current study. More studies are required to determine if these scaffolds can effectively protect the patient from aneurysm rupture over the long-term after transplantation. Additional efforts should be also made to study the effect of detergent-based decellularisation protocols on ECM remodelling in the transplanted AVCs. 5. Conclusions From our findings, we conclude that decellularisation methods differ considerably in their cell removal efficacy and valve matrix component preservation. Detergent-based decellularisation solutions were more effective in removing the cells that are responsible for the immunogenicity of the semilunar tissues while preserving the ECM. Our newly developed detergent-based method produced an excellent scaffold that fulfils two main requirements, acellularity and well-preserved ECM components. Enzyme-based methods altered the semilunar structure of the valvular and conduit sections. In addition to the ECM damage, the cells were not completely removed. Different behaviours of the valvular and conduit segments of the AVCs after treatment with the same methods were observed. Despite all the improvements in producing engineered tissues and organs, there is still a long way to go to achieve practical tissue-engineered AVCs that are able to substitute for native ones. Conflict of interests No conflict of interest exists in relation to the submitted manuscript. Financial disclosure There was no source of extra-institutional commercial funding or funding received from National Institutes of Health (NIH), Wellcome Trust, Howard Hughes Medical Institute (HHMI) and others. References [1] Holmes DR Jr, Mack MJ, Kaul S, Agnihotri A, Alexander KP, Bailey SR, et al. 2012 ACCF/AATS/SCAI/STS expert consensus document on transcatheter aortic valve replacement. J Am Coll Cardiol 2012;59(13):1200–54. [2] Cabrol C, Gandjbakhc I, Pavie A. Surgical treatment of ascending aortic pathology. J Card Surg 1988;3(3):167–80. [3] de Almeida AS, Picon PD, Wender OC. Outcomes of patients subjected to aortic valve replacement surgery using mechanical or biological prostheses. Rev Bras Cir Cardiovasc 2011;26(3):326–37.
[4] Tedder ME, Simionescu A, Chen J, Liao J, Simionescu DT. Assembly and testing of stem cell-seeded layered collagen constructs for heart valve tissue engineering. Tissue Eng Part A 2011;17(1–2):25–36. [5] Lichtenberg A, Tudorache I, Cebotari S, Suprunov M, Tudorache G, Goerler H, et al. Preclinical testing of tissue-engineered heart valves re-endothelialized under simulated physiological conditions. Circulation 2006;114(Suppl. 1): I559–65. [6] Zhou J, Fritze O, Schleicher M, Wendel HP, Schenke-Layland K, Harasztosi C, et al. Impact of heart valve decellularization on 3-D ultrastructure, immunogenicity and thrombogenicity. Biomaterials 2010;31(9):2549–54. [7] Somers P, de Somer F, Cornelissen M, Thierens H, Van Nooten G. Decellularization of heart valve matrices: search for the ideal balance. Artif Cells Blood Substit Immobil Biotechnol 2012;40(1–2):151–62. [8] Woessner JF Jr. The determination of hydroxyproline in tissue and protein samples containing small proportions of this imino acid. Arch Biochem Biophys 1961;93(2):440–7. [9] Takkenberg JJ, Rajamannan NM, Rosenhek R, Kumar AS, Carapetis JR, Yacoub MH, et al. The need for a global perspective on heart valve disease epidemiology. The SHVD working group on epidemiology of heart valve disease founding statement. J Heart Valve Dis 2008;17(1):135–9. [10] Siu SC, Silversides CK. Bicuspid aortic valve disease. J Am Coll Cardiol 2010;55(25):2789–800. [11] Vesely I. Heart valve tissue engineering. Circ Res 2005;97(8):743–55. [12] Erdbrugger W, Konertz W, Dohmen PM, Posner S, Ellerbrok H, Brodde OE, et al. Decellularized xenogenic heart valves reveal remodeling and growth potential in vivo. Tissue Eng 2006;12(8):2059–68. [13] Shinoka T, Breuer CK, Tanel RE, Zund G, Miura T, Ma PX, et al. Tissue engineering heart valves: valve leaflet replacement study in a lamb model. Ann Thorac Surg 1995;60(Suppl. 6):S513–6. [14] Shinoka T, Shum-Tim D, Ma PX, Tanel RE, Langer R, Vacanti JP, et al. Tissueengineered heart valve leaflets: does cell origin affect outcome? Circulation 1997;96(Suppl. 9):II-102-7. [15] Dohmen PM, Lembcke A, Holinski S, Kivelitz D, Braun JP, Pruss A, et al. Midterm clinical results using a tissue-engineered pulmonary valve to reconstruct the right ventricular outflow tract during the Ross procedure. Ann Thorac Surg 2007;84(3):729–36. [16] Quinn RW, Hilbert SL, Bert AA, Drake BW, Bustamante JA, Fenton JE, et al. Performance and morphology of decellularized pulmonary valves implanted in juvenile sheep. Ann Thorac Surg 2011;92(1):131–7. [17] Grabow N, Schmohl K, Khosravi A, Philipp M, Scharfschwerdt M, Graf B, et al. Mechanical and structural properties of a novel hybrid heart valve scaffold for tissue engineering. Artif Organs 2004;28(11):971–9. [18] Stock UA, Schenke-Layland K. Performance of decellularized xenogeneic tissue in heart valve replacement. Biomaterials 2006;27(1):1–2. [19] Kirali K. Stentless Bioprostheses for Aortic Valve Replacement in Calcific Aortic Stenosis. In: Aikawa E, editor. Calcific Aortic Valve Disease. Rijeka (Croatia): InTech; 2013. http://dx.doi.org/10.5772/46239. Chapter 14. [20] Meyer SR, Nagendran J, Desai LS, Rayat GR, Churchill TA, Anderson CC, et al. Decellularization reduces the immune response to aortic valve allografts in the rat. J Thorac Cardiovasc Surg 2005;130(2):469–76. [21] Meyer SR, Chiu B, Churchill TA, Zhu L, Lakey JR, Ross DB. Comparison of aortic valve allograft decellularization techniques in the rat. J Biomed Mater Res A 2006;79(2):254–62. [22] Yu BT, Li WT, Song BQ, Wu YL. Comparative study of the Triton X-100-sodium deoxycholate method and detergent-enzymatic digestion method for decellularization of porcine aortic valves. Eur Rev Med Pharmacol Sci 2013;17(16): 2179–84. [23] Liao J, Joyce EM, Sacks MS. Effects of decellularization on the mechanical and structural properties of the porcine aortic valve leaflet. Biomaterials 2008;29(8): 1065–74. [24] Grauss RW, Hazekamp MG, van Vliet S, Gittenberger-de Groot AC, DeRuiter MC. Decellularization of rat aortic valve allografts reduces leaflet destruction and extracellular matrix remodeling. J Thorac Cardiovasc Surg. 2003;126(6): 2003–10.
R. Khorramirouz et al. / Advances in Medical Sciences 59 (2014) 299–307 [25] Tudorache I, Cebotari S, Sturz G, Kirsch L, Hurschler C, Hilfiker A, et al. Tissue engineering of heart valves: biomechanical and morphological properties of decellularized heart valves. J Heart Valve Dis 2007;16(5):567–73. discussion 574. [26] Cebotari S, Mertsching H, Kallenbach K, Kostin S, Repin O, Batrinac A, et al. Construction of autologous human heart valves based on an acellular allograft matrix. Circulation 2002;106(12 (Suppl. 1)):I63–8. [27] Schenke-Layland K, Vasilevski O, Opitz F, Ko¨nig K, Riemann I, Halbhuber K, et al. Impact of decellularization of xenogeneic tissue on extracellular matrix
307
integrity for tissue engineering of heart valves. J Struct Biol 2003;143(3): 201–8. [28] Crapo PM, Gilbert TW, Badylak SF. An overview of tissue and whole organ decellularization processes. Biomaterials 2011;32(12):3233–43. [29] Booth C, Korossis S, Wilcox H, Watterson K, Kearney J, Fisher J, et al. Tissue engineering of cardiac valve protheses I: Development and histological characterization of an acellular porcine scaffold. J Heart Valve Dis 2002;11(4): 457–62.
Contents lists available at ScienceDirect
Advances in Medical Sciences journal homepage: www.elsevier.com/locate/advms
Original Research Article
Effect of three decellularisation protocols on the mechanical behaviour and structural properties of sheep aortic valve conduits Reza Khorramirouz a, Shabnam Sabetkish a, Aram Akbarzadeh a, Ahad Muhammadnejad b, Reza Heidari a, Abdol-Mohammad Kajbafzadeh a,* a
Pediatric Urology Research Center, Section of Tissue Engineering and Stem Cells Therapy, Children’s Hospital Medical Center, Tehran University of Medical Sciences, Tehran, Iran Cancer Research Center, Iranian Cancer Institute, Department of Pathology, Tehran University of Medical Sciences, Tehran, Iran
b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 28 February 2014 Accepted 13 August 2014 Available online 26 August 2014
Purpose: To determine the best method for decellularisation of aortic valve conduits (AVCs) that efficiently removes the cells while preserving the extracellular matrix (ECM) by examining the valvular and conduit sections separately. Material/methods: Sheep AVCs were decellularised by using three different protocols: detergent-based (1% SDS + 1% SDC), detergent and enzyme-based (Triton + EDTA + RNase and DNase), and enzyme-based (Trypsin + RNase and DNase) methods. The efficacy of the decellularisation methods to completely remove the cells while preserving the ECM was evaluated by histological evaluation, scanning electron microscopy (SEM), hydroxyproline analysis, tensile test, and DAPI staining. Results: The detergent-based method completely removed the cells and left the ECM and collagen content in the valve and conduit sections relatively well preserved. The detergent and enzyme-based protocol did not completely remove the cells, but left the collagen content in both sections well preserved. ECM deterioration was observed in the aortic valves (AVs), but the ultrastructure of the conduits was well preserved, with no media distortion. The enzyme-based protocol removed the cells relatively well; however, mild structural distortion and poor collagen content was observed in the AVs. Incomplete cell removal (better than that observed with the detergent and enzyme-based protocol), poor collagen preservation, and mild structural distortion were observed in conduits treated with the enzyme-based method. Conclusions: The results suggested that the detergent-based methods are the most effective protocols for cell removal and ECM preservation of AVCs. The AVCs treated with this detergent-based method may be excellent scaffolds for recellularisation. ß 2014 Medical University of Bialystok. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.
Keywords: Aorta Heart valve Aortic conduit Acellular Sheep
1. Introduction Valvular heart diseases are important causes of mortality. Congenital and acquired valvular dysfunction is frequently treated by heart valve replacement, which is a common procedure worldwide [1]. Aneurysmal rupture is one of the leading causes of death, with an incidence of 3–4% [2]. However, the current treatments for aortic valve defects in children are not satisfactory,
* Corresponding author at: Pediatric Urology Research Center, Section of Tissue Engineering and Stem Cells Therapy, Children’s Hospital Medical Center, Tehran University of Medical Sciences, No. 62, Dr. Qarib’s Street, Keshavarz Boulevard, 1419433151 Tehran, Iran. Tel.: +98 21 66565400; fax: +98 21 66565400. E-mail address: [email protected] (A.-M. Kajbafzadeh).
and the incidence of regurgitation and stenosis are more common in children than in older patients. Children and neonates treated with mechanical valve prostheses can be confronted with significant postoperative complications due to the lack of appropriately sized prostheses. Therefore, to minimise probable postoperative complications, ideally, in terms of cell removal and mechanical properties, a decellularised aortic valve conduit (AVC) should be used as a substitute for juvenile patients. Surgical treatment of ascending aortic aneurysms accompanied with AV regurgitation is still challenging, and the management is a debatable matter. Therefore, alternative treatments are needed to reduce the incidence of this life-threatening event. Mechanical and bio-prosthetic valves are the two conventional substitutes for dysfunctional heart valves. However, the application
http://dx.doi.org/10.1016/j.advms.2014.08.004 1896-1126/ß 2014 Medical University of Bialystok. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.
300
R. Khorramirouz et al. / Advances in Medical Sciences 59 (2014) 299–307
of these replacement valves is associated with several disadvantages, including their inability to grow, which is a particular problem in patients that are still growing, structural valve deterioration, the need for long-term anticoagulation therapy, immunological responses, and the absence of remodelling [3]. To overcome these limitations, decellularised heart valve conduits can be used as alternatives with physiological functions that are similar to those of natural valves, with the least immunogenic potential [4]. The process of engineering AVCs includes an effective method for decellularisation of both the valve and conduit sections for further in vitro or in vivo cell seeding. A critical point of the decellularisation process is the preservation of the extracellular matrix (ECM) in both sections. The goal of decellularisation is maximum cell and nuclei removal to minimise the cellular debris remaining in the AVCs and greatly lessen the adverse immune responses, inflammation, fibrous scarring, and calcification [4–7]. A practical scaffold should be capable of self-repair and remodelling, be durable, and not induce rejection. Therefore, it is necessary to determine the best decellularisation protocol for complete cell removal and preservation of the tissue microstructural features of the AVCs. In this study, we compared three different protocols for the decellularisation of AVCs, and we introduced an ideal method that strikes a balance between maximum cell removal and preservation of tissue microstructure, with optimal biochemical properties to avoid possible complications encountered after transplantation. 2. Material and methods 2.1. Tissue preparation The ethical committee of Tehran University of Medical Sciences approved this study. Twelve sheep, with a mean body weight of 47 5 kg, were sedated by an injection of xylazine (0.15 mL per 10 kg body weight; IM). The animals were anesthetised by administration of atropine sulphate (11 mg per 10 kg body weight), propofol (5 mg per kg body weight), and diazepam (0.27 mg per kg body weight). Hearts were harvested under sterile conditions and placed in Hank’s balanced salt solution (HBSS) containing antibiotics (penicillin, gentamycin, streptomycin, and amphotericin). AVCs were precisely dissected and decellularised. Harvested AVCs had a thin rim of subvalvular muscle tissue and a short arterial segment. A total of 12 AVCs were included in the study, and they were randomly divided into 3 decellularisation groups and a control group. 2.2. Decellularisation process Nine AVCs were treated with 3 different decellularisation protocols (detergent-, enzyme-, and detergent and enzyme-based), with 3 AVCs per group. Before starting each treatment, all valves and conduits were placed in PBS for 90 min, and the solution was changed every 30 min to rinse the tissues. Then, each group was treated as described below. All materials were purchased from Sigma–Aldrich (Belgium) unless stated otherwise. 2.2.1. Protocol 1 (1% SDS + 1% SDC) In this detergent-based decellularisation method, AVCs were treated with 1% (w/v) sodium dodecyl sulphate (SDS) and 1% (w/v) sodium deoxycholate (SDC) dissolved in distilled water for 48 h at room temperature. Then, they were washed with distilled water 2 times at 4 8C for 12 h each. In the final stage of the decellularisation procedure, the AVCs were washed in phosphate buffered saline (PBS) (137 mmol/L NaCl, 2.7 mmol/L KCl, 1.78 mmol Na2HPO42H2O, and 2 mmol/L KH2PO4, pH 7.4) for 24 h by changing the solution every 12 h. This step was performed to eliminate the applied detergents.
2.2.2. Protocol 2 (1% Triton + EDTA + RNase and DNase) In this detergent and enzyme-based method, the AVCs were placed in 1% (w/v) Triton X-100 with a combination of 1 mmol/L ethylenediaminetetraacetic acid (EDTA), 20 mg/mL RNase, and 200 mg/mL DNase (dissolved in PBS) at 37 8C for 24 h. In the second step, samples were incubated in HBSS (137 mmol/L NaCl, 5.4 mmol/L KCl, 0.5 mmol/L KH2PO4, 4.1 mmol/L NaHCO3, and 0.33 mmol/L Na2HPO4 without Ca, Mg, and phenol red) at 4 8C for 72 h. The solution was changed every 12 h (6 times). 2.2.3. Protocol 3 (0.1% Trypsin + RNase and DNase) All the steps in this protocol were similar to those in protocol 2. The only difference was the use of 0.1% (w/v) Trypsin (TS) instead of Triton X-100. All the above-mentioned steps were conducted under continuous shaking on a mechanical shaker at 50 revolutions per minute (rpm). 2.3. Histological examination and DAPI staining To evaluate and compare the effectiveness of our decellularisation protocols in removing the cellular components of both the valvular and conduit sections, native and decellularised AVCs were cut into 3 mm 4 mm sections and fixed in 10% neutral buffered formalin (Merck, Darmstadt, Germany) at pH 7.4 for 48 h at room temperature. Then, they were washed in distilled water, dehydrated in a graded series of ethanol, embedded in paraffin, and cut into 5–8-mm thick sections. To visualise remnant deoxyribonucleic acids (DNA), the sections were stained with 40 ,6-diamidino-2-phenylindole (DAPI; Biotium, Inc.). To enhance fluorescence, the DAPI solution was diluted to 30 nM in PBS, and 300 mL was pipetted directly onto all samples. After incubation in the dark for 30 min and rinsing with distilled water, the slides were evaluated with a fluorescent microscope. Images were taken with a Nikon digital camera (DXM 1200). Picrosirius red and Russell–Movat pentachrome staining were also used for collagen typing and histological evaluation of the ECM. For Picrosirius red staining, paraffin-embedded blocks were cut into 5-mm slices. The sections were deparaffinised and hydrated in graded series of alcohol. The slides were stained for 1 h in Picrosirius red solution (0.1% Sirius red in saturated picric acid), followed by two washes of 0.5% acetic acid without counterstaining. Under polarised microscopy, type I collagen fibres are seen as thick, strongly birefringent, yellow-orange fibres, and type III fibres are seen as thin, weakly birefringence, greenish fibres. Different connective tissue constituents are highlighted by pentachrome staining. Using this technique, collagen, elastin, muscle, mucin, and fibrin can be differentiated. For Movat pentachrome staining, paraffin-embedded blocks were cut into 5-mm slices, deparaffinised, and hydrated in a graded series of alcohol. Briefly, mucin was stained with Alcian blue 8GS and converted to insoluble blue pigment by treatment with alkaline alcohol; nuclei and elastic fibres were stained black with Verhoeff’s haematoxylin; fibrinoid, fibrin, and muscle were stained red with Crocein scarlet-acid fuchsin solution; and collagen and reticular fibres were stained yellow with Saffron. 2.4. Scanning electron microscopy (SEM) Valvular and conduit sections of AVCs treated with the 3 decellularisation protocols as well as fresh control specimens were fixed with 2.5% glutaraldehyde (Merck, Darmstadt, Germany). Then, they were washed in PBS 3 times for 60 min each to remove the glutaraldehyde. Both native and decellularised samples were fixed with 1% osmium tetroxide, 0.8% potassium
R. Khorramirouz et al. / Advances in Medical Sciences 59 (2014) 299–307
301
ferrocyanide, and 1% tannic acid. Then, the samples were completely dehydrated using a graded series of ethanol (30%, 50%, 70%, 90%, and 100%; Merck) for 10 min each. Finally, the samples were sputter coated with gold to improve the conductivity of electrons and to visualise the surfaces via SEM (Hitachi-54160, Japan). Finally, all the samples were examined in a Jeol JSM-6340F Field Emission Scanning Electron Microscope. The acceleration voltage was adjusted to 10 kV, and a working distance of 8 mm was used. Secondary and backscattered electrons were used to obtain the images.
endothelial layer in the intima layer with interstitial cells and smooth muscle cells in the media layer. The collagen fibres in the media layer were stained by Movat pentachrome staining. Elastin fibres were also present in the media layer. Picrosirius staining showed the presence of collagen type I in the media layer (Figs. 1A and 2A). DAPI staining showed cellularity in all 3 layers (Fig. 3A and B), and SEM examination showed an intact ultrastructure within the endothelial layer (Figs. 4A and 5A).
2.5. Mechanical properties A tensile-test device (Hct 400/25; Zwick/Roell, Germany) was used to compare the tensile strength of the native and decellularised conduit sections. Both longitudinal and transitional tensile strength was measured in 2 separate tests. To calculate the physical properties of the longitudinal (maximum strength and Young’s modulus) and transitional fibres (force/displacement), samples were cut into 4 mm 4 mm strips. The specimens were clamped in sample holders with a constant elongation rate of 0.1 mm/s (6 mm/min) at room temperature. Each sample was subjected to mounting uniaxial tensile testing until the tissue tore and the load disappeared, as demonstrated by the device. The maximal point indicated by the device was considered the maximum pressure tolerance. Four samples from each group were tested, and the results were averaged.
H&E and DAPI staining confirmed that the detergent-based method was the most effective protocol for removing cells and intracellular debris from all parts of the AVCs. The samples were well decellularised in the intima, media, and adventitious layers, with relative structural integrity. The ECM in both the valvular and conduit sections was satisfactorily preserved. Pentachrome staining of the AVCs showed preservation of elastin and collagen fibres, especially in the media layer. Pentachrome staining of the conduits demonstrated mild a decrease in elastin content compared to the control samples. Picrosirius red staining of valvular and conduit sections showed collagen preservation in the media layer (Figs. 1B, 2B, 3C and D). Under SEM, no cellularity or structural distortion were detectable in either the inflow or outflow sides of the valves and conduits in the decellularised constructs. Therefore, the ultrastructure was preserved after the decellularisation procedure (Figs. 4B and 5B).
2.6. Hydroxyproline content
3.3. Protocol 2: (1% Triton + EDTA + RNase and DNase)
The collagen content of the valvular and conduit portions of the native and decellularised AVCs was quantitatively measured by hydroxyproline evaluation. The collagen content of 4 replicates of the native and decellularised heart valve samples was analysed. A 10-mg/mL 4-hydroxy-L-proline stock solution was diluted to make hydroxyproline standards (0.75–25 mg/mL). The collagen content per wet weight of the native and decellularised samples was assessed by measuring the total amount of hydroxyproline according to a previously described method [8]. Tissue samples were hydrolysed in 5 mL of 6 N HCl at 110 8C for 14–16 h. In the next step, the samples were incubated in 500 mL of chloramine-T solution (0.14 g chloramine-T, 2 mL of distilled water, and 8 mL of citrate/acetate buffer) at room temperature for 20 min. One millilitre of Ehrlich’s reagent (10.13 g of p-dimethylaminobenzaldehyde, 41.85 mL of 1-propanol, and 17.55 mL of 70% perchloric acid; Fluka Chemicals) was then added to the specimens. The resulting mixture was incubated at 65 8C for 20 min, and then the absorbance at 546 nm was measured with a spectrophotometer (BioTek Synergy HT1 microplate reader). The hydroxyproline concentration was calculated as the mg of hydroxyproline per g of wet tissue (mg/g wet tissue). All measurements were expressed as mean standard error of the mean (SEM). Data were analysed using SPSS version 16 (SPSS Inc., Chicago, IL, USA). Differences between groups were analysed by one-way ANOVA and Duncan’s tests. A p value less than 0.05 was considered statistically significant.
In the conduits and in each layer of the AVs treated with this protocol, cells and nuclei were observed, especially in the media layer by both H&E and DAPI staining. Pentachrome staining of the AVs showed good collagen preservation in all 3 layers. In the conduit portion, the collagen and elastin fibres were well preserved in the media layer (Figs. 1C, 2C, 3E and F). SEM examination of the AVs confirmed the distortion of the ultrastructure, with multiple fenestrations as well as a patchy cell distribution. However, cells remained in the conduits and the ultrastructure was well preserved with no media distortion (Figs. 4C and 5C).
3.2. Protocol 1: (1% SDS + 1% SDC)
3.4. Protocol 3: (0.1% Trypsin + RNase and DNase) The worst results were observed in the AVCs treated with this protocol. Picrosirius staining revealed poor collagen type I staining in both the valves and conduits. The results of the Pentachrome staining were in agreement with the Picrosirius staining regarding the collagen content in all 3 layers. Furthermore, the elastin fibres were completely eliminated (Figs. 1D and 2D). Using this enzymatic decellularisation method, the AV samples were well decellularised in all 3 layers as confirmed by DAPI staining (Fig. 3G and H). However, the conduits were not well decellularised in all 3 layers, although the remaining cellularity was less than that observed with the Triton-based protocol. Mild structural distortion and pore formation in the valvular and conduit sections was confirmed by SEM evaluation (Figs. 4D and 5D).
3. Results 3.5. Mechanical properties 3.1. Control cellular AVCs A normal meshwork of structural proteins was observed under light microscopy. Ultrastructure examination by SEM also showed a confluent monolayer of endothelial cells covering both the inflow and outflow tracts. This pattern is referred to as cobblestone morphology. H&E staining of the cellular AVCs showed a confluent
All decellularised aortic conduits underwent tensile testing to evaluate the resistance to biomechanical forces. Protocol 1 showed the following mechanical properties: maximal longitudinal strength = 0.39 MPa, Young’s modulus = 0.7 MPa, and transitional strength = 42 N/mm. However, the biophysical properties of the native conduits were as follow: maximal longitudinal
302
R. Khorramirouz et al. / Advances in Medical Sciences 59 (2014) 299–307
Fig. 1. H&E, Red Sirius (light microscope), Red Sirius (fluorescence microscope), and Pentachrome staining of AVs. (A) Control. (B) Decellularised valve with SDS 1% and SDC 1%. (C) Decellularised valve with Triton–EDTA–RNase and DNase. (D) Decellularised valve with Trypsin and RNase and DNase.
strength = 0.47 MPa, Young’s modulus = 0.81 MPa, and transitional strength = 43 N/mm). Conduits treated by using protocol 2 exhibited the following biophysical properties: maximal longitudinal strength = 0.3 MPa, Young’s modulus = 0.6 MPa, and transitional strength = 33 N/mm. The conduits treated by using the third protocol had the following biophysical properties: maximal longitudinal strength = 0.19 MPa, Young’s modulus = 0.39 MPa, and transitional strength = 20 N/mm. Overall, at strain smaller than 0.5, protocol 3 is closest to the native tissue, but its final strength is much lower than the native tissue. The final strength of protocol 1 is closest to the native tissue, but is much stiffer at small strains and getting softer when strain increases. Moreover, at small displacement, protocol 2 is the closest to the native tissue (Fig. 6). However, regarding the fact that the modulus is the slop of each stress-strain curve and the slop of each curve is changing with strain; the overall averaged modulus is not a good value to compare. 3.6. Hydroxyproline evaluation The hydroxyproline content of the conduit sections treated by using protocol 1 (0.66 0.01 mg/mg), protocol 2 (0.69 0.01 mg/ mg), and protocol 3 (0.65 0.02 mg/mg) was significantly higher (p < 0.05) than that of the native tissue (0.54 0.02 mg/mg).
The collagen content of the AVs decellularised with protocol 1 (0.24 0.1 mg/g) and protocol 2 (0.18 0.008 mg/g) was significantly higher (p < 0.05) than that of the native AV (0.16 0.01 mg/g) (Fig. 7). 4. Discussion In the current study, the characteristics of AVCs treated by using distinct decellularisation methods were compared to determine the optimal approach for cell removal and ECM preservation. The comparative approach of this study, in which we separately examined the valvular and conduit sections of decellularised AVCs obtained from the same decellularisation solutions, is a concept that has not been previously described. In addition, in the current study, we aimed to determine the best AVC decellularisation protocol for complete cell removal and to provide greater insight into the initial functional state of the decellularised AVC. Our results demonstrated that a detergent-based method has gained credibility because it completely removed the cells while preserving the ECM in both the valve and conduit sections relatively well which may reduce the probable postoperative complications. Cardiovascular disease is still the leading cause of death worldwide. In 2006, over 300,000 heart valve surgeries were performed, and this number is expected to triple by 2050 [9]. However, their effects on public health are significantly
R. Khorramirouz et al. / Advances in Medical Sciences 59 (2014) 299–307
303
Fig. 2. H&E, Red Sirius (light microscope), Red Sirius (fluorescence microscope), and Pentachrome staining of conduit sections. (A) Control. (B) Decellularised conduit with SDS 1% and SDC 1%. (C) Decellularised conduit with Triton–EDTA–RNase and DNase. (D) Decellularised conduit with Trypsin and RNase and DNase.
understated. Owing to these alarming statistics, an urgent call to action is necessary. Several decellularised tissues and organs treated by using different decellularisation protocols have been applied in tissue engineering and regenerative medicine. The efficacies reported in the literature for the methods that are most commonly used to generate decellularised heart valves differ widely. In addition, concomitant decellularisation of valvular and conduit segments of sheep AVCs has not been previously reported. Abnormalities of the aortic media can result in dilation of the proximal ascending aorta. This dilation is the most common congenital defect of the bicuspid aortic valve [10]. Decellularised heart valves can play a crucial role in the treatment of congenital valvular heart diseases. They are capable of growth, repair, and remodelling which makes them a practical substitute for valvular transplantation, especially in the paediatric population [11,12]. In early studies, attempts were made to construct engineered valves from polyglycolic acid fibre matrix with myofibroblasts and endothelial cells, the results of which were satisfactory in developing leaflets with similar functional and morphological properties compared with native structures [13,14]. Later studies introduced tissue-engineered heart valves treated with decellularisation processes, and investigated the fate of seeded cells in these manipulated tissues, with promising results both in vitro and in vivo [5,15,16]. Despite increasing interest among researchers to develop bioengineered heart valves, there are still many challenges in this field, and greater insight into the behaviour of AVCs after
decellularisation is needed. Various approaches have been used to develop valves with satisfactory properties, including construction of a 3D valvular structure using resorbable synthetic polymers, the development of decellularised xenogeneic tissues followed by direct implantation or in vitro cell seeding, and fabrication of hybrid heart valves from decellularised xenogeneic tissues enhanced by bioresorbable polymers [17,18]. These decellularised scaffolds can replace mechanical valves in patients confronted with several complications, such as systemic embolisation, bleeding, and valve obstruction due to thrombosis or pannus formation [19]. An effective decellularisation method for heart valves must remove the cellular and nuclear components that are responsible for evoking an immune response and causing subsequent damage to the valvular structure. It is worth noting that the damage induced by the decellularisation techniques is much less than the damage incurred by the recipient’s immune response to the transplanted tissue-engineered grafts [20]. In the study by Steven et al., 3 different decellularisation protocols were used for AVs, including detergent, enzymatic, and osmotic lysis methodologies. In vitro assays demonstrated that the detergent-based methodology was the most effective decellularisation protocol in terms of cell removal and preservation of the ECM [21]. In another study, porcine aortic valve leaflets were treated with 2 different decellularisation methods, including a Triton X-100/SDC and a detergent–enzyme digestion method. The results demonstrated that the Triton X-100/SDC method was better for cell removal and collagen fibre preservation [22]. In the study by Somers et al.,
304
R. Khorramirouz et al. / Advances in Medical Sciences 59 (2014) 299–307
Fig. 3. DAPI staining. (A) Control group of AVs. (B) Control group of conduit. (C) Decellularised valve treated with protocol 1. (D) Decellularised conduit treated with protocol 1. (E) Decellularised valve treated with protocol 2. (F) Decellularised conduit treated with protocol 2. (G) Decellularised valve treated with protocol 3. (H) Decellularised conduit treated with protocol 3.
4 different decellularisation methods were applied to porcine AVs by using Trypsin, osmotic, Trypsin-osmotic, or detergent-osmotic solutions. The results showed that the Trypsin-osmotic method was the most effective for nearly complete cell removal [7]. In one study in 2008 [23], 3 different decellularisation methods were used for the AV leaflets: SDS, Trypsin, and Triton X-100. The results demonstrated that the tri-layered structure of the leaflet decellularised with SDS was more similar to the tri-layered structure of the native AV, and the fibrosa layer was better stained compared to the other treated AVs. A certain amount of damage to the ECM will inevitably occur when the cells detach from the surrounding matrix in each of the decellularisation procedures. Some authors have reported that Trypsin-based decellularisation protocols [24] lead to extensive destruction of the elastin elements in the ECM of the heart valves [21]. Other studies showed satisfactory decellularisation of heart valves with ECM preservation after treatment with 0.1% Trypsin [7,25–27] or DNase and RNase [24]. This may be due to the differences in the origin of the semilunar valves (rat, porcine, and sheep) or the concentration of the consumed enzymes and the duration of enzymatic digestion [27,28]. In one recent study, an AVC decellularisation protocol with SDS in the presence of protease inhibitors was used. Histological analysis demonstrated that the major structural components were maintained [29]. In the current study, treatment with 1% Triton X-100 was the least effective method, in terms of cell removal, compared with the other decellularisation protocols. One of the goals of this study was to compare the effect of different decellularisation solutions on cell removal and ECM preservation in both the valvular and conduit sections. The durability of the AVC scaffolds, with respect to the increased mechanical stress on the conduit section, was also discussed in the current study. In the current study, hydroxyproline evaluation of the AVCs showed increased collagen content in all the treated samples compared to the native samples. Aortic samples from AVCs treated with each of the protocols had higher collagen content that the conduit samples, which may be due to their
Fig. 4. Scanning electron microscopy of AVs. (A) Control. (B) Decellularised valve with protocol 1. (C) Decellularised valve with protocol 2. (D) Decellularised valve with protocol 3.
R. Khorramirouz et al. / Advances in Medical Sciences 59 (2014) 299–307
305
Fig. 5. Scanning electron microscopy of conduit segments. (A) Control. (B) Decellularised valve with protocol 1. (C) Decellularised valve with protocol 2. (D) Decellularised valve with protocol 3.
naturally higher collagen content. We also demonstrated that among all the samples, those treated with the enzyme-based protocol had the lowest collagen content in the conduit and valvular segments. Our findings suggested that detergent-based methods using 1% SDS and 1% SDC (w/v) were the most effective decellularisation protocols for removing all cell types in the 3 layers of the AV leaflets and conduits. This method may be considered a practicable procedure to obtain an empty matrix for further in vivo evaluations. Accordingly, the ECM structure of both parts of the conduits was well preserved, with no loss or disruption of the major structural components. This scaffold can be considered as an acceptable and reliable substitute for transplantation of AVCs, especially in patients suffering from ascending aortic aneurysms and dissection. In contrast, treatment of conduits with the enzymebased protocol led to a notable deterioration of the ECM and incomplete decellularisation of the treated AVs. Although some limitations seem to exist, more studies are currently in progress to evaluate the efficacy of the present method of AVC decellularisation for surgical treatment of annuloaortic ectasia, dissections, and ascending aortic aneurysms.
A biocompatible decellularised AVC could be useful in patients with ascending aortic aneurysm, elongation, and severe AV incompetence. However, more studies should be performed to fully confirm this method of decellularisation as the gold standard for the treatment of AVC diseases. It should also be mentioned that the lack of degenerative traits makes these AVCs a promising alternative for further AV transplantation. This study demonstrated the state of the art in tissue engineering techniques for the development of decellularised AVCs from an experimental point of view. The results of the current study estimated the availability and feasibility of using AVC scaffolds for the clinical surgery. Additionally, growing and adaptively decellularised AVCs can be manufactured, and they will be able to prevent reoperations, especially in children. Although many issues regarding decellularised heart valve tissues still need to be resolved, our results provide new information on the safety of decellularised AVCs for transplantation. The findings of the current study will have a significant impact on future investigations in the field of aortic heart valve tissue engineering. Nevertheless, to ensure that a completely
Fig. 6. Comparison of biomechanical properties. (A) Transitional tensile strengths in native conduit, decellularised conduit with protocol 1, decellularised conduit with protocol 2, and decellularised conduit with protocol 3. (B) Longitudinal tensile strengths in native conduit, decellularised conduit with protocol 1, decellularised conduit with protocol 2, and decellularised conduit with protocol 3.
306
R. Khorramirouz et al. / Advances in Medical Sciences 59 (2014) 299–307
Fig. 7. Biochemical assays. (A) Hydroxyproline contents of native and decellularised AVs. (B) Hydroxyproline contents of native and decellularised conduits. Result of collagen analysis. Each column represents the mean value Standard Error.
cell-free matrix has been achieved that could be used for in vivo implantation; a DNA-assay should be performed, which is one of the limitations of the current study. More studies are required to determine if these scaffolds can effectively protect the patient from aneurysm rupture over the long-term after transplantation. Additional efforts should be also made to study the effect of detergent-based decellularisation protocols on ECM remodelling in the transplanted AVCs. 5. Conclusions From our findings, we conclude that decellularisation methods differ considerably in their cell removal efficacy and valve matrix component preservation. Detergent-based decellularisation solutions were more effective in removing the cells that are responsible for the immunogenicity of the semilunar tissues while preserving the ECM. Our newly developed detergent-based method produced an excellent scaffold that fulfils two main requirements, acellularity and well-preserved ECM components. Enzyme-based methods altered the semilunar structure of the valvular and conduit sections. In addition to the ECM damage, the cells were not completely removed. Different behaviours of the valvular and conduit segments of the AVCs after treatment with the same methods were observed. Despite all the improvements in producing engineered tissues and organs, there is still a long way to go to achieve practical tissue-engineered AVCs that are able to substitute for native ones. Conflict of interests No conflict of interest exists in relation to the submitted manuscript. Financial disclosure There was no source of extra-institutional commercial funding or funding received from National Institutes of Health (NIH), Wellcome Trust, Howard Hughes Medical Institute (HHMI) and others. References [1] Holmes DR Jr, Mack MJ, Kaul S, Agnihotri A, Alexander KP, Bailey SR, et al. 2012 ACCF/AATS/SCAI/STS expert consensus document on transcatheter aortic valve replacement. J Am Coll Cardiol 2012;59(13):1200–54. [2] Cabrol C, Gandjbakhc I, Pavie A. Surgical treatment of ascending aortic pathology. J Card Surg 1988;3(3):167–80. [3] de Almeida AS, Picon PD, Wender OC. Outcomes of patients subjected to aortic valve replacement surgery using mechanical or biological prostheses. Rev Bras Cir Cardiovasc 2011;26(3):326–37.
[4] Tedder ME, Simionescu A, Chen J, Liao J, Simionescu DT. Assembly and testing of stem cell-seeded layered collagen constructs for heart valve tissue engineering. Tissue Eng Part A 2011;17(1–2):25–36. [5] Lichtenberg A, Tudorache I, Cebotari S, Suprunov M, Tudorache G, Goerler H, et al. Preclinical testing of tissue-engineered heart valves re-endothelialized under simulated physiological conditions. Circulation 2006;114(Suppl. 1): I559–65. [6] Zhou J, Fritze O, Schleicher M, Wendel HP, Schenke-Layland K, Harasztosi C, et al. Impact of heart valve decellularization on 3-D ultrastructure, immunogenicity and thrombogenicity. Biomaterials 2010;31(9):2549–54. [7] Somers P, de Somer F, Cornelissen M, Thierens H, Van Nooten G. Decellularization of heart valve matrices: search for the ideal balance. Artif Cells Blood Substit Immobil Biotechnol 2012;40(1–2):151–62. [8] Woessner JF Jr. The determination of hydroxyproline in tissue and protein samples containing small proportions of this imino acid. Arch Biochem Biophys 1961;93(2):440–7. [9] Takkenberg JJ, Rajamannan NM, Rosenhek R, Kumar AS, Carapetis JR, Yacoub MH, et al. The need for a global perspective on heart valve disease epidemiology. The SHVD working group on epidemiology of heart valve disease founding statement. J Heart Valve Dis 2008;17(1):135–9. [10] Siu SC, Silversides CK. Bicuspid aortic valve disease. J Am Coll Cardiol 2010;55(25):2789–800. [11] Vesely I. Heart valve tissue engineering. Circ Res 2005;97(8):743–55. [12] Erdbrugger W, Konertz W, Dohmen PM, Posner S, Ellerbrok H, Brodde OE, et al. Decellularized xenogenic heart valves reveal remodeling and growth potential in vivo. Tissue Eng 2006;12(8):2059–68. [13] Shinoka T, Breuer CK, Tanel RE, Zund G, Miura T, Ma PX, et al. Tissue engineering heart valves: valve leaflet replacement study in a lamb model. Ann Thorac Surg 1995;60(Suppl. 6):S513–6. [14] Shinoka T, Shum-Tim D, Ma PX, Tanel RE, Langer R, Vacanti JP, et al. Tissueengineered heart valve leaflets: does cell origin affect outcome? Circulation 1997;96(Suppl. 9):II-102-7. [15] Dohmen PM, Lembcke A, Holinski S, Kivelitz D, Braun JP, Pruss A, et al. Midterm clinical results using a tissue-engineered pulmonary valve to reconstruct the right ventricular outflow tract during the Ross procedure. Ann Thorac Surg 2007;84(3):729–36. [16] Quinn RW, Hilbert SL, Bert AA, Drake BW, Bustamante JA, Fenton JE, et al. Performance and morphology of decellularized pulmonary valves implanted in juvenile sheep. Ann Thorac Surg 2011;92(1):131–7. [17] Grabow N, Schmohl K, Khosravi A, Philipp M, Scharfschwerdt M, Graf B, et al. Mechanical and structural properties of a novel hybrid heart valve scaffold for tissue engineering. Artif Organs 2004;28(11):971–9. [18] Stock UA, Schenke-Layland K. Performance of decellularized xenogeneic tissue in heart valve replacement. Biomaterials 2006;27(1):1–2. [19] Kirali K. Stentless Bioprostheses for Aortic Valve Replacement in Calcific Aortic Stenosis. In: Aikawa E, editor. Calcific Aortic Valve Disease. Rijeka (Croatia): InTech; 2013. http://dx.doi.org/10.5772/46239. Chapter 14. [20] Meyer SR, Nagendran J, Desai LS, Rayat GR, Churchill TA, Anderson CC, et al. Decellularization reduces the immune response to aortic valve allografts in the rat. J Thorac Cardiovasc Surg 2005;130(2):469–76. [21] Meyer SR, Chiu B, Churchill TA, Zhu L, Lakey JR, Ross DB. Comparison of aortic valve allograft decellularization techniques in the rat. J Biomed Mater Res A 2006;79(2):254–62. [22] Yu BT, Li WT, Song BQ, Wu YL. Comparative study of the Triton X-100-sodium deoxycholate method and detergent-enzymatic digestion method for decellularization of porcine aortic valves. Eur Rev Med Pharmacol Sci 2013;17(16): 2179–84. [23] Liao J, Joyce EM, Sacks MS. Effects of decellularization on the mechanical and structural properties of the porcine aortic valve leaflet. Biomaterials 2008;29(8): 1065–74. [24] Grauss RW, Hazekamp MG, van Vliet S, Gittenberger-de Groot AC, DeRuiter MC. Decellularization of rat aortic valve allografts reduces leaflet destruction and extracellular matrix remodeling. J Thorac Cardiovasc Surg. 2003;126(6): 2003–10.
R. Khorramirouz et al. / Advances in Medical Sciences 59 (2014) 299–307 [25] Tudorache I, Cebotari S, Sturz G, Kirsch L, Hurschler C, Hilfiker A, et al. Tissue engineering of heart valves: biomechanical and morphological properties of decellularized heart valves. J Heart Valve Dis 2007;16(5):567–73. discussion 574. [26] Cebotari S, Mertsching H, Kallenbach K, Kostin S, Repin O, Batrinac A, et al. Construction of autologous human heart valves based on an acellular allograft matrix. Circulation 2002;106(12 (Suppl. 1)):I63–8. [27] Schenke-Layland K, Vasilevski O, Opitz F, Ko¨nig K, Riemann I, Halbhuber K, et al. Impact of decellularization of xenogeneic tissue on extracellular matrix
307
integrity for tissue engineering of heart valves. J Struct Biol 2003;143(3): 201–8. [28] Crapo PM, Gilbert TW, Badylak SF. An overview of tissue and whole organ decellularization processes. Biomaterials 2011;32(12):3233–43. [29] Booth C, Korossis S, Wilcox H, Watterson K, Kearney J, Fisher J, et al. Tissue engineering of cardiac valve protheses I: Development and histological characterization of an acellular porcine scaffold. J Heart Valve Dis 2002;11(4): 457–62.
