Triton X-100 combines with chymotrypsin: A more promising protocol to prepare decellularized porcine carotid arteries

Abstract

Background:

Morbidity and mortality of cardiovascular diseases are increasing in recent years. To solve these problems, vascular transplantation has become a common approach. Decellularization has been a hot spot of tissue engineering to prepare vessel substitutes for vascular transplantation. However, there is no established canonical protocol for decellularization thus far.

Objective:

To further understand the decellularization effect of decellularization protocols and the causal relationship between decellularization and mechanical properties.

Methods:

Three decellularization protocols including two chemical protocols based on SDS and Trypsin respectively and a combination of Triton X-100 with chymotrypsin were adopted to obtain decellularized porcine carotid arteries in our study. After decellularization, histological analysis, scanning electron microscopy and mechanical tests were performed to evaluate their efficiency on removing of cellular components, retention of extracellular matrix and influence on mechanical properties.

Results:

All these decellularization protocols used in our study were efficient to remove cellular components. However, SDS and trypsin performed more disruptive effect on ECM structure and mechanical properties of native arteries while Triton X-100 combines with chymotrypsin had no significant disruptive effect.

Conclusions:

Compared with decellularization protocols based on SDS and trypsin, Triton X-100 combines with chymotrypsin used in our study may be a more promising protocol to prepare decellularized porcine carotid arteries for vascular tissue engineering applications.

Keywords

Decellularization Triton X-100 chymotrypsin extracellular matrix sodium dodecyl sulfate trypsin

1. Introduction

Cardiovascular diseases are the leading cause of death in the world [1,2]. In 2012, 17.5 million people died from CVDs, representing 31% of all global deaths [3]. Vessel reconstruction has become a common approach to solve cardiovascular diseases [3]. Autologous and artificial blood vessels are commonly used vascular substitutes in cardiac and peripheral vascular surgery. The great saphenous vein and internal mammary artery are the most commonly used autologous vessels. However, previous diseases, operation history and inappropriate size limit their application [4]. Although artificial blood vessels such as ePTFE and Daron have obtained positive results when used in large diameter vessels, thrombosis, intimal hyperplasia and other complications often occurred when applied in small diameter vessels (<6 mm) [5]. Therefore, it is urgent to explore “off-the-shelf” vascular substitutes to meet the demands of vessel reconstruction.

Tissue engineering has attracted more and more attention in recent years for it can overcome limitations mentioned above and create ideal vessel substitutes for transplantation. Using decellularized arteries to prepare blood vessel substitutes has become an attractive option in the tissue engineering field [6,7]. By using decellularization methods, immunological materials such as cell components of the blood vessels were removed, and the decellularized vessels can take advantage of native arteries to create an ideal extracellular matrix (ECM) template that is thought to better mimic the extracellular surroundings for the next step of cell seeding [8,9]. Commonly used decellularization methods include physical, chemical and enzymatic methods, among which the most commonly used agents are ionic detergent sodium dodecyle sulfate (SDS), non-ionic detergent Triton X-100 and enzymatic agent Trypsin. Although these methods have been widely applied to prepare decellularized vessels, there is no established “gold standard” protocol for decellularization thus far. What’s more, there are also many drawbacks bothering people including long decellularization time, residual cell toxicity, destruction of extracellular matrix and weakened mechanical properties after decellularization. In order to further understand the decellularization effect of these agents and the causal relationship between decellularization and mechanical properties, three decellularization protocols were adopted to obtain decellularized porcine carotid arteries in our study. After decellularization, histological analysis, scanning electron microscopy and mechanical tests were performed to evaluate their efficiency on removing of cellular components, retention of extracellular matrix and influence on mechanical properties. All these results can provide an insight into the potential of these decellularization protocols in vascular tissue engineering.

2. Materials and methods

2.1. Animal blood vessels

Fresh porcine carotid arteries were obtained from a slaughterhouse immediately after pigs were slaughtered. After obtained from the slaughterhouse, arteries were stored in phosphate buffered saline (PBS) containing 100 U/ml penicillin and 100 g/l streptomycin and were transported to our laboratory at 4°C. After arrived at laboratory, outer membrane, adipose tissue and connective tissue of arteries were stripped immediately, then arteries were stored in PBS at 4°C for further processing, the overall storage time of arteries until processing did not exceed 24 h.

2.2. Decellularization procedures

All samples were equally divided into 4 groups: A, B, C and D. Group A includes fresh carotid arteries with no further treatment. Group B was processed according to the following steps: vessels were firstly immersed in 10 mmol/l Tris-HCL (pH7.4, Xingke Biological Technology Co. Ltd, Shanghai, China) containing 1% sodium dodecyl sulfate (SDS, Hanran Biological Technology Co. Ltd, Shanghai, China) and 0.02% ethylenediaminetetraacetic acid (EDTA, Kaiyue Chemical Co. Ltd, Baoding, China) while shaking for 60 h on the orbital shaker (TS-100, Qilinbeier Instrument Equipment Manufacturing Co. Ltd, Haimen, China) under 4°C. Then vessels were washed with PBS for 24 h and stored under −20°C for future use. Group C was processed as follows: vessels were firstly immersed in 10 mmol/l Tris-HCL containing 0.25% trypsin (Geyuan Tianrun Biotechnology Co. Ltd, Beijing, China) and 0.02% EDTA for 60 h wather bath under 37°C. After washed with PBS for 24 h, vessels were stored under −20°C for future use. Group D were decellularized by 1% Triton X-100 (Labest Biological Technology Co. Ltd, Beijing, China) combines with chymotrypsin. Briefly, vessels were thoroughly immersed in PBS containing 1% Triton X-100 with continuous shaking at 4°C for 60 h and then were immersed in PBS containing 1 mg/ml chymotrypsin for 3 h water bath under 37°C. Vessels were then washed with PBS for 24 h and stored under −20°C for future use.

2.3. Histological analysis

All samples were fixed in 10% neutral-buffered formalin for 24 h, and embedded in paraffin, cut into 5-μm sections. After removal of paraffin and re-hydration, HE staining was used to verify the absence of cellular elements, Masson’s trichrome and Elastica Van Gieson (EVG) staining were used for identification of collagen and elastin fiber, respectively.

2.4. Scanning electron microscopy

Nondecellularized and decellularized arteries segments were cut into open patches and were prepared by standard procedures of scanning electron microscopy (SEM). Briefly, patches were fixed with 1% glutaraldehyde solution (BASF SE, Ludwigshafen, Germany) in 0.1 M sodium cacodylate buffer (pH 7.4, BASF SE, Ludwigshafen, Germany) for 5 min. Then the samples were washed for 5 min with distilled water and dehydrated with 15 min exchanges in each of the 30%, 50%, 70%, 90%, and 100% aqueous ethanol and absolute ethanol. The samples were further immersed in hexamethyldisilazane (BASF SE, Ludwigshafen, Germany) for 10 min and air-dried overnight. Dried samples were mounted, sputter-coated with gold and examined with a scanning electron microscope (TM1000, Hitachi, Tokyo, Japan).

2.5. Mechanical properties

For burst pressure test, 50 mm length fresh and decellularized arteries were selected. One end of the blood vessel was connected to the clamp and closed tightly, and the other end was connected to a three-limb tube. Each end of the three-way tube was connected to pressure testing device and pressure injection device. PBS was injected into the vessel through the injection device at a speed of 5 ml/min and the pressure was displayed on the pressure gauge. Burst pressure was defined as the highest pressure measured when the vessel ruptured.

Suture retention test was performed according to the following descriptions. Briefly, one end of the vessel segment was clipped on a tension testing machine, and a 5-0 prolene suture was placed in the other end which was 2 mm from the vessel’s edge. Then the suture was pulled at a constant rate of 50 mm/min and the suture retention strength was measured until the suture teared out the vessel.

Mechanical properties of fresh and decellularized arteries were then measured by stress–strain test at room temperature as described in previous study [10]. Briefly, vessel samples were cut into particular shape about 20 mm in length and 4 mm in width before the test, and then each end of the samples were clamped by an electronic tension tester (DLL-5000, Caldecott Shanghai Machinery Equipment Co. Ltd, Shanghai, China). Then vessels were stretched at a rate of 50 mm/min until tensile failure occurred. The breaking strength, maximum strength and the elongation at break were recorded.

2.6. Statistical analysis

Statistical analysis was performed using SPSS 22.0 (IBM Inc, Armonk, NY, USA). All results are expressed as mean ± standard deviation (SD). One-way analysis of variance (ANOVA) was used to compare the average among different groups, and $P < 0.05$ was considered statistically significant.

3. Results

3.1. Histological analysis

Histological analysis was used to evaluate the removing of cellular components and retention of extracellular structure after decellularization. Results showed that cell nuclei and intact ECM can be clearly seen in fresh arteries (Figs 1(A), 2(A), 3(A)). After decellularization, cellular components were completely removed (Fig. 1(B), 1(C)), while major ECM components including collagen matrix (Fig. 2(B), 2(C)) and elastic fiber (Fig. 3(B), 3(C)) were all preserved in groups B and C. In group D, after decellularization by Triton X-100 only and before using chymotrypsin, although collagen and elastic fiber were well retained without severe disruption (Figs 2(D), 3(D)), plentiful nuclear materials can be still observed on the vessel wall (Fig. 1(D)). After using chymotrypsin, cellular components were completely removed (Fig. 1(E)) and the ECM was still well retained (Figs 2(E), 3(E)). However, more loss of ECM occurred in group B and C compared with group D after all the decellularization process, which were undoubtedly caused by decellularization and indicated that detergents used in group B and C were more disruptive than group D.

Fig. 1.

HE staining of arteries before and after decellularization. (A) Fresh carotid arteries. (B) Decellularized arteries from group B. (C) Decellularized arteries from group C. (D) Decellularized arteries prepared by Triton X-100 only from group D. (E) Decellularized arteries prepared by Triton X-100 and chymotrypsin from group D. Magnification, ×100.

Fig. 2.

Masson’s trichrome of arteries before and after decellularization. (A) Fresh carotid arteries. (B) Decellularized arteries from group B. (C) Decellularized arteries from group C. (D) Decellularized arteries prepared by Triton X-100 only from group D. (E) Decellularized arteries prepared by Triton X-100 and chymotrypsin from group D. Magnification, ×100.

Fig. 3.

Elastica Van Gieson staining of arteries before and after decellularization. (A) Fresh carotid arteries. (B) Decellularized arteries from group B. (C) Decellularized arteries from group C. (D) Decellularized arteries prepared by Triton X-100 only from group D. (E) Decellularized arteries prepared by Triton X-100 and chymotrypsin from group D. Magnification, ×100.

3.2. Scanning electron microscopy

SEM was performed on the cross section along the circumferential direction of the fresh and decellularized vessels. Results showed that in the fresh vessels, compact structure was present with intact cells embedded in the vascular wall (Fig. 4(A)). After decellularization, cells were no longer present while extracellular matrix fibers were all maintained, pores can be clearly seen in groups B and C (Fig. 4(B), 4(C)). In group D, after decellularization by Triton X-100 only and before using chymotrypsin, vascular structure were well retained without severe disruption, and cells can be still observed on the vessel wall (Fig. 4(D)). After using chymotrypsin, cells were completely removed and the ECM was still well retained (Fig. 4(E)). However, compared with group D, more disruption and fracture of ECM were observed in group B and C, which also indicated the greater disruptive effect of the detergents used in group B and C.

Fig. 4.

Scanning electron microscopy of arteries before and after decellularization. (A) Fresh carotid arteries. (B) Decellularized arteries from group B. (C) Decellularized arteries from group C. (D) Decellularized arteries prepared by Triton X-100 only from group D. (E) Decellularized arteries prepared by Triton X-100 and chymotrypsin from group D. Bar equals 2 μm.

3.3. Mechanical properties

Table 1 showed the test results of burst pressure and suture retention. After decellularization, burst pressure and suture retention of decellularized groups all decreased in a certain degree, while values in group B and C decreased the most and were significantly different from fresh arteries, and no significant difference was found between group D and fresh arteries.

Table 1
Burst pressure and suture retention of fresh and decellularized arteries

Group A Group B Group C Group D

Burst pressure (KPa) 310.33 ± 18.58 235.00 ± 8.89^* 207.00 ± 11.53^* 290.67 ± 7.64

Suture retention (N) 1.14 ± 0.10 0.66 ± 0.07^* 0.59 ± 0.10^* 0.98 ± 0.03

	Group A	Group B	Group C	Group D
Burst pressure (KPa)	310.33 ± 18.58	235.00 ± 8.89^*	207.00 ± 11.53^*	290.67 ± 7.64
Suture retention (N)	1.14 ± 0.10	0.66 ± 0.07^*	0.59 ± 0.10^*	0.98 ± 0.03

Results are expressed as the mean ± SD.

$P < 0.001$ , significantly different from the fresh arteries.

Results of stress–strain test were showed in Table 2. Results showed that after decellularization, breaking strength and maximum strength of group B and C were significantly decreased as compared with fresh arteries, while no significant difference was found between group D and fresh arteries in spite of strength degradation also occurred in group D. Elongation at break of decellularized groups all increased significantly compared with fresh arteries. All these mechanical test results indicated that decellularization did have adverse impact on the biomechanical properties of vessels and decellularization protocols used in group B and C performed more disruptive effect on mechanical properties of arteries than the protocol used in group D. Characteristics of all the groups were summarized in Table 3.

Table 2

Stress–strain test results of fresh and decellularized arteries

	Group A	Group B	Group C	Group D
Breaking strength (MPa)	2.20 ± 0.18	1.27 ± 0.07^*	1.12 ± 0.11^*	1.94 ± 0.07
Maximum strength (MPa)	2.28 ± 0.21	1.40 ± 0.11^*	1.23 ± 0.15^*	2.12 ± 0.07
Elongation at break (%)	115.48 ± 8.03	164.79 ± 9.90^*	183.86 ± 5.78^*	156.09 ± 5.01^*

Results are expressed as the mean ± SD.

$P < 0.001$ , significantly different from the fresh arteries.

Table 3

Characteristics of all the groups

Group A	Group B	Group C	Group D
Fresh arteries without any treatment, compact structure of ECM with intact cells, ideal mechanical properties	Decellularized arteries prepared by SDS, severely disrupted ECM without cellular components, poorer mechanical properties	Decellularized arteries prepared by trypsin, severely disrupted ECM without cellular components, the poorest mechanical properties	Decellularized arteries prepared by Triton X-100 and chymotrypsin, well preserved ECM without cellular components, similar mechanical properties as fresh arteries

4. Discussion

Tissue engineering is a new branch of science developed in 1980s, by which people can build alternative organizational alternatives. The first vascular tissue engineering scaffold was built by Weinberg and Bell in 1986, using a natural scaffold and bovine vascular cells [11]. Nowadays, the commonly used vascular tissue engineering scaffold materials include polyglycolic acid (PGA), polylactic acid (PLA) and its co-polymers, but their mechanical properties and degradation rate can’t meet the requirements of the physiological blood vessels, and will produce a large amount of acid metabolites after implantation [12]. Therefore, facing the continuing growth of vascular diseases morbidity and the urgent need for vessel reconstruction, searching for ideal vascular substitutes becomes imperative in the research of vascular tissue engineering scaffold.

Decellularization is a process to obtain ECM structure from natural origin which has attracted increased attention in tissue engineering. As biomaterials without immunogenicity, decellularized ECM can be used to create tissue-engineered alternatives for tissue or organ replacement and repair. Consensus has been accepted that both simple tissues and complicated organs can be decellularized for tissue engineering use, including, but not limited to, kidney, liver, bone and blood vessels [13–17]. Biological scaffolds derived from decellularized vessels have been widely studied in many years. Developing a small-diameter vascular graft of which all cellular materials are removed and structural components of ECM are preserved is the common goal of different decellularization protocols. Removing of cellular materials can reduce the immunogenicity of allogeneic or heterogenic tissues, while preserving of ECM can not only provide tissue structure and substrate for cell adhesion but also cues for cell migration, proliferation, differentiation [18]. Moreover, decellularized vessels can maintain mechanical properties of a native artery as a conduit for transplantation. Different approaches have been developed for tissue decellularization [19,20], but results with respect to the balance mentioned above are different [21,22]. In our study, we compared three different protocols to obtain decellularized vascular matrix, including two chemical protocols based on SDS and Trypsin respectively and a combination of Triton X-100 with chymotrypsin, and evaluated their efficiency on decellularization.

Various decellularization protocols are frequently used including physical, chemical, and enzymatic approaches and their combinations. Among these agents, sodium dodecyle sulfate (SDS) is a commonly used ionic chemical detergent and plays the role of decellularization by dissolving the cytoplasm and the cell nucleus. Compare to other detergents, SDS is more effective at removing cellular elements but can also bring severe disruption to ECM [23,24]. Enzymatic techniques have also been widely used to prepare decellularized tissues, among which trypsin is one of the most commonly used proteolytic enzyme. Although decellularization by trypsin can successfully remove cell and nuclear components, destruction of ECM is still unavoidable, which has been validated by several studies before [25,26]. In our study, results showed that both SDS and trypsin can obtain completely removing of cellular elements, for no residual cells were observed after decellularization. Although major ECM structure of decellularized vessels in our study was preserved, results also showed more loss of ECM in vessels treated by SDS and trypsin than the vessels treated in group D, which indicated more disruptive effect of SDS and trypsin as studies validated before.

As a moderate non-ionic detergent, Triton X-100 has been widely used in decellularization field. Mechanism of Triton X-100 mainly depends on disruption of lipid–lipid and lipid–protein interactions, without disruption of protein–protein interactions and hence cause little damage to ECM [27]. Several researchers considered that Triton X-100 was not an ideal decellularization detergent when used alone. Woods compared three extraction techniques in the development of porcine bone anterior cruciate ligament-bone grafts and found the least effective of Triton X-100 in removing of cellular extraction [28]. Dahl also found that Triton X-100 was ineffective in removing cellular components of porcine carotid arteries [29]. However, when combine Triton X-100 with other detergents or techniques, exciting results of effective decellularization can be achieved. Xiong used a combination of Triton X-100 and ammonium hydroxide followed by cell culture complete medium containing serum to improve cellular removal during the decellularization process and found completely removing of cellular components [3]. Ye combined Triton X-100 and trypsin-EDTA to prepare decellularized porcine aortic valve and complete removal of cellular components was also observed [30]. So in order to enhance the efficiency of Triton X-100, we combined Triton X-100 with chymotrypsin, a seldom used enzyme in previous decellularization studies, to prepare decellularized vascular matrix.

Chymotrypsin is a proteolytic enzyme extracted from cow or pig pancreas and functions as the endopeptidase by the mechanism of cutting off the carboxy terminal peptide chain of tyrosine and phenylalanine. As a proteolytic enzyme, it plays the role of hydrolysis by the similar mechanism with trypsin but has lower toxicity, less adverse reactions and a more effective capacity of decomposition. Given these advantages, we assume that chymotrypsin can be used as an efficient decellularization detergent. In our study, vessels were firstly immersed in PBS containing 1% Triton X-100, after decellularized by Triton X-100 for 60 h, vessels were decellularized by chymotrypsin. In order to avoid serious damage to ECM, processing time of chymotrypsin is limited to 3 h. Results revealed that after decellularization by Triton X-100 only and before using chymotrypsin, although collagen and elastic fiber were well retained without severe disruption, plentiful nuclear materials can be still observed on the vessel wall, indicated that decellularization by Triton X-100 only was not effective in removing cellular components. After using chymotrypsin, cellular components were completely removed and the ECM was still well retained, indicated that combination of Triton X-100 with chymotrypsin can be an effective remedy and was effective in completely removing cellular components and well preserving ECM.

Decellularized vessels are expected to function as native vessels that can withstand physiological pressure from the blood flow. Therefore, whether the mechanical properties can be maintained is also an essential evaluation factor of different decellularization protocols. Mechanical properties of native arteries are generally depended on the integrity of collagen and elastin, so maintaining the integrity of ECM is essential to prepare decellularized arterial scaffolds. Although ECM can be well preserved in the decellularized vessels, a fact that we can’t ignore is any decellularization steps aims to remove cells will slightly damage or alter the native structure of ECM and therefore influence biomechanical properties of decellularized vessels [31]. To minimize these undesirable effects rather than complete avoidance is the objective of different decellularization protocols. As evidenced by the results in our study, mechanical properties including burst pressure, suture retention, breaking strength and maximum strength of all decellularized groups decreased to some extent, indicated that decellularization did have adverse impact on the biomechanical properties of vessels, which was undoubtedly caused by the destruction of the ECM. Moreover, results also showed that mechanical properties of SDS and trypsin treated groups were significantly weakened after decellularization, while no significant difference was found between the fresh arteries and arteries decellularized in group D, indicated that SDS and trypsin were more disruptive when used as decellularization detergents to prepare decellularized arteries. Therefore, all above results suggested that Triton X-100 combines with chymotrypsin used in group D may be more appropriate for decellularization in preserving ECM and mechanical properties of decellularized vessels.

5. Conclusions

In our study, we used three decellularization protocols including SDS, trypsin and a combination of Triton X-100 and chymotrypsin to prepare decellularized porcine carotid arteries. Histological analysis, scanning electron microscopy and mechanical tests were performed to evaluate their potential as decellularization protocols in vascular tissue engineering. Results showed that all these decellularization protocols used in our study were effective to remove cellular components. However, SDS and trypsin performed more disruptive effect on ECM structure and mechanical properties of native arteries while Triton X-100 combines with chymotrypsin had no significant disruptive effect. Therefore, Triton X-100 combines with chymotrypsin used in our study may be a more promising protocol to prepare decellularized porcine carotid arteries for vascular tissue engineering applications.

Footnotes

Acknowledgement

This study was supported by the capital health research and development of special (Grant Number 2016-1-2012), Beijing, People’s Republic of China.

Conflict of interest

The authors have no conflict of interest to report.

References

Alexandar V Nayar

P.G.

Murugesan

Mary

Darshana P and Ahmed

S.S.S.J.

, CardioGenBase: A literature based multi-omics database for major cardiovascular diseases, PLoS One 10(12) (2015), e143188. doi:10.1371/journal.pone.0143188.

Pagidipati

N.J.

and Gaziano

T.A.

, Estimating deaths from cardiovascular disease: A review of global methodologies of mortality measurement, Circulation 127(6) (2013), 749–756. doi:10.1161/CIRCULATIONAHA.112.128413.

Xiong

Chan

W.Y.

Chua

A.W.

Feng

Gopal

Ong

Y.S.

et al., Decellularized porcine saphenous artery for small-diameter tissue-engineered conduit graft, Artif. Organs 37(6) (2013), E74–E87. doi:10.1111/aor.12014.

Pellegata

A.F.

Asnaghi

M.A.

Stefani

Maestroni

Dominioni

et al., Detergent-enzymatic decellularization of swine blood vessels: Insight on mechanical properties for vascular tissue engineering, Biomed. Res. Int. 2013 (2013), 918753. doi:10.1155/2013/918753.

Huang

A.H.

and Niklason

L.E.

, Engineering of arteries in vitro, Cell. Mol. Life Sci. 71(11) (2014), 2103–2118. doi:10.1007/s00018-013-1546-3.

Hwang

S.J.

Kim

S.W.

Choo

S.J.

Lee

B.W.

I.R.

Yun

H.J.

et al., The decellularized vascular allograft as an experimental platform for developing a biocompatible small-diameter graft conduit in a rat surgical model, Yonsei Med. J. 52(2) (2011), 227–233. doi:10.3349/ymj.2011.52.2.227.

Ishino

and Fujisato

, Decellularization of porcine carotid by the recipient’s serum and evaluation of its biocompatibility using a rat autograft model, J. Artif. Organs 18(2) (2015), 136–142. doi:10.1007/s10047-015-0819-z.

Quint

Kondo

Manson

R.J.

Lawson

J.H.

Dardik

and Niklason

L.E.

, Decellularized tissue-engineered blood vessel as an arterial conduit, Proc. Natl. Acad. Sci. USA 108(22) (2011), 9214–9219. doi:10.1073/pnas.1019506108.

Birchall

and Hamilton

, Tissue-engineered vascular replacements for children, Lancet 380(9838) (2012), 197–198. doi:10.1016/S0140-6736(12)60817-4.

10.

Zhu

G.C.

Y.Q.

Geng

Feng

Z.G.

Zhang

S.W.

et al., Experimental study on the construction of small three-dimensional tissue engineered grafts of electrospun poly-epsilon-caprolactone, J. Mater. Sci. Mater. Med. 26(2) (2015), 112. doi:10.1007/s10856-015-5448-9.

11.

Weinberg

C.B.

and Bell

, A blood vessel model constructed from collagen and cultured vascular cells, Science 231(4736) (1986), 397–400. doi:10.1126/science.2934816.

12.

Garcia

C.A.

and Mikos

A.G.

, In vitro degradation of thin poly(DL-lactic-co-glycolic acid) films, J. Biomed. Mater. Res. 46(2) (1999), 236–244. doi:10.1002/(SICI)1097-4636(199908)46:2<236::AID-JBM13>3.0.CO;2-F.

13.

Chen

Y.C.

Chen

R.N.

Jhan

H.J.

Liu

D.Z.

H.O.

Mao

et al., Development and characterization of acellular extracellular matrix scaffolds from porcine menisci for use in cartilage tissue engineering, Tissue Eng. Part C Methods 21 (2015), 971–986. doi:10.1089/ten.tec.2015.0036.

14.

Navarro-Tableros

Herrera

S.M.

Figliolini

Romagnoli

Tetta

and Camussi

, Recellularization of rat liver scaffolds by human liver stem cells, Tissue Eng. Part A 21 (2015), 1929–1939. doi:10.1089/ten.tea.2014.0573.

15.

Guan

Liu

Sun

Cheng

Luan

et al., Porcine kidneys as a source of ECM scaffold for kidney regeneration, Mater. Sci. Eng. C Mater. Biol. Appl. 56 (2015), 451–456. doi:10.1016/j.msec.2015.07.007.

16.

DiMuzio

and Tulenko

, Tissue engineering applications to vascular bypass graft development: The use of adipose-derived stem cells, J. Vasc. Surg. 45(Suppl A) (2007), A99–A103. doi:10.1016/j.jvs.2007.02.046.

17.

Schoeller

Lille

Stenzl

Ninkovic

Piza

Otto

et al., Bladder reconstruction using a prevascularized capsular tissue seeded with urothelial cells, J. Urol. 165 (2001), 980–985. doi:10.1016/S0022-5347(05)66588-3.

18.

Fitzpatrick

L.E.

and McDevitt

T.C.

, Cell-derived matrices for tissue engineering and regenerative medicine applications, Biomater. Sci. 3(1) (2015), 12–24. doi:10.1039/C4BM00246F.

19.

Crapo

P.M.

Gilbert

T.W.

and Badylak

S.F.

, An overview of tissue and whole organ decellularization processes, Biomaterials 32 (2011), 3233–3243. doi:10.1016/j.biomaterials.2011.01.057.

20.

Gilbert

T.W.

Sellaro

T.L.

and Badylak

S.F.

, Decellularization of tissues and organs, Biomaterials 27 (2006), 3675–3683. doi:10.1016/j.biomaterials.2006.02.014.

21.

Grauss

R.W.

Hazekamp

M.G.

Oppenhuizen

van Munsteren

C.J.

Gittenberger-de

G.A.

and DeRuiter

M.C.

, Histological evaluation of decellularised porcine aortic valves: Matrix changes due to different decellularisation methods, Eur. J. Cardiothorac. Surg. 27(4) (2005), 566–571. doi:10.1016/j.ejcts.2004.12.052.

22.

Schenke-Layland

Vasilevski

Opitz

Konig

Riemann

Halbhuber

K.J.

et al., Impact of decellularization of xenogeneic tissue on extracellular matrix integrity for tissue engineering of heart valves, J. Struct. Biol. 143(2) (2003), 201–208. doi:10.1016/j.jsb.2003.08.002.

23.

Pang

and Yang

, Histological evaluation and biomechanical characterisation of an acellular porcine cornea scaffold, Br. J. Ophthalmol. 95(3) (2011), 410–414. doi:10.1136/bjo.2008.142539.

24.

Deeken

C.R.

White

A.K.

Bachman

S.L.

Ramshaw

B.J.

Cleveland

D.S.

Loy

T.S.

et al., Method of preparing a decellularized porcine tendon using tributyl phosphate, J. Biomed. Mater. Res. B Appl. Biomater. 96(2) (2011), 199–206. doi:10.1002/jbm.b.31753.

25.

Zou

and Zhang

, Mechanical evaluation of decellularized porcine thoracic aorta, J. Surg. Res. 175(2) (2012), 359–368. doi:10.1016/j.jss.2011.03.070.

26.

Meyer

S.R.

Chiu

Churchill

T.A.

Zhu

Lakey

J.R.

and Ross

D.B.

, Comparison of aortic valve allograft decellularization techniques in the rat, J. Biomed. Mater. Res. 79(2) (2006), 254–262. doi:10.1002/jbm.a.30777.

27.

Seddon

A.M.

Curnow

and Booth

P.J.

, Membrane proteins, lipids and detergents: Not just a soap opera, Biochim. Biophys. Acta 1666(1–2) (2004), 105–117. doi:10.1016/j.bbamem.2004.04.011.

28.

Woods

and Gratzer

P.F.

, Effectiveness of three extraction techniques in the development of a decellularized bone-anterior cruciate ligament-bone graft, Biomaterials 26(35) (2005), 7339–7349. doi:10.1016/j.biomaterials.2005.05.066.

29.

Dahl

S.L.

Koh

Prabhakar

and Niklason

L.E.

, Decellularized native and engineered arterial scaffolds for transplantation, Cell Transplant. 12(6) (2003), 659–666. doi:10.3727/000000003108747136.

30.

F.L.

Z.Y.

and Zhang

B.R.

, Preparation of acellularized porcine heart valve and seeding of bovine aortic endothelial cells, Chinese Journal of Reparative and Reconstructive Surgery 17(6) (2003), 493–495.

31.

Fitzpatrick

J.C.

Clark

P.M.

and Capaldi

F.M.

, Effect of decellularization protocol on the mechanical behavior of porcine descending aorta, Int. J. Biomater. 2010 (2010), 620503. doi:10.1155/2010/620503.

Triton X-100 combines with chymotrypsin: A more promising protocol to prepare decellularized porcine carotid arteries

Abstract

Background:

Objective:

Methods:

Results:

Conclusions:

Keywords

1. Introduction

2. Materials and methods

2.1. Animal blood vessels

2.2. Decellularization procedures

2.3. Histological analysis

2.4. Scanning electron microscopy

2.5. Mechanical properties

2.6. Statistical analysis

3. Results

3.1. Histological analysis

Table 1 Burst pressure and suture retention of fresh and decellularized arteries Group A Group B Group C Group D Burst pressure (KPa) 310.33 ± 18.58 235.00 ± 8.89 * 207.00 ± 11.53 * 290.67 ± 7.64 Suture retention (N) 1.14 ± 0.10 0.66 ± 0.07 * 0.59 ± 0.10 * 0.98 ± 0.03

5. Conclusions

Footnotes

Acknowledgement

Conflict of interest

References

Table 1
Burst pressure and suture retention of fresh and decellularized arteries

Group A Group B Group C Group D

Burst pressure (KPa) 310.33 ± 18.58 235.00 ± 8.89^* 207.00 ± 11.53^* 290.67 ± 7.64

Suture retention (N) 1.14 ± 0.10 0.66 ± 0.07^* 0.59 ± 0.10^* 0.98 ± 0.03