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Decellularised and Chemically Cross linked Human Umbilical Vein as a Small Caliber Conduit--Scaffold for Vascular Tissue Engineering.

Introduction

As there is a continuous increase in the number of vascular procedures performed annually throughout the world the demand for blood vessel prosthesis is very great. Their performance is disappointing when used to replace small diameter arteries (<6 mm) where blood flow is low and resistance is high resulting in graft failure due to thrombosis and occlusion [1-3]. In the last several decades there has been little improvement in the long term patency of small caliber blood vessel prosthesis grafts [4-6]. At this juncture tissue engineering of viable small caliber blood vessel with the potential to grow, repair and remodel offers hope to millions requiring cardiovascular reconstruction surgeries [7-9]. But due to lack of suitable polymeric materials and fabrication techniques the attempt of scientists to tissue engineer an ideal small caliber vascular conduit with adequate mechanical strength as well as architecture akin to natural ECM has met with little success.

An alternate approach is to implant an acellular native tissue as scaffold and let the body act as cell source to seed the acellular matrix. Decellularised bovine jugular vein, porcine carotid artery and rat iliac arteries are some xenografts investigated to generate vascular grafts [10-12]. But the use of xenografts has several limitations like ethical issues, immunogenicity and the risk of pathogen transmission [13, 14].

The human umbilical vein (HUV) which is discarded after delivery has merits like easy availability, non-invasive method of collection and presence of natural ECM. Moreover the allogenic nature of the HUV will help to avoid immunogenic issues associated with xenografts and has been considered by researchers as a scaffold for tissue regeneration [15-17]. The goal of the present study was to subject the HUV to a tissue processing methodology for generating an "off the shelf" biological conduit that can be used as a small caliber blood vessel substitute in cardiovascular and peripheral bypass surgeries. This paper reports the single step cell extraction process employed to decellularise the HUV followed by treatment with one or more chemical crosslinkers to stabilize the glycosaminoglycans and crosslink the proteins in the acellular matrix and their characterization.

Materials and Methods

Collection of human umbilical cords

The protocol of this study was approved by the Institutional Review Board of Central Leather Research Institute, Chennai, India. Human umbilical cords (HUC) were harvested from healthy full term births delivered by cesarean section with the informed consent of the parents of the newborns in sterile containers with 0.9% saline processed under aseptic conditions within 2 hours of collection by washing extensively with sterile Phosphate Buffered Saline (PBS) Then they were rinsed in 75% ethanol for 30 seconds and again washed thoroughly with PBS.

Dissection of umbilical cords

First the cord was cut into 8cms segments. Each segment was cut open horizontally to expose the blood vessels. The umbilical vein was severed and removed from the surrounding Whartons Jelly as well as two arteries. From each end of the extracted HUV segment a 1 cm section was cut and discarded to ensure that both ends of the HUV were regular and uniform. The 6cms HUV segments were immediately subjected to a decellularization process or used for further analysis.

Decellularization of HUVs

Human umbilical vein segments were decellularised using the ionic detergent Sodium Dodecyl Sulphate (SDS). Briefly, the decellularization process involved immersing the 6cms long sections of HUVs in SDS solution of known concentration for 16 hours at room temperature. To extract the detergent, vein segments were rinsed thoroughly three times with distilled water and then washed for 15 min, 30 min and 1 hour durations in water. Then the segments were given two consecutive water washes of 24 hours duration each and placed in 75% ethanol for 24 hours to facilitate the extraction of lipids and any residual ampiphilic detergent. Finally the decellularised HUVs (dHUVs) were rinsed in distilled water followed by PBS and used immediately for analysis or stored in PBS containing antibiotics at 4UC up to 3 days before further experimentation.

Protein crosslinking of HUVs

To make the decellularised veins intended for implantation more resistant to degradation they were chemically crosslinked. Three reagents were selected for crosslinking, i.e 1) 0.6 % w/v dimethylsuberimidate (DS) in Tris buffer pH 9 2) 100 mM ethyldimethylaminopropylcarbodimide (EC) in 0.01 M PBS, pH 7.4 and 3) 0.01, 0.001 and 0.0001% v/v glutaraldehyde (GA) in water. Crosslinking treatment included incubation of 6 segments of dHUV each measuring 6 cm in length with the crosslinking reagent for 24 hours at room temperature. The three test groups were DS-dHUV, EC-dHUV and GA-dHUV. Native veins and decellularised but uncrosslinked veins both served as controls. After crosslinking, the veins were washed extensively with water and then incubated in lysine (5mM in PBS) for 3 hours at 37UC. Then the veins were washed with distilled water followed by PBS pH 7.4 and used for analysis. The shrinkage temperature (Ts) of controls and crosslinked HUVs were measured to assess the extent of mechanical stabilization of tissue afforded by various crosslinking reagents and their relative efficacies were compared.

Glycosaminoglycans (GAGs) stabilisation and protein crosslinking of HUVs

In another approach to augment the mechanical stability of the vein, a combination of two crosslinking agents was used to treat the vein. The first crosslinking agent was employed to stabilise the GAGs in the vein matrix and the second crosslinking agent was used to crosslink the proteins. For this, HUVs were first maintained in a GAG stabilizing solution of EC (100 mM w/v in 0.01M PB pH 7.4) for 24 hours at room temperature. Then the HUVs were washed thoroughly in water followed by PBS and placed in the second crosslinking solution for 24 hrs to fix the proteins. The second crosslinking solution was either glutaraldehyde or dimethyl suberimidate as explained in section 2.4 for crosslinking proteins. Thus two groups of GAG stabilized and protein crosslinked HUVs were prepared namely EC-DS-dHUV and EC-GA-dHUV. The Ts of both groups of veins along with controls were measured to determine the extent of mechanical stabilization afforded by this pre-implantation tissue processing methodology.

Characterization of decellularization

Histological examination by hematoxylin eosin staining

To determine whether decellularization had rendered the vein matrix acellular, nHUV and dHUV were fixed in 10% buffered formalin and left overnight at room temperature. eosin. The stained sections were observed under light microscope (Nikon Eclipse E600, Japan) and photomicrographs were taken.

DNA quantification

To evaluate the decellularization process quantitatively, the DNA contents of native and decellularised veins were determined by the diphenylamine assay method using calf thymus DNA as standard. Briefly, the DNA from tissues was extracted in 5% TCA, centrifuged and the supernatant was treated with diphenylamine reagent and the blue colour developed was measured at 600nm using a spectrophotometer.

Qualitative analysis for collagen

To verify that the decellularisation process had not affected the main component in the connective tissue of the vein, sections of nHUV and dHUV were prepared as explained earlier and the presence of collagen in the ECM was analyzed by von Giesons staining.

Scanning Electron Microscopy

For scanning electron microscopic examination both nHUV and dHUVs were fixed in 2.5% glutaraldehyde for 24 hours and then The tissue sections were mounted with luminal or abluminal surface up, sputter coated with palladium-gold and examined with FEI Quanta 200 Scanning Electron Microscope.

Biochemcial quantification of ECM components

Known quantities of six nHUV and dHUV sections were defatted in chloroform--methanol mixture and hydrolyzed in 6N HCl at 110UC for 12 hrs. The hydrolysates were washed with water, dried and made up to known volume with water. Aliquots from the samples were used for the estimation of ECM components which was expressed as pg/mg dry weight of tissue.

Collagen content

The amount of collagen in ECM was determined by the method of Woessner in which samples were first treated with Chloramine-T and then with p-dimethylaminobenzaldehyde for 30 mins at 60UC. The amount of hydroxyproline was measured at 557nm spectrophotometrically and the value was multiplied by a factor of 7.46 to arrive at the collagen content of the tissue. GAG content

The amount of GAGs in the ECM was determined by the method of Elson and Morgan in which samples in were first treated with acetylacetone followed by Ehrlish reagent. The colour developed was read spectrophotometrically after 30 mins at 530nm to calculate the hexosamine content of the tissue.

Biomechanical Testing

The biomechanical properties of nHUV, dHUVs and crosslinked HUVs were studied using Universal Testing Machine, Instron, UK. For axial testing 6cms segments of veins were stress loaded to failure at a constant speed of 10mm/min. The maximum load, tensile strength and percent elongation at rupture were recorded for veins in various groups.

In vitro enzyme degradation test to determine extent of crosslinking of HUVs

To understand the extent of crosslinking afforded by each crosslinking agent and compare the relative efficacies of various crosslinkers, the ability of crosslinked HUVs to resist in vitro proteolytic degradation was assessed. For this, 3 segments of HUVs in each group were defatted and incubated with collagenase type I from Clostridium hystolyticum in 0.01 M PBS, pH 7.4 (1mg per 100mg tissue) for 3 hrs. The enzyme digest was centrifuged and the amount of hydroxyproline in the pellet was estimated gas explained in 2.6.5.1 to determine the amount of undigested collagen. Both nHUV s and dHUV s served as control groups. There were five test groups namely, 1) DSdHUV 2) EC-dHUV 3) GA-dHUV 4) EC-DS-dHUV and 5) EC-GA-dHUV.

Statistics

All values were expressed as mean [+ or -] standard deviation (SD). Statistical analysis was performed with a one-way ANOVA to test the significance of each group with control and decellularised vein groups. A value of p < 0.05 was considered to be statistically significant. All statistics were performed by SPSS version 7.

Results

Gross morphology

Decellularised vein appeared pale and slightly more swollen than native vein but otherwise had consistency similar to fresh vein (Fig.1a). Crosslinked veins did not appear different from dHUV and had the integrity and handling characteristics comparable to the native vein.

Staining for cellularity

Fig. 1b and c are photomicrographs of HUV before and after decellularisation using 0.25% SDS respectively. Since post treatment with SDS concentrations e" 0.25% the HUV was completely devoid of cells and revealed a near normal morphology of the native vein, 0.25% w/v SDS was considered as the optimal concentration for converting HUV into an acelluar scaffold. For further work reported in this paper HUV was decellularised with 0.25%w/v SDS and used.

DNA content of dHUV

There was significant reduction in the amount of DNA present in dHUV when compared to native HUV (Table 1). The results showed that treatment with SDS and subsequent washings in water and ethanol were not only effective in causing cell lysis but also removed most of the cellular DNA from vein matrix.

Analysis of ECM of vein matrix

Von Giesons staining for collagen in HUV before and after decellularization is shown in Fig. 2a and b. The dHUV matrix showed positive staining which indicated that collagen fibres were well preserved throughout the vein matrix after decellularization. The results showed that the decellularization process did not adversely affect or deplete the collagen in the connective tissue of the dHUV.

Biochemical estimation of the amounts of collagen and GAGs before and after decellularization indicated that the cell extraction process had not depleted these components of the ECM. The results (Table 1) showed that collagen and GAGs contents of dHUV were higher than that of nHUV. This can be attributed to the fact that decellularisation resulted in loss of cell mass which in turn reduced the dry weight of the biological tissue. As a consequence, collagen and GAGs levels of acellular biological tissue were higher than those of native tissue although this increase was not significant.

Scanning electron microscopy

Fig. 3a is the SEM image of nHUV showing the nano fibrous network of the vein matrix which is desirable for tissue engineering. Fig.3b shows the lumen of the vein. The luminal surface of dHUV before decellularization and the same devoid of cells after treatment with 0.25% w/v SDS are shown in Fig.3c and d. Both light and SEM photographs revealed that the decellularization process converted the HUV into an acellular matrix.

Shrinkage temperatures of protein crosslinked HUVs

Shrinkage temperature studies were performed to assess the nature and extent of protein crosslinking and also compare the efficacies of three different crosslinkers (Fig.4). Decellularised vein had lower [T.sub.s] than that of nHUV. However crosslinking with GA (0.01 and 0.001% v/v) or EC increased the [T.sub.s] of dHUV significantly above that of nHUV. It was evident from the results that GA and EC afforded effective crosslinking of the decellularised biological matrix. However GA (0.0001 %v/v) and DS did not significantly increase the Ts of dHUV, The relative efficacies of chemical crosslinkers were in the order GA > EC > DS. The relative rate of crosslinking afforded by GA was concentration dependent and in the order of 0.01% > 0.001% > 0.0001% v/v. Based on these results in the case of glutaraldehyde crosslinking an optimum concentration of 0.001% v/v GA was selected for crosslinking the HUVs in further analysis.

GAG stabilized and protein crosslinked HUVs

The thermal shrinkage temperature values of HUVs which were first GAG stabilized and later protein crosslinked are shown in Fig.4. Overall, decellularised veins crosslinked with two agents had significantly higher Ts than nHUV and dHUV. The results showed that HUVs treated first with EC followed by GA (0.001%) had the highest Ts values among all treatments. Similarly the [T.sub.s] values of EC stabilized and DS crosslinked HUVs were higher than that of HUVs treated with DS alone and near comparable to Ts of HUVs crosslinked with GA (0.001%v/v) alone. The order of efficacy of crosslinking agents in the approach of GAG stabilization followed by protein crosslinking was (EC + GA) > (EC + DS).

Biomechanical Test

Biomechanical testing in the axial direction (Fig.5 a, b, c) revealed that mechanical stability decreased after decellularisation. When GA and EC were used either alone (crosslinking with single agent) or in combination (crosslinking with two agents) there was significant increase in tensile strength and maximum load bearing properties of dHUV. The improvement in biomechanical properties brought about by GA and EC when used either alone or together were near comparable. Although percent elongation of crosslinked HUVs was higher than nHUV and dHUV it was seen that among the five groups of crosslinked HUVs percent elongation decreased with increase in their tensile strength. This may be attributed to the fact that percent elongation decreases with increase in tensile strength. The order of improvement in biomechanical properties for HUVs crosslinked with a single agent was GA > EC > DS and for those crosslinked with two agents was (EC + GA) > (EC + DS).

In vitro resistance to enzyme degradation

The crosslinking efficacy of a reagent is reflected in its ability to confer proteolytic protection upon the tissue. The degree of proteolytic protection afforded by various chemical crosslinkers was conspicuous in the resistance to in vitro proteolysis demonstrated by crosslinked HUVs (Fig.6). For this purpose the amount of collagen remaining undigested in the tissue after in vitro exposure to collagenase for stipulated time period was measured. Greater the amount of crosslinking the tissue underwent, less collagen was released from the tissue by the proteolytic enzyme. All groups of crosslinked dHUVs had significantly higher resistance to collagenase digestion when compared to nHUV and uncrosslinked dHUV. The highest resistance to enzymatic digestion was shown by GA-dHUV followed by GA-EC-dHUV. It was seen that EC-dHUV, DSdHUV and EC+DS-dHUV gave near comparable results. The proteolytic protection provided by various crosslinkers was in the order GA > DS > EC (for single crosslinkers) and it was (EC+GA) > (EC+DS) for double crosslinkers. Crosslinking with GA alone or in combination with EC conferred multifold increase in resistance of veins to enzyme attack when compared to nHUV. Crosslinking with only EC/DS or both together (EC+DS) afforded twofold increase in resistance to enzyme attack which were near comparable.

Discussion

Hitherto various approaches like treatment with detergents and/ or enzymes, osmotic shock etc, have been employed for the decellularisation of biological tissues [18]. While the method of osmotic lyses does not remove the cellular DNA completely from the tissue, the enzymatic method has adverse effects on the extracellular components of the tissue. Moreover the remnants of the enzyme in the matrix will elicit adverse host immune reactions after implantation. [19].

In this work, SDS a common ionic detergent which is reported to lyse cells uniformly in all layers of the tissue was selected for decellularization. The mechanism of action of SDS is believed to cause less damage to cells than other enzymatic or mechanical methods [20]. Most of the works using SDS for the decellularization of biological grafts employed a two or multi-step extraction protocol in combination with other agents such as CHAPS buffer, detergents and enzymes like nucleases and/or trypsin. For example, one group reported that a SDS concentration as low as 0.1% w/v was effective for the decellularization of HUV but only with nuclease treatment [21]. There is one report where SDS alone has been used for the decellularization of human umbilical vein but a concentration as high as 1% w/v was employed [15]. .

In this work we have used SDS as the sole decellularising agent in a simple and effective single step protocol at a concentration as low as 0.25% w/v. This effectively removed cell components from HUV and at the same time preserved the structural integrity of the ECM. Schaner et al have reported to have achieved complete cell removal with 0.075% SDS in the case of human greater saphenous vein. [22]. However in our hands total cell and nuclear material removal from HUV was obtained at a concentration e" 0.25% SDS for HUV.

In this work tissue decellularization performed using 0.25% SDS had neither reduced the amounts of collagen and GAGs nor disrupted the collagen fibres in the ECM. This can be attributed to the fact that the concentration and the time period of exposure to the reagent were optimal for cell extraction from the HUV. The amount of collagen dictates the biomechanical behaviour of the vasculature. Since collagen content was well preserved in the acellular matrix the biomechanical properties were not altered much by decellularization as attested by mechanical testing study. Depletion of GAGs will have a negative effect on the viscoelastic behaviour of the tissue because water retention is one of their main functions. Our results showed that GAGs which are essential for binding an array of bioactive molecules and gel properties of the ECM have not been depleted by the decellularization process.

To reduce the antigenicity of the tissue we selected cross-linking agents of little or no toxicity i.e., EC and DS to crosslink the acellular HUV matrix (23). Glutaraldehyde was selected for comparison because although extensively employed as a crosslinker, its cytotoxicity and tendency to promote in vivo calcification has been of great concern for several years [24,25]. The chemical agent EC is water soluble and widely employed to crosslink proteinaceous matrices [26,27]. When compared to conventionally used GA and some other chemical reagents, EC does not remain as a part of the bond it generates and is excreted as water soluble urea derivatives having very low cytotoxicity. DS is a water soluble diimidoester used to cross-link primary amine groups of proteins and relatively safe when compared to GA [28,29]. It has minimum cross reactivity with other nucleophilic groups in the protein and the imidoamide reaction product does not alter the overall charge of the molecule and thereby the native conformation and activity of the protein is retained.

As stabilization of glycosaminoglycans improves durability and immune tolerance of the tissue by the recipient, in this work we also used a combination of two crosslinkers. This comprised of first stabilizing the GAGs using EC and later crosslinking the proteins using GA or DS in the tissue. While using a single crosslinker i.e. GA or DS involves mainly the proteins in the matrix, it is thought that the use of two crosslinkers i.e., EC and GA together or EC and DS together will address GAGs as well as proteins in the ECM of the vein matrix.

A GAG molecule is considered to be stabilized when a reagent generates at least one covalent bond between the GAG and another molecule of the tissue. Accordingly, the GAGs on the acellular HUV were stabilized (prior to crosslinking the extracellar proteins) by merely contacting with the water soluble EC in the absence of any other enhancers of carbodiimide activity. It is believed that EC forms a covalent link between a carboxyl group of a GAG and an amino group of an extracellular protein of HUV matrix. After stabilization of GAGs by treatment with EC, protein crosslinking was carried out with either GA or DS in this study.. As attested by the results of shrinkage temperature, tensile strength and in vitro enzyme resistance measurement studies it can be said that the methodology of GAG stabilization along with protein crosslinking of the acellular HUV had improved its physical stability and mechanical strength.

The best biomechanical behavior, physical strength and in vitro enzyme resistance profile was exhibited by GA crosslinked dHUV followed by EC crosslinked dHUV and EC+DS crosslinked dHUV. As cytotoxicity is a major deterrent in the usage of the conventional crosslinker GA, EC which gave best results next to GA can be considered for crosslinking the acellular HUV in future studies.

The crosslinked and cell free HUV developed in this study can be used as a conduit-scaffold for generating an endothelium in the laboratory by in vitro tissue engineering approach and then implanted as a vascular substitute [30]. Alternatively, by the in situ tissue engineering approach the same can be implanted as a bare vascular substitute at the specific location and the body allowed to act as an endogenous cell source to repopulate it and generate the endothelium [31].

While in vitro tissue engineering of endothelium in scaffolds for vascular grafting is very expensive, labour intensive, time consuming and carries the risk of infection, the in situ tissue engineering approach merely exploits the intrinsic regenerative potential of the human body to form an endothelium with endogenous cells. Hence currently there is a paradigm shift in perspective from in vitro to in situ tissue engineering as a more practical option for small caliber blood vessel reconstruction. The crosslinked acellular HUV can be used directly as a passive conduit-scaffold or linked to bioactive molecules like fibronectin, VEGF etc, which will favour cell recruitment and subsequent endothelialization of the scaffold in situ.

Conclusion

The human umbilical vein was subjected to a tissue processing methodology that comprised of single step--single reagent decellularisation and crosslinking with two chemical reagents which led to improvement of physical, biomechanical and in vitro enzyme resistance properties of the decellularised vein. Crosslinking of vein matrix with carbodiimide and dimethylsuberimidate can be considered as an alternative to crosslinking with glutaraldehyde. The chemically crosslinked acellular HUV shows promise as a 3D artificial biological small caliber conduit in vascular reconstructive surgeries via in vitro or in situ tissue engineering approaches.

Acknowledgement

This work was funded by the Department of Science and Technology, Government of India, under Women Scientist Scheme, grant number WOS-A-LS-02/2009. The authors gratefully acknowledge Dr. Vasundara Jagannathan, Dr. Padma Chandrasekhar and Mr. Tamizhmani of Devraj Manikchand Maternity Hospital, Sowcarpet, Chennai for providing the umbilical cords and thank Dr.Suguna Lonchin for help in biochemical analysis.

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R. Narayani and R. Rajaram

Biochemistry Department, Central Leather Research Institute, Adyar, Chennai 600020, India

Received 8 June 2017; Accepted 11 November 2017; Published online 31 December 2017

* Coresponding author: Dr. Narayani Ramamoorthy;

E-mail: narayani.rv@gmail.com

Caption: Figure 1: a) Gross appearance of dHUV b) HE staining of HUV before decellularization at 100x showing the presence of cells. c) HE staining of HUV after decellularization with 0.25%w/v SDS solution showing a cell free matrix at 100x

Caption: Figure 2: a) vons Giesons staining of collagen fibers in nHUV. b) Decellularized HUV was also positive for collagen fibers indicating that decellularization with 0.25%w/v SDS was optimal for removing cells without adverse effect on the main ECM component

Caption: Figure 3: SEM of HUV. a) Reveals the nano fibrillar architecture of nHUV matrix. b) Vessel lumen of native vein. c) Luminal surface of HUV before decellularization. d) Luminal surface of HUV after decellularization

Caption: Figure 4: Shrinkage temperature values of uncross-linked and cross-linked HUVs. Statistical analysis was made by One way ANOVA using Tukey's Multiple Comparison Test to compare the treated groups with control (N) and decellularized (De) groups. Values are expressed as means [+ or -] SD, (n = 3); * denotes p < 0.05 and 'a' denotes p < 0.001 compared to N; 'b' denotes p < 0.001 compared to De; 'ns' denotes not significant.

Caption: Figure 5: Mechanical testing of HUV; a) maximum load b) tensile strength c) percent elongation of control, decellularized (DE) and experimental groups crosslinked with glutaraldehyde (GA), ethyldimethylcarbodiimide (EDC), dimethylsuberimidate (DMS), ethyldimethylcarbodiimide and glutaraldehyde (EG) and ethyldimethylcarbodiimide and dimethylsuberimidate (ED). Statistical analysis was made using One way ANOVA to compare groups. Values are expressed as means [+ or -] SD (n = 6); * denotes p<0.05, ** denotes p< 0.01 and *** denotes p < 0.001 compared to control; $ denotes p < 0.05 and $$ denotes p< 0.01 compared to DE

Caption: Figure 6: Percentage of undigested collagen of uncrosslinked and crosslinked HUVs remaining after in vitro incubation with collagenase. Statistical analysis was made by one way ANOVA using Tukey's Multiple Comparison Test to compare the treated groups with control (N) and decellularized (De) groups. Values are expressed as means [+ or -] SD, (n = 3); 'a' denotes p < 0.001 compared to N; 'b' denotes p<0.001 compared to DE
Table 1: Biochemical analyses of native and decellularized
HUV

                Native HUY           Decellularized HUY
              (Mg mg dry wt)          (Mg. mg dry wt)

DNA         8.00 [+ or -]  0.70     0.562 [+ or -]  0 55
Collagen   368.28 [+ or -]  7.02   422.47 [+ or -]  37.89
GAG        11 29 [+ or -]  3 38     1216 [+ or -]  3 75
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Title Annotation:Original Article
Author:Narayani, R.; Rajaram, R.
Publication:Trends in Biomaterials and Artificial Organs
Date:Jul 1, 2017
Words:5262
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