Objectives: Endothelial cells harbor many antigenic determinants that may be targets for the immune system. The aim of this study was to determine the immunologic effects of decellularization, using 3 different methods, on xenograft rejection.

Materials and Methods: In a sterile plate containing phosphate-buffered saline, fresh sheep aortic heart valves were decellularized using 3 different enzymatic methods: with 900 μg/mL of collagenase at 40°C (method A), with 450 μg/mL of collagenase at 4°C (method B), and with 900 µg/mL of collagenase at 4°C (method C). Intact and decellularized valves were implanted subdermally into inbred male albino rabbits and extracted after 21 days (extra valve
pieces were also extracted after 60 days, as control samples, for assessing chronic rejection). Valves were histologically analyzed for inflammatory cell infiltration. Subendothelial structure integrity was determined using surface electron microscope.

Results: No inflammatory cell infiltration was seen around the decellularized valve with method A, and no subendothelial structure change was observed by surface electron microscope. Infiltration of immune cells involved in rejection was not seen around valves decellularized with method B, although the subendothelial structure was relatively preserved and valve stiffness was increased. With method C, we observed a foreign body-type reaction around the intact valve and the decellularized valve.

Conclusions: Method A is considered the optimal method of decellularization in our study, as this method significantly reduced the immune response to xenograft tissue, while maintaining subendothelial tissue.

Key words : Collagenase, Endothelial cells, Xenotransplant

Introduction

The endothelium is a single cell layer that lines the vessels and heart valves and serves as a barrier between the circulation and extravascular space. Endothelial cells (ECs) represent many different surface antigens. Circulating antibodies may attach to these antigens, and this attachment triggers a cascade of reactions that induce acute or chronic antibody-mediated rejection after graft transplant.^1,2

Because there is a critical and increasing shortage of human donors (ie, allograft tissue) for transplant, xenograft tissue may be used to replace or reconstruct malformed structures. There is no doubt that, in organ transplant, such as xenograft transplant, EC interactions are responsible for the initiation of many immune responses, which may result in rejection of the graft. Xenoreactive natural antibodies attach to donor EC antigens and activate recipient complement system, causing hyperacute rejection.^3,4

In a series of 80 consecutive cardiac transplants, a significant correlation was found between the occurrence of humoral rejection and the presence of anti-EC antibodies, as detected in a cell-based enzyme-linked immunosorbent assay.⁵ Consistently, in 1 retrospective study, 80% of patients positive for anti-EC antibodies experienced rejection compared with only 9% of patients negative for anti-EC antibodies.⁶ Anti-EC antibodies induce EC activation, which is marked by a prominent overexpression of adhesion molecules, suggesting an increase in EC-leukocyte adhesion.⁷ In addition, proinflammatory cytokines induce up-regulation of the expression of class I HLA antigens on ECs, which increases the binding of circulating antibodies, thus resulting in up-regulation of various proteins implicated in the rejection process.⁸

Another means of xenograft rejection is cell-mediated endothelialitis caused by T lymphocytes in the absence of antibodies.⁹ The decision to implant a tissue or mechanical heart valve is made based on many patient factors.¹⁰ The need for chronic anticoagulation therapy and continued patient somatic growth in the pediatric and young adult population limits widespread use of a mechanical valve in younger patients. Apart from the shortage of allograft heart valves, direct allograft and xenograft immune responses have restricted the use of allograft and xenograft tissues, as rejection after transplant may result in limited durability and thus necessitate reoperation. To reduce the frequency of a repeat procedure in younger patients, current research has mainly focused on scaffolds consisting of extracellular matrix (ECM) proteins produced by decellularization of xenografts or allografts.¹¹ The attempt to produce a nonimmunogenic substitute, undistinguishable from a native valve regarding mechanical strength, growth, and remodeling, has led to the introduction of various decellularized homograft or xenograft valve conduits in which the immunogenic cellular elements of the tissue have been removed.¹² Enzymatic removal of cells and cellular remnants significantly reduces immunologic reaction,¹³ whereas the intact ECM scaffold is expected to improve recolonization of decellularized conduits with native cells, as experimental evidence exists that the decellularized matrix becomes populated with functional native cells.^14-16

To our knowledge, this is the first study to analyze 3 different methods of heart valve tissue decellularization using collagenase enzyme. Immuno­logic responses against decellularized tissue after implantation, as well as the tissue construction, differed with these 3 methods. This raises the hope that an improved decellularization technique could lead to further significant reductions of immunogenicity, with the decellularized xenograft representing an advance in graft transplant.

Materials and Methods

Animals received humane care in accordance with the “Principles of Laboratory Animal Care” and the “Guide for the Care and Use of Laboratory Animals” (National Institutes of Health, Publication 85-23, revised 1985). An ethical committee on animal research at Shahid Beheshti University approved the protocol. In this study, the minimum number of animals was used.

Seven fresh sheep aortic heart valves were obtained from a local slaughterhouse, placed in phosphate-buffered saline (PBS), and immediately transported to our laboratory. On arrival, they were rinsed with normal saline and freed from adherent fat and myocardium. The valves were stored in PBS and penicillin-streptomycin solution at 4°C until the time of implantation.

In a trial and error process, we tested different settings of concentrations of collagenase and temperatures. Finally, we executed 3 optimal decel­lularization methods. For method A, we used a sterile plate containing PBS with 900 μg/mL of collagenase (collagenase type 2, No. 17101-015; GIBCO, Paisley, UK) in an incubator temperature of 40°C for 16 hours overnight (from 4 PM to 9 AM). For method B, we used a sterile plate containing PBS with 450 μg/mL of collagenase (also collagenase type 2 from GIBCO) in refrigerator temperature of 4°C for 16 hours overnight (from 4 PM to 9 AM). For method C, we used a sterile plate containing PBS with 900 μg/mL of collagenase (also collagenase type 2 from GIBCO) in refrigerator temperature of 4°C for 16 hours overnight (from 4 PM to 9 AM).

After decellularization, heart valves were rinsed thoroughly with PBS to remove residual cell debris and stored in an antibiotic solution (penicillin-streptomycin solution) until the time of implantation. The process was repeated 2 times to ensure complete decellularization despite preservation of the subendothelial structure. Before implantation, both intact and decellularized heart valve pieces were analyzed using surface electron microscope to assess the decellularization effectiveness and subendothelial structure. Each valve piece was thoroughly studied by light microscopy before implant to be sure that each part had the structure of an intact valve (each part containing decellularized valve leaflet tissue and peripheral annulus fibrosus).

Each valve was cut into 3 pieces, and each piece was then separately implanted subdermally into an inbred male Albino rabbit (1-1.5 kg). Our goal was to assess whether any immunologic responses respon­sible for transplant rejection were seen.

Briefly, the animal was anesthetized with 35 mg/kg ketamine and 3 mg/kg acepromazine, and a small incision was made parallel to the skin Langer lines in the medial side of right thorax between ribs 6 to 8, with the heart valve implanted subdermally through a small incision. The incision was then closed and checked for reincision.

Twenty-one days after implant, the implanted heart valve was extracted with its surrounding tissue and stored (4°C) until dissection for histologic studies by light microscopy. Samples were formalin-fixed, paraffin embedded, and serially sectioned for histologic examination, ensuring valve leaflets were visualized in all sections.

Intact and decellularized explanted heart valves and their surrounding tissue were rinsed thoroughly and examined by light microscope for the identification of rejection-related immunologic responses, and images were taken using a digital camera. All processes from excision of heart valve from sheep heart to decellularization and all surgery procedures were done in standard sterile conditions.

Results

To compare each enzyme-based method utilized to decellularize heart valve tissue, we conducted surface electron microscope studies. In the intact nondecellularized heart valves, there was an intact endothelial layer (Figure 1). Surface electron microscope revealed that each decellularization method affected EC removal differently. Decellularization activity following overnight incubation of 900 µg/mL collagenase in an incubator temperature of 40°C (method A) showed no sign of existing intact ECs and nuclei throughout the tissue and comparable distribution of elastin and collagen throughout the remaining tissue. The subendothelial structure appeared to be well maintained (Figure 2); however, 900 μg/mL collagenase in refrigerator temperature of 4°C (method C) was not effective in the complete removal of the EC layer (Figure 3). Although the use of 450 μg/mL collagenase in refrigerator temperature of 4°C (method B) removed the EC layer completely, less distinct preservation of normal-appearing subendothelial structure was shown (Figure 4). These results confirmed that method A was the most efficient method in removing the majority of donor ECs and thus eliminating rejection due to infiltration of inflammatory cells.

Sections of intact and decellularized heart valve tissues explanted at 21 days are shown in Figures 5 to 8. Most notable in these images are the intense inflammatory cell infiltration in the xenogeneic nondecellularized heart valves compared with that seen in the decellularized ones.

Histologic findings by light microscopy demo­nstrated that the intact valve implanted before decellularization showed acute severe rejection, including inflammation and inflammatory cell infiltration (Figure 5). The decellularized valve using method A indicated no signs of inflammation and inflammatory cell infiltration (Figure 6). Although the decellularized valve using method B indicated no signs of inflammation and inflammatory cell infiltration, the compression of subendothelial collagen fibers made it so dense that making tissue cuts for light microscopy assessment was very difficult (Figure 7). The decellularized valve using method C indicated acute mild rejection, including mild inflammation and inflammatory cell infiltration and thus mild rejection (Figure 8).

Discussion

Human organ transplant (allotransplant) has recently become a generally accepted treatment for many diseases that cause organ failure; however, the main complications regarding this promising treatment is lack of donor tissue.¹⁷ Xenotransplant (ie, transplant of tissue between different species) is considered a promising possible solution to overcome this problem.¹⁸ One of the most challenging issues regarding xenotransplant as the treatment choice is immunologic rejection.^19,20 Because of this, patients who receive xenotransplant may not have any symptomatic relief. Immunologic rejection regarding the matter of time and mechanism can be divided into hyperacute rejection, acute vascular rejection, acute T-cell-mediated rejection, and chronic rejection. Reaction of antibodies with endothelial surface antigens as the antidonor humoral response promotes coagulation, complement activation, and EC injury, which finally leads to xenograft thrombosis.²¹

Two different approaches have been introduced to alleviate reactive responses and immunologic rejection: altering the recipient immune system (eg, immunosuppression) or altering the graft tissue in a way to decrease or eliminate graft-versus-host immunologic interactions. As a result of systemic toxicity, current immunosuppression regimens are not acceptable choices for many recipients, especially young children.

Another more acceptable and less harmful approach is altering the graft tissue to reduce its immunogenicity. Different strategies have been used to reduce tissue graft immunogenicity, with the primary one being tissue decellularization.

An ideal valve substitute should be viable, nonimmunogenic, and undistinguishable from a native conduit regarding mechanical strength, growth, repair, and remodeling.¹² Theoretically, these conditions could be fulfilled by employing tissue decellularization techniques to produce viable, antigen-free, and hemodynamically stable tissue-engineered scaffolds that would support recolonization with native cells and thus perform equivalently to other tissue-engineered valves.

Various decellularization methods have been described in the literature, with the most successful being variations of detergent and enzyme extractions for both allograft²² and xenograft tissues.²³ Although most decellularization methods are successful in reducing tissue cellularity, some methods also alter tissue integrity.^22,24,25

Detergent and enzyme decellularization methods have been reported to be associated with ECM disruption and degraded basement membranes.^26,27 We investigated 3 different protocols to decellularize heart valve samples using collagenase enzyme. No immunologic reaction was seen in decellularized valves using methods A and B, whereas this was observed around the heart valve tissue when decellularized using method C, suggesting an incomplete removal of ECs, which was further confirmed by surface electron microscope.

In our study, surface electron microscope examination confirmed that decellularized heart valve samples using collagenase preserved the subendothelial structure of the tissue and ECM and basement membrane integrity, despite the need to further analyze heart valve strength and its hemodynamic performance in vivo. However, ECM alterations in decellularized valves may not be altogether negative, as it is hypothesized that some softening or loosening of the tissue structure may actually be necessary to encourage cell migration into the tissue and enhance recellularization.²⁸

Unfortunately, the decellularization process exposes the ECM, causing the graft to be highly thrombogenic.²⁶ Many previous studies have suggested that the ECM may provide a suitable environment for repopulation by host cells, which would provide the ECM with a regenerative capacity typical of native valves, thus improving xenograft durability and preventing thrombosis.^14,15,29,30 However, further studies are required to investigate the capacity of the matrix to be repopulated with host endothelial and interstitial cells.

In this study, 7 heart valves were dissected from 7 sheep hearts, with each cut into 3 pieces. Each valve piece was decellularized by methods A, B, and C. We tested different concentrations of collagenase and temperature many times, and each time the valve pieces were studied for rejection. These three methods showed the optimal settings reached from our assessments. Valve pieces were rinsed thoroughly before implant and before microscopic study to make sure that cells described as inflammatory cells were not cellular debris. One nondecellularized valve, as control, was also implanted, which ended in acute graft rejection after 21 days.

Each valve piece was thoroughly studied by light microscopy before implantation to prove that each part had an intact valve structure (each part containing decellularized valve leaflet tissue and peripheral annulus fibrosus). Each was then respectively implanted subdermally into male albino rabbits. This method of implantation allowed us, in case of any kind of graft rejection, to ensure that heterogeneity between species (sheep and rabbit) was not the principal causative factor of rejection.

Implanted tissues decellularized using methods A and B showed no signs of inflammation and mild inflammation in method C. We utilized light and electron microscopy because we mainly focused on gross features of rejection, and molecular and cellular processes involved in rejection were not considered. This limitation of our study may require further investigation. In addition, decellularized heart valves were implanted at the same time as control and explanted after 60 days to assess whether chronic rejection occurred; even after 60 days, no sign of rejection was seen.

Rabbits were used as the recipient animal. For our purpose, this was a good choice, with previous studies having xenotransplanted bovine-rabbit or porcine-rabbit systems.³¹ Because our final goal was to establish a xenotransplant-based heart valve for humans, the rabbit is a good choice because it is a fairly small animal and has appropriate size and antigenic properties.

One of the most widely used strategies to reduce graft immunogenicity is cryopreservation. The standard cryopreserved allograft became the choice of the right ventricular outflow tract reconstruction conduit in the mid-1980s. Development of cryo­preservation techniques considerably improved the availability and durability of valve allografts.

Although it is favored, cryopreserved valves are prone to shrinkage, leading to significant valve regurgitation. In most patients who receive a transplanted allograft valve, humoral antibodies develop against human leukocyte antigen that are specific to the transplanted tissue, and host antigen recognition and antibody development may be linked to early-onset tissue calcification and structural valve deterioration. To improve these shortcomings, decellularizing an allograft was a promising choice, leaving only connective tissue, which then may become repopulated with host cells.³²

Standard cryopreserved allografts are commonly used for right ventricular outflow tract reco­nstruction during a variety of congenital cardiac defect repairs.³² Despite numerous advantages, they have variable durability, especially in very young children, and frequently require repeat operations to replace failing conduits. Decellularized cryo­preserved heart valve allografts were developed to ameliorate the recipient immune response to the graft. A decellularized cryopreserved heart valve allograft repopulated by recipient cells would be capable of repairing and remodeling similar to native tissue, endowing it with increased durability. In 2010, Burch and associates compared the decellularized cryopreserved heart valve allograft with a standard cryopreserved valve in repairing right ventricular outflow tract. Despite the standard cryopreserved valves eliciting a more profound immunologic response than decellularized cryopreserved heart valve allografts, these allografts were superior to standard cryopreserved allografts with regard to only 1 parameter in 1 subset of patients.³³ Together, cryopreserved valves are promising choices for substituting defective heart valves, although they can trigger immunologic reactions for moderate-term periods, leading to patient reoperation.

As previously mentioned, it is hypothesized that the EC elements are the antigenic trigger of the reactive immune responses; hence, decellularization has been proposed to reduce the antigenicity of graft tissues.²⁶ Elimination of the immunogenic cellular components of the grafts (ie, endothelium) seems to be the best protocol to improve valve durability and functionality.

In 2005, Da Costa and associates showed that decellularized allografts had normal function for up to 18 months and showed important reduction of the immunogenic response when compared to cryo­preserved valves.³⁴ According to the previous study of Hawkins and associates, decellularized grafts elicited significantly lower levels of class I and class II HLA antibody formation after implant than did standard cryopreserved allografts. Early hemo­dynamic function of decellularized grafts was similar to that of standard cryopreserved allograft valves.¹³

Endothelial cell activation and death are the mainstays of acute humoral vascular xenograft rejection occurring within the first few weeks of transplant. Endothelial cell activation causes coagulation pathway switch from an anticoagulant into procoagulant state. It also promotes inflam­mation by dysregulation of a broad complex of adhesion molecules, cytokines, and chemokines that trigger and alter adhesion and trafficking of immune cells through the graft.^35,36

Endothelial cell activation due to immunologic responses to the xenograft organ is responsible for xenograft rejection shortly after transplant. The current challenge is to overcome the induced antidonor antibody production after implantation.³⁵ Here, we assessed the immunologic response to a heart valve xenograft after decellularization, showing this method as a promising research avenue for minimizing and eliminating immunologic rejection after xenotransplant.

The results of this study are promising and show that decellularization is a beneficial strategy for preparing a xenotransplant-based scaffold for further repopulation of recipient native ECs and represent the initial step toward the goal of developing a xenograft-based tissue-engineered heart valve, followed by additional tissue processing methods to enhance the durability and quality of heart valve grafts.

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