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Volume: 18 Issue: 3 June 2020

FULL TEXT

ARTICLE
Histologic Evaluations of Xenotransplanted Rabbit Knees by In Vitro-Propagated Human Amniotic Epithelial Cells: A Preclinical Study

Objectives: Human amniotic epithelial cells have multipotent differentiation capacity and are consi­dered as potential therapeutic cells for clinical use. This study represents the first published report on the evaluation of the safety and clinical feasibility of human amniotic epithelial cells for transplant into knee joints, serving as an initial step for subsequent therapeutic evaluations within arthritis clinics.

Materials and Methods: Our experimental design was based on subjecting groups of rabbits as a recipient model for human amniotic epithelial cell transplant into knee joints. Twenty rabbits received 200 µL sterile 0.9% sodium chloride solution containing 1 × 109 human amniotic epithelial cells/knee joint by intra-articular injection. Control groups received cell-free saline into knees, and some animals were not treated. After 10 days of xenotransplant, radiology scans and histologic sections of transplanted and nontrans­planted knees were examined and compared. Immunohistochemistry staining was also applied to detect tumor necrosis factor-alpha and interleukin 17 (as inflammatory and immuno-rejection markers) in knee sections.

Results: Similar to results shown in noninjected and saline-injected knees, all treated knees appeared normal, with no signs of acute immuno-rejection, no microbial colonization, no pain, no allergic reactions, no inflammation, and normal motion. Use of human amniotic epithelial cells appeared safe without risk of immuno-rejection or tumor formation in the transplanted knee joint.

Conclusions: Human amniotic epithelial cells can be safely transplanted into knee joints, encouraging a need for complementary research for further therapeutic evaluations of human amniotic epithelial cells for curing arthritis.


Key words : Arthritis, Regenerative medicine, Stem cell transplant

Introduction

The amniotic membrane (AM) is the innermost placental layer surrounding the fetus. It acts as a tissue barrier between mother and fetus during pregnancy. The immunomodulatory effects of AM have shown that the AM can prevent immunologic reactions between both sides. Thus, many clinical investigations have considered AM as an immu­nologic inert biomembrane for tissue engineering application and as an allograft for treating burns and ulcers, without risk of graft rejection.1

Several biologic characteristics of AM show it to be a great source of stem cells. Specifically, human amniotic epithelial cells (HAECs) show pluripotent, extended duplication capacity and express growth factors and anti-inflammatory cytokines. Furthermore, because placenta is known as medical waste after birth, HAECs are considered as an inexpensive source of stem cells without ethical concerns.2 In particular, amnion-derived stem cells have shown an ability to differentiate into chondrocytes when implanted into cartilage defects of nude rats.3

Regenerative medicine represents a direct way to improve the quality of life of patients with organ or tissue failure. The most important trend in rege­nerative medicine research is cell-based therapy, that is, treatment in which stem cells are transplanted into impaired tissue to improve the regeneration of new healthy cells. There are many investigations that have approached the use of stem cells in the treatment of osteoarthritis; these studies have mainly used mesenchymal stem cells, which are usually from adipose tissues or bone marrow.4,5 However, these studies still need further confirmatory trials regarding the safety and their therapeutic effect. In addition, these types of cells involve ethical problems for allotransplant.

The safety of allotransplant of stem cells is based on overcoming issues related to the risk of pathogenic transmission from donor to recipient, immunologic rejection, and the potential oncogenic activity of stem cells. A good practical technique would be in vitro propagation of stem cells sourced from virus-free donors, thus avoiding cross-infection hazards. Immunogenicity and oncogenicity eval­uations of stem cell transplant are important to allow prediction of their immuno-privilege, safety, and specifically into the target organ.

Akle and associates proved the lower immuno­genicity of HAECs by subcutaneous transplant of a monolayer of in vitro-propagated HAECs into 7 volunteers. In their study, they found no clinical signs of acute rejection among the volunteers. They reasoned that the HAECs did not express outer surface HLA-A, HLA-B, HLA-C, and DR antigens or β2-microglobulin.6

Evaluations of the potential therapeutic benefits of HAECs, as an intra-articular transplant for the clinical treatment of patients with osteoarthritis, have not yet occurred because their safety must be evaluated first. In this study, we aimed to preclinically evaluate the feasibility of HAECs with the use of an animal model. Our evaluation involved xenotransplant of HAECs into rabbit knee joints, followed by immuno-rejection and tumorgenicity evaluations by specific histologic tests.

Materials and Methods

Transplant preparation
Human amniotic epithelial cell preparation and counting were carried out according to previously published methodology.7 Briefly, human AM was separated from placenta obtained from virus-free mothers after elective cesarean delivery (including hepatitis B and C viruses and human immuno­deficiency virus). Membranes were then washed several times with 0.9% sterile sodium chloride solution to eliminate blood and tissue debris. Trypsin treatment was used to dissociate HAECs from AM. Human amniotic epithelial cells were then counted, placed into T-25 flasks (1 × 108 viable cells/flask), and maintained until confluence. Only healthy cultures of HAECs were subsequently harvested by trypsinization. Viable HAECs were counted and aliquoted into 200 μL of 0.9% sterile sodium chloride solution, with each aliquot containing 1 × 109 HAECs.

Experimental design
A total of 25 healthy New Zealand rabbits, each weighing approximately 2 kg, were provided by the Experimental Animal House of the National Central of Radiation Research and Technology (NCRRT), Egyptian Atomic Energy Authority. All rabbit experiments and care were conducted in accordance with the “Principles of Laboratory Animal Care” stated by the National Society for Medical Research. The experimental design of this work was approved by the NCRRT ethical committee.

Both knees from each rabbit received intra-articular injection. Group I (n = 10 animals) received injection of 200 μL of 0.9% sterile sodium chloride solution containing 1 × 109 HAECs in left knees and injection of saline only in right knees. Group II (n = 10 animals) received HAEC injection in right knees and injection of saline only in left knees. A positive control group was formed to determine the positive reactivity of interleukin 17 (IL-17) in the knee joint by immunohistochemistry staining. This was accom­plished by intra-articular injection of 10 ng IL-17 dissolved in 100 μL saline (this concentration was defined by Wang and associates8). In addition, 4 animals that received no injection were assigned as an environmental control group. All treated groups were observed daily, and data were recorded over 10 days until death. Individual rabbits were checked for allergic reactions, inflammation, pain, swelling, and motion complications. Radiologic scans were taken anteroposterior at 1 day before treatment and at 10 days after injection (before death).

Histologic examination
After animal death, both knees were cut and preserved in 10% formalin, and samples were sent for histologic evaluations to the pathology laboratory (Faculty of Veterinary Medicine, Cairo University, Cairo, Egypt). Femoral condyle, tibial plateau, and patellar cartilage were fixed in 4% paraformaldehyde (Leagene Bio., Beijing, China) for 3 days and decalcified in 10% Na2EDTA (Sigma-Aldrich, St. Louis, MO, USA) for 30 days.9 Paraffin sections (thickness of 5 μm) were then prepared and subjected to subsequent histologic examinations. Counterpart sections were divided into 3 comparable sets (Table 1). The scoring of positive and negative reactivity of knees sections with tumor necrosis factor alpha (TNF-α) and IL-17 antibodies were analyzed statistically using SPSS software (SPSS: An IBM Company, version 23, IBM Corporation, Armonk, NY, USA).

Results

Establishment of healthy human amniotic epithelial cell grafts
Healthy HAEC grafts were developed during the in vitro propagation of dissociated amniotic epithelia in a primary culture. Cultures reached confluence within 10 to 15 days after incubation; confluence during this time frame indicated the ability of cultured cells to double and have good establishment in flasks. It was noted that not all collected placenta resulted in successful establishment of HAEC cultures (overall success rate of ~85%). This may have been because of criteria related to the conditions of delivery. Furthermore, a microbial contamination into HAEC cultures would cause HAEC death; thus, all successfully propagated HAEC cultures were free of microbial contamination.

Clinical evaluation of transplanted knees
Both treated rabbit groups (groups I and II) showed good healthy motion after receiving HAECs in their knee joint until time of death (10 days after intra-articular transplant). In addition, no signs of allergic reactions or inflammation were noted. However, one knee (1/20; 5%) revealed some swelling at the site of injection with saline, which may have been as a result of an injection complication. Radiologic scans indicated the structural integrity of all knees, and no differences were shown between HAEC-treated and saline-treated knees (Figure 1 and Table 2).

Histologic finding
Histologic examinations of HAEC-treated and saline-treated knees of rabbits showed normal integrity of cartilage surfaces, which were separated by normal joint space. Examined sections showed no significant occurrence of complications after stem cell transplant (P = .253), including hyperplasia, tumors, or mononuclear cell localization into the treated knee joints. These findings indicated that HAECs showed no tumorigenic activity in the knee joints. Moreover, there were no cases of microbial colonization in treated knees (Figure 2).

Immunohistochemistry assay
The reactivities of specific antibodies to TNF-α and IL-17 antigens in the immunohistochemistry sections of rabbit knee joints were determined in HAEC-treated knees and saline-treated knees. We observed no significant reactivity in both groups (P = .253) compared with that shown in the positive-stained control group, which received IL-17 injection 10 days before death (Table 2, Figure 3, Figure 4, and Figure 5).

Discussion

Cell-based therapy, in which cellular material is injected into a patient, represents the most recent phase of the biotechnology revolution in medicine.10 The most challenging issues for the use of stem cell allografts in clinical applications are immuno-rejection, cost, and ethical concerns. Our study proposes that HAECs are a low-cost and an easy to obtain stem cell option without the challenges related to ethical concerns as this cell type is derived from placenta, which is considered as medical waste after birth.

There are several experimental and clinical trials that have proposed HAECs as therapeutic cells for treatment, including for spinal cord injury,11 several liver diseases,12 neurologic disorders,13 and lung injury.14 These uses are attributed to the multipotent capacity of HAECs to differentiate into the 3 germ layers. Previous data have shown that HAECs represent OCT-4, Nanog, SOX-2, and Rex-1 molecular markers. The expression of these markers proves the multipotent capacity of HAECs and their advantages for therapeutic use rather than the use of embryonic stem cells, which challenge ethical concerns.15 Furthermore, an in vitro investigation of HAEC immunogenicity verified that HAECs do not express HLA class II antigens, which is promising to bypass the immune system after in vivo transplant.6

There are no previous publications on the potential activity of HAECs when transplanted into knee joints for subsequent evaluations to treat patients with osteoarthritis. Consequently, this study aimed to confirm the safety and immunologic adaptive properties of HAECs after xenotransplant, in which an organism receives a graft from a different organism. This type of transplant involves the highest possibility of immuno-rejection among organisms. Therefore, the successfulness of xenotransplant of HAECs theoretically depends on their immuno-privilege properties.

Unpublished data have indicated that HAECs did not form tumors when injected subcutaneously or intra-muscularly into experimental rats. These experiments also recorded no signs of allergic reactions, inflammation, weight loss, or abnormal activities. In addition to these observations, previous investigations have recorded no abnormal growth of HAECs after in vivo transplant into various organs.11-14

In vitro propagation of HAECs ensures the harvesting of healthy cells without risk of microbial contamination. That is, the selection of cultures with well-established cells on the flask surface (HAECs successfully attached to cell culture flask surface and showing spindle shape 3 to 4 days after incubation) results in proper cells (HAECs reaching confluence 10 to 15 days after incubation, resulting in 1 × 108 of HAECs) that are free of the turbidity of microbial growth.

It is known that TNF-α and IL-17 are graft rejection markers that increase during localized transplant inflammation and immuno-rejection. In rats, levels can be detected by their specific antibody interactions with T-cell proteins localized into the rejected graft starting from day 3 posttransplant, which increase significantly during days 5 and 7.16

In our investigation of histologic and immuno­histochemistry findings, we found that HAEC transplant did not result in immunologic reactivity in the knee joint of rabbits. This proved their immuno-privilege property. Moreover, our preclinical study showed that in vitro-propagated HAECs were safe for transplant without risk of microbial conta­mination and did not result in allergic reactions in the knee joint.

Conclusions

Our preclinical study indicated the safety of in vivo transplant of HAECs into the knee joint, without risks of immuno-rejection and tumorigenic activity. These promising indications suggest the need for further confirmatory studies and therapeutic evaluations to determine whether HAEC transplant could be used to cure osteoarthritis.


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Volume : 18
Issue : 3
Pages : 375 - 381
DOI : 10.6002/ect.2019.0049


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From the 1Department of Health Radiation Research and the 2Department of Radiation Microbiology, National Center for Radiation Research and Technology (NCRRT), Egyptian Atomic Energy Authority (EAEA), Cairo, Egypt
Acknowledgements: The authors have no sources of funding for this study and have no conflicts of interest to declare. We give many thanks to Professor Kawkab A. Ahmed (Department of Pathology, Faculty of Veterinary Medicine, Cairo University) and to Associate Professor Michael. I. Michael (Department of Physiology and Biology Application, Atomic Energy Authority) for their technical help and support.
Corresponding author: Waleed A. Nemr, Egyptian Atomic Energy Authority, Cairo, PO 29 Nasr City, Egypt
E-mail: wnemr@hotmail.com