The role of fetal pancreatic islet cell transplantation in the treatment of type 2 diabetes mellitus

Abstract

Objectives

Diabetes mellitus has a negative impact on patients’ lives and is a significant medical and social problem. Due to the high prevalence of diabetes mellitus, shortage of donor materials, immune rejection of the pancreas and limited efficacy of existing treatment methods, the study of promising and more effective approaches to the treatment of this disease, such as transplantation of fetal pancreatic islet cells, becomes relevant. The aim of the study is to determine the efficacy and necessity of fetal pancreatic islet cell transplantation in the treatment of type 2 diabetes mellitus.

Methods

The study was carried out with the help of analytical-synthetic method, literature review and analysis of medical databases corresponding to the topic of work, clinical and experimental studies conducted by other authors were considered.

Results

As a result of this work, it was found that the use of fetal stem cell transplantation is an effective method in the treatment of diabetes. Studies confirm that this method reduces hyperglycaemia and NOMA index, increases c-peptide values without serious side effects on the background of treatment.

Conclusions

Fetal islet cells have advantages in cell culture, relatively low immunogenicity, effective engraftment, although they may produce less insulin relative to adult somatic stem cells. Transplanted islet cells are able to replace and renew the function of the recipient’s own pancreatic β-cells, and prevent their destruction. Fetal pancreatic islet cell transplantation is a promising treatment option for type 2 diabetes that can complement or replace existing therapies, improving patients’ glucose control.

Introduction

Type 2 diabetes mellitus (T2DM) is the most common form of diabetes, usually associated with insulin resistance and insulin production deficiency. As of 2016, among all diagnosed cases of diabetes, type 1 was defined in 5.8% of cases and 90.9% were T2DM [1]. By 2030, the prevalence of T2DM is expected to reach 7079 cases per 100 thousand people [2]. In the adult population of Kazakhstan, the prevalence of T2DM is 8% [3]. Treatment of diabetes mellitus (DM) consists of normalization of body weight, balanced diet, sufficient physical activity, and blood glucose control [4]. Stem cells (SC) have a unique ability to regenerate tissues and organs, as well as to differentiate into specialized cells, including cells of the pancreas [5]. The value of fetal porcine islet cell (FPIC) transplantation in the treatment of T2DM remains a subject of research, and this procedure is not yet a standard treatment for this form of diabetes.

Many studies and clinical observations demonstrate the potential of FPIC transplantation in the treatment of T2DM. Patients who have undergone this procedure often report improved blood glucose levels, reduced need for insulin therapy, and improved quality of life. The procedure can reduce the risks of hypoglycaemia and complications of DM. FPIC transplantation research will allow understanding the mechanisms by which transplanted cells can restore the normal function of the pancreas, will allow evaluating the efficacy and potential benefits of this treatment method, will be able to optimize the extraction technique to achieve the best results with FPIC transplantation.

Research into FPIC transplantation in the treatment of T2DM is ongoing, and some preliminary studies show the potential benefit of this procedure. O. Ulyanova et al. [6] studied the levels of transforming growth factor β1 (TGF-β1) in patients suffering from T2DM after fetal transplantation of SC of the pancreas. There were 10 patients under observation, half of the patients were taking tablet hypoglycaemic therapy or insulin, the other half received intravenous FPIC (16–18 weeks gestational age cells). After transplantation, TGF-β1 levels increased significantly and a decrease in glycated haemoglobin (HbA1c) levels after 3 months was observed [6].

A. Gaipov et al. [7] published the results of a long-term study of the effect of FPIC transplantation from an aborted foetus at 16–20 weeks gestation for the treatment of DM. The study was conducted with a large sample of patients in Kazakhstan using administrative health data. Mortality and complications associated with DM in patients who underwent FPIC transplantation were assessed, and the same parameters were studied in patients receiving conventional hypoglycaemic therapy. Patients receiving FPIC had a 48% lower risk of all-cause mortality relative to the conventional treatment control group. The FPIC group had better outcomes for coronary heart disease, diabetic neuropathy, neuropathy and diabetic foot, but experienced more frequent diabetic nephropathy [7].

Improvement of T2DM patients after FPIC transplantation was demonstrated by S. Tuganbekova et al. [8]. After FPIC transplantation (16–18 weeks of gestation), an increase in C-peptide from 2.24 ± 0.84 ng/L to 3.8 ± 1.25 ng/ml and a decrease in HbA1c in DM patients after 3 months of follow-up were observed [8]. There are numerous clinical studies demonstrating the potential of mesenchymal stem cells (MSCs) to improve the function of the pancreas and reduce insulin resistance in patients with T2DM [9–13]. On the other hand, FPIC-related studies represent scientific novelty in the context of T2DM treatment. FPICs are highly plastic and have the ability to differentiate into various cell lineages, including β-cells. The limited number of studies on the use of FPICs makes this area poorly understood and requires additional research efforts.

The aim of this study is to determine the efficacy of treatment of T2DM by transplantation of fetal porcine islet cells.

Materials and methods

This study was conducted by searching and then analysing available scientific electronic sources such as NCBI, PubMed, Scopus, Medscape, Elsevier, Google Scholar, Web of Science, UpToDate and other specialized internet resources. Keywords such as fetal pancreatic islet cells, fetal pancreatic stem cell transplantation, hyperglycaemia, type 2 diabetes mellitus, insulin resistance were used in the process of searching for the necessary information. The criteria for selecting medical studies and articles by age from information databases reached 35 years. Considering the speed of medical advances and future prospects in the study of pancreatic islet transplantation for the treatment of T2DM, eligible research papers from the past few years were included, taking into account the history. No restrictions were applied to including certain countries and languages in the article. Articles unrelated to the research topic and papers that did not contain accessible and reliable information were also not considered. Lack of access to certain studies, as well as the possibility of subjective influence when searching and analysing data, was also a limitation. In this paper, research materials, medical publications and journals, research articles, electronic libraries, comparative medical data on the specified topic were reviewed and analysed.

The criteria for inclusion in this material included information on transplantation of fetal pancreatic islet cells for the treatment of T2DM, data on the properties of stem cells and their role in transplantation to patients with T21D and T2DM, new technologies to improve the process of pancreatic islet cell transplant engraftment and preserve its function in the donor’s body, difficulties associated with islet cell implantation in recipients with diabetes mellitus, efficacy and safety of pancreatic islet cell transplantation in patients with T21D and T2DM, the effectiveness and safety of pancreatic islet cell transplantation in patients with diabetes mellitus, the effectiveness and safety of pancreatic islet cell transplantation in patients with T21D and T2DM. Articles on the treatment of T1D by islet cell transplantation were considered, and references to stem cells and diabetes mellitus were taken into account. The search strategy was performed using the specified keywords.

After selecting relevant articles and analysing the retrieved data, the study provided information on the use of pancreatic islet cell transplantation in the treatment of T2DM under experimental conditions, methods of preserving the graft in the recipient’s body. Data on the use of pancreatic islet cell transplantation in T1D and T2DM in clinical and preclinical conditions, advantages of islet cell therapy compared to conventional hypoglycaemic therapy are also highlighted. In addition, possible adverse events associated with pancreatic islet cell transplantation are described. Information about immunological difficulties after islet cell transplantation and the advantages of fetal stem cells in the treatment of T2DM is outlined. The efficacy, safety, as well as complications and side effects of using stem cells in the treatment of T2DM are analysed.

Results and discussion

Prospects of transplantation of islets of the pancreas

This article presents an important study involving a promising area of medicine — pancreatic islet cell transplantation for the treatment of T2DM. FPIC transplantation is a procedure in which isolated islet cells containing insulin-producing β-cells extracted from a fetus are transplanted into the pancreas of a patient with diabetes mellitus (DM). The procedure for isolation of fetal pancreatic islets involves a series of chemical and mechanical processes, which consist of cleavage by the enzymes collagenase and protease, stopping the enzymatic reaction and purification of the tissue, centrifugation to separate the islets from the acinar and ductal tissue of the pancreas. After culturing, insulin release test is performed [14].

The advantage of transplantation of fetal islet cells of the pancreas is restoration of normal pancreatic function and regulation of blood glucose level in patients with T2DM. The utilization of FPIC with respect to engraftment and reduced risk of immune response makes them more attractive for transplantation [15]. With the beginning of the use of islet of the pancreas transplantation, the procedure was performed by insertion into the liver through the portal vein, this access was the least invasive with a low bleeding rate [16]. The study of F. Cayabyab et al. [17] was to search for new transplantation sites to increase the success of islet cell therapy in the treatment of diabetes mellitus. Transplantation of islets of the pancreas through the portal vein remained preferable, limited survival of transplanted islets due to hypoxia, possibility of portal vein thrombosis prompted the search for more optimal transplantation sites. Various aspects such as vascularization, immunoprivilege and other factors affecting the success of transplantation were considered. The results showed that the anterior chamber of the eye, subcutaneous space, bone marrow, omentum, and muscle represented potential sites for FPIC transplantation [18]. Currently, parenteral intravenous drip injection of fetal stem cells using a protein-based preparation is the preferred method, which is convenient for both patients and physicians, with minimal time and material costs.

Islet transplantation can be applied to patients who do not fulfil the criteria for whole pancreas transplantation or who wish to avoid undesirable effects of the pancreas transplantation [19–22]. To date, FPIC transplantation is indicated in patients with severe type 1 diabetes (T1D) or post-kidney transplantation [23]. Patients can avoid insulin injections longer after transplantation of the whole of the pancreas compared to transplantation of islets of the pancreas [24]. However, the method of whole pancreas transplantation is limited by its complexity, high costs, availability of donor organs and risks for patients. The use of foetal tissue of the pancreas as a possible alternative has become more promising and relevant in this field [25].

SCs used to obtain pancreatic β-cells and further treatment of DM are derived from various tissues. Human SCs are located in the inner cell layer of the embryo (embryonic), in certain fetal tissues (foetal), placenta, umbilical cord (mesenchymal) and specialized adult tissues (somatic stem cells) [26]. Embryonic and foetal tissue cells have a higher in vitro culturability and show more intensive proliferation relative to mature tissue cells [27, 28]. Also, somatic stem cells can generate some cells specialized for a particular organ, which narrows their therapeutic potential [29]. For adults with DM, 2–3 donors are needed to maintain normal glycaemia without insulin, which severely limits transplantation options [30]. Pluripotent stem cells can be used to generate significant numbers of cells for transplantation, from half a billion to a billion suspended cells [31]. The minimum therapeutic quantity used to correct hyperglycaemia in mouse models was 2 million cells [32]. According to some sources, infusion of 700 thousand to 1 million donor islet cells is sufficient [33].

Immunological peculiarities insular cells

Studies show that FPICs have advantages over somatic stem cells, these advantages include better engraftment and a lower likelihood of leading to an immune response [34]. Embryonic allografts are less likely to cause a graft versus host reaction due to the immunological resistance of the developing foetus, making embryonic cell transplantation relatively safe for the donor, virtually eliminating immunosuppressive therapy and ensuring favourable graft engraftment. Fetal tolerance consists of increased levels of interleukin-10, which induces T cells [35]. Also, 10–20% of islet cells are lost during culturing, which are less inflamed and therefore less immunogenic at the end of the procedure [36]. FPIC have low histocompatibility antigen (HLA) expression and are also able to inhibit macrophage activation [37]. FPICs can exhibit immunomodulatory effects on the microenvironment in tissues, promoting a more favourable environment for self-tolerance and suppressing inflammatory responses, which also reduces the risk of rejection [38]. This is an important scientific aspect that makes FPIC transplantation more promising and less demanding of immunosuppression compared to transplantation of other cell and tissue sources. The study by W.Y. Chung et al. [39] shows a significant increase in cytokine concentration after allogeneic islet cell transplantation relative to autotransplantation.

The procedure of transplantation of specialized adult SC requires immunosuppressive therapy to prevent rejection of transplanted cells, which may lead to the risk of infection and other side effects [40]. In addition, immunosuppressants are toxic to β-cells of the pancreas [41].

Differentiation of insular cells

In the process of long-term SC cultivation, there is a possibility of genetic changes, including chromosomal and gene mutations, which may limit the use of transplantation of islets of the pancreas and affect the safety and efficacy of cell therapy [42]. Possible modalities for selective removal of undifferentiated SC include immunoselective depletion, pharmacological and gene therapy, and molecular tracking [43].

A. Salib et al. [44] describe the use of islets of the pancreas derived from human SC to model diseases associated with T2DM. Traditional cell culture lines do not fully reproduce the physiological properties of human β-cells and have limitations in studying genetic and developmental factors associated with DM pathogenesis. Based on pluripotent stem cells (hPCS), three-dimensional structured functional human pancreatic islet-like organoids (HILO) are being developed that are able to secrete insulin in response to glucose. These organoids provide an opportunity to study the genetic basis of T2DM and to develop new therapeutic approaches using genetic engineering and drug screening.

The study of defective cell marker genes and their association with β-cell development and function may provide valuable data for improving treatment approaches for T2DM and understanding its underlying mechanisms. F. AlRashidi and K. Gillespie [45] studied biomarkers circulating microRNA-375 and differentially methylated insulin cfDNA, which can be used to detect β-cell damage after transplantation of islets of the pancreas. They allow rapid real-time detection of β-cell death during and immediately after transplantation that can predict the clinical outcome of transplantation. The ability to differentiate fetal cells and the potential for further utilization depends on the technical status of pluripotency factors [46].

The ability to produce huge numbers of cells for transplantation from pluripotent SCs provides a solution to the problem of donor cell shortage in the treatment of DM. A.H. Shilleh and H.A. Russ [47] published a study, in which an alternative method of differentiation of cells from the pancreas is proposed. A promising method for mass gain of β-cells of the pancreas is transdifferentiation by induction of β-cell replication, so far only under experimental conditions. To guarantee the safety and ethics of SC research and application, it is necessary to improve differentiation procedures to obtain pure cell populations in order to detect possible undesirable effects such as tumour growth and metastasis [48].

β-cell maturation pancreas glands

Based on the available data, the timing, and mechanisms of human β-cell maturation have so far remained unknown due to the limited availability of the tissue to be tested. The first in vitro study provided data on the immaturity of primary β-cells of the pancreas of newborns, which are unable to maintain optimal insulin levels when glucose levels fall and are dependent on glucose concentration. The study was conducted on one neonate of 35 weeks of gestation. It was found that β-cells of the newborn are less sensitive to glucose levels relative to adult islets and become functionally mature by the age of 1 year [49]. Newborn islets showed similar adequate insulin secretion in response to glucose drop to that of mature islets when cyclic adenosine monophosphate levels were raised with forskolin. Only tolbutamide was effective in stimulating insulin secretion at low glucose levels, but its effects were not enhanced when glucose levels were raised [50]. This indicates the possibility of similar regulatory mechanisms in mature individuals and neonates. Thus, control of basal secretion and metabolic signalling is of particular importance for activation of insulin secretion in neonates and their becoming functionally developed.

One of the methods to improve the procedure of transplantation of islets of the pancreas is the identification of genes associated with β-cell maturation to isolate more mature β-cells from the pool of differentiated cells. Urocortin 3 (UCN3) is a human fetal gene that regulates insulin and glucose levels. Its expression has been found to begin well before the acquisition of functional maturation. Detection of β-cell function defects at the stage of differentiation will improve the process of maturation of islet cells of the pancreas [51]. The gap junction gene (Cx36) is secreted in large amounts by β-cells of mature rats’ relative to immature ones. Presumably, Cx36 is a marker of mature human β-cells [52].

K. Cheng et al. [53] studied the proliferation potential of epithelial cells of the human fetal pancreas and the ability to form β-cells producing insulin. It was observed that the precursors efficiently formed primary cultures, although their replication capacity was small. Transformation of cells by the catalytic subunit of human telomerase reverse transcriptase restored endocrine properties of the cells of the pancreas in vitro. In a further study on mice, the modified cells confirmed their ability to produce insulin and fulfil their function.

Efficiency and safety islets of the pancreas

The first encouraging study on the use of fetal cells published in 1988, the authors studied the effect of transplanting fetal substantia nigra and adrenal medulla into the caudate nucleus of patients with Parkinson’s disease. Fetal substantia nigra and adrenal medulla improved dopamine production, which improved coordination of movements, reduced tremor and improved the patients’ general mobility [54].

X.F. Hua et al. [55] studied the efficacy of insulin-producing cells derived from human embryonic stem cells in the correction of hyperglycaemia in mice model of diabetes with severe combined immunodeficiency (SCID)/diabetes without obesity (NOD). In the study, the researchers found that transplantation of insulin-producing cells derived from human embryonic stem cells resulted in lower blood glucose levels and correction of hyperglycaemia in SCID/NOD mice. This indicates the potential efficacy of using such cells to treat diabetes in humans. The study indicates the possibility of using SC to generate insulin-producing cells that can replace deficient β-cells of the pancreas in diabetes. This may represent a promising approach to the treatment and management of diabetes, helping patients to maintain normal glucose levels and prevent diabetes-related complications.

In a clinical trial of T2DM treatment with fetal SC, M. Demchuk et al. [56] involved 42 patients who were injected once with cryopreserved FPIC suspensions from human fetal cadavers at 5–8 weeks of gestation. The patients were followed up clinically for one year, and almost all patients were taking oral hypoglycaemic drugs. A few months after transplantation 64.3% of patients noted a 1.5-2-fold decrease in hypoglycemic doses, 92.9% of patients had a 2.4-2.6-fold decrease in homeostasis model assessment of insulin resistance (NOMA) after 5–7 months. 85.7% of patients noted a decrease in body weight, 83.4% had an improvement in blood pressure (BP), 81% of patients showed improvements in lipid metabolism.

The positive effect of T2DM treatment with pancreatic progenitor cells from embryonic SC was demonstrated by J.E. Bruin et al. Bruin et al. [57]. Immunodeficient T2DM was modelled in mice using an artificial high-fat diet. Twenty-four weeks after transplantation, an improvement in glucose tolerance was observed in one group of subjects. The second group was treated with transplantation in combination with anti-diabetic drugs. The second group of combined treatment showed positive results already after 12 weeks of treatment, body weight decreased, blood glucose index decreased. It is worth considering that the treatment success was shown in the studied mice with T2DM under immunodeficiency conditions.

Some studies suggest greater therapeutic success with transplantation of tissue-specific somatic stem cells. In a study by F.R. Bani Hamad et al. [58], by reviewing medical databases on the efficacy and safety of SC use in the treatment of T1D and T2DM, it was found that the most favourable treatment outcome was obtained with transplantation of hematopoietic bone marrow stem cells in T1D and bone marrow mononuclear cells in combination with mesenchymal stromal cells in T2DM.

In a study by W.J. Zhang et al. [59], the authors examined the efficacy of islets from progenitor cells of the human fetal pancreas in the treatment of DM. After transplantation of these islets in an animal model of diabetes or a diabetic patient, microscopy, biochemical and functional tests of the islets of the pancreas were performed. Growth of progenitor cells into endocrine cells of the pancreas was found to express insulin and contribute to the maintenance of normal glucose levels in nude mice with diabetes after transplantation. Reduction of kidney damage in diabetic nephropathy (DN) in rats by transplantation of progenitor cells of the human fetal pancreas was demonstrated by Y. Jiang et al. [60]. In the framework of the experiment, the progenitor cells of the pancreas were transplanted to rats with streptozotocin-induced DN. It was found that the level of blood glucose, 24-hour urine protein and urine albumin were lower in rats undergoing transplantation of progenitor cells relative to the control group.

In a safety study of the use of fetal cells in the treatment of diabetes mellitus, 56 patients who underwent allotransplantation of fetal renal suspension or placebo were examined [61]. No serious adverse events and complications of DM were detected among 44 patients. One case of meningioma was detected. The positive effect of combined therapy of fetal and haematopoietic stem cells in the treatment of DM was demonstrated by B. Arjmand et al. [62], mean glucose values were decreased when a mixed suspension was administered. More than 54% of patients could discontinue insulin injections one year after transplantation, 20% remained independent after two years. Many patients resume insulin treatment several years after islet cell transplantation [63].

A study by F. Rahim et al. [64] is a review and meta-analysis of studies related to the use of SC therapy for the treatment of patients with T1D and T2DM. The main focus is on analysing the risks and benefits of such treatment using metabolomics techniques. As a result of this work, it is found that stem cell transplantation improves glycosylated haemoglobin and dosed insulin requirements, but negatively affects c-peptide in the treatment of T2DM. In T1D, it improves c-peptide and glycosylated haemoglobin values but worsens insulin requirement.

In evaluating the efficacy and safety of stem cells in the treatment of T2DM, Shroff et al. found an improvement in 94.8% of patients by one Nutech scale (NFS) grade at the end of follow-up. 54% of patients showed the highest possible NFS scale score. The diagnostic parameters were improvement in insulin, glycated haemoglobin and reduction in the dose of sugar-lowering drugs [65]. G. Hwang et al. [66] also found that the use of stem cells in the therapy of T2DM promotes an increase in the functionality of β-cells. A similar study was conducted by H.M. Shahjalal et al. [67], it was found that embryonic and pluripotent cells are able to secrete less insulin relative to endogenous human stem cells after transplantation. The importance of creating methods for the production of islet structures with the possible creation of vascularized structures for use in regenerative therapy was also noted.

The study by J. Brown et al. [68] evaluated the ability of the fetal rat pancreas to grow and function after its transplantation to adult rats with diabetes, demonstrating a significant reduction in plasma glucose levels, improvement of pancreatic function and normalization of glucose metabolism after transplantation, which demonstrated the effectiveness of this approach for the treatment of diabetes in rats. In a systematic review of publications comparing the efficacy and safety of SC treatment and classical T1D and T2DM therapy, it was found that SC therapy significantly lowered fasting glucose and glycosylated haemoglobin values in all observations, c-peptide value was also significantly improved [69]. The results of SC treatment showed better results in relation to conventional drug therapy.

Advantage of insular cells with regard to traditional therapy

Fetal pancreatic islet transplantation has several advantages over sugar-lowering therapy in the treatment of T2DM. FPIC transplantation restores natural insulin production because of the ability of the transplanted cells themselves to release insulin [70]. This approach aims to restore the natural ability of the pancreas to produce insulin, which is impaired in T2DM. Hypoglycaemic drugs work by improving insulin sensitivity or increasing insulin secretion, but cannot fully restore natural production [71]. FPIC transplantation can provide long-term efficacy in the treatment of T2DM; immediately after integration into the recipient’s body, the islet cells produce insulin continuously, reducing the need for constant dose adjustments or multiple injections as with hypoglycaemic drugs. FPIC transplantation eliminates the need for insulin treatment [72]. The transplanted cells also respond to changes in blood glucose by releasing the necessary amount of insulin to normalize glycaemic levels, and overall glucose control is improved by preventing glucose spikes [73]. FPIC transplantation can improve the quality of life of patients by reducing or eliminating the need to take sugar-lowering drugs, which have financial costs and side effects in the form of hypoglycaemia and gastrointestinal disturbances [74]. Disease modification by FPIC transplantation is also possible; restoration of normal insulin production and hypoglycaemic control may slow disease progression, reduce the risk of complications and improve long-term outcomes compared to the use of hypoglycaemic drugs alone and lifestyle adjustments [75, 76]. An obstacle to the use of SCs in regenerative medicine is the difficulty in their detection and isolation due to unclear phenotype information. The most promising marker for progenitor cells is stem cell factor receptor C-kit and CD117 located on the cell membrane and used as a marker for their isolation [77].

Conclusions

This study provides insight into the advances in the use of fetal pancreatic islet cell transplantation in the treatment of T2DM. Most of the reviewed clinical studies confirm the positive results of pancreatic β-cell transplantation derived from SCs in the therapy of T1D and T2DM. With FPIC transplantation, patients have experienced improved glucose metabolism, increased endogenous insulin production, dose reduction or withdrawal of sugar-lowering drugs, improved lipid profile and overall performance without significant adverse effects or complications. In addition, some studies suggest improvement in patients’ quality of life, reduction in body weight and diabetes-related complications. Pancreatic β-cell transplantation has advantages with respect to hypoglycaemic medication and insulin injections. The islet cells can detect changes in glycaemia and produce the necessary amount of insulin. Constant insulin production reduces the need for multiple injections and reduces the risk of hyper- and hypoglycaemia. It was found that fetal stem cells have a high proliferation capacity, better engraftment ability, have immunomodulatory properties and can reduce inflammatory processes in the donor’s body, unlike specialized somatic cells, which have a limited number and are worse differentiated for transplantation. Pluripotent cells are able to produce less insulin relative to differentiated cells.

Overall, the findings are encouraging and point to the potential of fetal cells in the treatment of diabetes. Clinical therapy with fetal pancreatic islets may represent a promising avenue to combat T2DM. Although fetal pancreatic islet transplantation has advantages, it is important to note that this is still an evolving field with ongoing research and technical challenges. Further research and improvements are needed to optimize transplantation procedures and ensure long-term graft survival and safety.

Declarations

Conflict of interest

The authors declare that they have no conflict of interest.