Review Article - (2021) Volume 6, Issue 6
David Brownell1, Christophe Caneparo1, Stéphane Chabaud1 and Stéphane Bolduc1,2,3*
1Centre de Recherche en Organogénèse Expérimentale/LOEX, Regenerative Medicine Division, CHU de Québec Research Center, Quebec, QC, Canada
2Division of Urology, CHU de Québec- Université Laval, Québec, Québec, Canada
3Department of Surgery, Faculty of Medicine, Laval University, Quebec, QC, Canada
Received Date: June 03, 2021; Accepted Date: June 10, 2021 Published Date: June 17, 2021
Citation: Brownell D, Caneparo C, Chabaud S, Bolduc S (2021) Endothelialised Tissue Engineered Substitutes Produced by the Self- Assembly Approach for Implantation and Research: Past, Present and Future. Insights Biomed Vol.6 No.6:26.
Recent innovations in medicine include tissue engineering to produce tissues or organs for replacement/repair surgeries but also to serve as relevant tridimensional research models. For both applications, presence of microvascular networks inside the reconstructed tissues is necessary to obtain structures that are both complex and more complete. Indeed, rapid inosculation of graft to host is essential for positive clinical outcomes but pre-vascularized tissues are also a need to obtain thick tissue where diffusion of nutrients and oxygen cannot be only passive. In the last two decades, the self-assembly approach was developed at LOEX and has allowed breakthroughs in many organ/tissue reconstructions. This unique technique relies on the production of a stroma scaffold by the mesenchymal cells themselves without the need of exogenous materials. Endothelialisation of such tissues shows a great impact not only on graft reperfusion but also in the improvement of research models such as cancer and psoriatic models. The presence of a vascular or lymphatic network now opens the door to the development of complex and configurable models which may be available soon.
Tissue engineering; Endothelial cells; Self-assembly
In recent years, the Western world has been confronted with a shortage of adequate tissues for transplants to meet the needs of various pathologies [1]. Indeed, the increase in number of patients requiring repair or replacement of defective organs, mainly related to an ageing demographic and an increasingly sedentary lifestyle of the population, are combined with a decrease in the pool of available organs. This is due to the reasons mentioned above and to the tightening of the selection criteria for tissues suitable for grafting by regulatory agencies. At the request of clinicians, and often with their contribution, scientists have been led to develop innovative technologies to remedy this situation. Among these, tissue engineering seems particularly promising and has undergone significant development in recent decades, even if sometimes chaotic due to the gap between the scientific world and the financial and media worlds who do not think on similar time scales [2-4].
Tissue engineering is primarily based on the use of molecular scaffolding of synthetic or natural origin, onto which cells, potentially from the patient, can be seeded to recreate a functional organ/tissue [5,6]. One of the main pitfalls encountered by tissue engineering has been the ability to graft thick tissues. Indeed, the diffusion limit, which allows a passive exchange of nutrients and oxygen in one direction and of metabolic waste and carbon dioxide in the other, is variable but rarely allows the reconstruction of scaffolds with a thickness of beyond 300-350 μm. Most complex structures, however, require tissues of this thickness and beyond. This is the case when attempting reconstruction of any three dimensional organ. Failing to overcome this dilemma would restrict tissue engineering to small, thin tissues and eliminate the possibility of replacing entire organ [7].
Even if the host's vascular network is capable of invading the reconstructed tissue once grafted, which is not necessarily the case, particularly with synthetic materials; the rate of invasion of the tissue for optimal perfusion of the graft is rarely reached. Partial or total tissue necrosis may be observed. Several solutions have been considered, such as helping the host network to invade the graft more quickly by using proangiogenic growth factors such as Vascular Endothelial Growth Factor (VEGF), or by forming a capillary-like network inside the graft itself before implantation, which can connect to the host by inosculation [8-10].
Over the past twenty years, Dr. François A. Auger's group has developed at LOEX a new approach to reconstruct tissues by tissue engineering: the “self-assembly" technique (Figure 1) [11]. This technique, which relies on the capacity of stromal cells to produce, secrete and assemble their own extracellular matrix (ECM), makes it possible to build scaffolds very similar to native tissues without the need for exogenous biomaterials which may distance a model from the native tissue. Mechanical properties of transplant tissues are a critical factor for the transplant and for tissue functionality. These properties are fairly satisfactory when using the self-assembly technique [12-15]. In addition, these tissues demonstrated a histological organization and functionality of epithelial cells seeded on stromas, at a level very similar to that observed in native epithelia [12,16]. This is not always the case for epithelial cells when seeded on many biomaterials.
Figure 1: Schema of the reconstruction of tissue using the standard protocol of self-assembly.
The protocols developed from the self-assembly technique have made it possible to develop a whole range of tissues helpful to compensate for the lack of tissues suitable for grafting. Bilayered skin substitutes consisting of an epidermis made of keratinocytes and a dermis of fibroblasts with their ECM have been used for several years to treat severely burnt patients, but also for those with ulcers or children affected by epidermolysis bullosa [17-19]. Corneal tissues have also been developed to treat patients with blindness associated with corneal clouding following trauma [20]. Blood vessels have been designed simultaneously with skin substitutes, using a similar technique, obtaining a tubular shape by rolling the ECM sheet around a mandrel [21]. Other models are currently under development such as adipose tissue reconstructed from adipose-derived stem/stromal cells (ASC) to serve as filling tissues or biological dressings [22,23]. Bone tissues have also been derived from ASC [24]. More recently, genitourinary tissues, such as the ureters, bladder and urethra have been developed using tubular or flat models, and the vagina as flat models only [12,15,25,26].
In addition to the obvious clinical applications, fundamental study models have been derived from these reconstructed tissues to understand pathologies in a context close to native human tissue. Indeed, most studies are performed on inadequate models associated with a low potential for clinical translation [27]. Monolayers of cells in plastic Petri dishes generally do not make it possible to recreate either the complexity of the organization of the tissue in three dimensions, and therefore, the cell dynamics, polarity in particular, and hence differentiation, or the interactions between cells and the ECM, which a protein coating can very partially represent. Similarly, laboratory animals often show significant differences between species, humans included. The contribution of these models to the advancement of science should not be underestimated; nevertheless, biological complexity obliges us to recognize that it is necessary today to develop more sophisticated models and perhaps in the midterm, not only to recreate tissues in the laboratory but also to interconnect them, similarly to organ-on-chip technology, which is exciting but too simplistic given the complexity of realistic biological conditions.
In this context, the self-assembly technique has made it possible to study skin pathologies such as hypertrophic scars, systemic sclerosis (scleroderma), melanoma and basal carcinoma, psoriasis, epidermolysis bullosa, neurofibromatosis or skin manifestations of amyotrophic lateral sclerosis (ALS) [19,28-34]. Studies have also been carried out to better understand Fuchs’ dystrophy, which affects the corneal endothelium [35]. Models have also been developed to study pathologies of the genitourinary tract, such as bladder cancer, ketamine-induced cystitis, urinary tract infections, as well as infection of an immunocompetent vaginal mucosa model by the human immunodeficiency virus (HIV) [36- 38]. These various models could also serve as a solid basis for conducting permeability studies for the pharmaceutical industry as current in vitro models are often lacking in complexity.
Whether the objective is to reconstruct tissue for transplantation or to produce models that are as complete as possible, possibly composed of interconnected reconstructed tissues, vascular network formation within the 3D tissue is essential. For example, to study the early stages of metastasis or to measure the dynamics of diffusion of various factors within the tissue [39]. Several approaches have been attempted to recreate a network of capillaries within tissues produced by the self-assembly technique. Initially, human umbilical cord vein endothelial cells (HUVEC) or microvascular endothelial cells (MVEC) were seeded on the surface of ECM sheets which were then stacked to form an endothelialized stroma [40,41]. Although this approach may have given interesting results in the clinic by demonstrating that tissues produced with this approach were reperfused more quickly than non-endothelial tissues, the distribution of capillaries on the surface of the sheets was not optimal. A new technique was therefore introduced (Figure 2). The HUVEC/MVEC was seeded along with the stromal cells to produce the sheet and formed a capillary network throughout its entire thickness [42]. It was then possible to reseed endothelial cells and stromal cells on the ECM sheet to thicken it and homogenize the distribution of cells. It was also possible to stack reseeded sheets and have a network of capillaries harmoniously distributed in a relatively thick tissue.
Figure 2: Schema of the reconstruction of tissue using the “reseeding” variant protocol of self-assembly.
Limits
Even if the tissues produced by the self-assembly method, endothelialized or not, offer real advantages compared to substitutes produced with biomaterials, the fact remains that they have certain drawbacks that it is necessary to mention. Indeed two main problems can arise in the context of the use of tissues reconstructed with the self-assembly method: the first is related to the time required for the production of the tissues and therefore their costs, although significant efforts have been made to decrease these two factors [42,43]. As seen in Figures 1 and 2, the time to produce a complete substitute, i.e. with stroma and epithelium, takes about two months. For certain applications which require the production of tissues in an emergency, the technique therefore seems unsuitable even if work is in progress to evaluate the possibility of using decellularized matrices in order to halve the time required [44]. For other applications where time is not a critical factor, such as hypospadias correction surgeries which are performed on an elective basis, this factor is less impactful. The cost issue may, however, put off some surgeons. Nevertheless, if the treatment is evaluated in a long term point of view, the advantages of a bilayered tissue produced with the cells of patients and having a very strong histological and functional similarity can be an important advantage and thus reduce the costs subsequent to the surgery which are often important when using more classic approaches [17]. Another problem arises from the planar or circular configuration of the tissues produced. Some organs require more elaborate morphologies such as the lungs or kidneys, for example, and are difficult to reconstruct using the self-assembly technique. Nevertheless structures as complex as heart valves could be produced with this approach [45,46]. However, no techniques or mix of techniques should be ignored in order to provide the most suitable product to patients, for graftable tissue, or to scientists, for research models.
Nevertheless, many challenges remain to be overcome to recreate thick tissues, that of a thickness beyond the diffusion limit, or to produce an interconnected network of organs/tissues reconstructed by tissue engineering, using the self-assembly technique or others as appropriate. For example, it could be exciting to connect brain organoids with intestinal or bladder tissue due to the numerous pathologies linking the brain and these organs [47,48]. The hypothesis that cancer cells secrete messages that remotely alter the microenvironment of metastasis sites has been developed for many years. It has been revived over the past fifteen years by studies on exosomes [49,50]. Producing tissues from primary cancer sites and studying the diffusion of these exosomes through the bloodstream and their effect on metastatic sites could provide valuable information. It would also be interesting to understand why specific tissues are more inclined to receive circulating cancer cells where others are refractory. The self-assembly technique has also been shown to reconstruct lymphatic vessels, which play a role in these processes [51]. From a technical point of view, such an interconnected tissue network could be built. Several techniques reconstructed many organs/tissues, and blood vessels can be designed and easily interconnected for various calibres (Figure 3).
Figure 3: Schema of a potential model to study cancer metastases spreading from bladder to lungs and bones.
The medicine of the future has a high potential be a personalized medicine where each person can receive appropriate treatment for the pathology that affects them, adapted to each unique profile [52-55]. Contrary to what some people imagine, such a development would reduce health costs by avoiding the use of expensive drugs that may not prove useful for a specific person and therefore by shortening treatment times and reducing side effects. Unlike the models currently used, the development of complex research models where all aspects could be parameterized and controlled, unlike animal models, and this from a patient's own cells, will allow these feats that were still unimaginable a half-century ago but are today within our reach. Also, people will be able to have access to a range of tissues reconstructed in the laboratory with their own cells and that are therefore perfectly immune-compatible, having a degree of differentiation, and therefore functionality, as high as possible, allowing a return to a better quality of life. The development of tissue engineering for direct clinical applications and for the advancement of knowledge has the potential to change the face of medicine. It could be comparable to the discoveries of antibiotics and vaccines. Sky is the limit.
The authors declare no conflict of interest.
Ethical review and approval were waived for this study, due to no human subject or animal was used.
This study was supported by a Canadian Urological Association Scholarship (S.B.), a Canadian Institutes of Health Research Grant (#258229) (S.B.), the ‘Fonds de Recherche du Québec – Santé (FRQS) (CC).
The project is supported by the Quebec Cell, Tissue and Gene Therapy Network –ThéCell (a thematic network supported by the Fonds de recherche du Québec–Santé).
