Introduction and review of literature
1.2 Review of literature
1.2.3 Current limitations and emerging strategies in vascular tissue engineering
1.2.3.5 Acellular vascular grafts: bypassing cell incorporation
Since the beginning, when Weinberg and Bell (1986) proposed the idea of creating tissue- engineered vascular grafts using autologous primary vascular cells, research impetus in the field is focused on implementing a diverse array of cells fostering an off-the-shelf TEVG, as summarized in Table 1.1. Cell-laden grafts have performed better than cell-free grafts; but the former approach poses several impediments. For instance, culture and expansion of patient- specific vascular cells is a time-consuming and costly process, challenging its clinical feasibility.
Moreover, the diminished expansion ability of adult vascular cells restrains a broader adoption of this technique. Vascular progenitor cells are a suitable alternative, but their scarce availability hampers the idea. Subsequently, the focus shifted to various stem cells to obtain larger vascular cell populations via lineage-specific differentiation [85]. Direct seeding of patient-specific stem cells in TEVGs before implantation soon became tremendously popular in the field, considering their abundant availability and bypassing the need for culture expansion [86, 87]. Autologous BM- MNCs seeded TEVGs were the first to enter into clinic [36] and have long been a part of active clinical investigation [40]. Bone marrow and adipose tissue-derived MSCs also remain a substantial choice for researchers towards creating an off-the-shelf system [88, 89]. Nevertheless, it is believed that acellular grafts would presumably follow a shorter path towards clinical translation as compared to the seeded ones. Contemporaneously with cellular TEVGs, acellular grafts are also being investigated routinely. Three different approaches are broadly adopted: 1) Cell-free biodegradable polymeric grafts, 2) Decellularization of native blood vessels and 3) Decellularization of grafts prepared by in vitro cellular self-assembly (Figure 1.4).
Table 1.1. Cell types used for vascular tissue engineering applications.
Cell type Prominent sources Comments Ref.
Primary Vascular Cells Endothelial
cells
Human umbilical vein endothelial cells (HUVEC)
Microvascular endothelial cells (MVEC)
Biopsies from patient blood vessels
Naturally aligned along the direction of blood flow
Provide a natural anti-thrombotic surface
Endothelialized grafts with autologous cells prevent thrombosis
Patient-specific cells bypass the need for immunosuppressive therapy
Limited availability and long in vitro expansion time restrict their direct implementation for TEVGs
[28, 30, 59]
Smooth muscle cells
Human umbilical artery smooth muscle cells (HUASMC)
Naturally aligned circumferentially, provide contractile function and maintain elasticity
[3, 90, 91]
Biopsies from patient blood vessels
Responsible for rendering mechanical stability to a blood vessel via ECM secretion
Synthetic/proliferative phenotype leads to overgrowth and intimal hyperplasia
Engineered HAVs, which are in phase III clinical trials for hemodialysis access, are prepared from allogenic SMCs/PGA scaffolds
Fibroblasts Dermal fibroblasts
Biopsies from patient blood vessels
Naturally present in the outermost adventitial layer of blood vessels
Provide mechanical resilience by ECM production
Predominantly used for preparing cell- sheet based and fibrin embedded engineered vessels
[29, 30, 92, 93]
Vascular Progenitor Cells Perivascular
pericytes
Any tissue with capillary bed (muscle, fat, saphenous vein segments, etc.)
Cells found in microvasculature interspersed between endothelial cells
Have mesenchymal stem cells (MSC) like features
Secrete pro-angiogenic factors
Allogenic cells show immune compatibility
[94-96]
Endothelial progenitor cells (EPCs) and
endothelial colony forming cells (ECFCs)
Bone marrow
Adipose tissue
Human peripheral blood
Placenta
Perivascular cell fractions
Encompass a broad category of cells capable of differentiating into endothelial cells
[95, 97]
Stem Cells
Mesenchymal stem cells (bone marrow mononuclear cells, stromal vascular fraction and adipose- derived mesenchymal stem cells)
Bone marrow
Umbilical cord blood
Adipose tissue
Guide TEVG remodeling via paracrine signaling
Immunomodulatory
Immunocompatible
Due to the availability of abundant sources and easy processing, their implementation in TEVG markedly reduces graft fabrication time
MSC derived extracellular vesicles have shown remarkable therapeutic potential
Their secretome has angiogenic potential
[40, 44, 98]
Induced pluripotent stem cells (iPSCs)
Somatic cells (Fibroblasts)
Peripheral blood
Can be obtained in high quantity and differentiated into vascular cell lineage (ECs and SMCs)
Minimally or non-immunogenic
Allogenic implementation is feasible
[99, 100]
1.2.3.5.1 Cell-free biodegradable polymeric grafts
Notwithstanding the benefits of cell seeding, acellular polymeric grafts are one of the prodigious choices that pragmatically epitomize the most compelling candidate for clinical translation [5, 101- 103]. The advantageous aspects of implementing this approach are: 1) minimal batch to batch variation, 2) the whole process is automated, conferring better chances of reproducibility, 3) bypass the need of culturing cells and long-term maturation, 4) removing cellular component would confer immune compatibility, 5) avoid chances of disease transmission, 6) can be functionalized with a plethora of bioactive molecules to improve graft performance, 7) can be made readily available for patients in need. However, this technology lags in terms of lack of bioactivity, one of the key pre-requisites of TEVG success, generally conferred by cell seeding for cellular grafts. Seeded MSCs not only provide anti-thrombogenicity [104] but also help in graft remodeling [44]. Emerging technologies are now trying to imitate the effect of cell-seeding in acellular polymeric grafts by orchestrating their functionalization with bioactive moieties. This innovative approach could be the most suitable strategy for acellular TEVGs’ clinical translation, pending identification of a suitable combination.
1.2.3.5.2 Decellularization of native blood vessels
Allogenic and xenogenic blood vessels are suitable alternative for vascular reconstruction provided they are devoid of any cellular immunogenic materials orchestrating immune compatibility. Both allogenic and xenogenic vessels from various sources are under active investigation to explore their ability as a suitable grafting option. The main advantage of using native vessels is that it mitigates the limitations associated with the availability of autologous grafts. Moreover, they intrinsically contain the well-organized ECM proteins assisting in desired graft mechanics.
Notwithstanding these advantages, their immunogenicity remains the prime setback for wider clinical adoption [101].
Decellularized xenografts from porcine, bovine, ovine, and other rodent sources have been tested in either pre-clinical or clinical settings. Xenografts from bovine are clinically available for hemodialysis access (e.g., ProCol®, Artegraft®, SynerGraft®, Solcograft®), however, their performance is not satisfactory as they are prone to thrombosis and aneurysm formation [105].
Another commercialized xenograft is CorMatrix, derived from porcine SIS was also found to be inefficient for a low-flow high-pressure system as it showed significant stenosis [106]. From a clinical perspective, the clinical performance of xenografts has not been very successful. Research impetus is now shifted towards exploring various allografts with innovative modifications to improve their bioactivity and long-term performance while mitigating their immunogenicity.
1.2.3.5.3 Decellularization of grafts prepared by in vitro culture
Driven by the challenges associated with decellularization of native vessels and encouraging evidence suggesting the dependence of graft mechanics on structural organization of conserved ECM proteins, underlaid the idea of decellularizing in vitro bioengineered vessels. This strategy bypasses the need for autologous biopsy from the patient and leverages the capability of vascular cells’ self-assembly under in vitro conditions. Following methodologies described in foundational studies [33, 107], bioengineered vascular conduits were prepared by culturing bovine/porcine SMCs onto PGA mesh followed by long-term maturation in bioreactors. Furthermore, bioengineered HAVs entered into clinical trial studies, wherein they were implanted for hemodialysis access in a human patient cohort having an end-stage renal disease. Interestingly, HAVs barely exhibited any post-cannulation bleeding, and 1-year follow-up showed excellent secondary patency without dilatation [35]. In a subsequent study, the implanted HAVs were
investigated for host cell infiltration and remodeling over 200 weeks, revealing their transformation into living blood vessel [108]. Considering the prodigious performance of HAVs in pre-clinical and clinical studies for hemodialysis access, recently, they were implanted for arterial reconstruction (femoral-to-popliteal and above-knee bypass) in humans with great success [109, 110]. As of now, HAVs remain the most successful engineered biological vessel in human clinical trials.
In addition to HAVs comprising of allogenic SMCs and PGA mesh (wherein both of them are subsequently removed, leaving behind collagenous tubular framework), researchers are exploring the inherent self-assembling property of fibroblasts to prepare a completely biological graft. In a partial decellularization approach, TEBVs consisting of inner endothelium cultured on decellularized cell membrane and a living adventitia were developed by long term maturation of fibroblast cell sheets derived from human skin biopsies [111]. Notably, cultured fibroblasts are devoid of MHC II antigens, hence are minimally immunogenic. Allogenic, fibroblast-derived TEBVs were further rendered non-immunogenicity by dehydration in lieu of decellularization and clinically investigated in humans for hemodialysis access by implanting them as shunts. This case study revealed the clinical applicability of scaffold-free cell-derived TEBVs [112]. In recent work, the same research group has shown the production of yarns derived from cell-assembled sheets as true ‘bio’ material for versatile tissue-engineering applications [93].
In another effective approach, donor fibroblast cells were embedded in a tubular fibrin gel and subjected to pulsatile flow maturation for 5 weeks, followed by decellularization. Intriguingly, the resulting tubes had the growth potential post-implantation as evinced by their implantation replacing pulmonary arteries in a growing lamb model [113]. The application of cell-free biological graft was further extended to hemodialysis access in baboons [92]. Graft prepared from human neonatal fibroblasts (15 cm long, 6 mm diameter) were implanted in the axillary-brachial upper arm or axillary-cephalic position and explanted after six months. Explant analysis suggested graft recellularization with host cells with 60% primary patency with no signs of calcification and stenosis [92]. Outcomes of these studies spur the notion that acellular biological grafts can grow with the host age and undergo in vivo recellularization rendering natural-like vessel characteristics.
Clinical outcomes of this technology, wherein acellular grafts are prepared by long-term in vitro maturation, have made it the most advanced approach in the current scenario. As described earlier, the leading groups in the field have shown the remarkable potential of cell-free grafts for
hemodialysis access and arterial replacement. With the advent of technology, these grafts are already in different phases of clinical trials.
Figure 1.4. Acellular TEVGs. Scheme representing various methodologies adopted to obtain cell- free/acellular vascular grafts.