1
Inflammatory and regenerative processes in bioresorbable synthetic 1
pulmonary valves up to 2 years in sheep: Spatiotemporal insights 2
augmented by Raman microspectroscopy 3
4
De Kort B.J.
a,b
, Marzi J.
c,d,e
, Brauchle E.
c,d,e
, Lichauco A.M.
a,b
, Bauer H.S.
f
, Serrero A.
f
, 5
Dekker S.
a,b
, Cox M.A.J.
f
, Schoen F.J.
g
, Schenke-Layland K.
c,d,e,h
, Bouten C.V.C.
a,b
, Smits 6
A.I.P.M.
a,b *
7
8
a. Department of Biomedical Engineering, Eindhoven University of Technology, 9
Eindhoven, The Netherlands 10
b. Institute for Complex Molecular Systems (ICMS), Eindhoven University of Technology, 11
Eindhoven, The Netherlands 12
c. Department of Women's Health, Research Institute of Women's Health, Eberhard 13
Karls University Tübingen, Tübingen, Germany 14
d. NMI Natural and Medical Sciences Institute at the University of Tübingen, Reutlingen, 15
Germany 16
e. Cluster of Excellence iFIT (EXC 2180) “Image-Guided and Functionally Instructed 17
Tumor Therapies”, Eberhard Karls University Tübingen, Tübingen, Germany
18
f. Xeltis B.V., Eindhoven, The Netherlands 19
g. Department of Pathology, Brigham and Women's Hospital, Harvard Medical School, 20
Boston, MA, USA 21
h. Department of Medicine/Cardiology, Cardiovascular Research Laboratories, David 22
Geffen School of Medicine at UCLA, Los Angeles, CA, USA 23
*Corresponding author: Dr.ir. A.I.P.M. (Anthal) Smits; a.i.p.m.smits@tue.nl
Adress: 24
Eindhoven University of Technology, Department of Biomedical Engineering, P.O. Box 25
513; 5600 MB Eindhoven
26
was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
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2
Abstract 27
In situ heart valve tissue engineering is an emerging approach in which resorbable, off-28
the-shelf available scaffolds are used to induce endogenous heart valve restoration. Such 29
scaffolds are designed to recruit endogenous cells in vivo, which subsequently resorb 30
polymer and produce and remodel new valvular tissue in situ. Recently, preclinical studies 31
using electrospun supramolecular elastomeric valvular grafts have shown that this 32
approach enables in situ regeneration of pulmonary valves with long-term functionality in 33
vivo. However, the evolution and mechanisms of inflammation, polymer absorption and 34
tissue regeneration are largely unknown, and adverse valve remodeling and intra- and 35
inter-valvular variability have been reported. Therefore, the goal of the present study was 36
to gain a mechanistic understanding of the in vivo regenerative processes by combining 37
routine histology and immunohistochemistry, using a comprehensive sheep-specific 38
antibody panel, with Raman microspectroscopy for the spatiotemporal analysis of in situ 39
tissue-engineered pulmonary valves with follow-up to 24 months from a previous 40
preclinical study in sheep. The analyses revealed a strong spatial heterogeneity in the 41
influx of inflammatory cells, graft resorption, and foreign body giant cells. Collagen 42
maturation occurred predominantly between 6 and 12 months after implantation, which 43
was accompanied by a progressive switch to a more quiescent phenotype of infiltrating 44
cells with properties of valvular interstitial cells. Variability among specimens in the extent 45
of tissue remodeling was observed for follow-up times after 6 months. Taken together, 46
these findings advance the understanding of key events and mechanisms in material-47
driven in situ heart valve tissue engineering. 48
49
Keywords: Tissue-engineered heart valve (TEHV), in situ tissue engineering, 50
endogenous tissue restoration, biomaterial, foreign body response 51
52
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3
Introduction 53
Surgical or interventional valve replacement is the standard of care treatment for most 54
patients with severe symptomatic valvular heart disease, and this treatment improves 55
quality of life and prolongs survival. Surgical valve replacement with either mechanical or 56
tissue valve substitutes (the latter composed of animal or human tissue and thus often 57
called bioprostheses) generally yield favorable long-term outcomes; survival is 50-70% at 58
10-15 years following valve replacement [1]. Nevertheless, valve-related problems 59
necessitate reoperation or cause death in more than half of patients with substitute valves 60
within 10-15 years postoperatively [2], [3]. Mechanical valves induce platelet deposition 61
and blood coagulation, (i.e., thrombosis) necessitating lifelong anticoagulation to reduce 62
the risk of prosthetic valve-related blood clots in patients receiving them. In contrast, 63
bioprostheses have low potential for thrombosis. However, despite improvements in 64
tissue treatments intended to enhance durability, bioprostheses frequently suffer structural 65
valve degeneration, often resulting from calcification, which is particularly accelerated in 66
children and young adults [4]. Although transcatheter valve replacement technologies 67
have recently gained favor, owing to less invasive implantation and good short-term 68
results, their long-term durability is uncertain [5], [6]. Recently the principle of in situ tissue 69
engineering (TE), also known as endogenous tissue restoration (ETR), has emerged as a 70
promising alternative [7]–[9]. This approach utilizes the regenerative capacity of the 71
human body to transform a resorbable polymeric implant into a living functional valve, 72
directly in its functional site, or in situ. The resorbable graft functions as a suitable valve 73
immediately upon implantation, and subsequently serves as an instructive template for 74
progressive endogenous cell infiltration and tissue deposition [7], [10]. 75
Preclinical studies demonstrate the potential of in situ heart valve TE for pulmonary and 76
aortic valve replacements using varying materials, such as decellularized xeno- and 77
allogenic matrix (e.g. small intestine submucosa, SIS) [11]–[13], in vitro cultured and 78
decellularized matrices [14]–[17], and synthetic degradable polymers [18]–[21]. Promising 79
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4
functionality has been reported, as well as successful in situ restoration processes, 80
including rapid cellularization, collagen deposition, endothelialization and material 81
resorption. Recently, the first report on ongoing clinical trials using resorbable 82
supramolecular elastomeric valves has been published, showing highly promising results 83
when applied as pulmonary valved conduits for right ventricular outflow tract 84
reconstruction in pediatric patients [22]. However, the regulation of the regenerative 85
response in the complex in vivo environment remains poorly understood [23], and 86
unexplained and uncontrolled adverse remodeling events such as loss of valve function, 87
due to valve thickening and shortening, have been reported in preclinical studies 88
(reviewed in [9]). Additionally, heterogeneity in remodeling processes, such as cell 89
infiltration, graft resorption and ECM deposition, has been reported between valves, as 90
well as between leaflets within the same valve [24], [25]. These variabilities in outcome 91
emphasize the need for more in-depth knowledge of the events, kinetics and mechanisms 92
involved in in situ TE, in order to achieve effective tissue formation, limit the risk of 93
unpredicted (maladaptive) remodeling and ensure safe clinical translation. 94
The goal of this study was to map the long-term spatiotemporal processes of polymeric 95
graft resorption, scaffold-induced inflammation and tissue regeneration in resorbable 96
synthetic pulmonary valves in sheep. To that end, in depth retrospective analysis was 97
performed on explant material of a previously reported preclinical study [19]. In that study, 98
supramolecular elastomeric heart valve grafts (Xeltis Pulmonary Valved Conduits, XPV, 99
Xeltis, Eindhoven, Netherlands) were implanted at the pulmonary position in an ovine 100
model with follow-up time up to 24 months. It was demonstrated that with this graft design, 101
safety and functionality remained acceptable throughout the follow-up time, and clinical 102
health, blood values and systemic toxicity were not influenced by the device. Gross 103
morphological analysis showed generally pliable leaflets, with some local anomalies, such 104
as focal leaflet thickening or rolling of the free edge of the leaflet. The grafts were 105
populated by endogenous cells from 2 months on, in both the conduit and the leaflet of the 106
valves. In order to advance the understanding of how recruited cells and cellular 107
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interactions guide scaffold resorption and tissue formation in vivo, in the present study, 108
grafts from the previous in vivo study were analyzed using a comprehensive 109
immunohistochemistry (IHC) antibody panel [26] and Raman microspectroscopy [27][28]. 110
The antibody panel for IHC was previously developed and validated and consists of 111
antibodies to mark inflammatory cells, valvular interstitial cells (VICs), and extracellular 112
matrix components, such as proteoglycans, collagens and elastic fiber-associated 113
proteins [26]. Specifically, we assessed the presence and phenotype of inflammatory and 114
VIC-like cells, paracrine signaling factors, endothelialization and microvascularization, and 115
extracellular matrix components related to collagen and elastin deposition. 116
Complementary to that, Raman microspectroscopy was applied to measure the local 117
molecular composition of graft material and newly formed tissue in various locations of 118
longitudinal sections of the explanted valves. Spectroscopic techniques are relatively 119
simple, reproducible and nondestructive. Raman microspectroscopy is a vibrational 120
spectroscopic technique that probes a specific chemical bond (or a single functional 121
group), yielding molecular-level information of functional groups, bonding types, and 122
molecular conformation, thus providing specific information about biochemical 123
composition of tissue constituents and their microenvironments [29], [30]. Specifically, we 124
applied Raman microspectroscopy on longitudinal sections of the valve explants including 125
conduit and leaflet to assess the local chemical changes in the scaffold materials, 126
indicative of scaffold resorption, as well as the composition and maturation of collagen in 127
different regions of interest of the valved conduit and for various follow-up times (2, 6, 12, 128
and 24 months). The measured trends on the molecular level were correlated to events on 129
the cell and tissue level using IHC analysis, providing new insights into the spatiotemporal 130
events of inflammation, tissue formation and maturation, and scaffold resorption during 131
material-driven in situ heart valve tissue engineering. 132
133
was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint (whichthis version posted April 8, 2021. ; https://doi.org/10.1101/2021.04.06.438611doi: bioRxiv preprint