Development of small-diameter vascular grafts using patterned silk films recapitulating native arterial structure
3.4 Discussion
(Figure 3.13A, IV-VI). Retention of cells after rolling step verifies the applicability of this procedure.
Mechanical strength of the silk film based acellular vascular construct was determined in terms of burst strength so as to check if it sustains physiological blood pressure. Burst pressure ranged between 915-1260 mmHg for tubular constructs made up of different silk varieties under hydrated conditions and no significant difference was observed among different experimental groups (Figure 3.13B).
Figure 3.13. (A) Histological analysis of mature small diameter vascular construct. (I) Gross view of vascular graft. (II) Bright field image of graft cross section after day 1 of cell seeding depicting loosened multilayer structure. (III) Cross-section of matured vascular graft after 14 days of cell seeding showing maintenance of graft integrity films due to ECM secretion. Deparaffinized sections of matured graft were stained with Hoechst 33342 to locate cellular distribution of vascular cells - (IV) Bright field unstained, (V) Fluorescent microscopic Hoechst 33342 stained (blue color) and (VI) Merged view of the graft cross section. (B) Burst pressure of silk film based acellular vascular tubes.
it an apt choice for vascular tissue engineering application [306, 307]. Our overall aim for preparing SDVG is to biomimic the cellular geometry and architecture of native blood vessel.
Herein we used pattern silk films from three different silk varieties in combination with all three vascular cells. Patterned silk films were used as template for cell sheet engineering which would be advantageous over latter in many aspects discussed previously.
Integration of implanted graft with native body tissue is crucial for its successful implementation into various tissue engineering aspects. Among other properties, it requires a controlled rate of degradation so as to maintain equilibrium with the growth and maturation of native tissue in vivo. In this regard, silk serves as an ideal material due to its controlled slow rate of degradation whereas simultaneously maintaining tissue integrity. In vitro degradation profile of silk films from all three silk varieties suggested structural integrity in PBS and only ~6-8% wt.
loss was recorded even after 28 days. This long term integrity may be attributed to presence of β- sheet resulting from water annealing as evidenced by FTIR data. Structural integrity of silk logged in this study was in accordance with the prior reports. Moreover, lesser water holding capability of non-mulberry silk varieties is directly co-related with the presence of higher extent of hydrophobic amino acid residues [292, 308]. Hence, considering the structural inequity among different silk varieties, faster degradation of B. mori films might have resulted from its high water holding capability. It allows access to higher extent of proteolytic enzyme to interact with native structure. Enomoto et al. have demonstrated successful replacement of rat aorta with custom made silk based vascular graft with acceptable patency that was subsequently replaced by an artery like structure within 1 year tenure [189]. SF fibers retained more than 50% of initial mechanical strength two months after their in vivo implantation [309]. Degradation of biomaterials under in vivo conditions is a complex phenomenon that involves various synergistic pathways of biochemical and mechanical origin. In vitro enzymatic degradation may provide a clue regarding the functional interaction of biopolymer with the biological environment. Li et al. investigated the degradation response of porous silk films in the presence of protease XIV, collagenase 1A and α- chymotrypsin [310]. Study demonstrated maximum degradation in presence of protease XIV owing to non-specific activity of this enzyme towards chemical structure and amino acid sequence.
Mimicking in vivo conditions more closely entails concurrence of several enzymes (chymotrypsin, collagenase, etc.) with specific amino acid sequence for their activity. Instead of using several enzymes in order to speculate about in vivo biodegradation of silk films, we opted for protease
XIV despite its absence in human body. Several reports also attest that non-specific proteolytic activity of protease XIV would show higher degradation and might simulate the synergistic effect of several enzymes with specific activity [303, 309, 311]. This would help to understand the stability of biomaterial under harsh enzymatic in vivo conditions. Non-mulberry silk films were comparatively more stable than mulberry silk in presence of protease. Outperformance of P. ricini and A. assama films may be attributed to differential molecular weight of heavy and light chains or amino acid content present as compared to B. mori silk. It has been reported that B. mori SF contains multiple repeats of AGSGAG that accounts for nearly 55% of total protein making the crystalline structure [237]. Moreover 30.2% alanine and 45.9% glycine make up majority of protein structure. The methyl groups of alanine are known to be arranged outside the protein backbone and exposed to external microenvironment [236]. On the other hand, the non-mulberry P. ricini SF’s major constituent is poly alanine (47.9%) that remains in α-helix conformation and packed closely [308]. The differential composition of these two proteins might be the plausible reason for stability of non-mulberry silk owing to presence of greater percentage of hydrophobic amino acid residues leading to close packing and minimal exposure to external aqueous microenvironment. Additionally, AFM data suggested an increase in surface roughness of non- mulberry SF films that may contribute to better stability of these films [312]. Non- mulberry films (A. assama) also demonstrated superior mechanical properties and elasticity than that of mulberry silk. The distinctive feature of A. assama SF is presence of its poly alanine residues not intervened by any other amino acid. This feature makes it quite unique as compared to its other Saturniidae family members. Such characteristic properties mainly impart mechanical robustness to these films [239, 293].
An ideal biomaterial for any tissue engineering application should be minimally immunogenic. In this regard, we checked for mouse macrophage activity (in terms of TNF-α release) in response to silk films. The amount of TNF-α released in response to non-mulberry silk films were comparable with FDA approved B. mori silk [175]. Material safety was further confirmed by examining the in vivo immunogenic response (4 weeks) in a mice model for both mulberry and non-mulberry silk films. Tissue response towards silk films was assessed in terms of growing collagen fibrils orientation and fibroblast layers surrounding the silk films. Attached fibroblast layer and macrophages at tissue implant junction was observed for all silk varieties.
Very mild response was observed (extent of macrophage infiltration) for non-mulberry silk that
was almost analogous with the B. mori silk which is known to be less immunogenic than collagen [176]. Moreover, degradation of silk produces small peptides and amino acids that are utilized by the surrounding cells to carry out their metabolic activities [146].
We further checked for cellular metabolic activity and attachment of vascular cells on silk films. Percentage AlamarBlue reduction, that directly co-relates with cell viability suggested better cellular proliferation on non-mulberry silk films. Results were in accordance with AFM data indicating superior proliferative capacity on rougher surface of A. assama and P. ricini films. It may also be attributed to availability of integrin binding RGD motifs present on surface of non- mulberry A. assama and P. ricini silk films [292]. More interestingly, growth pattern of adventitial fibroblasts profoundly favored silk films as compared to standard tissue culture plates that clearly indicate the importance of surface roughness and presence of RGD motifs on cell attachment and proliferation. Surface topography and chemistry is known to alter the cellular response. Cells with spread-morphology and well developed actin cytoskeleton survive better than cells with round morphology [313]. Staining of actin cytoskeleton of aligned vascular cells with rhodamine- phalloidin revealed strong color intensity hence well-developed actin fibers onto non-mulberry silk films. This may also be attributed to RGD availability and roughness. These findings were in agreement with previous reports [291].
Engineering of functional vascular tissue demands well grown endothelial cell layer to serve as the hemocompatible surface, reducing the chances of thrombosis [314]. Taking all these aforementioned limitations into consideration, fabrication of tissue engineered blood vessel primarily demands hemocompatibility of the biomaterial. Motivation of using silk to fabricate vascular construct was acquired by virtue of its antithrombotic properties [315]. We investigated latter by analyzing the reaction of silk films towards platelet adhesion and activation (LDH activity). Results showcased least activation of adhered platelets on to silk films. Also, further processing for stabilization of the silk films against water, we adopted water vapor annealing over other methods owing to its superior hemocompatibility [316]. In native artery, endothelial cells remain in quiescent state and aligned in the direction of blood flow hence form a continuous lining.
These cells enter into the proliferative phase during an injury or diseased condition. One of the major hurdles in the field of vascular tissue engineering is proper endothelialization since most of the loosely attached cells detach under the influence of blood flow induced shear stress [317].
Under in vivo conditions after graft implantation, tissue engineered vascular grafts usually fail to
sustain the endothelial cell lining due to shear forces generated via blood flow [318]. Herein we have successfully shown a confluent monolayer of endothelial cells aligned along microgrooves on silk films. We hypothesize that parallel arrangement of cells along the flow direction might help them to resist shear forces and circumvent cell loss by presenting lesser surface area to the flow direction. Additionally, two layers of endothelial cell seeded films were wrapped to provide more congenial environment and reduce the chances of early stage thrombosis. Our findings of immunostaining also demonstrated strong expression of vWF in the cellular cytoplasm confirming the maintenance of endothelial cell phenotype on aligned silk film.
Another constraint of designing functional tissue engineered vascular graft is maturation of construct under pulsatile flow bioreactor for extended periods. Main aim behind this maturation is to align SMCs and extracellular matrix concentrically [319]. It also assists in transition from synthetic to contractile phenotype rendering superior strength and compliance of fabricated construct [320]. Major drawback of blood vessel maturation in a pulsatile bioreactor is that it requires long term (~3 months) maturation that reduces the chances of its clinical applicability.
Also during the long span of construct maturation might cause cellular senescence [321]. Herein, we tried to explore whether SMCs in a confluent cell sheet cultured on patterned silk films exhibit contractile phenotype. Positive staining of SMCs for calponin and α-SMA (contractile genes) clearly suggested that cellular patterning on silk films substantially induces phenotype transition of SMCs towards contractile nature. Moreover, the contractile phenotype of SMCs was verified by upregulation of contractile gene expression (SM-MHC and α-SMA). Results were in accordance with previous reports [79]. Pre fabrication alignment was achieved in shorter span of time (~3-6 days) thus allowing rapid assembly of vascular construct making it apt choice for clinical applications.
Cross sectional analysis of tubular vascular construct after maturation exhibited evenly distributed cell population. Different silk film layers were found to stick together. This might be attributed to ECM formation responsible for keeping the silk films together and maintaining the tubular construct. For confirming the aforementioned, we checked the deposition of two major ECM constituents- Collagen and Elastin. Results exhibited a time dependent increment of ECM deposition by SMCs cultured on patterned silk films. Amount of collagen was found to be almost doubled within 10 days. Black et al. (2008) reported that incorporation of ECM proteins (collagen and elastin) improve the modulus and other mechanical properties of sheets [322]. Hence, it is
quite reasonable to assume that ECM deposition would not only help in maintaining graft integrity but also it would improve the mechanical compliance of fabricated graft.
To reduce the possibility of structural loss during washing and processing steps during H&E staining protocol, we opted for Hoechst 33342 staining that directly stains cell nucleus with minimal washing steps. Tubular constructs without cell was considered as control. Cross sectioning of the latter construct although exhibited consecutively arranged film layers but distinguishable gap was observed between film layers. These findings suggest that prolonged maturation (~14 days) of film based construct even under static condition helps maintaining the structure by holding the films together where secreted ECM acts as glue. On further analysis of burst pressure of mature tubular constructs, the pressure values were around 10 times more as compared to physiological pressure (120/80 mmHg) and around 5 times higher than above pathological pressure (180-220 mmHg) [323]. This attests the mechanical suitability of silk film based vascular grafts fabricated in the current endeavor.
A noticeable aspect that is usually considered as missing link while developing SDVG is engineering of internal elastic lamina (IEL). It is a fenestrated proteinaceous barrier that works as a basement membrane for luminal endothelial cell lining and allows the exchange of various soluble factors crucial for cell functioning. Most of the tissue engineered vessels were not able to synthesize sufficient elastin so as to maintain IEL type layer and it is usually neglected while fabricating any such graft [324]. Irregularity in the IEL layer might lead to various diseased conditions like atherosclerosis since it fails to restrict the infiltration of macrophages into the intimal layer of artery [325]. We hypothesize that strategy followed in this work might enable us to circumvent the aforestated issue considering the analogy of silk film present in between the cell sheet layers with IEL. We anticipate that methodology developed herein would be advancement towards cell sheet based engineering and improve the clinical applicability of fabricated SDVG.