Field of Invention
The present invention broadly relates to the field of tissue engineering. In general the
present invention relates to the use of patterned silk films as vascular graft biomaterial. More
particularly the invention pertains to a biocompatible vascular graft made with combined
conventional template based approach with cell sheet engineering.
Background of the invention
Cardiovascular malfunctioning is one of the leading causes of death globally and
India is now being called 'the world capital of cardiovascular diseases'. The coronary artery
occlusion incidences are more prominent (~50% of total cardiovascular patients) along with
considerable domination of peripheral vascular pathology. A World Health Organization
(WHO) report states that 17.3 million people died of cardiovascular diseases in 2008 and this
number is expected to reach 23.3 million by 2030 [1]. Current surgical treatment methods
require an adequate supply of native or native like vascular constructs so as to replace the
diseased vessels. Autologous vessels, including saphenous and umbilical veins and mammary
arteries serve as 'gold standard' for coronary replacement but almost one-third of patients do
not have their veins appropriate for grafting due to pre-existing vein stripping, vascular
disease and prior vein harvesting [2-3]. The associated surgical cost and significant morbidity
rate pose additional limitations.
Considering these limitations, there exists an urgent need to find a suitable alternative
for the same. Tissue engineering, in this regard could serve as important tool for preparation
of readily available, functional and biocompatible vascular grafts through scaffold based
biomimetic approach. Synthetic materials like polyethylene terephthalate (Dacron) and
expanded polytetrafluoroethylene (ePTFE) are apt choice for large diameter (>6mm) vascular
grafts and have been implanted successfully in thoracic and abdominal aorta [4-5]. On the
contrary their use for small caliber vessels has shown to cause thrombosis, anastomotic
intimal hyperplasia and subsequent occlusion (reduced patency) due to compliance mismatch
and inflammation [6]. This necessitates the selection of a suitable biomaterial that could
withstand the continuous shear and vascular wall stretches with minimal energy loss and
supports the growth of vascular cells. Among others, silk represents its potential candidature
for vascular tissue engineering owing to its easy accessibility, ease in processing, morphology
control with immense modification options, extraordinary mechanical properties with
flexibility, tunable degradability and hemocompatability [7]. Further, amenability for
fabrication into various forms like film, fiber, gel, sponge and particles broadens its
usefulness and opens new portal of applications [8].
Reference can be made to US2014288638 (A1) which relates to a medical device,
particularly a vascular graft or an arteriovenous (AV) graft for haemodialysis. The medical
device comprises a layer of porous silk fibroin matrix and a layer of knitted silk fibers.
Reference can also be made to US2014309726 (A1) which relates to biodegradable
scaffolds for in situ tissue engineering, including a biodegradable polyester tubular core, a
biodegradable polyester electrospun outer sheath surrounding the biodegradable polyester
tubular core and/or a thromboresistant agent, such as heparin, coating the biodegradable
scaffold.

WO2013151463 teaches about a tissue-engineered vascular graft designed to be used
in cardiovascular surgeries, especially in coronary artery bypass grafting and peripheral
vessels reconstruction procedures. It employs a two-phase electrospinning technique to
fabricate a biodegradable polymer. graft composed of the porous tubular scaffold
supplemented by biologically active molecules, incorporated directly into the matrix walls in
order to promote regeneration process of the patient's own vessel wall.
US2010222863 (A1) discloses silk-containing stent grafts comprising an endoluminal
stent and a graft, wherein the silk induces the in vivo adhesion of the stent graft to vessel
walls, or, otherwise induces or accelerates an in vivo fibrotic reaction causing said stent graft
to adhere to vessel wall. Similarly US20010053931 discloses a stent-graft composite
intraluminal prosthesis comprising an adjustable tubular stent, defining opposed exterior and
luminal stent surfaces and a polymeric stent sheath covering at least the exterior surface
thereof. The polymer is selected from the group of polymeric materials consisting of
biological or genetically engineered spider silks, such as those derived from Nephila clavipes.
The silk includes bioengineered spider silks as well as silk-like polymers manufactured using
human proteins and blends of such silks with commonly used polymeric graft materials.
It is important to know that blood vessel is a layer by layer assembly of vascular cells
arranged in a unique fashion so as to sustain the shear forces induced via blood flow. Recent
endeavors implementing the use of silk for vascular tissue engineering application
demonstrated the fabrication of porous silk microtube using layer-by-layer deposition and gel
spinning methods [6, 9-10]. Such grafts although provides control over porosity but major
drawbacks with these grafts are the randomly arranged cells and their inability to maintain
functional cellular phenotype. Electrospun nanofibrous silk tubes certainly improved the prior
fabrication approach but maintenance of long term mechanical compliance and poor control
over mechanical and degradation properties imposed further limitations [11]. Silk composite
were then investigated for their supposedly improved properties. In this regard, tri layered
vascular grafts composed of elastin, polycaprolactone, silk and collagen were developed to
match the required mechanical properties [12]. Silk microtube encapsulating heparin have
been used as a carrier for vascular endothelial growth factor (VEGF) sustained release, with
concomitant hemocompatability and endothelialisation, thereby reducing the chance of
thrombosis [8, 13]. A recent study projected the use of hybrid protein polymers containing
silk and human recombinant tropoelastin to provide better tissue elasticity and extensibility
[14].
These 'top down approaches' are based on the use of spongy scaffold as a template
for engineering tubular construct followed by cell seeding in order to recapitulate compact
and organized tissue. However, limitations in terms of long term mechanical compliance can
be easily comprehended in situations that demand stretchable tube like structure with
millimeter range wall thickness. Developing native like cellular arrangement and maintaining
tissue integrity and complexity still remains a challenge in the field of vascular tissue
engineering. Further limitations include co-culture of vascular cells, remodeling capability,
long reproducible time and associated high cost.
Scaffold free approaches later came into existence that allows mimicking native
cellular alignment and tissue integrity with higher fidelity. Cell sheet engineering is a good
example of aforesaid technique. L.H eureux N et al.successfully demonstrated the fabrication

of beuman blood vessel using cell sheet engineering more than a decade ago [15]. They have
shown the subsequent rolling of sheets of smooth muscle cells (SMCs) and fibroblast cells
over an inert mandrel followed by endothelial cell seeding in the luminal surface. The
concept is although completely bio-based where one can precisely control the cellular and
extracellular matrix (ECM) alignment; the rolling of the highly delicate cell sheets itself is
tedious. In order to implement the concept of cell sheet engineering for preparing vascular
graft and making the rolling process more feasible, recently people have tried to combine the
principle of cell sheet engineering with the electrospinning technique [16-17]. In this
approach, aligned electrospun mat is used as a platform for cell seeding and after getting a
confluent cell sheet, mat is rolled over the mandrel. This combined approach made the rolling
process more facile but required cellular infiltration during the maturation process. Limited
infiltration of cells in electrospun scaffolds due to lesser pore size and inadequate surface
properties need additional measures for graft success [11, 18]. Using electrospinning machine
also made the process less cost effective.
In the present invention, an alternative approach by combining the cell sheet
engineering and patterned silk films to overcome the above mentioned limitations is
projected. A schematic representation of the methodology involved to fabricate small .
diameter vascular graft (SDVG) is shown in Fig.l. Several reports have attested the
applicability of Bombyx mori (B. mori) silk for various tissue engineering applications but
non-mulberry silk varieties like Philosamia ricini (P. ricini) and Antheraea assama {A.
assama) are unexplored. In this invention, the latter was used in comparison with B. mori
silk. This approach would serve as a suitable cell sheet platform simultaneously inducing the
functional contractile phenotype of SMCs (alignment induced phenotypic transition). Also, it
may be envisaged that cellular platform (silk film) would make the rolling process more
facile and would not require long term maturation in pulsatile bioreactor to obtain sufficient
mechanical strength and functionality. This consecutive rolling assembly is expected to
exactly mimic the native cellular alignment (EC aligned longitudinally along the blood flow
direction, SMCs and fibroblasts in the concentric arrangement improving mechanical
contractility).
Objectives of the invention
The main objective of the present invention is to provide a patterned silk film based
vascular graft.
Yet another object of the present invention is to provide a process of preparation of
vascular graft biomaterial from the non-mulberry silk worms (P. ricini and A. assama).
Yet another object of the present invention is to provide a silk based biocompatible
vascular graft made with combined conventional template based approach with cell sheet
engineering.
Summary of the Invention
The present invention proposes the use of patterned silk films as vascular graft
biomaterial and combined conventional template based approach with cell sheet engineering.

Highly explored mulberry silk (B. mori) is compared with the non-mulberry silk varieties (P.
ricini and A. assama). Films were physically characterized and checked for cellular
compatibility. Non-mulberry silk films presented superior characteristics in terms of thermal
stability, proteolytic degradation, cellular proliferation (-1.1-1.3 folds higher than mulberry
silk) and alignment of vascular cells. Immunogenicity of films was assessed in vitro by
considering macrophage response in terms of TNF-a secretion. It was -40.79-51.4% lesser
as compared to positive control after 7 days. In vivo subcutaneous implantation of films in
mice showed minimal fibrosis and inflammation after 28 days validating the material
suitability. All three vascular cells, viz. endothelial cells (ECs), smooth muscle cells (SMCs)
and fibroblasts were co-cultured in multilayered tubular construct. Pattern induced
alignment favoured functional contractile phenotype of SMCs and they strongly expressed
contractile markers calponin and a-SMA. Moreover, the expression of elastin in SMCs and
punctuated pattern of vWF in ECs layer further assures the potential candidature of silk
films. Burst strength of tubular construct ranged between 915-1260 mmHg, sufficiently
higher than the physiological pressure making it mechanically competent.
Accordingly the present invention provides patterned silk films as vascular graft
biomaterial.
In one general aspect, there is provided a process of preparation of vascular graft
biomaterial from the non-mulberry silk worms (P. ricini and A. assama).
In another general aspect, there is provided biocompatible vascular graft made with
combined conventional template based approach with cell sheet engineering.
Accordingly the present invention provides patterned silk films as biocompatible
vascular graft by utilizing silk from non-mulberry silk (P. ricini and A. assama) and the
process of preparation, the said process comprising the steps of-
i) Preparation of aqueous silk fibroin (SF) solutions
ii) Fabrication of silk films
iii) Isolation and culture of primary vascular cells
iv) Proliferation of vascular cells on SF films
v) Preparation of.silk film based vascular conduit
The details of the invention are set forth in the description below. Other features,
objects and advantages of the invention will be apparent from the description including
claims.
Brief Description of the Accompanying Drawings
Fig.1 Schematic representing the fabrication of patterned silk films (A) and rolling process of
vascular cell sheets to obtain tri-layered bio-mimicking tissue engineered small diameter
blood vessel (B).
Fig.2 Atomic force microscopic images of water vapor annealed patterned and flat films of
both mulberry and non-mulberry silk varieties.
Fig.3 Physical characterization of silk films. (I) Fourier transform infrared spectra of water

(A,B), P ricini (C, D) and B.mori (E,F) silkworms respectively. (II) X-ray diffractogram of
water vapor annealed silk films (A) B. mori, (B) P. ricini and (C) A.assama
Fig.4 Thermal analysis of silk films. (I) Diffrential scanning calorimetry (DSC) and (II)
Thermogravimetric analysis (TGA) studies.
Fig.5 Swelling (%) (A) and Degradation (B) profile of water vapor annealed silk films. (/P)
in the latter indicates the presence of protease. *p<0.05, **p<0.01
Fig.6 Growth profile of (A) Fibroblasts, (B) Smooth muscle cells and (C) Endothelial cells
cultured on silk films of different varieties obtained using Alamar blue dye reduction assay.
Fig.7 Production of TNF a by RAW 264.7 mouse macrophage cells in response to silk films.
Cells were stimulated by 1 wt% patterned silk films from different variety silk. Standard
tissue culture plate was considered as negative control whereas for positive control,
1000ng/ml lipopolysaccharide from E.coli was used. Amount of TNF a released was
calculated quantitatively from the standard curve plotted using recombinant TNF a. *p<0.05,
**p<0.01.
Fig.8 Bright field microscopic images of H&E stained sections showing in vivo
immunological response of silk films, P. ricini (A, B), A. assama (C, D) and B. mori (E, F)
retrieved 4 weeks post implantation from subcutaneous pocket of mice. (Black and white
arrows are indicating the location of film).
Fig.9 Phase contrast microscopic pictograph of confluent monolayer of vascular cells aligned
unidirectionally on patterned silk films. Inset images demonstrating rhodamine-phalloidin
stained (red color-alignment of actin cytoskeleton) and Hoechst 33342 stained (blue color)
fluorescent microscopic pictograph of vascular cells. White and black arrows are indicating
the direction of alignment. Scale bar 400 urn
Fig.10 Representative fluorescent microscopic images depicting phenotypic and functionality
of vascular cells cultured on A. assama patterned silk films. SMC's were stained with
contractile phenotype markers: Calponin (A, B) and a-SMA (C,D). Intracellular soluble
tropoelastin was also visualized using anti-elastin antibody (E) along with negative control
(F, without primary antibody). Endothelial cell functionality was confirmed by visualizing
the vWF expression. All phenotypic marker are stained with FITC tagged secondary antibody
(green fluorescence). Cells were further counterstained with rhodamine-phalloidin (red) and
Hoechst 33342 (blue color) to visualize actin cytoskeleton and nucleus respectively. White
arrows are indicating the direction of alignment.
Fig.ll Histological analysis of mature small diameter vascular construct. Deparaffinized
sections were stained with Hoechst 33342 to locate cellular distribution of vascular cells in
the prepared construct. White arrows are representing the stained nucleus (blue color) of
vascular cells. Lower panel is demonstrating the cross section of sticked silk films due to
extracelluar matrix secretion.
Fig. 12 Burst pressure of silk film based vascular tubes. Total internal pressure was increased
by flowing air using a syringe connected to a syringe pump at a controlled flow rate of

1ml/min. Pressure at tube failure was logged as burst pressure and recorded using digital
manometer. (n=4, p>0.05)
Fig. 13 (A) Set-up used to analyze the burst strength of vascular construct (In the inset is the
enlarged image of tubular specimen) and (B) Representative image of vascular graft
withholding the pressure of up to 908 mmHg.
Detailed Description of the Invention
Reference will now be made to the exemplary embodiments, and specific language
will be used herein to describe the same. It should nevertheless be understood that no
limitation of the scope of the invention is thereby intended. Alterations and further
modifications of the inventive features illustrated herein, and additional applications of the
principles of the invention as illustrated herein, which would occur to one, skilled in the
relevant art and having possession of this disclosure, are to be considered within the scope of
the invention. It must be noted that, as used in this specification and the appended claims, the
singular forms "a," "an," and "the" include plural referents unless the content clearly dictates
otherwise. All references including patents, patent applications, and literature cited in the
specification are expressly incorporated herein by reference in their entirety.
The present inventors have developed novel patterned silk films as vascular graft,
wherein the silk film is obtained from the non-mulberry silk (P. ricini and A. assama).
In one general aspect, there is provided a process of preparation of vascular graft
biomaterial from the non-mulberry silk cocoons of P. ricini and A. assama.
In another general aspect, there is provided biocompatible vascular graft made with
combined conventional template based approach with cell sheet engineering.
All the experiments as described below were carried out for n = 3 samples. For cell
viability analysis (alamar reduction assay), n = 4 samples were used. Data was reported as
mean ± standard deviation (S.D.). One-way analysis of variance (ANOVA) was performed to
measure the significance level among different groups followed by tukey's test. All statistical
analysis was performed using OriginPro 8 (Originlab Corporation, USA) at both 0.05 and
0.01 significance level.
1. Preparation of aqueous silk fibroin (SF) solutions
Silk fibroin (SF) protein was obtained from both mulberry (B. mori) cocoons and non-
mulberry (A. assama and P. ricini) silk glands as previously described procedures [19-20].
Briefly, small cocoon pieces of B. mori were degummed in boiling 0.02M sodium carbonate,
washed properly with distilled water and dried overnight at room temperature (RT). Dried
and degummed silk fibers were than dissolved in 9.3M LiBr and dialyzed against Milli-Q
water using dialysis membrane (MWCO 1200) for 3 days with successive water change. On
the other hand non-mulberry silk protein was isolated from silk glands of fully-grown fifth-
instar matured larvae of A. assama and P. ricini. Obtained protein was dissolved in 1% (w/v)
sodium dodecyl sulfate and dialyzed against Milli-Q water. The concentration of regenerated
silk solution obtained was determined by gravimetric method.

2. Fabrication of patterned silk films
Patterned (microgroove) silk films were fabricated by using grooved PDMS (poly-
dimethylesiloxane) substrates (Dow Corning Corporation, Midland, MI USA) prepared by
previously described soft lithography technique [21-22]. 1.5 ml of 1% (w/v) extracted
aqueous silk solution was poured onto 4x4 cm PDMS platforms and spread uniformly. Casted
SF solution was allowed to dry at RT overnight. Resulting 4-5 urn thick films were
subsequently subjected to water vapor annealing in a vacuum drying oven for 6h, 37°C in
order to induce P-sheet transition and making them water insoluble. After that films were
taken out from PDMS molds carefully and kept under sterile conditions till further use. A
schematic representation of the methodology involved in the present invention to fabricate
small diameter vascular graft (SDVG) is shown in Fig. 1.
Silk, being the ancient material having superior mechanical properties has long been
used as a suture material and still continues even after the availability of myriad of synthetic
materials in the market [34]. Apart from load bearing applications, silk is fast emerging as the
most promising material in the field of tissue engineering. Ability of silk to support the
growth of vascular cells projects it as a potential biomaterial for vascular tissue engineering
applications [6, 9, 35]. Mimicking the native geometry of blood vessel is also a crucial aspect
in the field of vascular tissue engineering. It requires directionally aligned cells to maintain
the cellular phenotype. Ability of silk to obtain desirable patterns even at micro and nano
levels makes it an apt choice for vascular tissue engineering application [36-37]. The main
aim of this invention for preparing SDVG is to biomimic the cellular geometry and
architecture of native blood vessel. Herein pattern silk films from three different silk varieties
were used 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
as discussed previously.
2.1 Characterization of patterned silk films
2.1.1. Atomic force microscopy
Surface roughness of water vapor annealed patterned and flat silk films was measured
in non-contact mode using atomic force microscope (Agilent Model 5500, USA) with tips
mounted on triangular cantilevers (spring constant 40 N/m, as specified by manufacturer). All
the measurements were performed at room temperature by selecting scanning area range of
20µx20µ.
Surface morphology of the various silk films prepared using regenerated SF after
water annealing was assessed by atomic force microscopy (Fig. 2). Patterned silk films made
up of non-mulberry silk exhibited sharp and distinct grooves whereas pattern on B. mori silk
films showed a plateau pattern. On analyzing the flat silk films, greater degree of roughness
on non-mulberry silk films was observed. Root mean square (RMS) roughness value was
44.70 nm with an average height of 331.18 nm for B. mori film. On the other hand RMS
roughness values for P. ricini and A. assama films were 92.73 nm and 94.53 nm with average
heights of 320.03 nm and 278.45 nm respectively, thereby representing greater extent of
roughness for non-mulberry silk varieties.

2.1.2. Fourier transform infrared (FTIR) spectroscopy
Fourier Transform Infrared (FTIR) spectroscopic analysis of water vapor- annealed
and untreated films was performed using an FTIR Spectrophotometer (Nicolet iS 10) in ATR
mode in the spectral region from 1100-1800cm"1 under absorbance mode. All spectra were
obtained with a resolution of 4cm"1 and 32 scans per spectra.
Secondary structure-transition of B. mori, A. assama and P. ricini regenerated SF
films was studied by FTIR spectroscopy (Fig. 3). For all samples, characteristic vibration
bands for peptide backbone between 1610-1660 cm"1 (amide I), 1510-1560 cm"1 (amide II)
and 1210-1260 cm"1 (amide III) were observed corroborating the presence of C=0 stretching,
N-H bending and C-N stretching, respectively [28]. Specific vibration frequency corresponds
to characteristic structural conformations (P-sheet and random coil/a-helix) for silk protein.
Absorption at 1648-1554 (amide I) and 1535-1542 is indicative of silk I conformation
whereas some studies report these values corresponding to random coil structure [29].
Regenerated SF from B. mori cocoon showed typical peaks at 1660 and 1549 cm"1 in the
amide I and amide II region suggesting its a-helix/random coil conformation. A similar peak
values were obtained for P. ricini Silk gland protein at 1660 and 1527 cm"1. Water vapor
annealing for 6 hours led to structural transition towards P-sheets conformation with signature
peaks at 1630 and 1530 cm-1 in amide I and amide II regions. Peak values within 1610-1630
cm"1 (Amide I) and 1510-1530 cm"1 (amide II) range are specific for silk II secondary
structure [30]. Water vapor annealed P. ricini SF films represented the characteristic peak
values at 1630 and 1529 cm"1 whereas for A. assama silk these peak values fall at 1626 and
1528 cm"1 suggesting P-sheet transition. A low intensity amide III band was also observed for
all samples ranging from 1222-1240 cm"1. The IR spectrum of regenerated silk protein
confirms the transition from native predominant random coil structure in untreated state to P-
sheet structures after water vapor annealing. Shifting of spectral peaks signifies the
rearrangement of hydrogen bonds in the SF protein moiety that converts it in to more stable
P-sheet secondary structures.
2.1.3. Wide angle X-ray scattering
The crystallinity of water vapor-annealed silk films was analyzed by X-ray diffraction
spectroscopy (Bruker D2 Phaser) under Cu radiation (30 KV, 40mA current having Ni
filtered) with a scanning rate of 1° per min. All scans extended from 5° to 50° in 29 with
Lynexeye detector system.
Conformational transition and crystallinity of regenerated SF was further analyzed
using wide angle X-ray diffraction study (Fig. 3-II). Previous studies suggest a-helix
conformation of SF at 20 values 11.8° and 22° whereas characteristic X-ray diffraction peaks
at 16.5°, 20.2°, 24.9°, 30.9°, 34.59°, 40.97° and 44.12° demonstrate the presence of P-sheets
[30-31]. X-ray spectra of SF protein isolated from B. mori cocoon and silk gland of A.
assama and P. ricini showed the characteristic peaks for 20 values at 11.8° and 16.5°, after
water vapor annealing for 6 hours. Minor peaks at 22.2° and 25.4° were also observed for all
silk protein samples confirming the presence of P-sheet structure. Data suggested that water
vapor annealing induces the conformational transition from silk I to silk II type. Significant
influence of water on the silk I structure is also evident from the data. These results were
consistent with prior reports where almost similar peaks were obtained for A. pernyi silk,

2.1.4. Thermal analysis
Water content of water vapor annealed dried silk films was detected using
thermogravimetric analysis (TGA, Pyris Diamond TG-DTA). 5mg of sample was used to
carry out analysis in nitrogen atmosphere. Sample was heated at constant rate of 10°C/min in
aluminum crucibles from 50°C to 700°C and % weight loss was recorded as a function of
time. A differential scanning calorimeter (Perkin Elmer Pyris Diamond DSC) was used to
study phase changes in silk films at higher temperatures. 3 mg of sample was heated in
aluminum crucibles from 50°C to 400°C with a dry nitrogen gas flow at a constant heating
rate of 10°C/min.
All the water vapor annealed SF films demonstrated three characteristic peaks (Fig. 4-
I). The first endothermic glass transition (Tg) peak was formed at around 80 C, indicating the
presence of water and the segmental movement of the SF protein molecules. The second
exothermic peak formed at around 224 C, indicates the movement of random coil and a-
helix conformations and crystallization of SF influenced by heat. The third endothermic peak
formed at around 300°C shows the thermal decomposition of ordered fibroin protein in all the
silk films [30]. The decomposition temperatures of B. mori silk were slightly lower than A.
assama and P. ricini films, a clear indication of improved thermal properties in the latter
films. An endothermic peak at around 290 C was obtained for B. mori silk whereas this peak
was at around 360°C for other two silk varieties. This signifies the induction of crystalline
structure in silk films which require higher activation energy for the breakage of covalent
bonds in P-sheet structures as compared to random coils structures.
The thermal behavior of B. mori, A. assama and P. ricini films was further studied by
TGA (Fig. 4-II). The thermogram demonstrates four steps of weight loss with significant
variation within the temperature range of 33-770°C. The initial weight loss step at around
100°C refers to the removal of absorbed water (loss of moisture). The second weight loss
step ranging from 250 to 400°C is imputed to the cleavage of peptide bonds and breakdown
of side chain groups of amino acid residues [30]. The third weight loss step ranging from 400
to 770°C is the decomposition stage as described earlier from the DSC results. The TGA
curves reveal that in the temperature region (400-770°C) for decomposition, the A. assama
silk films shows slightly more thermal stability (400- 770°C) than B. mori and P. ricini
treated films.
2.1.5. Swelling properties
Swelling properties were estimated by gravimetric method used conventionally [23].
Silk films of pre-determined weight prepared of 1% (w/v) SF were immersed completely in
phosphate buffer saline (PBS, pH 7.4) at 37°C. At pre-defined time points, swollen films
were weighed after soaking the excessive residual PBS using filter paper. The dry weight
(Wd) and wet weight (Ws) of silk films was recorded and swelling ratio was calculated
implying the following equation:
Swelling (%) = [(Ws-Wd)/Wd]xl00 [1]
Swelling behavior determines the structural integrity and stability. Rate of water

and-attained an almost plateau pattern within 2 hours owing to low protein concentration.
Maximum swelling was observed during first 20 min with a swelling ratio of 420 for all silk
varieties under consideration (Fig. 5A). Regenerated SF films of P. ricini and A. assama
silkworms exhibited swelling ratio of around 700 and 650 respectively whereas this value
was 550 for B. mori silk films. At later stages P. ricini films were found to uptake maximum
amount of water and it was significantly higher from B. mori films (p<0.05). On the other
hand, the swelling pattern of A. assama silk films did not vary from other two silk varieties.
2.1.6. In vitro enzymatic degradation
In vitro enzymatic degradation profile of silk films was studied in presence of
protease XIV isolated from 5. griseus (enzymatic activity 3.5 Umg-1, Sigma Aldrich). Initial
dry mass of all films was recorded. Silk films were than immersed in 2 ml of PBS (pH 7.4)
with and without 2 Uml-1 protease at 37°C. At predefined time points films were taken out of
the enzymatic solution and dried at 60°C for 24h. Enzyme treated dried films were weighed
and compared with the initial dried weight. For every 72 h, enzyme solution was replaced
with freshly prepared enzyme solution. Remaining mass fraction was calculated as:
% Mass remaining= (Mass at time t/initial mass) x100 [2]
In vitro degradation profile of regenerated silk films in PBS with and without protease
was studied till 28 days by monitoring the rate of weight loss. Results were reported as %
mass remaining with time (Fig.5B). Films were degraded more rapidly in presence of
protease whereas PBS alone did not impose such effect and silk films were found to maintain
their integrity for prolonged periods. 2-6% weight loss was observed in PBS for different silk
varieties. The differences in the degradation pattern were apparent in case of protease treated
SF films. A continuous time dependent degradation was observed for all silk film varieties. In
particular, B. mori silk film degraded maximally and lost up to 95.97±0.31 wt% after 21 days
and 97.85±1.20 % after 28 days. Also rate of degradation was highest for B. mori silk films.
Non-mulberry silk films were found to resist protease treatment till certain extent. Weight
loss of 43.05±1.97 % (2.27 fold decrease) and 66.70±3.78 % (1.46 fold decrease) was
observed for P. ricini and A. assama silk films respectively after 28 days. However initial rate
of degradation was much less for these two silk varieties. P. ricini silk films retained
85.45±1.17 % mass of the initial weight after 14 days whereas this value was 73.46±0.50 %
for A. assama silk film.
Integration of implanted graft with native body tissue is crucial for its successful
implementation in 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 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 -10%
wt. loss even after 28 days was recorded. This long term integrity may be attributed to
presence of P-sheet resulting from water annealing as evidenced by FTIR data.

2.1.7. Macrophage stimulation assay
Immunogenic response of SF films prepared using B. mori cocoons and A. assama/P.
ricini silk glands protein was assessed by estimating the tumor necrosis factor alpha (TNF-a)
release profile as previously reported [26-27]. TNF-a is a cell signaling cytokine that is
released primly from macrophages as a foreign body response causing inflammation. RAW
264.7, a mouse macrophage cell line, was used to analyze the immunogenic response of silk
films. Equal no. (~50,000cells/well) of cells were seeded on sterile protein coated 12 well
tissue culture plate. Uncoated wells with same cell density were considered as negative
control whereas wells containing 1000ng/ml lipopolysaccharide (LPS, from Escherichia coli,
Sigma Aldrich, USA) were taken for positive control. Culture media was collected at day 1
and day 7 for estimation of TNF-a production.
Tissue fabrication using cell sheet engineering principally came into existence to
nullify the chances of possible immune reaction or inflammation due to biodegradation of
biomaterial used as a template [15]. Ideally, neither the silk films nor the biodegraded product
(that is expected to be either disposed off from the system or utilized by the cells) should
elicit any immunogenic response. Silk being the natural biopolymer does not impart any of
the above mentioned threat as suggested by mouse macrophage activity (in terms of TNF a
release) in response to silk films since these values were comparable with FDA approved B.
mori silk films and in agreement with the previous reports [26].
Response of non-mulberry silk films was comparable with the B. mori silk. Material
safety was further confirmed by examining the in vivo immunogenic response (4 weeks) of
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 known to be less immunogenic
than collagen [33]. Moreover, degradation of silk produces small peptides and amino acids
that are utilized by the surrounding cells to carry out their metabolic activities [41].
2.1.8. Determination of TNF-a release
Amount of released TNF-a was quantified using Mouse TNF-a ELISA kit (life
technologies, sensitivity < 3 pg/mL) as per the manufacturer's instructions. Briefly, cell
culture supernatants were collected at pre-defined time points. For standard curve
preparation, 100µl of Ms TNF a (0-1000pg/ml) was added in duplicate in 96 well antibody
(against mouse TNF a) coated plate. Cell supernatant was diluted 2 fold with standard
diluents buffer and added in same manner as for standard. 50µl of biotinylated secondary
antibody was added into each well followed by incubation at RT for 90 minutes. After four
subsequent washings, wells were added with 100µl streptavidin-HRP working solution. Post
30 min. incubation period, wells were washed and incubated with 100µl stabilized
chromogen solution. Reaction was stopped after 20 min. by adding 100µl of stop solution and
absorbance was measured at 450 nm using multiplate reader (Tecan infinite M 200 pro).
Amount of TNF a released was calculated using standard curve values.
Immunogenicity of silk films was assessed by investigating the amount of TNF-a

variety silk membranes (Fig. 7). Both short and long term stimulatory effect was studied from
day 1 to day 7. Very low levels of TNF-a were observed at day 1 for different silk film
varieties and these values were comparable with polystyrene tissue culture plate. Slightly
elevated TNF-a levels were obtained for positive control (samples with LPS, p<0.05).
Moreover, at day 7, TNF-a release was increased 4 folds and ranged between 1100pg/ml-
1600pg/ml for different silk varieties. At this point also, these values were comparable with
polystyrene tissue culture plates. A rapid increase was although observed with positive
control. Response of macrophage to silk films of all three varieties was found to be in
acceptable range. These findings suggest the low immunogenicity of silk films in terms of
TNF-a release that enables their applicability as biomaterial for various tissue engineering
applications.
2.1.9. In vivo response to silk films
The animal experiments were performed following an ethical committee approved
protocol in accordance with Institutional Animal Ethical Committee (IAEC), West Bengal
University of Animal and Fishery Sciences (WBUAFS), West Bengal, India (Permit No.
Pharma/IAEC/36 dated 30.6.2014) in accordance with "Principles of laboratory animal care".
Swiss (I.B.) mice of 30-35 gm body weight and either sex were used to carry out the study.
Animals were undergone surgery under isoflurane (1-3% in oxygen and up to 5% for initial
induction), using a precision vaporizer. Patterned silk films of all three varieties were cut in
to 2x2 cm2 pieces and sterilized properly under UV radiation exposure. Sterile silk films were
implanted through a 0.5 cm incision in subcutaneous pocket of lateral side of thoraco-lumber
region and protected using a nonabsorbable nylon suture stitch. The healing process was
continuously monitored for any infection at the incision. No death was recorded during the
entire experimental duration. The animals were sacrificed after 4 weeks by cervical
dislocation. Implanted films were collected along with the surrounding tissue and stained
with haematoxylin and eosin for histological examination.
In order to better understand the immunogenicity of silk films and to assess the
implant integration, films were implanted in the dorsal subcutaneous pocket of mice.
Implanted material was retrieved after 4 weeks. Post retrieval, implanted films were stained
with H&E stain as per the standard protocol (Fig. 8). Occasional occurrence of immune cells
was observed nearby the film implants. 5-6 layers of fibroblast cell sheets were observed
along with milder immigration of macrophages for all silk films. Macrophages were limited
to film-tissue interface. In case of Eri (P. ricini ) film, implanted material lies in between
dermis and hypodermis which accounts for a peripheral low inflammatory reaction. However,
there is complete absence of monocyte, macrophage or giant cell. A. assama film section
depicted well organized adipose and muscular tissue which retained their structural integrity
without involving any inflammatory reaction. The material attached to the dermal and
subdermal tissues showed no infiltration of giant cells, macrophages and mononuclear cells.
Analysis of B. mori film suggested normal cellular proliferation and angiogenic reaction in
muscular and hypodermal tissues. The material revealed mild inflammatory reaction
consisting of few lymphocyte and macrophages. Orientation to the structural portion is quite
normal. Results were in accordance with previous reports stating the immunocompetancy of
pure silk films that has shown to induce lower immune response as compared to collagen
films [33]. A comparative immune response of non-mulberry silk varieties with FDA
approved B. mori silk validates the applicability of former for various tissue engineering
purposes.

3. Isolation and culture of primary vascular cells
Small part of descending porcine aorta was obtained from a local slaughterhouse and
kept in sterile, ice cold Dulbecco's phosphate buffered saline (PBS) for transportation. All
three vascular cell types were harvested as per the previously reported protocols [24-25].
Briefly, small part (~lcm) of aortic tube was cut. The outermost adventitial layer containing
fibroblasts was scraped followed by 0.018% collagenase digestion (Collagenase Type IA
from Clostridium histolyticum, lyophilized powder >125 CDU/mg solid, Sigma Aldrich) for
12 hours. Fibroblasts extruded from the tissue were plated on standard tissue culture plate.
Remaining part containing endothelial cell layer at the luminal side and SMCs' in the middle
layer (embedded in extracellular matrix) was cut open to expose the luminal surface.
Endothelial cells were harvested by scraping the inner part. SMCs' were harvested following
the similar protocol as described for fibroblasts. All three cells were maintained in high
glucose Dulbecco's Modified Eagle's Medium (DMEM; Gibco) supplemented with 10%
Fetal Bovine Serum (FBS, Gibco) and 1% antibiotic-antimycotic (Himedia) at 37°C in a
humidified incubator at 5% CO2 level. Media was replenished on regular basis to remove the
tissue debris and non-adhered cells. Primary cells were passaged twice after 60-80%
confluency and used at early passages (p4 to p8) for carrying out further experiments. It may
be mentioned here that commercially available cell source (e.g. humans) can also be used in
the process.
4. Proliferation of vascular cells on patterned silk films (Alamar assay)
SF (1% w/v) films from different silk varieties were evaluated for their ability to
support the proliferation of vascular cells. Films were conditioned with Dulbecco's Modified
Eagle Medium (DMEM, Gibco, USA) overnight prior to cell seeding. Equal number (~104
cells/cm2) of EC, SMC and fibroblasts were seeded on each film (n=4). Cell proliferation was
assessed using AlamarBlue® dye (Invitrogen, USA) reduction assay following the
manufacturer's instructions at 1, 4 and 7 days. Briefly, cell seeded silk films were incubated
with 10% (v/v) dye in culture media for 3 hours. Post incubation, 100µl of culture media
from each sample was read at 570/600nm using a multiplate reader (Tecan infinite M 200
pro). Results were reported as normalized value of % Alamar reduced at various time
intervals.
Alamar blue cell viability assay was used to investigate the proliferative index of
vascular cells on different variety of silk films (Fig. 6). Percentage reduction of Alamar blue
dye corresponds to cell viability and metabolism that is directly co-related with the number of
live cells. Regenerated SF coated and uncoated wells were analyzed for cellular proliferation
at day 1, 4 and 7. Growth profiles of vascular cells at day 7 clearly demonstrated better cell
attachment and proliferation on silk films from non-mulberry (P. ricini and A. assama) silk
varieties and-1.3 fold increase for SMCs,~l.l fold increase for fibroblasts,-1.15 fold increase
for ECs was observed as compared to B. mori silk. In particular, vascular adventitial
fibroblasts exhibited higher dye reduction on P. ricini and A. assama films than normal tissue
culture plate. Although P. ricini films outperformed during initial time periods (dayl to day
4) for all three cultured vascular cells but at day 7, proliferation values of vascular cells
seeded on A. assama films almost overlapped with P. ricini values (p>0.05).
Structural integrity of silk logged in this study was in accordance with the prior

custom made silk based vascular graft with acceptable patency that was subsequently
replaced by an artery like structure within 1 year tenure [5]. SF fibers retained more than 50%
of initial mechanical strength two months after their in vivo implantation [38]. 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. Among others, protease from S. griseus was opted to determine the degradation
of silk films due to its low specificity towards chemical structure [39]. 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. Additionally, AFM data suggested an increase in surface roughness of non-
mulberry SF films that may contribute to better stability of these films [40]. It is noteworthy
to mention here that commercially available cell source (e.g. humans) may also be used
instead of porcine cells in the present invention.
4.1. Cellular alignment and immunostaining
Vascular cells were seeded onto the patterned silk films at a density of 2xl04
cells/cm . Media was replenished every 2nd day. Cell growth was continuously monitored
under phase contrast microscope. After almost 80% confluent cell monolayer maturation,
cells were washed twice with phosphate buffer saline (pH 7.4) followed by Normal Buffer
formalin (NBF, Sigma Aldrich) fixing for 24 hours. NBF was removed with 3 subsequent
PBS washes. Formalin fixed cells were permeabilized with 0.1% TritonX-100 (in PBS,
Sigma Aldrich) for 15.min. In order to reduce the chances of non-specific binding, cells were
incubated with blocking buffer (1% bovine serum albumin and 0.3M glycine) for 1 hour at
RT. After three subsequent PBS washes, corresponding primary antibodies developed in
rabbit against anti vWF (abeam, 1:100 dilution) for endothelial cells, anti a-smooth muscle
actin (α-SMA, abeam, 1:300 dilution), anti calponin (abeam, 1:100 dilution), anti-elastin
(abeam, 1:300 dilution) for smooth muscle cells were used for cell specific marker detection.
FITC conjugated Goat anti rabbit IgG H&L (abeam) secondary antibody was implied for
fluorescent imaging. Cells were than counterstained with Hoechst 33342 (1:1000, Sigma
Aldrich) and rhodamine-phalloidin (1:40, Life Technologies, USA) for nucleus and
cytoskeleton respectively followed by imaging under an Inverted Fluorescent Microscope
(EVOS FL, Life Technologies, USA). Cells were washed properly with PBS before imaging
in order to reduce the background noise. Pattern direction and cytoskeleton alignment was
observed for cellular patterning.
Phase contrast microscopic images after one week of culture exhibited unidirectional
alignment of vascular cells on patterned silk films. Elongated cell morphology along the
direction of groove axis was observed. Smooth muscle cells responded comparatively faster
than other two cell types and aligned themselves parallel to groove direction within 24 hours.
Over extended period of time, a confluent monolayer culture of vascular cells was established
on all three varieties of silk films (Fig. 9). Magnified images demonstrated very low angle
between direction of pattern and major axis of cell. Elongated geometry of cells on pattern
silk films confirmed that microgroove imprinted silk films guide the cellular alignment

confirmed by presence of cell specific marker protein expression using
immunocytochemistry. Cells were plated on patterned silk films of all three varieties, keeping
initial cell density same (~lxl04 cells/cm2). After 7 days of culture, an almost confluent layer
of vascular cells was observed. Fluorescent microscopic images exhibited presence of
cytoplasmic distribution of vWF, a phenotypic marker for endothelial cells (Fig. 10 G, H).
Furthermore, distinguishable expression of calponin and α-SMA (early and mid-
differentiation marker of SMC) marked the maintenance of contractile phenotype of smooth
muscle cells (Fig. 10 A-D). Elastin production was also analyzed by staining intracellular
soluble tropoelastin of smooth muscle cells. Although we did not observe the prominent
fibrous elastin in the extracellular matrix of SMC culture but a distinct punctuated pattern in
the cellular cytoplasm does evidence elastin biosynthesis (Fig. 10 E, F). Presence of specific
marker positive cells suggested that patterned silk films supports the growth of vascular cell
types while maintaining the cellular phenotype.
The cellular metabolic activity and attachment of vascular cells on silk films was
checked. Percentage alamar 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 RGD motifs present on surface of A.
assama and P. ricini silk films [42]. 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 [43]. 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 [44].
5. Preparation of silk film based vascular conduit
Silk films were sterilized under ultraviolet light prior to cell seeding. An optimal
number of vascular cells (~4xl04 cells/cm2) were seeded onto 4x4 cm2 patterned film.
Approximately 70% confluent silk films were used for rolling. At first, endothelial cell
seeded film was rolled over the inert mandrel (3-6 mm diameter) using custom designed
rolling assembly. SMC seeded silk film was rolled over the endothelial layer considering the
concentric alignment. At last, fibroblast seeded silk film was used to cover the SMC layer.
This specific arrangement certainly mimics the native structure of blood vessel. Such rolled
films were held together onto the mandrel using a sterile silk thread. This assembly was
further submerged in the basal culture medium (High glucose DMEM supplemented with
10% FBS and 1% antibiotic-antimycotic solution) and maintained at 37°C in a humidified
incubator at 5% CO2 for maturation over 14 days. After maturation, thread was removed and
the vascular construct was slide out from the mandrel.
Engineering of functional vascular tissue demands well grown endothelial cell layer
that serves the purpose of hemocompatible surface thereof reducing the chances of
thrombogenesis [45].Taking all these aforementioned limitations into consideration,

biorkaterial. Motivation of using silk to fabricate vascular construct was acquired by virtue of
its antithrombotic properties [46]. Also, further processing for stabilization of the silk films
against water, water vapor annealing was adopted over other methods owing to its superior
hemocompatability [47]. 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 [48]. 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 [49]. Herein
the findings successfully shown a confluent monolayer of endothelial cells aligned along
microgrooves on silk films. It was hypothesized 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. The 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 [50]. It also
assists in transition from synthetic to contractile phenotype rendering superior strength and
compliance of fabricated construct [51]. 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 [52]. 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 a-SMA (contractile genes) clearly suggested that cellular patterning
on silk films substantially induces phenotype transition of SMCs towards contractile nature
which is in agreement with the previous reports [53]. 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.
5.1. Histological analysis of vessel structure
Immediately after harvesting (14 days), cell seeded vascular construct was cut into
small pieces (~lcm) and fixed in neutral buffer formalin (NBF) for at least 24 hours (4°C). It
was than dehydrated using grading ethanol solutions (50, 70, 90, 95, 100% v/v) followed by
paraffin embedding. 5 (a thick sections were cut and mounted onto glass slides. Cell
distribution was assessed by imaging the Hoechst 33342 stained sections as per the standard
protocol. Mature vascular conduit (after 14 days) was assessed for cellular distribution by
histology analysis. De-paraffinized sections were directly stained with Hoechst 33342 to
locate cells in the rolled and mature assembly. Fluorescent microscopic images revealed
homogenous distribution of vascular cells throughout the circular tissue section (Fig. 11). It
was also observed that mature vessel construct maintained a tubular morphology and cell
seeded film layers remained cohesively bound. Retention of cells after rolling step verifies
the applicability of this procedure.

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. To reduce the probability 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.
5.2. Burst strength of tubular construct
The burst strength of tubular construct was measured in hydrated conditions. Silk tube
of approximately 4 cm in length was connected to 50 ml syringe at one end while other one
was connected to traceable™ manometer pressure/vaccum gauge (Fisher Scientific™). This
assembly created a closed channel (Fig. 13). Syringe was fixed in a syringe pump and air was
perfused at a constant flow rate of 1ml/min. A continuous flow of air created positive
pressure inside the construct. Maximum pressure point at tube failure was recorded and
considered as burst pressure.
Mechanical strength of the silk film based vascular construct was determined in terms
of burst strength so as to sustain physiological blood pressure (Fig. 12). Burst pressure ranged
between 915-1260 mm Hg for tubular constructs made up of different silk varieties under
hydrated conditions and no significant difference was observed among different experimental
groups.
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 mm Hg) and
around 5 times higher than above pathological pressure (180-220 mm Hg) [54]. This attests
the mechanical suitability of silk film based vascular grafts fabricated in the current endeavor.
Advantages of the invention:
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 [55]. 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 [56]. We hypothesize that strategy followed in
this work might enable us to circumvent the afore-stated 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.

Current research work showcases a futuristic methodology to fabricate SDVG by
combining cell sheet engineering and implementation of patterned silk films to address the
challenges faced in this particular field. Pre cell seeded patterned silk films made the rolling
process more favorable simultaneously maintaining the tissue integrity. We successfully
demonstrated the formation of unidirectionally aligned monolayer of vascular cells on
patterned silk films expressing their functional phenotype and were metabolically active.
Constructs were mechanically suitable for bypass grafting since they withstood and did not
fail even at significantly higher values than physiological arterial pressure. Non-mulberry silk
varieties (A. assama and P. ricini) were superior to conventionally used B. mori silk in terms
of stability, vascular cell compatibility and did not elucidate any immunological response
when implanted in vivo in mice. Advantages of the present invention are enlisted below and it
is envisaged that future advancement in this direction would lead towards "off the shelf
clinical implementation of SDVG.
• Silk, unlike other natural ECM proteins, is easily accessible. There is no requirement of
using highly sophisticated techniques like chromatography, gel electrophoresis and other
precipitation techniques for protein extraction and purification. Also, there is no such
limitation of resource availability since tons of silk is produced every year and India is the
second largest producer in the world.
• Northeast silk varieties (A. assama and P. ricini) were projected for vascular tissue
engineering purpose and we observed positive results in terms of cellular compatibility
and alignment.
• Herein the inventors have combined the conventional template based tissue engineering
using thin patterned silk films with cell sheet engineering principle to exactly mimic the
native blood vessel cellular alignment. The novel vascular grafts have not only mimicked
the native architecture of blood vessel but also all three vascular cells (Endothelial cells,
Smooth muscle cells and Fibroblasts), arranged spatially in unique native like fashion
have been co-cultured.
• Tubular construct was obtained without using any sophisticated high end instrument like
electrospinning and 3D-printing and does not require long term maturation in pulsatile
bioreactor. Overall, the methodology is rapid, completely green and cost effective.
• This cell seeded silk film based rolling is advancement over cell sheet engineering that
reduces the vulnerability of cell sheet towards breaking during handling.
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CLAIMS
We claim:
1. A vascular graft construct comprising an elongate tubular body along an axis having at
least an open proximal and at least an open distal end, wherein the elongate tubular
body comprises plurality of superimposed concentric layers of rough micro-patterned
silk fibroin films.
2. A vascular graft construct as claimed in claim 1, wherein the elongate tubular body
comprises at least three concentric layers of superimposed rough micro-patterned silk
fibroin films.
3. A vascular graft construct as claimed in claims 1-2, wherein the three concentric layers
of innermost, middle and outermost rough micro-patterned silk fibroin films are seeded
with endothelial cell, smooth muscle cells and fibroblast cells, respectively.
4. A vascular graft construct as claimed in claims 1-3, wherein the rough micro-patterned
silk fibroin films are derived from the group comprising cocoons of mulberry silk worm
Bombyx mori and silk glands of fully-grown fifth-instar matured larvae of non-mulberry
North East wild silk worms Antheraea assama and Philosamia ricini.
5. A vascular graft construct as claimed in claims 1-4, wherein the burst pressure of the
vascular graft construct is ranging from 915-1260 nm Hg under hydrated conditions.
6. A vascular graft construct as claimed in claims 1-5, wherein the silk fibroin films are
micro-patterned in liner alignment and having rougher surface with roughness values
ranged between 68.16 nm - 78.26 nm and average height ranging from 145.02 nm -
250.36 nm, for better cell attachment, proliferation and alignment.
7. A vascular graft construct as claimed in claims 1-6, wherein the degradation weight loss
with time, of the rough micro-patterned silk fibroin films ranging from 43% - 98% in
presence of bodily enzyme after 28 days.
8. A process of fabricating a vascular graft construct as claimed in claim 1 comprising
rolling along an axis, of plurality of rough micro-patterned silk fibroin films on an inert
elongate tubular body to form an assembly followed by submerging the said assembly
in culture medium for maturation, wherein the plurality of micro-patterned silk fibroin
films are concentrically rolled and superimposed sequentially.
9. A process of fabricating a vascular graft construct as claimed in claim 8, wherein at
least three concentric layers of rough micro-patterned silk fibroin films are rolled and
superimposed sequentially.
10. A process of fabricating a vascular graft construct as claimed in claims 8-9, wherein the
three concentric layers of innermost, middle and outermost rough micro-patterned silk
fibroin films are seeded with endothelial cell, smooth muscle cells and fibroblast cells,
respectively.

11. A process of fabricating a vascular graft construct as claimed in claims 8-10, wherein
the rough micro-patterned silk fibroin films are prepared from aqueous silk fibroin
solution poured onto corrugated substrates made of grooved PDMS (poly-
dimethylsiloxane) followed by drying and separating the micro-patterned silk fibroin
films from the PDMS molds.
12. A process of fabricating a vascular graft construct as claimed in claims 8-11, wherein
the inert tubular body is a mandrel and is glided out from the said assembly after
maturation.