FORM 2
THE PATENTS ACT, 1970
(39 of 1970)
COMPLETE SPECIFICATION

1. TITLE OF THE INVENTION
"SYNTHESIS METHOD OF BIOINK FOR 3D PRINTING AND MOLDING OF
VASCULAR GRAFTS"
2. APPLICANT:
a) NAME: Dr. Meghnad G Joshi, Stem Plus Biotech Pvt Ltd.
b) NATIONALITY: Indian
c) ADDRESS: APPLICANT: Stem Plus Biotech Pvt. Ltd.
Sangli Miraj Kupwad Commercial Complex,
C/S No. 1317/2, Near Shivaji Maharaj Putla,
Bus Stand Road, Gaon Bhag, Sangli, MS - 416416,
India.
3. PREAMBLE TO THE DESCRIPTION
COMPLETE SPECIFICATION
The following specification particularly describes the invention and the manner in which it
is to be performed

Papers Related to work
1. Esmaeili S, Shahali M, Kordjamshidi A, Torkpoor Z, Namdari F, Samandari SS,
Ghadiri Nejad M, Khandan A. An artificial blood vessel fabricated by 3D printing for
pharmaceutical application. Nanomedicine Journal. 2019;6(3): 183-94.(1)
Summary:
In this study, thermoplastic polyurethane (TPU) composed of nanocrystalline
hydroxyapatite (HA) nanopowder was prepared using the extrusion technique to
construct the ABVs. X-ray diffraction (XRD) and scanning electron microscopy (SEM)
were used to investigate the optimum specimen. The narrowed arteries composed of
TPU composite with nanocrystalline HA nanopowder had proper chemical stability and
mechanical characteristics.
2. Li L, Qin S, Peng J, Chen A, Nie Y, Liu T, Song K. Engineering gelatin-based
alginate/carbon nanotubes blend bioink for direct 3D printing of vessel constructs.
International journal of biological macromolecules. 2020;145:262-71. (2)
Summary:
The hybrid bioink prepared with gelatin, sodium alginate and carbon nanotubes were
manufactured into cylindrical scaffolds through the collaboration between the vertical
directional extrusion of printing nozzle and axial rotation of stepper motor module.
Results demonstrated that the proper doping of carbon nanotubes could effectively
increase the mechanical properties of the composite scaffolds. Also, quantitative
experiments proved that a small amount of doping of carbon nanotubes had little effect

on cytotoxicity, and the constructs could meet the requirements of biomimetic vascular.
3. Song KH, Highley CB, Rouff A, Burdick JA. Complex 3D-molded microchannels
within cell-degradable hydrogels. Advanced Functional Materials.
2018;28(31):180I331.(3)
Summary:
Hydrogel is further engineered to undergo stabilization through a thiol-ene reaction
using di-lhiol crosslinkers, permitting (i) the washing of the ink to produce
microchannels and (ii) tunable properties depending on the crosslinker design. This
printing approach is used to investigate the influence of channel curvature on
angiogenic sprouting and increased sprouting at curved locations is observed when
compared to straight regions. Ultimately, this technique can be used for a range of
biomedical applications, from engineering vascularized tissue constructs to modeling in
vitro culture environments.
4. Xu Y, Hu Y, Liu C, Yao H, Liu B, Mi S. A novel strategy for creating tissue-engineered
biomimetic blood vessels using 3D bioprinting technology. Materials. 2018;11(9):158.
(4) Summary:

In this work, a novel strategy was developed to fabricate pre vascularized cell-layer
blood vessels in thick tissues and small-diameter blood vessel substitutes using three-
dimensional (3D) bioprinting technology. Mechanical properties of the molded scaffold
and found that its elastic modulus approximated that of the natural aorta. These findings
demonstrate the feasibility of fabricating different kinds of vessels to imitate the
structure and function of the human vascular system using 3D bioprinting technology.
5. Xu L, Varkey M, Jorgensen A, Ju J, Jin Q, Park JH, Fu Y, Zhang G, Ke D, Zhao W,
Hou R. Bioprinting small diameter blood vessel constructs with an endothelial and
smooth muscle cell bilayer in a single step. Biofabrication. 2020;12(4):045012.(5)
Summary:
This study aimed to fabricate a small diameter, heterogeneous bilayer blood vessel-like
construct in a single step with gelatin methacryloyl (GelMA) bioink using a 3D micro-
extrusion biomolded on a solid platform. GelMA was supplemented with Hyaluronic
acid (HA), glycerol and gelatin to form a GelMA bioink with good printability,
mechanical strength, and biocompatibility. The resulting 20 mm long, 4.0 mm diameter
lumen heterogeneous bilayer blood vessel-like construct closely mimics a native blood
vessel and maintains high cell viability and proliferation.

Prior Art:
Sr. Patent Name of the Inventors Particulars
no. Number
Month and
year patent
1 US201700297 Method for Keng-Liang OU The present invention relates to a
81 preparing artificial method for preparing artificial blood vessels, especially to a
02.02.2017 blood vessels method for preparing artificial blood vessels with an active POSS-PCU and stem cells.

2 WO20160663 Biologically MULLER, This invention concerns a
28 active Werner Ernst composition consisting of
06.05.2016 biomimetic Ludwig Georg biologically inert anionic polymers
tissue- SCHRODER, and biologically active polymers
engineered Heinrich- as a well as cross-linking calcium
blood vessels Christoph ions, that can be used for the
for small Wilhelm fabrication of biologically active
diameter Friedrich arlillcial blood vessels in
applications WANG,
Xiaohong particular in the small-diameter range.
3 WO20200262 Methods of STREHL, The present disclosure relates to
12 preparing Raimund methods of preparing
06.02.2020 personalized blood vessels personalized blood vessels, useful for transplantation with improved host compatibility and reduced susceptibility to thrombosis. Also provided are personalized blood vessels produced by the methods and use thereof in surgery.
4 CN10856799 3D Printing XUE WEI, The invention discloses 3D (three-
2 bio-ink for SONG dimensional) printing bio-ink for
108567992 quickly RONGGUANG quickly repairing injured blood
05.02.2021 repairing RUAN, vessels of vertebral columns and a
injured blood MIAOLIANG method for preparing the 3D
vessels of DAI JIAN, printing bio-ink, and relates to the
vertebral WANG field of 3D bio-printing. The
columns and method for preparing 3d printing bio-ink YONGZHOU method includes steps of preparing ink for shell materials; preparing ink for cell layers of inner layers; carrying out 3D printing preparation and the like
5 20142047991 Blood vessel ZHOU The utility model discloses a blood
8.4 molding device HUIXING vessel molding device for 3D
27.05.2015 for 3d biology printing LAN HAIMING biology printing, which belongs to the biology engineering technology and aims to solve the currently existing problems of bad blood vessel molding quality and short molding length when a blood vessel is molded by the 3D biology printing technology.
Field of the Invention:
The present invention relates to Synthesis of Bioink for 3D printing and molding technique
for generation of tissue engineered vascular grafts.
Background Art of the Invention:
Blood vessels (BV) perform the blood transportation from heart to every cell of the body and
vice-versa. The transportation of blood helps in providing oxygen and nutrients for growth
and repairing action (6). Blood vessels which exist outside the heart are collectively called as

peripheral vascular system. And another which rest in heart itself are central vascular system. Due to the deposition of calcium like salts or low-density lipoproteins the inner diameter of vessel decreases and it causes obstruction to the flow of blood (7). Endothelial proliferation responsible for the formation of clots which can complete or partial blockage of blood vessel due to hypercoagulability (8). The cardiovascular diseases are the major cause of increasing global mortality rate of about 523 million according to study done in 2019 (9). As a solution there are surgical techniques of grafting techniques available till date. In the allograft and xenograft, immunosuppressants should be provided continuously for reducing the possibility of graft rejection. Even though autografts do not have any immune reaction, there are chances of infection and scar formation at the position of removal of graft (9). Tissue engineering can be the key solution for replacing the graft with no side effects.
Synthetic or biosynthetic vascular grafts are appropriate candidates to overcome the shortage of autografts. Over time period synthetic grafts, biodegradable vascular grafts are able to provide mechanical support for tissue regeneration and be degraded and replaced by native tissue. (10). After implantation, biodegradable grafts will provide mechanical support and gradually degrade and replaced by tissue. Polylactic acid (PLA), Polyglycolic Acid (PGA), and poly (e-caprolactone) (PCL) are Common biodegradable polymers for vascular grafts manufacturing (11,12).
Our objectives were goat and porcine blood vessels would be an ideal material to generate ECM which can be combined with polymers to generate bioink for the 3D printing of blood vessel. In this work, goat, bovine and porcine blood vessels were decellularized followed by alkaline/acidic digested and incorporate with polymers such as polyvinyl alcohol (PVA), PLA and gelatin as a printable bioink. We investigated that the bioink preserved the intrinsic Extra Cellular Matrix (ECM) and attained viscosity for bioink formation. In this technology, we investigated printable bioink can attain well anatomical shape, biomechanical properties, biodegradability and wettability properties. This study investigated in ovo biocompatibility of 3D molded vascular graft as a scaffold. This study also investigated the heparin immobilization and platelet adhesion test of 3D printed/molded vascular garft. We also investigate the biocompatibility test in in vivo (rat model). This study investigated transplantation study in rat model. In this invention of 3D molded vascular graft, problems occurring during these mentioned techniques are addressed. 3D molded vascular graft is having no graft rejection after transplantation. This 3D molded vascular graft can be applied as a vascular graft substitute for clinical applications in the future. Objectives of Invention:
• Primary objective of the invention is to develop blood vessels specific Extra cellular matrix (ECM) by digesting goat, bovine and porcine blood vessels.
• Another primary objective of the invention is to generate bioink, combining synthetic polymer (PVA and PLA) and/or natural polymer (Gelatin) in the range of 60 to 110 °C temperature to generate bioink which consist of blood vessel specific ECM.
• Primary objective of the embodiment herein composite biomaterial which combines the properties of ECM of blood vessel mimicking the cellular environment and biomechanical properties of the blood vessels.
• Another primary objective of the invention is to use this bioink in 3D printing and molding of vascular graft.
• Another primary objective of the invention is to define in ovo biocompatibility of 3D printed/molded vascular graft.
• Another primary objective of the invention is to define morphological properties (SEM) and tensile biomechanical properties of the 3D molded vascular graft.
• Another primary objective of the invention is to define heparin loading in 3D molded vascular graft.

• Another primary objective of the invention is to define in vivo biocompatibility of the 3D molded vascular graft.
• Another primary objective of the invention is to define in vivo transplantation study of the 3D molded vascular graft.
• Another objective of the invention is use of 3D molded vascular graft for basic or research applications such as clinical application, in vitro toxicology.
Summary of Invention:
The various embodiments herein provide a bioink which combines the various components of
blood vessels extracellular matrix (ECM) incorporated in Polyvinyl Alcohol, polylactic acid
and gelatine to mimic the blood vessels ECM and mechanical properties of the native blood
vessels. The embodiment herein provides mechanical strength to support specific blood
vessels cells like, smooth muscles cell and endothelial cell growth.
Goat, bovine or porcine blood vessels brought from local slaughter house and cleaned in
distilled water before further use. After weighing of cleaned blood vessels washed in 70 %
alcohol by continuous agitation by shaker at 80 rpm for 15 min. After that distilled water
wash containing antibiotics and antifungal agents was given to blood vessels for another 15
min. on shaker at 80 rpm. Alkali digestion was carried out using 1- 5 N NaoH or KOH (1-10
g blood vessels/ml) at 40 to 80 °C for 24 to 48 hrs. Slurry was formed after complete blood
vessels digestion, and slurry was filtered with muslin cloth to remove non digested residuals.
pH of the slurry (Digested blood vessels) was maintained at neutral. PVA or PLA in 5 to 20
% concentration was added in neutral slurry, and allow it to dissolve at 60 to 100 °C for 36
to 48 hrs followed by to 2 - 10 % gelatine was added at 60 to 80°C for 24 to 48 hrs. The step
of polymerization of bio ink involves, i) The bio ink composite was subjected microwave
heating at 300-800 watt for 1-2 min followed by time of 10-20 min. Microwave heating was
done for 2-3 times, ii) The bioink composite was cooled down and kept in -40 to -80°C
overnight followed by microwave heating step. This step was repeated for 3-6 times and
prepared bioink used for further study.
Brief Description of the Drawings:
The objects, features and advantages will occur to those skilled in the art from the following
description of the preferred embodiment and the accompanying drawings in which:
Fig.l: Bioink preperation, molded assembly and manufacturing process of 3D printing of
vascular graft, according to one embodiment herein.
Fig.2: 3D printed/molded vascular graft Characterization A) Attenuated total reflection
(ATR) bioink, B) Contact angle, C) Spread ability of bioink, D) Mechanical testing of rat
abdominal aorta and 3D printed vascular graft according to one embodiment herein.
Fig.3: CAM assay: Biocompatibility of 3D printed/molded vascular graft. A) 3D printed
vascular graft implant on CAM area of chick, B) HE staining of CAM area, C) SEM analysis
of implanted 3D vascular graft on CAM, according to one embodiment herein.
Fig.4) A) Histological Evaluation of Heparin immobilization in vascular graft, i) Toluidine
blue stain for Heparin immobilization in vascular graft, ii) SEM study of Platelets adhesion
test.
B) Biocompatibility of 3D printed/molded vascular graft in rat model.
C) Histology of subcutaneous implanted 3D vascular graft in rat, according to one embodiment herein.
Fig.5) A) 3D printed/molded vascular graft transplant in rat model.
B) Histology of transplanted 3D printed vascular graft in rat femoral artery.
C) Immunohistochemistry study of Transplanted 3D printed vascular graft in rat
femoral artery.
Detailed Description: Synthesis of the bio ink:

The various embodiments herein provide bio ink which combines the components of blood vessels extracellular matrix (ECM) integrated in PVA, PLA and gelatine to mimic the cellular ECM and provide mechanical strength. According to one embodiment herein, the process of synthesis of blood vessels bio ink involves the following steps. Goat, bovine and porcine blood vessels cleaned in distilled water. After weighing of blood vessels, immersed in 70 % alcohol and put on shaker for agitation for 15 min. After that distilled water containing antibiotics and antifungal agents wash was given to blood vessels for another 15 min on shaker. Alkali digestion was carried out using 1-3N NaoH or KOH (1-5 g blood vessels/ml) at 40 to 80°C for 24 to 48 hrs. After complete blood vessels alkali/acidic digestion the slurry was filtered with muslin cloth to remove non digested residuals. pH of the slurry (digested blood vessels) was maintained at neutral. PVA or PLA in 5 to 20 % concentration was added in neutral slurry, and allow it to dissolve at 60 to 100 °C for 36 to 48 hrs followed by to 2 - 10 % gelatine was added at 60 to 80°C for 24 to 48 hrs. The step of polymerization of bio ink includes, I) The bioink composite was subjected microwave heating at 300-800 watt for 10-20 min followed by time of 2-5 min. Microwave heating was done for 2-3 times, ii) The bioink composite was cooled down and kept in -40 to -80°C overnight followed by microwave heating step. This step was repeated for 3-6 times and prepared bioink for used for further study and 3D printing of blood vessels. 3D printing of blood vessels manufactured by first created (Computer Aided Design) CAD blood vessels file then further stereolithography (STL) conversion of CAD blood vessels file and finally created G-code of STL blood vessels file. Over a period of several hours, this permits 3D printing vascular graft was to be built up in precise thickness. Another method used for preparation of vascular graft by deposition of bioink layer by layer on stainless steel needle, glass capillaries and needle as shown in Fig.l.
The following examples, according to preferred embodiments of the invention, demonstrate the features. However, it is understood that such examples are not to be interpreted as limiting the scope of the invention as defined in the claims. Example!:
According to one embodiment herein, the method of synthesis of bioink involves the following steps. Goat, bovine or porcine blood vessels (lg blood vessels/ml) was transferred for digestion using IN NaOH at 70 °C for 24 hrs. After complete digestion the slurry was filtered with sieve to remove unnecessary residuals. The neutral pH of the slurry was adjusted. Further, 5 % PVA was added to neutral slurry, and allow it to dissolve at 60 °C for 24 hrs followed by 1 % gelatine at 60 °C for 48 hrs. Bioink was stored in the temperature of 4 °C. The step of polymerization of bioink comprises the following steps. The bioink mixture was subjected to microwave heating at 400 watt for 2 min followed by interval of 5 min. The above microwave heating course of action was repeated for 3 times. The mixture was cooled down and kept at -40 °C overnight followed by microwave heating step. This step was repeated for 3 times and prepared bioink used for further study and application. 3D printing of vascular graft synthesized by first created (Computer Aided Design) CAD blood vessels file then further stereolithography (STL) conversion of CAD blood vessels file and finally created G-code of STL blood vessels file. Over a period of several hours, this permits 3D printing of blood vessels was to be built up in precise thickness.

Sr. Material Process Concentration Temperature °C Time in hrs
1 Goat, bovine or porcine blood vessels Slurry lg/ml Room Temperature
2. NaOH Digestion IN 70 24
3. The neutral ] pH of the slurry was adjusted
4. PVA Polymerization 5 % 60 24
5. Gelatine Polymerization 1% 60 48

6. Bio ink was stored at 4C
7. Microwave heating at 400 watt for 2 min followed by interval of 5 min for 3 times
8. The combination was cooled down and set aside in -40 °C overnight followed by microwave heating step. This step was repeated for 3 times.
9. 3D blood vessels: 1) Creating (Computer Aided Design) CAD blood vessels file, 2) Stereolithography (STL) conversion of CAD blood vessels file, 3) Creating G-code of STL blood vessels file 4) 3D Biomolded blood vessels
Swelling or water uptake ability of 3D molded vascular graft:
Swelling or water uptake ability of 3D molded material is important from the point of view to maintain three-dimensional structure in aqueous solution. 3D printed/molded vascular graft (n= 3) 1 to 1.05 gm with 1 cm in length was immersed in 30 to 40 ml distilled water (pH neutral) and incubated at 37 °C for 28 days. After a regular intermission (24h), the water-soaked sample was taken out from distilled water; surface water was blotted off by a tissue paper and reweighed until an equilibrium weight was reached. Swelling behaviours % value of 3D molded vascular graft such as 1% to 5 % as showed in Fig.2A. 3D molded vascular graft properties such as swelling and flexible uniqueness match with finest scaffold properties in aqueous solution.
Surface wettability (Contact angle) of 3D molded vascular graft:
3D molded vascular graft surface wettability properties significant in the tissue engineering and regenerative medicine in particular, important to maintain biophysical properties, cytocompatibility, and microstructure, and biodegradability, biocompatibility and attractive for cellular fate.Water drop was absorbed by the 3D molded vascular graft surface and showed no any contact angle. 3D molded vascular graft characterized as a super hydrophilic material. 3D molded vascular graft makes available hydrophilic exterior and zero measurable contact as showed in Fig.2B. XRD pattern of 3D molded vascular graft:
From the XRD pattern can determine crystalline phases of molded material. The 3D molded vascular graft was characterized by X-ray diffraction (XRD) (Fig.2C).The diffraction patterns were recorded with a XRD analyser using Cu Ka radiation. The XRD pattern of the 3D molded vascular graft revealed a prominent X-rays peak observed at around 20- 2theta (29) (Cu K-alpha) having an X-ray intensity between 5 to 15 counts (X-ray intensity ). XRD pattern of 3D molded vascular graft clearly indicates the crystalline nature of the 3D molded vascular graft. Example 2:
According to one embodiment herein, the method of synthesis of bioink comprises the
following steps. Goat, bovine or porcine blood vessels (10 g blood vessels/ml) were
transferred for digestion using IN KOH at 70 °C for 24 hrs. After complete digestion the
slurry was filtered with sieve to eliminate unnecessary residuals. The neutral pH of the slurry
was adjusted. Further, 10 % PVA was added to neutral slurry, and allow it to dissolve at 80°C
for 24hrs followed by 2 % gelatine at 60 °C for 48 hrs. Bioink was stored in the temperature
of 4 °C. The step of polymerization of bioink comprises the following steps. The bioink
mixture was subjected to microwave heating at 600 watt for 2 min followed by interval of 5
min. The above microwave heating course of action was repeated for 3 times. The mixture
was cooled down and kept at -40°C overnight followed by microwave heating step. This step
was repeated for 3 times and prepared bioink used for further study and application.

Sr. Material Process Concentration Temperature °C Time in hrs
1 Goat, bovine or porcine blood vessels Slurry l0g/ml Room Temperature
2. KOH Digestion IN 70 24

3. The neutral pH of the slurry was adjusted
4. PVA Polymerization 10% 80 24
5. Gelatin Polymerization 2% 60 48
6. Bioink was stored at 4 °C.
7. Microwave heating at 500 watt for 2 min followed by interval of 10 min for 3 times
8. The combination was cooled down and set aside in -40°C overnight followed by microwave heating step. This step was repeated for 3 times.
9. 3D printing of blood vessels: 1) Creating (Computer Aided Design) CAD blood vessels file, 2) Stereolithography (STL) conversion of CAD blood vessels file, 3) Creating G-code of STL blood vessels file 4) 3D Biomolded- print blood vessels
Spreadability of the bioink;
The printing reliability highly depends on the hydrophilicity and the viscosity of the bioink. It is reported that the application of the bioink also depends upon its spreading ability and print ability. The spread ability % value bioink: 10% to 40% as showed in Fig.2D. Biomechanical study:
Assessment of tensile Properties of 3D molded vascular graft and native rat abdominal aorta tensiometry was performed by tensometer. Samples were cut, measured and then the samples were clamped to the tensiometer. Applied force and the elongation of the 3D molded vascular graft and rat abdominal aorta was used to obtain a biomechanical data measurements. Ultimate tensile load (N), Tensile strength and Elongation of 3D molded vascular graft, native rat abdominal aorta shows in Fig.2E. Example 3:
According to one embodiment herein, the method of synthesis of bioink comprises the following steps. Goat, bovine or porcine blood vessels (5 g blood vessels/ml) was transferred for digestion using 2N NaOH at 80°C for 36 hrs. After complete digestion the slurry was filtered with sieve to eliminate unnecessary residuals. The neutral pH of the slurry was adjusted. Further, 10 % PVA was added to neutral slurry, and allow it to dissolve at 60 °C for 24 hrs followed by 5 % gelatine at 40 °C for 48 hrs. Bioink was stored in the temperature of 4°C. The step of polymerization of blood vessels bioink comprises the following steps. The bioink mixture was subjected to microwave heating at 800 watt for 1 min followed by interval of 10 min. The above microwave heating course of action was repeated for 3 times. The mixture was cooled down and kept at -80 °C overnight followed by microwave heating step. This step was repeated for 3 times and prepared bioink used for further study and application.
Sr. Material Process Concentration Temperature °C Time in hrs
1 Goat, bovine or porcine blood vessels Slurry 5g/ml Room Temperature
2. NaOH Digestion 2N 80 36
3. The neutral pH of the slurry was adjusted
4. PVA Polymerization 10 % 60 24
5. Gelatine Polymerization 5% 40 48
6. Bio ink was stored at 4"C
7. Microwave heating at 800 watt for 1 min followed by interval of 10 min for 3 times
8. The combination was cooled down and set aside in -80°C overnight followed by microwave heating step. This step was repeated for 3 times.
9. 3D blood vessels: 1) Cr Stereolithography (STL vessels file 4) 3D Biom eating (Computer Aided Design) CAD blood vessels file, 2)
,) conversion of CAD blood vessels file, 3) Creating G-code of STL blood
olded - print blood vessels
Biocompatibility of 3D molded vascular graft in chick embryo model:

The CAM assay allows in ovo vascularization of biomaterials planted on the surface. The CAM angiogenesis and biocompatibility after scaffold grafting which allows direct visualization and quantification. According to one embodiment herein, blood vessel has specific extra cellular matrix (ECM) that facilitates recruitment of different type cells. ECM is important to control the interactions between cells and microenvironment for guiding cellular fate. Biocompatibility of the 3D molded vascular graft graft checked by CAM model (n30). Zero hr. fertilized black leghorn chicken eggs (Gallus gallus) were obtained from local hatchery and cleaned with 70% alcohol. Fertilized chicken eggs were incubated at 37 °C and 80% humidified ambiance in household incubator. Test and control groups (nlO) were used in the experiments. The 3D molded vascular graft was cut into rectangular shapes (5 mm><10 mm) sample. On 4th day of the incubation, an eggs window was opened and 3D molded vascular graft were placed on chorioallantoic membrane (CAM) area. An eggs window was covered by sterile paraffin film and returned to the incubator. On day 4, 7, 10 and 13 the eggs were opened the chick embryos were observed using stereo-microscopy (Fig.3A). According to one embodiment herein, Chick embryo studies represent convenient and effective means for the integrated testing of the biocompatibility of 3D molded vascular graft implanted tissue were removed and collected in 10 % neutral buffered formalin. The adjacent CAM is also harvested and kept in 10% neutral buffered formalin for fixation. CAM was further processed for hematoxylineosine staining (HE). In the Fig.3A the 3D molded vascular graft showed satisfactory biocompatibility with CAM tissue without severe inflammatory response. HE study of CAM revealed that a differentiated cell with nucleus was better organized as shown in fig.3B. SEM analysis revealed cell attached on 3D printed vascular graft (Fig.3C). In this study, the functionalized grafted 3D molded vascular graft on to the chick embryo CAM to assay to evaluated angiogenesis significantly promote endothelial cell migration and in ovo. Loading of heparin on 3D printed/molded vascular scaffold
Heparin loading in a 3D printed/molded vascular graft was done by a similar method used by Kong X et al. vascular graft incubate at 37°C for 4 h. in 50 mL MES buffer (0.05 mol/L, pH 5.60) containing 100 mg EDC (l-(3-Dimethylaminopropyl)-3-ethylcarbodiimide) (Sigma), 100 mg heparin (Sigma) and 60 mg NHS (N-hydroxysuccinimide) (Sigma). After rinsing in disodium hydrogen phosphate solution (0.1 mol/L) for 2 h, sodium chloride solution (4 mol/L) for 24 h 3 4 times, and 24 h in distilled water for 3 3 times, the heparinized vascular graft was obtained. All heparinized grafts were sterilized by Ethylene oxide sterilization atmosphere (concentration of 321 g/m3) (RUGIKON Semi-Automatic KANC-125, Mumbai, India). Sterile grafts were stored in 0.9% NaCl +1% penicillin/streptomycin at 4°C for future use. Toluidine blue (0.05 mg/mL) was used for heparin content staining on heparin treated vascular graft slides (Fig.4A-i). It has been reported that heparin coating surfaces reduce platelet adhesion. After heparinization of biomaterials it prevents adsorption of nonspecific protein and improves cell attachment, proliferation and differentiation. Such modified surfaces have proved anticoagulant actions. Example 4:
According to one embodiment herein, the method of synthesis of bioink comprises the following steps. Goat, bovine or porcine blood vessels (3g blood vessels/ml) was transferred for digestion using 1.5 N NaOH at 70 °C for 24 hrs. After complete digestion the slurry was filtered with sieve to eliminate unnecessary residuals. The neutral pH of the slurry was adjusted. Further 5 % PVA was added to neutral slurry, and allow it to dissolve at 60°C for 36 hrs, followed by 10 % gelatine at 80°C for 24 hrs. Bioink was stored in the temperature of 4 °C. The step of polymerization of bioink comprises the following steps. The bioink mixture was subjected to microwave heating at 500 watt for 2 min followed by interval of 20 min. The above microwave heating process was repeated for 3 times. The mixture was cooled

down and kept at -80°C overnight followed by microwave heating step. This step was repeated for 6 times and prepared bioink used for further study and application.

Sr. Material Process Concentration Temperature °C Time in hrs
1 Goat, bovine or porcine blood vessels Slurry 3g/ml Room Temperature
2. NaOH Digestion 1.5 N 70 24
3. The neutral pH of the slurry was adjusted.
4. PVA Polymerization 5% 60 36
5. Gelatin Polymerization 5% 80 24
6. Bioink was stored at 4 °C.
7. Microwave heating at 500 watt for 2 min followed by interval of 20 min for 3 time s
e heating step.
8. The mixture was cooled down and kept in -80 °C overnight followed by microwave This step was repeated for 3 times

9. 3D molded of blood vessels: Bioink deposited layer by layer on stainless steel needle or glass capillaries.
Platelet adhesion test
3D printed/molded vascular grafts were used in the platelet adhesion lest. 10 ml human whole blood was collected by venous puncture from healthy donor with informed consent. For obtaining platelet-rich plasma (PRP), blood was added into the centrifuge tube and centrifuged at 2000 rpm for 10 min. Before performing platelet adhesion test, the samples (Heparin treated and non-heparin treated) were washed with PBS. Samples were added into the 24-well plate then 200 pL of PRP were added to samples and incubated at 37 °C for 2 h. After incubation, the samples were rinsed in PBS three times and fixed with 2.5 wt% glutaraldehyde at 4 °C overnight. For SEM analysis samples were dehydrated with different concentrations of alcohol for 15 min each and sufficiently dried in a freeze dryer. A platelet adhesion test was performed to evaluation the effect of surface modification with heparin coating for the risk of thrombosis. As shown in Fig.4A-ii SEM analysis of the morphologies of the platelets adhered on the non- heparin coated 3D printed/molded vascular garft surface to study the thrombogenicity of the modified surfaces. Many platelets were found on the non-heparin treated 3D printed/molded vascular garft surfaces. Platelets not adhered to 3D printed/molded vascular garft suggesting improved hemocompatibility. Biocompatibility of 3D molded vascular graft in in vivo.
In vivo models (i.e., animal models) that is accessible for exploring and advancing therapeutic options for tissue regeneration. In vivo models are essential for biocompatibility and mechanical properties and its support to neo vascular network of implant materials. In vivo experiments were performed according to guidelines of the Institutional Ethical Committee (IAEC) (Ref-6/IAEC/2017), D Y Patil Education Society, Kolhapur, India. Wistar rats obtained from Central Animal House, D Y Patil Education Society, Kolhapur, India. The animals were acclimatized for a week before the surgical procedure. Rat (male/female) weighing 200-250 gm were anesthetized by injection Inj. Thiosol (Thiopentone injection, NEON 173251 Neon Laboratories Ltd, Mumbai, India), intraperitoneal route (45 mg/kg/body wt.)(13). 3D molded vascular graft (n=3) implanted subcutaneously (Fig. 4B). An incision was closed with 3-0 prolene surgical sutures. After 14 days (Fig. 4C-b), implanted scaffolds were excised and fixed in 10% formalin buffer for further histological analysis. In Fig.4C histology of subcutaneously implanted graft showed cell were attached on surface of the 3D printed graft. Example 5:
According to one embodiment herein, the method of synthesis of bioink comprises the following steps. Goat, bovine or porcine blood vessels (5.5 g blood vessels/ml) was

transferred for digestion using 3N NaOH at 37 °C for 48 hrs. After complete digestion the slurry was filtered with sieve to eliminate unnecessary residuals. The neutral pH of the slurry was adjusted. Further,20 % PVA was added to neutral slurry, and allow it to dissolve at 80°C for 48 hrs followed by 10 % gelatine at 80°C for 48 hrs. Blood vessels bioink was stored in the temperature of 8°C. The step of polymerization of bioink comprises the following steps. The bioink mixture was subjected to microwave heating at 800 watt for 2 min followed by interval of 20 min. The above microwave heating process was repeated for 3 times. The mixture was cooled down and kept at -40°C overnight followed by microwave heating step. This step was repeated for 6 times and prepared bioink used for further study and application.
Sr. Material Process Concentration Temperature °C Time in hrs
1 Goat, bovine or porcine blood vessels Slurry 5.5 g/ml Room Temperature
2. NaOH Digestion 3N 37 48
3. The neutral pH of the slurry was adjusted.
4. PVA Polymerization 20% 80 48
5. Gelatin Polymerization 10% 80 48
6. Bioink was stored at 8°C.
7. Microwave heating at 800 watt for 2 min followed by interval of 20 min for 3 times
8. The mixture was cooled down and kept in -40°C overnight followed by microwave heating step. This step was repeated for 3 times
9. 3D molded of blood vessels: Bioink deposited layer by layer on stainless steel needle or glass capillaries.
Transplantation surgery
Rats were anesthetized with Inj. Thiosol (45 mg/kg/body wt.) by intraperitoneal route. Inj. Heparin (Caprin, Samarth Lifesciences Pvt Ltd, Himachal Pradesh, India) (100 U/kg) was administered intraperitoneally before surgery and half dose (50 U/kg) everyone hour, was administered. At the inguinal site, oblique incision was taken, and skin and fat tissue were cut to expose femoral vessels. The femoral artery was dissected and separated from the femoral vein. At the proximal and distal region micro clamp was applied in the femoral artery and between the clamps, 7-8 mm arterial segment was excised. The 3D molded vascular graft in the length of 10 mm was used to substitute for the excised artery segment in the femoral artery. End-to-end anastomosis was done with 8-0 monofilament sutures (Fig.5A). After confirmation of no leakage from the anastomosis site, micro-clamps at the distal and proximal regions were removed. The fat tissue was sutured by 7-0 prolene and the skin was closed in an interrupted manner. During the surgical procedure, an animal's body temperature was maintained at 37 °C by using a heating pad. After surgery animal was returned to the cage and allowed to freely access food and water. After 30 days experimental rat were euthanized and implanted 3D printed/molded vascular grafts were excised and fixed in 10% formalin buffer for further histological analysis. Histological analysis showed in fig.5B. In histology study found that, cells were recruited in inner and outer surface of 3D molded vascular graft. Immunohistochemistry study (Fig. 5C) revealed that endothelial cell and smooth muscles cells were recruited in transplanted 3D printed vascular graft.

We Claim:
1. A method of manufacturing artificial three-dimensional [3D] small diameter vascular graft
substitute comprising:
(a) Optionally, Extra Cellular Matrix (ECM) can be derived from goat, bovine or porcine blood vessels.
(b) Liquid ECM can be obtaining by using alkali digestion followed by neutralization.
(c) A second ingredient is use of bio-polymer such as gelatin as cross linker subject.
(d) A third ingredient is use of physiologically suitable polymer such as poly vinyl
acid (PVA) or gelatin as a binder.
2. A process of synthesis of 3D molded vascular graft comprises the following steps.
(a) First step: Goat, bovine or porcine blood vessels cleaned in distilled water. After weighing of blood vessels, wash given in 70 % alcohol by put on shaker for 1-2 hrs. Additional wash given by distilled water containing antibiotics and antifungal agents for 15 min. on shaker. Finally blood vessels were transferred for digestion. Alkali digestion was carried out using 0.5-3 N NaoH or KOH (1-10 g blood vessels /ml) respectively at 58 to 120 °C for 24 to 48 hrs.
(b) Second step: After complete blood vessels alkali digestion, the slurry was filtered with 16 layered muslin cloth to remove non digested and unwanted residuals. Then the pH of the slurry (Digested blood vessels) was maintained from 6 to 8 pH.
(c) Third step: Further, 5 to 20 % PVA was added to neutral slurry, and allow it to dissolve at 60 to 120°C for 24 to 48 hrs.
(d) Fourth step: 1 to 10 % gelatin at 60 to 100 °C for 24 to 48 hrs.
(e) Fifth step: The step of polymerization of bioink comprises, i) The bioink composite was subjected microwave heating at 700-900 watt for 1-3 min followed by time of 5 min. Microwave heating was done for 4-10 times, ii) The bioink composite was cooled down and kept in -80 °C overnight followed by microwave heating step. This step was repeated for 4-10 times and prepared bioink for used for further study.
(f) Sixth step: 3D printing of blood vessels synthesized by first created (Computer Aided Design) CAD blood vessels file then further stereolithography (STL) conversion of CAD blood vessels file and finally created G-code of STL blood vessels file over a period of several hours, this permits to print small diameter blood vessels.
3D molded of blood vessels: 1 deposited layer by layer on stainless steel needle or glass capillaries or needle.
3. The method of claim 1-2, composite bioink.
4. Characterized by comprising hydrophilic synthetic polymer PVA having an interaction, the function having a cross linked hydrophilic biological polymer gelatin, along with blood vessels ECM can form spontaneously blood vessels specific bio functional components.
5. The process according to claim 1-2, wherein the analysis of a plurality of rheological characteristics of the bio ink illustrate hydrophilic property imparted by the PVA and gelatin component.
6. The method according to claim 1, wherein the ECM component of the 3D molded vascular graft provides a scaffold mimicking environment for cells, and wherein PVA and gelatin component provides tensile support.
7. The method of claim 1 and 2, further comprising the step wherein artificial bioink is deposited by 3D extrusion bio printing to manufacture scaffold or vascular graft or implant medical object.
8. The method of claim 1-6 3D molded vascular graft, with or without human cells,
comprising entire blood vessels specific ECM.

9. The method according to claim 1-7, wherein the investigation of biocompatibility study,
cell attachment and cell migration in transplantation study of the 3D printed/molded vascular
graft.
10. The method of claim 1-8, wherein said 3D molded vascular graft, applied to subjects
damaged/injured/defected of blood vessels or for medical object.

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