3D Bioprinting for Regenerative Medicine: A Review of Current Research in India | Volume VI Issue I | Author : Ms. Shrishti Pandey |

 Abstract:

3D bioprinting, a revolutionary technology in tissue engineering and regenerative medicine, enables the precise fabrication of biological structures through layer-by-layer deposition of bioinks. Our goal is to bio-manufacture tissues and organs in vitro, motivated by the need for precise tissue models for organ transplantation.  In the past few decades, 3D bioprinting has been used extensively to create a variety of tissues and organs, including skins, vessels, hearts, and more. These can serve as in vitro models for pharmacokinetics, drug screening, and other purposes in addition to providing a basis for the ultimate goal of organ replacement.This technique has significantly advanced the creation of complex tissues and organs, facilitating drug testing, disease modeling, and organ transplantation. Despite being in its nascent stages, the technology offers promising solutions to the global organ donor shortage and paves the way for patient-specific therapies. Historical developments in bioprinting, from its inception in 1986 to recent breakthroughs like FRESH techniques, have expanded its applications in clinical research and therapeutic interventions. In India, initiatives such as the establishment of 3D Bioprinting Centres of Excellence highlight the country’s growing focus on this domain. The technology’s applications span drug development, toxicity testing, tumor modeling, and prosthetic medical devices, with potential to transform healthcare delivery. However, challenges such as material limitations, regulatory hurdles, high costs, and ethical concerns persist. Indian legal frameworks, including the Drugs and Cosmetics Act and the Patents Act, partially address 3D bioprinting but require updates to accommodate this rapidly evolving field. By overcoming these barriers, 3D bioprinting could redefine regenerative medicine, enhancing patient outcomes and addressing critical medical needs.

Keywords: Bioprinting, Regenerative Medicine, Bioink, Organ Transplantation, Drug Development.

 Introduction:

 Over the past few decades, the fields of tissue engineering and regenerative medicine which aim to create functioning tissue-constructs that imitate native tissue for the repair and/or replacement of damaged tissues or entire organs have advanced quickly. By enabling the creation of three-dimensional, functioning biological structures, 3D bioprinting signifies a principle change in biomedical engineering. In contrast to conventional tissue engineering methods which includes the formation of an interphase between cell, scaffolds and growth factors, 3D bioprinting enables a precise layer-by-layer deposition of bioink consisting of living cells, biomaterials, and bioactive molecules and creates exact tissue structures by combining cutting-edge printing technologies with Computer-Aided Design (CAD). This method creates living tissues and organs using 3D printing technology, which may be used for disease modelling, drug testing, and transplantation. Although this technology is still in its infancy, it has the potential to transform regenerative medicine, enhance drug testing models, and alleviate the global organ donor shortage. However, there are still several challenges that need to be overcome to unlock the full potential of 3D bioprinted tissues and organs.The main ingredients in bioprinting are biomaterials, which can be synthetic or natural materials that include living stem cells. They are also known as bioinks. When bioinks are compatible with biological systems, any tissue or organ in the human body can be restored, reinstated, or augmented at any time.

 First, a digital model or blueprint is created, usually using information from medical imaging tests like MRI or CT scans. The bioprinter uses this model as a guide to build up the desired structure by depositing layers of biological material, frequently in the form of a bioink.^[1]Typically, bioinks are made up of a combination of cells and a hydrogel, a supporting substance that gives the cells a framework to develop and multiply. The foundational basis of bioprinting is cells, which determine the properties of the bioprinted tissue or organ. Currently available three-dimensional bioprinting technology can create relatively simple tissues including blood arteries, cartilage, and skin. The intricacy of their structures and the requirement for exact organisation of several cell types make the development of more complex organs like the liver and heart still extremely difficult.

Because of the intricacies of their cell architectures, medical research has always faced difficulties in the tissue regeneration of hollow organs. The current state of the research underlying tissue engineering and 3D bioprinting will be covered in this brief review. This review will also cover the current legal challenges and prospects, as well as the application of these complicated 3D-printed organs.

Fig:1

[Source:https://pmc.ncbi.nlm.nih.gov/articles/PMC9088731/#Sec1]

 History:

Charles W. Hull coined the phrase “3D printing” in 1986. Using a thin layer of materials that were successively printed layer by layer with UV light to create a three-dimensional structure, he called the process “stereolithography.”^[2] In 1988, Klebe used a regular Hewlett-Packard (HP) inkjet printer to deposit cells utilizing cytoscribing technology, marking the first demonstration of bioprinting.^[3] Forgacs and co-workers concluded in 1996 that apparent tissue surface tension served as a quantitative indicator of tissue cohesion and was the macroscopic expression of molecular adhesion between cells.^[4]Odde and Renn used laser-assisted bioprinting for the first time in 1999 to create complexly shaped analogues by depositing living cells.^[5]Human cells were seeded onto a scaffold that was directly printed in the shape of a bladder in 2001.^[6]Landers et al. described the first extrusion-based bioprinting method in 2002; it was subsequently marketed as “3D-Bioplotter.”^[7] In 2003, Wilson and Boland modified an HP ordinary inkjet printer to create the first inkjet bioprinter.^[8]3D tissue made entirely of cells without a framework was created that same year. In 2006, living cells were deposited using electrohydrodynamic jetting.^[9]Norotte et al. used bioprinting to create scaffold-free vascular tissue in 2009.^[10] The following years saw the release of numerous novel bioprinting items, including tissue integration with the circulatory system in 2014, articular cartilage and artificial liver in 2012, and so on.^[11]

Fig:2

[Source:  https://www.sciencedirect.com/science/article/pii/S1818087619311869#sec0002]

Later, biomaterials were added to this technique to create 3D-printed cell scaffolds. The most recent tissue regeneration 3D printing method utilizing cellular biology, dubbed “FRESH” or “Freeform Reversible Embedding of Suspended Hydrogels,” was developed by Professor Adam Feinberg and his colleagues at Carnegie Mellon University and enabled the subsequent breakthrough. By printing soft biomaterials inside a gelatin bag, this method was able to get beyond the limitations of soft, low viscosity bio-inks and print more intricate organ systems like the heart and lungs.^[12] Researchers are creating functional 3D-printed human organ structures using a variety of bioprinting methods and techniques.

Fig:3

[Source: https://pmc.ncbi.nlm.nih.gov/articles/PMC10261138/figure/fig4/]

Significant turning points in the development of bioprinting:-^[13]

• Cell growth on a 3D prefabricated structure -1998
• Techniques based on syringe-based nano-depositions -2000.
• Use of hydrogels to fuse cells to create organs was first proposed -2003
• Inception of the phrase “bioprinting” -2004
• Cell encapsulation in hydrogels -2006
• Drug binding and bioprinting in -2009
• Bioprinting of cardiac tissue -2012
• Coil-based human ear bioprinting -2013
• Using HeLa cells for tumor bioprinting research -2014
• Using encapsulated primary neurons,brain-like structures were bioprinted -2015
• Creation of an integrated tissue and organ bioprinter -2016
• Bioprinting of ovarian and thyroid tissue of mice -2017
• Advent of 4D bioprinting, which will allow bioprinted components to transform as per the environment -2020
• Indian Institute of Science (IISc) in Bengaluru opened the first 3D bioprinting Centre of Excellence in partnership with Swedish bioprinters CELLINK -2022

Applications of 3D – Bioprinting:

 Developments in bioprinting are being driven by a number of factors, including the medical needs of ageing populations; the growing unmet demand for organ donors; the trend towards non-animal testing of medicines using 3D cell culture platforms; clinical needs in wound care and joint repair, and replacement operations. The following are some of the different clinical applications:^[14]

• Tissue modelling for medication discovery and development

Antibiotics, antifungals, and antivirals have long been used to treat infectious disorders when the host’s immune system is unable to combat the infection on its own. Consequently, the key to the successful design and development of effective medications is an understanding of the host’s reaction to pathogen invasion as well as the various interactions that take place between the host and the pathogen. As a result, 3D models may be a superior platform for developing medications and vaccines than 2D culture systems since they more accurately replicate the microenvironment that exists in the organism.^[15]

• Toxicity testing for drugs

In addition to improving the product model, 3D printing and high-throughput processes can shorten the time to market, lower production costs, and shorten manufacturing times. Using 3D-bioprinted tissue models for high-throughput drug testing helps achieve this. By replicating the in vitro reaction to drug administration, these 3D-bioprinted tissues provide the most precise possible replication of the target organ on which the medications will act, facilitating a quicker evaluation of the outcomes.^[16]

• Tissue engineering for prosthetic medical devices and regenerative medicine

The use of bioprinting, which creates transplants from the patient’s own cells and eliminates the need for a donor or the use of various immunosuppressants that may cause adverse side effects, is increasingly being regarded as a potential solution to this issue. Three primary methods are biomimicry, autonomous self-assembly, and microtissue building blocks are taken into consideration while creating tissues via bioprinting. A thorough understanding of the structure and interactions of the various tissues or organs that need to be produced is necessary in order to replicate them. Information about the 3D structure and its operation at the cellular, tissue, organ, and organism levels is provided by medical imaging technologies.^[17]

• Organ donation

The main strategy for treating organ failure has been organ donation, in which donors give their organs for transplantation in an effort to save lives. However, there is a worldwide shortage of organs because demand is significantly higher than supply. This problem may be resolved by 3D bioprinting, a cutting-edge tissue engineering technique that makes it possible to create customised, functional organs in a lab.

• Tumor Studies

3D-engineered tissue printing can be very beneficial in the fight against cancer, just like pharmacokinetic research. Current methods for researching various tumours may not be as effective as they may be because, during in vitro investigations, cells may fall dormant or even get new mutations while growing in the lab. Because 3D bioprinting technology allows for the creation of tumour models that replicate the conditions that cells in tumours are subjected to such as hydrostatic pressure, shear stress, compressive stress, and forces it is the best choice for the development of antitumor therapies. These factors are crucial for the control of tissue and cellular behaviour.^[18]

Fig:- 4

[Source: https://pmc.ncbi.nlm.nih.gov/articles/PMC9088731/figure/Fig3/]

It has been suggested that 3D printing can help with a number of aspects of healthcare, such as surgical planning, personalised medication, and diagnostics (using medical imagery to create models that aid in visualisation). Although they are probably farther in the future than other 3D printing applications, bioprinting techniques have the potential to upend existing organ and tissue donation systems. Nowadays, 3D printing is being researched or used in a number of therapeutic settings. Therefore, 3D printing may have an impact on a variety of health issues.

Challenges in 3D Bioprinting Technology:

 The following are the challenges faced by 3D Bioprinting Technology:-^[19]

 Layer by layer, components are produced throughout the 3D printing process. The part structure may disintegrate when the layers split under stress.

• Since the majority of printable 3D materials cannot be recycled, 3D printing is not adaptable enough to work with most raw materials.

• In order to manufacture huge volumes, 3D printing uses a lot of energy. As a result, it works best for production in small quantities.

• 3D printers release highly volatile chemical compounds that are poisonous and carcinogenic, and they can lead to major health issues like nausea, throat discomfort, and organ damage.

• The cost of 3D printing supplies and equipment is higher than that of conventional equipment.

• Numerous 3D-printed items require post-processing, which varies depending on a number of variables such the part’s size and the intended use of the final product.

• In addition to encouraging the creation of microstructures, the proliferation of biomaterials and bioprinting technologies can also result in a massive workload for researchers who are examining theoptimization of biomaterials and process parameters as well as the assessment of the effects they have during the printing process. As a result, the scientific research equations or mathematical models are inefficient, as previously stated.

• The majority of bioprinted tissues still lack certain functional components, such as the nervous system, lymphatics, vascular, and several supporting cell types, while being geometrically complicated on a macroscale.

• One ensuing query is whether the bioprinter is regarded as a medical device in and of itself or merely as a tool used in manufacturing of medical products.

Fig:5

[Sourcehttps://www.frontiersin.org/files/Articles/589171/fmech-06-589171-HTML/image_m/fmech-06-589171-g009.jp]

Regulations Governing 3D Bioprinting in India:

 Medical regulations in India fall into twelve categories that cover a range of topics, including licenses and certifications, periodic reports and returns, and more.

Given that the primary applications of 3D printing will be in the manufacturing of organs, tools, and, lastly, pharmaceuticals, the section that follows addresses Indian laws governing these fields.

 

1. Drugs and Cosmetics Act, 1940

The objective of the Drug and cosmetic act 1940 and Rules 1945 (Amendment, 2016) is the Regulation of new drug/cosmetics, to assist its manufacture, import and marketing in India with high standards and assured safety for human health care application through licensing. It also regulates Ayurvedic, siddha and Unani drug manufacture and import.

Section 3(b) of the Act defines the term [“Drug” as –

(i) all medicines for internal or external use of human beings or animals and all substances intended to be used for or in the diagnosis, treatment, mitigation or prevention of any disease or disorder in human beings or animals, including preparations applied on human body for the purpose of repelling insects like mosquitoes;

         (ii) such substances (other than food) intended to affect the structure or any       function of human body or intended to be used for the destruction of 6 (vermin) or insects which cause disease in human beings or animals, as may be specified from time to time by the Central Government by notification in the Official Gazette;] ^[20]

[(iii) all substances intended for use as components of a drug including empty gelatin capsules; and

        (iv) such devices intended for internal or external use in the diagnosis, treatment, mitigation or prevention of disease or disorder in human beings or animals, as may be specified from time to time by the Central Government by notification in the Official Gazette, after consultation with the Board ;]

 

When 3D bioprinted tissues and organs are developed for medical uses like organ transplantation, wound healing, or regenerative therapies, they can fall under this broad description.Under this clause, bioinks with cellular components that are intended for particular medicinal uses may also be governed as medications.

Section 10 of the Act regulates the import of drugs into India to ensure their safety, quality and efficacy. It prohibits the import of Substandard or Misbranded Drugs, Adulterated Drugs, Drugs which are prohibited in India, Drugs without Proper Labelling and, Unsafe Drugs.[It states that:

From such date1 as may be fixed by the Central Government by notification in the Official Gazette in this behalf, no person shall import—

 

• any drug or cosmetic which is not of standard quality;

 

• any misbranded drug or misbranded or spurious cosmetic;(bb)any adulterated or spurious drug;

 

• any drug or cosmetic for the import of which a licence is prescribed, otherwise than under, and in accordance with, such licence;

 

• any patent or proprietary medicine, unless there is displayed in the prescribed manner on the label or container thereof the true formula or list of active ingredients contained in it together with the quantities thereof;

 

• any drug which by means of any statement, design or device accompanying it or by any other means, purports or claims to cure or mitigate any such disease or ailment, or to have any such other effect, as may be prescribed;

 

(ee)any cosmetic containing any ingredient which may render it unsafe or harmful or use under the directions indicated or recommended;

• any drug or cosmetic the import of which is prohibited by rule made under this Chapter:

Provided that nothing in this section shall apply to the import, subject to prescribed conditions, of small quantities of any drug for the purpose of examination, test or analysis or for personal use:

Provided further that the Central Government may, after consultation with the Board, by notification in the Official Gazette, permit, subject to any conditions specified in the notification, the import of any drug or class of drugs not being of standard quality.][21]

This clause can be used in relation to the importation of bioinks, biomaterials, and 3D bioprinters, all of which are necessary for creating bioprinted tissues and organs. It requires that these imported supplies and machinery adhere to legal specifications.

Rule 122 A of the Drugs and Cosmetics Rules, 1945, deals with the procedure for obtaining approval from the Central Drugs Standard Control Organization (CDSCO) for the import and manufacture of new drugs in India.^[22]

Rule 122 DA under the Drugs and Cosmetics Rules, 1945, deals with the requirements for conducting clinical trials of new drugs in India, as outlined framed under the Drugs and Cosmetics Act, 1940.^[23]

2. Medical Devices Rules, 2017

The Medical Devices Rules, 2017 (also known as “the Rules”) will apply to and be put into effect for medical devices manufactured with 3D printers starting in 2018. A medical device, as defined by the Rules, is:

“(A) substances used for in vitro diagnosis and surgical dressings, surgical bandages, surgical staples, surgical sutures, ligatures, blood and blood component collection bag with or without anticoagulant covered under sub clause (i), (B) substances including mechanical contraceptives (condoms, intrauterine devices, tubal rings), disinfectants and insecticides notified in the Official Gazette under sub-clause (ii), (C) devices notified from time to time under sub-clause (iv), of clause (b) of section 3 of the Act; Explanation: For the purpose of these rules, substances used for in vitro diagnosis shall be referred as in vitro diagnostic medical device.”^[24]

As a result, these rules categorise gadgets into several classes and concentrate on their safety and quality control.According to §3 of the Drugs and Cosmetics Act of 1940, the Central Government may add 62 3D printed devices in these regulations by publishing a notice in the Official Gazette. It is expected that the Central Government will release the relevant announcement under this to incorporate additive manufacturing within its purview once it becomes widely used.

3. Indian Patents Act, 1970

Patent law concerns pertaining to 3D printers can be divided into two categories: first, issues pertaining to the replication of patented goods, and second, obtaining patents for 3D printed goods or procedures. According to Section 48 of the Patents Act, grants the patent holder exclusive rights to make, use, sell, or distribute the patented invention within the territory of India.^[25]

 

Section 2(1)(j) of the Indian Patents Act, 1970 defines the term “invention” as

“a new product or process involving an inventive step and capable of industrial application.”^[26]

In order to promote innovation and avoid the monopolisation of natural biological materials, the section makes sure that only really novel and useful components of 3D bioprinting are eligible for patenting.

Section 3 of the Indian Patents Act, 1970 provides a list of inventions and subject matter that are excluded from patentability. It provides for certain exceptions in the sub clauses of this section.

Section 3(b): Morality exclusions

It prohibits the patenting of inventions that are against morals or public order, or that harm the environment, human, animal, or plant life, or both. It states that: “an invention the primary or intended use or commercial exploitation of which could be contrary to public order or morality or which causes serious prejudice to human, animal or plant life or health or to the environment”.[27]

Section 3(c): Bars any invention that is of natural origin.

It prohibits the patentability of naturally occurring chemicals, and the guidelines issued by the Indian Patent Office states that the substances shouldn’t be “directly isolated from nature.[28]

Section 3(j) states that “plants and animals in whole or any part thereof other than micro organisms but including seeds, varieties and species and essentially biological processes for production or propagation of plants and animals;”^[29]

This clause makes it difficult to patent bioprinted tissues or organs because the finished result, such an organ or tissue, cannot be patented since it imitates naturally occurring biological things, which are excluded by Section 3(j).

4. The Transplantation of Human Organs and Tissues Act, (THOTA)1994

Its objective is to provide a legal structure that promotes moral and transparent organ transplantation while protecting the rights of both donors and recipients. The Act bans the trade in organs, creates regulations for the approval and control of organ donation, and imposes penalties for its violations.

Section 2(p) of the Act defines the term “transplantation” as “grafting of any human organ from any living person or deceased person to some other living person for therapeutic purposes.”^[30]

An illustration of the same can be seen in Section 3 of the Act which prescribes the requirement for taking the authorisation of a living donor before removing any organs for transplantation.^[31]This presupposes that organs will be obtained from a deceased or living individual, ruling out the potential of producing organs through additive manufacturing.

Another illustration of it is Section 10 of the Act, which discusses the registration of hospitals which engage in “removal, storage or transplanting of any human organ or tissue or both”.^[32]

This clause also exempts hospitals that bioprint organs from registration requirements, meaning they are not subject to regulation. Bioprinting must obviously be incorporated into the Act, which is based on this concept of transplantation.

The COVID-19 epidemic will undoubtedly increase 3D Bioprinting use. With 3D bioprinting, any medication, remedy, preventive vaccine, or relief materials may be swiftly put into mass production. Now, it is a good opportunity for India to think about a comprehensive 3D bioprinting policy or even the rules that should apply to 3D printing. It might serve as the template for international standards. The all-encompassing policy ought to cover: 

a)purchase of 3D printers and scanners;

1. b) manufacturing processes using 3D printers;
2. c) quality of input material and final product;

d)classification of computer-aided design (CAD)/Digital file, and whether it is a good or      a service, which will determine its sale, distribution and taxation;

1. e) product sale and distribution, including intermediary liability;
2. f) Governing body and single window clearance for businesses;
3. g) standardisation;
4. The Bureau of Indian Standards (BIS), India’s national standards organisation, should specifically include 3D bioprinting in its mandatory registration program for printers. [33]

1. In addition to standards for the finished result, BIS ought to think about creating distinct standards for the input units used in 3D bioprinting.

• The Central Drugs Standard Control Organisation, which establishes guidelines for medications produced in India and imported into the country, ought to establish guidelines for medications and medical equipment that are 3D printed.

5. Biotechnology Regulatory Authority of India Act, 2009

The Biotechnology Regulatory Authority of India (BRAI) Bill, 2009, proposed but not enacted, aimed to control the use of genetically modified organisms (GMOs) in accordance with the parliamentary bill’s requirements introduced in 2013 by the Ministry of Science and Technology. The objective of the Bill is to “promote” the safe use of modern biotechnology. Since India had ratified the Cartagena Protocol, which requires the establishment of a Regulatory Body, BRAI.

The goal of BRAI is to offer a one-stop shop for the scientific evaluation of the risks associated with all biotech products, including those used in the industrial, agricultural, health, and environmental sectors. Along with ensuring environmental and human health safety, it would also assist India in keeping up with regulatory requirements in light of the biotechnology industry’s rapid advancement.

The Bill sets up an independent authority, the Biotechnology Regulatory Authority of India, to regulate organisms and products of modern biotechnology. BRAI will regulate the research, transport, import, containment, environmental release, manufacture, and use of biotechnology products.^[34]

One of the Demerits of the bill was that the Bill does not specify any liability for damage caused by a product of biotechnology.Therefore, it will remain open to the courts to determine liability arising out of any adverse impact of modern biotechnology.

 Conclusion:

 3D bioprinting holds transformative potential in advancing regenerative medicine, offering innovative solutions to pressing healthcare challenges such as organ shortages and personalized treatment. The review underscores the significant strides made in bioprinting technology, from early stereolithography to advanced FRESH techniques. India’s involvement, reflected through research initiatives and regulatory efforts, demonstrates its readiness to harness this technology’s potential. Despite remarkable progress, challenges such as structural limitations, environmental impact, and ethical dilemmas remain. Regulatory frameworks like the Drugs and Cosmetics Act and THOTA provide partial guidance but require adaptation to include bioprinting-specific innovations. Addressing these gaps will demand interdisciplinary collaboration between scientists, policymakers, and industry stakeholders. Future efforts should focus on overcoming technical and legal challenges, fostering innovation, and ensuring ethical compliance. By doing so, 3D bioprinting can revolutionize tissue engineering and organ transplantation, ultimately improving lives and shaping the future of medicine.

In recent years, 3D bioprintingwhich blends biomanufacturing with extra manufacturinghas made tremendous strides, from proof-of-concept prints to intricate multi-material tissue-like structures. However, there are still some restrictions pertaining to bioprinting methods, cellular sources, choosing biomaterials, etc., that must be addressed in subsequent studies.

The evolution, process, classification, common bioprinters, bioinks, andabove allrepresentative studies of 3D bioprinting are covered in this text, along with the benefits and drawbacks of various strategies. Although there are still a number of obstacles to overcome before bioprinting may be fully used in therapeutic settings, several extremely encouraging advancements have been made in recent years.The use of 3D bioprinting technologies to construct skin, bone, cartilage, vasculature, organoids, etc., demonstrates a wide range of potential applications, and the integration of bioprinting and pharmaceutical sciences indicates a new avenue for future study. 3D bioprinting is expected to develop further and move from structural resemblance to functional realisation.

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^[2]Hull CW. Inventor, “Apparatus for production of three-dimensional objects by stereolithography”, Google Patents; 1986. US Patent 4,575,330.    

^[3]Robert J. Klebe,Cytoscribing: A method for micropositioning cells and the construction of two- and three-dimensional synthetic tissues, 179(2) ECR, 362-73 (1988).

^[4]R.A. Foty& C.M. Pfleger, Surface tensions of embryonic tissues predict their mutual envelopment behavior, 122(5) Development, 1611-1620 (1996).

^[5]D.J. Odde& M.J. Renn, Laser-guided direct writing for applications in biotechnology, 17(10) Trends in Biotechnology, 385-389 (1999).  

^[6] K. Karzyński& K. Kosowska, Use of 3D bioprinting in biomedical engineering for clinical application, 34(1) Medical Studies, 93-97 (2018). 

^[7]R. Landers & U. Hubner, Rapid prototyping of scaffolds derived from thermoreversible hydrogels and tailored for applications in tissue engineering, 23(23) Biomaterials, 4437-4447 (2002).    

^[8] W.C. Wilson Jr. & T. Boland, Cell and organ printing 1: protein and cell printers, 272(2) Anat Rec Part A, 491-496 (2003).

^[9]S.N. Jayasinghe & A.N. Qureshi, Electrohydrodynamic jet processing: an advanced electric-field-driven jetting phenomenon for processing living cells, 2(2) Small, (2006).                                                                                                

^[10]C. Norotte& F.S. Marga, Scaffold-free vascular tissue engineering using bioprinting, 30(30) Biomaterials, (2009).                                                                                                              

^[11]B. Duan, State-of-the-art review of 3D bioprinting for cardiovascular tissue engineering, 45(1) ABE, 195-209 (2017).

^[12]Mirdamadi E &Tashman JW, FRESH 3D bioprinting a full-size model of the human heart, 6(11) ACS Biomater Sci Eng., 6453–6459 (2020).

^[13]Mendoza-Cerezo L & Jesús MR, Evolution of bioprinting and current applications, 9(4) Int J Bioprint, 742 (2023).

^[14]Panja N, Maji S, Choudhuri S, 3D Bioprinting of Human Hollow Organs, 23(5) AAPS PharmSciTech, 139(2022).

^[15]Joseph JS &Malindisa ST, Two-dimensional (2D) and three-dimensional (3D) cell culturing in drug discovery, 2 Cell Culture, 1-22 (2018).

^[16]Vaidya M, Startups tout commercially 3D-printed tissue for drug screening, 21(1) NM, 2 (2015).

^[17]Murphy Sv& Atala A, 3D bioprinting of tissues and organs, 32(8) Nat Biotechnol, 773–785 (2014).

^[18]Butcher DT & Alliston T, A tense situation: Forcing tumour progression, 9(2) Nat Rev Cancer, 108–122 (2009).

^[19]Panja N, Maji S, Choudhuri S, 3D Bioprinting of Human Hollow Organs, 23(5) AAPS PharmSciTech, 139(2022).

^[20]The Drugs and Cosmetics Act, 1940,§3(b).

[21]Drugs and Cosmetics Rules, 1945, §10.

^[22]Drugs and Cosmetics Rules, 1945, Rule 122 A.

^[23]Drugs and Cosmetics Rules, 1945, Rule 122 DA.

^[24]Medical Devices Rules, 2017, Rule 2.

^[25] The Indian Patents Act, 1970, §48.

^[26]The Indian Patents Act, 1970, §2(1)(j).

[27]The Indian Patents Act, 1970, §3(b).

[28]The Indian Patents Act, 1970, §3(c).

^[29]The Indian Patents Act, 1970, §3(j).

^[30]The Transplantation of Human Organs and Tissues Act, 1994, §2(p).

^[31]The Transplantation of Human Organs and Tissues Act, 1994, §3.

^[32]The Transplantation of Human Organs and Tissues Act, 1994, §10.

[33]Bureau of Indian Standards, The National Standards Body of India, FMCS – Certification https://bis.gov.in/index.php/product-certification/products-under-compulsory-certification/scheme-ii-registration-scheme/

^[34]The Biotechnology Regulatory Authority of India Bill, 2013, Bill No.57 of 2013, §4 (August 18, 2014).