Biomimetic 3D scaffolds for regenerative medicine

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Biomimetic 3D scaffolds for regenerative medicine: Opportunities and challenges

By

Name: Sanjida Hussain ID: 18146101

A thesis submitted to the School of Pharmacy in partial fulfillment of the requirements for the degree of Bachelor of Pharmacy

School of Pharmacy Brac University

June 2022

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Declaration

It is hereby announced that

1. The thesis submitted is my own unique work while accomplishing degree at Brac University.

2. The thesis does not contain material recently published or composed by an outsider with the exception of where this is properly cited to through full and exact referring to.

3. The thesis does not contain material which has been acknowledged, submitted, for some other degree at a university or other institution.

4. I have confessed all primary sources of help.

Student’s Full Name & Signature:

Sanjida Hussain 18146101

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Approval

The thesis titled “Biomimetic 3D scaffolds for regenerative medicine: Opportunities and challenges” submitted by Sanjida Hussain (18146101) of Spring 2018, has been accepted as satisfactory in partial fulfillment of the requirement for the degree of Bachelor of Pharmacy.

Examining Committee:

Supervisor:

(Member)

_______________________________

Dr. Md. Abul Kalam Azad Assistant Professor, School of Pharmacy

Brac University

Program Coordinator:

(Member)

_______________________________

Namara Mariam Chowdhury Lecturer, School of Pharmacy

Brac University

Deputy Chair (Member)

Departmental Head:

(Chair)

_______________________________

Dr. Hasina Yasmin Professor, School of Pharmacy Brac University

_______________________________

Dr. Eva Rahman Kabir

Professor and Dean, School of Pharmacy Brac University

07.06.2022

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Ethics Statement

This study does not involve any human and animal trial.

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Abstract

The field of tissue engineering and regenerative medication (TERM) has flourished during the recent years for the recovery and recreation of pathologically changed tissues. The improvement of biomimetic permeable scaffold is fundamental for effective tissue designing which can be created from different traditional and rapid prototyping methods, based upon the sort of materials or kind of pore structures required. The purpose is to deliver tissue like materials which can ultimately perform like the original tissues. The most up to date techniques based on the 3D way of behaving and multi-cell co operations of local tissues for additional utilization for in vitro model handling are too illustrated. Finished and progressing preclinical examination for TE applications utilizing scaffolds, difficulties and upcoming possibilities of examination in this area are additionally introduced. This survey also emphasis where the TERM field might be going and how to screen such a wide consistently extending field.

Keywords: Scaffold, regenerative medicine, tissue engineering, polymers, biomimetic, extracellular matrix, fabrication.

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Dedication

I dedicated this paper to my grandmother parents and my project supervisor, Dr. Md. Abul Kalam Azad.

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Acknowledgements

Firstly, I would want to express gratitude toward Almighty for his limitless favor in endeavor to engage me with the strength and readiness to fulfil this project task.

I would want to convey my earnest thanks to my project and academic supervisor, Dr. Md. Abul Kalam Azad (Assistant Professor, School of Pharmacy, Brac University) for his significant direction and energy all through this project. He was really a wellspring of guidance and help me throughout my review and project composing. I'm unbelievably obliged for his valuable criticism and ideas all through my analysis that helped me a great deal to finish this project work fluently.

I would likewise want to offer my respectful thanks to Dr. Eva Rahman Kabir (Professor and Chairperson, School of Pharmacy, Brac University) for her devotion, commitment and direction towards the student as well as the department.

At last, I would want to offer my gratefulness to my parents who continue to motivate me to go past my cutoff points. I could not have possibly come this far without their persistent prayers and unlimited love. I would likewise want to thank every individuals who, at whatever point

required, have assisted me with their most extreme capacities.

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List of Figures

Figure 1: Freeze drying method (Ghalia & Dahman, 2016). ... 11

Figure 2: Electro spinning of scaffold fabrication (Lu et al., 2013). ... 13

Figure 3: Schematic representation of gas foaming technique (Koyyada & Orsu, 2021)... 14

Figure 4: The schematic diagram of solvent-casting particulate-leaching techniques (H. Liu & Webster, 2007). ... 17

Figure 5: 3D scaffold fabrication by using 3D bio printer (M. Du et al., 2015). ... 21

Figure 6: Steriolithography fabrication for polymeric scaffold (Mondschein et al., 2017)... 23

Figure 7: A schematic diagram of the FDM process (Zein et al., 2002). ... 26

Figure 8: Selective laser sintering process (Y. Du et al., 2017). ... 27

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List of Tables

Table 1: Comparative analysis of cell/tissue behavior under 2D and 3D culture condition (Geckil et al., 2010). ... 7

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List of Acronyms

ECM Extracellular matrix TE Tissue engineering

PHBV Poly (hydroxybutyrate-co-valerate) PLA Poly lactic acid

PCL Poly ε-caprolactone BMSC Bone marrow stem cell BCP Biphasic calcium phosphate TIPS Thermal induced phase separation BMPs Bone morphogenetic proteins SFF Solid free form

HMWHA High molecular weight hyaluronic acid

BMMSCs Bone Marrow-Derived Mesenchymal Stem Cells bFGF Basic fibroblast growth Factor

RWSF Regenerate wild Antheraeapernyi silk fibroin

EDCNHS N-ethyl-N'-[3-dimethylaminopropyl] carbodiimide/N-hydroxy succinimide

RP Rapid prototyping 3DP 3D printing,

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xiv FDM Fused deposition model

SLS Selective laser sintering DIW Direct ink write

MRI Magnetic resonance imaging CT Computed tomograph

PDMS Polydimethylsiloxane

HUVEC Human umbilical vein endothelial cells HLFs Human lung fibroblasts

Me-HA Methacrylated hyaluronic acid Me-Gel Methacrylated gelatin

PEG-DA Poly (ethylene glycol) - diacrylate HAVIC Human aortic valvular interstitial cell HMVEC Human micro vascular endothelial cell PRP Platelet rich plasma

TGF-β3 Transforming growth factor-𝛽 GAG Glycosaminoglycan

PEGT Poly (ethylene glycol) terephthalate PBT Poly (butylene terephthalate

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xv CS Chondroitin sulfate

PU Polyurethane HA Hyaluronic acid

MSCs Mesenchymal stem cells GT Gelatin

PCL Polycaprolactone

bACs Bovine articular chondrocytes MSNPs Mesoporous silica nanoparticles

dBECM Decellularized brain extracellular matrix eEPC Embryonic endothelial progenitor cells RPE Retinal pigment epithelium

BM Bruch's matrix

TMP Tympanic membrane perforations PDGF Platelet derived growth factors DBM Demineralized Bone Matrix

DFDBA Demineralized freeze-dried bone allografts REPs Regenerative endodontic procedures UC-MSCs Umbilical cord mesenchymal stem cells

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xvi FGF Fibroblast growth factor

VEGF Vascular endothelial growth factor OPCs Oligodendrocyte progenitor cells FDA Food and Drug Administration ISO International Standards Organization

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Chapter 1 Introduction 1.1 3D Scaffolds

3D scaffolds work as transitory substrates for supporting and directing tissue arrangement in different in vitro and in vivo tissue recovery settings. Research in this space makes frameworks to develop and shape tissues in three dimension. Scaffolds utilized in tissue designing copy the regular extracellular matrix (ECM) and aid in cell grip, movement, multiplication, tissue production, and 3D association. The framework should be totally biodegradable so the platform breaks down altogether and nontoxically as it is supplanted by tissue (Richbourg et al., 2019).

Implantable 3D frameworks are utilized for rebuilding and recreation of various physical imperfections of significant organs and working tissues. It gives a layout to the remaking of imperfections while advancing cell connection, expansion, extracellular matrix production, rebuilding of vessels, nerves, muscles, bones, and so forth. The alloplastic (synthetic material used as a choice of tissue graft)bioactive frameworks guarantee the mechanical strength of the tissue as well as act as a conveyance medium for bioactive particles (cytokines, restrainers, drugs, anti- toxins so on) and layouts for connecting hereditarily transduced cells setting up new places for tissue recovery and morphogenesis. At the same time, 3D platforms able to act like tissue mould duplicating primary complication of alive tissues. So, along with the biomaterial the large scale, miniature, and nano-engineering of the frameworks are of prime significance. Now-a-days, much exploration are done to foster different kind of modern biomaterials and compounds with upgraded endurance, multiplication and printability. Because of extra alteration, bio mimicry approach execute different cell parts (hormones, development factors, ECM proteins, and so on) to imitate

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2 living tissue is utilized to upgrade cell pointing or ECM production. Besides, biomaterial frameworks are utilized for conveying remedial agents like proteins, development factors, drugs (Nikolova & Chavali, 2019).Scaffolds are produced using natural and artificial biomaterials.

Natural framework biomaterials incorporate collagen, chitosan, and decellularized tissues.

Engineered biomaterials incorporate polymers, for example, poly (L-lactic corrosive) (PLA), poly (L-lactic-co-glycolic corrosive) (PLGA), and poly (ε-caprolactone) (PCL). Mainly natural biomaterials are frequently utilized as frameworks but they experience the bad effects of batch variation. Engineered or artificial polymers are all the more effectively and reliably imitated utilizing manufacturing details. Also they are frequently hydrophobic and less precisely imitate the in vivo microenvironment of cells (Richbourg et al., 2019).For 3D scaffolds, porosity and pore structure show a significant part in cell entrance and mechanical powers. For bone tissue designing, frameworks have been planned with trabecular bone-like porosity or through trial to decide ideal porosity for cell improvement (Fernandez-Yague et al., 2015). To speed up the endogenous recovery process, particularly in broad or irreversible injuries, various systems are utilized for incorporation of progenitor cells (after in vitro extended) solely/ mixes with naïve or manufactured frameworks. Stem cells via various sources are typically utilized because of their capacity to keep up with homeostasis in solid tissues and separate when initiated under infection. Tissue-explicit undifferentiated cells can recover the tissue from which they are segregated. After the injury, a course of natural occasions like progenitor cells relocation, chemokine and growth variables discharge happens for fixing obliterated tissue. The cycles might be imitated, animated and constrained along mixing it up of bioactive moieties, for example, development factors, peptides, genes, antibodies, tranquilizes or ECM. These materials s are imprinted physically/chemically (by electrostatic powers, aquaphobic communications and H2 securities) to the scaffolds Thereby, the

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3 designed composite platforms imitate regular flagging and fix occasions and produce reasonable microenvironment for attachment, expansion, separation of stem cells that recover the tissue (Nikolova & Chavali, 2019)

1.2 Regenerative medicine

Restorative remedy is the method of substituting or retrieving human cells, tissues or organs to reestablish or build up ordinary outcome (Mcphail et al., 2020).For instance, a person with type 1 diabetes can't deliver insulin. All things considered, day by day insulin infusions are needed to hold glucose levels within proper limits. Regenerative medication looks to address this by recovering the islets of Langerhans, which permit the person to make insulin. This would mean no more insulin infusions and a getting back to ordinary sugar digestion. In early days it was seen in regenerative medication wanted to design tissues also body parts exteriorly means ex vivo tissue designing. Moreover, developing adult tissues and body part exteriorly has identified most difficult also challenging so developing methodologies for business are restricted. But Restorative drug has extended techniques for vivo recovery of tissues (Mcphail et al., 2020). Regenerative medication tries to supplant tissue or organs that have been damaged by illness, injury, or intrinsic issues, versus the present clinical methods that centers basically healing the side effects. This methodologies utilize human being as biosensor to increasing the inborn capacity to recover and cure. Platforms, cells and bioactive variables are the three elements which was utilized in this strategies. Cells serve a focal job in regenerative medication since Relocated cells are utilized in recovering newly tissues, adjust insusceptible reaction alter local cell conduct via paracrine signaling. Bioactive elements utilized to alter cell practices furthermore incorporate development factors, cytokines, chemicals, also other molecules. Also these elements are utilized in vitro to monitor cell conduct too also in vivo as therapeutics to adjust restorative method. Platforms give

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4 a 3 layered construction for tissue recovery. They are planned at miniature size to monitor cell practices like separation and migration. Platforms are utilized acellularly or along with bioactive elements and cells. An expansive scope of pathway is utilized to produce platforms including 3 layered printing, electro spinning, redoing hydrogels and decellularizing tissue (Mcphail et al., 2020).As an encouraging area of current era, recovering drug is trying to reform structure along with working ability of wound tissues and body part. The prime goal of this approach is to search solution for healing earlier lethal injury and infections

1.3 Tissue engineering of tissue scaffold

Tissue designing is a methodology where organically viable frameworks are embedded in the body at the site where new tissue is to be shaped. When the scaffold is in the geometric shape of the tissue that needs to be formed and attracts the cell then a new tissue with desired shape will produced. Tissue designing spotlights on the making of counterfeit tissues and organs for relocate by using cells, biomaterials, biochemical variables, and actual elements to keep up with, improve or supplant organic tissues. In a tissue designing model, cell are gathered from a creature and filled in cell culture media, frequently with strong scaffold builds giving construction and backing in vitro (Nardo et al., 2017).Tissue engineering (TE) has significantly progressed over the most recent 10 years, giving the potential for recovering pretty much every tissue and organ of the human body. It is a multidisciplinary field concentrates on the substitution of harmed or missing body tissues with organically viable substitutes. TE is focused on the designing of utilitarian tissues and organs by refined living cells on appropriate permeable substrates in view of biocompatible biomaterials or by the utilization of appropriate biomaterial substrates ready to enlist endogenous cells and guide their conduct, once implanted .The aim of the biomaterial substrate is to imitate and additionally support existing natural cycles, and capacity as a counterfeit extracellular lattice

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5 (ECM).Biomaterial plan and determination are difficult in the advancement of designed tissues because it involves the following functions

(1) Give a three-layered (3D) structure where cells can create a utilitarian tissue with fitting design and capacity

(2) Allow adequate vehicle of gases, supplements, and suitable bioactive particles (cell adhesion peptides, growth factors) to permit cell endurance, multiplication, also differentiation (3) Improve cell biomaterial collaborations, cell grip, and ECM installation

(4) Give impermanent mechanical help adequate to endure in vivo powers applied by the encompassing tissue, while keeping a predefined structure for tissue development.

Biomaterials for designing tissues and organs can be manufactured or biologic, degradable or no degradable, according to the particular applications. Naturally determined materials (collagen), engineered biodegradable (polylactic corrosive (PLA) and manufactured no biodegradable (polytetrafluoroethylene, polyethylene (PE) terephthalate) polymers are the fundamental kinds of polymers utilized as biomaterials. Natural polymers have the better biologic acknowledgment and collaborations with the cells, while manufactured polymers are created under controlled conditions for an enormous scale with assumed and renewable mechanical and actual features like rigidity, versatile modulus, corrosion rate, and microstructure. Synthetic polymers are frequently less expensive than natural ones, can be delivered in huge amounts, and have a more extended timeframe of usability. In addition, numerous financially accessible engineered polymers show physicochemical and mechanical features practically identical to those of organic tissues (Nardo et al., 2017).

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1.4 Problems with 2D ECM /scaffold

For more than a century, conventional 2D cell culture was utilized in drug discovery where cells are developed on flat dishes streamlined for cell connection and development. These days, 2D cell culture models are yet utilized to test cell drug reactions to drug applicants. There are some restrictions in this model though 2D cell culture is the most acknowledged model. The primary restriction is that the cells here developed as a monolayer on flat petri plates. This firm stage offer unnatural development energy and cell connections (Joseph et al., 2018).Cells and tissues are refined in vitro on 2D substrates do not copy cell development in vivo, and also unable to reveal some certain tissue-explicit genes and proteins at levels practically identical to those found in vivo.

For example, it has been found that cell-drug interaction in a 2D culture framework don't address the genuine working mechanism in vivo. Consequently, 2D culture is not fitting to be utilized in vitro drug testing models. This is because the cells and tissues in vivo are submerged inside a 3D organization comprising a complex extracellular climate with a profoundly permeable Nano topography, while a 2D culture framework is too easy to even consider mirroring the local environment (Geckil et al., 2010).In recent times many significant work by specialists are delivered to upgrades better in vitro cell culture models that take after in vivo conditions. Three- layered cell model are result of such innovation that better copy tissue physiology in multicellular organic entities. That’s why 3D cell model strategies are gaining quick growth in contribution of drug discovery.

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1.5 Superiority of biomimetic 3D scaffolds

Comparative analysis of cell/ tissue behavior under 2 D and 3 D culture condition.

Feature 2D 3D

Tissue specific architecture Poor Rich

Cell Morphology Expanded Contracted

Cell motility Fast Slow

Cell growth Directional All direction

Cell proliferation High Poor

Cell polarization Partially Full

Apoptosis Initiated Tissue like

Fluid perfusion 1 D 3 D

Metabolic rate High Poor

Cell adhesion Poor Strong

Signaling and diffusion Asymmetric Close to symmetric

Extracellular matrix remodeling Poor or absent Present

Table 1: Comparative analysis of cell/tissue behavior under 2D and 3D culture condition (Geckil et al., 2010).

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8 Three layered cell culture strategies have been utilized in various phases of drug discovery including infections displaying, target determination, approval, screening, target choice, strength profiling and harmfulness evaluation. 3D culture models act as the same way to the cells in vivo, and are accordingly utilized in the beginning phase of the drug discovery process, particularly in cytotoxicity tests like MTT, Flow Cytometry etc. The most efficient cell-based strategies with 3D scaffold are cell endurance, expansion, flagging and movement it was seen that cells act distinctively in 3D conditions contrasted with 2D ones. Many cancer treatment which seem satisfactory in 2D could not stand in clinical development. Three-layered scaffolds cell guarantee to overcome the barrier between conventional 2D cell culture furthermore in vivo creature models.

Studies have shown that cell reaction to medicate treatment in 3D cell culture are more like what happens in vivo contrasted with 2D cell culture. A variety of examinations show that cells refined in 3D models are more counteractive to anticancer medications than those in 2D models. For instance, the cell durability of ovarian disease cells in 3D model after paclitaxel treatment was diminished by 40%, while a similar treatment prompted 80% diminished cell durability in 2D (Joseph et al., 2018).

1.6 Objectives

• Study the development of effective therapies for regenerative diseases for which limited therapeutic options exist

• Analyze the fabrication techniques for building three dimensional biomimetic scaffolds.

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Chapter 2 Methodology

This review paper has been conducted based on recent and relevant research papers and articles from high impactful journals .A comprehensive search has been performed though peer reviewed journals, articles and official reports. To enrich the review paper, fundamental and additional information have been gathered from various books also profoundly significant articles and ongoing publications are searched based on the topic related keywords. Following search engines have been used to collect data for this paper- Research Gate, Google Scholar, PubMed, Science Direct, Elsevier etc. in which the major publications include – Nature, Journal of Medicine, and Science etc. In depth screening of the journals followed by narrowing down to the most recent and relevant ones was done to create an ideal review on the role of 3D bio mimetic scaffolds in regenerative medicine.

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Chapter 3

Fabrication techniques of scaffolds 3.1 Conventional techniques

A multiple number of manufacturing methods are accessible to make 3D frameworks by utilizing engineered along with nature derived biomaterials. These methods intend to make frameworks to imitate the physiological microenvironments. In every conventional innovations, at first scaffolds are fabricated then accompanied by cultivating of the cells in the framework (Bajaj et al., 2014).Firstly, frameworks were grown traditionally for medicine conveyance, however also utilized in 3D cell medium with regards to TE. The conventional strategies for scaffold manufacturing like freeze drying , electro spinning, gas foaming ,solvent casting and practical leaching and thermal induced phase separation are planned to characterize the shape and pore size of scaffold. However all these methods have pros and cons regarding the scaffold interior plan or availability of the void space (Eltom et al., 2019).

A brief description of these traditional methods are given below:

3.1.1 Freeze drying

This method is also known as lyophilization which utilize a manufactured polymer to be disintegrated in a proper solution that includes three significant stages: frozen the mixture at a reduced temperature (−70°C to −80°C), iced object is situated in a low pressurized room where ice is taken out by straight sublimation and greater part of the thawed aqua in the substance is taken out by solidification. Over the most recent twenty years, the freeze-drying strategy has been broadly researched for the manufacture of three-layered permeable frameworks for tissue engineering. For example, methacrylamide-adjusted gelatin-2-hydroxyethyl methacrylate

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11 permeable platforms have been created by the freeze-drying method. Adhesion and multiplication of human mesenchymal stem cells on both permeable and nonporous polysaccharide-based scaffolds have additionally been examined (Lu et al., 2013).This procedure was applied for a 3D platform manufacture utilizing chitosan nanoparticles. Also, the development of chitosan-alginate bio composites including fucoidan for a bone tissue by lyophilization was investigated. Moreover, their platform has promising properties on porosity furthermore water ingestion (Eltom et al., 2019).Another innovation is researchers incorporated HA nanoparticles into poly(hydroxybutyrate-co-valerate) (PHBV) polymer to fabricate osteoconductive composite scaffolds and they were found highly porous with interconnectivity(Sultana & Wang, 2008).

Figure 1: Freeze drying method (Ghalia & Dahman, 2016).

Advantage: Freeze-drying procedure is an appropriate technique for biomedical exercise since the utilization of aqua and ice chunks rather than a natural dissolvable during scaffold creation It has the ability of blocking elevated climate condition enable to diminish the function of incorporated

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12 organic variables. The hole proportion can be altered by controlled and modified chilling technique (Eltom et al., 2019).

Disadvantage: Despite the fact that numerous benefits, the extensive timescales, the development of little and unusual size pores and high energy utilization limits its application for tissue designing .This strategy additionally utilizes cytotoxic solvents for blending the polymer which can be deadly for the cells(Bajaj et al., 2014). Consequently, the manufactured platform should be washed more than once to eliminate the dissolvable and to limit cell demise To beat these problem, scientists proposed different frosting condition (−10°C to−70°C) and incorporation of additional strengthening move to improve pace development of ice chunks (Eltom et al., 2019).

3.1.2 Electrospinning

Electrospinning or electrostatic turning is an exceptionally appealing ectrohydrodynamic strategy for developing polymer arrangements or melts as nanofibrous non-woven frameworks (Aldana &

Abraham, 2017).It is also a popular method that is able to create strands via solution by utilizing power. This method is significant for creating nanofibrous frameworks in TE (Eltom et al., 2019).A standard framework requires four significant parts: metallic needle, needle force, elevated voltage force source and stable compiler. The electric field strength defeats the surface pressure of the drop and produces a charged fluid jet that is then lengthened and whipped ceaselessly by electrostatic aversion until it is saved on the grounded authority. The solvent vanishes in the process and the jet cements to shape a nonwoven fibrous layer (Roseti et al., 2017). Electro spinning can empower fiber creation utilizing engineered, natural and combining both of them.

Some normal manufactured polymers utilized for electro spinning the frameworks are poly lactic acid (PLA), poly ε-caprolactone (PCL) and so on. Although the inborn hydrophobicity of such engineered materials might make complication in cell connection but mixing these manufactured

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13 substance with native polymers like collagen or gelatin can further develop the ability of not being toxic to cell. For example ,PCL-gelatin (1:1) grid platforms are observed as more qualified for bone marrow stem cell (BMSC) entrance rather than authentic PCL nanofibres (Dutta et al., 2017).To get the advantages of engineered and native polymers, chitosan-build mix nanofibers were manufactured by electro spinning utilizing chitosan and manufactured compostable polymers like poly lactic acid (PLA) (Eltom et al., 2019).

Figure 2: Electro spinning of scaffold fabrication (Lu et al., 2013).

Advantage: The electro spinning strategy has the flexibility to deal with a wide scope of materials in request to create frameworks with claimed permeability with surface shape, incorporating filaments ranging between microns to nanometer. The scientists discovered brilliant multifunctional platforms which could possibly be used to fix and recover bone imperfections and wounds. The inclusion of bioactive elements made the nanofibers, the significant component more useful (Roseti et al., 2017).

Drawback: A principle burden of electro spinning is the utilization of natural solvents. Although electro spinning is a speedy and basic way to deal with creation of different kinds of nanofibrous frameworks yet there is a major issue to design frameworks with complex constructions for some,

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14 tissue designing applications. In addition, development of platforms containing a homogeneous conveyance of pores should be a great concern(Lu et al., 2013).

3.1.3 Gas foaming

A bio fabrication method where a polymeric platform soaked at immense pressure by a foaming specialist like CO2, N2 / H2O. As a result of thermodynamic unsteadiness gas froth are shaped for polymer development (Bajaj et al., 2014).Studies showed that plates of PLGA was created utilizing gas foaming. Also a permeable biphasic calcium phosphate (BCP) framework was build utilizing gas-frothed polyurethane as a format. The biocompatibility of BCP framework enables its utility for bone separation and recovery was affirmed by in vivo and in vitro examination. The supercritical gas foaming innovation permits the fuse of heat sensitive drugs and biological specialists but only 10-30% of the pores are interconnected (Hutmacher, 2000).

Figure 3: Schematic representation of gas foaming technique (Koyyada & Orsu, 2021).

Advantage: The greatest benefit of this method for tissue designing applications is the utilization of moderately inactive frothing specialists without the inclusion of cytotoxic solvents (Bajaj et al.,

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15 2014).This method produces structures with a pore size of 30 to 700 μm and a porosity up to 85%

(Eltom et al., 2019).

Drawback: The disadvantage of this method is the acquired scaffold may possess shrink hole shape/solid polymeric skin. Another limitation is incorporation of unnecessary hotness during molding to tackle this issue, scientists advanced the method to obtain an extremely permeable item with modified permeable connection. In vitro investigations have demonstrated the attachment of cultivated cells to the framework while taking on 3D tissues (Eltom et al., 2019).

3.1.4 Thermal induced phase separation (TIPS)

The phase detachment method can be prompted thermally or by a nonsolvent has been used to manufacture permeable layers or foams for filtration and partition purposes. This brings out platforms having diverse orifice shape that isn't appropriate in creating tissue designing frameworks as they mainly required uniform pore structure. The TIPS process happens when a homogeneous polymer arrangement turns out to be thermodynamically unsteady under specific temperature circumstances also divides into multiphase framework spaces, including a polymer- poor stage and a polymer-rich stage. Subsequently, the polymer-rich stage gets harden while the polymer-lean stage transforms into pores because of dissolvable evacuation. The solid fluid phase division is utilized to inspire dissolvable crystallization from a polymer arrangement by reducing the temperature and initiate pores build up after evacuation of dissolvable crystals. In the fluid fluid stage division method, polymer arrangements with a high temperature structure a bicontinuous construction (for both stage) (Lu et al., 2013).Many studies focused TIPS method to develop polymeric three dimensional scaffolds. Before that this strategy has been broadly applied

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16 in drug conveyance to manufacture microspheres as they permits the blending of drug and biological specialists for example, bone morphogenetic proteins (BMPs) into the polymer grid .Normally pore sizes of up to 100 m can be reproducibly manufactured by this method. So scientists started to working in this regard to manufacture polymer and polymer/HA examples with a porosity up to 95% (Hutmacher, 2000).Different manufactured polymeric nanofibers have been created by this techniques for tissue designing applications. In the investigation animated mesoporous silica nanoparticles were made to utilize as micro carriers for dexamethasone stacking (Roseti et al., 2017).Recently, many polymers have been created into 3D permeable frameworks like chitosan frameworks with nanofibrous or micro fibrous structures have been arranged where the porosity and fiber size of the frameworks can be managed by phase detachment temperature and chitosan concentration that will be a possible device for tissue engineering(Lu et al., 2013).

Advantage: TIPS holds extraordinary potential in creating 3D nanofibrous platforms with identical pore shape via double or different stage partition method than electro spinning. Moreover, this procedure can be utilized along with other manufacture strategies like solid free form (SFF) in TE. Low temperatures favor the accumulation of bioactive particles. This strategy can be used for the development of the thermoplastic translucent polymer platform (Eltom et al., 2019).

Drawbacks: However the primary disadvantage of this procedure is that restricted materials (only used for thermoplastic) can be utilized in fabrication and lack of resolution (Eltom et al., 2019).

3.1.5 Solvent casting and practical leaching

A dissolvable mixed with consistently dispersed salt bits of a specific proportion is utilized to disintegrate the polymer arrangement. The dissolvable vaporizes departing a lattice holding salt bits and this polymer network is drenched in aqua to permit filtering of the salt particles to frame

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17 a design with high porosity (50%-90%) (Eltom et al., 2019).Solvent casting in mix with particle leaching turns out effective only for slim layers or three dimensional examples with very thin areas or it is impossible to eliminate the solvent particles from inside the polymer lattice. Scientists use the above-depicted innovation to manufacture permeable sheets and covered them to three dimensional constructions (Hutmacher, 2000).Specialists created nanoHA-nylon composite frameworks showing porosity similar to real bone. Though the nanoHA structure is exceptionally similar to real bone, its fragility and below par mechanical strength restricts its utilization for the healing of deformities .Nylon shows great biocompatibility with magnificent mechanical features, though the corrosion is hard to dominate. The two outcomes blending in a platform substance perhaps be a decent applicant for bone substitution because of its histocompatibility, osteoconductivity and osteoinductivity (Roseti et al., 2017).

Figure 4: The schematic diagram of solvent-casting particulate-leaching techniques (H. Liu &

Webster, 2007).

Advantage: This procedure is moderately simple and cheap. One of the fundamental advantages of this strategy is that the developed framework has huge permeability with the ability of adjusting

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18 the orifice length for suitable fitting regarding 3D cell formation. Different specialists have applied this method in the platform manufacture for various purposes, like the mixing of natural polymers or the coordination of bioactive particles into the scaffolds (Eltom et al., 2019).

Disadvantage: The primary burden of the strategy is the utilization of cytotoxic natural solvents for creation of these frameworks. The framework should be more than once washed to guarantee total evacuation of the solvents and limit cell death (Bajaj et al., 2014). Another drawback is this method is tedious as just slim layers can be utilized. Also the layering of permeable sheets permits just a set number of interconnected pore structure. The other limitations include broad utilization of profoundly poisonous solvents, time needed for dissolving, remaining particles in the polymer framework, uneven pores shape, and inadequate interconnectivity (Hutmacher, 2000).

3.2 Rapid prototyping (RP) techniques

RP techniques termed as a bunch of assembling method able to create clear shapes straightforwardly by using computer aided design (CAD) except requiring explicit information.

RP frameworks accumulate powder, fluid, and sheet materials and layer wise, RP device can deliver substances utilizing slim level cross segments (Eltom et al., 2019).With the help of CAD of harmed body stuff or bones from victims MRI initiates exact size framework production which make it particularly helpful for bone fix while overcome the challenges observed in customary strategies (Dutta et al., 2017).The fundamental advantage of these procedures is being able to create customer preferred and victim-explicit framework appropriate for 3D printing (3DP), fused deposition model (FDM), selective laser sintering (SLS), and stereolithography are some of the major process (Eltom et al., 2019).

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3.2.1 Bioprinting

Bioprinting is an attractive 3DP method, enable to rapidly fabricate complex structures in a top- down approach over broad length scales. Here bio ink are set onto a substrate also termed as bio paper in a layer-by-layer method to create 3D structures analogous to tissues or organs (Bajaj et al., 2014). Bioprinting is the most recent progression in tissue designing that could help in making scaffolds of targeted morphological shapes. Each print layer which is just 20 micron makes it conceivable to accomplish large scale geometry while duplicating miniature engineering of organs and tissues. It empowers customized medication by utilizing the specialized type of cell development and the innovations of 3DP can be ordered into two sorts, specifically, acellular and cell develops. In acellular bioprinting, the platform and biomaterial can be generated without a cell during the printing system and convey a higher exactness and more noteworthy shape intricacy since it has less obstructive manufacture conditions than techniques requiring the cell feasibility support. In case of cellular bioprinting cells and other bio agents are coordinates with the material during the creation method to manufacture living tissue develops. Now a days, there are various methods of 3DP among which micro extrusion, laser-aided, and inkjet printing are the most generally involved techniques for the designing of organic materials (Eltom et al., 2019).Current center is subsequently on the generation of viable printing instruments and materials (bio inks) that are fit for delivering biomimetic platforms. Fast prototyping additionally makes it conceivable to make pore size slope in a solitary step. The pore size inclination frameworks improve cultivating productivity from ~35% to ~70% in homogeneous platforms under static culture conditions. But, these methods are helpful when the natural synthetic substances are viable with nature inferred bio-polymers (Dutta et al., 2017).

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20 Drop based Bioprinting

A well-known method of building scaffolds and biomaterials with intricate 3D structures demand quicker or same gelation time like the droplet dismissal hour that it makes harder to apply to numerous polymer frameworks .This cycle was applied subsequently to print endothelial cells and fibrin framework at the same time to produce micro vascular structures .Although it provides a quick technique to manufacture huge constructions, demand of quick polymerization energy restricts the utilized polymers (Bajaj et al., 2014).

Extrusion Bioprinting

Bio fabrication with DIW (direct ink write) strategies is a proficient technique regarding colloidal gatherings/effective oxide gatherings production by using a pre polymer ink arrangement containing the monomers, cross-linker particles and inciter. In the course of cycle, UV brightening origin act in accordance with nozzle to photopolymerize the toner to its ultimate shape. Extrusion bioprinting has correspondingly been utilized to build complex 3D structures for tissue designing.

For instance, utilizing this strategy with agar-bar cell spheroids to make counterfeit vascular tube shaped grafts that showed heterogeneous cell appropriations. However extrusion techniques are smother and viable with more polymers but not quick like drop based. The extrusion interaction can restrict the ultimate design and geometry of the polymer (Bajaj et al., 2014).

3.2.2 3d printing

3DP is a course of making apparatuses and utilitarian model elements straightforwardly from the PC models.3DP strategy is carry out by putting the talc substance in layers and the particular combination of talc using "inkjet," at the place of cement printed.3D printing is a valuable creation strategy regarding TE that has exact power over framework shape at the micrometer stage. The

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21 pattern of poly (dopamine) overlays for 3D printing poly (lactic acid) frameworks for bone restoration were identified by many researches.As well as advancing bone formation, coverings were displayed for further cell bond development with multiplication. Upgraded 3DP innovation has been created to work on the delivered framework to copy the particular tissue Though its prosperity includes the capacity to imitate the regular tissue pattern stringently and the mechanical attributes of the framework, the created framework by 3DP strategy have restricted imitating the nanoscale extracellular network features of the tissue they plan to supplant (Eltom et al., 2019).

Figure 5: 3D scaffold fabrication by using 3D bio printer (M. Du et al., 2015).

3.2.3 3d plotting

3D plotting rely on the area of biomedical science like several RP strategies where platform is made layer by layer additionally permits manufacture of exceptional complicated structures .Here, a polymeric glue with intended consistency can be handled at aimed temperature (Dutta et al., 2017).

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3.2.4 Photolithotherapy

Photolithography strategies utilize photons to move the mathematical states of a veil to a light delicate exterior. For biomedical exercise this technique is utilized either to make a 2D framework for cells development or to encoded in a 3D organization of polymers (Bajaj et al., 2014).

Advantages: It has the capacity to consistently encoded cells with heat reduction all over the platform. So, this strategy is significantly utilized in tissue designing to make 3D scaffold for refined various cell categories like hepatocytes, fibroblast, endothelial cells and cardiovascular immature microorganisms.

Disadvantage: By consuming the occurrence luminescence photo initiator makes free radicals to start the chain response that can be cytotoxic to the cells based on portion particularly when the polymerization is performed in cells presence (Bajaj et al., 2014).

3.2.4.1 Mask based Photolithotherapy

In this method a designed mask is employed to enlighten particular area of a polymer. In Mask based photolithography the pre polymer solution are given exposure to UV light. Both natural (altered) and engineered polymers can be photo polymerized utilizing this technique(Bajaj et al., 2014).

Advantages: This method permits for controlled breeding of various cell catagory in 3D hydrogels. By utilizing this strategy researchers create oligo (PEGfumarate): PEG diacrylate (PEGDA) hydrogels with excessive dimensional target. Essential ligament fibroblasts and marrow stromal cells were encoded in these hydrogels and exhibited a serious level of feasibility above 14 days.

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23 Disadvantage: One of the significant problem is the absence of computerization. So photomasks should be physically altered and adjusted after each layer for complex frameworks. Furthermore, different photomasks should be pre-assembled, which make this method pricey and tedious (Bajaj et al., 2014).

3.2.4.2 Setriolithography

The CAD method utilized via RP corporation in biomedical administration almost for ten years.

The fundamental functioning rules is: The pattern of the created construction/platform initially evolved by a 3D computer sketching operating system. In case of exceptionally unpredictable 3D plans, magnetic resonance imaging (MRI) /computed tomography (CT) can likewise be applied.

Stereolithography technique is fundamentally used to make strong, 3D target by successively printing a slim film of (UV) reparable substance in layers (Bajaj et al., 2014).This strategy is applied in the development of framework constructions of complex plan at high resolution by utilizing poly (D, L-lactate)/ poly (D, L-lactide-co-e-Caprolactone) build resin (Eltom et al., 2019).

Figure 6: Steriolithography fabrication for polymeric scaffold (Mondschein et al., 2017).

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24 Advantage: As a mask less innovation, SL doesn't need creation of an actual cover which lessen both the expense and time needed for platform creation. Likewise, with this computerized method, there is exact command over the thickness of the platform which permits the fast creation of designs (Eltom et al., 2019).Also this procedure can be utilized to research cell communications in 3D microenvironments. It has the ability to beat the difficulties related to wastage in subtractive creation strategies. Also it has high resolution power with consistency in pores interconnectivity.

Disadvantage:

Albeit numerous kinds of exploration exhibited achievement in scaffold plan, this technique additionally possess restrictions simultaneously of photo polymerization. It requires enormous measures of macromolecules and postpolymerization therapy to further develop macromolecule transformation (Eltom et al., 2019).

3.2.4.3 Multiphoton lithography

In mask less lithography, an engaged laser or confocal magnifying lens has been displayed to offer high parallel (x-y) goal yet little to zero power over the pivotal (z) bearing. To beat this restriction, multiphoton-based methodologies have been discovered, which can restrict the photochemical responses in 3D. While in single-photon assimilation frameworks, a fluorophore is invigorated by one photon of a particular energy level, in multiphoton (or two-photon) processes, various photons of lower energy are expected to energize the fluorophore this is known as the multiphoton (or two- photon) impact. For multiphoton lithography, the fluorophore is supplanted by a photo initiator to produce a free extremist, which can be utilized in a large number of the photo polymerization techniques(Bajaj et al., 2014).

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25 Advantage: It offers an interesting technique to design assorted functionalities inside 3D hydrogels This strategy is great to make high-goal (∼1-μm) self-supporting structures, however it has additionally been used as a designing strategy inside HA hydrogels to immobilize biotinylated BSA which is bovine serum albumin that can be identified with neutravidin and biotinylated peptides (Bajaj et al., 2014).

Drawbacks: This strategy is hard to operate.

3.2.5 Fused deposition model

In FDM, a solid polymer is projected into a hot expulsion nozzle to be softened furthermore expelled on the outer layer of 3D target utilizing a computer controlled expulsion and dismissal processes and the framework is produced using various layers of neighboring microfilaments. It has been used to handle thermoplastic biopolymers and utilized naturally decomposable polyesters while making nonwoven platforms which may assist the cells produced in TE. Also, many studies showed the flexibility of demonstrating new framework shape accompanied by controlled mechanical features (Eltom et al., 2019).

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26 Figure 7: A schematic diagram of the FDM process (Zein et al., 2002).

Advantage: The frameworks created by this strategy have showed amazing biodegradation and biocompatibility (Koyyada & Orsu, 2021).The primary benefits are huge permeability, mechanically robust, no poisonous liquid incorporation and adaptability in substance taking care of with proper handling.

Disadvantage: The need of established strands along with reliable size and substance features to take care between the tumblers and spout is the primary trouble of the FDM strategy. Moreover, it has not effective administration on biodegradable polymers except PCL (Roseti et al., 2017). To survive these disadvantages, many adjusted FDM processes have been proposed by utilizing low temperature dismissal used to manufacture composite scaffolds for the designing of bone tissue

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27 and they showed brilliant biocompatibility, biodegradation, and bone conductivity properties (Eltom et al., 2019).

3.2.6 Selective laser sintering

A strategy which utilizes laser as the power source to sinter powdered material characterized by a3D model in lean layers and has been used to make different materials like polymers, metals or ceramics .This method utilize extra high-sub-atomic mass polyethylene in framework creation whereas to manufacture bionanocomposite microspheres inside a poly (L-lactide) (PLLA) framework which assemble TE framework (Eltom et al., 2019).Some researchers manufactured modified permeable PEEK platforms with a trabecular microstructure which can be utilized in muscular applications due to its high strength, weakness obstruction and great biocompatibility.

The further created platform structure can be a good choice for craniofacial reorganization (Roseti et al., 2017).

Figure 8: Selective laser sintering process (Y. Du et al., 2017).

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28 Advantage: SLS is a valuable method since it has phenomenal client command on the created frameworks microstructures by adjusting different boundaries of this process. It is used to acquire the favored properties of the produced scaffold (Eltom et al., 2019).

• SLS gives a significant mechanical and surface morphological element and helps in controlling the porosity of the created parts as they mold the powder particles through sintering not by involving any dissolvable as binder.

• SLS is exceptionally appropriate in biomedical applications because of its low part develop time and great accuracy. SLS framework can straightforwardly utilize the CT and MRI information and can additionally produce modified inserts and practically evaluated gadgets for controlled medication conveyance in hours or days.

• Wide scope of materials are manufactured like ceramics, polymers, metals, polymer composites, and polymer-earthenware composites is one more essential benefit for the SLS frameworks. A portion of these polymers are new bio composite polymers like HA/PLGA.

• SLS is an exceptionally adaptable cycle regarding plan changes and its execution as it is a CAD-based cycle (Riza et al., 2020).

Disadvantage:

• Requirement of extra technique for eliminating infused talc at huge working temperature later handling the stage turn (Eltom et al., 2019).

• The unavoidable issue with SLS constructed parts is the chance of thermal movement because of twisting and shrinkage

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• For complex 3D permeable organization that is a fundamental necessity for the biomedical platforms, complete withdrawal of trapped powder is a critical limit and in some cases this is impossible even after post-processing activities.

• Creating those structures in high-temperature handling require exhibition of high laser power that can influence the bioactivity and harmfulness of the manufactured scaffolds (Riza et al., 2020).

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Chapter 4

Opportunities and challenges of 3D scaffolds in regenerative medicine 4.1 Applications

4.1.1 Skin

Skin, the biggest body part is vulnerable towards outer damage and bruise since it is our supreme protection level against ecological damages. The utilization of designed frameworks can be a procedure to advance skin restoration (Aldana & Abraham, 2017)Now a days skin regeneration gain higher attention as skin implants have obstacles in antigenicity and deficiency of movable tissues. Scientists discovered PDMS (polydimethylsiloxane) base, covered with sodium bicarbonate, a pH-adjusting cross-connecting specialist. At that point, layer wise collagen was printed to create a multifoled skin build. A few benefits of this creation technique incorporate the accompanying: the framework can be made on unpredictable surfaces as the cross-connecting specialist covering is conceivable, and different sorts of hydrogels can replace collagen, assuming them cross-linkable (Chung et al., 2020).Nano fibers which possess ideal natural circumstances could be a great answer for the improvement of skin tissue designed frameworks. To advance cell expansion while working with cell separation method for skin recovery, ecological and biocompatible platforms with biophysical climate was planned. Researchers have revealed, initially, the consideration of a glycosaminoglycan (GAG) in gelatin nanofibers. The cross-linked gelatin and chondroitin sulfate (CS) nanofibrous platforms were combined. CS widely influences a few cell exhibitions like movement, connection and multiplication of the cells.The appearance of GAGs might increment electrical conduction since the existence of cations in chondroitin sulfate salts (Aldana & Abraham, 2017). Moreover 3D printed keratinocyte-and fibroblast-installed

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31 collagen for skin tissue designing was also discovered. Laser-assisted bio printing (LaBP) with laser-incited onward move method was utilized to develop 3D frameworks. This strategy is worthwhile over other bio printing methods due to elevated intent with prominent cell thickness while cell printing (Chung et al., 2020).To get a perfect lesion bandage, gelatin electro spun platforms was created by mixing (Tan et al., 2015) chitosan, gelatin and shape memory polyurethane (PU). This framework allow advantageous mechanical conduct and aqua fume passing proportion. Hence, injury healing might potentially benefit through shape obsession helped simple handling and shape regeneration helped conclusion of broken injuries, which can be adjusted by prearranging (Aldana & Abraham, 2017).

4.1.2 Liver

Liver is a basic body part for multiple metabolic purposes. Though liver transfer has been exercised for a prolonged period but the method is expensive, low endurance ratio of victims and deficiency of organ donors. Scientists invented chitosan-gelatin cross breed scaffold was created to bio mimic the design of normal liver. An exceptionally permeable efficient design was manufactured by mixing 3D printing, micro replication, and freeze-drying procedures having characteristic fluidic channels also hepatic chambers. For that, SLA strategy was used to manufacture a resin model to project polydimethylsiloxane (PDMS). Chitosan-gelatin arrangement was project in PDMS form via freeze-drying to create permeable construction. Biodegradability and hepatocyte development affirmed via 1 week cell culture and significantly albumin emission and urea mixture were apparent, indicates the hepatocyte efficacy (Chung et al., 2020).Some researchers 3D printed PCL to a lattice like shape as an essential system to precisely uphold collagen bio inks (Lee et al., 2016).

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4.1.3 Heart valve:

Aortic valve infection is mainly a genuine cardiovascular infections that are typically cured by valves substitution. Numerous scientists concentrated on synthetic heart valves utilizing different polymeric substance like PGA, PLA collagen, and fibrin. That’s why, a 3D printing method can be applied to this exploration area for a long time. Hydrogels are favorable substance because of their physicochemical and mechanical steadiness while they are moisturized. Besides, hydrogels are porous for nutrients furthermore waste clearance (Chung et al., 2020).Some researchers manufactured human aortic valvular interstitial cell (HAVIC)- confined heart valve conduits with photograph cross linkable methacrylated hyaluronic acid (Me-HA) and methacrylated gelatin (Me- Gel) hydrogels where hydrogel channels consistency enhanced and adjusted by putting in various hydrogel fixations (Duan et al., 2014). Also heart valve frameworks was printed by the help of photograph cross linkable poly (ethylene glycol) - diacrylate (PEG-DA) in which two kinds of PEG-DA with various atomic weights were used manufacture the scaffolds to match the diverse mechanical features of aortic valves. Also it possess great flexible modulus and almost 100 percent cell endurance.(Chung et al., 2020).

4.1.4 Blood vessels:

In USA, coronary artery bypass implanting medical procedures are carry out in huge number every year. But the disadvantages like graft harms throughout reaping system, giver illness increase the demand of synthetic blood vessels to beat the contemporary drawbacks. A perfect counterfeit blood vessel should possess biocompatibility, antithrombogenicity, stability and similar consistency with primary thickness of the natural one local (Mosadegh et al., 2015). A biomimetic 3D micro vascular network was manufactured based on hydrogel network. Furthermore, HUVECs refined in the vascular passage, effectively adjusted according to the direction of flow. It was

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33 affirmed by the gene expression examination that the3D printed vascular passage possess great capacity for tissue designing administration (Chung et al., 2020).Fibrin is a characteristic polymer shaped by polymerization of fibrinogen and thrombin, available in human blood to aid the injury recuperating method.3D printed human micro vascular endothelial cell (HMVEC) - cultivated bio ink of thrombin and calcium ion solution into a fibrinogen substrate. The framework was made out of fibrin passage with adjusted HMVECs, and 21 days of cell culture affirmed cylindrical shape development (Chung et al., 2020).Poly(propylene fumarate)- build aorta joint manufactured by means of advanced light stereolithography strategy exhibited probability to plan victim-explicit aorta joints. Moreover, the framework had the option to affirm biological active features in vivo with similar mechanical force contrast to human aorta (Melchiorri et al., 2016).

4.1.5 Bone:

Bone tissue engineering (BTE) refers the get together of bone constructions by consolidating bone cells and frameworks, offers a promising open door for bone recovery in a characteristic manner (H. Liu & Webster, 2007).Generally, designed bone utilizes an ECM type substance, cells, along with elements to manage cell conduct to elevate cell separation to calcium-saving cells lastly bone development. The ECM assumes a significant part in regulating cell adhesion, expansion, separation, and bone development. Consequently, it has now been grounded that an ECM which impersonates both the micro and nano-climate of regular bone will fundamentally work on biological reactions (Wang et al., 2016).Making tissue structure that imitate the bone in both construction and capacity has been a challenge. This is the place where BTE becomes possibly the most important factor and the latest things of regenerative medication have concentrated on the formation of 3D-framework with the assistance of biomaterials and cells that can imitate the ECM, assist the development of new tissue/bone, and able to degrade the new created bone. Different

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34 strategies meet up to build permeable platform to recover tissues/organs and furthermore for the controlled and designated arrival of bioactive specialists in tissue designing applications (Preethi Soundarya et al., 2018). Lu et al. used bone arrangement upgrading innovation combining solid material and different growth variables with platelet rich plasma (PRP).PRP is a growth factor that contains various autologous thrombocytes that increment bone recovery. Also, platelet derived growth factors are significant during granulation as enhance the acceptance of osteoblastic antecedents. As of late, PRP hydrogels have been utilized in maxillofacial medical procedure bone joining strategies by grip with cancellous bone particles. Therefore, it is significant to apply framework that evoke the ideal growth factor impacts (H. D. Kim et al., 2017). Oest et al. created poly (l-lactide-co-d, l-lactide) platforms with bone morphagic protein (BMP-2) and transforming growth factor-𝛽 (TGF-β3) and embedded the frameworks into an 8 mm rodent segmental imperfection model. They affirmed that the gathering of platforms with BMP-2 and TGF-β3 showed upgraded bone development contrasted with the benchmark group. In recent days, the blend of biodegradable polymer platform, calcium phosphate mixtures and growth factors connected with bone arrangement was used to recover bone tissue successfully in an in vivo model (S. H. Kim et al., 2018). A permeable hyaluronic acid frameworks have been utilized for BMP-2 conveyance for bone development furthermore, in vitro outcomes proposed constant deliverey for controlled periods in a functioning structure. Conventional framework creation strategies, for example, solvent casting, molecule filtering, gas frothing, particle leaching, phase separation and electro spinning have some drawbacks. For instance, generally, they lack careful command over pore dimension, nano-scale harshness, positive mechanical features and interconnectivity. In contrast to customary platform creation strategies, 3D printing innovation has arisen as a remarkable strategy than can definitively regulate such features and can be used to figure out

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35 implantable substance of the specific geometry as the bone crack at the clinical bedside for instant implantation (Wang et al., 2016).

4.1.6 Trachea:

Tracheal design rebuilding and scaffold creations are additionally in high interest as43% of infants victims experience tracheostomy respiratory capture. It is assuming if the victims are upheld by a transitory scaffolds for three years, the air tract development can normally solve the sickness.

Morrison along with some other researcher were successful to create a customized and compostable tracheal rib via SLS. PCL talc were blended with hydroxyapatite for utilized as a streaming specialist for the laser sintering method (Morrison et al., 2015). Moreover a 3D printed PCL frameworks via FDM was invented for tracheal recovery where platform was covered with MSC-cultivated fibrin to upgrade biological activity. In vivo research affirmed precisely durability of platforms and ready to recreate windpipe inside about two months of implantation (Chung et al., 2020).Another group of scientists created a circle shape cartilaginous windpipe framework via 3D bio printing with the help of advanced light handling method. Chondrocytes confined in methacrylated silk fibroin, compels cross linking conceivable via UV light. This cell-packed hydrogel platform exhibited uniformly dispersed cells and ligament tissue development in vitro (S. H. Kim et al., 2018). The artificially altered silk fibroin ink was additionally printed to heart, lung, and vascular shapes assuring the effectiveness of bio ink and printing strategy in different tissue designing applications.

4.1.7 Cartilage:

Cartilage injury extraordinarily happens in osteoarthritis, rheumatoid joint inflammation, and various traumas. Such deformities can prompt significant clinical outcomes since the restricted natural potential for healing. To beat this restriction, fix of cartilage damage is a big test in surgical

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36 procedures (Aldana & Abraham, 2017).The ECM of cartilage is made up of type II collagen and GAG, which permits to manage articulation of chondrocyte aggregate and affect chondrogenesis (Chung et al., 2020).As compared to bone, ligament has limited natural healing capacity since internal design restrains nutrients and progenitor cells to move toward the imperfection area.

Frameworks, the fundamental components of three-layered (three dimensional) cartilage development, perform a vital parts in coordinating cartilage recovery. Somewhat recently, natural and manufactured polymeric electro spun platforms have drawn an extraordinary interest because of their capacity to mirror ECM (Aldana & Abraham, 2017). Poly (ethylene glycol) terephthalate/poly (butylene terephthalate) (PEGT/PBT) block copolymer was 3D printed via FDM method to manufacture network like organized framework. The framework was cultivated with bovine articular chondrocytes (bACs) to create ligament like tissue in vivo and also possess mechanical features like the natural articular ligament. Scientists were successful to 3D print ligament frameworks with aqua as a printing