Nanomedicine NANOSC 403_607_707 Lectures Fall 2024 PDF

10/29/2024 Chapter 3: Nano-Scaffolds and Tissue Engineering (Source: Textbook + Slides + Provided 4 Research Articles) 1 1 Tissue engineering (TE) with stem cells is an interd...

10/29/2024 Chapter 3: Nano-Scaffolds and Tissue Engineering (Source: Textbook + Slides + Provided 4 Research Articles) 1 1 Tissue engineering (TE) with stem cells is an interdisciplinary field involving life sciences, medicine, materials science & Nanoscience/technology. The end objectives/targets of TE are: 1. To improve tissue function or heal tissue defects. 2. Replace diseased or damaged tissue/ organ TE is a necessity because: 1. The short supply of donors’ tissues and organs 2. The need to minimize immune system response/rejection of implanted organs by using our own cells or novel ways to protect transplant. 2 2 1  10/29/2024 Regenerate, Repair and Replace Regenerate – Identify the cues that allow for regeneration without scarring – Like growing a new limb. Repair – Stimulate the tissue at a cell or molecular level, even at level of DNA, to repair itself. Replace – A biological substitute is created in the lab that can be implanted to replace the tissue or organ of interest 3 Seed onto appropriate scaffold Remove cells with suitable growth from the factors and cytokines body Expand number in culture Re-implant Place into engineered tissue culture repair damaged site 4 2  10/29/2024 Tissue engineering typically involves four key components as illustrated in the figure below; (a) Selected and isolated cells (progenitor or stem cells from different origins), (b) Biomaterial scaffolds which may be natural or synthetic, to provide a platform for cell function, adhesion and transplantation, (c) Signaling molecules such as proteins and growth factors deriving the cellular functions of interest, (d) Bioreactors that support a biologically active environment for cell expansion and differentiation such as cell culture. A schematic illustration of the four key components of tissue engineering A progenitor cell is a biological cell that, like a stem cell, has a tendency to differentiate into a specific type of cell, but is already more specific than a stem cell and is pushed to differentiate into its "target" cell. 5 EXTRACELLULAR MATRIX (ECM) The ECM promotes a unique microenvironment that fosters tissue organization control the ECM -> control the tissue Consists of a 3D array of protein fibers and filaments embedded in a hydrated gel of glycosaminoglycans ECM molecules – Glycosaminoglycans – Proteoglycans – Proteins Proteins in ECM act as either structural members or adhesion sites Structural proteins Adhesion proteins Collagen Fibronectin Elastin Laminin 6 3  10/29/2024 Design Considerations of TE Scaffolds For a scaffold to act as ideal ECM, it has to be: 3-dimensional Biocompatible Biodegradable Porous Proper surface chemistry Matching mechanical strength of ECM Promotes natural cells proliferation and aggregation normally to form tissue Accessibility to components Commercial Feasibility Demonstrates enhanced Vascularization 7 Strategies to enhance Vascularization of TE Scaffolds Schematic illustration of blood vessel formation promoted by including growth factors (a) or by seeding endothelial cells (d) into the polymer scaffold. 8 4  10/29/2024 Surface characteristics of TE Scaffolds The surface of TE scaffolds is the initial and primary site of interaction with surrounding cells and tissues. Therefore, both physicochemical and topographical surface characteristics of scaffolds are vital parameters in controlling and affecting cellular adhesion and proliferation. The scaffolds should be designed in such a way to facilitate their attachment. For this reason, scaffolds with relatively large and accessible surface area are advantageous in order to accommodate the number of cells required to replace or reinstate tissue or organ functions. 9 Surface characteristics of TE scaffolds can be selectively improved by various approaches including thin film deposition and immobilizations of adhesive biomoieties such as RGD peptides, growth factors (like bFGF, EGF), insulin, fibronectin and collagen. This modification can enhance the biocompatibility of the scaffold and consequently, cells can specifically recognize the scaffold. The adhesive biomoieties can either be covalently linked, electrostatically absorbed, or self-assembled on the surface of hydrogel scaffolds. 10 5  10/29/2024 Some approaches for selective enhancement of surface characteristics of TE scaffolds toward increasing surface-cells attachment and controlled release of regulatory growth factors 11 Why we apply Nanotech in TE? Cells on microfibrous scaffolds have a polarized relationship, with one side of the cell attached to the scaffold, the other exposed to physiological media. In comparison, it is likely that cells are more naturally constrained by nanofibrous scaffolds. 12 6  10/29/2024 Some natural polymer- based Scaffolds for TE applications Several scaffolds have been developed from natural polymers for tissue engineering applications. These natural polymers include for instance, polynucleotides, polypeptides, and different polysaccharides. 13 For Example: Collagen fibers are one of the most popular natural polymer-based scaffolds in TE applications. As shown in the figure below, these collagen fibers are formed particularly through self-aggregation and crosslinking (through pyridinium crosslinks) of collagen molecules in a hydrated environment. Schematic illustration showing the basic structure of collagen hydrogel fibers. 14 7  10/29/2024 Natural polymers-based Scaffolds for TE applications Advantages: - Ease of biological recognition, including presentation of receptor- binding ligands and the susceptibility to cell-triggered proteolytic remodeling and degradation. Drawbacks: - The complexities associated with purification, immunogenicity and pathogen transmission. 15 Some synthetic polymers- based scaffolds for TE applications PEG: poly(ethylene glycol), PEGDA: poly(ethylene glycol) diacrylate, PLA: poly(lactic acid), PEO: poly(ethylene oxide), PVA: poly(vinyl alcohol), PHEMA: poly(hydroxyl-ethyl methacrylate), IPN: interpenetrating polymeric network, SMC: smooth muscle cell, Dex-MA-LA: methacrylated dextran-graft-lysine, Gel- MA: methacrylamide-modified gelatin 16 8  10/29/2024 Example: Schematic illustration of the self-assembled peptide- amphiphiles (SAPs) functionalized with cell adhesion ligand (RGD) into fibrous crosslinked scaffold for bone tissue engineering applications. 17 Some Common Types of TE Scaffolds 1. Nanoporous -Polymeric Scaffolds 2. Injectable Microparticles Scaffolds 3. Nano-Fibrous Scaffolds 4. 3D-Printed/Bioprinted Scaffolds 18 9  10/29/2024 1. Porous Nano-Polymeric Scaffolds Examples of Polymeric Materials-based Nano-Scaffold Used in TE Natural Materials 1. Alginate (Spinal cord injuries, myocardial TE) 2. Collagen (Skin, bone, blood vessel, corneal and cardiac TE) 3. Gelatin (Liver, Cardiac, bone TE) 4. Hyalurosan (Skin, Brain injury and joint repair related TE) 5. Fibrin (Wound healing, ear cartilage TE) 6. Chitosan (Cell encapsulation,cartilage TE) Synthetic Materials 1. Polylactic-co-glycolic acid (PLGA) Cardiac & adipose TE, 2. Polyurethane (PU) Cardiac TE 3. Polyglycerol sebacate (PGS) Cardiac TE 19 2. Injectable Microparticles Scaffolds Eg.: Use of injectable porous scaffold microspheres for cartilage TE 20 10  10/29/2024 3. Nano-fibrous Scaffold Advantages of Nanofibers as Scaffolds for TE: - Ultrafine continuous fibers - High surface to volume ratio - High porosity and variable pore size distribution - Mimicing extracellular matrix in vivo. A number of methodologies exist for the fabrication of NFbs however, many of these methods involve toxic metal catalysts and chemicals which exclude the final products Fabrication Techniques of Nanofibers: from use as in vivo therapeutics. (a) Electrospinning Here we will focus only on the three most (b) Self-Assembly popular nanofiber and relatively safe synthesis technologies: electrospinning, self- (c) Phase Separation assembly and phase separation. 21 (a) Electrospinning This process involves the ejection of a charged polymer fluid in form of nanofibers onto an oppositely charged collecting surface. Multiple polymers can be combined Fiber diameter and scaffold architecture are easy to control 22 11  10/29/2024 The process involves the application of high voltage to a liquid droplet of the desired material, which subsequently becomes charged. The three-dimensional nature of the droplet is stretched due to electrostatic repulsion counteracting surface tension and a stream of liquid emerges from the droplet surface. The exact point of this emergence is known as the Taylor cone. In order for fiber formation to occur, the molecular cohesion of the liquid must be at a critical level or the end result will not be fiber spinning but rather electrospray. 23 23 (b) Self-Assembly Cells interact with their external environment through cell surface receptors that often specifically recognize and bind to extracellular matrix (ECM) proteins such as collagen and fibronectin. These interactions promote migration, cell division and in some cases differentiation to generate mature lineages in a tissue-specific manner. A recapitulation of the ECM environment for tissue engineering purposes is therefore thought to be advantageous for cell function as it pertains to therapeutic applications. Much effort was spent recently at attempting to recreate the ECM through the manufacture of various matrices often containing ECM-localized proteins, perhaps some of the most promising advances have come in the form of non-covalently self-assembled peptide-rich nanofibers. 24 24 12  10/29/2024 Self-assembly is the formation of an organized structure or pattern from pre-existing disorganized components as a result of specific local interactions and without external direction. In 1995 Matthew Tirrell at the University of Minnesota developed a peptide- amphiphile (PA)-based system that allows for the self-assembly of thermally stable protein-based two- and three-dimensional architectures. An amphiphile is defined as a molecule containing a polar water-soluble head attached to a hydrophobic carbon tail and it is the amphiphilic nature of this system that promotes both self-assembly and biological compatibility. The polar peptide heads were derived from known collagen ligand sequences represented as: 25 25 This sequence is quite similar to the human alpha 1 (IV) 1263–1277 collagen ligand sequence. The hydrocarbon tail was originally a dialkyl chain later replaced and elongated by other researchers with a monoalkyl chain which resulted in significantly increased thermal stability. Both versions readily assembled into a triple helix three-dimensional formation at the liquid interface of aqueous solvents. The resulting self-assembled scaffolds showed enhanced cell adhesion, migration and proliferation and it was postulated that this is due to the similarity between the PA structures and the natural ECM. 26 26 13  10/29/2024 Diagrammatic illustration of a peptide amphiphile and its selfassembly into nanofibers. 27 27 The preparation of these nanofibers involves the reduction of cysteine residues present within the polar peptide heads to free thiol groups. These groups promote self-assembled cylindrical nanofiber formation below pH 4.0. They used the standard reducing agent dithiotheritol (DTT) and followed by acidification to drive nanofiber self-assembly. The resulting fibers were roughly 5–8 nm in diameter and up to several microns in length. In addition, mineralization of hyaluronic acid (HA) crystals on the surface of these nanofibers was successfully accomplished to mimic ECM of bones. 28 28 14  10/29/2024 Other studies by the Northwestern researchers characterized the correlation between alkyl length and pH sensitivity and its effects on selfassembly. This led to the introduction of three unique methods for the formation of PA-based self-assembled nanofiber platforms: 1. pH-controlled self-assembly, 2. Drying on surface self-assembly, and 3. Divalent-ion-bonded self-assembly. 29 29 Self-assembly was noted to be reversible and this reversibility was dependent upon pH. Time sequence of pH-controlled PA self-assembly and disassembly. (Upper) From left to right a peptide amphiphile dissolved in water at pH 8 is exposed to HCl vapor. As the acid diffused into the solution a gel phase is formed, which self- supports upon inversion (Far Left). (Lower) The same gel is treated with NH4OH vapor, which increases the pH and disassembles the gel, returning it to a fully dissolved solution. 30 30 15  10/29/2024 Fabrication of various peptide materials Self-assembling peptides (SAP) form a three- dimensional scaffold woven from nanofibers ~ 10 nm in diameter 31 Self-Assembling Peptide Scaffolds for Tissue Engineering Applications SAPNS heals the brain in young animals. SAPNS allows axons to regenerate through the lesion site in brain. PNAS March 28, 2006 vol. 103 no. 13 5054-5059 32 16  10/29/2024 (c) Phase separation Powder Scaffold Foam This process involves dissolving of a polymer in a solvent at a high temperature followed by a liquid–liquid or solid–liquid phase separation induced by lowering the solution temperature. Capable of wide range of geometry and dimensions include pits, islands, fibers, and irregular pore structures Simpler than self-assembly Advanced Drug Delivery Reviews Volume 59, Issue 14, 10 December 2007, Pages 1413-1433 33 It is a technique by which nanofibrous foams are produced that are very similar in morphology to naturally occurring collagen present within the ECM. It has many advantages as relative simplicity and the lack of equipment needed in comparison to either electrospinning or self-assembly. Developed in 1999 by researchers Peter Ma and Ruiyun Ma at the University of Michigan (thermally induced liquid-liquid phase separation). Nanofibers’ morphology in this method is influenced by many factors such as the types of polymer and solvent used as well as temperature ranges applied during the process. 34 34 17  10/29/2024 Porosity of NFbs can also be controlled with the addition of porogens such as sugar and salt to create a macroporous three- dimensional scaffold of nanofibrous foam. SEM micrographs of a PLLA fibrous matrix. 35 35 4. 3D-Printed/Bioprinted Scaffolds A novel approach in TE that is based on layered strand- or dropwise deposition of cell-laden hydrogels. Rapid & beneficial in developing 3D scaffolds with a predesigned external shape and internal morphology, and also with definite cell placement. It involves multiple printing heads, each containing a specific cell type and/or hydrogel, which enables printing of heterogeneous constructs. 36 18  10/29/2024 (a)Organ/tissue printing using fiber deposition. (b)Macroscopic view of the dual graft (heterogeneous tissue formation in a printed construct implanted subcutaneously in mice) at 6 weeks; dashed line represents the transition zone between (left) printed MSCs in Matrigel and (right) EPCs in Matrigel/hematoxylin and eosin staining, scale bar = 200 mm. Image was adapted from. 37 Comparison Process Ease Advantages Limitations Cost effective Large scale fibers (a) Electrospinning Easy Long continuous fibers No control over 3D Tailorable mechanical pore structure properties, size & shape Produce fiber on lowest Lack of control (b) Self-assembly Difficult ECM scale (5-8 nm) Limitation on polymers Tailorable mechanical Lab scale (c) Phase separation Easy prop. production Batch to batch Limitation on consistency polymers Batch to batch consistency Lab scale (d) 3D- Easy production Printing/Bioprinting Limitation on polymers 38 19  10/29/2024 Biomimetic electrospun nanofibers TE scaffolds A variety of fascinating structures resembling natural objects (e.g. lotus leaf, silver ragwort leaf, rice leaf, honeycomb, polar bear fur, spider webs, soap-bubble, etc.) have been successfully biomimicked via electrospinning 39 39 Other General Fabrication Techniques of TE Scaffolds Schematic illustration of Emulsification technique for fabrication of hydrogel particles scaffolds for tissue engineering applications 40 20  10/29/2024 Photolithography 41 41 Gas foaming/Salt leaching –> Porous Scaffolds 42 21  10/29/2024 Microfluidics Micromolding 43 Three- dimensional organ/tissue printing A novel approach in TE that is based on layered strand- or dropwise deposition of cell-laden hydrogels. Rapid & beneficial in developing 3D scaffolds with a predesigned external shape and internal morphology, and also with definite cell placement. It involves multiple printing heads, each containing a specific cell type and/or hydrogel, which enables printing of heterogeneous constructs. 44 22  10/29/2024 Three-Dimensional Printing in Tissue Engineering & Pharmaceutics By Dalia Hamza Submitted to Prof.Dr. Ibrahim El Sherbiny 45 Introduction 3D printing is a manufacturing process in which an object is created layer by layer using a machine, known as a 3D printer, and CAD software that instructs the 3D printer on how much material to deposit and where to deposit it. 3D printing is a promising field that enables fabrication of objects from personalized specific computer aided designs, while employing automated processes and standardized materials as building blocks. It is considered the twenty-first century manufacturing technology and widely used due to its superiority over the traditional manufacturing techniques: Advantages: It offers a high level of control over the architecture of the fabricated objects. It guarantees reproducibility. It enables scaleup and standardization 46 23  10/29/2024 Different TYPES of Additive Manufacturing 47 What is the difference between? 3D Printing (1984) 3D Bioprinting (2010)  A generalized term that encompasses  Refers specifically to the printing of the printing of various materials such live cellular material, usually mixed in as polymers, plastics, ceramics, metals, with a polymer (i.e. biomaterial) of and composites. choice.  A 3d printer can be used outside of a  A 3d bioprinter should be used inside laboratory environment. a lab and have some type of sterilizable printing area or chamber.  A 3d printer designed to print materials like polymer resins, metal, plastic and  A 3d bioprinter designed to print rubber (Printing Inks or Materials). biological materials, or bioinks (Printing Inks or Materials). 48 24  10/29/2024 4D Printing/Bioprinting (2013/2014) 5D Printing/5D Bioprinting (2016)  Five-axis 3D printing is an extension of 3D printing  4D printing uses the ability of shape and where the print head has the ability to move functionality transformation over time around from 5 different angles due to a mobile when exposed to an intrinsic/external plateau. This allows creating curved layers which stimuli. are stronger than the traditional 3D printed flat layers. Furthermore, this means that curved-  4D bioprinting is a specialized extension shaped products or implants with improved of 3D bioprinting that aims at strength can be produced reconstructing the biochemical and biophysical composition, as well as the  Five-axis 3D printing is currently of high interest hierarchical morphology of various because it addresses some of the challenges tissues using stimuli-responsive associated with regular 3D printing. Thus, due to its biomaterials and cells. ability to build an object from several directions, stronger parts can be produced. Multi-material printers will also be developed. 49 50 25  10/29/2024 Motivation for 3D Printing in Pharmaceutics and Tissue Engineering Need for individualized dosing Need for personalized biomaterials, tissues, organs, disease models 51 Why we need to use 3D Bioprinting in Tissue Engineering?  Bioprinting allows the spatial arrangement of cells, materials and biologically active factors. So, this technique provide a high level of biomimicry by recreating the complexity of tissues and organs, and they can be upscaled for manufacturing and production.  High spatial and temporal resolution is important for the fabrication of complex tissues for therapies and for the creation of 3D in vitro models to investigate biological processes. Traditional approaches to fabricating engineered scaffolds, such as porogen leaching or gas foaming, do not allow for the simultaneous incorporation of biologically relevant signals and cells with high spatial control. 52 26  10/29/2024 Why we need to use 3D Bioprinting in Tissue Engineering?  Enable us to control the internal architecture of the fabricated tissues/organs. This means we can fabricate them with high accuracy.  Enable us to control the complexity. This means we can fabricate complex tissues and organs using multiple materials.  Guarantee standardized and repeatable outcome that enable us to have personalized constructs and multiple replicates of tissue models so saving more time and cost for your application. 53 Complexity of Tissue Engineering Complexity increase in this direction Tissue Mode Role Example Biomechanical Bone Structural Cartilage Physical Barrier Tendon Support Blood Vessels Produce soluble, diffusible Chemical Pancreas biochemicals Liver Complex tissue function (absorption, Physiological Kidney chemical release) Brain 54 27  10/29/2024 Why we need to use 3D Printing in Pharmaceutics? 55 Why we need to use 3D Printing in Pharmaceutics?  For the development of precision medicines according to the need of individual patient and their susceptibility to certain diseases.  For the fabrication of drug delivery systems (DDS) with tailored doses, multiple drugs and/or controlled drug release kinetics. The personalization of the 3D printed DDS can be achieved through the customized design of the DDS, including the use of dosage forms with various geometries, the scaling of the dosage form dimensions and the formulation of compartmentalized dosage forms. 56 28  10/29/2024 Why we need to use 3D Printing in Pharmaceutics?  3D printer could develop variety of novel drugs like transdermal patches, pills, and sustained release formulations carrying multiple active ingredients in one dose. Thus a single dose could cure multiple disease designed at the point of treatment. This increases patient compliance with least side effects giving a revolutionary change in field of drug design and treatment options. Also, using this technology several doses can be combined into one dosage form which suits the patient’s demography.  3D Printing could help to support the synthesis of a range of different molecules on a small scale, particularly useful for those medication with high cost or poor stability. For example, it allow production of high-cost drugs, such as ‘orphan drugs’ that are developed for rare diseases (affecting less than 1 in 2000 people in Europe). 57 Typical Steps for 3D Printing/Bioprinting: Steps Software  1- Computer-aided design 1. Digital model generation (CAD) – Modeling 2. Printing Parameter Selection and Set-up  2- Computer-aided manufacturing (CAM) – Slicing 3. Printing Process  3-Computer-assisted modelling 4. Post-Printing Process which is highly dependent on the specific 3D printer used, the specific materials used and the specific application 58 29  10/29/2024 Common 3D Printing Techniques: 59 59 Stereolithography (SL/SLA) -  With this process 3D solid 1986 objects are produced in a multi- layer procedure through the selective photo-initiated cure reaction of a polymer. These processes usually employ two distinct methods of irradiation.  The 1st method is a mask-based method in which an image is transferred to a liquid polymer by irradiating through a patterned mask. The 2nd method is a direct writing process using a focused UV beam produces polymer structures. 60 30  10/29/2024 Selective Laser Sintering (SLS) - 1988  This technique uses a laser emitting infrared radiation, to selectively heat powder material just beyond its melting point. The laser traces the shape of each cross-section of the model to be built, sintering powder in a thin layer.  After each layer is solidified, the piston over the model retracts to a new position and a new layer of powder is supplied using a mechanical roller. 61 Fused Deposition Modeling (FDM) - 1991  By this process, thin thermoplastic filaments or granules are melted by heating and guided by a robotic device controlled by a computer, to form the 3D object. The material leaves the extruder in a liquid form and hardens immediately. The previously formed layer, which is the substrate for the next layer, must be maintained at a temperature just below the solidification point of the thermoplastic material to assure good interlayer adhesion. 62 31  10/29/2024 Powder Bed Fusion (Binder Jetting) - 1993  The process deposits a stream of microparticles of a binder material over the surface of a powder bed, joining particles together where the object is to be formed. A piston lowers the powder bed so that a new layer of powder can be spread over the surface of the previous layer and then selectively joined to it. The process is repeated until the 3D object is completely formed. 63 Power Resoluti Starting Technique Principle Speed Advantages Disadvantages Source on (mm) Material Filament Cheap, easy to use and Reduced speed and Material extrusion FDM and deposition Heat Average 0.1-0.3 (Thermoplastic readily available; multi- accuracy; support polymer) material printing needed Potential toxicity; Liquid Laser scanning and requires post Photo- UV induced curing Laser High precision and processing and support; SLA (VAT Beam Average 0.025-0.125 crosslinkable accuracy mechanical polymer Polymerization) properties decrease over time Highly dependent on Thermoplastic Laser scanning and particle size of polymer, metal heat induced Laser No need for support; starting material; SLS sintering (Powder Beam Average 0.1-0.12 and ceramic recyclable feed material mechanical bed fusion) properties vary Metal Powder Low mechanical Binder Drop-on-demand Chemical Polymer No need for support; properties; low Very Fast 0.089-0.12 powder and Jetting binder jetting Energy liquid binder multi-material printing selection of materials 64 32  10/29/2024 Fabrication of 3D-printed solid oral dosage forms (e.g., tablets and capsules) 3D printing can bring the following advantages to tablet/capsule manufacturing: 1. Tailored Dose 2. New Geometries and Designs 3. Accelerated Disintegration 4. Mini-Dispenser Unit 5. Integration with Healthcare Network 6. On-demand manufacturing 65 66 33  10/29/2024 67 Fabrication of transdermal drug delivery systems: 3D-printed microneedles 68 34  10/29/2024 69 Fabrication of 3D-bioprinted tissue/organ-on-chips (Single/Multi): 70 35  10/29/2024 71 Limitations and Future Directions of 3D-Printing in Tissue Engineering and Pharmaceutics General Challenges Outlook in Tissue Engineering Field Outlook in Pharmaceutics Field 72 36  10/29/2024 Limitations and Future Directions: Less cost-competitive at higher volumes “3D printing is the best option when a single (or only a few) parts are required at a quick turnaround time and a low-cost or when the part geometry cannot be produced with any other manufacturing technology.” “3D printing offers great geometric flexibility and can produce custom parts and prototypes quickly and at a low cost, but when large volumes, tight tolerances or demanding material properties are required traditional manufacturing technologies are often a better option.” Post-processing & support removal Printed parts are rarely ready to use off the printer. These usually require one or more post-processing steps. For example, support removal is needed in most 3D printing processes. 3D printers cannot add material on thin air, so supports are structures that are printed with the part to add material under an overhang or to anchor the printed part on the build platform. When removed and they often leave marks or blemishes on the surface of the part they came in contact with. These areas need additional operations (sanding, smoothing, painting) to achieve a high quallity surface finish. 73  Take a lot of time, trial and error to actually get good 3D prints  Require a little bit more setup  Expect to have prints fail  Loud, fumes that result during printing. Some fumes needs to be avoided  This technology is still in its infancy. So, each printing modality possesses its own limitations, including, but not limited to, process resolution, scalability, and availability of compatible bioinks/inks.  After approximately two decades, the possibilities and limitations in the 3D printing of pharmaceuticals have started to form, which has also been the driving force for reviewing regulatory documents to provide up-to-date guidelines for the pharmaceutical manufacturers. Nevertheless, it is expected to take several years, before the legal aspects of pharmaceutical printing are clearly understood.  The technological challenges of pharmaceutical 3D printing are mainly related to the limited availability of suitable materials, the incompatibility of the drugs with the printing conditions and the need for post-processing.  Innervation and vascularization for 3D bio-printed tissues and organs.  Long-term storage of 3D bio-printed tissues and organs without loss of function. 74 37  10/29/2024 References: 1- Palmara, G., Frascella, F., Roppolo, I., Chiappone, A. and Chiadò, A., 2021. Functional 3D printing: Approaches and bioapplications. Biosensors and Bioelectronics, 175, p.112849. 2-Sandler, N. and Preis, M., 2016. Printed drug-delivery systems for improved patient treatment. Trends in pharmacological sciences, 37(12), pp.1070-1080. 3-Jain, A., Bansal, K.K., Tiwari, A., Rosling, A. and Rosenholm, J.M., 2018. Role of polymers in 3D printing technology for drug delivery- an overview. Current pharmaceutical design, 24(42), pp.4979-4990. 4-Awad, A., Trenfield, S.J., Goyanes, A., Gaisford, S. and Basit, A.W., 2018. Reshaping drug development using 3D printing. Drug discovery today, 23(8), pp.1547-1555. 5-Elahpour, N., Pahlevanzadeh, F., Kharaziha, M., Bakhsheshi-Rad, H.R., Ramakrishna, S. and Berto, F., 2021. 3D printed microneedles for transdermal drug delivery: A brief review of two decades. International Journal of Pharmaceutics, 597, p.120301. 6-Bilal, M., Mehmood, S., Raza, A., Hayat, U., Rasheed, T. and Iqbal, H.M., 2021. Microneedles in smart drug delivery. Advances in wound care, 10(4), pp.204-219. 7-Elkasabgy, N.A., Mahmoud, A.A. and Maged, A., 2020. 3D printing: An appealing route for customized drug delivery systems. International Journal of Pharmaceutics, p.119732. 75 38