Tissue engineering......................pptx

Tissue engineering......................pptx

1. TISSUE ENGINEERING METHODS AND APPLICATIONS

2. What is ‘ TISSUE ENGINEERING ’…

3. Tissue Engineering is... “an interdisciplinary field that applies the principles of engineering and life sciences towards the development of biological substitutes that restore, maintain, or improve tissue function or a whole organ”

4. The term Tissue Engineering (TE) was first presented to the broad scientific community in 1993 by Langer and Vacanti . Tissue engineering aims to restore tissue and organ function by employing biological and engineering strategies to clinical problems. The functional failure of tissues and organs is a severe and costly healthcare problem, as their replacement is limited by the availability of compatible donors .

5. Why Tissue Engineering is Important • Supply of donor organs cannot keep up with demand. • Other available therapies such as surgical reconstruction, drug therapy, synthetic prostheses, and medical devices aren't always successful. • It will eliminate any risk of organ rejection because the new organ would be made from the person's own tissue. • It repairs tissues, organs, and bones successfully • Victims of organ/tissue defects will not have to suffer

6. Principle of Tissue Engineering: The general principles of tissue engineering involve combining living cells with a natural/synthetic support or scaffold that is also biodegradable to build a three dimensional living construct that is functionally, structurally and mechanically equal to or better than the tissue that is to be replaced.

7. Regenerative medicine  is a broad field that includes tissue engineering but also incorporates research on self-healing – where the body uses its own systems, sometimes with help foreign biological material to recreate cells and rebuild tissues and organs.  The terms “tissue engineering” and “regenerative medicine” have become largely interchangeable, as the field hopes to focus on cures instead of treatments for complex, often chronic diseases. This field continues to evolve. In addition to medical applications, non-therapeutic applications include using tissues as  biosensors  to detect biological or chemical threat agents, and tissue chips that can be used to test the toxicity of an experimental medication. Examples include cell therapies (the injection of stem cells or progenitor cells); immunomodulation therapy (regeneration by biologically active molecules administered alone or as secretions by infused cells); and tissue engineering (transplantation of laboratory grown organs and tissues).

8. The Triad of Tissue Engineering Tissue engineering applications typically involve the combination of three pillars: cells, signals, and scaffolds , which represent the “triad of tissue engineering”.

9. Approaches in Tissue Engineering Incorporating the three elements of tissue engineering needs a good scaffolding technique. Over the years, different approaches have been developed, resulting in scaffolds that can support the cells and encourage tissue growth after implantation.

10. Process of Tissue Engineering

11. Step 1: • Get Tissue Sample (Cells) from the body. • Patients own cells • Researches have to break tissue apart using, enzymes that digest the extracellular material that normally holds cells together. • Cells need structure, nutrients, and oxygen Scaffold Step 2: GROWING CELLS INTO NEW TISSUE Cells need a scaffold, For tissue regeneration Scaffold: It gives cells structure on which they need to grow, without them cells are free floating, cannot connect with each other, communicate or form tissue. Scaffold is biocompatible and biodegradable Scaffolds provide the structure that cells need for a certain period of time until they have formed enough tissue to have their own structure. Scaffold dissolves once structure of cells is formed.

12. Step 3: IMPLANTING NEW TISSUE • Bioengineered Tissue Implants Regenerate Damaged Knee Cartilage Science Daily. • Cartilage was removed from 23 patients with an average age of 36 years. • After growing the cells in culture for 14 days, the researchers seeded them onto scaffolds made of esterified hyaluronic acid, grew them for another 14 days on the scaffolds, and then implanted them into the injured knees of the study patients. • Cartilage regeneration was seen in ten of 23 patients, including in some patients with preexisting early osteoarthritis of the knee secondary to traumatic injury. • Maturation of the implanted, tissue-engineered cartilage was evident as early as 11 months after implantation.

13. The potential of stem cells capable of self-renewal- can divide and renew themselves for long periods unspecialized cells that can differentiate into other types of cells

14. SCAFFOLDS : Cells are often implanted or 'seeded' into an artificial structure capable of supporting three-dimensional tissue formation. These structures, typically called scaffolds usually serve at least one of the following purposes: Allow cell attachment and migration. Deliver and retain cells and biochemical factors. Enable diffusion of vital cell nutrients and expressed products. Exert certain mechanical and biological influences to modify the behavior of the cell phase.

15. Requirements for scaffolds • High porosity and an adequate pore size • Biodegradability rate at which degradation occurs has to coincide as much as possible with the rate of tissue formation • Inject ability

16. DIFFERENT TYPES OD SCAFFOLD FABRICATION TECHNIQUES The conventional tissue engineering scaffold production techniques include thermally induced phase separation (TIPS), fiber bonding, electrospinning, solvent casting and particulate leaching, membrane lamination, freeze-drying, and gas foaming . The recent development in scaffold fabrication mainly comprises integration with computer-aided design (CAD) software such as stereolithography , bioplotting , solvent-based free forming, combination modelling technique, fused deposition modelling, 3D printing, and selective laser sintering (SLS) . The techniques retain the ability to maintain pore structures, cell–cell interaction, reduction in mechanical instability, and control over mitigation of the cellular matrix.

17. Scaffold Fabrication : Scaffold fabrication is a critical step in tissue engineering as it provides the structural support necessary for cell attachment, proliferation, and differentiation. There are several methods used to fabricate scaffolds: 3D Printing : Also known as additive manufacturing, 3D printing allows for the precise layer-by-layer deposition of materials to create scaffolds with complex geometries and controlled porosity. This technique enables customization of scaffold properties such as pore size, interconnectivity, and mechanical strength.

18. 2. Electrospinning : Electrospinning involves the use of an electric field to draw polymer solutions or melts into ultrafine fibers. These fibers are collected to form nanofibrous scaffolds with a high surface area-to-volume ratio, which can closely mimic the native extracellular matrix (ECM) architecture. 3. Decellularization : Decellularization is a technique where cellular components are removed from natural tissues, leaving behind an acellular scaffold composed of the ECM. These scaffolds retain the native tissue's biochemical composition and mechanical properties, making them suitable for tissue engineering applications.

19. Cell Seeding Techniques: Once scaffolds are fabricated, cells need to be introduced onto or into them to initiate tissue formation. Several cell seeding techniques are employed: Static Seeding : In static seeding, cells are typically pipetted or seeded directly onto the scaffold surface and allowed to adhere through gravity or gentle agitation. While simple and straightforward, static seeding may result in uneven cell distribution. Dynamic Seeding: Dynamic seeding involves the use of bioreactor systems where scaffolds are placed in a chamber with controlled flow conditions. This allows for better distribution of cells throughout the scaffold, enhancing cell attachment and proliferation.

20. 3. Perfusion Seeding: Perfusion seeding takes dynamic seeding a step further by continuously perfusing cell suspension through the scaffold using a pump system. This method ensures uniform cell distribution and nutrient delivery, promoting cell viability and tissue formation.

21. Tissue Culture Methods : Tissue culture techniques are employed to maintain cells or tissues in vitro under controlled conditions. This is essential for studying cellular behavior, tissue development, and for producing functional tissue constructs. Key tissue culture methods include: 2D Cell Culture: In 2D cell culture, cells are typically grown as monolayers on flat surfaces such as tissue culture plates. While simple and widely used, 2D culture lacks the three-dimensional context present in vivo, which may affect cell behavior and function. 3D Cell Culture: 3D cell culture involves culturing cells in three-dimensional environments that mimic the in vivo tissue architecture more closely. This can be achieved using hydrogels, scaffolds, or spheroid culture methods, providing cells with a more physiologically relevant environment. Bioreactor Culture: Bioreactors are dynamic culture systems that provide controlled conditions such as temperature, pH, oxygen levels, and mechanical stimulation. Bioreactor culture allows for the scaling up of tissue constructs and the simulation of physiological conditions, making it suitable for tissue engineering applications.

22. Advantages and Disadvantages of different types of scaffolds fabrication techniques for tissue engineering application.

23. Various forms of 3D scaffolds

24. APPLICATIONS OF TISSUE ENGINEERING The main goal of tissue engineering is to regenerate and replace human tissues and organs through a combination of biological, clinical, and engineering approaches. Replacing/Regenerating Target Organs • Skin : Tissue-engineered skin aims to restore barrier function to patients for whom this has been severely compromised, e.g., burn patients. Skin is a widely explored engineered tissue, and several commercial skin products are available, e.g., Epicel ® by Genzyme is a product based on autologous cells grown to cover a wound; Apligraf ® by Organogenesis is a dual layer skin equivalent with keratinocytes and fibroblasts on collagen gel; Dermagraft ® by Advanced Tissue Science uses a similar approach with dermal fibroblast on resorbable polymer, among many others . These products treat burns, ulcers, deep wounds, and other injuries. Collagen, fibrin, hyaluronic acid, and poly(lactic glycolic acid) are mainly used in skin substitute matrices. Keratinocytes, melanocytes, and fibroblasts compose the majority of cells in skin tissue, thus expanding and transplanting these cells within biocompatible matrices is key to successful skin regeneration .

25. Liver : Liver transplantation is end-stage treatment for many liver diseases. Several factors including drug use, alcohol abuse, and viruses like Hepatitis can cause acute liver failure. It is important to completely replace the damaged liver or support the patients that wait for donor organs or suffer from chronic liver diseases with tissue-engineered livers . Intense efforts exist to develop a bridging device that can support a patient’s liver function until a donor is available, e.g., dialysis, charcoal hemoperfusion , immobilized enzymes or exchange transfusion. Several extracorporeal systems use patients’ own cells in a hollow-fiber, spoutedbed or flat-bed device, which reduce the chance of immune rejection. Several bioartificial livers (BAL) have been developed and designed to flow patient’s plasma through a bioreactor that houses/maintains hepatocytes sandwiched between artificial plates or capillaries.

26. Pancreas : Diabetes is the fifth highest cause of death . One type of diabetes is type 1 diabetes mellitus (T1DM), an autoimmune disease which destroys the insulin-secreting cells of the pancreas. T1DM is relevant to tissue engineering, since it can be treated by replacing the destroyed pancreatic islet cells. Techniques for pancreatic tissue engineering aim to release insulin from transplanted islets into the blood to restore normal blood glucose levels. Three main approaches are used: a tubular membrane that encapsulates islets and connects to blood vessels; hollow fibers containing islets embedded in a polymer matrix; and encapsulation of islets in microcapsules . The membranes used in the perfusion devices and coatings in microcapsules are developed from biocompatible polymers that allow insulin to diffuse into the bloodstream, while protecting the cells from destruction by immune cells. An insufficient source of islet cells presents a challenge, but recent advances with stem cells may overcome this.

27. Heart Many patients are left with damaged or malfunctioning cardiac tissue that lead to arrhythmias and diminished cardiac output. Tissue engineering is actively pursing treatments for myocardial infarction, congenital heart defects, and stenotic valves through regenerating cardiac tissues. Heart valves are developed by transplanting autologous cells onto a scaffold, growing and maturing the cell-seeded scaffold, and finally transplanting the valves into the patient. Decellularized heart valves consist of extracellular matrix which is repopulated with host cells, but they can potentially produce severe immune response . Alternatively, biomaterial-based heart valves are designed from various natural materials, e.g., collagen, fibrin, and synthetic polymers, e.g., PLGA, poly( hydroxy butyrate) . Biomaterial-based heart valves have advantageous characteristics, including malleability and improved mechanical strength. Cells for cardiovascular applications are usually obtained from donor tissues, e.g , peripheral arteries with mixed populations of myofibroblasts and endothelial cells, as well as established cell lines of myofibroblasts and endothelial cells. There are several successful applications of tissue-engineered heart valves for both in vitro and in vivo models. Additionally, efforts have been made to regenerate myocardium by scaffold-based approaches with natural and synthetic materials

28. Blood vessels: Poly( tetrafluoro ethylene) PTFE and Dacron® grafts have traditionally been used as vascular grafts, particularly for large diameter vessels. However, these grafts are largely unsuccessful for small diameter blood vessels, due to thrombogenicity and compliance mismatch. Tissue engineering strategies therefore provide great opportunities for development of blood vessels. Decellularized arteries with well-preserved extra cellular proteins have been repopulated with cells both in vitro and in vivo to generate blood vessels . Polymer-based scaffolds are often used as a template to guide the regeneration of blood vessels. Also, recently developed microfluidic and microfabrication techniques allow the complex architecture of a blood vessel with defined microstructures to be realized . In an alternative approach, sheets of smooth muscle cells and extracellular matrix are generated under flow conditions in the absence of scaffold, and are subsequently rolled over a support to mature into a vessel structure . An emerging approach focuses on combining endothelial cells with mesenchymal stem cells or smooth muscle cells to help stabilize the blood vessel and help aid in the vessel pruning process . It is important to note that formation of new blood vessels is critical for engineering any tissue to supply nutrients and oxygen to cells.

29. Nervous system: Generation of nerve tissues is a major focus in tissue engineering . Injuries in the central nervous system (CNS) are often accompanied by permanent functional impairment, unlike peripheral nervous system (PNS) damage where the axons are able to re-extend and re-innervate, leading to functional recovery . This is because the environment of the damaged site in CNS blocks the regeneration of neurons. Peripheral nerve grafts that include synthetic or biological substrates have been developed to act as a bridge to guide the nerve regeneration process. To restore the structures lost in the disorganization of axons during injury it is critical to build a bridge that spans the lesion gap with all the morphological, chemical, and biological cues that mimic normal tissue.

30. Bone : There are numerous clinical applications that can benefit from bone regeneration therapies including spinal fusion to alleviate back pain, temporomandibular joint reconstruction to alleviate jaw pain, and restoration of contour and shape within reconstructed craniofacial bone. Significant progress in the development of bone regeneration therapies has been achieved, yet large critical-sized defects which do not heal remain a significant clinical challenge. Although several inorganic-, polymeric-, and hybrid-based scaffolds have been examined to engineer bone, it has been difficult to develop a material that displays optimal mechanical properties and degradation kinetics for bone repair . It is clear that the scaffolds for bone regeneration should have an interconnected macroporosity to allow three-dimensional bone growth throughout the scaffold. Implantation of empty macroporous scaffolds that are devoid of cells typically do not improve the healing response though combining biodegradable scaffolds with cells is a commonly explored strategy

31. Other Applications of Tissue Engineering There are several non-traditional, yet useful and important applications of tissue engineering strategies. In particular, the field of drug screening is in need of advanced in vitro methods for the assessment of the activity and toxicity of drugs before clinical studies are initiated . Since most drugs are metabolized in the liver, microscale hepatocyte systems may allow high throughput screening for liver toxicity . Furthermore, humans or animals “on a chip” are in development. These chips are essentially several reactors with different cell types connected in series which can stimulate the pathway of a chemical substance through several parts of the body, such as the liver, brain, and fat. In the future, tissue engineering-based drug testing and toxicity assays can provide an alternate strategy to reduce the use of in vivo animal testing. Moreover, an artificially engineered tissue-engineered system can provide enhanced control of tissue microenvironments, both in physiological and pathological conditions (e.g., cancer tumor), contributing to the understanding of complex tissues and organs

32. Tissue engineering is a young field that utilizes cells, biomaterials, physical signals (e.g., mechanical stimulation), biochemical signals (e.g., growth factors and cytokines), and their combinations, to engineer tissues. The most common application of tissue engineering is to create tissues that can be used to repair or replace tissues in the body suffering partial or complete loss of function. However, tissue engineering has started to find new applications such as the development of extracorporeal life support units (e.g., bioartificial liver and kidney), in vitro disease models, tissues for drug screening, smart diagnosis, and personalized medicine.

33. Year Achievements Reference 1907 Dr. Harrison Ross first observed living developing nerve fiber Harrison Rose et al., 1907 1948 First artificial Kidney was synthesized Gauvin et al., 2011 1988 3D positioning of cells Klebe RJ , 1988 1993 Term "Tissue engineering" was defined Klebe RJ ,1988 1994 Biofabrication Fritz Monika,1994 1997 Commercially available skin Mac Neil ,2007 2002 Commercially available bone Parikh, 2002 2003 Patent for bio-printer Mironov et al., 2003 2008 Decellularized organ Ott et al., 2008 2010 10-year-old child saved Kalathur et al.,2010 2015 Development of soft tissue Pati et al ., 2015 2019 Development of new stereolithographic process for multi-vascular networks Grigoryan et al.,2019 Timeline for the milestone in Tissue engineering.

34. Cao, Vacanti et al ., (1997) The Vacanti mouse ear was created using a biodegradable polymer scaffold shaped like a human ear. The scaffold was seeded with chondrocytes, which are cartilage cells, and implanted under the skin on the back of a laboratory mouse. Over time, the chondrocytes proliferated and synthesized new cartilage tissue, resulting in the growth of an ear-shaped structure.

35. Different tissue-engineered organs. Scaffold prepared from synthetic biodegradable polyglycolic acid (PLA) in the shape of a 3-year-old auricle. Scaffolds implanted subcutaneously on the back of an immunodeficient mouse. First trachea organ transplant using human's bone marrow stem cells. Constructed artificial bladder seeded with human bladder cells and dipped in a growth solution. Bioengineered kidney that mimics the function of a normal kidney the control of the urinary system and blood filtration. Tissue-engineered heart valve using human marrow stromal cells.

36. Challenges in Tissue Engineering In fact, autologous, allogeneic, and xenogeneic cells are all potential sources, and each of these can be subdivided into stem cells (adult or embryonic) or differentiated cells. Since they all have their own advantages and disadvantages (immune reaction, differentiation, etc.), the choice of the right source for the cells and their culture is a challenge by itself. The choice of scaffold biomaterials is not an easier task either. The scaffolds must actually respond to both the structural and functional requirements of the body. It must be biocompatible and should be able to communicate with the ECM while at the same time providing the needed mechanical support. While natural materials have better biocompatibility and biodegradability, synthetic ones usually present stronger mechanical properties. Another important challenge in tissue engineering is related to the transportation of nutrients and waste secretion in the engineered tissue . Since the majority of tissues rely on blood vessels to transport oxygen and nutrients, the 3D engineered tissue needs to be vascularized with a vascular capillary network . Finally, a major challenge is still present, namely mass production and commercialization of the engineered tissues. Specific manufacturing conditions and quality control strategies need to be ensured.

37. The Future of Tissue Engineering The last few decades have witnessed major steps in health care, leading to improved surgical procedures and better management of diseases. Consequently, the aforementioned advancements have led to an increased demand for tissues and organs. The ultimate goal of tissue engineering is to bridge the constantly growing gap between organ demand and availability by producing complete organs . Stem cells will continue to be investigated for their differentiation potential, and more applications will be developed in the future. The ultimate goal would be to engineer immune-transparent stem-like cells with clear protocols, enabling their committed differentiation to targeted tissues. Developments in basic and applied science related to the fabrication of tissue engineering scaffolds will be a major future target. Potential limitations of such scaffolds will always be the shortage of supplies (e.g., scaffolds from allogeneic sources), potential immunoreactions, and ethical concerns (e.g., scaffolds from xenogeneic origins). In future, it is expected that new biomaterials will be developed incorporating selected molecules to address targeted tissues. Moreover, many basic science studies will be conducted to identify the effects of molecules on cells and determine the right degradation rate and material properties (porosity, mechanical properties, and structural properties) suitable for each tissue engineering applications.

38. Advantage of tissue engineering Tissue engineering provides long term and much safer solution than other options. The traditional transplantation complications are minimized. The donar can be patients himself or herself. The need for donar tissue is minimal. Immuno suppression problem can be minimized. The presence of residual foreign material can be minimized. Disadvantage of tissue engineering Cell isolation and preparation, biomaterial of nutrients transport and transplantation is very complex process. It is difficult to achieve cell diffrentiation into desired cell type and ensuring their nutrient supply after implantation into the body. There may be obstacles to growing cells in sufficient quantities. The time necessary to develops cells in culture before they can be used and possible lack of function at the donar site are some other limitations.

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