TISSUE DEVELOPMENT WITH TISSUE ENGINEERING APPROACH

PRESENTED BY

FELIX CHIBUZO OBI (20144610) MSc.

SUPERVISOR: PROFESSOR S. ISMET DELILOGLU GURHAN

INTRODUCTIONTissue Engineering is the development and practice of combining scaffolds, cells, and suitable biochemical factors (regulatory factors or Signals) into functional tissues. The goal of tissue engineering is to assemble functional constructs that restore, maintain, or improve damaged tissues or whole organs.

Cells are the building blocks of tissue, and tissues are the basic unit of function in the body. Generally, groups of cells make and secrete their own support structures, called extracellular matrix. This matrix, or scaffold, does more than just support the cells; it also acts as a relay station for various signaling molecules. Thus, cells receive messages from many sources that become available from the local environment. Each signal can start a chain of responses that determine what happens to the cell. By understanding how individual cells respond to signals, interact with their environment, and organize into tissues and organisms, Tissue Engineers are now able to manipulate these processes to amend damaged tissues or even create new ones.

STEM CELLS TECHONOLOGY Stem cells are undifferentiated biological cells that are capable of

differentiating into specialized cells and can divide through Mitosis to produce more stem cells. Most current strategies for tissue engineering depend upon a sample of autologous cells from the diseased organ of the host.

However, for many patients with extensive end-stage organ failure, a tissue biopsy may not yield enough normal cells for expansion and transplantation. In other instances, primary autologous human cells cannot be expanded from a particular organ, such as the pancreas. In these situations, pluripotent human embryonic stem cells are envisioned as a viable source of cells because they can serve as an alternative source of cells from which the desired tissue can be derived.

Embryonic stem cells exhibit two remarkable properties: the ability to proliferate in an undifferentiated but pluripotent state (self-renew), and the ability to differentiate into many specialized cell types. They can be isolated by immunosurgery from the inner cell mass of the embryo during the blastocyst stage (4-5 days after fertilization), and are usually grown on feeder layers consisting of mouse embryonic fibroblasts or human feeder cells. More recent reports have shown that these cells can be grown without the use of a feeder layer, and thus avoid the exposure of these human cells to mouse viruses and proteins. These cells have demonstrated longevity in culture by maintaining their undifferentiated state for at least 80 passages when grown using current published protocols.

ORGAN DEVELOPMENT FROM EMBRYONIC STEM CELLS

Human embryonic stem cells have been shown to differentiate into cells from all three embryonic germ layers (Endoderm, Mesoderm and Ectodrem) in vitro. In addition, as further evidence of their pluripotency, embryonic stem cells can form embryoid bodies, which are cell aggregations that contain all three embryonic germ layers, while in culture, and can form teratomas in vivo.Due to the Ethical concern of the Human Embryonic stem cell, some Tissue Engineers are working with Mesenchymal Stem cell (MSCs) to develop Tissues, such Tissue have no risk of rejection by the body.

STEN CELLS TECHNOLOGY CONT.

NEW DEVELOPMENT IN EMBRYONIC STEM CELL

SCAFFOLDS IN TISSUE ENGINEERINGA scaffold is a material that can be formed in the shape of tissue that

needs to be replaced. The scaffold can be biologically derived or a synthesized material.  The scaffold material must be biologically compatible for human implantation. The scaffold is typically impregnated (seeded) with a patient’s cells before implantation.

Typically, Scaffolds are synthesized from Biomaterials. These Biomaterials replicate the biologic and mechanical function of the native Extracellular Matrix (ECM) found in tissues by serving as an artificial ECM. As a result, biomaterials provide a three-dimensional space for the cells to form into new tissues with appropriate structure and function, and also can allow for the delivery of cells and appropriate bioactive factors (e.g., cell adhesion peptides, growth factors), to desired sites in the body. Because the majority of mammalian cell types are anchorage dependent and will die if no cell-adhesion substrate is available, biomaterials provide a cell-adhesion substrate that can deliver cells to specific sites in the body with high loading efficiency. Biomaterials can also provide mechanical support against in vivo forces such that the predefined three-dimensional structure is maintained during tissue development. Furthermore, bioactive signals, such as cell-adhesion peptides and growth factors, can be loaded along with cells to help regulate cellular function.

The ideal biomaterial should be biocompatible in that it is biodegradable and bioresorbable to support the replacement of normal tissue without inflammation. Furthermore, the biomaterial should provide an environment in which appropriate regulation of cell behavior (e.g., adhesion, proliferation, migration, and differentiation) can occur such that functional tissue can form. Cell behavior in the newly formed tissue has been shown to be regulated by multiple interactions of the cells with their microenvironment, including interactions with cell-adhesion ligands and with soluble growth factors.

Generally, three classes of biomaterials have been used for engineering tissues: naturally derived materials (e.g., collagen and alginate), Acellular tissue matrices (e.g., bladder submucosa and small intestinal submucosa), and synthetic polymers (e.g., polyglycolic acid (PGA), polylactic acid (PLA), and poly(lactic-co-glycolic acid) (PLGA). These classes of biomaterials have been tested in respect to their biocompatibility. Naturally derived materials and acellular tissue matrices have the potential advantage of biologic recognition. However, synthetic polymers can be produced reproducibly on a large scale with controlled properties of their strength, degradation rate, and microstructure.

Synthetic Breast ScaffoldsFigure a & b: Synthetıc ScaffoldsFıgure c & d: Acellular Scaffolds

SCAFFOLDING APPROCHES IN TISSUES ENGINEERINGThere are basically four major scaffolding approaches for tissue engineering.(Fig. 1). Table 1 (on next slide) highlights some of the working principles and the characteristics of these approaches.

Scaffolding approach (1) Pre-made porous

scaffolds for cell seeding

(2)Decellularized extracellular matrix for cell seeding

(3) Confluent cells with secreted extracellular matrix

(4) Cell encapsulated in self-assembled hydrogel

Raw materials

Processing or fabricating technology

Strategy to combine with cells

Strategy to transfer to host tissues

Synthetic or natural Biomaterials

Incorporation of porogens in solid materials; solid free-form fabrication technologies; techniques using woven or non-woven fibers

Seeding

Implantation

Allogenic or Xenogenic Tissue

Decellularization technologies

Seeding

Implantation

Cells

Secretion of extracellular matrix by confluent cells

Cells present before extracellular matrix secretion

Implantation

Synthetic or natural biomaterials able to self-assemble into hydrogels

Initiation of self-assembly process by parameters such as pH and temperature

Cells present before self-assembly

Injection

Table 1: Characteristics of different scaffolding approaches in tissue engineering

Scaffolding approach (1) Pre-made porous

scaffolds for cell seeding

(2)Decellularized extracellular matrix for cell seeding

(3) Confluent cells with secreted extracellular matrix

(4) Cell encapsulated in self-assembled hydrogel

Advantages

Disadvantages

Preferred applications

Most diversified choices for materials; precise design for microstructure and architecture

Time consuming cell seeding procedure; inhomogeneous distribution of cells

Both soft and hard tissues; load-bearing tissues

Most nature-simulating scaffolds in terms of composition and mechanical properties

Inhomogeneous distribution of cells, difficulty in retaining all extracellular matrix, immunogenicity upon incomplete decellularization

Tissues with high ECM content; load-bearing tissues

Cell-secreted extracellular matrix is biocompatible

Need multiple laminations

Tissues with high cellularity, epithelial tissues, endothelial tissues, thin layer tissues

Injectable, fast and simple one-step procedure; intimate cell and material interactions

Soft structures

Table 1: Characteristics of different scaffolding approaches in tissue engineering (Cont)

Pre-made porous scaffolds for cell seedingScaffolds are made of degradable biomaterials and these has become the most commonly used and well-established scaffolding approach. This approach represents the bulk of biomaterial research in tissue engineering, leading to enormous efforts in development of different types of biomaterials and fabrication technologies.Many types of biomaterials can be used to make porous scaffolds for tissue engineering provided that a fabrication technology compatible with the biomaterial properties is available

Decellularized ECM from Allogenic or Xenogenic Tissues for cell seedingAcellular ECM processed from allogenic or xenogenic tissues are the most nature-simulating scaffolds, which have been used in tissue engineering of many tissues including heart valves, vessels, nerves, tendon and ligament. This scaffolding approach removes the allogenic or xenogenic cellular antigens from the tissues as they are the sources for immunogenicity upon implantation but preserves the ECM components, which are conserved among species and therefore well tolerated immunologically. Specialized decellularization techniques are developed to remove cellular components and this is usually achieved by a combination of physical, chemical and enzymatic methods. In brief, cell membranes are lysed by physical treatments such as freeze-thaw cycles or ionic solutions such as hypo or hypertonic solutions before separating the cellular components from the ECM by enzymatic methods.

Cell sheets with self-secreted ECMCell sheet engineering represents an approach where cells secrete their own ECM upon confluence and are harvested without the use of enzymatic methods. This is achieved by culturing cells on thermo-responsive polymer, such as poly(N-isopropylacrylamide) coated culture dish until confluence. The confluent cell sheet is then detached by thermally regulating the hydrophobicity of the polymer coatings without enzymatic treatment. Such approach can be repeated to laminate multiple single cell layers to form thicker matrix.

Cell encapsulation in self-assembled hydrogel matrixEncapsulation is a process of entrapping living cells within the confines of a semi-permeable membrane or within a homogenous solid mass. The biomaterials used for encapsulation are usually hydrogels, which are formed by covalent or ionic crosslinking of water-soluble polymers. Many types of biomaterials including natural and synthetic hydrogels can be used for encapsulation provided that the conditions inducing the hydrogel formation or the polymerization are compatible with living cells. Encapsulation has been developed over several decades and the predominating use is for immunoisolation during allogenic or xenogenic cell transplantation.

A Synthetic Hydrogel

GROWTH FACTORS IN TISSUE DEVELOPMENT

A growth factor is a naturally occurring substance capable of stimulating cellular growth, cellular growth, proliferation, healing, and cellular differentiation. Usually it is a protein or a steroid hormone. Growth factors are important for regulating a variety of cellular processes.

Growth factors typically act as signaling molecules between cells. Examples are cytokines and hormones that bind to specific receptors on the surface of their target cells.

They often promote cell differentiation and maturation, which varies between growth factors. For example, bone morphogenetic proteins stimulate bone cell differentiation, while fibroblast growth factors and vascular endothelial growth factors stimulate blood vessel differentiation (angiogenesis).

TISSUE DEVELOPMENTBasically Tissue development begins with building a scaffold from a wide set of possible sources, from proteins to plastics. Once scaffolds are created, cells with or without a “cocktail” of growth factors can be introduced. If the environment is right, a tissue develops.  In some cases, the cells, scaffolds, and growth factors are all mixed together at once, allowing the tissue to “self-assemble.” 

 

Another method to create new tissue uses an existing scaffold. The cells of a donor organ are stripped and the remaining collagen scaffold is used to grow new tissue. This process has been used to bioengineer heart, liver, lung, and kidney tissue. This approach holds great promise for using scaffolding from human tissue discarded during surgery and combining it with a patient’s own cells to make customized organs that would not be rejected by the immune system.

CONCLUSION Tissue engineering efforts are currently underway for

virtually every type of tissue and organ within the human body. Because tissue engineering incorporates the fields of cell transplantation, materials science, and engineering, personnel who have mastered the techniques of cell harvest, culture, expansion, transplantation, and polymer design are essential for the Successful application of this technology. Various engineered tissues are at different stages of development, with some already being used clinically, a few in preclinical trials, and some in the discovery stage. Recent progress suggests that engineered tissues may have an expanded clinical applicability in the future because they represent a viable therapeutic option for those who require tissue replacement. More recently, major advances in the areas of stem cell biology, tissue engineering, and nuclear transfer techniques have made it possible to combine these technologies to create the comprehensive scientific field of regenerative medicine.

REFERENCEShttp://www.nibib.nih.gov/science-education/science-topics/tissue-engineering-and-regenerative-medicine

http://www.regenerativemedicine.net/Tissue.htmlhttp://rsif.royalsocietypublishing.org/content/

8/55/153http://jasn.asnjournals.org/content/

15/5/1113.full.pdf+htmlhttp://www.ncbi.nlm.nih.gov/pmc/articles/

PMC2587658/http://en.wikipedia.org/wiki/Growth_factorJohn P, Fisher A, Mikos J, Tissue Engineering. CRC

Press, Taylor & Francis GroupProfessor S. Ismet Deliloglu Gurhan Tissue

Engineering Lecture Note, Department of Biomedical Engineering, Near East University.

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