The Tissue Engineering And Regenerative Medicine

Published Date: 02 Nov 2017

1. Discuss the importance of mechanotransduction in tissue engineering. What factors should be considered when biomechanically engineering functional tissues.

The purpose of tissue engineering is to repair or replace tissues and organs by delivering cells, scaffolds, DNA and proteins. Previous studies have planned that for the repair of tissue defects, remote and expanded cells should be inserted within biodegradable three dimensional (3D) scaffolds [1]. Four main factors such as cell types, scaffolds, biochemical factors and mechanical forces, need to be considered when biochemically engineering functional tissues. It is also evident that physical factors such as cell differentiation, proliferation, gene expression and signal transduction control biological processes [2].

Extracellular matrix (ECM) is the supporting material for cell adhesion, growth and differentiation. ECM also provides mechanical support to tissues [3]. ECM is composed of collagen, elastic fibres, glycosaminoglycans (GAG) and glycoproteins. These components are synthesised by cells inside the ECM and their functions are activated by mechanical stimulation [4]. Mechanotransduction is the process by which cells in the body transfer mechanical stresses into biochemical signals to control their function. The significant component of the mechanotransduction process is the ECM and the initial responses to mechanical stimuli are recorded at the boundaries of cell-ECM contacts [4]. Cells regularly experience mechanical stimuli. Shear-stress, compression and tension are parameters of physical stimuli. These factors modify the organisation and distribution of structural components within cells. They also transform into biochemical inputs that adapt signalling networks between cells [4].

Fluid flow devices have been developed to study mechanotransduction in vitro because fluid shear stress plays an important role in mechanotransduction. An example is the adaptation of blood vessels to changes in blood flow [5]. In blood vessels, enhanced blood flow, during exercise, leads to expansion of the vessel, to guarantee a constant blood pressure. This response depends on the endothelial cells, which sense the increased blood flow and produce intercellular messengers such as prostaglandins [5]. The smooth muscle cells around the vessel relax, to allow the vessel to increase in diameter, due to the reaction of the messengers.

Recent research is focused on recognizing cellular mechanisms that contribute to the mechanotransduction response. Extensive cellular components including cytoskeleton, adhesion complexes and ion channels have been implicated as the primary mediators of mechanotransduction. These factors suggest that the creation of successful tissue engineering implants depend on the control of mechanical forces [6]. In a recent study it has been established that nanoscale topographies were able to accelerate human mesenchymal stem cells (MSCs) to construct bone material in vitro, without the need of osteogenic supplements [7].

Ion channels are associated with several studies involving chondrocyte mechanotransduction. Mechanical stimulation resulted in intracellular Ca2+ waves which were reduced by gadolinium. Gadolinium blocks stretch-activated channels [8]. It has been shown in previous studies that pressure effects various ion transport pathways. Pressurization of the gas phase above cells in culture to 16 kPa stimulated membrane depolarization in chondrocytes. This reaction can be eliminated by tetrodotoxin, a Na+ channel blocker [9].

Integrins are important mediators of cell-matrix interactions and should be considered when biomechanically engineering functional tissues. They act as cellular mechanosensors due to the connection between the extracellular matrix (ECM) and the actin cytoskeleton [8]. A previous study showed that α5β1 integrin, actin cytoskeleton and several kinases (an enzyme that relocates phosphate groups from high energy donor molecules to particular substrates) are involved in the response of chondrocytes to mechanical strain. Therefore a integrin regulated interleukin (IL-4) secretion pathway has been planned to initiate chondrocyte mechanotransduction [10].

Cytoskeleton is another important factor of mechanotransduction [11]. If there is a lack of cytoskeleton, then the cadherins do not function as cell adhesion molecules and do not transmit mechanical signals [12]. Mechanical stretching of rat vascular smooth muscle cells stimulates extracellular signal kinase. This process must take place in an microfilament network [13].

The major signalling pathways involved in gene regulation by mechanical loads are shown in Figure 1. The mechanical loads produce mechanotransduction pathways via cell and nucleus deformation due to hydrostatic pressure, compression and electromagnetic signals [14].

DRAW DIAGRAM FROM REFERENCE 1.

2. Discuss the molecular basis of gene therapy and the use of viral gene delivery systems for the treatment of human disease. What effect did the death of Jesse Gelsinger have on this technology and why?

There are two types of gene therapy. They are germ line gene therapy and somatic gene therapy. In somatic gene therapy genetic material is inserted in the target cells, but the alteration is not passed along to the next generation. In germ line gene therapy the modified gene will be passed on to the next generation. This difference is important because legislation today only allows gene therapy on somatic cells [15]. Cancer is the most common disease treated by gene therapy. 60% of all ongoing clinical gene therapy trials worldwide involve cancer, followed by monogenetic and cardiovascular diseases (Figure 2) [15].

Classical gene therapy requires that cloned genes are established in the patient's cells in order to defeat the disease. Therefore this therapy involves investigating diseased cells within human tissue [16]. In many gene therapies the cells which are being analysed are healthy immune system cells. This process is known as immune system-mediated cell killing. Another process is delivering gene products from cells at a isolated location [16]. In this process genes may be targeted primarily to a certain type of tissue. At the same time the gene products may be distributed to an inaccessible position. There are two methods which can be used to transfer genes. They include the transfer of genes into patient cells inside the body (in vivo) and the transfer of genes into patient cells outside the body (ex vivo) [16].

In vivo gene transfer the cloned genes are transferred completely into the tissues of the patient. If cells cannot be cultured in vitro in adequate numbers (brain cells) then this is a patient's only option for treatment [16]. Liposomes and specific viral vectors are gradually being used for this function. Ex vivo gene transfer involves cloned genes being grown in culture which then transform into cells (figure 3). The cells that grow successfully in culture are selected and then expanded by cell culture in vitro (inside a laboratory). Once this process is complete they are then introduced into the patient [16]. Cells that need to be treated are collected from the patient and grown in culture. They are then introduced into the same patient. These cells are known as autologous and are used to avoid immune system rejection. This method is only appropriate in patients whose tissues can be removed from the body. The tissues are then transformed genetically and implanted into the patient where they will survive for numerous years (skin cells) [16].

Mammalian virus vectors have been the preferred choice for the transportation of genes because of their prominent efficiency of transduction into cells. Oncoretroviral vectors are RNA viruses that can synthesize a corresponding DNA structure due to transcriptase [17]. Retroviruses distribute a nucleoprotein complex into the cytoplasm of diseased cells. Retroviruses are very efficient at transferring DNA into cells which may lead to a permanent cure for a disease. Currently 60% of all approved clinical protocols use retroviral vectors for gene delivery [17]. Adenoviruses are DNA viruses that cause an infection in the upper respiratory tract, with a very high transduction efficiency. They are an accepted delivery system in gene therapy and have numerous advantages. They can be produced at very high concentrations in culture and they are able to contaminate non-dividing human cells. Endocytosis is the process involving the entry of adenoviruses into cells [17].

Recent studies have showed that lactic acid bacteria (LAB) can be used as vaccine delivery vectors. The process is carried out using plasmids which articulate bioactive compounds at mucous membranes. They may then be able to generate a suitable immune response [18]. LAB offer numerous advantages over current delivery systems, such as eliminating the use of toxic agents. Strains have also been identified within LAB that offer defence against degradation during transfer through the gastrointestinal tract [19].

Non-viral delivery systems for gene therapy are now being used due to the safety concerns of recombinant viruses for viral gene delivery systems. The death of Jesse Geslinger created a negative effect on gene therapy. In 1999, 18-year old Jesse Gelsinger took part in a gene therapy clinical trial at the University of Pennsylvania in Philadelphia [15]. His body did not produce enough ornithine transcarbamylase (OTC), a liver enzyme that is required for the removal of excessive nitrogen from amino acids and proteins [15]. Jesse's immune system reacted to a high dose of adenovirus, however he died four days later because of multi-organ failure. This case had a negative impact on gene therapy because he became the first patient to die due to the use of a viral gene delivery system [15].

CREATE OWN PIE CHART (http://onlinelibrary.wiley.com/doi/10.1002/jgm.2698/pdf)

Figure 22.3. In vivo and ex vivo gene therapy.

DRAW ABOVE DIAGRAM FOR FIGURE 3.

3. Describe what you feel are the common roadblocks (i.e. hurdles) in the commercialisation of tissue engineering/regenerative medicine products for clinical use. What do you consider to be the most difficult and also the easiest hurdles in taking a technology to full product development?

Recent research has been carried out to address the specific factors that prevent the commercialisation of tissue engineering/regenerative products [20]. Start-up companies, development stage companies and established companies all experience the same hurdle, which is accessing capital for funding additional research required for commercialisation to take place [21]. Previous studies have shown that investors consider three barriers when deciding what tissue engineering/regenerative products to provide funding for. The three obstacles are regulatory pathway simplicity, clinical explanation of the technology and doubts about reimbursement [22]. When considering regulatory pathway simplicity, investors should contemplate the features of the product, its classification (tissue or product), its method of action and pre-clinical in vitro bench research [22]. Sponsors prefer to invest in therapeutic areas such as cardiovascular and renal or biotechnology/medical products [22]. Sponsors also consider the company's life cycle and factors such as risk tolerance, exit strategy and investment strategy priorities. There are few tissue engineering/regenerative companies that have attained commercial status which may explain why they do not consider public investors for funding [23]. Figure 4 below shows the key factors of research in regenerative medicine products for the marketplace.

Clinical trials and biomarker development are further hurdles which are required in taking a technology to full product development. Clinical trials need to be improved so that results are generated quickly [24]. In order to analyse the results from pre-clinical and clinical studies, biomarkers need to be developed quicker. Biomarkers can also distinguish a certain product [24]. This research also reported that companies found it difficult to distinguish their finished products so that the development of quality controls and product release specifications could be facilitated [24].

Companies who wish to achieve successful product commercialisation, must produce research strategies which include market analysis and provide evidence on the clinical need of their product [25]. A product development plan will interest the financial communities to provide funding and achieve product regulatory approval. If companies can familiarise themselves with the regulatory process and consider the important factors for tissue engineering/regenerative products, then this will help improve their commercialisation strategy [25].

Previous studies have shown that standards are needed to ensure stability and quality of regenerative medicine products. Standards would be one of the easiest hurdles in taking a technology to full product development [26]. In 1997 a voluntary company who develops standards was created with the support of the American Society for Testing and Materials (ASTM) [26]. Companies have a higher chance of taking their product to full product development if there is a large enough market and the product is easy to manufacture. Two product strategies which can be used by companies include mass market approach and specialised market [27]. Mass market approach involves marketing a product at a low price which will generate high volume of sales. Specialised market involves marketing a product at a high price, however this will generate low volume of sales [27]. This research also showed that investors would be more interested in regenerative products that tackled an unmet clinical need. The future of regenerative medicine should focus on products that are easier to commercialise and provide a large scale production, such as allogeneic products, which are genetically different but belong to the same species [27].

DRAW DIAGRAM ABOVE FOR FIGURE 4: http://www.oulu.fi/spareparts/ebook_topics_in_t_e_vol4/abstracts/hellman.pdf

4. A number of synthetic polymeric materials with various different properties are available for medical applications like prostheses, implants and tissue engineering matrices. Discuss how the modification of synthetic polymers with peptides such as the RGD motif may overcome the problem of inadequate interaction between polymer and cells, leading in vivo to foreign body reactions, such as inflammation, infections and aseptic loosening.

Synthetic polymers can be biocompatible, degradable, conductive and responsive to stimuli, however they are not capable of the folding and recognition properties that peptides contain [28]. Previous studies have shown that specific amino acid chains bind to receptors on cell surfaces and incite cell adhesion [29]. Fibronectin which is an extracellular matrix protein, contains arginine-glycine-aspartic acid (RGD), therefore cells will attach to the surface and reduce the cell binding activity of fibronectin. This process is important when cultured cells are undergoing adhesion [29]. Polylactic acid (PLA) scaffolds can lead to improved adhesion of osteoblast cells but also supported growth, differentiation and enhanced formation of bone-like tissues. This is due to the plasma of PLA scaffolds being immobilised with RGD [30].

Current research is focused on improving biomaterials by reducing protein absorption, enhancing specific proteins and modifying polymers. These factors will help overcome the problem that exists between polymer and cells which leads to in vivo foreign body reactions [31]. Using proteins has its disadvantages such as the proteins need to be secluded and disinfected from other organisms because they may increase the risk of infection. Infections will increase the degradation of the protein being used [32]. These disadvantages can be resolved by using RGD motifs. RGD motifs as well as other cell recognition motifs demonstrate higher stability in sterile conditions. They are also cost effective and can select one of the cell adhesion receptors [33]. RGD peptides should be attached to synthetic polymers by functional groups like hydroxyl. There are many synthetic polymers that do not have functional groups, therefore they have to be attached to RGD peptides by blending or co-polymerisation methods [34]. Figure 5 below shows the chemical RGD sequence. TALK ABOUT In vivo evaluation of RGD functionalized polymers FROM REFERENCE 34.

DRAW ABOVE DIAGRAM FOR FIGURE 5. Reference 34.