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Regenerative medicine is a field of medicine keen to treatments in which stem cells are induced to differentiate into the specific cell type required to repair and regenerate damaged tissues. Bone Tissue Engineering (BTE) stated as the implantation of a scaffold seeded with appropriate population of seed cells and growth factor at the bone defect site. Bone is one of the hardest tissues of the human body and is the main constituent of the skeleton. Its role is to provide structural support to the body and protects vital organs and consists of bone marrow, where the blood cells are formed. Bone is a dynamic material as it is able to undergo self-repair, changes with aging and immobilisation.
In human, a bone marrow aspiration yields 50 mL of whole bone marrow. Bone marrow derived mesenchymal stem cells (MSCs) have been recommended as an appropriate selection for cell-based tissue engineering therapies because of their ease of aspiration and isolation (Ballas et al., 2002) , highly proliferation capacity (Bruder et al., 1997a; Haynesworth et al., 1992a; Banfi et al., 2002) and have self-renewal capacity with the potential to differentiate into mesodermal lineages and therefore can form cartilage, bone, adipose tissue, tendons, ligaments, skeletal muscle and the stroma of connective tissue. A small number of cells are removed from the body and they are screened for phenotype and increased in number through proliferation. These cells are seeded onto porous scaffolds together with growth factors to enhance proliferation. The seeded scaffolds are placed in culture to further increase cell number. Osteogenic differentiation of MSCs in vitro is induced by the presence of dexamethasone, ascorbic acid and β-glycerol phosphate (Jaiswal et al., 1997). Finally, the regenerated tissue is implanted into the site of damage to integrate with the natural tissue.
Figure 1: Tissue Engineering Cycle
Different types of scaffold have been used to achieve different goals. Typically, an ideal scaffold should have meet several design criteria, for example the scaffolds should be biocompatible, biodegradable, suitable surface chemistry for cell attachment and proliferation, the material should be reproducibly processable into three-dimensional structure, and mechanically strong. Poly(lactide-co-glycolide) (PLGA) and bioactive glasses were chosen to be the scaffold materials.
Simplicity in design should also mean that the bioreactor is quick to assemble and disassemble. A flow perfusion bioreactor could conceivably allow marrow stromal osteoblasts to proliferate throughout a cultured scaffold through enhanced nutrient delivery/waste removal and also enhance osteoblastic differentiation through added mechanical stresses.
STEM CELLS FOR BONE TISSUE ENGINEERING
In the early life and growth, stem cells have the remarkable potential to develop into many different cell types in the body. Stem cells differ from other cell types as all stem cells have three general properties, regardless of their source. The properties are:
Capable of renewing themselves through mitotic cell division after long periods of inactivity (also known as cell proliferation)
Unspecialized as they does now have any tissue-specific structures
Ability to give rise to specialized cell types (also known as cell differentiation)
With characteristics consistent with cells of various tissues, such as muscles or nerves, stem cells can now be grown and transformed into specialized cells through cell culture. In some adult tissue, such as gut and bone marrow discrete population of stem cells are regularly divide to repair and replace worn out or damaged tissues caused by normal wear or tear, injury and disease.
Bone Marrow Derived from Mesenchymal Stem Cells (MSCs)
Mesenchymal Stem Cells (MSCs) were originally enriched in the bone marrow, which made up of about 0.001%-0.01% of total population nucleated cells in bone marrow. Bone marrow is the flexible tissue found in the inner part of bones. In the field of bone tissue engineering, the non-hematopoietic fraction of bone marrow can be a valid source of stem cells. In the 1950s, bone marrow was discovered to contain at least two types of stem cells, such as:
Hematopoietic stem cells, forms all types of blood cells in the body
Bone marrow stromal stem cells (also known as MSCs)
MSCs are an example of adult stem cell and they are multi-lineage stromal cells that can differentiate into a variety of cell types by in vitro or in vivo into bone cells (osteoblasts), cartilage cells (chondrocytes), fat cells (adipocytes) and other kind of connective tissue cells such as those in tendons. Figure 1 shows the differentiation of MSCs.
Figure 1: Mesenchymal Stem Cells Differentiation
MSCs can produce different variety of cells belonging to our skeletal tissues, where these specialized cells have their own characteristics shapes, structures and functions, such as bone, cartilage and fat. Figure 2 shows the bone cells made from MSCs. MSCs are easily accessible from patient's multiple tissues, such as bone marrow, adipose tissue and peripheral blood.
Figure 2: Bone cells made from MSCs
MSCs are thought to have promising potential in therapeutics. Autogenetic MSCs are feasible without a risk of immune rejection. The safety of potential bone repair methods was investigated and used in early clinical trials because of the ability of MSCs to differentiate into bone cells. Possible treatments for localized skeletal defects showing the damage at particular place in the bone was then observed.
The purpose of a cell isolation procedure is to maximize the yield of functionally viable, dissociated cells in as high purity as possible. Density gradient centrifugation is the most widely used method to isolate MSCs from human bone marrow. It is a method that enables the separation of particles, either on the basis of their buoyancy density or their rate of sedimentation. Figure 3 shows the example of density gradient centrifugation technique.
Figure 3: Density Gradient Centrifugation
The cell fraction to be separated is placed on top of the layer and centrifuged. Bone marrow sample is carefully loaded onto the premade Percoll gradient and centrifuged for 30mins at speed 400xg. Percoll has been widely used for separating cells, organelles and viruses and contains suspension of colloidal silica particles, which have a diameter if 15-30nm).
Table 1: Application for Density Gradient Media
Cell sorter and flow cytometer were used to identify MSCs. Cell sorter can rapidly separate the stem cells from other cells present in a tissue whereas flow cytometer is obtained by different type of marker. Number of molecular markers can be used to identify a particular type of stem cell.
In order to fast-track the pace of discovery, flow cytometry combined with the growth of new antibodies can expand cell population purity and provide faster multiparametric analysis. Nowadays, cell surface markers are used to phenotype and isolate stem cells. Figure 4 shows the Flow Cytometric Analysis of MSCs, where the MSCs from bone marrow were analysed for expression of surface markers. MSCs expressed the known positive CD29, CD44 and CD90 markers whereas CD34 and CD45 were not detected on these cells as the markers known to be negative.
Figure 4: Flow Cytometric Analysis of Mesenchymal Stem Cells
Cell Expansion Medium
The medium initiate to be most suitable for studies of this type was a 1:1 mixture of Dulbecco's Modified Eagle's Medium (DME) and Ham's F-12 Nutrient Mixture. This mixture was found to support the growth of a wide range of mammalian cells under serum free conditions when supplemented with hormones, growth factors and additional nutrients.
Dulbecco's Modified Eagle Medium (DMEM) is a medium consist of the basic ingredients for cell growth, such as amino acids, glucose, pH indicator, salts and vitamins. This medium contains phenol red to identify changes in pH. It gives a red colour to the medium at neutral pH, where the medium turns yellow when the pH is too low and turns magenta when the pH is too alkaline. Nutrient Mixture F-12 act as supplement to the cells and consists of inorganic salts, amino acids, vitamins and so on.
Growth-phase classification is a very important factor for controlling and optimizing the performance by stimulating osteoblast precursors to proliferate. Hence, the following statements are assumed:
All cells are in the growth phase
Cells double with every cell cycle
Growth factor is a naturally occurring substance capable of stimulating cellular growth, proliferation and cellular differentiation. For example, bone morphogenic proteins (BMPs) stimulate bone cell differentiation; basic fibroblast growth factor (bFGF) stimulates blood vessel differentiation and transforming growth factor-beta (TGF-β) controls proliferation and cellular differentiation. Figure 5 shows that cells cultured in vitro undergo four major phases of growth. The curve is appearing slowly along the line and stabilizing. During the lag phase, the rate of plant growth is slow while rate of growth then increases gradually during the exponential phase. However, after some time the growth rate decreases slowly due to limitation of nutrients, which constitutes the stationary phase.
Figure 5: Different phases observed during the in vitro cell culture
To estimate how long it will take to produce sufficient cells for the therapy to be successful, the following assumptions are done. 2-109 amount of cells is needed for bone therapy. Typical bone marrow donation is 10ml (Shiplu, et al., 2012) while the number of cells in 10ml is 4- (Aplenc, et al., 2002). By assuming 0.001% (Sung, et al., 2012) of all bone marrow nucleated cells is MSCs, 4000 MSCs is to be used from the sample. For the time expansion, 24 hours for one division (Promo Cell, 2012) is assumed.
Exponential growth (Sundararajan, V., 2010) is expressed as:
where X is the number of cells at time
is the initial number of cells seeded
is the specific growth rate constant with unit
By calculating the approximate t-value will be 13 days.
Cell differentiation is a process in which a generic cell develops into a specific type of cell in response to specific triggers from the body or the cell itself. This is the process which allows a single celled zygote to develop into a multicellular adult organism which can contain hundreds of different types of cells. Cell Differentiation can be done in two stages, which are α-MEM induction medium and Osteogenic Differentiation. Typical osteogenic differentiation media includes:
Dexamethasone stimulates alkaline phosphatase activity and enhanced matrix mineralization
Ascorbic acid enhances proliferation without loss of differentiation potential
β-glycerophosphate provides mineralization to modulate osteoblastic activities by promoting a bone-like mineral phase
The differentiation of MSCs in vitro largely depends on the culture conditions, for examples:
Maintained at 37°C
5% CO2, 95% O2 in a humidified atmosphere
The extracellular matrix (ECM) is also critical to tissue structure because it provides attachment sites for cells and relays information about the spatial position of a cell. Assuming differentiation and depositing of extracellular matrix occurs as one stage, the time typically is 21 days (Shiplu, et al., 2012). However, with the reaction conditions, extensive osteogenesis can be achieved in 18 days.
The purpose for scaffold is to enable cells attachment and migrate onto or within the scaffold and cell proliferation and differentiation (Vats et al., 2003). There are many types of materials that can be used to make a scaffold such as natural, synthetic, ceramic and composites. An ideal scaffold should have following properties:
Three dimensional and highly porous with an interconnected pore network for cell growth and flow transport of nutrients and metabolic waste
Biocompatible, biodegradable and bioresorbable with a controllable degradation or resorption rate to match cell or tissue growth in vitro and in vivo to facilitate remodelling
Suitable surface chemistry for cell attachment proliferation and differentiation
Mechanical properties to match those of the tissues at the site of implantation
Scaffold chosen is a porous PLGA-bioglass composite scaffold. The scaffold designed is a 3D porous structure that allows the MSCs to seed onto the highly porous structure of the scaffold. From a biological perspective, it makes sense to combine polymers and bioceramics to fabricate. It is a combination of a naturally occurring polymer and biological apatite.
Our composite foam tries to imitate the complexity of bone tissue. Bone has as well polymeric (collagen) and inorganic (hydroxyapatite) component, whose structure contributes to bone's high toughness. The structural relationship between the mechanical properties of bone material and its component phases at the various levels of hierarchical structural organization is shown in Figure 6. This hierarchically organized structure has an irregular, yet optimized, arrangement and orientation of the components, making the material of bone heterogeneous and anisotropic.
Figure 6: Hierarchical Structural Organization of Bone
Mechanical and Biological Advantages of the Composite Foam
Bone is a composite of collagen and hydroxyapatite. Bone is strongest in compression, weakest in shear stress and intermediate in tension.
Mechanically strong, with similar mechanical properties to cancellous bone:
Young's modulus: 51 MPa (50-500 for bone), much lower for other composites (Chen, 2008)
Compressive strength: 0.42 MPa (2-12 for bone), usually lower for other composites (Chen, 2008)
High porosity (75%) and lower pore size (100 μm) than for many other materials (Pamula, 2011)
Slow but adjustable degradation kinetics depending on composition and structure. Degradation from 1 to 6 months (Lee, 2007), can cope with the several weeks period time necessary for bone tissue regeneration
Good biological properties (Pamula, 2011):
enhancement of bone marrow cell proliferation
osteoinductive properties, promotion of osteoblast and osteoclast formation
enhancement of HA crystallisation
Properties can be tailored to specific functions, controllable degradation rate
Induce bone-bonding, vascularization and mechanically strong
Degradation produces products that may inhibit function
Stiff and brittle
Calculations for the Surface Area of Scaffold
Assuming cells are circular with diameter of 10-10-6m.
Area of cell calculated based on formula, is 7.85-10-7 cm2.
Since the minimum cell count must be 2-109, so the number of cells per cm2 is:
= 1.27-106 cm2
Hence, the surface area of scaffold is 1570 cm2.
-Scaffold is submerged in cell suspension in tank.
- left for 24 hours for cells to attach.
Sizing of the Scaffold Structure
There are few assumptions should be made before calculating the size of the scaffold structure:
Pore diameter of 100 µm
Scaffold porosity (É¸) of 0.75
From the calculation obtained from Section 3.4, the surface area of scaffold required is 1570 cm2. Therefore, void volume (Vv) is equal to 3.93x10-6 m3. Thus, total volume is calculated as 5.23x10-6 m3 or 5.23 cm3.
After a period of time the tissue fills the pore, at this point a diffusive flux of nutrients to the cells may be assumed at static conditions. The diffusivity of the structure can be modelled as that of a hydrogel. The tissue size will be the same size as the scaffold size (5.23 cm3), which is adequate for numerous medical applications.
Modelling of Static Conditions
In general, the material balances equation is shown as below:
Accumulation = Input-Output-Consumption
where Accumulation = 0
Input - Output =
Consumption = R
Hence, the general material balances equation obtained is
Assuming C0 is feed concentration and Cmin is minimum concentration to sustain a cell, then if the value of L is less than 5.23m (assuming a cubic structure) then cell necrosis will occur.
BIOREACTORS IN BONE TISSUE ENGINEERING
A tissue engineering bioreactor can be defined as a device that uses mechanical means to influence biological processes, to stimulate cells and encourage them to produce extracellular matrix (ECM) in a shorter time period and in a more homogeneous manner than would be the case with static culture. Bioreactors can be used in tissue engineering applications to overcome problems associated with traditional static culture conditions, improve cellular distribution and accelerate construct maturation (Freed, et al., 2006). In general, bioreactors are designed to perform at least one of the following five functions (Vunjak-Novakovic, et al., 1998, Bancroft, et al., 2003):
provide a spatially uniform cell distribution
maintain the desired concentration of gases and nutrients in culture medium
facilitate mass transport to the tissue
expose the construct to physical stimuli
provide information about the formation of 3D tissue
In contrast convective mixing (spinner flasks) and convective flow (flow perfusion) can improve initial cell seeding and homogeneity, and thereby improve tissue architecture (Freed, et al., 2006). Several different bioreactors have been designed for tissue engineering application to improve mass transfer into larger tissue constructs. Among these are pinner flask, culture dishes and flasks, compression, strain and flow perfusion. All of these bioreactors rely on forced media flow through and/or around the scaffold to provide nutrient and waste exchange within the scaffold. One bioreactor design that improves mass transfer at the interior of three-dimensional scaffolds is the flow perfusion bioreactor
Flow Perfusion Bioreactor
Flow perfusion bioreactor (Figure 8) is a promising in vitro strategy to engineer bone tissue as they supply oxygen and nutrients needed and tries to emulate flow of blood in the body. This bioreactor improves mass transfer at the interior of three-dimensional scaffolds and applies an osteoinductive mechanical stimulus to osteoblasts within large porous three-dimensional scaffolds.
Flow of nutrients
Scaffold construct with cells
Figure 8: Schematic Diagram of Flow Perfusion Bioreactor
Generally, this bioreactor consists of a pump which is use to perfuse medium continuously through the interconnected porous network of the seeded scaffold (Figure 9) and a scaffold chamber joined together by tubing, with the presence of a media reservoir. Scaffold is kept in position across the flow path of the device while media is perfused through the scaffold, hence enhancing fluid transport. Flow is directed through the scaffold structure passing through the pores, providing nutrients to the cells as well as removing waste from the cultured cells.
Figure 9: Flow Perfusion Culture
The perfusion bioreactor offers enhanced transport of nutrients throughout the entire scaffold by mitigating both external and internal diffusional limitations because it allows medium to be transported through the interconnected pores of the scaffold.
Bioreactor Design Requirements
To investigate the use of flow perfusion culture for bone tissue engineering, there are several requirements for a successful flow perfusion system design, such as:
Deliver the flow through the scaffolds, minimizing the non-perfusing flow that goes around each cultured scaffold, which offers many advantages over the mixing provided by a reactor such as a spinner flask.
The constructs cultured must have a repeatable, controllable, and consistent rate of flow as consistency must be maintained to correctly draw conclusions about the results obtained for comparisons both within and between experiments.
Ability to be sterilized and maintained in this sterile condition throughout the culture period as contamination can alter the results, causing incorrect conclusions to be made.
The system should be reasonable to operate. The more complicated and troublesome a system becomes, the more room for extraneous factors to unintentionally complicate the effects and thereby alter the results.
Mechanical forces play an important role in defining the structure of natural tissue such as bone and a wide diversity of laboratory devices have been established in mechanical conditioning of cell and tissue cultures. Flow perfusion bioreactor generates a more functional construct that have been conditioned to loads they will encounter in vivo. Pulsatile flow pattern changes the flow rate between an upper and lower limit, which enhances the cellular response over steady state (Kavlock, 2008). Compressional conditioning increases mineralisation, though this may result in infection, and limits the scaffolds that can be used due to the compressional force. As bone cells are known to be stimulated by mechanical signal, the amount of shear stress experienced by cells cultured in a flow perfusion system can be varied simply by varying the flow rates through the system. When the flow increases oestrogenic differentiation, proliferation mineralisation and spatial arrangement of the extracellular matrix on the scaffold, shear stresses will be provided.