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Scaffold is an important and useful biomaterial that wide been apply in tissue engineering nowadays. Although it just looks like a simple thing and small in shape but it able to gives support to the target cell group and behaviour like an alignment to the cell in a period of time. To achieve the optimal goal of scaffold in tissue engineering field even in medical field, a scaffold must be biodegradables and biocompatible which does not brings any negative feedback or reaction to the surrounding cells and tissue. Besides, the scaffold should be able to withstand certain level force and pressure since the scaffold may implanted at the bone of our body as target area. Therefore the shape, internal properties and the mechanical strength of the scaffold will be the most important features in affect of the performance for the scaffold. In this thesis, it is discussing on the most current fabrication technique of scaffold in how to overcome the conventional fabrication technique somewhere to compare the advantages by using the high advance technology technique fabrication. In addition, it also compares among the high advance technology technique by two different concept of the technique but in same collection, there are FDM and 3DP in SFF technique.
Tissue engineering is a combination field of knowledge in biology, material and medicine which all apply into engineering field. It has become important nowadays due its potential as a field of method to repair, enhancing or replace and also maintaining the cell and tissue functions (Langer R, 1993). The application field can be done by tissue engineering is wide enough; in term of practical it is always associated and emphasis into reparative usage of tissue which mean repairing or replace the portion of the organs in our body either in vitro or in vivo. Sometime, tissue engineering is defined as regenerative medicine to gives our body replacement of tissue by using the stem cells.
Generally, tissues engineering can be categorized into two categories in reparative field as in vitro construction of cell or tissues outside the body and in vivo alteration of cell growth which perform inside the body. No matter it is in vitro or in vivo; tissue engineering has applying the concept of scientific principles to make a design, modification, and construction for the living cell and tissues (Berthiaume & Yarmus, 2003). For the in vitro, isolation has been performing by using the enzymatic dissociation of donor tissue. Some called as 'bioartificial tissues' is been developed from this natural tissue and used to be an alternative for the organ transplantation. It can be useful to study the tissue morphogenesis and its function more deep and pratical through this method. In another hand, in vivo tissue engineering mostly affect on the alteration of tissue growth by implanted some biomaterial like scaffold to enhance and increase the growth ability of tissue around that region. This scaffoding process will promoted the connection for those damaged cells and tissue to perform adhesion process again thoughout the scaffodl surface thus improve the regenerative rates of the tissue. Therefore, tissue engineering can be summarize into three major components (Bell, 2000; Sipe, 2002) as in figure 1.1 below.
Figure 1. Three component of tissue engineering
Scaffold in Tissue Engineering
Scaffold is a supporting biomaterial structure mostly been used in implantation. It is an artificial material and can be applying into either in vivo or ex vivo conditions. The scaffold generally been divided into two types, as two dimensional scaffold and three dimensional scaffold. Three dimensions scaffold give more advantages than two dimension scaffold in term of scaffold porous matrix hence provide the increment in flexibility and functionality for its whole structure (Chen, 2008). Biocompatibility and biodegradable are the most important features of the scaffold no matter there are two dimensions or three dimensions. A biocompatible scaffold will not give a negative response or reflection to the surrounding area after performs the reaction in microenvironments of the cell. For the biodegradable scaffold, it wills breakdown into its original material normally is belonging to nature material and also coincide with the rates of tissue formation as many as possible. Furthermore, a biodegradable scaffold will prevent surgical removal after the process which benefit to the patients nowadays as well. Therefore, basically a high porous scaffold is serving to provide the support for cells to perform adhesion and migration. Besides, the biodegradability of the scaffold provides a structure integrity to surrounding cells and tissue meanwhile they are building their own matrix. Moreover, scaffold with those features are able to enhance the biochemical factors and environment thus improve and modify the cell phase as well as the tissue. The figure 1.2 has shows the relationship of important features for three dimensional scaffold in order to gives the desire advantages.
Figure 1. The relationship of important features for three dimensional scaffolds
Conventional techniques of Scaffold fabrication
A scaffold basically fabricate through two types of material, there are either natural or synthetic material to produce constant microarchitecture in pores structure. Natural-delivered materials are more promising in the early of research for the scaffold material, but in order to increase the feasibility finding more materials for clinical applications thus the trend has been change to concerns on other than natural materials that's come out with synthetic materials in scaffold (Wakitani, 1994).
Solvent-casting and particulate leaching
Solvent casting and particulate leaching are the one of the basic methods to produce a scaffold with regular porosity. In solvent casting, the material will dissolve into suitable organic solvent follow by casting into a desired shape of mold with porogen particles. The organic solvent normally will be evaporating after the mixing process and leave behind the material matrix and porogen throughout the region (Sachlos & Czernuszka, 2003). The composite structure then will undergoes particulate leaching process by mean fully immerse into suitable liquid bath to dissolving the porogen inside the scaffold in the mold. A porous structure material call as scaffold is obtained at the end of process with the porosity which depends on the porogen sizes and its respect ratio to the materials. The general advantage from this method of fabrication is producing the scaffold in a simplest way without any complexity of achievement. In another hand, there are some limitation somewhere for the shapes and thickness of the scaffold itself. It means the scaffold thickness must achieve some level to fulfil the desired porosity. Same for the shapes, this method will not process a complex shape of scaffold and typically produce a scaffold in tube or flat shapes. And lastly a disadvantage is that the risk of solvent may affect on the activity of the cell cause death due to toxicity of the organic solvent (Chen, 2008).
Gas foaming is an alternative fabrication method aim to replace used of organic solvent as mention in previous method; solvent casting and particulate leaching method. There is a new technique using gas as a porogen to replace the need of solid porogen. The gas foaming method is started by fabricate a disc shape scaffold material through compression molding together with heated mold. The solid disc shape scaffold material is then exposing to the high pressure of carbon dioxide inside a chamber. And the chamber is adjusted so that the pressure is gradually restored back to atmospheric level after a period of time. A pore structure scaffold is formed at the end of process due to the result of existing gas bubbles along the gas foaming process by mean the carbon dioxide has abandon the scaffold material after restoring to normal atmospheric level condition thus giving a constant range of pore size (Sachlos & Czernuszka, 2003). The only disadvantages in gas foaming are difficulty of cell seeding and migration due to unconnected pores structure and also the excessive heat used in the compression process that limit the incorporation of bioactive molecules (Antonios & Johnna, 2000).
Phase separation is defined as a technique same with SCPL method which does not using a solid form porogen. It is based on a phase separation concept in thermally rather than incorporation porogen concept (Antonios & Johnna, 2000). Requirement for the phase separation is that using a liquid solvent typically having a low melting point which is easy to sublime in the reaction. By adding small quantity of water and lower the temperature of suspension may induce a layer separation between the material and porogen. Since the porogen will be turn into solid form first compare to the materials then it will forcing the materials into the interstitial spaces of the porogen. A small porous scaffold is formed after perform, the vacuum drying process to eliminate the solvent which is the ice solvent evaporation (Chen, 2008; Sachlos & Czernuszka, 2003). .
Emulsification and freeze-Drying
Emulsification and freeze-drying technique basically are similar to phase separation method and does not using a solid porogen. At first the material of then scaffold will dissolve into suitable solvent and water to form an emulsion. The emulsion is then casting into a mold and in the same time it will placing into a liquid nitrogen tank to give frozen effect before it separated into two phases. Since the emulsion is in one phase of solution only thus forcing the materials access into the in the interstitial spaces of the porogen. And when the freeze-dying process is performs to remove solvent and water from the emulsion then it will leave a solidified porous structure scaffold at the end (Sachlos & Czernuszka, 2003). The advantages of this technique are the ability to producing small pores and saving time in term of time consuming in preparation compare to SCPL method.
Polycaprolactone & Hydroxyapatite
Polycaprolactone is defined as a group of polyester normally consists of physical properties in low melting point, approximately 60 degree Celsius and glass transition temperatures around negative 60 degree Celsius (Liu, 2007). It is a useful polymer and prepared through the ring opening polymerization of Îµ-caprolactone after performing chemical synthesis process from crude oil. There are some catalyst are added into the synthesis reaction to enhance the polymerization process and yield high molecular weight polymers. The reaction will provide with heat along the process until a high permeability polycaprolactone product is success forming at the end (Moore & Saunders, 1997). The product is resist in water even immerse in oil and some others solvent with standard mechanical strength in order to withstand the force and compression in some condition. Thus, polycaprolactone has wide been used in biomedical field as a biodegradable polymer especially for implantation due to its internal degradable properties and do not influence to cells or tissue during in vivo testing plus exhibit low toxicity all the ways (Liu, 2007).
Hydroxapatite is kind of natural bioceramic material which able to support bone ingrowth and integrate bone structure. It is calcium apatite based mineral form normally been found in bone or teeth of human body. Generally, hydroxyapatite are highly bioactive and biocompatible due to its osteoconductive and osteoinductive behaviors (webster, 2000; smith 2004). Therefore, it has common been used as spacers, filler in polymer-based bone substitutes, and bone graft substitutes in orthopedic and maxillofacial applications to gives enhancement to the bonding with natural bone (Gibson,2002; Engin, 1999; Azevedo, 2003). Besides, hydroxyapatite also had been applied in coating of metallic implant which will alter the surfaces properties to avoid isolation reaction from the tissue in the surrounding of implantation sites.
A composite scaffold fabricated through the combination of Polycaprolactone and Hydroxyapatite materials is called as PCL/HA scaffold which aim to reduce the weakness of PCL material in term of mechanical strength and increase the ability in promote the cells attachment for whole scaffold material. Theoretically, it can be achieve due to the osteoconductive and osteoinductive behaviors of HA plus HA itself is not stable thermally and cannot success in a long period load bearing application. Therefore through the combination with PCL, it will take the both advantages from those materials to enhance the further usage of the scaffold which has been aim in the bone engineering field. In the early stage of the experiment, the PCL/HA scaffold has shown a favorable interactions between the cell and scaffold material for the bony tissue (Azevedo, 2003; Daiwon, 2004; Rizzi, 2001). Therefore, an assumption can be making which PCL/HA scaffold is a potential material in bone tissue engineering applications in the future.
Fabrication of Polycaprolactone & Hydroxyapatite Scaffold
A scaffold with polycaprolactone mixing hydroxyapatite has been successful fabricated through the conventional methods. Particulate leaching, solvent casting and gas foaming methods like what have mention in previous chapter are able to produce a PCL/HA scaffold as final product, but with some limitation. The first limitations are the thickness and the shape of the fabricate scaffolds. Conventional fabrication techniques are not able to produce a scaffold in complex way since there is limitation in the shape of mold casting which just can gives a rod or tube shape. The thickness of the foam structure scaffold play an important role in determines the diffusion constraints level of the cells. It mean the cells cannot goes through deeply region of the scaffold if there is too thick cause by the lack of oxygen and nutrients and then the cell colonization is exist where also become a barrier to block the cells continue to goes further thus reduce the mass transfer of the interior section. Besides, the interconnection of the pores in the scaffold is very weak and almost depends on the connection between material and porogen. In addition, the control of pore sizes is difficult in the scaffold and the sizes just can be categories in a range of sizes but not in a specific or desired dimension. Furthermore, the porogen which must used in the conventional technique may not be fully remove from the scaffold due to the thickness limitation and only scaffold with thin layer is able to perform this action. The usage of organic solvent in conventional technique also seems to be a big challenge due to the risk of toxicity. As conclusion, conventional techniques are unable to control the pore geometry as well as the pore sizes distribution and also the internal constructions within the scaffold thus produce a spatial distribution product at the end (Sachlos & Czernuszka, 2003). To solving these problems, the trend has been changed into using high technology techniques to produce desired shapes and generate perfect interconnection within the scaffold to increase the efficiency of diffusion and mass transfer for oxygen and nutrients to overcome the limitation of conventional fabrication problems.
Solid freeform fabrication
Solid freeform fabrication or rapid prototyping is a high technology collection technique used to automatic and precisely control those parameters which cannot be solve by conventional techniques. Be specific, it is manage to modified the morphology of the internal structure in the scaffold by fabricated three dimensional objects through layer or additive manufacturing process. The three dimensional objects is designing by using a computer aided design software and the data is insert into the SFF machine to finish the fabrication process. Although there are a lots of techniques can be use in SFF but there are two major strategies technique current have success been used in SFF to fabricate the PCL/HA scaffold. There are Fused Deposition Modeling and Three Dimensional printing technique in SFF.
Fused Deposition Modelling
FDM is one of the major SFF technique used to fabricate a scaffold in advance. The concept been apply in fused deposition modeling is just like what have mention in SFF that using additive principles used of a thermoplastic filament supply the materials from a chamber to an extrusion nozzle. A nozzle tip will melt and extrude the materials in layers for both horizontal and vertical direction on the support bed. This process was repeated until forming the whole product at the end as same like what have been drawing through the computer aided design software. Therefore, interconnection structure problem can be easy be solved through this technique since we can design a shape that consist of constant or uniform pore sizes to enhance the adhesion of the cell in the scaffold. Moreover, it has provided the cell with sufficient oxygen and nutrient due to the optimal thickness of the scaffold thus provide the critical solution for conventional technique. In addition, FDM machine will model all the movement automatically after key in all the required parameters and gives the desired products after the material has hardens directly after the extrusion layer by layer thus reduce the possibility of human errors. Normally the FDM material is acrylonitrile butadiene styrene polymer but it may be change to desired polymer according to the application. If FDM machine is used to fabricate a mold first instead of fabricate for the scaffold itself, then we call this method as indirect FDM technique.
Three dimensional Printing
3DP also been defined as a common SFF technique to fabricate a Tissue Engineering scaffold. Its principle is to creating three-dimension object through the inkjet printing concept in successive layers to fabricate a desired scaffold. The whole process is started from creating a design of scaffold through the computer aided design software and then key into the 3DP machine to run it automatically. The machine consists of an ink-jet head used to delivering the chemical binder through the liquid adhesive supply and the material powder is supply by a powder delivery system pump. There is a piston at the bottom of the 3DP machine to increase or decrease the position of the powder bed after layering with those powders. And the powder bed is the place where the binders dissolve into the powder material. The 3DP technique is having the advantages that are fast and simple among the rapid prototyping methodologies. In contrast, there is an additional technique called as indirect 3DP technique that can also be used to fabricate scaffold. It using same concept by mean designing through the computer aided design software and run by the 3DP machine. The different is the computer aided design software used to design a mold for the scaffold instead of design the scaffold itself. There is an experiment has success to prove that the indirect 3DP technique able to overcome some limitation of direct 3DP technique. The limitation is included the nozzle of 3DP just able to give a small droplets in the deposition process which has limited the diameter of extruded material thus cannot provide a scaffold with optimal porosity (Lee, 2005).
Objectives of thesis
Nowadays the scaffold has wide been used in medical and tissue engineering field. And Polycaprolactone & Hydroxyapatite scaffold is having greatest potential in tissue engineering field especially for the bone tissue application. Therefore, a lot of experiments have been carried out toward it not only to fabricate the scaffold but also figure out the ways to enhance the fabrication methods in advance. In this thesis, it will carry out the high advance technologies technique to fabricate the PCL/HA scaffold.
The objectives of the study are as below:
To fabricate a Three Dimensional Polycaprolactone/Hydroxyapatite scaffold by using different techniques of Solid Freeform Fabrication.
To compare the mechanical strength properties of the scaffolds by different techniques of Solid Freeform Fabrication.
In vitro testing for the scaffolds.
Fabrication of scaffolds
(i) Indirect Fused Deposition Modeling
The experiment is start from preparing the materials, PCL and HA. PCL is direct bought from sigma with 900 Molecular weight and HA is prepared by our own. HA is obtains from a bovine bone, which has keep in the refrigerator before the experiment start. Then the bovine bones are cut into few parts and just leave the bones with either tibia or femur part only. The remaining tibia or femur of the bovine is undergoes boiling process for three times, one hours for the first time follow by forty five minutes and twenty minutes. After that all the bones is put into an oven in 60 degree Celsius for 96 hours. The bones can be start sawing after that period to obtain the HA powder. Then the HA powder is keep into a bottle. A three dimensional design of a mold with pore sizes of 500µm is draw by using the computer aided design software; Solid Work software. The design data is then uploads into the FDM machine and the required parameters are filled. The FDM machine read through the design and fabricated the mold automatically in a range of time. The mold is taken out from the FDM machine and immersed into distilled water. In another way round, the HA powder is dissolve into solvent like acetone to form a solution. And the solution is putting into a round flask and undergoes stirring around 3 hours through the mechanical stirrer. PCL solution with 900 MW is dissolves into HA/solvent solution to get a homogenous solution which is in the slurry form. Then the stirring process is repeated for 3 more hours. The slurry solution is removed from the flask and placing into the mold and let for 24 hours in room temperature to be solidified. The scaffold is takeout from the mold after 24 hours and immerse again into the distilled water. Lastly, the scaffold is dry at room temperature.
(ii) Indirect 3D Printing
A Same material of PCL and HA materials are using in this indirect 3DP fabrication. The experiment is start by designing a three dimensional mold with pore sizes of 500µm which is optimal pore size for bone ingrowth through the computer aided design software; Solid Work software. Then the design data is uploads into 3DP machine to undergo fabrication process. The mold that success been fabricated through the 3DP machine is immersed into the distilled water. Again, the HA powder is dissolve into solvent like acetone to form a solution. And the solution is putting into a round flask and undergoes stirring around 3 hours through the mechanical stirrer. PCL solution is then slowly added into the HA/solvent solution and the mechanical stirring is repeated for another 3 hours. A homogenous solution in the slurry form is obtained. The mold is taken out from the distilled water and the slurry solution is place into the mold follow by the solidification for 24 hours. The scaffold is removed from the mold after 24 hours and immersed into distilled water. Lastly, the scaffold is dry at room temperature.