Fabrication Of Polycaprolatone And Hydroxyapatite Scaffold Biology Essay

Scaffold is an of import and utile biomaterial that broad been apply in tissue technology today. Although it merely looks like a simple thing and little in form but it able to gives support to the mark cell group and behavior like an alliance to the cell in a period of clip. To accomplish the optimum end of scaffold in tissue technology field even in medical field, a scaffold must be biodegradables and biocompatible which does non convey any negative feedback or reaction to the environing cells and tissue. Besides, the scaffold should be able to defy certain degree force and force per unit area since the scaffold may engraft at the bone of our organic structure as mark country. Therefore the form, internal belongingss and the mechanical strength of the scaffold will be the most of import characteristics in affect of the public presentation for the scaffold. In this thesis, it is discoursing on the most current fiction technique of scaffold in how to get the better of the conventional fiction technique someplace to compare the advantages by utilizing the high progress engineering technique fiction. In add-on, it besides compares among the high progress engineering technique by two different construct of the technique but in same aggregation, there are FDM and 3DP in SFF technique.


Tissue technology is a combination field of cognition in biological science, stuff and medical specialty which all apply into technology field. It has become of import presents due its possible as a field of method to mend, heightening or replace and besides keeping the cell and tissue maps ( Langer R, 1993 ) . The application field can be done by tissue technology is broad plenty ; in term of practical it is ever associated and accent into reparative use of tissue which mean mending or replace the part of the variety meats in our organic structure either in vitro or in vivo. Sometime, tissue technology is defined as regenerative medical specialty to gives our organic structure replacing of tissue by utilizing the root cells.

By and large, tissues technology can be categorized into two classs in reparative field as in vitro building of cell or tissues outside the organic structure and in vivo change of cell growing which execute inside the organic structure. No affair it is in vitro or in vivo ; tissue technology has using the construct of scientific rules to do a design, alteration, and building for the life cell and tissues ( Berthiaume & A ; Yarmus, 2003 ) . For the in vitro, isolation has been executing by utilizing the enzymatic dissociation of donor tissue. Some called as ‘bioartificial tissues ‘ is been developed from this natural tissue and used to be an option for the organ organ transplant. It can be utile to analyze the tissue morphogenesis and its map more deep and pratical through this method. In another manus, in vivo tissue technology largely affect on the change of tissue growing by implanted some biomaterial like scaffold to heighten and increase the growing ability of tissue around that part. This scaffoding procedure will advance the connexion for those damaged cells and tissue to execute adhesion procedure once more thoughout the scaffodl surface therefore better the regenerative rates of the tissue. Therefore, tissue technology can be summarize into three major constituents ( Bell, 2000 ; Sipe, 2002 ) as in figure 1.1 below.

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Figure 1. Three constituent of tissue technology

Scaffold in Tissue Engineering

Scaffold is a back uping biomaterial construction largely been used in nidation. It is an unreal stuff and can be using into either in vivo or ex vivo conditions. The scaffold by and large 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 increase in flexibleness and functionality for its whole construction ( Chen, 2008 ) . Biocompatibility and biodegradable are the most of import characteristics of the scaffold no affair there are two dimensions or three dimensions. A biocompatible scaffold will non give a negative response or contemplation to the environing country after performs the reaction in microenvironments of the cell. For the biodegradable scaffold, it wills breakdown into its original stuff usually is belonging to nature stuff and besides coincide with the rates of tissue formation every bit many as possible. Furthermore, a biodegradable scaffold will forestall surgical remotion after the procedure which benefit to the patients nowadays every bit good. Therefore, fundamentally a high porous scaffold is functioning to supply the support for cells to execute adhesion and migration. Besides, the biodegradability of the scaffold provides a construction unity to environing cells and tissue meanwhile they are constructing their ain matrix. Furthermore, scaffold with those characteristics are able to heighten the biochemical factors and environment therefore better and modify the cell stage every bit good as the tissue. The figure 1.2 has shows the relationship of of import characteristics for three dimensional scaffold in order to gives the desire advantages.

Figure 1. The relationship of of import characteristics for three dimensional scaffolds

Conventional techniques of Scaffold fiction

A scaffold fundamentally fabricate through two types of stuff, there are either natural or man-made stuff to bring forth changeless microarchitecture in pores construction. Natural-delivered stuffs are more promising in the early of research for the scaffold stuff, but in order to increase the feasibleness happening more stuffs for clinical applications therefore the tendency has been change to concerns on other than natural stuffs that ‘s come out with man-made stuffs in scaffold ( Wakitani, 1994 ) .

Solvent-casting and particulate leaching

Solvent casting and particulate leaching are the one of the basic methods to bring forth a scaffold with regular porousness. In solvent casting, the stuff will fade out into suited organic dissolver follow by projecting into a coveted form of cast with porogen atoms. The organic dissolver usually will be vaporizing after the commixture procedure and go forth behind the stuff matrix and porogen throughout the part ( Sachlos & A ; Czernuszka, 2003 ) . The composite construction so will undergoes particulate leaching procedure by mean to the full immerse into suited liquid bath to fade outing the porogen inside the scaffold in the cast. A porous construction stuff call as scaffold is obtained at the terminal of procedure with the porousness which depends on the porogen sizes and its regard ratio to the stuffs. The general advantage from this method of fiction is bring forthing the scaffold in a simplest manner without any complexness of accomplishment. In another manus, there are some restriction someplace for the forms and thickness of the scaffold itself. It means the scaffold thickness must accomplish some degree to carry through the coveted porousness. Lapp for the forms, this method will non treat a complex form of scaffold and typically bring forth a scaffold in tubing or level forms. And in conclusion a disadvantage is that the hazard of dissolver may impact on the activity of the cell cause decease due to toxicity of the organic dissolver ( Chen, 2008 ) .

Gas foaming

Gas foaming is an alternate fiction method purpose to replace used of organic dissolver as reference in old method ; solvent casting and particulate leaching method. There is a new technique utilizing gas as a porogen to replace the demand of solid porogen. The gas foaming method is started by fabricate a phonograph record form scaffold stuff through compaction modeling together with heated cast. The solid phonograph record form scaffold stuff is so exposing to the high force per unit area of C dioxide inside a chamber. And the chamber is adjusted so that the force per unit area is bit by bit restored back to atmospheric degree after a period of clip. A pore construction scaffold is formed at the terminal of procedure due to the consequence of bing gas bubbles along the gas foaming procedure by intending the C dioxide has abandon the scaffold stuff after reconstructing to normal atmospheric degree status therefore giving a changeless scope of pore size ( Sachlos & A ; Czernuszka, 2003 ) . The lone disadvantages in gas foaming are trouble of cell seeding and migration due to unconnected pores construction and besides the inordinate heat used in the compaction procedure that limit the incorporation of bioactive molecules ( Antonios & A ; Johnna, 2000 ) .

Phase Separation

Phase separation is defined as a technique same with SCPL method which does non utilizing a solid signifier porogen. It is based on a stage separation construct in thermally instead than incorporation porogen construct ( Antonios & A ; Johnna, 2000 ) . Requirement for the stage separation is that utilizing a liquid dissolver typically holding a low thaw point which is easy to sublimate in the reaction. By adding little measure of H2O and lower the temperature of suspension may bring on a bed separation between the stuff and porogen. Since the porogen will be turn into solid signifier foremost compare to the stuffs so it will coercing the stuffs into the interstitial infinites of the porogen. A little porous scaffold is formed after perform, the vacuity drying procedure to extinguish the dissolver which is the ice solvent vaporization ( Chen, 2008 ; Sachlos & A ; Czernuszka, 2003 ) . .

Emulsification and freeze-drying

Emulsification and lyophilization technique fundamentally are similar to phase separation method and does non utilizing a solid porogen. At first the stuff of so scaffold will fade out into suited dissolver and H2O to organize an emulsion. The emulsion is so projecting into a cast and in the same clip it will puting into a liquid N armored combat vehicle to give frozen consequence before it separated into two stages. Since the emulsion is in one stage of solution merely therefore coercing the stuffs entree into the in the interstitial infinites of the porogen. And when the freeze-dying procedure is performs to take dissolver and H2O from the emulsion so it will go forth a coagulated porous construction scaffold at the terminal ( Sachlos & A ; Czernuszka, 2003 ) . The advantages of this technique are the ability to bring forthing little pores and salvaging clip in term of clip devouring in readying comparison to SCPL method.

Literature Review

Polycaprolactone & A ; Hydroxyapatite

Polycaprolactone is defined as a group of polyester usually consists of physical belongingss in low thaw point, about 60 degree Celsius and glass passage temperatures around negative 60 degree Celsius ( Liu, 2007 ) . It is a utile polymer and prepared through the ring opening polymerisation of Iµ-caprolactone after executing chemical synthesis procedure from rough oil. There are some accelerators are added into the synthesis reaction to heighten the polymerisation procedure and output high molecular weight polymers. The reaction will supply with heat along the procedure until a high permeableness polycaprolactone merchandise is success organizing at the terminal ( Moore & A ; Saunders, 1997 ) . The merchandise is resist in H2O even immerse in oil and some others solvent with standard mechanical strength in order to defy the force and compaction in some status. Therefore, polycaprolactone has broad been used in biomedical field as a biodegradable polymer particularly for nidation due to its internal degradable belongingss and do non act upon to cells or tissue during in vivo proving plus exhibit low toxicity all the ways ( Liu, 2007 ) .

Hydroxapatite is sort of natural bioceramic stuff which able to back up bone ingrowth and incorporate bone construction. It is calcium apatite based mineral signifier usually been found in bone or dentition of human organic structure. Generally, hydroxyapatite are extremely bioactive and biocompatible due to its osteoconductive and osteoinductive behaviours ( Webster, 2000 ; smith 2004 ) . Therefore, it has common been used as spacers, filler in polymer-based bone replacements, and bone transplant replacements in orthopaedic and maxillofacial applications to gives sweetening to the bonding with natural bone ( Gibson,2002 ; Engin, 1999 ; Azevedo, 2003 ) . Besides, hydroxyapatite besides had been applied in coating of metallic implant which will change the surfaces belongingss to avoid isolation reaction from the tissue in the surrounding of nidation sites.

A composite scaffold fabricated through the combination of Polycaprolactone and Hydroxyapatite stuffs is called as PCL/HA scaffold which purpose to cut down the failing of PCL stuff in term of mechanical strength and increase the ability in promote the cells fond regard for whole scaffold stuff. Theoretically, it can be achieve due to the osteoconductive and osteoinductive behaviours of HA plus HA itself is non stable thermally and can non success in a long period burden bearing application. Therefore through the combination with PCL, it will take the both advantages from those stuffs to heighten the farther use of the scaffold which has been aim in the bone technology field. In the early phase of the experiment, the PCL/HA scaffold has shown a favourable interactions between the cell and scaffold stuff for the bony tissue ( Azevedo, 2003 ; Daiwon, 2004 ; Rizzi, 2001 ) . Therefore, an premise can be doing which PCL/HA scaffold is a possible stuff in bone tissue technology applications in the hereafter.

Fabrication of Polycaprolactone & A ; Hydroxyapatite Scaffold

A scaffold with polycaprolactone blending hydroxyapatite has been successful fabricated through the conventional methods. Particulate leaching, solvent casting and gas foaming methods like what have reference in old chapter are able to bring forth a PCL/HA scaffold as concluding merchandise, but with some restriction. The first restrictions are the thickness and the form of the fabricate scaffolds. Conventional fiction techniques are non able to bring forth a scaffold in complex manner since there is restriction in the form of cast casting which merely can gives a rod or tubing form. The thickness of the froth construction scaffold play an of import function in determines the diffusion restraints degree of the cells. It mean the cells can non goes through deeply part of the scaffold if there is excessively thick cause by the deficiency of O and foods and so the cell colonisation is exist where besides go a barrier to barricade the cells continue to goes farther therefore cut down the mass transportation of the interior subdivision. Besides, the interconnectedness of the pores in the scaffold is really weak and about depends on the connexion between stuff and porogen. In add-on, the control of pore sizes is hard in the scaffold and the sizes merely can be classs in a scope of sizes but non in a particular or coveted dimension. Furthermore, the porogen which must used in the conventional technique may non be to the full take from the scaffold due to the thickness restriction and merely scaffold with thin bed is able to execute this action. The use of organic dissolver in conventional technique besides seems to be a large challenge due to the hazard of toxicity. As decision, conventional techniques are unable to command the pore geometry every bit good as the pore sizes distribution and besides the internal buildings within the scaffold therefore produce a spacial distribution merchandise at the terminal ( Sachlos & A ; Czernuszka, 2003 ) . To work outing these jobs, the tendency has been changed into utilizing high engineering techniques to bring forth coveted forms and generate perfect interconnectedness within the scaffold to increase the efficiency of diffusion and mass transportation for O and foods to get the better of the restriction of conventional fiction jobs.

Solid freeform fiction

Solid freeform fiction or rapid prototyping is a high engineering aggregation technique used to automatic and exactly control those parametric quantities which can non be solve by conventional techniques. Be specific, it is manage to modified the morphology of the internal construction in the scaffold by fabricated three dimensional objects through bed or linear fabrication procedure. The three dimensional objects is planing by utilizing a computing machine aided design package and the information is insert into the SFF machine to complete the fiction procedure. Although there are a tonss of techniques can be use in SFF but there are two major schemes technique current have success been used in SFF to manufacture the PCL/HA scaffold. There are Fused Deposition Modeling and Three Dimensional printing technique in SFF.

Fused Deposition Modeling

FDM is one of the major SFF technique used to manufacture a scaffold in progress. The construct been apply in amalgamate deposition mold is merely like what have reference in SFF that utilizing linear rules used of a thermoplastic fibril supply the stuffs from a chamber to an bulge nose. A nozzle tip will run and squeeze out the stuffs in beds for both horizontal and perpendicular way on the support bed. This procedure was repeated until organizing the whole merchandise at the terminal as same like what have been pulling through the computing machine aided design package. Therefore, interconnectedness construction job can be easy be solved through this technique since we can plan a form that consist of changeless or unvarying pore sizes to heighten the adhesion of the cell in the scaffold. Furthermore, it has provided the cell with sufficient O and food due to the optimum thickness of the scaffold therefore provide the critical solution for conventional technique. In add-on, FDM machine will pattern all the motion automatically after cardinal in all the needed parametric quantities and gives the coveted merchandises after the stuff has hardens straight after the bulge bed by bed therefore cut down the possibility of human mistakes. Normally the FDM stuff is acrylonitrile butadiene cinnamene polymer but it may be alteration to want polymer harmonizing to the application. If FDM machine is used to manufacture a cast first alternatively of fabricate for the scaffold itself, so we call this method as indirect FDM technique.

Three dimensional Printing

3DP besides been defined as a common SFF technique to manufacture a Tissue Engineering scaffold. Its rule is to making three-dimension object through the inkjet printing construct in consecutive beds to manufacture a coveted scaffold. The whole procedure is started from making a design of scaffold through the computing machine aided design package and so identify into the 3DP machine to run it automatically. The machine consists of an ink-jet caput used to presenting the chemical binder through the liquid adhesive supply and the stuff pulverization is supply by a pulverization bringing system pump. There is a Piston at the underside of the 3DP machine to increase or diminish the place of the pulverization bed after layering with those pulverizations. And the pulverization bed is the topographic point where the binders dissolve into the pulverization stuff. The 3DP technique is holding the advantages that are fast and simple among the rapid prototyping methodological analysiss. In contrast, there is an extra technique called as indirect 3DP technique that can besides be used to manufacture scaffold. It utilizing same construct by average planing through the computing machine aided design package and run by the 3DP machine. The different is the computing machine aided design package used to plan a cast for the scaffold alternatively of design the scaffold itself. There is an experiment has success to turn out that the indirect 3DP technique able to get the better of some restriction of direct 3DP technique. The restriction is included the nose of 3DP merely able to give a little droplets in the deposition procedure which has limited the diameter of extruded stuff therefore can non supply a scaffold with optimum porousness ( Lee, 2005 ) .

Aims of thesis

Nowadays the scaffold has broad been used in medical and tissue technology field. And Polycaprolactone & A ; Hydroxyapatite scaffold is holding greatest potency in tissue technology field particularly for the bone tissue application. Therefore, a batch of experiments have been carried out toward it non merely to manufacture the scaffold but besides figure out the ways to heighten the fiction methods in progress. In this thesis, it will transport out the high progress engineerings technique to manufacture the PCL/HA scaffold.

The aims of the survey are as below:

To manufacture a Three Dimensional Polycaprolactone/Hydroxyapatite scaffold by utilizing different techniques of Solid Freeform Fabrication.

To compare the mechanical strength belongingss of the scaffolds by different techniques of Solid Freeform Fabrication.

In vitro proving for the scaffolds.


Fabrication of scaffolds

( I ) Indirect Fused Deposition Modeling

The experiment is start from fixing the stuffs, PCL and HA. PCL is direct bought from sigma with 900 Molecular weight and HA is prepared by our ain. HA is obtains from a bovine bone, which has keep in the icebox before the experiment start. Then the bovine castanetss are cut into few parts and merely go forth the castanetss with either shinbone or femur portion merely. The staying shinbone or thighbone of the bovine is undergoes boiling procedure for three times, one hours for the first clip follow by 40 five proceedingss and 20 proceedingss. After that all the castanetss is put into an oven in 60 degree Celsius for 96 hours. The castanetss can be start sawing after that period to obtain the HA pulverization. Then the HA pulverization is keep into a bottle. A three dimensional design of a cast with pore sizes of 500Aµm is draw by utilizing the computing machine aided design package ; Solid Work package. The design information is so uploads into the FDM machine and the needed parametric quantities are filled. The FDM machine read through the design and fabricated the cast automatically in a scope of clip. The cast is taken out from the FDM machine and immersed into distilled H2O. In another manner unit of ammunition, the HA pulverization is dissolve into dissolver like propanone to organize a solution. And the solution is seting into a unit of ammunition flask and undergoes stirring about 3 hours through the mechanical scaremonger. PCL solution with 900 MW is dissolves into HA/solvent solution to acquire a homogeneous solution which is in the slurry signifier. Then the stirring procedure is repeated for 3 more hours. The slurry solution is removed from the flask and puting into the cast and allow for 24 hours in room temperature to be solidified. The scaffold is takeout from the cast after 24 hours and plunge once more into the distilled H2O. Last, the scaffold is dry at room temperature.

( two ) Indirect 3D Printing

A Same stuff of PCL and HA stuffs are utilizing in this indirect 3DP fiction. The experiment is start by planing a three dimensional cast with pore sizes of 500Aµm which is optimum pore size for bone ingrowth through the computing machine aided design package ; Solid Work package. Then the design information is uploads into 3DP machine to undergo fiction procedure. The cast that success been fabricated through the 3DP machine is immersed into the distilled H2O. Again, the HA pulverization is dissolve into dissolver like propanone to organize a solution. And the solution is seting into a unit of ammunition flask and undergoes stirring about 3 hours through the mechanical scaremonger. PCL solution is so easy added into the HA/solvent solution and the mechanical stirring is repeated for another 3 hours. A homogeneous solution in the slurry signifier is obtained. The cast is taken out from the distilled H2O and the slurry solution is topographic point into the cast follow by the hardening for 24 hours. The scaffold is removed from the cast after 24 hours and immersed into distilled H2O. Last, the scaffold is dry at room temperature.



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