ABSTRACT
Abundant tissue-to-tissue interfaces, present between bone, tendon and muscle, demonstrate mechanical and structural gradients from soft tissue to hard tissue, which permits cellular communication and load transfer. These allow joint motion and stabilisation and are susceptible to damage through abnormal activity or trauma. A major concern in the field of orthopaedic tissue engineering is the development of engineered bone and soft tissues with biomimetic properties that facilitate tissue regeneration and host tissue integration, as traditional surgical therapies are limited due to donor scarcities and immunological reactions. This project aims to investigate tissue interface development through integration of structural and mechanical gradients. This study involved designing and modelling scaffolds using the software Autodesk AutoCAD 2016 and manufacturing engineered constructs, using the 3D printing technique, and carefully examining scaffolds to refine successive complex models. The performance of the 3D printed scaffold will be tested analysing the cellular response and collecting experimental data in the near future.
Keywords: 3D printing; scaffold; tissue engineering; structural gradient
INTRODUCTION
Tissue Engineering is the branch of Regenerative Medicine that aims to fabricate functional tissue for clinical application and drug testing.[1] It is defined as the combination of cells, biochemical factors and engineering materials to improve or replace biological functions with the scope of stimulating the formation of damaged tissue.[2] Musculoskeletal defects, especially correlated to tissue-to-tissue interfaces, resulting from trauma, tumours or abnormal development necessitate surgical interventions to restore normal tissue function. This however, has associated limitations like pathogen transfer, donor shortages and insufficient supply of autograft tissues.[3] Among the strategies available in fabricating engineered scaffolds, three-dimensional (3D) printing holds a remarkable promise in the providing optimal characteristics for medical applications. In tissue engineering, the ideal scaffold is a three-dimensional biocompatible structure on which cells could migrate and proliferate to form functional tissue and potentially support host tissue integration.[25]
Tissue-to-Tissue Interfaces
Interfaces are surfaces forming common boundaries between musculoskeletal tissues (Figure 1). The musculoskeletal system is comprised of connective tissues that connects adjacent bones (ligament), articulating joints (cartilage) or a muscle to a bone (tendon) to consent transmission of load without creating stress concentrations that would cause tissue damage.[4] Tissue-to-tissue interfaces are essential for coordinated joint motion and musculoskeletal activity.[5] Working on the principles of tissue engineering interfaces like ligament, tendon and cartilage have been successfully created both in vivo and in vitro but the inability of attaining biological fixation (functional integration with the host environment) and the surrounding homogeneous tissue posed considerable obstacles.[6] The fabrication of functionally graded scaffolds aims to overcome these barriers in the upcoming years. The regeneration of tissue interfaces entails the assembly of engineered constructs that will provide chemical and mechanical indications, resulting in biologically precise cellular differentiation.[2]
Figure 1: Schematic representation of the various tissue-to-tissue interfaces present in the human body.[7]
Functionally Graded Scaffold
In regenerative medicine, a scaffold is a growth-directing structural element that provides a platform for cell adherence and supports the differentiation of the desired cell population, direct cellular communication, and promote the formation of functional tissue, once implanted into the body.[8] The production of engineered cellular constructs is the preferred method over surgical therapies as the patient’s own cells are manipulated, which minimises immunological reactions and transmission of diseases. Various materials are available to fabricate scaffolds, depending on the desired strength, flexibility, porosity, and the type of tissue. Hydrogels are considered most suitable for soft tissue and biopolymers used for 3D printing are suitable for hard tissues.[9] Scaffold functionality depends not only on the fabrication technique and on the choice of material, but also on the geometry and inner architecture of the structure, surface properties and characteristics like biodegradability, biocompatibility, and osteoconductivity.[25]
Functionally graded scaffolds (FGS) are porous biomaterials where porosity changes in space with a specific gradient. Low porosity regions offer high mechanical strength, high porosity regions promote, cell adhesion, support cell growth, proliferation and differentiation.[10] Recent studies conducted by Antonio et al. in 2016 showed that the bone formation is maximised utilising a FGS rather than a construct with homogeneous porosity.[10] Natural tissues and interfaces have a gradient porous structure, so it is important to match mechanical strength and stiffness between porous scaffold and the target tissue/interface. The porosity and pore size effects cell behaviour, differentiation potential and mechanical properties. Although higher porosity and pore size may facilitate nutrient and oxygen delivery, or enable more cell ingrowth, the mechanical properties of the scaffold will be compromised due to the large void volume, which will result in a weak structure.[11] Theoretically, scaffolds should have sufficient mechanical strength to maintain integrity until the target interface regenerates, but there should also be adequate space for cells to proliferate and to enable the transport of nutrients and removal of wastes.[11]
The success of an engineered structure depends on the ability to induce surrounding tissue to invade, grow and replace the implanted material.[3]
Technique: 3D Printing
Among all the technologies present, 3D printing has gained particular attention due to its ability to print porous engineered constructs, with defined structural properties and interconnected porosity.[12] In 3D printing, the structure is obtained layer by layer according to the designed computational model, which provides greater control of the architecture and physical properties.[13]
In 2009, only 18% of the patients that were registered for the human organ transplant in United States of America (USA) received the organ they needed to survive. Organ transplants tend not be beneficial to alleviate injuries as complications may arise if the donors tissue cells are not compatible to the patient, which causes severe immunological reactions. Using additive manufacturing, scientists are developing techniques to print living organs that could reduce or completely eliminate the organ transplant shortage, giving everyone an equal second chance.[14]
In this research project, Fused Deposition Modelling (FDM) technique was used to print the designed structures. The FDM method builds objects layer-by-layer from the bottom up by heating and extruding thermoplastic filament, polylactic acid (PLA) in this case, through a small nozzle whose size may vary according to the resolution of the model required.
The aim of the project was to design, model and 3D print a complex functionally graded scaffold with biomimetic properties that resemble the interfaces, developing a new design using the software Autodesk AutoCAD 2016.
Methods
The three-dimensional scaffold designs were created using the software Autodesk AutoCAD 2016.
First, simple models were designed and printed; then more complicated ones were fabricated changing different parameters like porosity, pore size, addition of extruded channels, height, addition of pores, until the final design was obtained.
The most important stages to fabricate the functionally graded scaffold were as follows:
- Designing the structure on Autodesk AutoCAD 2016
- Exporting the model into an .stl file (stereolithography file)
- Importing the .stl file in Makerware or Cura (depending on software compatible to the 3D printer used)
- Optimising the settings on the 3D modelling software, either Makerware or Cura
- Saving the file on the SD card of the printer
- Printing the model on the 3D printer
Computer-Aided Design (CAD)
The first stage of manufacturing the engineered structure was designing the model using a computer-aided design, also called CAD, which was used to generate virtual 2D or 3D models.
CAD is mainly used to assist creation, manipulation, analysis or optimisation of a design.[15].
Once the design was finalised, it was exported into a stereolithography file, commonly known as .stl file, and saved. The stereolithography file is a file format compatible to majority of the computer-aided software, as it reduces technical issues caused when the 3D model is sliced into 2D layers and prepared to print.
3D Printing
The second stage involved optimising the settings and printing using the software accompanied with the 3D printer (Figure 2) used. The majority of the printing was done on MakerBot replicator dual using the accompanied Makerware software.
This printer was limited in nozzle size and hence could not print to the scaled scaffolds utilised for scanning electron microscopy.
Therefore, the samples examined using light microscopy and scanning electron microscopy were scaled down to 12×6 mm (diameter × height) cylinders and printed using Ultimaker 2+ through the 0.4 nozzle that resulted in a high resolution. To print the desired model, a stereolithography was first imported to the software Makerware and the model was positioned on the computer-generated platform. Then, printing settings were optimised to generate the 3D scaffolds. These settings included support, raft, printing and plate temperature.
PLA was fed from a large coil, through a moving, heated printer extruder head. Molten material is forced out of the print head\’s nozzle and was deposited layer-by-layer to generate a 3D object.
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| A. MakerBot | B. Ultimaker 2+ |
Figure 2: 3D printers utilised in the research.
Polylactic acid, the thermoplastic filament used to print the structures, melts at around 180-230℃ and its glass transition temperature in this study is around 60-65℃.
Heating the build plate is important to ensure that the first extruded filament layer is attached to the platform thus preventing warping and improving print quality. When printing with the Ultimaker 2+ the build plate temperature was from 60℃ to 65℃ and the fan speed was decreased from 100% to 80% (which is the air blown through the nozzle to solidify the extruded layers as are laid down). After optimisation, the functionally graded scaffolds were easily fabricated using the Ultimaker 2+.
Imaging
Following the completion of the printing, the next step comprised analysing the samples manufactured through light compound microscopy and electron scanning microscopy (SEM). The specimens were too large to be examined using the light compound microscope but too small to be positioned on the platform. A glass plate was used as a base to place the sample. The final design was scaled down to 12×6 mm to allow analysis.
The investigation under the SEM comprises several stages, as listed below:
- The samples were mounted onto the scanning electron microscopes SEM stubs with carbon adhesive tape to attach them on the stage
- Samples were coated with a layer 0f 10 nm gold
- Samples were imaged in a scanning electron microscope
It was necessary to optimise the settings and ensure that the sample was not damaged during the procedure.
Results
The results suggested that fabricated 3D printed scaffold (Figure 3) exhibit structural, and hence mechanical gradients (Table 1).
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| A. 2D wireframe mode | B. X-Ray mode |
Figure 3: Final functionally graded scaffold
Digital representations of the designed 3D functionally graded scaffold.
3D Printing
Table 1 summarises the relative dimensions of the 3D scaffold designs. The diameter of all the engineered constructs is 20 mm and the height is 10 mm. Pore sizes range from 0-1.8 mm depending on the specific design. Scaffold B and C have uniform pores with the same size as opposed to scaffold D whose pore size alters from 1.10 mm to 1.80 mm.
Mathematical Analysis of 3D Printed Designs
Figure 4 demonstrates how the gradient of pores and channels changes in space.
Figure 4: Mathematical representation.
Graphs shows how porosity changes in space with a specific gradient.
Imaging
As a first step towards creating the functionally graded scaffold, the characteristics it should possess were explored. It was important to obtain uniform balance between structural properties and porosity.
Figures 5-7 represent the vision captured under the light microscope and the scanning electron microscope.
Images show the pore structure (Figure 5), characteristics of scaffold surface topography (Figure 6), the effect of the pattern of the thermoplastic filament deposition (Figure 7) and the inner architecture which, in future experiments, will be investigated for cell proliferation.
Figure 5: Pore structure of functionally graded scaffold.
Larger pores seen from the bottom of the FGS captured with the light microscope.
Figure 6: Illustration of surface topography.
Image was captured from the lowermost side of the scaffold using a light microscope. Pore structure appears to be smooth (arrow indicates surface structure).
Figure 7: Pattern of filament deposition.
The surface of the FGS was captured with the light microscope and appears to be a rough; visible the pattern of the deposition of the filament.
Different settings were altered such as the accelerating voltage, working distance and the probe current, to produce the best possible image under the scanning electron microscope. SEM imaging was performed to investigate the surface of the FGS. Figures 8-12 represent images captured using scanning electron microscopy.
Figure 8: Inner architecture of the FGS.
This shows the inner surface of the functionally graded scaffold, which possesses a generally smooth surface.
Figure 9: Transverse vision.
It exhibits the top vision of the longitudinally sliced engineered biostructure
Figure 10: Porous gradient.
This shows the change in gradient of the horizontal channel of the FGS.
Figure 11: Pore.
The physical appearance of the top small pore at microscopic level.
Figure 12: Filament conformation. This expresses the inner architecture of the PLA filaments of the scaffold.
Discussion
Implications of findings
The final design was a result of increasing the level of complexity of the previous models. The diameter of the vertical pores decreases from 4 mm (4000 μm) to 1 mm (1000 μm). Particular attention was given to the suitability of the functionally graded scaffolds for medical applications. The relative size of a human cell varies from 20-30 μm from which it can be predicted that the cells will easily migrate and proliferate in the scaffold as there is sufficient space for movement, cellular communication and transport of nutrients. Supposing the average size of a human cell being 2016 μm ,the following can be assumed:
Number of cells that could travel through
the bigger pore:
4000 μm ∶ 20 μm = 800
Number of cells that could travel through
the smaller pore:
1000 μm ∶ 20 μm = 50
As shown with the calculation above, the porosity gradient will allow different number of cells to populate the scaffold design.
Materials utilised and their purpose
PLA was chosen as the thermoplastic polymer since it is biodegradable (starts to degrade once exposed to the environment within six months), flexible and easily printable. It is made from renewable resources like starch, sugarcane and was introduced to replace petrochemical-based polymers; the plastic is typically used in medical implants, food packaging and rapid prototyping where the form is more important than the function, like in this case where the efficiency of the engineered structure depends on its physical appearance.[17]
Cellular properties explained
When cells adhere to the surface of a scaffold, a sequence of physico-chemical reactions will occur between cells and the scaffold.[18] Immediately after a scaffold is implanted into an organism or comes into contact with cell culture environments, protein adsorption to its surface occurs which mediates the cell adhesion.
Cells can adhere to surfaces that bind active compounds for cell signalling (through proteins called integrins), proliferation and differentiation. Focal adhesion, interaction between the cell population and the engineered biostructure, depends on various aspects like surface topography (roughness and smoothness), surface hydrophobicity and stiffness.[19] The surface hydrophobicity is the contact angle between the cell and the biostructure.
Studies conducted by Chang H. et al. (2011) demonstrated that the higher the contact angle, the more hydrophobic the surface is, which is not beneficial in this study because it rarely promotes cell adhesion and proliferation.[20] In future work, the surface hydrophobicity of the functionally graded scaffold could be assessed by measuring the contact angle through water spread of a droplet on the surface.[19] Material surface topography is another important factor influencing cell adhesion and behaviour. Roughness modulates the biological response of tissues in contact with the implant. Material surface roughness has a direct influence in vitro as well as in vivo on cellular morphology, proliferation, and phenotype expression.[20] The response of cells to roughness is different depending on the cell type. For larger cells, such as osteoblasts and mesenchymal stem cells, macroscopic descriptions of the surface roughness could be reasonable as there are more regions of contact between the cell and the scaffold that support cell adhesion, differentiation and proliferation. The roughness of the biomaterial has a major impact in the adhesion and cellular behaviour and exerts direct influence in both in vivo and in vitro. Smooth surface and rough surface have different contact areas with molecules and cells and this difference in contact influences the kind of biological units’ links, and therefore conformation and function. In most of the cases cells prefer rough surface to smooth ones, due to the fact that rough surfaces favor proliferation.[21] For low friction applications, such as implants of orthopaedics joints, biomaterials with detailed finishes are preferred.[22] After the surfaces of the final scaffold design were examined under the microscope, it was concluded that they are predominantly smooth, but the pattern of the thermoplastic filament deposition renders them rough which theoretically should stimulate cell adhesion. The bottom side of the scaffold is smooth compared to the top because as the first layer was extruded it retained the topography of the 3D printer platform, which was smooth. When the smaller pores were analysed it was discovered that they do not have a circular conformation, but rather an irregular conformation, which increases the level of roughness and increases the expected success of the construct once implanted into the body. On a macroscopic level, the overall shape of the scaffold provides boundaries for tissue regrowth.[23] On a microscope level, the material provides a framework and capillary networks for local cell growth and tissue organisation, permitting cell attachment, distribution and proliferation within a controllable microenvironment.[24] A study conducted by Freed et al. (1999) evidenced that a common problem encountered when using scaffolds in tissue engineering is the rapid cells attachment and proliferation on the outer edge of scaffold which restrict cell penetration to the scaffold center.[23] Another important factor to consider is the rate of the thermoplastic filament degradation, which needs to be directly proportional to the rate of the desired cell proliferation, resulting in a balance between surface of the scaffold degrading as the interface regenerates.
Limitations
Difficulties were encountered when scaling down the scaffolds because the size of the nozzle was not small enough to print the smaller channels; different settings were changed until the target design was printed. One limitation was that the printer could replicate the smaller pores only if the diameter of the cylinder was 12 mm or above which resulted it being a large sample to be examined under a scanning electron microscope. Therefore, it was decided to use large specimens and not examine the entire sample but just specific points or regions.
The 3D printing technique, especially the fabrication of functionally graded scaffolds, has the capacity to reduce the transmission of diseases and severe immunological reactions as opposed to metal and ceramic implants, because once implanted into the human body it does not require a successive surgical intervention to remove it. This reflects in being economically and clinically advantageous.
Although it is an intriguing technique, one of the drawbacks is the volume of waste generated. When the extruder starts heating the thermoplastic filament, a huge quantity of the filament is pre-extruded, generating a lot of waste filaments. Additionally, when optimising the settings, a lot of test samples are printed which do not go on to be used. Future work could include assessing the efficiency of the printed construct using biological characterisation and chemical characterisation.
Future work
The cellular response and cellular communication will be analysed. If similar opportunities will arise in the future, it will be intriguing to explore the role of hydrogels in the fabrication of scaffolds and use the bio printer as an alternative to the 3D printer.
Conclusion
This project resulted in the fabrication of functionally graded scaffolds demonstrating structural gradients. It is anticipated that regenerative medicine strategies could regenerate interfaces highly prone to injuries and offer a better quality of life.
It is emphasised that the product of this research project will be tested biologically and chemically, and the experimental data collected will provide direction for future work.
Acknowledgments
I gratefully acknowledge extreme support and guidance from Dr. Yvonne Reinwald from Nottingham Trent University. Sincere thanks to everyone who collaborated with me to the completion of the project.
References
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All figures, unless stated otherwise, by Ravinder Kaur.
About the Author
Ravinder Kaur moved to the UK in 2016 and she currently attends a Sixth Form in Nottingham. This research project was offered through the Nuffield placement scheme and carried at Nottingram Trent University. Passionate about science, she has successfully completed the project.