3D-Printed Porous Scaffolds Application in Bone Implants

Bone defect treatments remain a clinical challenge as they require synthetic bone scaffolds with strong mechanical and biological properties. There is a need for adequate exchange of waste and nutrients between the implanted bone scaffold and surrounding tissue. 3D-printed polymeric scaffolds can be designed in such a way that ensures that there is a uniform distribution of pore sizes and wall shear stress for proper nutrient exchange along with consistent cell growth and differentiation. Polycaprolactone (PCL) is a thermoplastic polymer mostly employed in bone tissue engineering due to its biocompatibility, biodegradability, processability, and tunable mechanical properties [1]. PCL has proven to produce effective bone cell growth in the form of PCL-based composite scaffolds in combination with a variety of metal, polymer, and ceramic materials [2]. PCL alone has insufficient osteogenic ability and mechanical strength therefore it’s often combined with Zinc metal alloy [3] or hydroxyapatite ceramic with polyethylene glycol polymer [4].

This project aimed to evaluate and demonstrate the effectiveness of 3D-printed porous bone scaffolds in supporting the proliferation of osteoblast cells. The primary goal was to assess how a scaffold design provided by 3D-Orthobiologic Solutions (3DOS) promotes cell proliferation and cell viability. This involved tracking how adipose-derived stem cells (ADSCs) populate the scaffold, differentiate, and interact with polycaprolactone (PCL) alone and in its composite form with an osteoconductive ceramic. After a series of experiments on optimizing the porous scaffolds’ material composition, layer thickness, and chemical treatment were done, it was found that none of the 3D-printed scaffolds were cytotoxic and were able to grow and differentiate into osteoblast cells over a span of 28 days based on fluorescence imaging and assays relevant to osteogenic differentiation.

References:

[1]A. Fallah et al., “3D printed scaffold design for bone defects with improved mechanical andbiological properties,” Journal of the Mechanical Behavior of Biomedical Materials, vol. 134, p.105418, Oct. 2022, doi: 10.1016/j.jmbbm.2022.105418
[2] M. Gharibshahian et al., “Recent advances on 3D-printed PCL-based composite scaffolds forbone tissue engineering,” Front Bioeng Biotechnol, vol. 11, p. 1168504, Jun. 2023, doi:10.3389/fbioe.2023.1168504.
[3] S. Wang et al., “3D-Printed PCL/Zn scaffolds for bone regeneration with a dose-dependent effecton osteogenesis and osteoclastogenesis,” Mater Today Bio, vol. 13, p. 100202, Jan. 2022, doi:10.1016/j.mtbio.2021.100202.
[4] C. C, H. P, P. A, P. Aj, A. F, and Y. J, “Characterisation of bone regeneration in 3D printed ductilePCL/PEG/hydroxyapatite scaffolds with high ceramic microparticle concentrations,” PubMed, 2021,Accessed: Sep. 26, 2024. [Online]. Available: https://pubmed.ncbi.nlm.nih.gov/34806738/

Hi everyone !

My name is Joelle Nguyen and I’m a senior majoring in bioengineering. I participated in OSCAR’s URSP this spring semester to conduct a research project on 3D-printed scaffolds for bone implants. Here’s a table of contents to capture what I will be going over today in this video presentation.

Starting with background, there is a clinical challenge for bone defect treatments as bone implants require synthetic scaffolds with strong mechanical and biological properties. These scaffolds require an adequate exchange of nutrients between the implanted bone scaffold and surrounding tissue and should be able to withstand any external forces or wall shear stress which bone implants usually encounter. 3D-printed polymeric scaffolds pose a potential solution, more specifically 3D-printed scaffolds using polycaprolactone (PCL) – composite blends. PCL-composite blends have proven effective to promote bone growth according to multiple researchers. PCL is a commonly used polymer in bone engineering due to its biocompatibility and ease in manufacturing. Composite blends with PCL combine PCL with a variety of metals, polymers or ceramic materials since PCL alone has insufficient osteogenic ability and mechanical strength necessary for bone growth.

My project aims to evaluate and demonstrate the effectiveness of 3D-printed porous scaffolds made of a composite blend which combines PCL with an osteoconductive ceramic and optimize the scaffold design’s porosity and thickness for future applications. The porous scaffold designs were provided by a 3D Orthobiologic Solutions or 3DOS for short.

Now I’ll go more in depth on the methodology of my research project. First let me start with my research timeline. So back in February there were some preparations made for setting up the experimental design and gathering all the necessary supplies. Then I started with my first experimental trial in late February where I wanted to compare how the thicknesses of the scaffold had an impact on cell proliferation. The next experimental trial started in mid March where I wanted to see how thickness and treating PCL-printed scaffolds with 5 M of NaOH affected cell proliferation & differentiation. Based on literature, NaOH worked to enhance the hydrophilicity of PCL and create a more rough surface for improved cell attachment. The third experimental trial was to compare the osteogenic cell growth of porous scaffolds made with PCL to porous scaffolds made with the composite blend after confirming which experimental conditions showed most cell proliferation in previous trials. Finally I have been working on the data analysis which includes counting all the cells imaged under fluorescence microscopy and performing one-way ANOVA on the data I’ve collected not only on the cell counts but from the multiple assays I’ve done throughout the semester.

The general procedure for each trial looked like the following where I’d sterilize the samples, seed the samples with 20,000 cells/mL of adipose-derived stem cells, stain the samples for fluorescence imaging, and perform assays on specific days. After all is done I work on cell counting and statistical analysis using one way ANOVA to compare between groups.

So I’ll only be discussing the results from Experiment #1. I have only a few fluorescence images here for the Days 1, 4, and 7 where the top row is of the non-porous PCL scaffold and the bottom row is the porous PCL scaffold. The blue circles are cell nuclei and the branches you may see are the cytoskeleton of the cell. These images give me a good idea of how many cells there are on each scaffold as well how well they are spreading throughout the sample.

Next is another set of images for Day 14 and 28 except this time there is green staining done to depict the amount of osteocalcin released from the cells. Osteocalcin is a protein released by osteoblast cells during bone formation. After imaging all samples under the fluorescence microscope, The general trend I found was that there is an increase in average cell count as the amount of layers increases for the scaffold designs. Through a one-way ANOVA analysis it was found that there wasn’t a significant difference in average cell count between the porous and nonporous scaffold designs with the same number of layers, which signifies that a similar amount of cells grow between nonporous and porous samples.

However, cell count does not completely capture what’s happening on the sample. Therefore, I conducted additional assays to determine what kind of cells are present on my samples: are there still adipose-derived stem cells or have these stem cells differentiated into osteoblast cells? First I’d observe the osteocalcin area on the fluorescence images. You can see in this graph that a porous scaffold with 5 layers or 1 mm depth showed the highest osteocalcin area in comparison to other scaffold designs, indicating that the cells imaged may be osteoblast cells. This is reaffirmed by the calcium assay done on Day 14 and 28 as porous scaffolds made with 5 layers, specifically on Day 28, showed a statistically significant concentration of calcium on the sample in comparison to other samples.

Onto the conclusion! Based on experiment 1, scaffolds printed with more layers showed an increase in cells, but there was no statistically significant difference between the number of cells on non-porous and porous scaffolds with the same amount of layers. A statistically significant difference in calcium deposition was observed for the porous layer 5 compared to other samples, meaning that porous scaffolds showed earlier differentiation of stem cells to osteoblast cells. Next steps include completing a statistical analysis for Experiments #2 and #3, testing scaffold designs with smaller pores, and testing additional scaffolds printed from the composite blend filament. Much more information will be provided on my poster during the OSCAR celebration on May 6th. Be sure to stop by if you’d be interested to learn more about how my research turned out.

I’d like to end my presentation with acknowledgements. I’m so grateful for all the support I received throughout this semester. Thank you for listening to my presentation !