Three-dimensional biofabrication of nanosecond laser micromachined nanofibre meshes for tissue engineered scaffolds

doi:10.12336/biomatertransl.2023.02.005

Biomaterials Translational ›› 2023, Vol. 4 ›› Issue (2): 104-114.doi: 10.12336/biomatertransl.2023.02.005

• RESEARCH ARTICLE • Previous Articles Next Articles

Three-dimensional biofabrication of nanosecond laser micromachined nanofibre meshes for tissue engineered scaffolds

Ross H. McWilliam¹, Wenlong Chang², Zhao Liu³, Jiayuan Wang³, Fengxuan Han³, Richard A. Black¹, Junxi Wu¹, Xichun Luo², Bin Li³, Wenmiao Shu¹^,^*()

1 Department of Biomedical Engineering, University of Strathclyde, Glasgow, UK
2 Centre for Precision Manufacturing, Design, Manufacturing & Engineering Management, University of Strathclyde, Glasgow, UK
3 Orthopaedic Institute, Department of Orthopaedic Surgery, The First Affiliated Hospital, Soochow University, Suzhou, Jiangsu Province, China

Received:2023-01-27 Revised:2023-04-19 Accepted:2023-06-20 Online:2023-06-28 Published:2023-06-28
Contact: Wenmiao Shu E-mail:will.shu@strath.ac.uk
About author:Wenmiao Shu, will.shu@strath.ac.uk.

Abstract

Abstract:

There is a high demand for bespoke grafts to replace damaged or malformed bone and cartilage tissue. Three-dimensional (3D) printing offers a method of fabricating complex anatomical features of clinically relevant sizes. However, the construction of a scaffold to replicate the complex hierarchical structure of natural tissues remains challenging. This paper reports a novel biofabrication method that is capable of creating intricately designed structures of anatomically relevant dimensions. The beneficial properties of the electrospun fibre meshes can finally be realised in 3D rather than the current promising breakthroughs in two-dimensional (2D). The 3D model was created from commercially available computer-aided design software packages in order to slice the model down into many layers of slices, which were arrayed. These 2D slices with each layer of a defined pattern were laser cut, and then successfully assembled with varying thicknesses of 100 µm or 200 µm. It is demonstrated in this study that this new biofabrication technique can be used to reproduce very complex computer-aided design models into hierarchical constructs with micro and nano resolutions, where the clinically relevant sizes ranging from a simple cube of 20 mm dimension, to a more complex, 50 mm-tall human ears were created. In-vitro cell-contact studies were also carried out to investigate the biocompatibility of this hierarchal structure. The cell viability on a micromachined electrospun polylactic-co-glycolic acid fibre mesh slice, where a range of hole diameters from 200 µm to 500 µm were laser cut in an array where cell confluence values of at least 85% were found at three weeks. Cells were also seeded onto a simpler stacked construct, albeit made with micromachined poly fibre mesh, where cells can be found to migrate through the stack better with collagen as bioadhesives. This new method for biofabricating hierarchical constructs can be further developed for tissue repair applications such as maxillofacial bone injury or nose/ear cartilage replacement in the future.

Key words: 3D biofabrication, electrospinning, hierarchical scaffold, micromachining, tissue engineering

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Figures/Tables 7

Figure 1. Design and assembly process for fabricating a 3D construct. (A) CAD model with 3D construction and surrounding box and cylinders. (B) Section of the array in InkScape showing different 2D slice shapes used to construct the pyramid. (C) Laser cut alignment slices with an appropriate number designating order. (D) Shape slices on the bulk material to be cross-referenced with alignment slice and placed accordingly on the construct. (E) Schematic of the steps involved in the assembly process, where accurate 3D reconstructions can be created from the 2D slices: Step 1 - placement of alignment slice 29 on the rig using the four holes; Step 2 - Deposition of a couple of drops of alginate solution using a syringe; Steps 3 and 4 - placement of the appropriate shape slice corresponding with alignment slice 29; Step 5 - starting the process again with alignment slice 28. This process continues until all slices are placed. 2D: two-dimensional; 3D: three-dimensional; CAD: computer-aided design. Created with Inventor? Vension 2018.

Figure 2. Examples of 3D demonstrators assembled according to the novel fabrication method. (A) Camera image of a pyramid. (B) Camera image of a successfully printed cube. (C) Camera image of a successfully printed hemisphere. (D) Three scale models of a human ear (200 μm slices) of three different sizes. (E) The corresponding ear with greater resolution (100 μm slices), which has excellent fidelity for anatomical size and accurately replicates the complex features of the human ear. Scale bars: 10 mm (A–C, E), 50 mm (D).

Figure 3. Desktop scanning electron microscope image of an electrospun polycaprolactone nanofibre mesh to be micromachined and used in in vitro testing. An average nanofibre diameter was 1.801 μm (n = 10) was found. Scale bars: 50 μm.

Figure 4. Morphological assessment of the perforated micromachined fibre mesh. (A, B) First cytotoxicity test of the 500 μm micromachined PLGA mesh. (A) Light Microscopy image of the micromachined array showing good circularity. (B) Live/dead image showing excellent cell viability and the holes clearly defined. (C–F) Light microscopy images of the 200 (C), 300 (D), 400 (E), and 500 (F) μm hole arrays, which show good circularity and even distribution. (G–J) Epifluorescent microscope images of live ADSCs on the micromachined fibre mesh with 200 (G), 300 (H), 400 (I), and 500 (J) μm hole arrays, with excellent cell viability seen across all samples, and ‘bridging' of cells across. (K–M) Light microscope images of three selected micromachined electrospun PCL fibre mesh samples, all micromachined with the same setting but with minor variances as a result of the non-uniform thickness of the PCL nanofibre mesh. Scale bars: 500 μm. PCL: polycaprolactone; PLGA: polylactic-co-glycolic acid.

Figure 5. The average diameter of the micromachined perforations in the electrospun fibre mesh samples. (A) The actual average hole diameter of each array (n = 3 holes) compared to the intended hole diameter of the PLGA samples, where the average diameter can be seen to be close to the intended diameter and a clear difference between each sample type seen. (B) The average diameter of each hole (n = 3 holes) in three representative micromachined electrospun PCL fibre mesh samples, with a wider variance of values around the intended 500 μm hole diameter due to using the same laser settings on the non-uniform thickness of the PCL nanofibre mesh. 3D: three-dimensional; PCL: polycaprolactone; PLGA: polylactic-co-glycolic acid.

Figure 6. Live/dead staining of ADSCs on the alginate and collagen stacks. (A–F) Inverted epifluorescent microscope images after live/dead staining of ADSCs on the alginate stacks. (A) Slice 1 (top slice) showing some good cell viability on day 1. (B) Slice 2 (middle slice) showing very limited cell presence indicating no cell mobility through the construct on day 1. (C) Slice 3 (bottom slice) showing no cells and thus no mobility through the cross-linked alginate on day 3. (D) Slice 1 (top slice) showing good cell viability with some stain intake by the alginate on day 7. (E) Slice 2 (middle slice) showing limited cell viability and thus mobility on day 7. (F) Slice 3 (bottom slice) showing no cell viability and thus no mobility through the alginate even on day 7. (G–L) inverted epifluorescent microscope images after live/dead staining of ADSCs on the collagen stacks. (G) Slice 1 (top slice) showing good cell viability on day 1. (H) Slice 2 (middle slice) showing good cell viability and therefore cells must be able to travel through on day 1. (I) Slice 3 (bottom slice) showing good cell viability and thus demonstrating the mobility of cells through the collagen and the micromachined fibre mesh even after 1 day. (J) Slice 1 (top slice) showing good cell viability on day 4. (K) Slice 2 (middle slice) showing good cell viability and therefore cells must be able to travel through on day 4. (L) Slice 3 (bottom slice) showing good cell viability and thus demonstrating the mobility of cells through the collagen and the micromachined fibre mesh on day 4. Scale bars: 500 μm. ADSC: adipose derived stem cell.

Table 1. Actual versus the predicted height of the cube, pyramid and hemisphere, along with the ratio of the discrepancy

Shape	Expected height (mm)	Actual height (mm)	Ratio
Cube	18.00	24.77	1.376
Hemisphere	12.60	16.87	1.350
Pyramid	15.40	21.62	1.404

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Three-dimensional biofabrication of nanosecond laser micromachined nanofibre meshes for tissue engineered scaffolds

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