\documentclass[10pt]{article}

\usepackage{fullpage}
\usepackage{setspace}
\usepackage{parskip}
\usepackage{titlesec}
\usepackage[section]{placeins}
\usepackage{xcolor}
\usepackage{breakcites}
\usepackage{lineno}
\usepackage{hyphenat}





\PassOptionsToPackage{hyphens}{url}
\usepackage[colorlinks = true,
            linkcolor = blue,
            urlcolor  = blue,
            citecolor = blue,
            anchorcolor = blue]{hyperref}
\usepackage{etoolbox}
\makeatletter
\patchcmd\@combinedblfloats{\box\@outputbox}{\unvbox\@outputbox}{}{%
  \errmessage{\noexpand\@combinedblfloats could not be patched}%
}%
\makeatother


\usepackage{natbib}




\renewenvironment{abstract}
  {{\bfseries\noindent{\abstractname}\par\nobreak}\footnotesize}
  {\bigskip}

\titlespacing{\section}{0pt}{*3}{*1}
\titlespacing{\subsection}{0pt}{*2}{*0.5}
\titlespacing{\subsubsection}{0pt}{*1.5}{0pt}


\usepackage{authblk}


\usepackage{graphicx}
\usepackage[space]{grffile}
\usepackage{latexsym}
\usepackage{textcomp}
\usepackage{longtable}
\usepackage{tabulary}
\usepackage{booktabs,array,multirow}
\usepackage{amsfonts,amsmath,amssymb}
\providecommand\citet{\cite}
\providecommand\citep{\cite}
\providecommand\citealt{\cite}
% You can conditionalize code for latexml or normal latex using this.
\newif\iflatexml\latexmlfalse
\providecommand{\tightlist}{\setlength{\itemsep}{0pt}\setlength{\parskip}{0pt}}%

\AtBeginDocument{\DeclareGraphicsExtensions{.pdf,.PDF,.eps,.EPS,.png,.PNG,.tif,.TIF,.jpg,.JPG,.jpeg,.JPEG}}

\usepackage[utf8]{inputenc}
\usepackage[T2A]{fontenc}
\usepackage[polish,greek,english]{babel}



\usepackage{float}








\begin{document}

\title{Development of a low-cost bioprinter based on a Kossel delta 3D printer
platform}



\author[1]{Sidne  Fanucci}%
\author[1]{Earl  Prinsloo}%
\affil[1]{Rhodes University}%


\vspace{-1em}



  
  \date{June 17, 2024}


\begingroup
\let\center\flushleft
\let\endcenter\endflushleft
\maketitle
\endgroup





\selectlanguage{english}
\begin{abstract}
The core of bioprinting related research aims to reduce the gap between
ex vivo cell cultures and in vivo cellular tissue models to further its
application within the biomedical field. While additive manufacturing is
touted as disruptive technology, bioprinter equipment costs exceed
limited resource budgets of many research laboratories restricting the
scope for further development for biomedical research and potential
medical application. In line with this, a relatively low-cost bioprinter
(SidneV1) was successfully designed and manufactured using a low-cost,
commercially available FDM Delta 3D printer as a prototype base with a
successfully custom designed and manufactured micro-extrusion printhead.
Printing accuracies assessed were 65\% (for width measurements) and 64\%
(for height measurements). This study aimed to demonstrate a way to
achieve low-cost bioprinting and hopefully pave the way for future
system modifications and refinements such that this technology becomes
more accessible to under-funded research groups around the world.
Although these findings are preliminary, further optimization of
printing parameters, bioink formulations and sterilization techniques
will allow for the engineering of viable, physiologically relevant
tissue models using low-cost bioprinting technology.%
\end{abstract}%



\sloppy


The core of bioprinting related research aims to reduce the gap
between\emph{ex vivo} cell cultures and \emph{in vivo} cellular tissue
models to further its application within the biomedical field. While
additive manufacturing is touted as disruptive technology, bioprinter
equipment costs exceed limited resource budgets of many research
laboratories restricting the scope for further development for
biomedical research and potential medical application. In line with
this, a relatively low-cost bioprinter (SidneV1) was successfully
designed and manufactured using a low-cost, commercially available FDM
Delta 3D printer as a prototype base with a successfully custom designed
and manufactured micro-extrusion printhead. Printing accuracies assessed
were 65\% (for width measurements) and 64\% (for height measurements).
This study aimed to demonstrate a way to achieve low-cost bioprinting
and hopefully pave the way for future system modifications and
refinements such that this technology becomes more accessible to
under-funded research groups around the world. Although these findings
are preliminary, further optimization of printing parameters, bioink
formulations and sterilization techniques will allow for the engineering
of viable, physiologically relevant tissue models using low-cost
bioprinting technology

\textbf{Keywords:} 3D Printing, additive manufacturing, tissue
engineering, bioprinting.

\textbf{Specifications table}\selectlanguage{english}
\begin{longtable}[]{@{}ll@{}}
\toprule
\begin{minipage}[b]{0.47\columnwidth}\raggedright\strut
Hardware name\strut
\end{minipage} & \begin{minipage}[b]{0.47\columnwidth}\raggedright\strut
SidneV1 (SV1)\strut
\end{minipage}\tabularnewline
\midrule
\endhead
\begin{minipage}[t]{0.48\columnwidth}\raggedright\strut
Subject area\strut
\end{minipage} & \begin{minipage}[t]{0.48\columnwidth}\raggedright\strut
Engineering and Material Science Biological Sciences (e.g., Microbiology
and Biochemistry) Educational Tools and Open-Source Alternatives to
Existing Infrastructure\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.48\columnwidth}\raggedright\strut
Hardware type\strut
\end{minipage} & \begin{minipage}[t]{0.48\columnwidth}\raggedright\strut
Three-dimensional printer modifications Syringe extruder\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.47\columnwidth}\raggedright\strut
Open-Source License\strut
\end{minipage} & \begin{minipage}[t]{0.47\columnwidth}\raggedright\strut
GPL\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.47\columnwidth}\raggedright\strut
Cost of Hardware\strut
\end{minipage} & \begin{minipage}[t]{0.47\columnwidth}\raggedright\strut
\textbf{\$ 1 007,52}\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.47\columnwidth}\raggedright\strut
Source File Repository\strut
\end{minipage} & \begin{minipage}[t]{0.47\columnwidth}\raggedright\strut
https://doi.org/10.17605/OSF.IO/7TQKB\strut
\end{minipage}\tabularnewline
\bottomrule
\end{longtable}

\textbf{Introduction}

\emph{Context}

Three-dimensional \emph{in vitro} human tissue models have the potential
to act as models for disease which will not only reduce the use of
animals as models for disease but may potentially increase turnover time
associated with new drugs undergoing pharmaceutical trials {[}1{]}. The
purchase and maintenance costs of commercially available bioprinters
have meant this technology is inaccessible to most research labs whether
in developed economies or developing countries {[}2{]}. There are,
however, many facets of this cutting-edge technique (bioprinting) that
are continuously being improved for its application, including the
reduction in associated cost, design of bioinks, design of extruder
systems and the ability to sustain post-printed viability of cells
within the 3D structures {[}3,4{]}. General access to desktop \&
open-source additive manufacturing technologies has seen advancement in
this field in recent years. Arguably, this has spurred the bioprinting
revolution.

Extrusion based bioprinting has been largely focused on the manufacture
of 3D tissue constructs or scaffold due to its many advantages over
other methods, including, but not limited to; high cell viability
{[}5{]}, flexible geometric shapes {[}6{]}, ability to incorporate
multiple biomaterials and cell types {[}7{]}, both homogenous and
heterogeneous structures can be created {[}6,8{]}. In extrusion based
bioprinting, the bioink is extruded out of a nozzle tip to form a
continuous line structure driven by either pneumatic pressure or
mechanical pistons. The extruded product is referred to as filaments
instead of droplets {[}5,6,8{]}. A three-axis/cartesian automatic
extrusion system is typically used in this type of bioprinting, equipped
with a fluid dispensing nozzle {[}9{]}. This method has been extensively
reported in many studies {[}10--16{]}. Most systems make use of standard
plastic syringes which provide many benefits including; wide
availability, low cost, aseptic, pyrogen free and compatibility with a
range of needle sizes used as print nozzle heads {[}17{]}. Although the
most widely employed method, it is not without its limitations. Most
syringe-based extruders are designed to incorporate the fluid reservoir
into the extruder carriage which is solely responsible for the high mass
typically associated with this approach. A heavy extruder carriage can
cause various issues during the printing process by affecting speed and
resolution which may cause compromises in geometries of the printed
constructs {[}12{]}. Reducing the volume of the fluid reservoir could
provide a solution to the increased mass, however this will negatively
impact the ability to print complex and larger constructs. Other larger
volume systems have utilized the Bowden approach to minimize the weight
of the extruder carriage. This approach makes use of Bowden tubing to
connect the fluid reservoir (which is completely removed from the
extruder carriage) to the nozzle. These systems are typically
pneumatically driven which then brings in another separate set of
limitations such as; poor retraction, the need for vacuum during
printing and unstable extrusion pressures {[}12{]}. The ability of the
printer to retract is important during the printing process as this
prevents material from dripping during non-extruding moves which in turn
reduces printing fidelity of the construct {[}12{]}. Pusch \emph{et
al.}(2018) have addressed the two major concerns of extruder carriage
weight and inability to retract using the Large Volume Extruder (LVE)
design. This design utilizes the Bowden tube approach with stepper
motors and a lead screw driven extruder. The use of stepper motors is
suggested to achieve retraction through straightforward reversal of
direction and to apply constant pressure using a standard syringe
{[}18{]}.

The RepRapPro (RRP) Paste extruder utilized in this study also uses a
Bowden tube approach along with a lead screw driven extruder system
which makes use of stepper motors. As with the LVE approach, attaching
the RRP paste extruder to the printer frame places all the weight on the
printer frame and not the extruder carriage. This adds minimal payload
to printer movements and should in turn maximize the print speeds and
reduce any vibration of the nozzle during printing which may lead to
nozzle dripping.

\emph{Design Criteria}

Overall, the bioprinting process has multiple facets to consider which
have been described under one of the three pillars required for
successful tissue engineering -- hardware, wetware and software
considerations {[}19{]}. The outcome should include low operational
cost, ability to use a wide variety of materials, allow for fine
deposition of materials with high viscosities, maintain high cell
viabilities, reduced maturation time of printed constructs and finally,
minimal handling of the constructs. Here, we discuss the criteria
required for the hardware development process applied in this study. The
overall design and development process outlined in this study is
specifically for the printing of cell free scaffolds which then provides
the necessary foundation for future cell-laden bioink bioprinting.

\emph{1.2.1 Choice of Printer \& RepRapPro (RRP) Paste extruder}

A commercially sourced, RepRap based Delta 3D printer kit was selected
for modification to include a hydrogel paste extruder in the place of
the original thermoplastic extruder. The high printing precision and
accuracy expected of delta printers led to the choice for eventual
cell-free scaffold bioprinting {[}20{]}. The ANYCUBIC Kossel Linear Plus
Delta printer used in this project was based off the popular Kossel
RepRap delta printer designed by Johann C. Rocholl. All documents and
development kits for the Kossel Delta are available online {[}L1{]} with
many components of the model made readily available from Thingiverse
{[}L2{]} . This allows for successful modification to be made to the
printer at relatively low-cost. The firmware supplied with the Delta
printer is the open-source Marlin Firmware (originally developed by
Scott Laheine, {[}L3{]}). This firmware allows for modifications
allowing for paste extrusion with the Delta hardware which allows for a
relatively quick transition from thermoplastic extrusion to paste
extrusion.

Delta printers contain an effector plate where the extruder nozzle sits
and moves around the build volume, but the extruder stepper motor is
situated outside the printing space, attached to the frame. The extruder
nozzle must therefore be lightweight and secured properly onto the
effector plate to prevent unwanted movement. Suspending the extruder
nozzle above the bed arguably eliminates any contamination from
particles caused by friction during print moves. The dimensions of the
extruder carriage and the mass allowed on the axis that holds the
extruder therefore dictate the maximum dimensions of the hydrogel paste
extruder.

Furthermore, the kit used in this study was selected because of the
linear rail-based motion control allowing for increased XYZ precision
required for bioprinting. The ball bearing design of the linear rail
system allows for a more precise motion control compared to the linear
rod systems, typically found in desktop 3D printers. There is a
considerable reduction in binding occurrences (ball bearings getting
caught on the rail during a movement) which contributes significantly to
a much smoother printing movement and creates a jerk-free print. The
stepper motors (NEMA17 with 1.8 \selectlanguage{english}step angle) used in conjunction with the
linear rails also contribute significantly to the increased precision
found in this system, where micro-stepping allows for more controlled
moves.

With such a highly application specific technology such as bioprinting,
the commercially available bioprinters tend to be available at a very
high cost. This has forced many research groups to develop their own
bioprinters based on their specific application requirements whilst also
maintaining a low overall build cost. This also allows for the
functionality of the printer to be expanded over time, as required. As
in this case, where a bioprinter initially designed for cell-free
bioprinting of scaffolds may be easily modified for future cell-laden
bioink printing. In this study a low cost commercially available 3D
printer was modified to accommodate for bioprinting with cell-free
hydrogels. This provided for majority of the printer parts at a
relatively low cost. All additional parts were either 3D printed using
thermoplastics or purchased at a low cost. Although the linear rails are
considerably more expensive than smooth rod systems, the increase in
printing precision is imperative to successful bioprinting and so
outweighs the potential overall increase in build cost.

\textbf{Methods \& Materials}

\textbf{Design description.}

An Anycubic Kossel Linear Plus Delta 3D printer kit was selected for
modification. Based on the criteria for bioprinting, both the hardware
and software of the Delta printer was successfully modified to
accommodate for tissue engineering purposes. The manufacture and
assembly of the final delta bioprinter can be separated into three main
parts; (1) the hardware modification of the 3D printer frame, (2) the
replacement of the thermoplastic extruder for a cell-free hydrogel paste
extruder, and (3) the software modifications for hydrogel paste
extrusion. Majority of the added components were 3D printed -- making it
inexpensive and easily modified as necessary. A leadscrew driven
hydrogel deposition concept is employed within the system and due to the
modular nature of the extruder parts, the RRP system easily allows for
adjustments regarding the use of different syringe diameters and
volumes.

All 3D printed parts mentioned were printed using an ANYCUBIC Kossel
Delta Linear Plus 3D printer following the same general printing
parameters as listed: Layer height: 0.1 mm, Fill density: 100 \%, Print
speed: 80 mm/s, Printing temperature: 210~\selectlanguage{english}C, 100 \% Flow, No support
structures unless necessary and No platform adhesion. Poly-L-lactic acid
(PLA) was used as the printing material (1.75 mm diameter eSUN PLA+ 3D
filament). No additional post-print processing was required. The 3D
modelling software SketchupMake2017 (v.17.2.2555) was used to design all
printed parts (unless otherwise specified) and the slicing software Cura
(v.15.04.2) used to prepare the design files for further 3D printing.

\emph{Bioprinter Frame (Hardware) modifications}

\textbf{Enclosure:} The printer was enclosed using both printed and
non-printed parts (Fig 1a). The side frames and mid-sections (later
referred to as bioprinter frames, windows, and doors) were designed and
files sent for manufacture to a Perspex laser cutting service (M\&D
Creations, Makhanda, South Africa) using 3 mm thickness clear Perspex
sheets. The 3D printed parts consist of three separate corners.

\textbf{Board box:} The Anycubic Tri-gorilla controller board was moved
from under the build plate (as per printer design) to outside the
system; shown in Fig 1b. Wiring was adjusted to accommodate for the new
board position. A board box was designed, and 3D printed to accommodate
for the new location of the board. Furthermore, a few changes were made
to the electronic board such that the hotbed power input cables and fans
were removed.

\textbf{Bed platform} : A new bed platform was designed, and 3D printed,
to hold two 220 mm diameter glass plates of 2 mm thickness (Fig 1b). The
bed platform with the glass in place was designed to cover most of the
base of the printer with a few openings to allow for air flow to reduce
any pressure build-up.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image1/image1}

\end{center}
\end{figure}



\textbf{Fig. 1. CAD files used for encasing of the printer,
repositioning of the electronic board and new printing platform.}

\emph{Extruder Modifications}

The syringe extruder was based off the RepRapPro Paste Extruder design
(Licensed under the GPL; all files available from the RepRapPro Ltd
Github repository at https://github.com/reprappro/Paste-extruder). The
syringe-based extruder designed in this project is made up of printed
parts, non-printed parts, and electronic components. Figure 2 below
shows the full syringe-based extruder, including 6 parts printed
directly from the RepRapPro Extruder design (available online). The part
labelled ``MOUNTING BRACKET'' was custom designed and 3D printed. The
extruder was assembled as per instructions given on the RepRapPro
website{[}L1{]}.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image2/image2}

\end{center}
\end{figure}

\textbf{Fig. 2. Labelled CAD file of the syringe-based extruder model.}

A Luer-lock effector plate adaptor was designed to attach to the
original effector plate provided with the Delta printer and incorporates
a male Luer-lock connection fitting and a Bowden tube connection fitting
(Fig. 3).\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image3/image3}

\end{center}
\end{figure}

\textbf{Fig. 3. Labelled CAD file of the Luer-lock effector plate
adapter. A: Full assembly with effector plate and needle nozzle, B:
Front view, C: Top view, D: Bottom view, E: Side view.}

\emph{Marlin Firmware (Software) Modifications}

Pronterface (printrun-2015031011 https://github.com/kliment/Printrun )
host interface was used for controlling the printer X-, Y- and Z-
movements as well as the extrusion motor. Default Pronterface settings
were used at 250~000 baud rate. The firmware was modified to accommodate
for hydrogel extrusion as described throughout the following sections.

\textbf{Key strengths to the overall design approach}

\begin{itemize}
\tightlist
\item
  The open-source nature of the project, including the use of the
  Anycubic Kossel Delta 3D printer and the GPL licensed RepRapPro Paste
  extruder allows for personal modifications at a low cost.
\item
  The delta style 3D printer allows for a user-friendly modification
  process and requires few bigger modifications made to convert from
  thermo plastic to hydrogel printing.
\item
  Making a few simple changes to the extruder and nozzle design will
  potentially allow for a multitude of different bioinks and
  cross-linkers to be used.
\item
  This system is easily manufactured and more economically feasible
  compared to other commercially available bioprinters.
\end{itemize}

\textbf{Design files}

\emph{Bioprinter hardware design files}\selectlanguage{english}
\begin{longtable}[]{@{}lllll@{}}
\toprule
Design file name & Design file name & File type & Open source license &
Location of the file\tabularnewline
\midrule
\endhead
Enclosing components & Printed Corners & CAD \& STL file & LGPL &
https://doi.org/10.17605/OSF.IO/7TQKB\tabularnewline
& Frames & CAD \& STL file & LGPL &
https://doi.org/10.17605/OSF.IO/7TQKB\tabularnewline
& Window & CAD \& STL file & LGPL &
https://doi.org/10.17605/OSF.IO/7TQKB\tabularnewline
& Door & CAD \& STL file & LGPL &
https://doi.org/10.17605/OSF.IO/7TQKB\tabularnewline
& Handle & CAD \& STL file & LGPL &
https://doi.org/10.17605/OSF.IO/7TQKB\tabularnewline
& Glass Lid & CAD \& STL file & LGPL &
https://doi.org/10.17605/OSF.IO/7TQKB\tabularnewline
Electronic board & Box & CAD \& STL file & LGPL &
https://doi.org/10.17605/OSF.IO/7TQKB\tabularnewline
& Lid & CAD \& STL file & LGPL &
https://doi.org/10.17605/OSF.IO/7TQKB\tabularnewline
Bed platform & Platform supports & CAD \& STL file & LGPL &
https://doi.org/10.17605/OSF.IO/7TQKB\tabularnewline
\bottomrule
\end{longtable}

\textbf{Frames:} The frames were manufactured in Perspex and secured
onto the printer frame using M3 x 5 mm screws and T-nuts (Fig. 4).

\textbf{Window:} These are the mid-sections of the fully enclosed
printer that attach to the Perspex frames using M3 x 8 mm screws and
nuts (Fig. 4). These pieces were manufactured in Perspex.

\textbf{Door and Handle:} The door was manufactured in Perspex and
attached to the one side of the printer frame using two hinges (Fig. 4).
A handle was designed and added to the door for ease of use.

\textbf{Printed corners:} The corners were designed in such a way to
allow sliding on to the aluminum extrusions without the need for screws
(Fig. 5). The corners allowed for complete enclosure of the system (Fig.
5).

\textbf{Glass lid:} A glass lid was designed and manufactured in order
to complete the full enclosure of the printer (Fig.6). Six M4 x 5 mm
screws and six M4 T-nuts were used to secure the glass into place (two
screws on each side). Three additional aluminum extrusions were added to
the top of the printer (Fig. 6b) with 10 x 10 x 300 mm dimensions to
create additional height and secured onto the existing extrusions using
six 3D printed parts and twelve M4 x 5 mm screws and twelve M4 T-nuts
(Fig.6). Nine 3D printed pieces were designed to fit into the aluminum
extrusions and complete the finished look (Fig.6).

\textbf{Electronic board box and lid:} The Tri-gorilla electronic board
was moved to outside the printer and the board box designed to house it.
The board box consists of two parts: the box and the lid (Fig. 7). The
electronic board was secured in place using two M3 x 10 mm screws and
nuts (Fig. 7) and the box then secured onto the side of the printer
using two M4 x 5 mm screws and T-nuts (Fig. 7).

\textbf{Bed platform supports:} The platform consists of six separate
pieces; designed to hold two 220 mm diameter glass plates, with the top
plate designed to hold a standard size petri dish (Fig. 7). The printed
pieces were secured onto the printer's aluminum extrusions using two M3
x 25 mm cap-head screws and two M3 T-nuts per piece (Fig. 7).\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image4/image4}

\end{center}
\end{figure}

\textbf{Fig. 4. Labelled image of fully assembled and enclosed Delta 3D
printer. Door handle shown was assembled using Perspex.}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image5/image5}

\end{center}
\end{figure}

\textbf{Fig. 5. Top view of fully assembled and enclosed Delta 3D
printer. Insert highlighting how the corners were assembled onto the
aluminum extrusions.}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image6/image6}

\end{center}
\end{figure}



\textbf{Fig. 6. CAD files of glass lid system designed to complete
enclosure of the bioprinter. A: CAD file of all parts assembled (blue
part indicating glass piece), B: Exploded image of all parts
incorporated, including added aluminum extrusions and 3D printed parts,
C: Method by which parts are assembled. All grey colored parts shown in
the image indicate original parts.}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image7/image7}

\end{center}
\end{figure}

\textbf{Fig. 7. Designed, manufactured, and assembled bed platform and
electronic board box. Printed and assembled platform pieces holding two
glass plates (vertical arrows indicating M3 x 25 mm cap-head screws used
to secure pieces to aluminum extrusions. Larger red arrow showing newly
positioned electronic board. The inset provided (i, ii, iii) shows the
designed board box at various angles.}

\emph{Extruder hardware design files}\selectlanguage{english}
\begin{longtable}[]{@{}llll@{}}
\toprule
Design file name & File type & Open source license & Location of the
file\tabularnewline
\midrule
\endhead
Top strengthener & CAD \& STL file & GPL &
https://github.com/reprappro/Paste-extruder\tabularnewline
Mounting bracket & CAD \& STL file & GPL &
https://github.com/reprappro/Paste-extruder\tabularnewline
Driven gears & CAD \& STL file & GPL &
https://github.com/reprappro/Paste-extruder\tabularnewline
Drive gear & CAD \& STL file & GPL &
https://github.com/reprappro/Paste-extruder\tabularnewline
Frame & CAD \& STL file & GPL &
https://github.com/reprappro/Paste-extruder\tabularnewline
Drive Block & CAD \& STL file & GPL &
https://github.com/reprappro/Paste-extruder\tabularnewline
Bearing holder & CAD \& STL file & GPL &
https://github.com/reprappro/Paste-extruder\tabularnewline
Luer lock effector plate adapter & CAD \& STL file & LGPL &
https://doi.org/10.17605/OSF.IO/7TQKB\tabularnewline
\bottomrule
\end{longtable}

\textbf{Top strengthener:} This component further secures the system in
place, particularly the syringe. It is secured onto the frame using the
same M3 x 35 mm screws used for attaching the bearing holders onto the
frame. The top strengthener protects the syringe from moving, once
secured into place for printing (Fig. 8b).

\textbf{Mounting bracket:} The mounting bracket is used to secure the
fully assembled extruder onto the aluminum frames of the bioprinter
(using M4 x 15 mm screws and T-nuts) (Fig. 8b).

\textbf{Drive gear and driven gears:} These components are used together
with the NEMA17 stepper motor to rotate the threaded bars and therefore
move the drive block (Fig. 8b). The drive gear mounts onto the NEMA17
motor shaft and is designed to be self-centering on the motor shaft. The
driven gears are mounted onto adjacent corners of the motor face
(meshing with the drive gear in the center) using two M2 nuts. The M5 x
95 mm threaded bars are secured in the center of the driven gears using
two M5 nuts (Fig. 8b).

\textbf{Frame:} This is the main component of the extruder. The NEMA17
motor is secured onto the main printer frame along with the bearing
holders and drive block. The design allows for a syringe to be secured
in place whilst the plunger side is secured to the drive block (Fig.
8b).

\textbf{Drive block:} This component is secured to the end of the
syringe plunger and is responsible for enabling actuation of the syringe
plunger (extrusion and retraction). Two M5 x 95 mm threaded bars are
used (one on either side of the drive block) which control the movement.
These are secured into place using M5 nuts (Fig. 8b).

\textbf{Bearing holders:} The bearing holders are attached to the
extruder frame and included to allow the rotation of the threaded bars
which move the drive block (Fig. 8b). They are secured into place using
623 bearings, M3 nuts and M3 washers.

\textbf{Luer lock effector plate adaptor:} This component completes the
extruder by connecting the mounted syringe extruder to the effector
plate and nozzle (Fig. 8b,c). The design incorporates a male Luer-lock
connection fitting and a Bowden tube connection fitting (Fig. 8c). It
was secured onto the standard effector plate using four M3 x 8 mm screws
and M3 nuts (Fig. 8c).\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image8/image8}

\end{center}
\end{figure}

\textbf{Fig. 8. Printed and assembled syringe-based extruder including
the effector plate adaptor. A: Fully 3D printed and assembled syringe
extruder highlighting the RepRapPro Syringe extruder (B) and the
Luer-lock effector plate adaptor (C) including labelled parts.}

\emph{Marlin software design files}\selectlanguage{english}
\begin{longtable}[]{@{}llll@{}}
\toprule
\begin{minipage}[b]{0.22\columnwidth}\raggedright\strut
\textbf{Design file name}\strut
\end{minipage} & \begin{minipage}[b]{0.22\columnwidth}\raggedright\strut
\textbf{File type}\strut
\end{minipage} & \begin{minipage}[b]{0.22\columnwidth}\raggedright\strut
\textbf{Open source license}\strut
\end{minipage} & \begin{minipage}[b]{0.22\columnwidth}\raggedright\strut
\textbf{Location of the file}\strut
\end{minipage}\tabularnewline
\midrule
\endhead
\begin{minipage}[t]{0.24\columnwidth}\raggedright\strut
Bioprinter marlin firmware 2019\strut
\end{minipage} & \begin{minipage}[t]{0.24\columnwidth}\raggedright\strut
.ino\strut
\end{minipage} & \begin{minipage}[t]{0.24\columnwidth}\raggedright\strut
GPL\strut
\end{minipage} & \begin{minipage}[t]{0.24\columnwidth}\raggedright\strut
https://doi.org/10.17605/OSF.IO/7TQKB forked from
https://github.com/MarlinFirmware/Marlin\strut
\end{minipage}\tabularnewline
\bottomrule
\end{longtable}

\textbf{Basic settings:}

\textbf{Endstop inverting:} the firmware was edited to invert the x-min
and y-min endstop logic. This was done to accommodate for the Delta
printer endstop set up.

\#define X\_MIN\_ENDSTOP\_INVERTING \emph{false} set to \#define
X\_MIN\_ENDSTOP\_INVERTING \emph{true}\#define
Y\_MIN\_ENDSTOP\_INVERTING \emph{false} set to \#define
Y\_MIN\_ENDSTOP\_INVERTING \emph{true}

\textbf{LCD character setup:} the firmware script was edited from a
Japanese character display setup to a western character display.
\#define DISPLAY\_CHARSET\_HD44780 \emph{JAPANESE} set to \#define
DISPLAY\_CHARSET\_HD44780 \emph{WESTERN}

\textbf{Thermal Settings:}

\textbf{Bed minimum and maximum temperature:} both initial lines in the
script were changed to 0 degrees Celsius. This was done as the hotends
were removed from the bed platform during modification of the printer
into a bioprinter.

\#define BED\_MINTEMP \emph{5} set to \#define BED\_MINTEMP
\emph{0}\#define BED\_MAXTEMP \emph{120} set to \#define BED\_MAXTEMP
\emph{0}

\begin{enumerate}
\tightlist
\item
  \textbf{Cold extrusion:} The script was changed to allow cold
  extrusion. \#define PREVENT\_COLD\_EXTRUSION set to \emph{//} \#define
  PREVENT\_COLD\_EXTRUSION
\item
  \textbf{Extruder minimum/maximum temperature:} As with the bed
  platform temperature settings, both were changed to 0 as the extruder
  hotend and thermistor were removed during modification. \#define
  EXTRUDE\_MINTEMP \emph{170} set to \#define EXTRUDE\_MINTEMP\emph{0}
\item
  \textbf{Preheating bed and extruder:} These temperatures were also set
  to 0 as there was no need for preheating, without hotends available.
  \#define PREHEAT\_1\_TEMP\_HOTEND \emph{180} set to \#define
  PREHEAT\_1\_TEMP\_HOTEND \emph{0}\#define PREHEAT\_1\_TEMP\_BED
  \emph{70} set to \#define PREHEAT\_1\_TEMP\_BED \emph{0}
\end{enumerate}

\textbf{Mechanical Settings:}

\begin{enumerate}
\tightlist
\item
  \textbf{Printable radius:} The printable radius was reduced during the
  modification to accommodate for the smaller printing environment
  required. \#define DELTA\_PRINTABLE\_RADIUS \emph{116} set to \#define
  DELTA\_PRINTABLE\_RADIUS \emph{30}
\item
  \textbf{Y and Z probe offset from extruder:} The Y and Z probe offsets
  were changed to accommodate for the smaller, newly included extruder
  nozzle. \#define Y\_PROBE\_OFFSET\_FROM\_EXTRUDER \emph{-10} set to
  \#define Y\_PROBE\_OFFSET\_FROM\_EXTRUDER \emph{0}\#define
  Z\_PROBE\_OFFSET\_FROM\_EXTRUDER \emph{-3.5} set to \#define
  Z\_PROBE\_OFFSET\_FROM\_EXTRUDER \emph{-18.5}
\item
  \textbf{Invert Extruder motor direction:} The extruder stepper motor
  inversion was changed to false as it was extruding correctly. \#define
  INVERT\_E0\_DIR \emph{true} set to \#define
  INVERT\_E0\_DIR\emph{false}
\item
  \textbf{Z-Home position:} The distance between the nozzle and the
  printing platform was changed regularly to accommodate for the
  different extruder used and the different length needles. \#define
  MANUAL\_Z\_HOME\_POS \emph{295.2} set to \#define MANUAL\_Z\_HOME\_POS
  \emph{116.5}
\end{enumerate}

\textbf{Movement Settings:}

\textbf{Axis steps per unit:} These values were changed based on the
Stepper motor used. \#define DEFAULT\_AXIS\_STEPS\_PER\_UNIT \{ 80, 80,
80, \emph{96} \} set to \#define DEFAULT\_AXIS\_STEPS\_PER\_UNIT \{ 80,
80, 80,\emph{200} \}

\textbf{Maximum/Default/Retraction/Travel Acceleration:} All movement
related script was changed to reduce the speed of the various movements
during printing.

\#define DEFAULT\_MAX\_ACCELERATION \{ \emph{3000, 3000, 3000, 3000} \}
set to \#define DEFAULT\_MAX\_ACCELERATION \{ \emph{1000, 1000, 1000,
1000} \} \#define DEFAULT\_ACCELERATION \emph{2000} set to \#define
DEFAULT\_ACCELERATION \emph{1000}\#define DEFAULT\_RETRACT\_ACCELERATION
\emph{2000} set to \#define DEFAULT\_RETRACT\_ACCELERATION
\emph{1000}\#define DEFAULT\_TRAVEL\_ACCELERATION \emph{2000} set to
\#define DEFAULT\_TRAVEL\_ACCELERATION \emph{1000}

\textbf{Default E-Jerk:} The E Jerk value is the minimum speed change
that requires acceleration. This was reduced in order to create smoother
print movements whilst printing smaller and finer constructs.

\#define DEFAULT\_EJERK \emph{5.0} set to \#define
DEFAULT\_EJERK\emph{1.0}

\textbf{Bill of Materials}

The complete bill of materials for the final Bioprinter manufactured is
listed in Table 1. The original 3D Delta printer used for modification
was assembled according to manufacturer's instruction and all parts used
in the final bioprinter model were from the original 3d printer, unless
mentioned in the Bill of materials Table 1.

\textbf{Build Instructions}

Assembly of commercially available ANYCUBIC Kossel Delta 3D printer

For ease of access to build materials, an Anycubic Kossel Linear Plus
Delta 3D printer was purchased from DIYelectronics (Durban, South
Africa). All parts were provided within the build kit and assembly of
the printer was done as per supplier's instructions. The printer
technical specifications are outlined in Table 2.

Firmware Flashing

The Tri-gorilla control board (integrated with the function of the
Mega2560+ramps 1.4; main control chip: ATMEGA256016AU), on the assembled
delta printer, required installing (i.e. flashing) software (firmware)
specifically written for controlling and monitoring the 3D printer.
Open-source Marlin Firmware (v.1.1.0) was first used for flashing the
board; available on the manufacturers' website {[}L4{]}. Firmware
installation was performed through the Arduino Integrated Development
Environment (IDE) desktop software (v.1.8.1.0, {[}L5{]}). The printer
was first connected to a PC using a USB cable (USB 2.0-A Male to USB-B
Male cable), the Arduino.ino file was then opened using the Arduino
software environment. In the Arduino environment, Tools \textgreater{}
Board \textgreater{} Arduino MEGA 2560 serial connection was selected.
The Firmware was subjected to several changes as described in detail in
section 1.1. Within the Arduino environment, the
\emph{Configuration.h}tab was used for the software modifications. The
``upload'' option was then selected to flash the Tri-gorilla board. The
final modified firmware is available at
https://doi.org/10.17605/OSF.IO/7TQKB.

Hardware modification

Based on the criteria for bioprinting, the delta printer was modified to
accommodate for tissue engineering application purposes. The methods of
modifications to the hardware are explained throughout the following
sub-sections.

Enclosure of the printer

The printer was encased to maintain sterility during future bioprinting.
All parts used were designed using CAD software and manufactured in PLA
(corners), Perspex (sides) or Glass (lid). Assembly was as follows;

Corners:

1) Printed corners were pushed onto the aluminium frame.

Sides and door:

1) Each frame was secured to the aluminium extrusions of the printer
using six M3 x 10mm cap-head screws, six M3 T-nuts and twelve M3
washers.

2) The windows, for the two sides of the printer, were secured onto the
newly attached frames using four M3 x 8mm cap-head screws, four M3 nuts
and eight M3 washers.

3) The door handle was fitted onto the door without the need for
fasteners.

4) The door was assembled onto the frame of the front facing side of the
printer using two hinges, ten M3 x 10mm cap-head screws, ten M3 nuts and
twenty M3 washers.

Lid:

\begin{enumerate}
\tightlist
\item
  Additional aluminum extrusions were secured to each existing extrusion
  using 3D printed parts and twelve M4 x 5mm screws and twelve M4
  T-nuts.
\item
  Glass was secured onto the top of the new extrusions using six M4 x 5
  mm screws and six M4 T-nuts.
\item
  The finishing pieces were added simply by sliding into the new
  extrusion grooves.
\item
  Electronic board box
\end{enumerate}

The Tri-gorilla electronic board (assembled according to manufacturer's
instruction) was moved to outside the printer. A board box was designed
and manufactured in PLA using a standard Delta 3D printer. The designed
and printed board box consists of two separate pieces: the box and the
lid. Assembly was as follows;

1) All wires were disconnected from the Tri-gorilla electronic board and
the board removed from its original fixture (under the print bed).

2) The board was secured onto the newly designed and printed board box
using two M3 x 10mm cap-head screws.

3) The board box was secured onto the aluminium extrusions of the
correct side of the printer using two M4 x 5mm cap-head screws and two
M4 T-nuts.

4) All wires were fed through the aluminium extrusions (from within the
printer) into the board box and reconnected in their respective places.

Bed platform

A print bed platform was designed and printed using a standard Delta 3D
printer using PLA as extrusion material. The platform consists of six
separate pieces; designed to hold two 220 mm diameter glass plates, with
the top plate designed to hold a standard size petri dish. The printed
pieces were secured onto the printers aluminium extrusions using two M3
x 25mm cap-head screws and two M3 T-nuts per piece.

Extruder

The original thermoplastic extruder was removed from the effector plate
and replaced with a newly designed syringe-based extruder. Assembly of
the syringe-based extruder was carried out exactly as described online
{[}L6{]}. Instructions up until attachment of the power-supply mounting
bracket were followed exactly as described. The following changes were
made from there;

Assembly onto mounting bracket and printer

1) The assembled drive block was attached to the mounting bracket using
four M3 x 35mm cap-head screws and four M3 nuts.

2) The mounting bracket with attached drive block was attached to the
printer frame aluminium extrusions using two M4 x 15mm screws with four
M4 T-nuts.

3) The NEMA17 stepping motor was connected to the E0 port of the
Tri-gorilla electronic board using a standard 4-pin extruder cable.

Assembly of Luer-lock effector plate adaptor

A ``nozzle'' was designed for the extrusion of biomaterials using the
syringe-based extruder system. It was designed to attach to the original
effector plate provided with the Delta printer. The nozzle was designed
to incorporate a male Luer-lock connection fitting and a Bowden tube
connection fitting. Assembly went as follows;

1) The printed adaptor piece was secured onto the effector plate using
four M3 x 8mm cap-head screws and four M3 nuts.

2) The bowden tube was pushed into the bowden tube connection fitting
and secured into place using insulating tape and silicone paste.

3) A Luer-lock syringe needle was then fastened into place using the
male Luer-lock connection fitting part of the adaptor piece.

Addition of syringe, top strengthener and tubing

1) A 5 ml Luer-lock syringe was added to the assembled and mounted
syringe extruder.

2) The printed top strengthener was fitted onto the mounted syringe
extruder using the threaded bar used to secure the motor to the frame
and the two M3 x 35mm cap-head screws positioned on the other end of the
frame.

3) The bowden tube was attached to the syringe using a Luer-lock
adaptor, silicone sealant and insulating tape. The other end of the
tubing was attached to the Luer-lock nozzle piece.

\textbf{Operation Instructions}

Software requirements

\begin{itemize}
\tightlist
\item
  The bioprinter was connected to the PC using a USB cable (USB 2.0-A
  Male to USB-B Male cable)
\item
  An 18G needle was added to the nozzle of the bioprinter (Luer-lock
  fit)
\item
  Pronterface (printrun-2015031011\textsuperscript{,3}
  https://github.com/kliment/Printrun ) software was initiated and used
  to manually count the Z home position. This depends on the 18G needle
  length used. Briefly, once the printer was connected to the software,
  the homing option was initiated. The distance between the needle
  nozzle and the printing platform was then calculated using the Z-axis
  10 mm, 1 mm and 0.1 mm movement arrows within the software. This was
  repeated several times until the exact distance between bed platform
  and nozzle after homing was determined.
\item
  The printer was disconnected from Pronterface and the correct Marlin
  firmware file was opened in the Arduino Environment.
\item
  The firmware was changed, in the \emph{Configuration.h} tab of the
  Arduino environment, to include the correct Z position calculated.
\item
  The edited firmware was uploaded onto the printer by clicking
  ``upload'' button in the Arduino environment.
\item
  The new Z home position was tested by, once again, connecting to
  Pronterface (printrun-2015031022).
\item
  Setting up slicing software
\end{itemize}

\begin{itemize}
\tightlist
\item
  Cura (v.15.04.2) software was initiated.
\item
  The printer was manually set up for desired bioprinter parameters
  starting with Machine settings. For this study, the following machine
  settings were chosen: E-steps per 1mm filament = 0, Maximum width =
  60, Maximum depth = 60, Maximum height = 50, Extruder count 1, No
  heated bed, Machine centre 0,0 Yes, GCode Flavor Marlin/Sprinter, All
  printer head size options set to 0.0, Serial port Auto, Baudrate Auto.
\item
  The desired bioprinting parameters were set up next, such as: Layer
  height = 0.1, Shell thickness = 0.4, Disable retraction, Fill density
  100\%, Print speed = 10, Printing temperature set to 0, No support or
  platform adhesion, Filament diameter = 1 and flow = 150. In the
  ``Advanced'' tab, all speeds were set to 5.
\item
  The desired STL file was uploaded into Cura (v.15.04.2).
\item
  The Gcode was saved and loaded onto an SD card which was then inserted
  into the printer awaiting print.
\item
  Cell-free Bioprinting
\end{itemize}

\begin{itemize}
\tightlist
\item
  The tubes and printer environment were cleaned and sterilized
  thoroughly using 70 \% (v/v) ethanol. The sterilizing solution was
  manually fed through the tube several times using a sterile syringe.
\item
  The cell-freebioink solution was prepared (heating, cooling, mixing
  etc.)
\item
  The bioink was then added to a 5 ml sterilized syringe and placed into
  the frame of the extruder with the top strengthener added to secure it
  into place.
\item
  One end of the sterilized tube was attached to the bioink syringe, and
  the other end fastened to the top of the effector plate (via the
  luer-lock adaptor).
\item
  A newly sterilized 18G Luer-lock needle was added to the bottom
  section of the effector plate adaptor.
\item
  A sterilized petri dish was added to the printing platform where the
  glass plates permit.
\item
  The bioprinter was once again connected to the PC using a USB cable
  (USB 2.0-A Male to USB-B Male cable)
\item
  Using Pronterface (printrun-2015031033\textsuperscript{,3}
  https://github.com/kliment/Printrun ) software, the M302 g-code
  command was entered to allow for cold extrusion and the extruder motor
  turned on to extrude until the bioink was fed through the sterilized
  tube to the tip of the nozzle needle.
\item
  The bioprinter was then disconnected from the PC.
\item
  The LCD display on the bioprinter was used to navigate to the saved
  GCode files on the inserted SD card.
\item
  Printing was initiated.
\item
  Once completed, the bioprinter door was opened using aseptic technique
  and cross-linker solution was added to the construct (if required).
  The construct was left to cross-link for 2 minutes followed by removal
  of the cross-linker solution.
\end{itemize}

\textbf{Results}

\textbf{Validation and Characterization}

The concept was assessed to test initial printability using a 6 \% (w/v)
sodium alginate (dissolved in sterile double distilled water) (see
Appendix 1 for a detailed protocol). Cell-free bioprinting experiments
were performed initially using an 18G syringe needle as the nozzle.
Subsequent analysis on optimal parameters were then carried out. All
materials used were purchased from Sigma-Aldrich Pty. Ltd. (South
Africa), unless otherwise specified.

Proof of concept: does the extrusion mechanism work as intended and is
it capable of printing geometrically complex constructs?

The computer aided design (CAD) software SketchupMake2017 (v.17.2.2555)
was used to design a grid-like structure for initial printability tests.
Dimensions include: Height = 19.80 mm

Width = 19.80 mm, Layer height = 2 layers (0.2 mm). Cura (v.15.04.2) was
used as the slicing software as well as for the parametrization of the
printing process. The printing parameters include Layer height (1 layer
= 0.1 mm), Shell thickness (0.4 mm), No retraction, Bottom/Top thickness
(0.4 mm), Fill density (100\%), Print speed (10mm/s), Flow rate (150\%),
Nozzle diameter (18G needle).\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image9/image9}

\end{center}
\end{figure}

\textbf{Fig. 9. Assessing printability.; (i) CAD file of construct, (ii)
Post-printing photographs of the printed construct; printed using a
syringe-based extruder, (iii) Post-printing accuracy (\%) calculated for
the printed alginate constructs (using height and width). Scale bars
represent 5 mm. Data points are means; error bars represent SEM for n =
6.}

Using the measurements of height and width (mm), of both Fig.9(i) and
Fig.9(ii), the printing accuracies (\%) were calculated for the
assembled hydrogel extruder (Fig.9(iii)). Printing accuracies (\%) for
height and width were calculated at 64.13 \% and 64.58 \%, respectively.
The settings chosen turned out to be suitable for experimental trials
based on cell-free 6 \% (w/v) concentrated hydrogel inks, hence these
parameters were kept for the remaining experiments except for flow rate
and needle gauge optimization studies.

Optimization studies: Does needle gauge and flow rate affect
printability?

Needle gauge

The printing accuracy (\%) was determined using the following equation;

\begin{equation}
\text{Printing\ Accuracy\ }\left(\%\right)\frac{\text{Experimental\ Value}}{\text{Theoretical\ value}}x\ 100\nonumber \\
 \end{equation}

A 6\% (w/v) alginate solution was used as extrusion material to print
the designed constructs with subsequent cross-linking in
CaCl\textsubscript{2} solution for 2 minutes. Images were taken of the
cross-linked constructs and measurements determined with ImageJ software
(v1.4.6.r). Prints were repeated three times for each needle gauge
tested (15G, 18G, 23G and 25G). The flow percentage used in the
experiment was 100\%. This experiment aimed to determine the effect of
nozzle diameter (using different needle gauges) on the printed construct
shape quality, under certain constant parameters (flow rate = 100\%,
print speed = 10 mm/s).

Kahl \emph{et al.} (2019) found that the needle diameters are essential
in optimizing printing resolutions such that; the larger the needle
diameter, the smaller the resolution and therefore the lower the
printing accuracy. The choice in needle diameter, length and shape (i.e.
conically shaped or cylindrical) has additional consequences in terms of
cell viabilities. Findings suggest shorter, conically shaped needles
allow for higher cell viabilities, post-printing, compared to
cylindrically shaped needles {[}11,22,23{]}. These findings have major
implications in cell-laden bioprinting approaches but little
implications in cell-free approaches, as the cells are seeded onto the
bioprinted constructs post-printing.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image10/image10}

\end{center}
\end{figure}

\textbf{Fig. 10. Assessment of printability using different nozzle
diameters. A: Post-printing accuracy (\%) calculated for alginate
constructs printed using various nozzle diameters (15G, 18G and 23G),
25G not included in graph. B: Post-printing photographs of alginate
constructs printed using (ii) 25G, (iii) 23G, (iv) 18G and (v) 15G
needle gauge. (i) CAD file for printed construct. Scale bars represent
5mm. Data points are means; error bars represent SEM for n=3. No
statistical significance was found between groups (p = 0.0522; p
\textgreater{} 0.05).}

Figure 10 shows the images of the printed constructs (post-printing and
cross-linking), printed using various needle gauges; 15G, 18G and 23G.
The inner diameters for each needle gauge are as follows; 15G = 1.372
mm, 18G = 0.838 mm, 23G = 0.337 mm. A final needle gauge was included in
the experiment; 25G (inner diameter = 0.260 mm), however, the printed
constructs produced were not uniform and therefore printing accuracies
based on filament width were unable to be calculated; Fig.
10b(ii)\textbf{.} The printing accuracies (\%) produced for each needle
gauge experiment include; 136.53 \%, 86.01 \% and 129.93 \% for 15G, 18G
and 23G, respectively. Although no statistical significance was found
between groups, the 18G needle produced a printing accuracy closest to
100 \% (Fig. 10a).

Flow rates

The spreading ratio was determined at different flow percentages (100\%,
125\%, 150\%, 200\%) using the following equation:

\begin{equation}
Spreading\ ratio=\frac{\text{Filament\ diameter}}{\text{Nozzle\ diameter}}\nonumber \\
 \end{equation}

The flow percentages listed are extrusion multipliers. These values
instruct the printers to extrude more material during the prints.
Post-printing images were taken immediately after printing. Calcium
chloride was not added to the printed constructs for cross-linking. A
nozzle diameter of 0.838 mm (18G needle) was used for the syringe
extruder based on the results obtained from the needle gauge
printability experiment. Three constructs were printed for each flow
percentage tested. An average of the width, measured at various
positions along the filament, of the printed filaments were divided by
the needle diameter which remained constant throughout the experiment;
0.838 mm. The constructs were printed using 6\% (w/v) alginate hydrogel
with no post-printing cross-linking methods employed.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image11/image11}

\end{center}
\end{figure}

\textbf{Fig. 11. A comparison of hydrogel printability. A: Post printing
spreading ratio's (filament diameter/needle diameter) determined for
various flow rates using 6\% (w/v) alginate. B: Post printing
photographs of printed constructs for each flow rate (i) 100\%, (ii)
125\%, (iii) 150\% and (iv) 200\%. Data points are means; error bars
represent SEM for n = 3. Asterix represents statistical significance (p
= 0.0363; p \textless{} 0.05), no Asterisk shows no statistical
significance (p \textgreater{} 0.05). Scale bars represent 5 mm.}

Figure 11 shows a positive correlation between flow percentage and
spreading ratios obtained. The spreading ratios obtained for 100 \% and
200 \% flow percentage groups showed a statistically significant
difference; p = 0.0363; p \textless{} 0.05, whereas no other statistical
significance was found between groups. Similarly, Kahl \emph{et
al.}(2019) shows the higher the extrusion rate, at a constant nozzle
diameter, results in lower shape fidelity of the printed construct.
Images in Fig. 11 show the inconsistency in filament structure produced
when printing at a 100 \% flow percentage, even though a low spreading
ratio was noted. Due to the complex nature of bioprinting and the
necessity to print complex geometries at extremely high resolutions, the
final printed construct must be highly precise and accurate. A high
spreading ratio is, therefore, an undesirable characteristic and further
optimizing must take place in order to accommodate for this {[}24{]}.

\textbf{Conclusions}

The overall aim to create a low-cost bioprinter from a commercially
available thermoplastic 3D delta printer was achieved. The final
bioprinter designed and manufactured consists of an already established
syringe-based extruder, incorporated with minor alterations to fit the
delta style 3D printer, and an easily built commercially available Delta
printer with added enclosure and easily modifiable firmware. Converting
the thermoplastic Delta 3d printer into a bioprinter cost a total of \$
1,007.52, making it affordable to most research laboratories. This
system allows for the printing of geometrically complex structures using
cell-free hydrogel based bioinks and can be easily modified (i.e.,
firmware parameters) for other paste extrusion materials and future
cell-laden hydrogel bioprinting. There is, however, much room for
improvement where this study only aims to provide the much needed and
promising foundation work for what could be a fully functional low-cost
bioprinter with multiple applications. The modifications made to the
hardware during the conversion from 3D printer to bioprinter were
successful and allowed for an increased sterile environment for the
sensitive nature of bioprinting, as set out as a priority criterion. To
further increase sterility, one improvement would include the addition
of HEPA-filtered laminar air flow to the system. Smaller
sterility-related improvements may include the addition of UV lights to
the system and re-designing of the internal hardware components (i.e.,
extruder carriage) into fully enclosed and easily cleaned singular
components.

Securing the extruder carriage to the frame of the printer had the
desired effect of removing the excessive weight from the effector plate
which then allowed for smoother printing movements and higher printed
construct quality. Another important design criterion stipulated was
that of producing highly complex shapes with precise geometry. This
criterion was partially met but remains an area that requires much
improvement. Lowering the extruder carriage closer to the nozzle would
aid in reducing the distance between the syringe feedstock and the
nozzle which in turn allows for more control over the extrusion of the
bioinks. More control over the extrusion means less possibility of under
or overextrusion; gaps forming within the construct versus leakage of
bioink during travel moves, respectively.

Overall, the results achieved with the final bioprinter prototype model
were satisfactory and provide the much-needed foundation work for future
optimization studies. Incorporating the use of cell-laden bioinks and
assessing the different modes of cell-based bioprinting will be employed
in future work, along with the assessment of various bioink formulations
and cross-linking methods. While Delta style bioprinters are
commercially available (Pensees Vitarix and VitarixW) the systems are
registered design closed-source premium systems. The leading goal of the
SV1 was toward the development of an open-source system.. Relative to
commercial systems the substantial cost reduction presented may prove to
be impactful to research laboratories in lower resourced economies as
well as in developed countries.

\textbf{Acknowledgements}

The authors acknowledge the South African Medical Research Council for a
Self-Initiated Research grant, National Research Foundation of South
Africa (NRF) for a Competitive Support for Unrated Researchers (UID:
129400) and Rhodes University for research and student funding.

\textbf{Conflict of interest}

Authors have no conflict of interest relevant to this article.

\textbf{Human and animal rights}

Not applicable.

\textbf{References:}

{[}1{]} S. Melissaridou,, E. Wiechec,, M. Magan,, M.V. Jain,, M.K.
Chung,, L. Farnebo,, K. Roberg, Cancer Cell Int. 19(1) (2019) 1--10.
10.1186/s12935-019-0733-1.

{[}2{]} J. Mason,, S. Visintini,, T. Quay, CADTH Issues Emerg. Heal.
Technol. (2016).

{[}3{]} R. Jamee,, Y. Araf,, I. Bin Naser,, S.K. Promon, Regen. Ther. 18
(2021) 133--45. 10.1016/j.reth.2021.05.006.

{[}4{]} B. Yenilmez,, M. Temirel,, S. Knowlton,, E. Lepowsky,
Bioprinting (January) (2019) e00044. 10.1016/j.bprint.2019.e00044.

{[}5{]} E.S. Bishop,, S. Mostafa,, M. Pakvasa,, H.H. Luu,, M.J. Lee,,
J.M. Wolf,, G.A. Ameer,, T.C. He,, R.R. Reid, Genes Dis. 4(4) (2017)
185--95. 10.1016/j.gendis.2017.10.002.

{[}6{]} F. You,, B.F. Eames,, X. Chen, Int. J. Mol. Sci. 18(7) (2017)
1597. 10.3390/ijms18071597.

{[}7{]} H. Ding,, R.C. Chang, Appl. Sci. 8(3) (2018).
10.3390/app8030403.

{[}8{]} Z. Xia,, S. Jin,, K. Ye, SLAS Technol. Transl. Life Sci. Innov.
(2018) 247263031876051. 10.1177/2472630318760515.

{[}9{]} F. Liu,, C. Liu,, Q. Chen,, Q. Ao,, X. Tian,, J. Fan,, H. Tong,,
X. Wang, Int. J. Bioprinting 4(1) (2018) 1--15. 10.18063/ijb.v4i1.128.

{[}10{]} Z. Zheng,, J. Wu,, M. Liu,, H. Wang,, C. Li,, M.J. Rodriguez,,
G. Li,, X. Wang,, D.L. Kaplan, Adv. Healthc. Mater. 7(6) (2018) 1701026.
10.1002/adhm.201701026.

{[}11{]} J.A. Reid,, P.A. Mollica,, G.D. Johnson,, R.C. Ogle,, R.D.
Bruno,, P.C. Sachs, Biofabrication 8(2) (2016) 025017.
10.1088/1758-5090/8/2/025017.

{[}12{]} K. Pusch,, T.J. Hinton,, A.W. Feinberg, HardwareX 3 (2018)
49--61. 10.1016/J.OHX.2018.02.001.

{[}13{]} W. Jia,, P.S. Gungor-Ozkerim,, Y.S. Zhang,, K. Yue,, K. Zhu,,
W. Liu,, Q. Pi,, B. Byambaa,, M.R. Dokmeci,, S.R. Shin,, A.
Khademhosseini, Biomaterials 106 (2016) 58--68.
10.1016/j.biomaterials.2016.07.038.

{[}14{]} T.J. Hinton,, Q. Jallerat,, R.N. Palchesko,, J.H. Park,, M.S.
Grodzicki,, H.-J. Shue,, M.H. Ramadan,, A.R. Hudson,, A.W. Feinberg,
Sci. Adv. 1(9) (2015) e1500758--e1500758. 10.1126/sciadv.1500758.

{[}15{]} C. Amza,, A. Zapciu,, D. Popescu, Technologies 5(3) (2017) 50.
10.3390/technologies5030050.

{[}16{]} B. Wijnen,, E.J. Hunt,, G.C. Anzalone,, J.M. Pearce, PLoS One
9(9) (2014) e107216. 10.1371/journal.pone.0107216.

{[}17{]} L. Banovi\selectlanguage{polish}ć,, \selectlanguage{english}B. Vihar, J. Open Hardw. 2(1) (2018) 1--10.
10.5334/joh.6.

{[}18{]} J.W. Tashman,, D.J. Shiwarski,, A.W. Feinberg, HardwareX 9
(2021) e00170. 10.1016/j.ohx.2020.e00170.

{[}19{]} S. Fanucci,, M.W. Barwick,, E. Prinsloo, South African Med. J.
109(8b) (2019) 34. 10.7196/samj.2019.v109i8b.13838.

{[}20{]} B.M. Schmitt,, C.F. Zirbes,, C. Bonin,, D. Lohmann,, D.C.
Lencina,, A. Da Costa Sabino Netto, Mater. Res. 20 (2017) 883--6.
10.1590/1980-5373-mr-2016-1039.

{[}21{]} M. Kahl,, M. Gertig,, P. Hoyer,, O. Friedrich,, D.F. Gilbert,
Front. Bioeng. Biotechnol. 7(July) (2019) 1--12.
10.3389/fbioe.2019.00184.

{[}22{]} T. Billiet,, E. Gevaert,, T. De Schryver,, M. Cornelissen,, P.
Dubruel, Biomaterials 35(1) (2014) 49--62.
10.1016/j.biomaterials.2013.09.078.

{[}23{]} A. Faulkner-Jones,, S. Greenhough,, J. A King,, J. Gardner,, A.
Courtney,, W. Shu, Biofabrication 5(1) (2013).
10.1088/1758-5082/5/1/015013.

{[}24{]} A.C. Daly,, S.E. Critchley,, E.M. Rencsok,, D.J. Kelly,
Biofabrication 8(4) (2016) 045002. 10.1088/1758-5090/8/4/045002.

\textbf{List of linked webpages:}

{[}L1{]} https://reprap.org/wiki/Kossel

{[}L2{]} https://www.thingiverse.com/

{[}L3{]} https://github.com/MarlinFirmware/Marlin

{[}L4{]} http://www.anycubic3d.com/support/show/594034.html

{[}L5{]} https://www.arduino.cc/en/Main/Software

{[}L6{]}
http://reprapltd.com/reprappro/documentation/building-the-paste-extruder/index.html

\textbf{Tables and Figures:}

\textbf{Table 1: Bill of Materials for the final Bioprinter build (given
in South African Rands and United States Dollar).}\selectlanguage{english}
\begin{longtable}[]{@{}llllllllllll@{}}
\toprule
\textbf{Designator} & \textbf{Designator} & \textbf{Component} &
\textbf{Number} & \textbf{Cost per unit} & \textbf{Cost per unit} &
\textbf{Total cost} & \textbf{Total cost} & \textbf{Total} &
\textbf{Total} & \textbf{Source of materials} & \textbf{Material
type}\tabularnewline
\midrule
\endhead
& & & & \textbf{South African Rand (ZAR)} & \textbf{United States Dollar
(USD)} & \textbf{South African Rand (ZAR)} & \textbf{United States
Dollar (USD)} & \textbf{South African Rand (ZAR)} & \textbf{United
States Dollar (USD)} & &\tabularnewline
3D printer modified for bioprinting & 3D printer modified for
bioprinting & Anycubic Kossel 3D Delta printer with all components &
1,00 & 6999.95 & 804,00 & 11999,95 & 804,00 & R 6999.95 & \$ 447,19 &
\emph{www.Diyelectronics.co.za} & Other\tabularnewline
3D printed parts & Bioprinter & Corners (A1, A2, A3) & 3,00 & 73,99 &
4,96 & 221,97 & 14,87 & R 573,42 & \$ 38,42 &
\emph{www.Diyelectronics.co.za} & PLA\tabularnewline
& Electronic board box & Box & 1,00 & 37,00 & 2,48 & 37,00 & 2,48 & & &
&\tabularnewline
& & Lid & 1,00 & 37,00 & 2,48 & 37,00 & 2,48 & & & &\tabularnewline
& Bed platform & Support type 1 & 3,00 & 22,20 & 1,49 & 66,59 & 4,46 & &
& &\tabularnewline
& & Support type 2 & 3,00 & 22,20 & 1,49 & 66,59 & 4,46 & & &
&\tabularnewline
& Extruder & Top strengthener & 1,00 & 22,20 & 1,49 & 22,20 & 1,49 & & &
&\tabularnewline
& & Frame & 1,00 & 22,20 & 1,49 & 22,20 & 1,49 & & & &\tabularnewline
& & Drive gear & 1,00 & 11,10 & 0,74 & 11,10 & 0,74 & & &
&\tabularnewline
& & Mounting bracket & 1,00 & 22,20 & 1,49 & 22,20 & 1,49 & & &
&\tabularnewline
& & Driven gears & 2,00 & 11,10 & 0,74 & 22,20 & 1,49 & & &
&\tabularnewline
& & Drive block & 1,00 & 11,10 & 0,74 & 11,10 & 0,74 & & &
&\tabularnewline
& & Bearing holders & 2,00 & 11,10 & 0,74 & 22,20 & 1,49 & & &
&\tabularnewline
& Nozzle & Luer-lock effector plate adaptor & 1,00 & 11,10 & 0,74 &
11,10 & 0,74 & & & &\tabularnewline
Perspex parts & Bioprinter Frame & Frame (B1, B2, B3) & 3,00 & 182,85 &
12,25 & 548,55 & 36,75 & R 1279,95 & \$ 85,76 & Private Manufacturing &
Perspex\tabularnewline
& Bioprinter sides & Window (C1, C2) & 2,00 & 182,85 & 12,25 & 365,70 &
24,50 & & & &\tabularnewline
& Bioprinter door & Door (C3) & 1,00 & 182,85 & 12,25 & 182,85 & 12,25 &
& & &\tabularnewline
& & Handle (C4) & 1,00 & 182,85 & 12,25 & 182,85 & 12,25 & & &
&\tabularnewline
Mechanics & Bioprinter Frame & M3 cap-head screws & 18,00 & 1,86 & 0,12
& 33,46 & 2,24 & R 603,39 & \$ 40,43 & \emph{www.Diyelectronics.co.za} &
Metal/Other\tabularnewline
& & M3 T-nuts & 18,00 & 0,29 & 0,02 & 5,20 & 0,35 & & & &\tabularnewline
& & M3 Washers & 36,00 & 0,68 & 0,05 & 24,31 & 1,63 & & &
&\tabularnewline
& Bioprinter door & M3 cap-head screws & 10,00 & 1,86 & 0,12 & 18,59 &
1,25 & & & &\tabularnewline
& & M3 nuts & 10,00 & 0,29 & 0,02 & 2,89 & 0,19 & & & &\tabularnewline
& & M3 Washers & 20,00 & 0,68 & 0,05 & 13,50 & 0,90 & & &
&\tabularnewline
& & Hinges & 2,00 & 75,00 & 5,03 & 150,00 & 10,05 & & & &\tabularnewline
& Bioprinter sides & M3 cap-head screws & 6,00 & 1,86 & 0,12 & 11,15 &
0,75 & & & &\tabularnewline
& & M3 T-nuts & 6,00 & 0,29 & 0,02 & 1,73 & 0,12 & & & &\tabularnewline
& & M3 Washers & 12,00 & 0,68 & 0,05 & 8,10 & 0,54 & & &
&\tabularnewline
& Electronic board box & M3 cap-head screws & 2,00 & 1,86 & 0,12 & 3,72
& 0,25 & & & &\tabularnewline
& & M4 screws & 2,00 & 0,29 & 0,02 & 0,58 & 0,04 & & & &\tabularnewline
& & M4 T-nuts & 2,00 & 1,42 & 0,10 & 2,84 & 0,19 & & & &\tabularnewline
& Bed platform & M3 cap-head screws & 12,00 & 1,86 & 0,12 & 22,31 & 1,49
& & & &\tabularnewline
& & M3 T-nuts & 12,00 & 0,29 & 0,02 & 3,47 & 0,23 & & & &\tabularnewline
& Nozzle & M3 cap-head screws & 4,00 & 1,86 & 0,12 & 7,44 & 0,50 & & &
&\tabularnewline
& & M3 nuts & 4,00 & 0,29 & 0,02 & 1,16 & 0,08 & & & &\tabularnewline
& & M3 Washers & 8,00 & 0,68 & 0,05 & 5,40 & 0,36 & & & &\tabularnewline
& Extruder & M3x35mm cap-head screws & 3,00 & 2,15 & 0,14 & 6,44 & 0,43
& & & &\tabularnewline
& & M3 nuts & 11,00 & 0,29 & 0,02 & 3,18 & 0,21 & & & &\tabularnewline
& & M3 nyloc nut & 3,00 & 1,36 & 0,09 & 4,08 & 0,27 & & &
&\tabularnewline
& & M3 plain washer & 14,00 & 0,68 & 0,05 & 9,45 & 0,63 & & &
&\tabularnewline
& & 623 bearings & 4,00 & 31,57 & 2,12 & 126,28 & 8,46 & & &
&\tabularnewline
& & M5x95mm cap-head screw & 1,00 & 7,25 & 0,49 & 7,25 & 0,49 & & &
&\tabularnewline
& & M5 nut & 10,00 & 0,62 & 0,04 & 6,23 & 0,42 & & & &\tabularnewline
& & M3x8mm cap-head screw & 10,00 & 2,14 & 0,14 & 21,44 & 1,44 & & &
&\tabularnewline
& & M3x50mm threaded bar & 2,00 & 47,66 & 3,19 & 95,33 & 6,39 & & &
&\tabularnewline
& & M4 cap-head screw & 2,00 & 2,51 & 0,17 & 5,03 & 0,34 & & &
&\tabularnewline
& & M4 T-nut & 2,00 & 1,42 & 0,10 & 2,84 & 0,19 & & & &\tabularnewline
Miscellaneous & Bed platform & Circular glass bed (? \selectlanguage{english}220 mm) & 1,00 &
77,99 & 5,23 & 77,99 & 5,23 & R 280,95 & \$ 18,82 & PGglass &
Glass\tabularnewline
& & Circular glass bed (? \selectlanguage{english}220 mm with ? \selectlanguage{english}96 mm centre cut out) & 1,00 &
77,99 & 5,23 & 77,99 & 5,23 & & & &\tabularnewline
& Nozzle & 200mm Bowden tube & 1,00 & 79,99 & 5,36 & 79,99 & 5,36 & & &
Pharmacy & Plastic/other\tabularnewline
& & 18G Luer lock syringe needle & 1,00 & 19,99 & 1,34 & 19,99 & 1,34 &
& & &\tabularnewline
& Extruder & 5 ml Luer lock syringe & 1,00 & 24,99 & 1,67 & 24,99 & 1,67
& & & &\tabularnewline
Electronics & Extruder & NEMA 17 stepping motor & 1,00 & 299,95 & 20,10
& 299,95 & 20,10 & R 299,95 & \$ 20,10 & \emph{www.Diyelectronics.co.za}
& Other\tabularnewline
& & 4-pin extruder head cable & 1,00 & 0,00 & 0,00 & 0,00 & 0,00 & & &
&\tabularnewline
& & & & & & & & \textbf{R 15 037,61} & \textbf{\$ 1 007,52} &
\textbf{TOTAL} & \textbf{TOTAL}\tabularnewline
\bottomrule
\end{longtable}

\textbf{Table 2: Anycubic Kossel Delta 3D printer technical
specifications.}\selectlanguage{english}
\begin{longtable}[]{@{}ll@{}}
\toprule
Make & Anycubic\tabularnewline
Model & Kossel Linear plus\tabularnewline
Software input formats & .STL, .OBJ, .AMF\tabularnewline
Software output formats & GCODE\tabularnewline
Technology & Fused Deposition Modelling\tabularnewline
Frame & Delta\tabularnewline
Year & 2017\tabularnewline
Print area & \selectlanguage{greek}Φ\selectlanguage{english}230 x 300 mm\tabularnewline
Max. bed temperature & 100@\selectlanguage{english}C\tabularnewline
Nozzle size & 0.4 mm\tabularnewline
Max. nozzle temperature & 260@\selectlanguage{english}C\tabularnewline
Max. Z-axis resolution & 0.1 -- 0.4 mm\tabularnewline
Z-axis accuracy & 0.0025 mm\tabularnewline
X/Y-axis accuracy & 0.0125\tabularnewline
Max. print speed & 60 mm/s\tabularnewline
Max. travel speed & 60 mm/s\tabularnewline
Printable materials & PLA, ABS\tabularnewline
Interface & LCD display\tabularnewline
Connectivity & SD card\tabularnewline
Printer size & 380 mm x 680 mm\tabularnewline
Printer weight & 7 kg\tabularnewline
Power input & AC 110-220V AC, 50/60Hz\tabularnewline
Firmware & Marlin\tabularnewline
Board & Tri-Gorilla control board\tabularnewline
\bottomrule
\end{longtable}

\textbf{Appendix}

Detailed protocol for preparing a 6 \% (w/v) Alginate bioink

A 6\% (w/v) alginate solution was prepared by dissolving alginic acid
sodium salt in double distilled water (ddH\textsubscript{2}O) under
continuous stirring on a hot plate. The alginate was prepared and used
immediately before each experiment. A 0.3 M
CaCl\textsubscript{2}solution was prepared and used for cross-linking of
the alginate post-printing.

\selectlanguage{english}
\FloatBarrier
\end{document}

