Materials and Manufacturing Processes

ISSN: (Print) (Online) Journal homepage: https://www.tandfonline.com/loi/lmmp20

3D Printing of Shrimp Derived Chitosan with HAp

as a Bio-Composite Scaffold

N Pradeep, C Chandrasekhara Sastry, Lc Brandão & B.S. Meennakshi

To cite this article: N Pradeep, C Chandrasekhara Sastry, Lc Brandão & B.S. Meennakshi (2021):
3D Printing of Shrimp Derived Chitosan with HAp as a Bio-Composite Scaffold, Materials and
Manufacturing Processes, DOI: 10.1080/10426914.2021.1973032

To link to this article: https://doi.org/10.1080/10426914.2021.1973032

Published online: 07 Sep 2021.

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MATERIALS AND MANUFACTURING PROCESSES
https://doi.org/10.1080/10426914.2021.1973032

3D Printing of Shrimp Derived Chitosan with HAp as a Bio-Composite Scaffold

a
N Pradeep , C Chandrasekhara Sastryb, Lc Brandãoc, and B.S. Meennakshid
a
Faculty, Mechanical Engineering Department, Main Campus: CEG, Anna University, Chennai, Tamil Nadu, India; bFaculty, Mechanical Engineering
Department, Indian Institute of Information Technology Design and Manufacturing Kurnool, Andhra Pradesh, India; cFaculty, Centre for Innovation in
Sustainable Manufacturing, Mechanical Engineering Department, Federal University of São João del Rei, Minas Gerais, Brazil; dDepartment of
Physiology, Government Medical College, Chengalpattu, Tamil Nadu, India

ABSTRACT ARTICLE HISTORY

India is the world’s 3rd largest shrimp producer at the same stint the shrimp industry generates 40%–70% Received 17 June 2021
waste of the catch volume. In this research, chitosan from shrimp (Penaeus indicus) waste was extracted at Accepted 13 August 2021
75% degree of deacetylation and was brought down to powder form. Parallelly, a wet chemical precipita­ KEYWORDS
tion method was adopted to produce hydroxyapatite suspension. The Chitosan powders were blended 3D; biocomposite; cell;
with the HAp suspension and finally ball milled to achieve composite powders of particle size 0.020 mm culturing; chitosan;
having mesoporous nature (3.17 nm) when quantified with BJH model. The scaffold was designed environment;
considering four design models namely the hollow cube, dumbbell, dual cone and honeycomb models hydroxyapatite; printing;
with 62–67% of porosity. Post FE simulation analysis and design optimization using Pugh’s matrix revealed scaffold; waste; reduction
that the honeycomb model was superior considering the mass (g), porosity (%), compressive stress (MPa)
and the level of complexity in mesh array. The composite powders were preheated up to 200°C and then
the laser with spot of 0.5 mm width sinters the composite powder forming a scaffold as per design using
the Selective Laser Sintering (SLS) process. The final part was subjected to cell viability assay using MG63
osteoblast cell line resulted in an increased cell level on the sixth day.

Introduction
colossal flexibility and being biodegradable.[4] Also, chitosan
India, being the top shrimp producer and exporter across the powder provides sites for complex calcium formation, owing to
globe with a market value of 150 crores producing 6,50,000 better compatibility between HAp powdered particles. Latest
tons in the year 2020 despite the COVID-19 pandemic.[1] The developments have been the application of HAp and polymers
shrimp market is expected to reach 1,500,000 tons by 2026. (organic) in nature,[5–10] i.e., collagen,[6] chitosan[7] and other
During processing the aquaculture sector approximately pro­ organic polymers, [8–10] where nanocomposites (HAp) are pre­
duces 106 million tons/annum of waste, a large portion of the pared extensively. These extensively prepared nano-
waste is bound to treat the soil in the form of manures or composites have shown extensive bioactivity and simultaneous
further treated to feed the animals and the remains are dumped equity in the mechanical strength of 40 MPa. Though many
in the coastal area that would create ecological imbalance. In organic polymers, find extensive usage in the clinical field,
such circumstances, the Indian shrimp processing industries chitosan stands out as a bone substitute, due to its minimal
produce more than 150,000 ton as wastage and this waste can toxic nature, colossal flexibility and being biodegradable.[9]
be used to extract minerals, antioxidants, chitin, chitosan, and Also, chitosan powder provides sites for complex calcium for­
proteins[2,3] for various applications. Though many organic mation, owing to better compatibility between HAp powdered
polymers, find extensive usage in the clinical field, chitosan particles. Scientists and researchers across the globe used
stands out as a bone substitute, due to its minimal toxic nature, mechanochemical reactions with the application of ball mill

CONTACT Pradeep N natarajanpradeep11@gmail.com Faculty, Mechanical Engineering Department, Main Campus: CEG, Anna University, Chennai, Tamil
Nadu, India
© 2021 Taylor & Francis
2 N. PRADEEP ET AL.

to produce HAp nanocomposites. The drawback of this pro­ design. The advantage of using SLS in scaffold fabrication
cess involves high time duration to obtain at nano-scale.[10,11] has already been studied by various researchers. The selec­
So, a combination of wet precipitation method along with tion of machining parameters determines the quality of
pelletization, provides a useful way of synthesizing polymer scaffold with no warpage, shrinkage, having desired surface
HAp inorganic nanocomposites. This technique was applied topography. The parameters such as laser fill power, laser
for chitosan-HAp nanocomposite preparation, in order to scanning speed, laser spot width, scan fill space, orientation,
obtain novel bone substitutes, which have a close correlation bed temperature determine the quality of the scaffold.[17]
to bones analogous in nature. Among those, the most predominant parameters suitable for
From the evolution of fetus to a full-grown adult, the bones nano-composite HAp scaffold are the laser power, laser scan
undergo from being cartilage molds to full-grown structures speed, scan fill space, bed temperature.[17–19]
adapting with age, lifestyle, eating habits and exercise. There The survey helped us to understand the individual nature
are three categories of cells in the body, which bring about of the materials and the possibility of fabricating a metal and
changes in the ones: osteocytes, osteoclasts and osteoblasts.[12] or polymer scaffold using 3D printing with commercially
The osteoblasts are bone forging cells that engender the matrix available materials while no authors reported producing
that makes up the bone. The matrix part of the bone is made of materials from shrimp waste with lab-prepared HAp and
organic material inclusive of molecules such as collagen pro­ turning them into a bio-composite scaffold using ‘3D print­
tein fibers, which administer bone the required flexibility, ing’ which is the novelty of this research article. In this work,
resilience and presence of calcium (Ca2+), phosphate (PO4-) Chitosan was generated from shrimp waste shells and then
ions provide appended rigidity. The molecules (matrix) are blended in HAp suspension obtained from the wet chemical
built and enveloped due to osteoblasts for discharge into the method, further processed to obtain powder particles using
extracellular environment. These discharged molecules react the ball milling setup. The best scaffold design from four
with one another to form austere flexible bone tissues called design models i.e., hollow cube, dumbbell, dual cone and
osteoid that harden the formation of bone.[12,13] The bones in the honeycomb models with porosity level within 62–67%
the body are naturally designed in three-dimension with an was selected after understanding the model’s versatility
inorganic-organic nanocomposite in which crystals of hydro­ upon compressive load, and obtain an optimal structure
xyapatite (HAp, Ca10(PO4)6(OH)2) are garnered on collagen. viable for scaffold using the FE analysis. The final processed
The advantage of this type of structure, is a combination of two scaffold design was then fabricated availing the modified
factors, the colossal mechanical strength of the bone mineral selective laser sintering with a lab-made Chitosan-HAp inor­
and flexibility due to the presence of fibrous form collagen.[1] ganic-organic polymer composite. The fabricated scaffold was
The ceramic powders that exhibit biological properties and subjected to cell viability, to understand its biocompatibility
affinity are termed bioactive nanocomposites/ceramics. nature using MG-63 cell assay.
Crystalline apatite, wallostonite, sinter HAp, bioglass (Na2
O-CaO-SiO2-P2O5) are few bioactive ceramic materials found
in nature. Amongst them, only a few ceramic powders bond Materials and methods
directly to the contemporary living bone in the human body. In Preparation of Chitosan suspension
the effective case of artificial powders, an envelope of fibrous
tissues is carried out, to encapsulate the powder in the area of Chitosan was extracted from the collected shrimp (Penaeus
a bone defect.[14] The bonding nature of the ceramics being indicus) waste shells after thorough wash to remove dirt particles
bioactive toward the bone has generated interest in the medical from the shells. The washed shells are then dried in a hot air
community as this creates a rigid and irritation-free fixation oven at about 80°C for 3 days and later dried and grounded into
between the scaffold/implant and the bone. Though most of fine powder as shown in Fig. 1. Chitosan suspension was then
the ceramic biomaterials have their application in the field of prepared with 125 gram of chitosan powder in 0.3 mol/L acetic
orthopedics, for repair or fixing of damaged bone tissues, acid. This dissociated powdered acetic solution was magnetically
a serious limitation exists due to their brittle nature, minimal stirred for 35 minutes. This solution was adjusted in the presence
toughness and modulus of elasticity.[15] The combination of of ammonia aqueous solution, to obtain a pH value at 9.0–10.2.
inorganic and organic nanocomposites provides a design From Fig. 1(a) It was observed that the spectral peak falls at
(novel in nature) that can be molded and shaped into con­ the wavenumber 1656 cm−1 depicts the amide-1 group and the
toured desirable sizes and shapes as specified by the clinical vibrational peaks at 1550 cm−1 depicts the N-H bending of the
field. amide II group. Similar spectral bands were confirmed by
Almost any materials can be tailored (by design) with various researchers.[20–22] This confirms the presence of
desired mechanical properties, bio-chemical properties Chitosan extracted from shrimp shells.
using 3D printing technology. The design of the scaffold is
based on the design thinking of the researchers. Various
Preparation of HAp suspension
designs like honeycomb, auxetic, s-shaped, I-shaped scaffold
designs have been proved to be an optimal design viz. A wet chemical precipitation method was carried out,[23,24] to
attaining all design considerations such as porosity, mesh carry out HAp powder synthesis as depicted in Fig. 2(a). In
complexity, load-bearing capacity, optimization, etc.[16,17] comparison to pellet formation and sintering, this provided better
Selective Laser Sintering (SLS) is one among other additive efficacy, to attain HAp powder of Ca/P ratio of 1.64, with an
manufacturing processes for fabricating scaffolds by tailored accession of 12 grams of Ca and 18.89 grams of NH4OH+PO43-.
 MATERIALS AND MANUFACTURING PROCESSES 3

Figure 1. Preparation of Chitosan and its (a) FT-IR plot with wavenumber against %transmission and SEM micrograph (inset) of Chitosan flakes at ×500.

Figure 2. (a) HAp powder synthesis (b) FTIR at elevated temperature.

The powders are brought down to a particle size of < 0.1 µm achieve mean particle size, stability in the solution and equal
after ball milling. Figure 2(b) depicts the spectral peaks stretch­ size distribution. The slurry was withdrawn and dried in the
ing at 1149–1000 cm−1 and at 956 cm−1 exhibiting phosphate furnace for 6 mins at 60°C and then milled for 10 hours using
functional group which is a characteristic peak for HAp. ball milling process in order to obtain a fine particle composite
Similarly, the observed bending of peaks between 556 cm−1 to powdered. After the completion of milling cycle, the powder
620 cm−1 confirms the presence of HAp.[25,26] HAp suspension grains are transferred to a nonstick vessel and stirred at dis­
was then prepared by mixing the obtained HAp powders with parate temperatures of 35 to 75°C of a duration of 36 hours. The
ethanol in the ratio of 1 gms:60 ml at room temperature. obtained slurry undergoes sieve analysis and the residue was
thoroughly mixed with ultrapure water before the pH reaching
the achromatic neutral point. The precipitate formed was again
Preparation of composite Chitosan-HAp powder dried in a furnace at 60°C for 36 hours. The same process was
The prepared Chitosan solution was mixed with the HAp sus­ carried until 300 gms of composite powder is prepared as shown
pension solution resulting in a composite formation at 1:3 ratio. in Fig. 3. The TEM micrograph exhibiting needle like shape of
The composite solution was ultrasonicated for 30 minutes[27] to the composite Chitosan-HAp powder imaged on 50 nm scale.
4 N. PRADEEP ET AL.

Figure 3. Preparing HAp/Chitosan composite powder (a) TEM of Composite suspension, (b) Ball milling set-up and (c) Chitosan/HAp powder.

Table 1. Design specification for the models.

Structure Hollow cube Dumb bells Honeycomb Dual cone
Porous structure is modeled by creating a
Hollow
Horizontal and vertical opening Space around the circular hexagon and Space around the tapered circular
Description channels beam holes at faces beam.
Complexity Medium Medium Medium Medium
Porosity (%) attained from 62.3 63.1 62.9 66.3
ABACUS
Size l x b x h mm3 16 x 16 x 25 15 x 15 x 25 15 x 16 x 25 15 x 15 x 25
Total Mass (Grams) 17.04 15.97 17.15 14.6

Figure 4. 3D- Scaffold design models.

 MATERIALS AND MANUFACTURING PROCESSES 5

Design models for scaffold fixed as shown in Table 2 enabled to fabricate the composite
powdered scaffold with no warpage using the modified SLS
For promoting osteoconduction, a biocompatible structure was
machine.
designed based on predominant criteria’s such as porosity,
The powders were preheated in a bin up to 200°C and then
stress, minimum wall thickness, and the element size and
the CO2 laser source of 100 W was used to fuse the powder
shape. Research studies have proved that porosity in the
particles in a nitrogen atmosphere with laser beam speed of
range of 60% to 80% is desired to accelerate the osseointegra­
1600 m/s at 45° build direction[29] to decrease the anisotropic
tion process.[28] The stress component plays its part in main­
properties of the composite material.
tenance and implant longevity/survival. A minimum wall
thickness fabricated availing SLS is 1 mm and hence created
3D elements with a size of 3 × 3 × 3 mm3. The various design Preparation of scaffold for cell culture
models and its description availing the above conditions are
For the manufactured scaffold to exhibit bioactivity form,
tabulated in Table 1 and Fig. 4.
the scaffold was scaled down to 9 × 4 mm for an easy fit
into cell culture petri dish. The CAD model of the cell
culture petri dish was modeled and assembled to validate
Modified SLS machine
the honeycomb structured specimen fit inside the cell cul­
The SLS machine (DTM-Sintersation 2500+, USA) was mod­ turing petri dish, as indicated in Fig. 5. The scaled-down
ified for conscious consumption of biomaterials with scaffold was soaked in simulated body fluids (SBF) to
a miniaturized 2.5-liter bin system with 80 mm piston and reproduce in vitro bioactivity. The specimens were
double chambers for powder supply with separate control immersed in 40 ml of simulated body fluids (Na+ 142.0,
movements using stepper motors. The movements were Mg2+, K+ 5.0, Ca2+ 2.5, Cl− 147.80, HPO42-, HCO3− and
directly connected to the existing piston cylinder of the SO42- m.mol/L for a span of 10 days. The SBF solution was
Sintersation 2500+ and the parameters were administered by buffered at a pH value of 7.50 with 55 m.mol/L tris hydro­
extant sinterstation software and this enabled the building xymethyl-aminomethane ((CH2OH)3CNH2) and sparse
scaffold to its intended shape and size. The preliminary experi­ amounts of hydrolic acid (HCl), keeping the temperature
mental trials adopting Response Surface methodology (RSM)- constant at 38°C.[30] After complete soaking for 10 days, the
based design of experiments by varying fill scan space, laser specimen was cloistered out of the SBF chamber, cleaned
spot width and keeping other parameters such as laser power, with distilled water and oven-dried in a sealed enclosure.
laser beam speed, bed temperature, and stepper motor speed Dulbecco’s altered Eagles’ medium (DMEM) was acquired
from PAN Biotech augmented with 10% FBS (Invitrogen Life
Technologies) 1% penicillin and streptomycin it was stored at
4°C. Once the media becomes acidic indicated by color change
Table 2. Parameters suitable for building composite scaffold.
(pink to yellow), the cells were taken and given a media change.
Parameters Value
DMEM was taken from 4°C and brought down to room tem­
Laser Power (CO2) 100 W
Laser Beam Speed 1600 mm/s @ 45°
perature. The working area of the laminar air hood was ster­
Bed temperature 200°C ilized with spirit and then the hood was UV sterilized for about
Fill Scan Space 0.2 to 0.3 mm 15 minutes.[31] The media was discarded from the flask and the
Laser spot 0.5 mm width
Stepper motor 140 m/s
cells were cleaned twice with 1 ml of PBS (Phosphate buffered
saline). About 3–4 ml of fresh DMEM media was added to the

Figure 5. 3D model of petri dish preparation and the sample dimension.

6 N. PRADEEP ET AL.

cells and kept inside the incubator with culture conditions

(37°C/5% CO2). The coalescent cells were washed twice with
1 ml of 1xPBS. The cells were trypsinized with 1 ml of trypsin
and kept inside the incubator with culture conditions (37°C/5%
CO2) for about 5–10 minutes till the cells come off as sheets.
The cells were ascertained under an inverted microscope for the
appearance of detached cells. About 1 ml of thawed DMEM
media was added to the trypsinized cells and gently pippetted to
avoid clumping of cells. About 1 ml of the final volume of cells
was pippetted out and reseeded in a fresh UV sterilized culture
flask. The flask was labeled appropriately. To both the flasks, 3–
4 ml of DMEM media was added and kept inside the incubator
with culture conditions (37°C/5% CO2). Samples were sterilized
by UV for 30 minutes. After that samples kept in 24 well tissue
culture plates. MG63 osteoblast Cells were seeded on 24 well
tissue culture plates with 50,000 cell density and for control
wells, MG63 cells were added for the 1st day, 3rd day and 5th day.
The cell-cultured scaffold was analyzed for visual analysis at the
6th-day post-stabilizing with 3% formaldehyde and by adding
GFP 238 amino acids to modify the cell color with bright green
under the fluorescence microscope.

Results and discussion

FE simulation & design optimization
FE simulation was carried out to compare the compressive
stress developed under uniaxial compression applied loads
using the CREO software tool. The nodes at the bottom surface
of the scaffold design were constrained and a maximum com­
pressive load of 2000 N was loaded in the z-direction[14] as
indicated in Fig. 6.
The maximum stress developed on the hollow cube, dumb­
bells, honeycomb and dual cones are presented in Fig. 7. The
stress plots were captured by setting the legend 0 MPa to 160
MPa. From the FE simulation, it was observed that the
Honeycomb model exhibited less stress to the applied load Figure 6. Boundary constraint and applied load: FEA model.
due to its equal distribution of loads across the nodes and
also due to the 62.19% porosity and mass of 17.15 gms. It is
also observed that the stress was higher for hollow cube model subtract the “-” score from the “+” score to obtained the total
< dumbbell model < dual cone model when compared to the score. The honeycomb concept was selected for fabricating the
honeycomb model. All the models except the honeycomb may scaffold owing to its high total score.
fail when applying shear force due to the absence of diagonal
elements.[32]
Morphological study
A Pugh Matrix (PM) design criterion was availed to identify
the optimum design. PM is a category of matrix diagram that Powder particles
accords to the correlation of the available design models and In the Scanning Electron Microscope (SEM) image depicted in
provides an optimal model based on the put-forth criteria[33] Fig. 8(a), a uniform and spherical shaped inorganic mineral
such as porosity and compressive stress. Another advantage it was ascertained which is due to the frequent stirring of com­
holds the area of providing a degree of qualitative optimization posite suspension using magnetic stirrers and less agglomera­
of substitute concepts through the genesis of hybrid amalgam tion was obtained with HAp and chitosan matrix owing to the
options. The PM matrix design selection and evaluation are ultrasonication striation.
provided in Tables 3 and 4. The porosity level within the powder ensures interconnectiv­
The evaluation was carried out by keeping, the hollow cube ity across the scaffold. Hence, the porosity size (Ø) was quantified
as a standard design in correlation with other design concepts. using Bruker Emmet-Teller (BET) analysis as shown in Fig. 8(b).
Based on the value each criterion of those three concepts was Chitosan of 0.1 gm and Composite powder of 0.1 gm were
assigned with “S” (same as the standard concept) or “+” (better analyzed for porosity volume and size using the BJH model by
than the standard concept) or “–” (lower than the standard passing nitrogen gas. The adsorption level of the powder(s)
concept). After that “+” and “–” signs were counted and determines the value as tabulated in Table 5. It was observed
 MATERIALS AND MANUFACTURING PROCESSES 7

Figure 7. FE simulation analysis on various scaffold models.

Table 3. Design criteria for model selection. Table 4. Pugh matrix for model selection.
Hollow Criteria Hollow Cube Dumb Bells Honeycomb Dual Cone
Cube Dumb Bells Honeycomb Dual Cone Mass (g) S + - +
Mass (g) 17.04 15.97 17.15 14.6 Porosity in % S + + +
Porosity in % 62.3 63.1 62.9 66.3 VonMises Stress (MPa) S - + -
Compressive Stress 110.36 146.9 82.19 152.04 Mesh array comlexity S S + S
(MPa) Total “+” 0 2 3 2
Attained via. FE Total “-” 0 1 1 1
Simulation Total score 0 1 2 1
Mesh array comlexity Medium Medium High Medium
8 N. PRADEEP ET AL.

Figure 8. Chitosan blended HAp composite powders (a) SEM micrograph and (b) Pore size distribution.

Table 5. BJH pore distribution model for composite powder. agglomeration of the composite under the influence of N2
Powder Pore Volume Pore size adsorption. The advantage of having such mesoporous materials
(0.1 gm) (Cm3/g) (nm) extends to the strong loading of drugs for drug delivery
Chitosan 0.510 4.86
Chitosan and HAp composite powder 0.503 3.71
application.[32]

Scaffold
that the composite powder particles are mesoporous with avg.
porosity diameter of 3.71 nm. Also, the chitosan powder reported The fabricated composite scaffold availing SLS process was
having 23% increased porosity diameter. The decrease in poros­ analyzed with atomic force microscope (AFM) to evaluate
ity of the composite powder may be due to the increased the surface roughness of the 3D printed scaffold.

Figure 9. Photograph of the composite scaffold and its corresponding AFM image.

Figure 10. Cell culturing study on composite scaffold (a) DMEM medium plot for 2nd 4th and 6th days (b) Visual photo of control and sample medium (c) SEM
morphology and (d) Fluorescence image of live cells on 6th day.
 MATERIALS AND MANUFACTURING PROCESSES 9

The AFM image of the composite scaffold is depicted in temperature, and stepper motor speed fixed to fabricate
Fig. 9. indicates a surge in the surface roughness in the the composite powdered scaffold with no warpage and to
range of Ra 500–512 nm that will facilitate the adhesion of curtail the anisotropic properties of the composite mate­
cells across the scaffold resulting in better osteointegration/ rial using the modified SLS machine.
conduction.[33] (6) The AFM image of the composite scaffold indicates
a surge in the surface roughness which will help the
protein cells to spread over the scaffold resulting in
Cell culturing
better osteointegration/conduction.
The scaffold was subject to cell culturing and inspected for (7) Inspection under the fluorescence microscope for cell
6 days in DMEM medium as shown in Fig. 10(a,b) exhibiting activity indicated positive live cells adhering to the scaf­
controls and samples plot for 2nd 4th and 6th day with increased fold on the 6th day, subjected to cell viability assay using
cell activity. The SEM morphology as depicted in Fig. 10(c) MG63 osteoblast cell line after DMEM treatment. From
shows the cytoplastic projections along with a quasi-2D mesh SEM analysis, cytoplastic projections along with a quasi-
spread across the scaffold which is due to the porosity and the 2D mesh spread across the scaffold. This shows that the
surface roughness of the fabricated scaffold. The fluorescence HAp/Chitosan scaffold fabricated with SLS processes will
microscope enabled to observe the adhering positive live cells be a suitable candidate for biomedical application.
on the 6th day as shown in Fig. 10(d). This shows that the HAp/
Chitosan scaffold fabricated with SLS processes will be
a suitable candidate for biomedical application. ORCID
N Pradeep http://orcid.org/0000-0002-2388-000X
Conclusions
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