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.

Submit your article to this journal

View related articles

View Crossmark data

Full Terms & Conditions of access and use can be found at

https://www.tandfonline.com/action/journalInformation?journalCode=lmmp20
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
India, being the top shrimp producer and exporter across the
globe with a market value of 150 crores producing 6,50,000
tons in the year 2020 despite the COVID-19 pandemic.[1] The
shrimp market is expected to reach 1,500,000 tons by 2026.
During processing the aquaculture sector approximately pro­
duces 106 million tons/annum of waste, a large portion of the
waste is bound to treat the soil in the form of manures or
further treated to feed the animals and the remains are dumped
in the coastal area that would create ecological imbalance. In
such circumstances, the Indian shrimp processing industries
produce more than 150,000 ton as wastage and this waste can
be used to extract minerals, antioxidants, chitin, chitosan, and
proteins[2,3] for various applications. Though many organic
polymers, find extensive usage in the clinical field, chitosan
stands out as a bone substitute, due to its minimal toxic nature,
colossal flexibility and being biodegradable.[4] Also, chitosan
powder provides sites for complex calcium formation, owing to
better compatibility between HAp powdered particles. Latest
developments have been the application of HAp and polymers
(organic) in nature,[5–10] i.e., collagen,[6] chitosan[7] and other
organic polymers, [8–10] where nanocomposites (HAp) are pre­
pared extensively. These extensively prepared nano-
composites have shown extensive bioactivity and simultaneous
equity in the mechanical strength of 40 MPa. Though many
organic polymers, find extensive usage in the clinical field,
chitosan stands out as a bone substitute, due to its minimal
toxic nature, colossal flexibility and being biodegradable.[9]
Also, chitosan powder provides sites for complex calcium for­
mation, owing to better compatibility between HAp powdered
particles. Scientists and researchers across the globe used
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
cess involves high time duration to obtain at nano-scale.[10,11]
So, a combination of wet precipitation method along with
pelletization, provides a useful way of synthesizing polymer
HAp inorganic nanocomposites. This technique was applied
for chitosan-HAp nanocomposite preparation, in order to
obtain novel bone substitutes, which have a close correlation
to bones analogous in nature.
From the evolution of fetus to a full-grown adult, the bones
undergo from being cartilage molds to full-grown structures
adapting with age, lifestyle, eating habits and exercise. There
are three categories of cells in the body, which bring about
changes in the ones: osteocytes, osteoclasts and osteoblasts.[12]
The osteoblasts are bone forging cells that engender the matrix
that makes up the bone. The matrix part of the bone is made of
organic material inclusive of molecules such as collagen pro­
tein fibers, which administer bone the required flexibility,
resilience and presence of calcium (Ca2+), phosphate (PO4-)
ions provide appended rigidity. The molecules (matrix) are
built and enveloped due to osteoblasts for discharge into the
extracellular environment. These discharged molecules react
with one another to form austere flexible bone tissues called
osteoid that harden the formation of bone.[12,13] The bones in
the body are naturally designed in three-dimension with an
inorganic-organic nanocomposite in which crystals of hydro­
xyapatite (HAp, Ca10(PO4)6(OH)2) are garnered on collagen.
The advantage of this type of structure, is a combination of two
factors, the colossal mechanical strength of the bone mineral
and flexibility due to the presence of fibrous form collagen.[1]
The ceramic powders that exhibit biological properties and
affinity are termed bioactive nanocomposites/ceramics.
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
directly to the contemporary living bone in the human body. In
the effective case of artificial powders, an envelope of fibrous
tissues is carried out, to encapsulate the powder in the area of
a bone defect.[14] The bonding nature of the ceramics being
bioactive toward the bone has generated interest in the medical
community as this creates a rigid and irritation-free fixation
between the scaffold/implant and the bone. Though most of
the ceramic biomaterials have their application in the field of
orthopedics, for repair or fixing of damaged bone tissues,
a serious limitation exists due to their brittle nature, minimal
toughness and modulus of elasticity.[15] The combination of
inorganic and organic nanocomposites provides a design
(novel in nature) that can be molded and shaped into con­
toured desirable sizes and shapes as specified by the clinical
field.
Almost any materials can be tailored (by design) with
desired mechanical properties, bio-chemical properties
using 3D printing technology. The design of the scaffold is
based on the design thinking of the researchers. Various
designs like honeycomb, auxetic, s-shaped, I-shaped scaffold
designs have been proved to be an optimal design viz.
attaining all design considerations such as porosity, mesh
complexity, load-bearing capacity, optimization, etc.[16,17]
Selective Laser Sintering (SLS) is one among other additive
manufacturing processes for fabricating scaffolds by tailored
design. The advantage of using SLS in scaffold fabrication
has already been studied by various researchers. The selec­
tion of machining parameters determines the quality of
scaffold with no warpage, shrinkage, having desired surface
topography. The parameters such as laser fill power, laser
scanning speed, laser spot width, scan fill space, orientation,
bed temperature determine the quality of the scaffold.[17]
Among those, the most predominant parameters suitable for
nano-composite HAp scaffold are the laser power, laser scan
speed, scan fill space, bed temperature.[17–19]
The survey helped us to understand the individual nature
of the materials and the possibility of fabricating a metal and
or polymer scaffold using 3D printing with commercially
available materials while no authors reported producing
materials from shrimp waste with lab-prepared HAp and
turning them into a bio-composite scaffold using ‘3D print­
ing’ which is the novelty of this research article. In this work,
Chitosan was generated from shrimp waste shells and then
blended in HAp suspension obtained from the wet chemical
method, further processed to obtain powder particles using
the ball milling setup. The best scaffold design from four
design models i.e., hollow cube, dumbbell, dual cone and
the honeycomb models with porosity level within 62–67%
was selected after understanding the model’s versatility
upon compressive load, and obtain an optimal structure
viable for scaffold using the FE analysis. The final processed
scaffold design was then fabricated availing the modified
selective laser sintering with a lab-made Chitosan-HAp inor­
ganic-organic polymer composite. The fabricated scaffold was
subjected to cell viability, to understand its biocompatibility
nature using MG-63 cell assay.
Materials and methods
Preparation of Chitosan suspension
Chitosan was extracted from the collected shrimp (Penaeus
indicus) waste shells after thorough wash to remove dirt particles
from the shells. The washed shells are then dried in a hot air
oven at about 80°C for 3 days and later dried and grounded into
fine powder as shown in Fig. 1. Chitosan suspension was then
prepared with 125 gram of chitosan powder in 0.3 mol/L acetic
acid. This dissociated powdered acetic solution was magnetically
stirred for 35 minutes. This solution was adjusted in the presence
of ammonia aqueous solution, to obtain a pH value at 9.0–10.2.
From Fig. 1(a) It was observed that the spectral peak falls at
the wavenumber 1656 cm−1 depicts the amide-1 group and the
vibrational peaks at 1550 cm−1 depicts the N-H bending of the
amide II group. Similar spectral bands were confirmed by
various researchers.[20–22] This confirms the presence of
Chitosan extracted from shrimp shells.
Preparation of HAp suspension
A wet chemical precipitation method was carried out,[23,24] to
carry out HAp powder synthesis as depicted in Fig. 2(a). In
comparison to pellet formation and sintering, this provided better
efficacy, to attain HAp powder of Ca/P ratio of 1.64, with an
accession of 12 grams of Ca and 18.89 grams of NH4OH+PO43-.

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
after ball milling. Figure 2(b) depicts the spectral peaks stretch­
ing at 1149–1000 cm−1 and at 956 cm−1 exhibiting phosphate
functional group which is a characteristic peak for HAp.
Similarly, the observed bending of peaks between 556 cm−1 to
620 cm−1 confirms the presence of HAp.[25,26] HAp suspension
was then prepared by mixing the obtained HAp powders with
ethanol in the ratio of 1 gms:60 ml at room temperature.
Preparation of composite Chitosan-HAp powder
The prepared Chitosan solution was mixed with the HAp sus­
pension solution resulting in a composite formation at 1:3 ratio.
The composite solution was ultrasonicated for 30 minutes[27] to
MATERIALS AND MANUFACTURING PROCESSES 3
achieve mean particle size, stability in the solution and equal
size distribution. The slurry was withdrawn and dried in the
furnace for 6 mins at 60°C and then milled for 10 hours using
ball milling process in order to obtain a fine particle composite
powdered. After the completion of milling cycle, the powder
grains are transferred to a nonstick vessel and stirred at dis­
parate temperatures of 35 to 75°C of a duration of 36 hours. The
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
dried in a furnace at 60°C for 36 hours. The same process was
carried until 300 gms of composite powder is prepared as shown
in Fig. 3. The TEM micrograph exhibiting needle like shape of
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.

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

that the composite powder particles are mesoporous with avg.
porosity diameter of 3.71 nm. Also, the chitosan powder reported
having 23% increased porosity diameter. The decrease in poros­
ity of the composite powder may be due to the increased
Scaffold
The fabricated composite scaffold availing SLS process was
analyzed with atomic force microscope (AFM) to evaluate
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.

The AFM image of the composite scaffold is depicted in
Fig. 9. indicates a surge in the surface roughness in the
range of Ra 500–512 nm that will facilitate the adhesion of
cells across the scaffold resulting in better osteointegration/
conduction.[33]
Cell culturing
The scaffold was subject to cell culturing and inspected for
6 days in DMEM medium as shown in Fig. 10(a,b) exhibiting
controls and samples plot for 2nd 4th and 6th day with increased
cell activity. The SEM morphology as depicted in Fig. 10(c)
shows the cytoplastic projections along with a quasi-2D mesh
spread across the scaffold which is due to the porosity and the
surface roughness of the fabricated scaffold. The fluorescence
microscope enabled to observe the adhering positive live cells
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.
Conclusions
The advantage of using i) chitosan extract from shrimp waste, ii)
lab-produced bio-compatible HAp material resulted in produ­
cing a bio-composite material that enabled to 3D print a bio-
compatible composite scaffold in a cleaner environment by
contributing toward reducing the environmental pollution.
The following can be concluded from the research study of 3D
printed shrimp derived chitosan-HAp composite scaffold mate­
rial and its effect on MG63 cell viability assay viz., four design
models i.e., hollow cube, dumbbell, dual cone and the honey­
comb models having porosity level in the range of 62–67%.
(1) The composite HAp powder is synthesized availing wet
perception method. Chitosan is generated from shrimp
waste shells, further processed by mechanochemical ball
milling. Furthermore, the composite powder was char­
acterized by availing FT-IR and TEM analysis to ensure
composition and morphology.
(2) The ratio of 1:3 chitosan and HAp suspensions was
mixed and its influence under magnetic stirring and
ultrasonication resulted in lesser agglomerated powder
particles. Post-mechanical ball milling resulted in pow­
der particle size of <0.1 µm. The FT-IR analysis was able
to corroborate the presence of Chitosan and HAp cor­
responding to the spectral peaks.
(3) The SEM/TEM morphology enabled to view the spheri­
cal shape of the composite powder, while the BJH model
enabled to quantify the porosity volume (0.503 cm3/g),
the porosity diameter (3.17 nm) and the mesoporous
nature of the prepared composite powder particles.
(4) From the FE simulation and Pugh’s matrix design selec­
tion method, the honeycomb model was found to be
superior with 62.9% porosity and exhibited less stress of
82.19 MPa when loaded with 2000 N, which was further
fabricated under modified SLS technique.
(5) The preliminary experimental trials are done by varying
fill scan space, laser spot width and keeping other para­
meters such as laser power, laser beam speed, bed
MATERIALS AND MANUFACTURING PROCESSES 9
temperature, and stepper motor speed fixed to fabricate
the composite powdered scaffold with no warpage and to
curtail the anisotropic properties of the composite mate­
rial using the modified SLS machine.
(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
better osteointegration/conduction.
(7) Inspection under the fluorescence microscope for cell
activity indicated positive live cells adhering to the scaf­
fold on the 6th day, subjected to cell viability assay using
MG63 osteoblast cell line after DMEM treatment. From
SEM analysis, cytoplastic projections along with a quasi-
2D mesh spread across the scaffold. 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
References
[1] Reitz, W. Bioceramics: Materials and Applications Edited by
G. Fisher, A. Clare, and L. Hench. Mater. Manuf. Processes. 2007,
12(4), 743–744. DOI: 10.1080/10426919708935181.
[2] Uribe-Lam, E.; Treviño-Quintanilla, C. D.; Cuan-Urquizo, E.;
Olvera-Silva, O. Use of Additive Manufacturing for the
Fabrication of Cellular and Lattice Materials: A Review. Mater.
Manuf. Processes. 2021, 36(3), 257–280. DOI: 10.1080/
10426914.2020.1819544.
[3] Reiser, J.; Hartmaier, A. Elucidating the Dual Role of Grain
Boundaries as Dislocation Sources and Obstacles and Its Impact
on Toughness and Brittle-to-ductile Transition. Sci. Rep.- UK.
2020, 10(1), 1–18. DOI: 10.1038/s41598-020-59405-5.
[4] Al-Amin, M.; Abdul Rani, A. M.; Abdu Aliyu, A. A.; Bryant, M. G.;
Danish, M.; Ahmad, A. Bio-ceramic Coatings Adhesion and
Roughness of Biomaterials through PM-EDM: A Comprehensive
Review. Mater. Manuf. Processes. 2020, 35(11), 1157–1180. DOI:
10.1080/10426914.2020.1772483.
[5] Liu, S.; Huang, D.; Hu, Y.; Zhang, J.; Chen, B.; Zhang, H.;
Zhou, W.; Tong, R.; Li, Y.; Zhou, W. Sodium Alginate/collagen
Composite Multiscale Porous Scaffolds Containing Poly (ε-
caprolactone) Microspheres Fabricated Based on Additive
Manufacturing Technology. RSC. Adv. 2020, 10(64),
39241–39250. DOI: 10.1039/D0RA04581K.
[6] Ghosh, S.; Ghosh, S.; Pramanik, N. Bio-evaluation of Doxorubicin
(Dox)-incorporated Hydroxyapatite (Hap)-chitosan (CS)
Nanocomposite Triggered on Osteosarcoma Cells. Adv. Compos.
Hybri. Mater. 2020, 3(3), 303–314. DOI: 10.1007/s42114-020-
00154-4.
[7] Ghiasi, B.; Sefidbakht, Y.; Mozaffari-Jovin, S.; Gharehcheloo, B.;
Mehrarya, M.; Khodadadi, A.; Uskoković, V.; Ranaei Siadat, S. O.;
Uskoković, V. Hydroxyapatite as a Biomaterial–a Gift that Keeps
on Giving. Drug. Dev. Ind. Pharm. 2020, 46(7), 1035–1062. DOI:
10.1080/03639045.2020.1776321.
[8] Ranjan, N.; Singh, R.; Ahuja, I. P. S.; Rahman, M.; Ramakrishna, S.
PLA-HAp-CS-Based Biocompatible Scaffolds Prepared through
Micro-Additive Manufacturing: A Review and Future
Applications. 3D Print. Addit. Manuf. 2020, 209–229. DOI:
10.1007/978-981-15-5424-7_10.
[9] Neacsu, I. A.; Serban, A. P.; Nicoara, A. I.; Trusca, R.; Ene, V. L.;
Iordache, F. Biomimetic Composite Scaffold Based on Naturally
Derived Biomaterials. Polymers. 2020, 12(5), 1161. DOI: 10.3390/
polym12051161.
10 N. PRADEEP ET AL.
[10] Zima, T.; Bataev, I.; Smirnov, A. Fabrication of Surface-modified
One-dimensional Titania Nanostructures in the Presence of
Chitosan. Mater. Manuf. Processes. 2014, 30(5), 611–615. DOI:
10.1080/10426914.2015.1004693.
[11] Abate, K. M.; Nazir, A.; Yeh, Y. P.; Chen, J. E.; Jeng, J. Y. Design,
Optimization, and Validation of Mechanical Properties of
Different Cellular Structures for Biomedical Application. Int.
J. Adv. Manuf. Tech. 2020, 106(3), 1253–1265. DOI: 10.1007/
s00170-019-04671-5.
[12] Uribe-Lam, E.; Treviño-Quintanilla, C. D.; Cuan-Urquizo, E.;
Olvera-Silva, O. Use of Additive Manufacturing for the
Fabrication of Cellular and Lattice Materials: A Review. Mater.
Manuf. Processes. 2021, 36(3), 257–280. DOI: 10.1080/
10426914.2020.1819544.
[13] Liu, J.; Hu, H.; Li, P.; Shuai, C.; Peng, S. Fabrication and
Characterization of Porous 45S5 Glass Scaffolds via Direct
Selective Laser Sintering. Mater. Manuf. Processes. 2013, 28(6),
610–615. DOI: 10.1080/10426914.2012.736656.
[14] C Feng, P.; Niu, M.; Gao, C.; Peng, S.; Shuai, C. A Novel Two-step
Sintering for Nano-hydroxyapatite Scaffolds for Bone Tissue
Engineering. Sci. Rep.- UK. 2014, 4(1), 1–10. DOI: 10.1038/
srep05599.
[15] Prakash, C.; Singh, S.; Pabla, B. S.; Sidhu, S. S.; Uddin, M. S. Bio-
inspired Low Elastic Biodegradable Mg-Zn-Mn-Si-HA Alloy
Fabricated by Spark Plasma Sintering. Mater. Manuf. Processes.
2019, 34(4), 357–368. DOI: 10.1080/10426914.2018.1512117.
[16] Aliyu, A. A. A.; Abdul-Rani, A. M.; Ginta, T. L.; Rao, T. V. V. L. N.;
Selvamurugan, N.; Roy, S. Hydroxyapatite Mixed-electro
Discharge Formation of Bioceramic Lakargiite (Cazro3) on Zr–
Cu–Ni–Ti–Be for Orthopedic Application. Mater. Manuf.
Processes. 2018, 33(16), 1734–1744. DOI: 10.1080/
10426914.2018.1512122.
[17] Deng, J.; Li, P.; Gao, C.; Feng, P.; Shuai, C.; Peng, S. Bioactivity
Improvement of Forsterite-based Scaffolds with nano-58S
Bioactive Glass. Mater. Manuf. Processes. 2014, 29(7), 877–884.
DOI: 10.1080/10426914.2014.921712.
[18] Deepachitra, R.; Nigam, R.; Purohit, S. D.; Kumar, B. S.;
Hemalatha, T.; Sastry, T. P. In Vitro Study of Hydroxyapatite
Coatings on Fibrin Functionalized/pristine Graphene Oxide for
Bone Grafting. Mater. Manuf. Processes. 2015, 30(6), 804–811.
DOI: 10.1080/10426914.2014.994758.
[19] Peng, H.; Wang, K.; Huang, Z. An Injection Molding Method to
Prepare Chitosan-zinc Composite Material for Novel
Biodegradable Flexible Implant Devices. Mater. Manuf. Processes.
2019, 34(3), 256–261. DOI: 10.1080/10426914.2018.1532580.
[20] Kumari, S.; Rath, P. K. Extraction and Characterization of Chitin
and Chitosan from (Labeo Rohit) Fish Scales. Procedia Mater. Sci.
2014, 6, 482–489. DOI: 10.1016/j.mspro.2014.07.062.
[21] Ashassi-Sorkhabi, H.; Bagheri, R.; Rezaei-moghadam, B.
Sonoelectrochemical Synthesis of ppy-MWCNTs-chitosan
Nanocomposite Coatings: Characterization and Corrosion
Behavior. J. Mater. Eng. Perform. 2015, 24(1), 385–392. DOI:
10.1007/s11665-014-1297-9.
[22] Hariharan, K.; Chandrasekhara Sastry, C.; Padmanaban, M.;
Gideon Ganesh, M. Experimental Investigation of Bioceramic
(Hydroxyapatite and Yttrium Stabilized Zirconia) Composite on
Ti6Al7Nb Alloy for Medical Implants. Mater. Manuf. Processes.
2020, 35(5), 521–530. DOI: 10.1080/10426914.2020.1711929.
[23] Correia, R. N.; Magalhaes, M. C. F.; Marques, P. A. A. P.;
Senos, A. M. R. Wet Synthesis and Characterization of Modified
Hydroxyapatite Powders. J. Mater. Sci. Mater. Med. 1996, 7(8),
501–505. DOI: 10.1007/BF00705432.
[24] Huang, Z.; Zhou, K.; Zhang, D. Porous Hydroxyapatite Scaffolds
with Unidirectional Macrochannels Prepared via Ice/fiber-tem­
plated Method. Mater. Manuf. Processes. 2014, 29(1), 27–31.
DOI: 10.1080/10426914.2013.840909.
[25] Baji, A.; Wong, S. C.; Srivatsan, T. S.; Njus, G. O.; Mathur, G.
Processing Methodologies for Polycaprolactone-hydroxyapatite
Composites: A Review. Mater. Manuf. Processes. 2006, 21(2),
211–218. DOI: 10.1081/AMP-200068681.
[26] Nguyen, N. T.; Nguyen, B. H.; Ba, D. T.; Pham, D. G.; Van Khai, T.;
Nguyen, L. T.; Tran, L. D. Microwave-assisted Synthesis of Silver
Nanoparticles Using Chitosan: A Novel Approach. Mater. Manuf.
Processes. 2014, 29(4), 418–421. DOI: 10.1080/
10426914.2014.892982.
[27] Zhou, W. Y.; Lee, S. H.; Wang, M.; Cheung, W. L.; Ip, W. Y.
Selective Laser Sintering of Porous Tissue Engineering Scaffolds
from Poly (L-lactide)/carbonated Hydroxyapatite Nanocomposite
Microspheres. J. Mater. Sci. Mater. Med. 2008, 19(7), 2535–2540.
DOI: 10.1007/s10856-007-3089-3.
[28] Gogheri, M. S.; Kasiri-Asgarani, M.; Bakhsheshi-Rad, H. R.;
Ghayour, H.; Rafiei, M. In Vitro Corrosion Behavior and
Cytotoxicity of Polycaprolactone–Akermanite-Coated Friction-
Welded Commercially Pure Ti/AZ31 for Orthopedic
Applications. J. Mater. Eng. Perform. 2020, 29(9), 6053–6065.
DOI: 10.1007/s11665-020-04952-1.
[29] Harris, M.; Potgieter, J.; Ray, S.; Archer, R.; Arif, K. M. Preparation
and Characterization of Thermally Stable ABS/HDPE Blend for
Fused Filament Fabrication. Mater. Manuf. Processes. 2020, 35(2),
230–240. DOI: 10.1080/10426914.2019.1692355.
[30] Grigolato, L.; Filippi, S.; Cantarella, D.; Lione, R.; Moon, W.;
Rosso, S.; Savio, G. Concept Selection and Interactive Design of
an Orthodontic Functional Appliance. Int. J. Interact. Des. Manuf.
2020, 1–6. DOI: 10.1007/s12008-020-00743-z.
[31] Sahu, K. K.; Modi, Y. K. Investigation on Dimensional Accuracy,
Compressive Strength and Measured Porosity of Additively
Manufactured Calcium Sulphate Porous Bone Scaffolds. Mater.
Technol. 2020, 1–12. DOI: 10.1080/10667857.2020.1774728.
[32] Wang, X.; Zeng, D.; Weng, W.; Huang, Q.; Zhang, X.; Wen, J.;
Jiang, X. Alendronate Delivery on Amino Modified Mesoporous
Bioactive Glass Scaffolds to Enhance Bone Regeneration in
Osteoporosis Rats. Artif. Cells. Nanomed. Biotechnol. 2018, 46(2),
171–181. DOI: 10.1080/21691401.2018.1453825.
[33] Marrese, M.; Guarino, V.; Ambrosio, L. Atomic Force Microscopy:
A Powerful Tool to Address Scaffold Design in Tissue Engineering.
J. Funct. Biomater. 2017, 8(1), 7. DOI: 10.3390/jfb8010007.