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Alginate-poly(amino acid) extrusion printed scaffolds for tissue engineering

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Alginate-poly(amino acid) extrusion printed

scaffolds for tissue engineering applications

Sudipto Datta, Ankita Das, Pranabesh Sasmal, Sumant Bhutoria, Amit Roy
Chowdhury & Pallab Datta

To cite this article: Sudipto Datta, Ankita Das, Pranabesh Sasmal, Sumant Bhutoria, Amit Roy
Chowdhury & Pallab Datta (2018): Alginate-poly(amino acid) extrusion printed scaffolds for tissue
engineering applications, International Journal of Polymeric Materials and Polymeric Biomaterials,
DOI: 10.1080/00914037.2018.1539988

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INTERNATIONAL JOURNAL OF POLYMERIC MATERIALS AND POLYMERIC BIOMATERIALS
https://doi.org/10.1080/00914037.2018.1539988

Alginate-poly(amino acid) extrusion printed scaffolds for tissue engineering

applications
Sudipto Dattaa, Ankita Dasa, Pranabesh Sasmala, Sumant Bhutoriab, Amit Roy Chowdhurya,c, and Pallab Dattaa
a
Centre for Healthcare Science and Technology, Indian Institute of Engineering Science and Technology, Shibpur, Howrah, India; bAlfatek
Systems, Kolkata, India; cDepartment of Aerospace Engineering and Applied Mechanics, Indian Institute of Engineering Science and
Technology, Shibpur, Howrah, India

ABSTRACT ARTICLE HISTORY

Alginate is the most commonly explored biomaterial used for extrusion printing though it pro- Received 10 August 2018
vides limited cell-material interactions. Thus, bioink functionality is improved by blending with pol- Accepted 21 October 2018
y(amino acid) polymers at low ratios for extrusion bioprinting. Poly-l-lysine (PLL) and poly(glutamic
KEYWORDS
acid) (PGA) affected the shapes of extruded constructs, with best accuracies obtained for an
Alginate; bioink; extrusion
alginate:PGA ratio of 1:5. Both modifications showed increase in cell proliferation and adhesion as bioprinting; osteoblast
printed constructs over pure alginate. Osteoblast cell culture showed PGA scaffolds to effect a differentiation; poly
dose-dependent increase in alizarin red, demonstrating a facile method to obtain 3 D plotted con- amino acids
structs for bone tissue engineering.

GRAPHICAL ABSTRACT

1. Introduction along with ability to form stable conjugates and complexes

Poly(amino acids) (PAA) are an attractive class of bioco-
maptible polymers[1, 2], which offer unique opportunities to
combine the tailored degradation properties of synthetic pol-
ymers (like poly(lactic acid)) with functionalization possibil-
ities of natural polymers (like chitosan, alginate). More
importantly, PAA can mimic extracellular matrix proteins,
possess low toxicity and biodegradability. These properties
with drugs, nucleic acids, vaccines and proteins have made
them attractive candidates for several biomedical applica-
tions[3]. PAA degrade in body by combination of dissolution
and enzymatic cleavage of peptide bonds. However, their
exploitation in tissue engineering applications as stand-alone
scaffolds is constrained by the poor mechanical properties of
the polymers. Nevertheless, their presence as components of

CONTACT Pallab Datta contactpallab@gmail.com; pd@chest.iiests.ac.in Centre for Healthcare Science and Technology, Indian Institute of Engineering
Science and Technology, CHST, Heaton Hall, Shibpur, Howrah 711103, India.
Color versions of one or more of the figures in the article can be found online at www.tandfonline.com/gpom.
ß 2018 Taylor & Francis Group, LLC
2 S. DATTA ET AL.
extracellular matrix of several tissues can serve as inspiration
for design of functionalized, biomimetic and bioinstructive
scaffolds for tissue engineering. Indeed, several studies have
shown the positive influence of PAA in bone, cartilage and
nervous regeneration[4–8]. Degradation products of PAA are
amino acids- essential nutrients for metabolism of several cell
types, adding to the interest of these materials in tissue engin-
eering. Poly(glutamic acid) (PGA), polylysine, poly aspartic
acid are some of the polyamino acids which have received
attention for fabrication of tissue engineering scaffolds such
as gels, electrospun membranes, films and sponges[9–13].
For tissue engineering scaffolds, fabrication attempts are
now directed towards biofabrication or techniques which
allow for simultaneous deposition of living cells with hydro-
gel polymers[14, 15]. These techniques overcome the limita-
tions of conventional scaffold fabrication, allowing improved
cell-cell or cell-material interactions. Extrusion-based bio-
printing is one such technique which has evolved rapidly to
allow for fabrication of several in vitro tissue models and
constructs. This method is based on the principle of depos-
ition of cell/hydrogel ink under pneumatic- or piston-driven
pressure. Especially for tissue engineering, extrusion bio-
printing or bioplotting offers the advantages of high depos-
ition and/or printing speed, scalability, affordable
instrumentation, and more importantly, ability to 3 D print
a wide array of inks[16, 17]. Though extrusion methods allow
3 D printing of constructs at lower resolutions compared to
other droplet-based or laser-based modalities, it becomes the
method of choice for screening and optimizing biological
properties of various hydrogel inks due to the ease of oper-
ation. However, successful extrusion 3 D printing also
requires optimization of the ink properties with respect to
parameters such as viscosity, yield behavior and gel-
ation time[18–20].
To become an effective ink for extrusion 3 D printing or
plotting, polymer hydrogels are expected to possess flowabil-
ity under low dispensing pressures, ability to undergo gel-
ation at physiological conditions in optimum time, maintain
dimensional stability of printed constructs as per design and
possess mechanical integrity of constructs and favorable bio-
logical properties for specific applications. Alginate under-
goes instant gelation in ionic solutions of calcium (Ca2þ)
containing salts like calcium chloride, calcium sulfate or cal-
cium carbonate[21–23]. However, alginate hydrogels do not
allow for adequate cell-material interactions, impeding their
proliferation and differentiation. In addition, rapid gelation
of alginate has also been attributed to inhomogenous gel-
ation and erratic degradation behavior. There has been,
therefore, a strong interest to improve the 3 D plotting-
printing properties of alginate hydrogels for tissue engineer-
ing applications.
Amongst different approaches used for improving print-
ing with alginate hydrogels, modifications of alginate (by
means of RGD conjugation of oxidized alginate)[24] and
blending of other hydrogels such as collagen, gelatin,
Pluronic etc with alginate[25–28] have been reported. In syn-
thesis of modified alginate, the major problem encountered
is addition of an additional synthetic step, concomitant
requirement of characterization and more importantly,
change in molecular weight of native alginate, potentially
affecting its printability. In blending approach, high enough
concentrations of blending polymers are required, which
shifts the gelation conditions beyond the physiologically-
amenable conditions. Therefore, there exists a scope for
improving biocompatibility of alginate without significantly
altering the gelation conditions. In this context, despite the
advantages of PAA as biological macromolecules, blending
of alginate with these polymers to obtain improved inks has
not been yet attempted in literature.
The present investigation hypothesizes that PAA blending
at low concentrations can improve biological properties of
alginate hydrogels without affecting its printability conditions.
Since PAA comprise a diverse class of polymers, two repre-
sentatives PAA- (poly-l-lysine (PLL) and PGA were selected
for the study. While PLL has been successfully blended with
alginate for cell encapsulation, PGA has found role as bio-
functional scaffolds elsewhere. Viscosity of the bioinks was
studied to ascertain changes in physiological parameters, fol-
lowed by extrusion printing and characterization of printed
constructs for print fidelity and mechanical properties.
Responses of osteoblasts are evaluated on printed alginate-
PAA constructs to observe the changes in biological response.
2. Materials and method
2.1. Preparation of alginate
PAA (AP) inks: Alginic acid sodium salt (viscosity 2000 cP,
2%), PLL (MW 30,000–70,000) and PGA (MW
15,000–50,000) were obtained from (Sigma-Aldrich, USA).
Ink solutions were prepared at various concentrations using
ASTM Type III Grade water (Wasserlab, Spain) as per
details given in table 1. Solutions were allowed to mix over-
night under mechanical stirring (Scilogex, USA, 200 rpm)
under room temperature (25  C).
2.2. Physico-chemical characterization of AP inks
Hydrogel inks were characterized for pH/conductivity
(Thermo Oakton meter), surface tension SURFTENS 4.5
instrument (OEG GmbH, Germany) and viscosity (Lamy
Rheology, France, Spindle L-4, constant torque of 6 rpm, 60 s)
at 25  C. The vial inversion method was used to measure the
gelation time of the inks. The alginate-PAA blend solutions
were mixed in glass vials with 0.5 M CaCl2 (1 mL) and sealed.
The glass vial was inverted every 30 s (held for 15 s) after
Table 1. Physico-chemical properties of different alginate:poly(amino acid)
blend bioinks.
Composition
(% w/v)
Sample Alginate PAA Viscosity (Pa s) Surface Tension (N/m)
A5 5 0 9.77 ± 0.02 72 ± 1.5
A5PLL0.5 5 0.5 8.56 ± 0.01 53 ± 0.1
A5PLL1 5 1 7.19 ± 0.22 49 ± 0.1
A5PGA1 5 1 8.97 ± 0.01 53 ± 0.4
A5PGA2 5 2 6.03 ± 0.02 47 ± 1.2
 INTERNATIONAL JOURNAL OF POLYMERIC MATERIALS AND POLYMERIC BIOMATERIALS
Figure 1. The lattice structures created for fabrication of scaffolds.
addition of CaCl2 to observe the time taken for the solutions
to stop flowing- designated as gelation time of the inks[29].
2.3. Extrusion printer
A printer, developed by M/s Alfatek Systems, Kolkata and
described in previously[30], was used for ink dispensing. 3 D
models were sliced using open source software, Cura (ver-
sion 15.04.5, Ultimaker, Netherlands) while G-codes files to
control machine movements was generated using
Pronterface (version 2014.03.10, Printrun).
2.4. Scaffold design
Structures of 34 mm 34 mm lattice were created using
sketch-up and saved in .stl format. The solid design was
then sliced by CURA version 15.04.4. A mesh design was
further created by adjusting the fill density to 19% as shown
in Figure 1. The input parameters were taken as follows:
layer height- 0.1 mm, strand thickness- 2.0 mm, strand-to-
strand distance- 4.4 mm, with layer-upon-layer orientation
and Z-layer stacking- 3.961 mm.
Further parameters: shell thickness- 0.8 mm, bottom/top
thickness- 0.6 mm, printing speed- 50 mm/s, filament diam-
eter- 1.75 mm, flow(%)  300 and nozzle size- 0.41 mm. The
design was saved as G-Code files and communicated to
the printer.
2.5. Printing of AP inks
The AP inks were charged onto disposable syringe, attached
to a nozzle of 0.41 mm diameter. Extrusion printing was car-
ried out at a head movement speed of 50 mm/s at 25  C
temperature. Point-to-point printing was adopted to print
each dot as per design dimensions. The hydrogel inks were
printed on a petriplate (BOROSIL) using CaCl2 solution at
0.5 M concentration for crosslinking. Post-printing, struc-
tures were washed with 95% for 10 min three times and vac-
uum-dried at 40  C. The AP inks were prepared as follows.
PAA were weighed and dissolved in ASTM Type II water
(Wasserlab, Spain) to obtain the designated wt% of PAA sol-
utions under magnetic stirring. After obtaining complete
3
dissolution, 5 g of alginate was added to the solution. The
blend solutions were stirred overnight under mechanical
stirring to get homogenously blended solutions and loaded
into dispensing syringes.
2.6. Scaffold characterization
Images of scaffolds were captured through Nikon digital
camera (NIKON DS-Fi1c). Pore area (PA), and strut length
(SL) of each printed constructs were calculated by using the
software AxioVision SE64 Rel. 4.9.1. Attenuated Total
Reflectance-Fourier transform infrared spectroscopy
(Shimadzu, IR Affinity, Japan) was used to confirm chemical
composition of scaffolds in range 4000–400 cm1.
2.7. Structural accuracy
Quantitative evaluation of the extrusion-printed constructs
was performed by using the accuracy metrics proposed in
literature. For each group, five constructs were printed and
measurements recorded for the total printed solid area was
compared with designed area (for each strands) to get per-
centage printing accuracy[31].
 
AobtAdes
Accuracy of printing ð%Þ ¼ 1  100 (1)
Ades
2.8. Mechanical characterization of AP scaffolds
An universal testing machine with 125 N load cell (Tinius
Olsen 5KT, Tinius Olsen, UK) was used to determine the
uniaxial tensile properties of the scaffold. 34 mm 34 mm
5 mm sample dimensions were used and gripped with
gripped with clip-type sample holder. Samples were loaded
at crosshead speed of 0.1 mm/min. Three samples for scaf-
fold each bioink formulation scaffolds were tested. Before
testing each sample, samples were preconditioned upto 10%
strain for ten cycles to observe for structural integrity. The
stress at break of the samples was recorded as the ultim-
ate stress.
2.9. Cell culture study
Scaffolds of dimensions 10 mm 10 mm, and 100 mm in
thickness were fabricated for studying the response of MG63
cells. Cells were cultured in Dulbecco’s modified Eagle’s
medium (DMEM) (Gibco, USA) and supplemented with 1%
penicillin-streptomycin antibiotic solution and 10% fetal
bovine serum. Cells at the concentration of 104 mL1 were
seeded on UV-sterilized scaffolds and kept at 37  C
and 5% CO2. Cell viability was measured on day 1 by 3-
[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide
(MTT) assay (EZcountTM MTT Assay Kit) in a 96 well plate.
MTT reagent was added in serum free high glucose DMEM
after 24 h of cell cultured upon the scaffolds and were incu-
bated for 4 h. Formazan crystals formed were solubilized in
solubilization buffer and absorbance at 570 nm and 670 nm
4 S. DATTA ET AL.
Figure 2. Photographic images of different alginate:poly(amino acid) fabricated lattice scaffolds (a) A5 (b) A5PLL0.5 (c) A5PLL1 (d) A5PGA1 (e) A5PGA2 (in vertical
view) and (f) A5 (g) A5PLL0.5 (h) A5PLL1 (i) A5PGA1 (j) A5PGA2 (in lateral view).
was recorded using Multi-Scan Go (Thermo Fisher, Finland)
and the percentage of cell viability was calculated according
to the manufacturer’s protocol. To evaluate cell proliferation,
the same assay procedure was repeated on day 3. The cell
viability is reported normalized to initial solid surface area
of each sample, so that the toxicity of the samples due to
the ink components can be compared without scaffold geo-
metries influencing the interpretation.
2.9. Alizarin red assay
Approximately, 1  105 MG-63 cells were seeded upon the
scaffolds and cultured for 3 days in serum free high glucose
DMEM. The scaffolds were stained with 40 mM Alizarin
Red S for 15 min at room temperature. The stained scaffolds
were dissolved in 10% acetic acid for 1 h and the absorbance
was taken at 405 nm in triplicate.
2.10. Statistical analysis
All measurements were taken in triplicate, unless otherwise
stated and results are expressed as mean ± SD A one-way
analysis of the variance with Bonferroni as a post hoc test
was used to assess the significance of the differences between
the results. Results with a P value <.05 were considered sig-
nificantly different from each other.
3. Results and discussion
3.1. Physico-chemical properties of AP solutions
Scaffold-based extrusion printing has emerged as a promis-
ing method to fabricate anatomically-correct tissue engi-
neered constructs and in vitro drug assay models. However,
hydrogel components of the hydrogel should possess
favorable rheological and crosslinking properties for hassle-
free printing and be able to provide mechanical and func-
tional cues for the cells to proliferate and differentiate.
Alginate, the most commonly used ink with good bioprint-
ability, however, exhibits very limited cell-material interac-
tions. Therefore, PAA have been blended in this work with
alginate to improve the biological properties of inks.
Concentrations of 0.5 and 1% PLL, and 1 and 2% PGA were
selected such that the resultant pH of blends remained in
the physiologically amenable range (6–8), as the addition of
PLL increased the blend ink pH while PGA decreased the
pH with concentration. All AP inks showed a decrease in
viscosity due to addition of PAA and the measured values
were 9.77 ± 0.02, 8.56 ± 0.01, 7.19 ± 0.22, 8.97 ± 0.01, and
6.03 ± 0.02 Pa s respectively for A5, A5PL0.5, A5PL1, A5PG1
and A5PG2. Thus, all blend inks exhibited a decline in vis-
cosity compared to pure alginate, which is in agreement
with published literature results on blending poly(ethylene
oxide) with alginate[32]. Nevertheless, the viscosity of blends
lied in the recommended range (0.3–30 Pa s) for printing
applications[33]. On the other hand, surface tension of all
PLL and PGA containing blends were also reduced com-
pared to pure alginate. This may be explained by the amphi-
philic nature of the PAA, which are known to cause a
reduction in surface tension. Along with viscosity, surface
tension is an important principal physicochemical property
of the solutions which can affect bioprintability[34, 35], and
indeed, manipulation of physico-chemical properties can be
exploited to produce structures with different geometrical
accuracies[36]. The results of the physicochemical properties
are summarized in Table 1. Addition of PAA to the inks
also affected the gelation times of the blends, which showed
gelation times of 30 s, 45 s, 60 s, 30 s, 45 s. It was seen that
PLL-alginate inks were gelled slower compared to PGA-
alginate gels while pure alginate demonstrated fastest
 INTERNATIONAL JOURNAL OF POLYMERIC MATERIALS AND POLYMERIC BIOMATERIALS 5

Table 2. Accuracy analysis of alginate:poly(mino acids) scaffolds.

Samples Strut Diameter (mm) Pore Area (mm2) Perimeter (mm) Printing Accuracy (%)
A5 1.2 ± 0.3 18.5 ± 4.6 16.6 ± 1.8 85 ± 0.6
A5PLL0.5 1.3 ± 0.2 10.0 ± 2.2 12.6 ± 1.0 86 ± 1.5
A5PLL1 1.5 ± 0.8 7.19 ± 1.0 11.4 ± 0.9 81 ± 0.9
A5PGA1 1.5 ± 0.3 14.5 ± 3.7 15.4 ± 1.8 87 ± 0.3
A5PGA2 2.0 ± 0.9 10.6 ± 3.6 13.3 ± 2.3 78 ± 0.7

gelation. The difference between the behaviors of two amino

acid polymers may be due to crosslinking of PGA along
with alginate by the divalent cations[36]. Together, the gel-
ation time should not be too short and surface tension very
high, to facilitate the inks to adopt the strut geometries as
designed. On the other hand, a high viscosity or high gel-
ation times will not allow the inks to flow larger distances
before solidifying thus reducing geometrical distortions but
will result in enhanced process times. Enhanced process
times will also be associated with greater cell deaths. Bioink
characterization with respect to these parameters provides
vital information for development of structurally-accurate
printed constructs. All the AP formulations were printed
with well-demarcated shapes but with varying shape fidelity.
As seen from Figure 2a–e and quantified in Table 2, accur-
acy of constructs was in the order A5PGA1 > A5PLL0.5>
A5 > A5PLL1 > A5PGA2, though viscosity decrease was in
the order A5> A5PGA1 > A5PLL0.5 > A5PLL1 > A5PGA2.
Thus, strut size increased and pore area decreased as the
concentration of either PLL or PGA was increased in the
formulation. This may be expected based on decrease in
both the viscosity and surface tension of the blends com-
pared to pure alginate, which increases flowability of the Figure 3. Chemical nature of the fabricated scaffolds observed in FTIR spectra
(a) A5 (b) A5PLL0.5 (c) A5PLL1 (d) A5PGA1 (e) A5PGA2.
inks allowing them to occupy more space. On the contrary,
crosslinking/gelation of inks prevent further flowability[36].
In both PAA cases, at highest concentrations (i.e. A5PLL1
AND A5PGA2), inks were more flowable and distorted the the range of 3000–3600 cm1 due to stretching vibration of
square shaped design in printed structures. For the hydroxyl bond (C-OH) and 1610 cm1 due to stretching
A5PLL0.5 and A5PGA1 scaffolds, the marginal reduction in vibration of carbonyl functional group (CO-) of carboxyl
viscosity was compensated by lower surface tension, which group. The stretching vibration of (–C-O-C-) of pyranosyl
also enables the inks to assume a square shape with straight ring and C-O stretching with contribution from (C-O-H)
edges compared to a more spherical A5 (due to higher sur- deformation appears in the range of 1000 and 1300 cm1.
face tension). In between A5PLL0.5 and A5PGA1, it was Newly formed absorption peak attributed to 1411 cm1
seen that the crosslinking time played a determinant role, as (COO)sym was further observed in PGA-blend constructs.
A5PGA1 exhibited faster crosslinking preventing over-flow Due to the formation of amide bond the absorption spectra
and hence, higher structural accuracy. Examination of the was formed at 1781 cm1. Broadening and increase in inten-
lateral dimensions of the scaffolds (Figure 2f–j) also show sity of spectrum at 1278 cm1 in PGA scaffolds with
that structure of A5PLL0.5 and A5PLL1 flowed and were increase in PGA content due to C-O stretching from (C-H-
not stable in z-direction, whereas A5PGA1 showed the clos- O) deformation was also observed confirming the chemical
est fit to the input z-dimensions. The decrease in ink viscos- nature of scaffolds[36].
ity and effect on structure of printed construct has also been In particular a broad spectrum near 3000–3600 cm1
observed within gelatin-alginate blended scaffolds[36]. From coincided the placement of amide (N-H) A bond. The
this analysis, it was evident that for best printing A5PGA1 absorption bands at 1644 and 1411 cm1 are the strong
and A5PLL0.5 provides the best construct accuracies, and amide I and amide II were due to the intra-molecular attrac-
increase in concentrations affected the scaffold shapes. tion between the hydroxyl (O-H) group of alginate and
amine (N-H) group of glutamic acid. This confirmed the
immobilization of the PGA in the constructs. The other
3.2. Characterization of printed AP scaffolds
peaks of the PGA scaffolds showed no difference with
The FTIR spectra of alginate, alginate-PLL and alginate-PGA respect to those of the alginate, which indicated that PGA
printed constructs were shown in Figure 3. In all scaffolds, was very likely fixed through a mode of non-chemical
characteristic vibrational modes of alginate were observed at interaction.
6 S. DATTA ET AL.
Figure 4. Mechanical properties of fabricated alginate:poly(amino acid) scaffolds.
In PLL scaffolds, the electrostatic interaction between
positively charged –NH3þ of PLL and negatively charged
(–COO) of alginate leads to conjugation and formation of
an amide bond between them, as a result asymmetric bend-
ing of NH3þ in PLL at 1638 cm1 is shifted to 1605 cm1 in
the blends due to amide bond formation. Peaks near
1605 cm1 corresponded to the strong amide I and
1402 cm1 with sharp intensity is associated with amide II.
The amide A (N-H) bond near 3000–3600 cm1 in PLL
coincided with a broad spectrum[37, 38]. The intra-molecular
hydrogen bonding in 3 D scaffold can take by random coil
formation between alginate and PLL, alpha helical structure
between amide group of PLL and antiparallel beta sheet
structure between amide groups of PLL.
Figure 4 shows the mechanical strength of the printed
scaffolds. The results show that there is a decrease in tensile
strength with increasing concentration of PAAs. For the
samples, A5, A5PLL0.5, A5PLL1, A5PGA1 and A5PGA2
corresponding ultimate stress values obtained were 520 KPa,
490 KPa, 440 KPa, 210 kPa and 200 kPa respectively. Though,
the mechanical properties of blend constructs decreased
compared to pure alginate, the decrease were more promin-
ent in PGA compared to PLL constructs. Electrostatic inter-
actions between the NH2 group of PLL and COO group of
alginate may be responsible for the higher mechanical prop-
erty of PLL scaffolds compared to PGA. Elsewhere, the
PGA-alginate hydrogels have also shown similar reduction
in mechanical properties accompanied by changes in cross-
linking densities[39], while subtle reduction in mechanical
properties of alginate-PLL microcapsules has also
been reported[40].
3.3. Cellular compatibility of AP scaffolds
AP inks were evaluated for their biocompatibility by culture
of osteosarcoma cells on the printed scaffolds. Scaffolds were
sterilized by UV radiation, following reports which have
shown ethanol and UV treatment as terminal sterilization
methods to cause minimal damage to mechanical properties
Figure 5. Biocompatibility of alginate:poly(amino acid) scaffolds with MG63
cell lines.
Figure 6. Mineralization of alginate:poly(amino acid) scaffolds observed by
quantification of Alizarin Red staining.
of alginate scaffolds, compared to other methods[41]. UV
treatment was selected as certain reports have shown it can
be used effectively as a crosslinking mechanism to obtain
mechanically strong extrusion printed alginate structures [42]
as well as minimizes the need of washing the scaffolds
repeated to remove ethanol traces. Further, sterilization time
has kept below 25 min, to ensure that substantial degrad-
ation of alginate scaffolds compromising mechanical stabil-
ity, are prevented[43]. Cell viability on day 1 on A5PGA1,
A5PLL0.5, A5PLL1, and A5PGA2 scaffolds was found to be
significantly different from A5 as depicted in Figure 5 (p <
.05). A significant difference between A5PLL0.5 and A5PLL1
was also observed showing that PLL can provide a concen-
tration-dependent increase in cell adhesion. On the other
hand, PGA incorporation also showed enhanced cell viabil-
ity but there was no difference between A5PGA1 and
A5PGA2, indicating quantitative structure-property relations
may not be conserved with all PAAs. However, on both
days of measurement, all samples showed progressive
increase of cell viability indicating all AP scaffolds supported
cell proliferation.
The functionality of cells was investigated by means of
alizarin red assay indicating the calcium mineralization of
the scaffolds and represented in Figure 6. It was evident that
calcium deposition was higher only with the PGA scaffolds,
 INTERNATIONAL JOURNAL OF POLYMERIC MATERIALS AND POLYMERIC BIOMATERIALS
and no significant difference between A5, A5PLL0.5 and
A5PLL1 was observed. On the other hand there was a sig-
nificant difference between A5PGA1 and A5PGA2, as well
as between both of these scaffolds in comparison to A5. The
observation also suggests that certain PAA as bioink compo-
nent can promote the osteogenic differentiation of cells.
As bioprinting research advances, the exploration of func-
tional bioinks is going to gain momentum. Extrusion-based
3 D printing or plotting is a scalable bioprinting modality,
which yields constructs with mechanical properties suitable
for implantation. Though alginate posses several advantages
for facilitating bioprinting, the cell-material interactions are
limited. Our results showed that blending of poly-l-lysine
can improve the osteoblast proliferation and adhesion in
alginate bioinks. Elsewhere, PLL have been shown to
improve functionalization of scaffolds for cartilage and skin
tissue engineering[4, 44, 45]. The mechanism of improved
chondrogenesis has been suggested to be increase in N-cad-
herin expression[4]. In addition, we found improved osteo-
genesis with PGA as bioink component, which may be due
to the crucial role in biomineralization in vivo[46–48]. Thus
alginate-poly(amino acid) biomaterials are developed as bio-
mimetic scaffolds for bone, skin and cartilage tissue engin-
eering applications. Compared to conventional scaffolds,
printed scaffolds can be customized to conform to patient
defect size and can be developed to contain multiple cell
types in multiple layers. The technique can be further
extended to perform bioprinting with cell-laden bioinks thus
allowing fabrication of multilayered constructs, with better
cell-matrix interactions[49]. Compared to alginate inks
blended with other biopolymers like gelatin, PAA-alginate
offer the advantage of not altering the crosslinking condi-
tions[28], but providing bioactive cues for regeneration.
4. Conclusions
We presented results showing the feasibility of using poly(a-
mino acids)- poly-L-lysine and poly(glutamic acid) as com-
ponents for fabricating alginate-based printed constructs.
Our results showed that poly(amino acids) can be blended
with alginate at low proportions so as to not significantly
changes the physico-chemical properties required for print-
ing like viscosity and gelation conditions. The concentration
of blend polymers can be manipulated to obtain structures
with desired shape fidelities. PAA-alginate constructs further
provide PAA concentration-dependent changes in cellular
behavior like cell proliferation and differentiation. Thus
depending upon the anatomical structures to be regenerated,
PAA concentration variation may be a facile method to
improve biological properties of alginate constructs.
Acknowledgments
Authors acknowledge financial assistance received from SERB,
Governement of India, through grant no. SB/FTP/ETA/003-2014.
7
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