Accepted Manuscript

Preparation, characterization and in vitro test of composites poly-

lactic acid/hydroxyapatite scaffolds for bone tissue engineering

Francesco Carfì Pavia, Gioacchino Conoscenti, Silvia Greco,

Vincenzo La Carrubba, Giulio Ghersi, Valerio Brucato

PII: S0141-8130(18)31297-2
DOI: doi:10.1016/j.ijbiomac.2018.08.007
Reference: BIOMAC 10251
To appear in: International Journal of Biological Macromolecules
Received date: 14 April 2018
Revised date: 25 July 2018
Accepted date: 2 August 2018

Please cite this article as: Francesco Carfì Pavia, Gioacchino Conoscenti, Silvia Greco,
Vincenzo La Carrubba, Giulio Ghersi, Valerio Brucato , Preparation, characterization
and in vitro test of composites poly-lactic acid/hydroxyapatite scaffolds for bone tissue
engineering. Biomac (2018), doi:10.1016/j.ijbiomac.2018.08.007

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 ACCEPTED MANUSCRIPT

Preparation, characterization and In vitro test of composites Poly-Lactic Acid

/Hydroxyapatite scaffolds for Bone Tissue Engineering

Francesco Carfì Pavia1,2,*, Gioacchino Conoscenti1, Silvia Greco1, Vincenzo La Carrubba1,2,

Giulio Ghersi3, Valerio Brucato1,2

1
DICAM, University of Palermo, Viale delle Scienze building 8, 90128 Palermo

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ATeN center, University of Palermo, Viale delle Scienze building 18A, 90128 Palermo

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STEBICEF, University of Palermo, Viale delle Scienze building 16, 90128 Palermo
*

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Corresponding authors. E-mail addresses: francesco.carfipavia@unipa.it (F. Carfì Pavia)

Keywords: Hydroxyapatite, Tissue Engineering, Scaffold, Osteoblasts

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Abstract

In this work, the possibility to produce composite Poly-L-lactic acid (PLLA)/Hydroxyapatite

(HA) porous scaffolds via Thermally Induced Phase Separation (TIPS) for bone tissue
engineering applications was investigated. Several PLLA/HA wt/wt ratios (95/5, 90/10,
70/30, 50/50, 34/66) were tested and the as-obtained scaffolds were characterized via
Scanning Electron Microscopy, Wide Angle X-Ray Diffraction, Thermogravimetric analysis,

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Gas Pycnometry, Differential Scanning Calorimetry (DSC) and mechanical compression test.
Morphological analysis revealed an open structure with interconnected pores and HA particles

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embedded in the polymer matrix. Finally, cell cultures were carried out into the composite
scaffolds in order to evaluate the effect of HA on the proliferation and differentiation of

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osteoblastic cells, showing a higher alkaline phosphatase activity on composite scaffolds
compared to neat PLLA ones.
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1. Introduction

Tissue engineering is an interdisciplinary field that applies the principles of engineering and

life science toward the development of biological substitutes that restore, maintain and or

improve tissue function [1]. The production of an engineered tissue starts by the design and

the formation of a structure able to support the migration and growth of cells that will

originate the new tissue. Those structures are characterized by an interconnected pore network

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able to guide, after the degradation of the scaffold, the implanted cells to form a new tissue

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showing a well-integrated structure. [2,3]

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The use of scaffolds for bone regeneration has been widely investigated in the last two

decades. The literature highlights how bone repair and regeneration appears to be a very
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challenging target, indeed, osteo-articular tissues exhibit a hierarchical structure with a

relevant level of complexity. [4–6] Bone fractures are one of the most common forms of
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injury. For example, in the United States, the estimated billed charges for bone diseases are

over $ 2,500,000 each year and approximately 500,000 vertebral fractures annually occur.
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Furthermore, in patients who are managed with various bone grafts, the failure rate ranges
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from 16% to 50% [7]. The major concerns are: the frequent necessity of a second site of

surgery, limited supply, inadequate size and shape and morbidity associated with donor site.
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Tissue engineering offers a promising new approach to repair bone fractures with bone loss,
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fractures that do not heal, and fractures due to bone tumours. [8,9]

Since it was discovered that the bone tissue of mammals contains about 60% (based on dry

weight) of ceramic Hydroxyapatite (HA) [10], several studies have been carried out in order

to produce implantable materials containing calcium phosphate [11]. As a matter of fact, HA

supports the attachment, differentiation, and proliferation of significant cells, in particular

osteoblasts and mesenchymal cells [12]. HA presents a chemical structure comparable to the

mineral component in natural bone, demonstrating good osteoconductivity [13]. Moreover, it

has been shown that HA particles, when incorporated within biodegradable polymers,

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enhance the mechanical properties of a scaffold making it mimicking as close as possible

bone mechanical strength [14].

Many researchers have carried out biological in vitro studies on scaffolds containing HA

using osteoprogenitor cells by analysing the biocompatibility and osteoinductivity through

proliferation and differentiation assays. In particular, alkaline phosphatase (ALP) activity is

one of the widely employed assays for highlighting the osteoblastic differentiation [15]

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[13,16,17].

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Thermally Induced Phase Separation (TIPS) technique allows one to obtain a wide span of

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morphologies, above all in terms of pore size, simply by varying one or more experimental

parameters [18]. Previous studies demonstrated the possibility to produce porous scaffold
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with different morphologies by adopting TIPS technique starting from a ternary solution

(polymer, solvent, non-solvent) by changing the polymer concentration, the solvent and the
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cooling rate or phase separation path. [19–22]

Several papers describe the possibility to embed HA in a polymer network via TIPS, starting
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from a binary mixture polymer-solvent where HA is the filler. However, in those cases, the
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so-obtained scaffolds show an irregular and anisotropic structure, probably due to the lack of
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experimental parameters to tune [23–25].

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In this work, the possibility to produce composite Poly-L-lactic acid (PLLA)/Hydroxyapatite

porous scaffolds via TIPS, starting from a ternary solution (where HA particles where
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suspended) was investigated. Several PLLA/HA ratios were tested: 95/5, 90/10, 70/30, 50/50,

66/34 wt/wt. The as-obtained scaffolds were characterized via Gas Pycnometry, Scanning

Electron Microscopy (SEM), Compression test, Wide Angle X-Ray Diffraction (WAXD) and

Differential Scanning Calorimetry (DSC) analysis. Furthermore, in vitro cell culture studies

with MC3T3-E1 pre-osteoblastic cells were carried out on composite PLLA/HA scaffolds.

Cell viability and ALP activity were investigated during 27 days of culture to determine the

long term HA effects on cellular behaviour.

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2. Materials and methods

2.1. Materials

Poly-L-lactic-acid (PLLA, Resomer® L 209 S), 1,4 dioxane (Sigma-Aldrich, Munich,

Germany) and double distilled water were utilized as polymer and solvents to prepare the

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ternary solution. HA particles, produced as described elsewhere [26], were kindly provided by

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Professor Licciulli from University of Salento. Granulometric analysis of HA was performed

with a laser granulometer Mastersizer 2000 (Malvern Instruments Ltd).

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2.2. Scaffold preparation
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A suspension made of a homogeneous ternary solution (composed by PLLA, dioxane, water)

and HA particles was prepared, with a constant dioxane to water weight ratio of 87/13 wt/wt.
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The concentration of PLLA was 4% wt and different PLLA/HA ratios (95/5, 90/10, 70/30,

50/50, 34/66) were tested. The thermal protocol was designed and set up as follows. The
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solution was initially kept at 60°C. Then the temperature was suddenly lowered to 30°C, a
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temperature within the metastable region [27], for 10 minutes. Later, a quench by direct pool
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immersion in an ethyl alcohol bath at a temperature of -25 °C was performed. The foams
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obtained were subjected to washing in deionised water and drying at 35 °C under vacuum, in

order to completely remove any remaining solvent trace.

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2.3. Characterization of PLLA/HA scaffolds

2.3.1. Porosity

The porosity was evaluated by liquid displacement method and gas pycnometer [28]. For

liquid displacement method, ethanol was used as wetting agent, as it penetrates easily into the

foam and does not induce shrinkage nor swelling [29,30]. First, the density of the ethanol was

accurately measured with a densimeter. Dry samples were cut in cylindrical shape (15 mm

diameter, 15 mm height) and their weights were recorded. Finally, air was removed from the

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sample in a vacuum process to force ethanol into the pores of the foams and then the wet

scaffolds were weighted again.

The porosity was obtained by:

𝑃2 −𝑃1
𝜌𝑒𝑡
Φ= ∗ 100 [1]
𝑉𝑏

where  is the porosity of the scaffold, P1 is the weight of the dry scaffold weight, P2 is the

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wet scaffold weight (after immersion in ethanol), ρet density of ethanol and Vb the bulk

volume of scaffold.

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Porosity results obtained via ethanol displacement were compared via Boyle’s pycnometer

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[31]. Five samples (bulk volume about 2 cm3) for each condition were analysed. Via gas
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pycnometry, a calculation of the volume of the skeleton of a porous solid was possible. Once

known the bulk volume, the real volume was attained and, dividing those two values, the
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porosity value was derived, as ε=V/Vb, where V is the skeletal volume measured by

pycnometer and Vb is the bulk volume. The adopted gas for this type of measurement was
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Helium.
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2.3.2. SEM analysis

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The morphology of the obtained foams was analysed by scanning electron microscopy (SEM)
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through a SEM-FEI QUANTA 200F on sample cross section fractured in liquid nitrogen and

gold sputtered (Sputtering Scancoat Six, Edwards) for 120s under argon atmosphere before
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imaging. SEM images were exported as 24-bit image files using the tagged image file format

(tiff) for further analysis.

2.3.3. Wide-angle X-ray analysis

The crystalline structure of PLLA/HA scaffold was investigated by Wide-angle X-ray

analysis (WAXD). The measurements were carried out by means of a Panalytical X’Pert

Powder Diffractometer with 2θ angle ranging from 5° to 70°, with a step angle and a step time

of 0.15° and 10 s, respectively. The voltage was 40 kV and the tube current was 30 mA.

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2.3.4. Thermogravimetric analysis

Thermogravimetric analysis (TGA) was carried out on samples (initial weight about 30 mg)

by utilizing a Netzsch STA 449 F1 Jupiter. After an initial stabilization, the temperature was

increased at 10°C/min from 30°C to 500°C.

2.3.5. Differential Scanning Calorimetry analysis

The melting temperatures and melting enthalpies of the samples were extrapolated by

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thermograms obtained with a Setaram 131 evo Differential Scanning Calorimeter (DSC). The

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samples, after an initial stabilization at 30 °C for 15 minutes, were heated from 30 to 210°C at

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a rate of 10°C/min, under a nitrogen gas flow of 1 ml/min.
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2.3.6. Mechanical testing

The scaffolds were mechanically tested in compression mode by using an Instron 3365 (UK).
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Disc-shaped sample (diameter 10 mm, thickness 2 mm) were tested applying a crosshead

speed of 0.5 mm/min. The compressive modulus was defined as the slope of the initial linear
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zone in the plot. At least five specimens were tested for each sample.
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2.4 In vitro cell cultures

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Pre-osteoblastic MC3T3-E1 cells (Sigma Aldrich) were cultured in Dulbecco’s modified

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Eagle’s medium (DMEM, Sigma Aldrich) supplemented with 10% of fetal bovine serum, 1%

of glutamine and 1% of streptomycin/penicillin in 75 cm2 cell culture flasks incubating at

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37 °C with 5% CO2. Pure PLLA and PLLA/HA scaffolds with a weight ratio of 95/5 were

reduced to circular-shaped samples (diameter: 5 mm, thickness 1 mm). The samples were

sterilized under vacuum in a 70% ethanol solution. After removing ethanol, scaffolds were

abundantly rinsed in sterile PBS 1X (Euroclone) for 2 hours and then treated with Micro-

fibrillary rat-tail type I collagen (BD Biosciences) in 0.02N acetic acid at the concentration of

100 µg/ml for 90 minutes. Finally, the scaffolds were washed in complete medium to activate

the collagen mesh formation thanks to neutral pH shift. Cells were detached from the culture

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flasks with the standard protocol using the tripsyn/EDTA solution and counted through a

Burker chamber. Thereafter, 25 µl of cellular suspension at the concentration of 104 cells/µl

were kindly inoculated into each scaffold. After an incubation at 37°C and 5% CO2 for 90

minutes to promote cell adhesion, each scaffold was transferred into a well of a 24 well plate

and fresh medium was added. The culture medium was replaced every three days up to 27

days in order to maintain constant the pH and the nutrients level for the whole experiment

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[32].

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2.4.1 Cell viability assay

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Cell proliferation on scaffold of PLLA and PLLA/HA was evaluated through viability assays

employing Cell counting Kit 8 (Sigma Aldrich); a sensitive colorimetric kit containing WST-
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8, a salt reduced by mitochondrial dehydrogenases to orange formazan, evaluated in

absorbance at 450 nm. Since the number of living cells is directly proportional to the value of
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absorbance, a standard curve was created to convert the absorbance in terms of cell number.

After scaffolds transfer into other wells (to avoid the quantification of cells adhered to well),
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each sample was incubated at 37°C and 5% CO2 for 3 hours with 500 µl of 1:10 diluted
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reagent in fresh medium. Finally, the medium was collected and analysed with a
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spectrophotometer at absorbance of 450 nm. The assays were carried out at 0, 7, 14, 21 and 27
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days of culture in triplicate for each time. Not seeded scaffolds were used as negative controls

in each measurement.
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2.4.2 Alkaline phosphatase activity

The osteoblastic differentiation process was investigated with SensoLyte pNPP Alkaline

Phosphatase Assay kit (Anaspec, AS-72146) measuring the alkaline phosphatase activity,

marker expressed during osteoblastic maturation. In particular, incubating the protein extracts

with the enzymatic substrate, p-nitrophenyl phosphate (pNPP), a colorimetric reaction caused

by dephosphorylation occurs, bringing to the formation of a yellow product, p-nitrophenol.

The spectrophotometric measure through absorbance at 405 nm reveals the ALP activity. The

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cell seeded scaffolds were collected, washed twice with PBS and, according to the kit

protocol, incubated with a washing buffer. Successively, samples were grinded and 100 µl of

0.2% Triton X-100 lysis buffer was added to each scaffold at 4°C for 10 minutes. After a

centrifugation at 2500 g at 4°C for 10 minutes, supernatant was harvested and 50 µl of the

extracts containing alkaline phosphatase was incubated at 37°C for 60 minutes with 50 µl of

substrate for 1 hour into wells of a 96 well plate. Finally, the reaction was stopped with 50 µl

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of the stop solution and the colorimetric determination of the product was analysed at 405 nm.

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The measurements were performed at 0, 7, 14, 21 and 27 days in triplicate. Data are expressed

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in ng/cell using a standard curve to convert absorbance values in ng of alkaline phosphatase.

2.5 Statistical analysis

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Group data were compared using one-way analysis of variance, and when applicable, data

were compared using the Student’s t test. P value < 0.05 was considered significant.
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3. Results and Discussion

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3.1 Particles characterization

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From the SEM micrographs shown in Figs. 1A and 1B it is possible to observe the HA
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particles employed in this study. A granulometric analysis, carried out on three different
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batches of HA particles, has revealed an average size distribution shown in Figure 1C. A

bimodal distribution with nanometric particles (100 nm) and micrometric particles (10-100
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m) was observed.

3.2 Scaffold characterization

Fig. 2A shows SEM micrographs of the cross sections of PLLA/HA scaffolds prepared with a

70/30 PLLA/HA ratio wt/wt. From the micrograph it is possible to appreciate that the liquid-

liquid phase separation process has correctly taken place, since a porous structure with

interconnected pores can be observed. Fig. 2B shows the same scaffold at a higher

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magnification: it is easy to observe the HA particles integration into the polymer matrix. The

same pore morphology and pore size were found in the scaffolds prepared with a 50/50

PLLA/HA ratio. In this case, as expected, the presence of HA is more evident (see Fig. 3).

Fig. 4 shows SEM micrographs of 90/10 PLLA/HA scaffolds prepared keeping constant the

demixing time (10 minutes) and changing the demixing temperature (25, 30 and 35 °C). From

the micrographs it is possible to appreciate that also in the presence of HA, the higher is the

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demixing temperature, the higher is the pore size according with several our previous work

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where neat PLLA was utilized [27,33]. Very similar results were obtained also at different

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PLLA/HA ratios (data not shown)

Porosity is defined as the percentage of void space into a solid. Large and interconnected
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pores are a necessary feature to allow migration and proliferation of osteoblasts mesenchymal

cells for bone tissue engineering [34].

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A porosity evaluation via Boyle’s porosimeter and liquid displacement of the composites

scaffolds produced is reported in Fig. 5, from which it can be noticed that, in both cases, the
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increase of HA content leads to lower values of porosity, probably because a portion of open
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pores is occupied by HA particles. It is relevant to underline that also a high amounts of HA

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(66% in weight), gives rise to scaffolds with a high degree of porosity (89-90%), an essential
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feature for cell proliferation and tissue regeneration within the devices.
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3.3 HA integration and quantification

In Fig. 6 the WAXD pattern of PLLA/HA foams is shown and highlighted. Pure PLLA

pattern presents two peaks located at 16.5 and 19 degrees, respectively (oval “a”). Mineral

Hydroxyapatite shows a set of typical peaks around 30° (oval “b”) and around 50° (oval “c”).

It is easy to notice that when HA content increases, the two peaks of PLLA reduced. In

accordance with the present results, previous studies have demonstrated that the differences in

spectra originate from interactions of the filler with the PLLA matrix [29,35–37]. This

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analysis, corroborated by experimental evidence of a uniform particle distribution within the

polymeric matrix provided by SEM images (figs 2 and 3 ), confirmed the effective and stable

HA incorporation on a microscopic scale.

The major part of scaffold production techniques involves several steps to reach the final

device and, often, the concentration of filler in the scaffold is different if compared to the

nominal concentration. On the opposite, via ternary TIPS technique, porous and

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interconnected composite foams were fabricated in a single step. Fig. 7 shows the results of

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TGA measurement of neat polymer, neat filler and the composite foam. In figure it is reported

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only the range of temperature from 150 °C to 450 °C, since nothing occurs before and after

this range.
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At 400°C pure PLLA an almost complete scaffold degradation was observed, whereas no

weight loss were recorded in pure HA. The curves show a good accord with the amount of
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filler within the initial solution, with an average difference of about 10-15%. Furthermore, the

curves show an improved thermal stability when the filler percentage is increased, witnessed
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by higher initial decomposition temperatures and degradation temperatures. This also accords
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with earlier observations [38], which showed the positive effect of HA on the thermal stability
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of the composites, due to the interface interaction between HA and the polymer [39,40].
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Fig. 8 reports the enthalpies of melting and the melting temperatures of the composite

scaffolds. As expected, the melting enthalpy decreases when the HA content increases. For
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example, an enthalpy of about 55 J/g was observed for pure PLLA scaffold, whereas an

enthalpy of 15 J/g was recorded for those with a PLLA/HA ratio of 33/66, which is in line

with the weight content of PLLA within the composite (about 1/3 of the melting enthalpy of

the neat PLLA). On the other hand, a drop of melting temperature is observed when HA

particles are added. The decrease of melting point in PLLA/HA composite materials was

reported in a recent works [41] and can be explained by inferring that the intercalation of HA

particles may hinder the crystallization of the polymer matrix inducing the formation of more

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defective crystals that exhibiting a lower the melting temperature[42]. An alternative

explanation, provided by Lee at al., is that the melting temperature drop is also a possible

outcome of the presence of voids in the composite material [43] although, in our case, this

hypothesis does not seem to be supported by other experimental evidences.

3.4 Mechanical testing

Representative compressive stress–strain curves, indicating elastic–plastic deformation are

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given in Fig. 9A. The calculated compressive moduli are shown in Fig. 9B as a function of

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HA content. The PLLA scaffold had a compressive modulus of 1.9 ± 0.7 𝑀𝑃𝑎. The addition

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of HA into the scaffold improved the mechanical properties. The modulus significantly

increased (more than double) when the HA amount reached about 30% of the composite. Also
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the other samples containing HA show an increase of the Young’s modulus with respect to

neat PLLA, although no significant differences were recorded when increasing HA content.
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Mechanical properties of the porous system depend on several parameters such as: porosity,

pore size, amount of filler, thermal history and/or other parameters concerning the foaming
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process. It makes a meaningful comparison of literature data rather difficult [44]. It is

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reasonable to speculate that, despite an increase of HA contents, above a certain threshold of

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HA content (50%), the distribution and orientation of particles within the structure plays a key
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role in determining the complex foamed composite’s architecture (as witnessed by the lower

melting point), which may lead to a decrease in Young’s modulus. An interesting explanation
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was provided by Xiao et al where a similar trend was encountered in PLLA-HA composite

scaffold produced via TIPS starting from a binary solution: HA can uniformly disperse in the

whole scaffolds and improve the compressive strength of composite scaffolds through

connecting and reinforcing the wall of PLLA. However, the HA will easily agglomerate

together in the process of phase separation, as the amount of microspheres excesses the

opportune degree in the scaffolds [45]. Indeed, it is mandatory to underline that the main goal

of adding nanoparticles in PLLA was not only to improve the mechanical properties, but,

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above all, to enhance bioactivity [46]. Biomechanical studies have already proven the

feasibility of using scaffolds with relatively modest mechanical properties in clinical

applications, such as revision knee arthroplasty [47].

3.5 Cell viability and ALP activity

In order to investigate about the effect of HA toward the osteoblasts differentiation, MC3T3-

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E1 pre-osteoblastic cells were seeded on composite scaffold. For this study were utilized the

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95/5 PLLA/HA samples in order to verify if the smallest amount of incorporated HA could

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promote a differentiation effect.

CCK-8 assay was carried out to study cell viability of cells seeded on pure PLLA (used as
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control) and PLLA/HA scaffolds. The absorbance values have been translated in terms of

number of cells through a standard curve previously built. Cell number as a function of days
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of culture on both kinds of sample, is reported in Fig. 10A. From the whole culture time taken

in consideration, on both substrates, viable cell number had regularly grown from about 1*105
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to 4.5*105 cells, reaching the maximum peak. In the last week a slight drop towards about
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3.5*105 cells was observed. This behaviour could be explained by supposing the reaching of
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cell confluence at 21 days, followed by a physiological detachment due to the lack of

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available surface. The trends overlap for both scaffolds, PLLA and PLLA/HA, demonstrating

a comparable biocompatibility.
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As marker of osteoblastic differentiation, alkaline phosphatase (ALP) activity was monitored

in both substrates. The histogram in Fig. 10B shows the ALP activity as nanograms of ALP

normalized with number of cells. From 7 to 14 days, the data revealed an increase in ALP

activity, despite the fact that PLLA and PLLA/HA values were not significantly different due

to the standard deviation calculated. At 21 and 27 days the presence of ALP in PLLA/HA

resulted considerably increased with respect to pure PLLA.

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It seems evident that both substrates promote cell growth at the same level and HA seems to

address the osteoblastic cells toward the maturation through a significant raise in ALP activity

after 21 days when cell confluence is reached.

4. Conclusions

In this work composite PLLA/HA porous scaffolds were obtained via TIPS in a single step

starting from a polymer/solvent/non-solvent solution. Based on the results, it was possible to

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state that the presence of the HA did not influence the phase separation process in terms of

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final scaffold morphology. As a matter of fact, a porous and interconnected structure was

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observed in the bulk of the scaffolds. SEM analysis revealed the presence of HA particles and

their integration in the polymer matrix was confirmed by several characterization techniques.
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Mechanical tests have highlighted an increase of Young’s modulus of composites scaffolds

with respect to pure PLLA scaffolds when relative low amount of filler is embedded in the
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matrix. In vitro cell cultures studies demonstrated that HA particles did not influence the cell

viability of mouse preosteoblastic MC3T3-E1 cells. Furthermore, an addressing towards the

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osteoblastic maturation was observed into the scaffold containing HA particles, witnessed by
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a more than twofold level of ALP after 21 days of culture. Having considered these
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introductory biological data, composite scaffold containing HA can be used for additional
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analysis to evaluate in a deeper way the cellular response in terms of bone formation.

Some limitations of the present study were summarized as follows: the cell cultivation in the
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scaffold was limited to 28 days in this study and to the 95/5 PLLA/HA scaffolds. Particularly

osteoblast might need a longer time for their complete differentiation to osteocyte. Moreover

the reason for which the Young modulus decreases at higher HA percentages would require a

more in deep study.

However, the contribution of this research was to demonstrate that it is possible to produce

composite scaffolds (utilizing a wide range of polymer/filler ratios) with a single step

protocol, and to control accurately the scaffold morphology, above all in terms of pore

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dimensions. All things considered it was realistic to address the potential of these devices

towards their successful use in bone tissue engineering applications.

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Figures Caption:

Figure 1: SEM micrographs of the HA particles at (a) lower (3000X) and (b) higher
magnification (16000X); (c) Size distribution of the particles

Figure 2 - SEM micrographs of the scaffold prepared with a 70/30 PLLA/HA ratio. a) lower
magnification (800X); b) higher magnification (3000X).

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Figure 3 - SEM micrographs of the scaffold prepared with a 50/50 PLLA/HA ratio. a) lower
magnification (800X); b) higher magnification (3000X).

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Figure 4 - SEM micrographs of the scaffold prepared with a 90/10 PLLA/HA ratio at different
demixing temperatures, keeping constant the demixing time. a) 25 °C; b) 30 °C; c) 35 °C.
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Figure 5 - Porosity evaluation of composite scaffolds at different PLLA/HA weight ratios
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Figure 6 - WAXD pattern of composite PLLA/HA scaffolds at different polymer/filler ratios.

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Figure 7 - TGA analysis of PLLA/HA foams at different polymer/filler ratios.

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Figure 8 - DSC analysis of different PLLA/HA foams.

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Figure 9 – A) Representative stress-strain curves of three different composite scaffolds. B)

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Young’s modulus of the PLLA/HA scaffolds, *p < 0.05.

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Figure 10 - A) MC3T3-E1 viability assays on scaffolds of PLLA (blue) and PLLA/HA (red)
expressed as number of viable cells during 27 days. B) ALP activity at 7, 14, 21 and 27 days.
Error bars represent means±SD for n=3 (*P<0.05). The data are normalized using not seeded
scaffolds as negative control.

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