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Synthesis and characterization of nanocomposite scaffolds based on triblockcopolymer of L-lactide, ε-caprolactone and nano-hydroxyapatite for bonetissue engineering

Bahman Torabinejad, Jamshid Mohammadi-Rovshandeh, Seyed MohammadDavachi, Ali Zamanian

PII: S0928-4931(14)00263-XDOI: doi: 10.1016/j.msec.2014.05.003Reference: MSC 4627

To appear in: Materials Science & Engineering C

Received date: 29 January 2014Revised date: 16 March 2014Accepted date: 3 May 2014

Please cite this article as: Bahman Torabinejad, Jamshid Mohammadi-Rovshandeh,Seyed Mohammad Davachi, Ali Zamanian, Synthesis and characterization of nanocom-posite scaffolds based on triblock copolymer of L-lactide, ε-caprolactone and nano-hydroxyapatite for bone tissue engineering, Materials Science & Engineering C (2014),doi: 10.1016/j.msec.2014.05.003

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Synthesis and characterization of nanocomposite scaffolds

based on triblock copolymer of L-lactide, ε-caprolactone and

nano-hydroxyapatite for bone tissue engineering

Bahman Torabinejad a,b

, Jamshid Mohammadi-Rovshandeh c, Seyed Mohammad

Davachi d, Ali Zamanian

a

a Nanotechnology and Advance Materials Department, Materials and Energy Research Center, P.O. Box: 31787-

316, Karaj, Alborz, Iran

b School of Polymer Chemistry, College of Science, University of Tehran, P.O. Box 14155-6455, Tehran, Iran.

c Caspian Faculty of Engineering, College of Engineering, University of Tehran, Rezvanshar, P.O. Box 43841-

119, Guilan, Iran

d School of Chemical Engineering, College of Engineering, University of Tehran, P.O. Box 11365-4563,

Tehran, Iran

torabinejad@ibb.ut.ac.irr: Corresponding autho

Abstract: The employment of biodegradable polymer scaffolds is one of the main

approaches for achieving a tissue engineered construct to reproduce bone tissues, which

provide a three dimensional template to regenerate desirable tissues for different applications.

The main goal of this study is to design a novel triblock scaffold reinforced with nano-

hydroxyapatite (nHA) for hard tissue engineering using gas foaming/salt leaching method

with minimum solvent usage. With this end in view, the biodegradable triblock copolymers

of L-lactide and ε-caprolactone with different mol % were synthesized by ring opening

polymerization method in the presence of Sn(Oct)2 catalyst as initiator and ethylene glycol as

co-initiator. The chemical compositions of biodegradable copolymers were characterized by

means of FTIR and NMR. The thermal and crystallization behaviors of copolymers were

characterized using TGA and DSC thermograms. Moreover, nano-hydroxyapatite was

synthesized by the chemical precipitation process and was thoroughly characterized by FTIR,

XRD and TEM. Additionally, the nanocomposites with different contents of nHA were

prepared by mixing triblock copolymer with nHA. Mechanical properties of the prepared

nanocomposites were evaluated by stress-strain measurements. It was found that the

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nanocomposite with 30% of nHA showed the optimum result. Therefore, nanocomposite

scaffolds with 30% nHA were fabricated by gas foaming/salt leaching method and SEM

images were used to observe the microstructure and morphology of nanocomposites and

nanocomposite scaffolds before and after cell culture. The in-vitro and cell culture tests were

also carried out to further evaluate the biological properties. The results revealed that the

porous scaffolds were biocompatible to the osteoblast cells because the cells spread and grew

well. The resultant nanocomposites could be considered as good candidates for use in bone

tissue engineering.

Keywords: Biodegradability, L-lactide, ε-caprolactone, Nano-hydroxyapatite, Scaffolds, Cell

culture.

1. Introduction

Use of biodegradable polymers, with numerous biomedical applications, has been

progressively increased during the past years. Degradable polymeric biomaterials are the

most preferable choices for therapeutic devices including artificial prosthesis, controlled drug

release devices and 3D Scaffolds for tissue engineering [1]. Biodegradable polymers can be

generally classified into two groups. The first group is the natural based materials among

which polysaccharides (starch, alginate, chitin/chitosan, hyaluronic acid derivatives) or

proteins (soy, collagen, fibrin gels, silk) are the main materials. The second type is the

synthetic polymers which can be manufactured under restrained conditions exhibiting

reproducible mechanical and physical properties such as tensile strength, modulus and rate of

degradation [2].

Poly α-esters or aliphatic polyesters such as poly(lactide), poly(lactide-co-glycolide) and

poly(ε-caprolactone), and their synthesized copolymers are the most common synthetic

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biodegradable polymers which have been widely used for medical applications. They have

been commercialized due to their superior tissue compatibility and safety profile, both of

which are very important parameters concerning the human body. These polyesters possess

great mechanical properties and their rate of degradation can be simply modified as well as

their shapes. However, due to their hydrophobic surface, their cell recognition signals are low

[3,4]. Poly(L-lactide) (PLLA) is a semicrystalline thermoplastic which has been widely used

in biomedical purposes, such as bone fixation and surgical sutures because of its good

bioresorbability and biocompatibility, however, brittleness has limited its applications [5,6].

Poly(ε-caprolactone) (PCL) is relatively non-toxic and possesses sufficient mechanical

strength, biocompatibility, and thermal stability for scaffolding applications. A low glass

transition temperature about -60°C provides a rubbery consistency at room temperature,

offering a promising potential for load-bearing applications in tissue engineering.

Nevertheless, slow degradation rate, owing to its high hydrophobicity and crystallinity, is the

major limitation for this polymer [7]. Copolymerization of ε-caprolactone (CL) with different

lactones is an easy method to tailor the PCL properties to extend the range of its possible

applications [8]. Copolymerization of PCL and PLLA has modified PLLA mechanical

properties to be utilized in orthopedic bone defects repairs [9]. One of the most common

methods to synthesize these copolymers and obtaining high molecular weight products with

controlled microstructures is ring-opening bulk polymerization with coordination insertion

mechanism. Stannous octoate, Sn(Oct)2, is the most commonly used initiator in the

polymerization of cyclic esters due to its non-toxicity and high efficiency [10,11].

Several ceramic materials have been prevalently used to repair diseased or damaged hard

tissue for a long time. Materials such as bioglass, sintered hydroxyapatite and glass-ceramic,

also known as bioactive materials, instinctively bond to the living bone, and bone substitutes.

Synthetic ceramic materials based on calcium phosphate (CaP) particularly in the

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composition of tricalcium phosphate [TCP-Ca3(PO4)2], hydroxyapatite [HA-

Ca10(PO4)6(OH)2] and biphasic calcium phosphates (BCP, a mixture of HA and TCP) have

been widely studied due to the similarity to the chemical composition of bone [12–14].

One of the most interesting materials for orthopedic applications is hydroxyapatite (HA). HA

possesses structure and chemical composition very close to the natural bone mineral and

gives excellent biocompatibility, bioactivity, non-toxicity, non-inflammatory behavior and

non-immunogenicity. HA is also advantageous to be used as scaffold in the bone tissue

engineering due to its osteoconductivity, osteoinductivity and its extremely slow degradation

rate. It makes the bone regeneration faster, and also, directly bonds to the regenerated bones

without mediatory tissues, but because of brittleness and inflexibility, it is difficult to shape

HA limiting its applications. In addition, HA powders can immigrate from the implanted bulk

to the surface making the material unsuitable for use. These powders have high surface

energy, and thus, they will agglomerate easily and cannot be well dispersed [15–18].

Composites have shown to be more effective for enhancement of both mechanical properties

and bioactivity as compared to ceramic and polymers. In the past few years, the progress of

bioactive ceramic-polymer biocomposites, like HA-polymer composites, has gained a

phenomenal impetus in the orthopedic field for their bone analog design as well as good and

improved biological and mechanical performances to meet specific clinical requirements [19–

21]. In nanocomposites for bone repair, the bond between matrix and reinforcement is very

strong which can be ascribed to the higher surface area-to-volume ratio of nanoparticles.

Therefore, these nanocomposites show higher mechanical properties and improved cell

behavior compared to the conventional composites [22]. Natural bone tissue is a

biocomposite which is composed of nHA crystals dispersed in collagen matrix [20]. Lately,

synthesis and preparing nanocomposites comprising dispersed nano-hydroxyapatite in

biodegradable polymer matrices have been extensively studied [4,19–28]. Tissue engineering

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has emerged as a promising alternative approach to treat the loss or malfunction of a tissue or

organ without the limitations of current therapies [29]. In fact, tissue engineering and the

other independent regenerative medicines are the thriving area with new possible medical

cares for many more disease states. Porous scaffold is the main part of tissue regeneration and

repair. However, its fabrication with desirable properties usable in tissue engineering

applications remains a complex and challenging process [30,31].

Many of the fabrication techniques including solvent casting, salt leaching, electrospinning

and freeze drying require use of toxic organic solvents and elevated temperatures. The

residues that remain after completion of process can damage cells and adjacent tissues, and

may also denature the biologically active molecules incorporated within the scaffolds. The

gas foaming scaffold fabrication techniques require no toxic organic solvents and elevated

temperatures [31,32].

In the current study, at first, a random prepolymer of L-lactide and ε-caprolactone P(LLA-co-

CL) is synthesized by using stannous octoate (Sn(Oct)2) as catalyst and initiator and ethylene

glycol as co-initiator via ring opening polymerization to obtain a prepolymer with hydroxyl

groups in two ends (telechelic random prepolymer). After that, synthesized prepolymer is

polymerized with predetermined amounts of L-lactide for preparing triblock copolymers in

two different mole fractions. The structure and properties of these copolymers are

investigated by using a combination of nuclear magnetic Resonance spectroscopy (1HNMR

and 13

CNMR), differential scanning calorimetry (DSC), thermogravimetric analysis (TGA),

and Fourier transform infrared (FTIR). In the next step, nanosized hydroxyapatite is prepared

via precipitation technique [33]. These nano-particles are characterized by Fourier transform

infrared (FTIR), X-ray diffraction (XRD) analysis and Transmission electron microscopy

(TEM). Nano-sized HA was then added to the copolymer for preparing the nanocomposites

with different compositions. Finally, the novel scaffolds are prepared from triblock

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copolymers/Hydroxyapatite by gas foaming/salt leaching method which has minimum

solvent usage and their structures as well as their surface morphology are investigated by

scanning electron microscopy (SEM) before and after cell culture in details. The prepared

scaffolds can be used in bone tissue engineering applications.

2. Experimental

2.1. Materials

L-lactide monomers were synthesized from 90% L-lactic acid solution (Merck, Darmstadt,

Germany) according to the Gilding and Reed procedure [34]. At first, L-lactide monomers

were purified by several recrystallizations from dry ethyl acetate, and finally, were eluted by

dry diethyl ether to remove trace impurities. Then, L-lactide monomers were dried under

vacuum at room temperature for 24 hr. The catalyst, stannous octoate (Sn(Oct)2) (Sigma, st.

Louis, USA), was purified by vacuum distillation. ε-Caprolactone, ethylene glycol and all

other chemicals and solvents such as ethanol, ethyl acetate, chloroform, dichloromethane, n-

hexane, orthophosphoric acid (H3PO4), calcium hydroxide, ammonium hydroxide,

ammonium bicarbonate and dimethyl sulfoxide were purchased from Merck. Solvents were

further purified according to the established procedures if necessary [35].

2.2. Synthesis of the random prepolymer

In the first step, a 68:32 mol % P(LLA-co-CL) random prepolymer was synthesized in a

polymerization tube. A calculated amount of LLA and CL mixture, in a LLA:CL = 68 : 32

mol % ratio with specific amounts of ethylene glycol (EG) (monomers/EG molar ratio of

500) and Sn(Oct)2 (mole of reacting monomer/ mole of catalyst =5000) were placed into a

polymerization tube and kept under vacuum at 50°C for 2 hr, until all volatiles were removed.

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Then, the polymerization tube was sealed under vacuum and immersed in an oil bath at

120°C for three days. Subsequently, the tube was broken and the contents were completely

dissolved in chloroform and then reprecipitated in n-hexane. Finally, this prepolymer was

dried under vacuum at 50°C for 24 hr, and used as the central segment in the synthesis of

triblock copolymers. In these types of reactions, Sn(Oct)2 forms a complex with hydroxyl-

containing compounds initiating the ring-opening polymerization of lactones by a

coordination-insertion mechanism. In this case, EG acts as two-functional micro-initiator for

the polymerization of LLA and CL. The obtained random prepolymer has also two hydroxyl

functional groups, and it can be used for further polymerization with L-lactide in the presence

of Sn(Oct)2. The schematic of synthesis reaction of prepolymer is presented in Figure1.

Figure1. Polymerization reaction of P(LLA-co-CL) prepolymer.

2.3. Synthesis of the triblock copolymers

After purification of synthesized prepolymer, a calculated amount of LLA was added to

predetermined amounts of dried telechelic P(LLA-co-CL) prepolymer (LLA : prepolymer

molar ratios of 85 : 15 (TB1) and 95 : 5 (TB2)mol %) in a polymerization tube with a

magnetic stirrer. Then, the mixture was kept under vacuum at 50°C for 2 hr. In this step,

stannous octoate, Sn(Oct)2, was added to the mixture again (mole of reacting monomer/ mole

of catalyst was kept constant at 5000) without adding further ethylene glycol. The prepolymer

was dissolved in LLA monomers by primary heating the mixture to higher polymerization

temperature for a short time until the homogenous system was obtained. Under vacuum, the

glass tube was sealed and immersed in oil bath at 120°C for seven days. Finally, the glass

tube was broken, the contents was grinded, and for final purification they were subjected to

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high vacuum at ambient temperature for 24 hr. The polymerization reaction of PLLA-P(LLA-

co-CL)-PLLA triblock copolymers is schematically shown in Figure2.

Figure2. Polymerization reaction of PLLA-P(LLA-co-CL)-PLLA triblock copolymer

2.3. Synthesis of nano-hydroxyapatite:

HA nano-crystals were prepared based on the following reaction of calcium hydroxide with

orthophosphoric acid (H3PO4):

10 Ca(OH)2 + 6 H3PO4→ Ca10(PO4)6(OH)2 +18 H2O

In this method of synthesis, 100 mL of 0.3 M H3PO4 solution was added to an equal volume

of 0.5 M Ca(OH)2 solution at a drip rate of two drops per second, at room temperature. The

pH value was kept above 10 by the addition of ammonium hydroxide (NH4OH) during the

precipitation process. The resultant precipitate was left in the mother solution for five days.

At the end of the process, the precipitate was washed by deionized water and dried in an oven

at 110°C for 2 hr [36,37].

2.4. Fabrication of PLLA-P(LLA-co-CL)-PLLA/HA nanocomposites

According to the results which will be discussed in the next sections, TB1 was selected for

making nanocomposites. TB1/HA nanocomposite was prepared by solution casting method.

The weight percentages of nano-HA particles in nanocomposites were 0, 10, 20 and 30%. At

first, specific amounts of TB1 were dissolved in dichloromethane (CH2Cl2), and then, mixed

by a magnetic stirrer. Then, nHA powders were immersed in ethanol solution and

homogenized by ultrasonic for 10 min. After that, nHA and ethanol solution were added

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dropwise to the prepared polymer solution under stirring. Consequently, upon precipitating

the copolymers in ethanol, a white emulsion was obtained, which was further stirred for 6 hr

continuously. Finally, solvent was evaporated slowly from the emulsion during the process,

and PLLA-P(LLA-co-CL)-PLLA/nHA nanocomposites were precipitated [38,39].

2.5. Fabrication of PLLA-P(LLA-co-CL)-PLLA/HA nanocomposite scaffolds

TB1 (1.5 gr) was first dissolved in 20 mL of dichloromethane. Then, a solution which

includes the dispersed nHA in ethanol was slowly added to the polymer solution in various

weight percentages. The weight percentages of nHA in the nanocomposite scaffolds were 0,

10, 20 and 30%. Afterwards, ammonium bicarbonate salt particulates (weight ratio of salt/

(TB1 and nHA) were 7:3) were added to the mixture of TB1/nHA solutions. The particle size

of ammonium bicarbonate (NH4HCO3) was in the range of 200–300 μm, and subsequently, a

paste mixture TB1/nHA/salt was obtained, which was molded into cylindrical Teflon molds.

Finally, after partial evaporation of dichloromethane under atmospheric pressure, the samples

were placed into hot water for 5-10 min until bubbles were generated. Afterwards, the

samples were placed into cold water for 20 min, and then, were freeze-dried for several days

and stored at 220°C until use [39–41]. The porosity of polymer/HA nanocomposite scaffolds

was calculated through the following equation:

%1001

c

csPorosity

(1)

Where ρc is the density of initial nanocomposite with progen agent and ρcs is the density of

nanocomposite scaffold. These two densities were measured using a pycnometer and

determined by the gross weight and volume measurement of the specimens, respectively [42].

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2.6. Cell Culture

2.6.1. Degradation behavior of nanocomposite scaffolds

For in vitro degradation investigation, the polymers were subjected to an accelerated

hydrolysis at elevated temperatures and at normal temperature in two different conditions

(time temperature superposition). The specimens were immersed in a phosphate buffer at pH

7.4 and 70oC with stirring under an accelerated test. At normal test conditions for a period of

four months, the test was carried out at the same pH at 37oC near the human body

temperature [11,43].

2.6.2. MTT assay

Cell proliferation was analyzed by using MTT. This assay was based on the ability of live

cells to reduce a tetrazulium-based compound, MTT, to a purplish formazan product. Briefly,

nanocomposite scaffolds were washed with phosphate buffer, transferred into new plates

containing 5:1 ratio of media and MTT solution (5 mg/mL in Phosphate buffer), respectively,

and incubated for 2 hr at 37oC. After removing the culture media, 0.5 mL of dimethyl

sulfoxide (DMSO) as extraction solution was added. The scaffolds were washed extensively

by pipetting up and down repeatedly to allow total color release. The absorbance of the

supernatant was read with a microplate reader at 540 nm. Cell population was determined

through a standard curve that was established by using a known number of cells counted by a

Neubauer-counting chamber. This test was done according to the procedure presented by

Zund et al. who have developed a linear correlation between the cell number and the MTT

absorbency at 550 nm [44,45].

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3. Characterization

The compositions of prepolymer and triblock copolymers were characterized with a

combination of high-resolution 500MHz 1H-NMR and 125MHz

13C-NMR spectrometers (

1H

and 13

C NMR, Bruker, DRX series, Germany). Deuterated chloroform (CDCl3) and

tetramethylsilane (TMS) were used as a solvent and an internal standard, respectively. The

infrared spectroscopy was performed using FTIR (Bruker, Equinox 55(LS 101 series),

Germany) at a resolution of 4 cm−1

(averaging 50 scans) for recognition of functional groups

in copolymers. Thermal analysis was accomplished by differential scanning calorimetry

(DSC, Polymer laboratories, PL-800 series, England). For DSC, 5–12 mg prepolymer and

triblock copolymer samples were heated at 10°C/min under a nitrogen atmosphere over a

temperature range of -80 to 180°C to observe the glass transition temperature (Tg), melting

temperature (Tm) and heat of melting (ΔHm). Thermal stability of copolymers was

characterized by thermogravimetric analysis (TGA, Polymer laboratories, PL-800 series,

England). For TGA, 10-20 mg samples were heated over a temperature range of 25 to 700°C

at a heating rate of 10°C/min under a nitrogen atmosphere.

X-ray diffraction analysis (XRD) (Philips diffractometer-PW3710) and FTIR spectra (Fourier

transform infrared, Bruker, Equinox 55 spectrophotometer) (4000–400 cm-1

) were used to

further characterize nHA powders. Moreover, scanning electron microscopy (SEM, VEGA-

XMU, Tescan, Czech Republic) was used to observe the morphology of surface,

microstructure and cell growth in interior walls of nanocomposites and scaffolds. SEM

samples were gold coated prior to imaging. For investigation of the size of nano-

hydroxyapatite (nHA), Philips CM200 transmission electron microscope (TEM) with an

accelerating voltage of 200 kV was used. The stress-strain behavior, elastic modulus and

stiffness of the triblock copolymer (PLLA-P(LLA-co-CL)-PLLA with (LLA:prepolymer

molar ratio 85:15 mol%) and the nanocomposite scaffoldss were determined by using a

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mechanical testing machine (SMT-20, santam, Iran) according to ASTM D638. The cross-

head speed was set at 1 mm/min. The measurements were conducted at room temperature and

to verify the reproducibility, at least five specimens were characterized for each composition.

4. Results and discussion

4.1.2. FTIR, XRD and TEM results of nano-hydroxyapatite

To evaluate the chemical composition of nHA powder, FTIR spectrum was recorded and

shown in Figure3. PO43-

peaks of nHA at 565, 600, 1030 and 1091 cm-1

could easily be

detected. The OH- absorption peaks at 630 and 3569 cm

-1 of nHA were also appeared.

Moreover, a broad peak was appeared in the wave range of 3100-3600 cm-1

corresponding to

the presence of H2O in nHA powder [46,47]. The X-ray pattern of nano-HA showed the

presence of the major HA peaks such as (002), (211) and (300) at 25°, 31°, 32° 2θ angles as

shown in Figure4, and no secondary phase was detected, which confirmed the purity of

prepared nHA (JCPDS No. 09–0432) [48]. Figure5 shows the TEM images of particles which

are mostly agglomerated exhibiting needle shape morphology. The particle size was

calculated and the length and width scales were 50-100 and 25-30 nm, respectively.

Figure3. FTIR spectra of nano-hydroxyapatite (nHA)

Figure4. XRD pattern of nano-hydroxyapatite (nHA)

Figure5. TEM image of nano-hydroxyapatite (nHA) needle-like crystals

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4.1. FTIR results of prepolymer and copolymers

The FTIR spectrum of prepolymer and its triblock copolymers are shown in Figure6. In all

spectra, the carbonyl (C=O) and (C-O) absorption bands appeared at 1749 and (1185 and

1092) cm-1

, respectively. The peaks at 1454 and 1382 cm-1

are also corresponded to the -CH2

and -CH3 absorption bands related to PCL and PLA segments, respectively [48,49].

Figure6. FTIR spectra of prepolymer and triblock copolymers

4.2. NMR results of prepolymer and copolymers

The typical 1HNMR and

13C-NMR spectrum of a resulting prepolymer and TB1 as a

representive are shown in Figure7 and Figure8. According to the previous studies [7,48–50],

the resonance signals belonging to the two kinds of methine and methyl protons in the

repeating PLLA units were observed at 5.0-5.1 ppm (CH) and 1.60 ppm (CH3) in Figure7a.

The caprolactyl methylenic protons appear at 3.9- 4.0 δ ppm (CH2-O) and 2.25 δ ppm (CH2-

CO), and inner methylenic protons appear at 1.1–1.7 δ ppm. The copolymer composition of

the prepolymer was determined from their 1H-NMR spectra by rationing the peak areas

corresponding to the LLA methine protons at δ=5.0-5.1ppm and the CL ε-methylene protons

at δ=3.9-4.0ppm. Because of the random nature of prepolymer, the spectrum is noisy and the

corresponding peaks have been spilt to several peaks. The calculated composition (LLA:CL

mol%) of prepolymer is given in Table1 showing that the feed ratio is approximately the

same as 1HNMR derived ratio, which was expected since the copolymerization was

proceeded to near quantitative conversion. The13

CNMR spectrum of prepolymer is presented

in Figure8a. The CH and CH3 carbons and carbonyl carbon of lactide moiety were appeared

at δ=69.5 ppm, δ=17.2 ppm and δ=170 ppm, respectively. The inner caprolactyl methylenic

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carbons appear at 28.7, 25.94 and 24.98 ppm. The caprolactyl methylenic carbons appear at

64.55 ppm (CH2-O) and 34.53 ppm (CH2-CO). Moreover, the carbonyl carbons of ε-

caprolactone and lactide appear at 170 and 173.9 ppm, respectively, and finally, the carbonyl

carbons of ε -caprolactone are detected at 174 ppm. In random blocks, because of different

monomer sequences and chemical environments, several peaks attributed to the carbonyl

carbon in different sequencings are observed. Therefore, the presence of several peaks

attributed to carbonyl carbon in 1HNMR spectrum confirms that the resulting prepolymer has

random structure. The calculated compositions of triblock copolymers are also given in

Table1, which are quite close to the comonomer feed ratios. Figure7b clearly indicates that

the peak areas related to the LLA protons significantly increase to those of the CL protons in

triblock copolymer than prepolymer which is due to the incorporation of further LLA in the

prepolymer. According to Figure8b, the 13

CNMR spectrum of triblock is closely similar to

prepolymer but because of the low caprolactone mole ratio in triblock copolymer, the signal

intensities of caprolactyl carbons are weak as the signal of carbonyl carbon approximately

cannot be recognized. Moreover, it can be observed that the 13

CNMR signals of prepolymer

are split to several peaks due to the random structure. However, mentioned division has not

been appeared in triblock signals which will be a qualitative confirmation of block structure

in synthesized triblock.

Figure7. 1HNMR spectra of resulting polymers (a) Prepolymer (b) TB1

Figure8. 13

CNMR spectra of resulting polymers (a) Prepolymer (b) TB1

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4.3. Gel permeation chromatography (GPC) Results

GPC results of the copolymers can be seen in Table1 indicating that the TB1 has the higher

molecular weight and the TB2, because of the highest amount of lactide, has the lowest PDI

which describes the high tendency of lactide monomers to be together. Results show that the

triblock with higher amount of prepolymer has lower molecular weight and lower Tg due to

higher content of ε-caprolactone (CL). Furthermore, the results show that CL increases the

molecular weight without changing the dispersity of polymer chains and this will be helpful

due to its soft nature to be used as a material to strengthen the scaffold.

Table1. The characteristics and molecular weights of Prepolymer and triblock polymers

4.4. Thermal properties and crystallization behavior of prepolymer and copolymers

To study the crystallization behavior and thermal properties of the samples, DSC technique

has been utilized. Because most processing methods occur under non-isothermal conditions,

the understanding of polymer crystallization under dynamic condition is of considerable

importance [51]. The DSC thermograms of copolymers are presented in Figure9. The

characteristic parameters of non-isothermal crystallization exotherms such as the

crystallization peak (Tm), the relative degree of crystallinity (Xt), in terms of ∆Hm (Hm is the

enthalpy of melt), and width of half height of crystallization peaks (WHH) are reported in

Table2. Results of DSC thermograms for prepolymer and two triblock copolymers indicate

that with incorporation and increasing of PLLA side block length, the Tg, Tm and heat of

melting (ΔHm) values are increased. According to the previous studies, there is a direct

relationship between crystallinity and ΔHm, therefore, with increasing the PLLA block length

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the crystallinity is increased [52]. It also can be confirmed by the WHH that with increasing

the amount of PLLA, the rate of crystallinity is increased as TB2 has the higher WHH

compared to the others. The melting enthalpy of 100% crystalline PLLA [53] and PCL [54]

are considered 93 and 81.6 J /g, respectively. Although there is no data available for melt

enthalpy of PCL-co-PLLA, but according to the mixture law, they were calculated 89.35,

89.92 and 89.47 for prepolymer, TB1 and TB2, respectively; and as can be seen, they are

quite similar. Finally, the relative degrees of crystallinity have been reported in Table2. As

expected, by increase in PLLA the crystallinity also increases.

Figure9. DSC thermogram for Prepolymer and Triblock Polymers

Table2. Tm،Xt،∆Hc and Width of half height crystallization peak (WHH) of Samples

Thermal stability, and initial (TI) and final (Tf) degradation temperatures of samples were

investigated using TGA technique and the results are shown in Figure10 and Table3. Because

of the low thermal stability of lactide as compared to ε-caprolactone, with incorporation and

subsequent increasing of PLLA blocks in two ends of prepolymer, thermal stability of

triblock copolymer is decreased. Thermal degradation data could give some important

information about polymer melt processing. Some polymers that are nominated for using in

medical applications, like drug delivery systems and tissue engineering, form toxic and

harmful products for human body during the melt processing [10]. Thus, for determination of

‘‘processing window’’, the combination of TGA and DSC data may be employed

concurrently. Processing window is the range of processing safely without synchronic

thermal degradation. With increase in amount of PCL, initial degradation temperature

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exhibits a slight decrease but Tf increases. However, the amount of char increases in triblock

copolymers, and for TB2 this amount is 2.62 which is higher than TB1. According to NMR,

FTIR and thermal properties, TB1 showed better properties especially in crystallization rate.

It is quite flexible and for further medical applications the rate of crystallinity has to be lower

and the flexibility of polymer should be higher since nano-hydroxyapatite makes the polymer

slightly brittle. This phenomenon is expectable, thus TB1 has been selected for composite

and scaffold preparation studies. Furthermore, the processing window of TB1 is between 170

and 233°C.

Figure10. TGA and DTGA thermograms for Prepolymer and Triblock Polymers

Table3. The characteristics of the TGA curves in nitrogen atmosphere

4.5. Mechanical tests

Based on the previous results, TB1 has been chosen for preparing nanocomposites, and then,

mechanical properties have been measured to find the best nanocomposite for scaffold

preparation. The stress-strain curve, elastic modulus and stiffness are presented in Figure11.

Elastic modulus and stress-strain curve were reported and the stiffness was calculated as the

area of stress-strain curve in the linear region based on S=0.5×σ×ε in which σ and ε are yield

stress and elongation at yield, respectively.

Figure11. Mechanical properties of nanocomposites with different contents of nHA

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According to the Figure11a, it is observed from the stress-strain curve that with increasing of

nHA weight percent in the nanocomposites stress values have been increased, but strain

values have not shown any significant changes in samples containing nHA. As depicted in

Figure11b and Figure11c, the elastic modulus and stiffness of these materials are also

increased by the increase in nano content from 5 to 14 GPa in modulus which shows a triple

increase and 651 to 4675 MN/m in stiffness which shows nearly seven fold increase. It can be

realized that the behavior of triblock copolymer was shifted from ductile to brittle, and so,

stiffness and modulus increased due to the presence of inorganic fillers in polymeric matrix.

In conclusion, the chain mobility is restricted which can be due to the physical interaction

between organic fillers and polymeric chains as well as chemical bonding between hydroxyl

groups of nHA and functional groups of copolymers like (C=O) [10]. According to the

mechanical results, nanocomposite with 30% nHA has been chosen for scaffold preparation.

4.5. In-vitro degradation, MTT assay and Morphological Behaviors

The microstructure and morphology of the nanocomposites and nanocomposite scaffolds

before and after cell culture and interconnectivity of Scaffolds are investigated by using SEM

as shown in Figure12. According to Figure12a and Figure12b , the nHA particles are well

distributed within the polymeric matrix with minimum agglomeration. SEM image of

nanocomposite scaffolds also shows micro pores within the scaffolds. Pore size of

nanocomposite scaffolds are in the range of 200-300 µm and the calculated porosity is around

70%. As can be observed in Figure12c and Figure12d, micrographs of prepared scaffolds

show the pore interconnectivity. Nanocomposite scaffolds exhibit the regular internal

spherical pore structure. In fact, all the nanocomposite scaffolds displayed more uniform

cellular structures with large pores. These porous scaffolds can be used as a substrate for cell

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growth and survival factors [38]. Moreover, Figure12e Shows osteoblast cells attachment and

mineralization on nanocomposite scaffolds.

Figure12. SEM images of (a), (b) nanocomposite 30% nHA, scaffold 30% nHA (c) before

cell culture with interconnectivity (d) before cell culture (e) after cell culture

It can be observed that osteoblast cells are well adhered and proliferated on the pores of

scaffolds. The osteoblast growth on the nanocomposite scaffolds was determined at three and

seven days after seeding using the MTT assay. According to Figure13, nanocomposite

scaffolds with different ratios of nano-hydroxyapatite showed considerably higher cell

growth as compared to the pure polymeric scaffolds at the same time periods. The cell growth

in 3 and 7 days has been increased from 195 to 332 and 410 to 663 respectively. Moreover,

after seven days, osteoblasts showed a slight growth rate and the difference between 30%

content and 20% is quite insignificant.

Figure13. MTT Assay of cultured cells on Nanocomposite scaffold

The in-vitro degradations of TB1, TB1/10%, TB1/20% and TB1/30% were examined under

accelerated (70oC) and normal temperature conditions. The specimens were immersed in a

phosphate buffer at pH= 7.4, which was stirred in order for the solution to become

homogeneous at 70°C under accelerated conditions. At normal test conditions, the test was

carried out in the same pH at 37°C, near to the human body temperature. The transparent

samples became opaque after nearly 20-25 hours in 70°C and six to nine weeks at 37°C

which is indicative of water diffusion into the bulk of the copolymers and the beginning of

hydrolytic degradation of amorphous regions as the crystalline regions resist against the water

diffusion. According to Figure14, degradation time is different for the samples with different

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compositions but as the degradation time increases, the weight loss values are increased;

however, the nanocomposites show lower degradation rate in comparison with TB1. The

copolymers hydrolytic degradation mechanism is bulk degradation and when the water

uptake is increased, the degradation rate accelerates. Nevertheless, with introduction of nHA

and increase in nHA content, the degradation rate decreases which could be ascribed to the

alkaline nature of solution that acts as a physical barrier causing a decrease in the rate of

degradation [43] and increase in the crystallinity since the hydroxyapatite could act as

nucleation agent. It has also been reported that thermal stability in the presence of nHA could

increase [55]. According to the accelerated degradation, a 1.5-2 year complete degradation

can be expected from the 30% nHA since it takes 210 hr to completely degrade in 70oC. The

in-vitro test has been taken in only pH condition and if the in-vivo was employed in the

process, the result would have shown a degradation lower than that of in-vitro test in the

presence of enzymes and blood contents.

Figure14. Normal and accelerated in vitro degradation of TB1 and its nanocomposites

5. Conclusion

Triblock copolymer and nanocomposites of L-Lactide and ε-CL were successfully

synthesized. Thermal properties of triblock copolymers were shown to be dependent on block

length of PLLA side blocks and P (LLA-co-CL) middle block. It was found that changing the

length of PLLA and P (LLA-co-CL) blocks could vary the thermal properties of triblock.

Prepared copolymers of L-lactide and CL are proper candidates for use as biodegradable

scaffolds for soft tissue engineering due to their soft amorphous P (LLA-co-CL) blocks. The

obtained copolymers can also be suitable for making hard tissue engineering scaffolds due to

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the existence of LLA crystalline segment in copolymers. According to the mechanical

properties, with introducing nHA into the polymer matrix, triblock behavior shifts form

ductile to brittle and with increase in the nHA content, stiffness and elastic modulus were

increased. Due to the homogeneous dispersion of nHA as well as good interfacial adhesion,

the prepared nanocomposites are suitable for fabrication of scaffolds. An excellent porosity

was obtained for the fabricated scaffold and nHA fillers could induce osteoconductivity and

osteoinductivity to the nanocomposite scaffolds as compared with the pure polymeric

scaffolds. Thus, it can be proposed that these nanocomposite scaffolds are suitable as bone

substitute for bone tissue engineering applications.

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Figure1. Polymerization reaction of P(LLA-co-CL) prepolymer.

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Figure2. Polymerization reaction of PLLA-P(LLA-co-CL)-PLLA triblock copolymer

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Figure3. FTIR spectra of nano-hydroxyapatite (nHA)

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Figure4. XRD pattern of nano-hydroxyapatite (nHA)

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Figure5. TEM image of nano-hydroxyapatite (nHA) needle-like crystals

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Figure6. FTIR spectra of prepolymer and triblock copolymers

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Figure7. 1HNMR spectra of resulting polymers (a) Prepolymer (b) TB1

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Figure8. 13

CNMR spectra of resulting polymers (a) Prepolymer (b) TB1

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Figure9. DSC thermogram for Prepolymer and Triblock Polymers

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Figure10. TGA and DTGA thermograms for Prepolymer and Triblock Polymers

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Figure11. Mechanical properties of nanocomposites with different contents of nHA

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Figure12. SEM images of (a), (b) nanocomposite 30% nHA, scaffold 30% nHA (c) before

cell culture with interconnectivity (d) before cell culture (e) after cell culture

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Figure13. MTT Assay of cultured cells on Nanocomposite scaffold

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Figure14. Normal and accelerated in vitro degradation of TB1 and its nanocomposites

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Table1. The characteristics and molecular weights of Prepolymer and triblock polymers

Copolymer LLA/CL

(molar ratio)a

LLA/CL

(molar ratio)b

Mw

(gr/mol)c

Mn

(gr/mol)c

PDI

c

Prepolymer 68/32 65.5:34.5 104000 46600 2.23

Copolymer LLA/Prepolymer

(molar ratio)a

LLA/Prepolymer

(molar ratio)b Mw

c Mn

c PDI

c

TB1 85:15 81.5:18.5 212300 136100 1.56

TB2 95:5 94:6 201700 131800 1.53

a Feed ratio

bProduct ratio extracted From

1HNMR.

c GPC Technique

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Table2. Tm،Xt،∆Hc and Width of half height crystallization peak (WHH) of Samples

Samples Prepolymer TB1 TB2

Tg(oC) 40 44 46

Tm (oC) 125.9 170.8 178.5

∆Hm(J/g) 14.63 50.42 55

WHH (oC) 13.86 25.78 28.36

Xt (%) 16.37 56.07 61.47

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Table3. The characteristics of the TGA curves in nitrogen atmosphere

Copolymer Prepolymer TB1 TB2

TI (oC)

238.22 233.88 238.51

Tf(oC) 428.44 390.83 370.74

Tmax(oC) 334.84 321.48 329.44

Char (%) 1.73 2.38 2.62

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Highlights

Design of triblock polymer/nano hydroxyapatite scaffolds for tissue engineering.

Study on morphological, mechanical and thermal properties of synthesized triblocks.

Synthesize and characterization of nano-hydroxyapatite by chemical precipitation.

Scaffold preparation using gas foaming/salt leaching method using minimum solvent.

Well growth and spread of osteoblast cells in synthesized biocompatible scaffolds.