Injectable photocrosslinkable nanocomposite based on poly(glycerol ...

Research Article Injectable photocrosslinkable nanocomposite based on poly(glycerol sebacate) fumarate and hydroxyapatite: development, biocompatibility and bone regeneration in a rat calvarial bone defect model

Pr

oo

f

Aim: An injectable, photocrosslinkable nanocomposite was prepared using a fumarate derivative of poly(glycerol sebacate) and nanohydroxyapatite. Materials & methods: Polymers with varying physical and mechanical properties were synthesized. Furthermore, nanocomposites were developed using a homogenization process by combining nanohydroxyapatite within poly(glycerol sebacate) matrix via photocrosslinking and evaluated both in vitro and in vivo. Results & discussion: The nanocomposites were injectable, highly bioactive and biocompatible. Addition of nanohydroxyapatite led to enhanced mechanical properties with an ultimate strength of 8 MPa. The optimized nanocomposite showed good in vitro cell attachment, proliferation and differentiation of rat bone marrow-derived MSCs. The in vivo evaluation in rat calvarial bone defect model showed significantly high alkaline phosphatase activity and bone regeneration. Conclusion: This injectable, biocompatible and bioactive in situ hardening composite graft was found to be suitable for load-bearing bone regeneration applications using minimally invasive surgery. Original submitted 17 April 2012; Revised submitted 26 October 2012

or

KEYWORDS: biomineralization n bone tissue engineering n injectable bone graft n nanocomposite n nanohydroxyapatite n photocrosslinking n poly(glycerol sebacate) fumarate

microstresses to the emergent tissue. Elastomers encompass such desirable properties because they can provide stability and structural integrity within a mechanically dynamic environment without irritation to the host tissues [1] . Some elastomers are already reported in the literature including poly(1,3-trimethylene carbonate) [2] , star poly(e-caprolactone-co-d,l-lactide) [3] , poly(diol citrate) [4] and poly(glycerol sebacate) (PGS) [5] . PGS is a biodegradable, inexpensive, biocompatible elastomer that has been explored for tissue engineering applications including myocardial [6] and vascular [7] applications, as tissue adhesive [8] , and as nerve guide material [9] . However, it has certain drawbacks. For example, curing of PGS during synthesis requires high temperature and vacuum conditions, which limits its processing only for simple scaffolds and makes in vivo crosslinking impossible, thereby making it unsuitable for minimally invasive applications; PGS has been reported to produce leachates with significant cellular toxicity due to nonreacted carboxylic acid groups or carboxylic acids produced by aqueous hydrolysis of the PGS ester groups [10] . To overcome such drawbacks, a need has been expressed for the use of alternative

doi:10.2217/NNM.12.192 © 2013 Future Medicine Ltd

Nanomedicine (Epub ahead of print)

A ut h

In recent times, ‘minimally invasive surgery’ has gained popularity for bone regeneration, where malleable and injectable composite grafts (both hardening and nonhardening type) have been explored to minimize patient trauma and speed up recovery. These grafts are flowable in the form of liquids, pastes or fine particulate materials dispersed in gels, can be delivered using a small incision and eliminate the need to shape the material to adjust to implantation site, especially for irregular-shaped bone defects. The challenge while choosing a suitable polymer for these applications is to have a balance between the desired material properties of the polymer (injectability, biocompatibility) and its long-term use at the implant site (e.g., mechanical strength and degradation rate). In the exploration of such polymers, efforts in the materials’ design focus on in situ conversion of an injectable material into a gel or solid scaffold by various triggers or mechanisms including redox initiation, ionic initiation, thermal initiation, photoinitiation or self-crosslinking. In addition, scaffolds employed in engineered tissue constructs should adjust to the mechanical dynamics of the surroundings and the developing tissues, and transmit

Santosh Bodakhe‡1, Shalini Verma‡1,2, Kalpna Garkhal1, Sanjaya K Samal1, Shyam S Sharma3 & Neeraj Kumar*1 Department of Pharmaceutics, National Institute of Pharmaceutical Education & Research (NIPER), Sector 67, S.A.S. Nagar, Punjab, India 160062 2 Department of Orthopedic Surgery, UConn Health Center, Farmington, CT, USA 3 Department of Pharmacology & Toxicology, National Institute of Pharmaceutical Education & Research (NIPER), Sector 67, S.A.S. Nagar, Punjab, India 160062 *Author for correspondence: [email protected] ‡ Authors contributed equally 1

part of

ISSN 1743-5889

Bodakhe, Verma, Garkhal, Samal, Sharma & Kumar

of fumarylation in the polymer to optimize the desirable properties of the proposed nanocomposite bone graft. The biocompatibility of optimized polymer was evaluated and degradation behavior was studied under both in vitro and in vivo conditions. A bioactive nanocomposite was developed by addition of nHAP into the PGS fumarate (PGSF) matrix. The fabrication, material properties and in vitro bioactivity, as well as biocompatibility of the nanocomposite were studied. Furthermore, developed nanocomposite was evaluated for in vitro cell attachment, proliferation and differentiation behavior of rat bone marrowderived MSCs and tested in vivo in a rat calvarial bone defect model.

Methods

oo

chemistry approaches to decrease the enzymatic hydrolysis rate of the ester bonds or use of alkaline fillers (composite bone graft approach), with an expectation that their alkalinity can counteract the acidity of the PGS leachates [11] , as is reported for various polymers [12,13] . In the present work, both of these approaches have been used to modify PGS polymer. An in situ photocrosslinkable injectable polymer was developed and used to fabricate a nanocomposite with nanohydroxyapatite (nHAP) to form an injectable hardening composite bone graft. The composite bone graft approach intends to utilize the desirable properties of all components for fabrication of an ideal matrix that can mimic the nature of native tissue. Bone is considered to be a nanocomposite of mineral and protein. Use of nanocomposites has emerged as an efficient strategy to upgrade the structural and functional properties of synthetic polymers, especially while applying them for bone tissue engineering applications [14] . Inorganic–organic composites, especially those based on nanomaterials, aiming to ‘mimic’ the nanocomposite nature of real bone, combine the toughness of a polymer phase with the compressive strength of an inorganic one to generate bioactive materials with improved mechanical properties and degradation profiles. Recent developments in biomineralization have already demonstrated that nanosized crystals and particles play an important role in formation of hard tissues. It is also established that nanosized and nanocrystalline calcium orthophosphates can mimic the dimensions of the constituent components of bone [15] . nHAP has been used in bone tissue engineering, either alone or in the form of nanocomposites, due to its excellent bioactivity, osteoconductivity, biocompatibility and osseointegrative nature [16] . nHAP of 20 nm or less in size have been found to exhibit favorable cell viability and proliferation of mesenchymal stem cells (MSCs) due to efficient translocation into the cells through the cell membrane into the cytoplasm in a nonreceptor-mediated fashion [17] . Current work focuses on the development and evaluation of a bioactive, biocompatible, injectable, nanocomposite bone graft that allows in situ hardening by photocrosslinking and is suitable for minimally invasive surgery for load-bearing bone regeneration applications. This is the first report on modification of PGS by fumarylation and its use as an injectable nanocomposite bone graft. The synthesis was carried out to obtain a varying degree

f

Research eview Authors Article

A ut h

or

Pr

Synthesis & characterization of PGS fumarate polymer The PGS prepolymer was synthesized by polycondensation using a reported method with slight modif ication [5] . Equimolar glycerol and sebacic acid were melted at 120°C under inert atmosphere for 1 h. Thereafter, the pressure was reduced from 1 Torr to 40 mTorr and the reaction was allowed to proceed for 6–8 h (Figure 1A) . For synthesizing photocrosslinkable PGSF, PGS was dissolved in anhydrous dichlomethane (1:2 volume by volume) and the solvent was distilled off to half of its previous volume so as to remove traces of water, if any. Potassium carbonate (K 2CO3) was added to the above polymer solution as an acid scavenger. Furthermore, fumaryl chloride (Merck, Germany) was dissolved in anhydrous dichloromethane and added, drop‑wise, into PGS solution, and refluxed at 50°C for 24 h (F igure 1B) . After 24 h, unreacted K 2CO3 and byproducts such as KCl were removed by centrifugation for 15 min at 5000 rpm to obtain the final polymer, which was dried on a rotary evaporator and stored at -20°C. The reaction was carried out by using different ratios of PGS:fumaryl chloride to obtain polymers with varying physical and mechanical properties. Polymer was analyzed using a nuclear magnetic resonance (NMR) spectrophotometer (Bruker, Fällanden, Switzerland), Fourier transform infrared spectroscopy (FTIR; Perkin Elmer, MA, USA), x-ray diffraction (XRD; Bruker D-8 Advance; 3–60°; step size: 0.010°; step time: 1 s at 25°C), differential scanning calorimetry (DSC) 821e (Mettler Toledo, Greifensee, Switzerland; temperature range: -60–130°C; heating rate: 10°C/min under

doi:10.2217/NNM.12.192


future science group

Injectable photocrosslinkable nanocomposite for bone regeneration

Research Article

O OH

O

OH Sebacic acid

Glycerol

1) 120°C, 1 h O O n 2) 120°C, OR O 40 mTorr, 6–8 h Poly(glycerol sebacate) R=H/polymer chain

O

O O

OR Poly(glycerol sebacate) K2CO3 50°C, 24 h

O O

O O

O

n

n

O CI

CI O Fumaryl chloride O O

O O

O O

Poly(glycerol sebacate) fumarate

f

OH + HO

oo

HO

On

Where W0 is initial weight and Wt is final weight of the specimen at time t (n = 6). Tensile properties of photocrosslinked p oly mer ic sp e c i men s were s t ud ie d by TA.XT2i texture ana ly zer (Stable Microsystems, UK) using a reported procedure [18,19] where polymer specimens were pulled at a rate of 0.5 Nmin-1 up to a maximum static force of 18 N (n = 5) at room temperature. The specimens were first soaked in 100% ethanol for 24 h and then subsequently soaked in phosphate-buffered saline for 24 h prior to mechanical testing. The crosslink density (n) was estimated based on the theory of rubber elasticity, using E quation 2 :

or

nitrogen purging [80 ml/min]), TGA/ SDTA851e (Mettler Toledo, temperature range 25–800°C, heating rate 10°C/min) and gel permeation chromatography (Shimadzu LC-10AT VP HPLC pump and Shimadzu SIL-10AD VP refractive index detector with Styragel® HR3 column [Waters, MA, USA]).

Pr

Figure 1. Reaction scheme for the synthesis of poly(glycerol sebacate) and poly(glycerol sebacate) fumarate. (A) poly(glycerol sebacate); (B) poly(glycerol sebacate) fumarate.

A ut h

Photocuration of synthesized PGSF polymer For prepa ration of photocrosslinked PGSF specimens, 150 µl of photoinitiator bisacylphosphinoxide (BAPO; 300 mg/1.5 ml of dichloromethane) was added to a solution containing Irgacure® 2959 (Sigma-Aldrich, USA; 15 mg) and PGSF (1.5 g) in dichloromethane and mixed thoroughly. The mixture was poured into a petri dish and photocrosslinked under UV light (l = 315–380 nm) for 15 min. After crosslinking, the obtained films were cut into small cubes to obtain uniform specimens of approximately (5 × 5 mm)/50 mg each for further studies. For mechanical testing, polymer specimens of 30 × 2 × 4 mm (length × breadth ×height) were prepared. Swelling behavior of crosslinked polymer specimens was studied by incubating preweighed specimens in distilled water at 37°C. After 24 h, the swollen weights of the specimens were determined by the Equation 1: % swelling = 100 # ; future science group

^Wt - W0 h

W0

E

(1)

n = E0 3RT

(2)

Where, E 0 is Young’s modulus (Pa); R is universal gas constant (8.3144 J/mol K); T is absolute temperature (K) [20] . To evaluate the degradation behavior of photocured PGSF_0.95 (PGSF_x refers to PGSF prepared using 1:x ratio of PGS:fumaryl chloride) specimens, preweighed samples (n = 4) were placed in 20 ml of PBS (pH = 7.4) at 37°C and the medium was changed after every 3 days during the study period (60 days). At predetermined time points, the specimens were taken out, washed, dried and weighed to determine the mass loss. In addition, the pH of the degradation medium was measured at each time point.

www.futuremedicine.com

doi:10.2217/NNM.12.192


Table 1. Composition and nomenclature of tested nanocomposites. Sample code

Composition

PGSF C5 C10 C15 C20 C25 C30

PGSF_0.95 alone PGSF_0.95 + 5% nHAP PGSF_0.95 + 10% nHAP PGSF_0.95 + 15% nHAP PGSF_0.95 + 20% nHAP PGSF_0.95 + 25% nHAP PGSF_0.95 + 30% nHAP

PGSF: Poly(glycerol sebacate) fumarate; nHAP: Nanohydroxyapatite.

Injectability evaluation of precrosslinked nanocomposite Injec t abi l it y of t he precros sl i n ked nanocomposite was evaluated using TA.XT2i texture analyzer, equipped with a 5-kg load cell. This load was suitable for evaluation of the injectable nanocomposites and was used to provide a constant force of 4.9 kgf at which all the sample showed complete injectability. In this way, injectability was maintained while keeping the highest possible mechanical strength in the tested samples. Briefly, uncrosslinked PGSF polymer/precrosslinked nanocomposites were filled in a syringe and fitted with a 16-gauge needle (1.6-mm internal diameter). The syringe piston was placed in contact with the material without any air retention and a constant force of 4.9 kgf (~48 N) was vertically applied on the top of the plunger for 2 min. The mass of the material before and after injection was measured and the percentage injectability was given by the ratio of ‘mass expelled from the syringe’ to ‘total mass before injecting’. In addition, the maximum propulsion required for injection was also recorded as force of injection [21,22] .

or

Pr

oo

Synthesis & characterization of nHAP The wet chemical nanoprecipitation method was used to synthesize nHAP, where cetyl trimethyl ammonium bromide was used for particle size regulation [16] . The synthesized nHAP was characterized by attenuated total reflectance (ATR)-FTIR (Spectrum 1 with a Perkin Elmer Synthesis Monitoring System ATR accessory; 60 scans; 650–4000 cm-1 region), energy dispersive x-ray spectroscopy (NORAN System SIX version 2.0 x-ray microanalyzer with Super Dry II detector, Thermo Fischer Scientific), XRD (Bruker D-8 Advance; 10–60°; step size: 0.020°; step time: 4 s at 25°C), zetapotential (Zetasizer nanoZS, Malvern, UK) and TGA (Mettler Toledo TGA/SDTA851e ; temperature range: 25–1000°C; heating rate: 10°C/min).

30 × 2 × 4 mm (length × breadth ×height) were prepared.

f


A ut h

Fabrication of injectable photocurable nanocomposite Initially, polymer solution was prepared by mixing 150 µl of BAPO (300 mg/1.5 ml of dichloromethane) into a solution containing Irgacure 2959 (15 mg) and PGSF (1.5 g) in dichlorometha ne. Furthermore, a preweighed quantity of nHAP was added into dichloromethane (1:5 ratio) and sonicated (60 A: 10 min) to obtain a uniform dispersion (Table 1) . nHAP dispersion was then added to the polymer solution and homogenized at 5000 rpm for 15 min. The solvent was then rotary evaporated to obtain a precrosslinked, injectable nanocomposite. For preparing crosslinked nanocomposites, the precosslinked nanocomposite was poured in a petri dish and photocrosslinked under UV light (l = 315–380 nm) for 15 min using UV cabinet (Spectroline Model CM-10, Spectrolines corporation, NY, USA; tube light details: ModelENF-260/FE, 230 V, 50 Hz, 0.17 A). After crosslinking, the obtained films were cut into small cubes to get uniform specimens of approximately (5 × 5 mm)/50 mg each for further studies. For mechanical testing, different specimens of

doi:10.2217/NNM.12.192


Characterization & evaluation of photocured nanocomposites The chemical nature and molecular bond structure of samples were determined using ATR-FTIR in the range of 650–4000 cm-1 over 60 scans. The crystalline phase of samples was determined using XRD (Bruker D-8 Advance; 3-60°; step size: 0.010°; step time: 1 s at 25°C). Phase identification was achieved by comparing diffraction patterns with the International Centre for Diffraction Data (hydroxyapatite standard; JCPDS PDF card number 09-0432). Swelling and tensile properties of photocured nanocomposite specimens were studied by the procedure described in the section ‘Photocuration of synthesized PGSF polymer’. For determining the pH change in culture medium, photocured nanocomposite specimens (n = 4) were sterilized in ethanol and washed with sterile PBS. Specimens were soaked in 4 ml Dulbecco’s Modified Eagle Medium tissue culture medium and then placed in an incubator at 37°C in 5% CO2. The pH of the cell culture medium was measured at predetermined time points for 10 days and compared with control (medium alone). Culture medium was changed every third day to simulate standard tissue culture future science group


f

oo

Evaluation of optimized nanocomposite for bone formation using a rat calvarial bone defect model Wistar rats (200–250 g) were used in this study and were maintained in a temperaturecontrolled environment with ad libitum access to water and a standard laboratory pellet diet. Each animal was premedicated with an intraperitoneal injection of 0.3 mg/kg atropine and anesthetized with a combination of ketamine (80 mg/kg) and xylazine (10 mg/kg) administered intraperitoneally. After induction of anesthesia, the animal was fixed in stereotaxic apparatus and the scalp was infiltrated with 0.5% lidocaine with adrenaline (1:200,000) for local hemostasis. The incision sites and surrounding areas were shaved and disinfected with povidone iodine. An anterior–posterior, 2-cm long midline incision was made through the skin and muscle down to cranial vertex. After elevation of full-thickness skin–muscle–periosteal flaps, two full-thickness 5-mm parietal lesions were created using a slow-speed dental drill (Marathon‑3, Saeyang Company, Taegu, South Korea) attached with circular 5 mm trephine in calvarium under constant irrigation with sterile saline. This resulted in critical size defects that give rise to a fibrous nonunion when bone loss is not replaced. Attention was paid not to perforate the underlying dura mater and not to involve the sagittal suture. The defects were then loosely packed with marketed formulation (Artosal® granules [Aap Biomaterials Gmbh & Co KG, Germany], a synthetic bone graft composed of 60% hydroxyapatite and 40% tricalcium phosphate) or precrosslinked C30 nanocomposite, which was exposed to UV light (l = 315–380 nm) for 5 min to initiate

A ut h

or

Biocompatibility & mineralization of bone marrow-derived MSCs Animal selection and management, surgical protocols, and experimental procedures of biological evaluation were approved by the Institutional Animal Ethics Committee and the National Institute of Pharmaceutical Education and Research (SAS Nagar, India). MSCs isolated from rat bone marrow were used to evaluate the biocompatibility of crosslinked PGSF_0.95 and C30 specimens. MSCs were isolated by asceptically excising femurs and tibias of recently euthanized wistar rats (3000 cm-1

C=C stretch stretch C=C 1645 cm -1 1645 cm -1

PGSF PGSF C=C C=Ccis cisforform -1 700 700cm cm-1

C-PGSF C-PGSF

4000

3600 3200 2800 2400 2000 1800 1600 1400 cm-1

1200 1000

800

600 450

Figure 2. Spectra of poly(glycerol sebacate), poly(glycerol sebacate) fumarate and crosslinked poly(glycerol sebacate) fumarate. (A) 1H nuclear magnetic resonance spectra of PGS and PGSF; (B) FTIR spectra of PGS, PGSF and C-PGSF. PGS: Poly(glycerol sebacate); PGSF: Poly(glycerol sebacate) fumarate; C-PGSF: Crosslinked poly(glycerol sebacate) fumarate.


www.futuremedicine.com

doi:10.2217/NNM.12.192


was observed at >3000 cm-1. The peak at 700 cm-1 confirmed the presence of a fumarate group in cis configuration. A Decrease in intensity of OH stretching was observed since fumaryl moiety is substituted on free -OH group of PGS, which was confirmed by the disappearance of C-OH peaks of PGS. A relatively strong absorption band at 1160 cm-1 was due to asymmetric coupled vibrations of C-C(=O)-O and O-C-C groups in PGSF. The XRD spectrum of both PGS and PGSF showed broad halo patterns, indicating the amorphous nature of the synthesized polymers. An increase in the molecular weights of the obtained PGSF_0.8, PGSF_0.9 and PGSF_0.95 polymers is depicted in Table 2. Glass transition temperature of PGS and PGSF were observed at -35 and -47°C, respectively, while melting temperature of PGSF was observed at -7°C, which indicates a stable liquid state with a glassy nature at room temperature.

oo

by the appearance of peaks corresponding to -CH=CH- at d 6.8 ppm. 1H NMR data show that fumaryl chloride reacts preferentially with hydroxyl groups of glycerol compared with carboxylate groups of sebacic acid. This was confirmed by the increase of signal integral at d 5.2 ppm, corresponding to the protons from trisubstituted glycerol and the decrease of the signal integral at d 3.7 ppm, corresponding to protons from monosubstituted glycerol. Since a chemical shift of -CH=CH- protons of fumarate is below d 7.0 ppm, the fumarate group in copolymer is in cis-configuration, which is further supported by FTIR data. The FTIR spectrum of the PGS prepolymer indicated the presence of an ester linkage with ester carbonyl peak at 1735 cm-1, and characteristic absorption of C-O at 1160 cm-1 (Figure 2B) . Broad band at 3445 cm-1 was due to bonding OH stretch vibration. Peaks at 2931, 2856 and 1457 cm-1 corresponded to methyl group. Inclusion of fumaryl moiety was confirmed in FTIR spectrum of PGSF by emergence of alkene (C=C) stretching at 1645 cm-1. In addition, C-H stretch of sp2 carbon

f


Pr

Photocuration of PGSF In this study, a free radical initiator Irgacure 2959 was used which is safe and less toxic in 1.2

5

1

Stress (MPa)

2

or

3

A ut h

Swelling (%)

4

PGSF_0.8

PGSF_0.9

0.6 0.4 0.2

1 0

0.8

0

4

6

8

-0.2

PGSF_0.95

12

7

16

PGSF_0.8 PGSF_0.9 PGSF_0.95

6

10

5

8

4

6

3

4

2

2 0

14

pH

Average weight loss (%)

8

12

Time (s)

Sample code

14

10

1 0

10

40 20 30 Time (days)

50

60

0

Weight loss (%) pH

Figure 3. The effects of degree of fumarylation of synthesized poly(glycerol sebacate) fumarate polymers. Effect on (A) swelling behavior, (B) mechanical properties and (C) degradation studies of PGSF_ 0.95 polymer showing percentage average weight loss and change in medium pH with time. PGSF: Poly(glycerol sebacate) fumarate.

doi:10.2217/NNM.12.192




Pr

oo

f

fumarylation: 27%) and 1 MPa (PGSF_0.95; degree of fumarylation: 44%) (F igur e 3B) . Both fumarylation, as well as acrylation, have been reported in the literature to obtain photocrosslinkable polymers. In an earlier report on acrylate derivative of PGS, UTS was reported to be 0.36 and 0.5 MPa at 41 and 54% degree of acrylation, respectively [26] . Although acrylate groups undergo a faster reaction in comparison with fumarate groups due to the difference in their molecular weights, our results show that higher mechanical strength could be obtained in the presented fumarate derivate compared with the previously reported acrylate derivative at a given degree of fumarylation or acrylation, respectively. The better mechanical properties of PGSF might be due to the use of K 2CO3 instead of triethyamine; however, the current study does not aim to compare different methods for obtaining photocrosslinkable polymers. Strain to failure of the PGSF polymers ranged from 80 to 37.3% with an increasing degree of fumarylation from 11 to 44% (Table 2) . On the basis of these results, PGSF_0.95 was chosen for fabrication of nanocomposites since it provided maximum crosslinking density and high mechanical strength while maintaining injectability. In vitro degradation of PGSF_0.95 showed 12.15% weight loss after 60 days with a change of pH from 7.4 to 5.9 (Figure 3C) due to release of polymer degradation products such as sebacic acid, fumaric acid and glycerol into the degradation medium.

A ut h

or

comparison with other Irgacure derivatives [25] . At the same time, Irgacure 2959 is less reactive; hence it was combined with BAPO, which helped in improving the efficiency of cross-linking and resulted in scaffolds with desirable mechanical properties. Photocuration of PGSF initiates crosslinking through the fumarate unit and results in the formation of a new bond between polymer chains leading to disappearance of double bond in the crosslinked PGSF. This was confirmed by FTIR spectra that clearly showed a reduction in intensity of peaks specific to the C=C group of fumarate moiety (Figure 2B) . No change was observed in the XRD pattern of the polymer postcrosslinking and the crosslinked PGSF specimen showed a halo pattern, indicating its noncrystalline nature. At the same time, the DSC thermogram also suggests a noncrystalline glassy state of the polymer. The solubility of a PGSF polymer was significantly reduced postcrosslinking. The swelling properties of cross-linked PGSF specimens were studied in an aqueous environment at 37°C. At a low degree of swelling in water, polymer can maintain its mechanical properties once implanted. A higher swelling of polymer will result in proper coverage of defects uniformly, however, it leads to loss of mechanical strength; thus, an optimum swelling is required in filling non uniform defects so that it can provide a better defect coverage along with required mechanical strength, especially in case of minimally invasive surgery applications. The swelling ratio was reduced from 4.2 to 1.42 on increasing fumarylation from 11 (PGSF_0.8) to 44% (PGSF_0.95), however, crosslinking density increased from 67.26 to 134.55 mol/m3 (Figure 3A) . These results clearly indicate that degree of fumarylation dictates degree of crosslinking and is inversely proportional to swelling ratio of crosslinked PGSF specimens. Introduction of fumarate groups into the prepolymer facilitated an additional level of control so as to obtain a malleable, injectable polymer that could be crosslinked in situ to obtain a polymer with desirable characteristics; for example, high mechanical strength required for bone regeneration applications. Ultimate tensile strength (UTS) of the photocured PGSF was found to be linear and proportional to the degree of fumarylation and no deformation was observed after mechanical testing. Mechanical properties of photocured PGSF spanned from soft to relatively stiff where UTS increased from 0.5 MPa (PGSF_0.8; degree of fumarylation: 11%) to 0.78 MPa (PGSF_0.9; degree of

Research Article


Characterization & evaluation of nanocomposites Nanocomposite was fabricated by sonication of nHAP suspension in PGSF solution followed by homogenization. The homogenized suspension was a yellowish pasty material that was further processed under UV light for crosslinking to obtain the nanocomposite. XRD spectra of PGSF showed a halo pattern and nHAP showed a pure semicrystalline phase consistent with the diffraction pattern of standard HAP (JCPDS09–0432) with peak maxima at 25.9, 29, 30, 31.7, 32.3, 32.9, 40, 47 and 10.98° (Figure 4A) . These peaks could be attributed to miller indices 002 (c-axis), 210, 211, 102, 112, 300, 310, 222 and 100, respectively, indicating various planes of nHAP crystal lattice. Broad patterns around 002 and 211 planes also indicated that the crystallites were tiny in nature with much atomic oscillation. Crystallite size was calculated to be approximately 23 nm with the degree of crystallinity being 30.6%. The zeta-potential of the synthesized www.futuremedicine.com

doi:10.2217/NNM.12.192



PGSF_0.95 PGSF_0.95 nHAP nHAP C30 C30

T (%)

Lin (counts)

C10 C10

C20 C20

C20 C30 C30 nHAP nHAP

C10 C10 PGSF_0.95 PGSF_0.95

B

2-theta scale

cm-1

6 4 2 0

C10 C15 C20 C25 C30 Sample code

6

80

4

60

3.0

20 0 0 PGSF C5 C10 C15 C20 C25 C30 Sample code

7.0

2.5

pH

2.0

or

1.5 1.0

6.5

PGSF C10 C20 C30

6.0

0.5 0.0

C10 C20 Sample code

A ut h

PGSF

Lin (counts)

i

C30

*

*

5.5

1

5 Time (days)

10

ii

*

T (%)

*

G1

40

2

7.5

3.5 Swelling at 24 h (%)

8

120 100

Pr

PGSF C5

140

Ultimate strength Strain at break

Strain at break (%)

8

10

oo

Force (N)

10

f

Ultimate strength (MPa)

12

* *

*

*

*

2-theta scale

*

G2 cm-1

Figure 4. Nanocomposite characterization studies. (A) XRD spectra overlay, (B) FTIR spectra overlay, (C) injectability properties, (D) mechanical properties, (E) swelling characteristics, (F) pH change of culture medium in fabricated nanocomposites, (G)(i) XRD and (G)(ii) FTIR spectrum of mineral phase deposited on C30 nanocomposite surface after 14 days of incubation in simulated body fluid.

nHAP particles dispersed in water (0.1% w/v) was -3.8 mV. With increasing content of nHAP in nanocomposites, an increase in the intensity of peaks corresponding to semicrystalline nHAP doi:10.2217/NNM.12.192


was observed, whereas the intensity of broad halo pattern attributed to PGSF was reduced. This was further confirmed by FTIR spectra that showed an emergence in characteristic peaks of future science group


nHAP as the content of nHAP increased within nanocomposites from C10 to C30 (Figure 4B) . For a nanocomposite that can be photocured after injection at implant site, injectability is an important criterion, particularly for minimally invasive surgery applications.

The precrosslinked nanocomposites showed 100 % injectability at all tested nHAP concentrations, however an increase of nHAP content in the nanocomposite led to a linear and significant increase in overall force of injection (Figure 4C) . This indicated a uniform

1.2

* **

Control PGSF_0.95 C30

0.8

‡ ‡‡ ¶

† ††

§

0.6 0.4

f

Absorbance (OD)

1

Research Article

0 1

3

7

Time (days)

A ut h

or

Control

2500x

Pr

500x

oo

0.2

PGSF _0.95

C30

Figure 5. In vitro biocompatibility evaluation of control, poly(glycerol sebacate) fumarate_0.95- and C30-coated specimens using mesenchymal stem cells derived from rat bone marrow. (A) MTT assay; (B) morphological evaluation using scanning electron microscopy (day 7); (C) matrix mineralization (Ca deposition) in osteogenic medium using alizarin red staining (day 7; 10× magnification). Values represent mean ± standard deviation, n = 4. **p