Preparation and Characterization of Composite Blends Based on

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Preparation and Characterization of Composite Blends Based on Polylactic acid/Polycaprolactone and Silk Shiva Balali, Seyed Mohammad Davachi, Razi Sahraeian, Behzad Shiroud Heidari, Javad Seyfi, and Iman Hejazi Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.8b01254 • Publication Date (Web): 23 Oct 2018 Downloaded from http://pubs.acs.org on October 24, 2018

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Preparation and Characterization of Composite Blends Based on Polylactic acid/Polycaprolactone and Silk Shiva Balali a, Seyed Mohammad Davachi b,a,* , Razi Sahraeian c, Behzad Shiroud Heidari d,e,f, Javad Seyfi g, Iman Hejazi d a

Department of Chemical and Polymer Engineering, Faculty of Engineering, Central Tehran Branch, Islamic Azad University, Tehran, Iran. b Soft Tissue Engineering Research Center, Tissue Engineering and Regenerative Medicine Institute, Central Tehran Branch, Islamic Azad University, Tehran, Iran. c Composites Department, Faculty of Processing, Iran Polymer and Petrochemical Institute, P.O. Box 14975/112, Tehran, Iran. d Applied Science Nano Research Group, ASNARKA, Tehran, Iran. e Vascular Engineering Laboratory, Harry Perkins Institute of Medical Research, QEII Medical Centre, Nedlands and Centre for

Medical Research, The University of Western Australia, Perth, Australia. f School of Engineering, The University of Western Australia, Perth, Australia. g Department of Chemical Engineering, Shahrood Branch, Islamic Azad University, P.O. Box 36155-163, Shahrood, Iran. *Corresponding

Author. Email address: [email protected] (S.M. Davachi) Tel/Fax: +98 -21 – 44600056

Abstract Silk-reinforced polylactic acid/poly ɛ-caprolactone composites containing 1-7 wt% of silk fibers were fabricated through the melt-mixing method. The composites were then characterized by implementing Fourier transform infrared (FTIR), differential scanning calorimetry (DSC) and rheometry to investigate functional groups, thermal properties, rheological properties and intrinsic viscosities of each composite. The crystallinity of the composites was found to decrease upon addition of silk, while, both storage modulus (G’) and loss modulus (G’’) were increased which is an indication of interface bonding between the polymer and silk. The composite containing 5% silk fiber (PLACLS5) showed the optimum results. The composites’ morphological analysis was conducted by scanning electron micrograph coupled with energy dispersive X-ray (SEM-EDX) mapping to assess the fiber dispersion in the composite matrix. The contact angle measurements and in-vitro degradation performed to evaluate the hydrophilicity, free surface energy and hydrolytic degradation of the composites. The results implied that addition of higher contents of silk fiber could reduce the degradation duration of the composites, which is due to the high hydrophilicity of the fiber, uniform fiber dispersion within the matrix, the porous structure and consequently the hydrophilic behavior of the composites. These composites can be great alternatives for both soft and hard tissue engineering applications. Keywords: Polycaprolactone, Polylactic acid, Silk fiber, Biocomposites, Characterization. 1 ACS Paragon Plus Environment

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Introduction From the resource and environment viewpoints, biodegradable polymers have been at the focal point to address the predicament caused due to the massive use and disposal of petroleum-based plastics 1. The most widely studied biodegradable polymers are aliphatic polyesters (e.g. polylactic acid (PLA), poly ɛ-caprolactone (PCL), polyethylene oxide (PEO), poly(3hydroxybutyrate) (PHB), polyglycolic acid (PGA) and thermoplastic proteins 2. Being biodegradable and biocompatible, PLA is a thermoplastic with good mechanical properties as well. Within the recent years, synthesis, crystallization kinetics, morphology, mechanical properties, and biodegradability of PLA have been investigated in detail 1–5. According to the literature, substantial efforts have been made to alter the PLA’s properties via blending it with hydrophilic polymers, such as PCL or silk fiber. Being a ductile biodegradable polymer, PCL might be employed to modify the PLA behavior from rigid to ductile since the former has been chosen as a blending partner for the latter 2. However, to further improve some properties such as impact strength, PLA blends with more flexible polymers such as PCL have been developed 6. According to literature, PCL has been blended with PLA to achieve the improved mechanical properties 7–9, shape memory ability, thermal degradation 10 as well as biodegradability 11,12. Meanwhile, some additives and compatibilizers, such as starch 2, maleic anhydride 12, organoclay 13,

CaCO3 and SiO2 14 and natural fibers 8, have shown their dominant roles in providing

miscibility and desired properties for PLA/PCL composites. Among them, the culpability of the inorganic fillers in the polluting ecosystem from their production till end-use has been established. On contrary, the organic ones, like starch, are abundant, low-cost, renewable, and biocompostable making them desirable for usage 1. Silk fiber has been vastly consumed in manufacturing and engineering. Silk merges high tensile strength, toughness, and elasticity.

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Besides their stunning mechanical behavior, silk is relatively stable slow degrading (degradation temperature ≈ 265 ̊C) 15. Silk materials are promising candidates to be used for scaffolds in tissue regeneration. In vitro and in vivo experiments have indicated that silk proteins show a broad spectrum of benefits such as low degradation, wide pore-size distribution and elastic properties, low toxicity, the low/absence of immunoreactivity and the possibility to functionalize silk proteins with cell-adhesion domains 16. Removal of sericin from silkworm silk is necessary to prepare non-allergic and non-toxic silk based materials for medical and tissue engineering applications. A series of methods to extract and regenerate silk fibroin have been developed and several silk based materials such as silk porous scaffolds, silk films, hydrogels coatings and electrospun nanofibers have been processed from silk solutions 17. According to the Tsukada, the sericin will be degraded at 170-200 oC which will make the silk useful for medical and tissue engineering applications without the need of sericin removal in this temperature range

18.

However, if a porous scaffold is made of PLA, its mechanical properties would drop dramatically providing insufficient strength for the load-bearing applications. To counterbalance such issue, silk fiber sounds a good alternative as a reinforcement to improve the strength of PLA 19. Silk fiber has been payed many attention as a functional filler for PLA-based composites due to its unique benefits. The mechanical and thermal properties of the PLA/Silk biocomposites with regard to the length and weight content of silk fibers have been investigated by Cheung et al. The biocomposites were prepared by extrusion and injection molding method 20. Findings showed that the fiber length and weight content of silk fibers could be regarded as the key parameters which may greatly affect the hardness of the biocomposites. Moreover, the optimal values in terms of fiber length and weight content of 5 mm and 5 wt%, respectively, were reported to attain the maximum micro-hardness of the biocomposite. The electrospun PLA/silk


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fibroin-gelatin composite nanofibrous scaffolds were fabricated to evaluate the porosity, mechanical behavior and biocompatibilities 21. The scaffold made of PLA/silk fibroin-gelatin (50:50) showed a higher level of toughness and pliability and provided a proper environment for cell growth of mouse fibroblasts. Microcellular biodegradable PLA/Silk composite foams were also payed attention by researchers 22. In that study, silk fibroin loaded PLA composites were fabricated via solution casting method employing CH2Cl2 as the solvent. The composites and the neat PLA were then foamed by supercritical CO2. With respect to the PLA foam, the composite foams showed a decline in cell size and an enhancement in cell density at high silk content, which can be considered as the efficient scaffolds in tissue engineering. The biodegradation PLA\silk composites were also investigated as the potential materials for bone tissue engineering 19.

It was found that upon introduction of silk fiber, a higher stiffness and ductility and a faster

biodegradation of PLA were observed. Recently, the biodegradable crystalline silk nano-discs (CSNs) were used for the production of PLA/CSN nanocomposites via melt-extrusion 1. In that work, the effects of CSN on isothermal melt crystallization kinetics, spherulitic growth rates, morphology, and hydrolytic degradation of PLA were studied. Results showed that the degradation rate is decreased for PLA/CSN composites due to the hydrophobic character of CSN. Beside PLA/silk biocomposites, PCL-based composites containing silk fiber (or powder) have been also studied in some cases in recent years 23–25. However, the PLA/PCL composite containing silk fiber has not been studied yet. Both of PCL and PLA are relatively hydrophobic with the long degradation times. Hence, the polymers could be more hydrophilic for the shortterm degradation applications by using silk fibers. The main goal of this study is to prepare silkreinforced PLA/PCL composites (PLACLS) with various silk content in order to achieve a novel series of short-term biodegradable composites. Processing of the PLA, PCL and silk in 170oC



can somehow be a novel way to remove the sericin and make the composite useful for medical applications. In this regard, the PLACLS composites are characterized by different techniques including FTIR, DSC, SEM-EDX, rheometry, hardness, impact strength and contact angle measurement to understand the effect of silk fibers in the PLA/PCL blend. Moreover, the composites are subjected to an accelerated hydrolysis at elevated and normal temperatures under two different conditions to study the in-vitro degradation behavior of composites. Experimental Materials Poly L-lactic acid (PLLA) was synthesized from the purified L-lactide prepared by L-lactic acid 26,27.

The ring-opening polymerization was carried out for the dried L-lactide monomers by reactive

extrusion technique described elsewhere 28. The molecular weight (Mw) and polydispersity index (PDI) of the PLLA were 120,000 g.mol-1 and 1.78, respectively. Poly ɛ-caprolactone (PCL) with a molecular weight of 80,000 (Mn) was supplied by Sigma-Aldrich (Germany). Natural silkworm silk fibers (hereafter called ‘‘silk”) were procured from the local market and cut gently into short fiber fractions in the length of 5 mm which was the optimum size to make sure that the fiber was not stressed plastically during the fabrication process according to Cheung et al. 20. All the other chemical and solvents were the reagent grade from Merck and used as received. Polymer Processing Before the blending process, PLLA, PCL, and Silk were all dried at 80oC for 4 hours. PLLA hereafter called “PLA” was first melted in an internal mixer Rheomix Haake (USA) for 5 min, followed by the addition PCL and subsequent mixing with PLA for an extra 5 min. Eventually, silk was added at varying weight ratios (1-7%) for 10 min. Therefore, the overall mixing time was adjusted to be 20 min. According to previous studies the PLA and PCL ratio 70:30 with the


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optimum properties was used for the current work 2,6,10. The melt mixing was conducted at 170oC at a rotor speed of 60 rpm. The attained composites, as well as neat PLACL, were molded using a hot-press at 170°C by applying 14 bar pressure for 5 min

29,30.

Moreover, 1 g of PLA and PCL

were molded into disc-shaped samples using a hot-press at 170°C and 60°C respectively by applying 14 bar pressure for 5 min 29,30, while 0.5 g of silk fibers at room temperature was pressed for 5 min under 50 bar loading to prepare a flat sheet for contact angle measurement. The compositions of composites are listed in Table1. The general name of composites hereafter will be PLACLS composites. Table 1. Composition of PLACLS composites Silk Content Samples (wt.%) PLACL PLACLS1 1 PLACLS3 3 PLACLS5 5 PLACLS7 7

Characterization The infrared spectroscopy (FTIR-ATR) was carried out by a Bruker instrument (Bruker, Equinox 55LS 101 series, Germany) with the resolution of 4 cm−1 (averaging 50 scans). The differential scanning calorimetry (DSC) was performed using a Mettler-Toledo DSC1 Star System (Switzerland) equipped with a low-temperature accessory. The temperature scale was calibrated by the high-purity standards. The DSC measurements were performed at a heating rate of 10 °C/min, in a nitrogen atmosphere and at temperatures ranging from -100 to 270°C. The repeated heating scans were performed in order to verify the reproducibility of results. All the adjustments were carried out according to ASTM D3418. The degree of crystallinity (Xc) for samples was determined according to the following equation 29:



X c (%) 

H m  H c 100 wH m0

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(1)

Where ΔHm and ΔHc are the enthalpies of melting and cold crystallization, respectively. w and ∆𝐻0𝑚 are the weight fraction of poly(L-lactide) and melting enthalpy of 100% crystalline poly(Llactide) or PCL, respectively. The rheological behavior of composites were evaluated at 170°C via an Anton Paar Physica MCR501 (Graz, Austria) with a parallel-plate geometry according to ASTM D4440 under air atmosphere. The dynamic experiments were performed under oscillatory shear mode with dimensions of 25 mm (diameter) and 1 mm (gap). The frequency sweep measurements were carried out from 0.05 to 500 1/s. Prior to the frequency sweep measurement, the strain sweep at various frequencies was conducted to make sure that the applied strain did not exceed the limit of linear viscoelasticity. To ensure that there is no change in viscoelastic behavior n period of 1 hour, the time sweep test was also taken. The measurements of mechanical behavior of the composite blends were carried out by means of Elma Tensile tester (Iran) according to ASTM D638. The measurements were conducted at room temperature (25°C) and the cross-head speed was 5 mm/min. Charpy impact tests were carried out using a ZWICK 5102 impact testing machine (Germany) on the edgewise un-notched rectangular specimens (50×10×1.5 mm3) according to ASTM D256. The shore D hardness of each composite was measured by a ZWICK Materialprüfung testing machine according to ASTM2240. For all the mechanical properties, five measurements were considered for each sample and the average value was reported. To study the effect of silk on the wetting behavior of the composites, contact angle measurement was carried out at ambient conditions using the Sessile drop method on a Kruss-contact angle measurement system G10, Germany. The surface energy of the composites obtained according to ASTM D7490 with using water and diiodomethane. Measurements of each sample were conducted at least three


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times across the sample surface and after 10 seconds the angles were recorded. The surface free energy of every discrete component of samples were determined using Owens-Wendt by dispersive and polar surface energy (𝛾d and 𝛾p) with a set of test liquids on a solid surface, where s and l represent the solid and liquid surfaces, respectively, and ϴ is the contact angle of liquid droplet on the surface (Eq. 2 and 3). After obtaining γs for all the composites the differences based on silk contents can be compared. γs = γps + γds

(2)

γl(1 + cosθ) = 2 γds.γdl +2 γps.γpl

(3)

Knowledge of thermodynamic equilibrium was employed in the determination of the location of silk in the samples since the time of melt mixing was sufficiently long for the migration process to occur. The interfacial energy of every two components were obtained from their dispersive and polar parts of surface energies using harmonic-mean and geometric-mean equations reported in Eq. 4 and 5 where γ i is the surface energy of component i, γ12 is the interfacial energy of components 1 and 2, and d and p superscripts stand for dispersive and polar parts of surface free energy of the components, respectively 29,30. γd1γd2

γp1γp2

Harmonic Mean: γ12 = γ1 + γ2 ―4(γd + γd + γp + γp) 1

2

1

(4)

2

Geometric Mean: γ12 = γ1 + γ2 ―2( γd1γd2 + γp1γp2)

(5)

Finally, the wetting coefficient (ωa) has been used to predict the thermodynamic equilibrium distribution of silk in the polymer blend as shown in Eq. 6.

ωa =

γpolymer1/silk ― γpolymer2/silk

(6)

γpolymer1/polymer2

For the hydrolytic degradation, the sheet specimens (1×1×0.2 cm3) with the weight of 0.23 g were prepared by hot press. The specimens were incubated in 10 ml phosphate buffer solution (PBS) 8 ACS Paragon Plus Environment


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with pH 7.4 at 37 and 70oC while stirring. The specimens were periodically removed, dried, and weighed. The weight loss percentage was calculated with Eq. 7, where W0 and W are the initial and final weights of the samples, respectively. Weight loss percent =

𝑊0 ― 𝑊 W0

(7)

× 100

The SEM micrographs (FE-SEM Mira 3 Tescan, Czech) were taken from the surface and fractured surface of the composites broken in Liquid N2 at an accelerating voltage of 20 kV to observe the morphology of the composites. The fracture surface and upper surface of the composites was sputter coated with a 15nm layer of gold using K450X EMITECH (England). Results and Discussion FTIR investigation The FTIR spectra of the PLACL, silk, and PLACLS7, as representative of composites, are shown in Figure 1. The PLACL shows C=O stretching vibrations in ester groups related to the PLA and PCL at 1749 cm-1. The peak at 1082, 1099, 1132 and 1182 cm-1 belong to the vibrations of the CO-C bond in PLA and PCL. The peak at 1361 cm-1 is assigned to the methyl group of PLA, while the peak which appeared at 1452 cm-1 represents the CH2 group of PCL and CH bending in ester groups 28–30. A broad weak peak at 2942 cm-1 could be due to the possible OH groups bound to the ester groups and finally, the C-C peaks of PLACL can be observed at 867 cm-128,31. Silk shows three absorption peaks at 1619 cm-1 (amide I), 1513 cm-1 (amide II), 1234 cm-1 (amide III), which can be attributed to α-helix or random coil conformation, whereas the shoulder peak at 1441 cm-1 (amide II) and the peak at 1067 cm-1 (amide III) are attributed to β-sheet conformation

32.

PLACLS7 shows a shift in all the peaks due to the presence of silk which can be an indication of the conformation transition in silk structure during the process. According to Zhu et al., the 9 ACS Paragon Plus Environment

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intermolecular hydrogen bonds were formed by establishing interactions between the hydroxyl groups of amino acids on the silk chain and the carbonyl groups of the esters, resulting in the conformational transition of silk structure 33.

Figure 1. FTIR spectra of PLACL, PLACLS7, and Silk

The carbonyl group exhibits a peak at 1722 cm-1, while the peaks at 1099, 1132 and 1180 can be ascribed to the C-O-C bond of ester groups. The peaks at 1363 and 1457 cm-1 are attributed to the CH3 and CH2 groups of PLA and PCL respectively. All the characteristic peaks of PLA and PCL appeared in Figure 1 imply that the main structure of PLA and PCL has not been modified in the course of the blending process, while few peaks are added to the PLACLS7 and intensity of some peaks also increased. The peaks at 1292 and 1238 cm-1 can be assigned to amide III groups of the silk in α-helix and β-sheet conformation respectively. By zooming into the PLACLS7 a weak peak at 1441 cm-1 can be seen which belongs to amide II β-sheet conformation, while the 1722 cm-1 peak shows an increase in the intensity which definitely could be due to the conformity of amide I αhelix conformation. Therefore, the shift in the absorption value of this peak can be related to this 10 ACS Paragon Plus Environment


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coincidence. Usually, various amide bands appeared in the wavenumber range of 600-1700 cm-1, are typical bands of polypeptides and proteins allowing their conformational characterization 34. A broad weak peak at 2942 cm-1 in PLACL becomes stronger and more intensified in PLACLS7 which could be due to the higher OH groups of ester groups and also NH stretching vibrations of the amide groups. Aside from the unchanged structure of the PLACLS7 upon addition of the silk, it can be seen that the PCL has been physically dispersed in the PLA matrix to make a partially miscible blend and silk dispersed in the miscible blend. Thermal properties To assess the thermal properties and crystallization behavior of the PLACLS composites, DSC method was performed and the results are reported in Figure2 and Table2. As can be seen the PLACL and all the composite blends show two melting temperature (Tm) and glass transition temperature (Tg), that, clearly shows the successful blending of these polymers, however in PLACL, which is a 70:30 blend of PLA and PCL, an increase of Tg can be seen which according to the mixture law the Tg will be shifted to the PLA with higher Tg. Interestingly, the Tg of PLA could not be detected due to the overlapping with Tm of PCL. Therefore, one could expect that the Tg of the PLA in the blends would be increased by the addition of PCL, which can be attributed to the plasticizing effect of PCL for the PLA phase

29.

The introduction of silk up to 5% into the

PLACL matrix reduces the Tg value of PCL phase which could be ascribed to the restricted macromolecular movements, while the PLACL chain segments are free from restraints

35.

However, in higher contents this Tg increases, which could be due to the agglomeration of silk in the blend. It has been previously found that a high fiber content can increase the possibility of fiber agglomeration resulting in regions of stress concentration which results in a failure for both


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mechanical and thermal properties 36,37. In the current work nearly in all the obtained data in 7% of silk increasing behavior due to agglomeration is observed.

Figure 2. DSC thermograms of PLACLS composites

PCL has a higher crystallinity comparing to the PLA and blending these to polymers will definitely decrease the crystallinity of the PCL and consequently affects the melting temperature of the PCL by decreasing it in the blend, therefore, PLACL shows lower melting temperature comparing to neat PCL. Upon addition of the silk to PLACL blend the composites showing a decreasing trend up to 5% and then an increase was observed in both Tm of PLA and PCL, which the decreasing trend is related to the good dispersion and interface bonding of silk in the PLACL matrix, while as mentioned earlier in higher contents the agglomeration could result in an increase in the Tm value. The melting enthalpy (∆Hm) and crystallinity (Xt) of the PLACLS composites decrease upon addition of silk, for which there are two main reasons for such behavior. On one hand, silk disrupted the regularity of the chain structures, leading to the increased free spaces between the polymer chains. On the other, due to the interfacial bonding and physical hindrance of the silk, the molecular movement of the polymer matrix is restricted and the polymer chains cannot fully 12 ACS Paragon Plus Environment

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incorporate in the growth of crystalline lamella. Therefore, the decreasing trend for crystallinity was observed however in higher contents it increases due to the agglomeration. There are very small increasing peaks right before Tm of the PLA phase, that might be due to the recrystallization of PLA crystals having a lower degree of perfection into α crystals with a higher degree of perfection 29,39,40. In another word, the less perfect crystals may find sufficient time to be melted and reorganized into crystals with higher structural perfection which melt again at higher temperatures 35,41. Table 2. Thermal properties, of PLACLS composites Tg

Tg

Tm

Tm

Samples

(PCL)

(PLA)

(PCL)

(PLA)

PLA 28 PCL 38 PLACL PLACLS1 PLACLS3 PLACLS5 PLACLS7

(oC) -62.81 -58.99 -62.94 -63.29 -63.84 -58.52

(oC) 52.94 -

(oC) 65.92 58.57 58.07 57.92 56.57 57.54

(oC) 164.56 168.91 168.58 168.29 166.24 167.04

1 melting

Coincides with Tm of PCL

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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∆Hm

∆Hm

Xc1

Xc1

WHH

WHH

(PCL)

(PLA)

(PCL)

(PLA)

(PCL)

(PLA)

(J/g) 87.61 27.42 25.28 24.03 22.25 23.10

(J/g) 39.29 31.04 29.81 28.76 27.43 28.08

(%) 62.89 62.61 60.49 57.50 53.24 55.28

(%) 42.25 48.14 45.79 44.18 42.14 43.13

(oC) 17.07 17.54 17.89 18.14 18.51 17.99

(oC) 16.23 16.45 16.94 17.19 17.48 16.58

enthalpy of 100% crystalline PLA and PCL are 93 J/g 28 and 139.3 J/g 38, respectively.

The reduced melting temperature and crystallinity of PCL in the blends, regarding the neat PCL, are in accordance with the previous reports and could be related to the used procedure for removal of the thermal history. Based on this thermal history removing process, samples were heated up to 120 oC which causes the PCL phase to be melted. Therefore, due to the slow rate of crystallization in PCL, its chains did not find sufficient time to form ordered chains 29,42. On the contrary, increase in melting temperature and also crystallinity of PLA in the blends compared to the neat PLA is due to similar structure polyester with higher crystallinity since the PCL crystals cool faster and act as nucleation agents for PLA crystals 43. The results clearly state that silk has a plasticizing effect since it has decreased the crystallinity and thermal properties, which previously also reported by Zhao et al. 35. Plasticizers mainly decrease the nucleation points 44, therefore according to the 13 ACS Paragon Plus Environment

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width of the half-height crystallization peak (WHH) data, an increasing trend for WHH up to the addition of 5% silk can be observed. The silk which acts as a plasticizer deducts the rate of crystallinity, which is in accordance with other obtained data. Based on the thermal studies it is clear that there is a good dispersion and also reasonable interface bonding between PLACL matrix and mainly PLA as it acts as the main matrix especially in 5% of silk, since a composite with poor interface bonding and dispersion, usually dissipates more energy and inhomogeneous thermal properties comparing to good interface bonding 35,45. Rheological Properties The rheological response of the PLACLS composites is illustrated in Figure 3. The strain sweep tests were performed at different frequencies to ensure that the applied strain is in the linear viscoelastic region. This measurement was performed at 170oC in the strain range of 0.01–100% as shown in Figure 3a and 3b. The strain of 0.1% was chosen for the frequency sweep in which the modulus is linear with a strain in the PLACL and all the composites. Upon addition of silk, both storage modulus (G’) and loss modulus (G’’) is increased which is an indication of interface bonding between the polymer and silk. G’’ shows higher values comparing to G’ which represents the viscous nature of composites against the strain, similar to the neat PLACL blend. Both PLA and PCL have shown the same behavior as reported elsewhere 28,29.



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Figure 3. Amplitude sweep of PLACLS composites (a) G’ (b) G’’

The dynamic frequency sweep measurements were performed to study the dynamics of PLACLS composites, and the results are illustrated in Figure 4. According to Figure 4a,b upon addition of silk both G’ and G’’ show an increasing trend, which could be due to the higher interactions between the PLACL and silk restricting the movements of polymer chains, especially at lower frequencies. The PLACL blend has a typical terminal relaxation behavior (𝐺′~𝜔2,𝐺′′~𝜔) which comes from the nature of PLA and PCL

38,

and the incorporation of silk modifies this behavior (𝐺′~𝜔0,𝐺′′~𝜔)

mostly in lower frequencies 46,47. It`s noteworthy to mention that the plateau of both modulus in lower frequency especially in composites with 3% and more, which somehow shows an independence versus angular frequency in this zone is an indication of the formation of physical crosslink in these composites which creates a yield stress. The G’ plateau in lower frequencies also shows the solid-like behavior, while based on the results the fine dispersion of the silk in the PLACL matrix could be the direct result of the formation of a network

38,48,49.

The complex

viscosity of the samples (Figure 4c) also enhanced upon addition of silk, while the introduction of silk has changed the behavior of PLACL completely. As can be seen the neat PLACL, shows a relatively unchanged behavior versus frequency, but the addition of silk has increased the complex viscosity and while the silk content increases the slope of the complex viscosity also increases. 15 ACS Paragon Plus Environment

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The rise in complex viscosity could be attributed to the formation of a network-like microstructure, that reduces the mobility of polymer chains 38.

Figure 4. Frequency sweep of PLACLS composites (a) G’, (b) G’’, (c) complex viscosity, (d) G’, G’’ intersections

Figure 4d shows the intersection of G’ and G’’ in the frequency sweep test. Similar to the strain sweep test G’ shows lower values comparing to G’’ in all the frequencies and G’ and G’’ show no intersection, in PLACL blend, therefore, a non-viscoelastic behavior during the process of the PLACL is expected, however upon addition of silk the composites show viscoelastic behavior. PLACLS1, PLACLS3, PLACLS5, and PLACLS7 show an intersection at a frequency of 1.08,



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10.8, 158 and 419, respectively. This increase in the intersection clearly states that the silk is dispersed in the PLACL matrix and shows solid-like behavior. PLACLS3 shows similar storage and loss modulus in some frequency zones, which clearly shows a more appropriate viscoelastic behavior in this composite. With the addition of silk, the G’ values start to show higher values and in PLACLS7 the composite clearly shows a more elastic or solid-like behavior. based on the frequency sweep tests the fine and homogeneous dispersion of silk in PLACL matrix can be seen, that shows the formation of a network.

Figure 5. The modified cole-cole curve for PLACL and PLACLS composites

Figure 5 shows the modified Cole-Cole curves plotted to clarify the structural differences between the PLACL and the composite systems at 170oC. The Cole-Cole plot is a logarithmic plot of G’’ versus G’ and in this method, the effect of frequency is omitted

50,51.

The black line is the equi-

modulus line which shows the G’= G’’ and crossing this line changes the viscoelastic behavior. Similar to previously rheological observations the PLACL shows a complete viscous nature while the addition of the silk has changed the composite natures and by addition of the silk the composites become more solid. PLACL and PLACLS1 show higher G’’ with respect to G’ in lower 17 ACS Paragon Plus Environment

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Biomacromolecules

frequencies while this behavior changes in contents higher than 1% silk showing that these three composites have much longer relaxation times (τ). It needs to be mentioned that the PLACLS7 also could see lower relaxation time comparing to the PLACLS5 as the curve becomes mostly like a line, which may be attributed to the possible agglomeration. The elasticity of the mentioned composites increases upon addition of silk; therefore, the relaxation times also show an increasing trend. These composites show higher melt elasticity and longer relaxation times and it makes them good alternatives for soft tissue engineering applications and drug delivery systems. The relaxation spectra were attained for the samples via plotting the G' and G" data versus the angular frequency at the reference temperature. From the relaxation spectra, the zero shear viscosity (𝜂0 = ∑𝜆𝑖𝐺𝑖), ∑𝜆2𝑖 𝐺𝑖

plateau modulus (G0N = ∑𝐺𝑖), mean relaxation time (𝜆 = ∑𝜆 𝐺 ) and the entanglement density (𝜈𝑒 = 𝑖 𝑖

𝜌𝑎

𝑀 𝑒, 𝑀 𝑒 =

𝜌𝑅𝑇 𝐺0𝑁

) of the composites were calculated

52,53.

Table 3 shows the attained

parameters from the relaxation spectra. The entanglement density (𝜈𝑒) is defined as the number of entanglement junctions per unit volume. R, T, ρ and ρa denote the ideal gas constant, temperature, melt density and the amorphous density, respectively. ρ and ρa for PLLA are 1.248 and 1.290 g/cm3 31 and for PCL are 1.2 and 1.02 g/cm3 54, respectively. For the PLASCL blend, ρ and ρa are calculated based on the mixture law and are 1.2336 and 1.209 g/cm3, respectively. The zero-shear viscosity of composites is increased upon addition of silk which is due to the formation of the network caused by interfacial bonding of PLACL and silk. The plateau modulus and entanglement density of the composites were all enhanced up to the addition of 5% silk, which clearly is a sign of formation of the physical network, however, it shows a decrease in 7% silk. A higher zero viscosity and a decrease in plateau modulus and entanglement density in PLACLS7 clearly is a sign of silk agglomeration and somehow separation of the matrix and silk 55,56. The mean relaxation 18 ACS Paragon Plus Environment


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time, the ratio of the elastic to viscous response, also increases up to 5% silk and decreases in higher contents. Based on the previous claims the increase could be due to the formation of the physical network, while the reduction can be caused by the formation of the silk agglomeration and phase separation, which results in higher viscose response and lower relaxation time. Table 3. Viscoelastic parameters and in-vitro degradation of PLACLS composites

Samples

PLACL PLACLS1 PLACLS3 PLACLS5 PLACLS7

𝜼𝟎

𝐆𝟎𝐍

𝝀

𝝂𝒆

In-vitro Degradation at 37oC

In-vitro Degradation at 70oC

×103 (Pa.s)

×103 (Pa)

(s)

×10 (gr.mol/cc)

(Month)

(hour)

0.76 11.3 232 657 1910

2.11 3.15 303 387 364

3.83 7.8 15.9 16.3 12.9

0.05 0.08 7.5 9.6 8.2

20 16 13 11 9

288 264 216 192 168

Finally, the time sweep test, which can be seen in Figure 6, was done to make sure that there is no change in the overall properties of neat PLACLS and PLACLS7 loaded the highest amount of silk in 1h.

Figure 6. Time sweep and stability of G’, G’’ and complex viscosity over time under constant oscillation


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Biomacromolecules

A substantial increase in the rheological response of PLACLS7 comparing to PLACL is observed. Storage and loss modulus, as well as complex viscosity, show approximately linear behavior during the time. Therefore, one could conclude that the rheological response of the samples remains nearly unchanged during a one-hour period. As can be seen based on the rheology tests PLACLS5 shows the optimum rheological results, while these results are also in accordance with the thermal studies. In-vitro degradation of composites To investigate the in-vitro degradation, the PLACLS composites were subjected to an accelerated hydrolysis at elevated and normal temperatures under two different conditions 44,57. The samples were submerged in PBS at pH of 7.4 and temperature of 70oC under stirring within an accelerated process. Under normal test conditions, the measurement was conducted at the same pH at 37oC. Figure 7 shows the in-vitro degradation of PLACLS composites in both normal and accelerated conditions. It could be observed that as the silk content increases, the hydrolytic degradation becomes faster comparing to neat PLACL which is clearly due to the hydrophilic character of silk which absorbs water. PLA and PCL are both semi-crystalline polyesters and they have a degradation time of 18 and 24 months, respectively. Therefore, diffusion of PBS or any other water-based liquids into their bulk is very difficult

29,43,57.

The degradation of these polyesters

usually starts with swelling and hydration, then the ester bonds start to break and the soluble degradation products diffuse and finally the polymer disappears 58. In the accelerated test, nearly all the samples except PLACLS7 showed very small changes in their weight after 5 h at 70oC and their milky color remained unchanged, however, after just 10 h the samples with higher contents of silk started to become whiter and the change in the color was quite obvious. PLACL shows an initial decrease which is due to swelling of the PLA and PCL as reported in previous studies 29,43,57, 20 ACS Paragon Plus Environment


however after a period of time and nearly 72 h the blend started to degrade. The main breakage of ester bonds and diffusion of the PBS happened after 120 h which in comparison with neat PLA and PCL is much lower which could be due to the interface of the blend. PLACL showed 50% degradation after 168 h and completely degraded after 288 h. Upon addition of silk, the degradation rate increased as the silk reduces the crystallinity by acting as a plasticizer and the PBS diffused into the bulk of the composites and the interfaces of PLACL and silk. It needs to be mentioned that upon introduction of silk the swelling was not observed, which can be due to the interfacial bonding of the blend and the silk, while the hydrophilic nature of the silk could be the main cause for such behavior since the silk absorbs the PBS and facilitates the degradation process. PLACLS7 shows complete degradation after 168 h while the PLACLS1 completely degraded after 264h.

Figure 7. In-vitro degradation of PLACLS composites (a) Normal condition, (b) Accelerated condition

It appears that the incorporation of silk leads to a faster degradation of the amorphous part, allowing a reduction in the molecular entanglements and an enhancement in chain mobility leading to faster degradation of the PLACLS composites. Moreover, the silk induced porous structure to the matrix for a faster rate of water intake due to its strong hydrophilic character 19. According to Zhao et al., the surface of composites is rougher than that of the neat polymer prior to the degradation and the rough surfaces promote the degradation process. After degradation, silk 21 ACS Paragon Plus Environment

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Biomacromolecules

exposes on the surface of the composites due to the hydrolysis of PLACL. Therefore, the speed of degradation after initial degradation will increase more as the composites become cracked and more PLACL are hydrolyzed. In another words, the hydrolysis not only takes place on the surface but also at the interface between silk and PLACL matrix, which allow the PLACL chains in the composites to be more sensitive to the hydrolytic degradation. Upon addition of silk and good dispersion, the PBS diffusion happens much better. The incorporation of silk could accelerate the hydrolytic degradation of the composites and the interactions between silk and PLACL matrix might be decreased upon increasing the silk content. The weight loss of the composites is mainly due to hydrolyzed PLACL and released silk fiber 35. The same trend was observed at 37oC and with an addition of silk content, the composites exhibit a lower degradation time comparing to 20month degradation of neat PLACL. PLACLS1 shows a 16-month degradation time while the PLACLS7 with highest silk content shows a 9-month degradation time. The in-vitro degradation at 37oC and 70oC are summarized in Table 3. According to the in-vitro results, the composites could be the promising alternative for a wide range of medical applications. For instance, PLACLS1, PLACLS3 and specially PLACLS5 due to optimum overall properties can be applied to liver tissue engineering, since an adult human liver cell has a turnover time of 10 to 16 months 59,60.

These composites can also be used for bone tissue engineering since the time for regeneration

of these cells also is in the obtained range 29,30,57. Therefore, the shortened degradation rates benefit tissue engineering, especially for liver and bone tissue engineering. Mechanical Properties Stress-strain curves of neat PLA and PCL, PLACL blend and PLACLS composites are depicted in Figure 8 and the related data are reported in Table 4. PLA is a polymer with very high elastic modulus and a brittle structure as it shows a very low elongation at break (2.7%) and PCL shows



an elastic behavior with a very high elongation at break (228%) comparing to PLA. It is shown that by the addition of PCL into PLA, elongation at break of PLACL blend is increased. while a considerable decrease in the elastic modulus and tensile strength can be observed compared to neat PLA, therefore blending can modify the brittle structure of PLA. According to the data, tensile strength and elastic modulus enhanced upon addition of 5% silk, while the elongation at break shows a decreasing trend, most probably because of the strong interactions between silk and polymer matrix, which is emanated from a lessened free-volume and restricted molecular mobility of the polymer chains

41,61.

The qualitative observations infer that the presence of silk in the

polymer matrix resulted in the fragility of the composites and the elongation at break results further confirmed the observations, as the values of elongation decreased from 20 to 12.79% with an increase in silk content. In higher contents of silk (PLACLS7), elastic modulus and tensile strength both drastically decreased, which could be due to the agglomeration of silks, porous structure and lack of affinity between the polymer and silk phases 29.

Figure 8. Stress vs strain curves of PLACLS composites


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Biomacromolecules

The variations of impact strength and hardness with different contents of silk are shown in Table4. The PLACL neat blend exhibits a lower amount as compared with that of PLA whereas the addition of silk enhanced the impact strength of composites compared with the neat PLACL, which could be due to the network formation of polymer chains by microdrawing and good adhesion could easily distribute the energy, while the fracture performance in PLACLS composites is further substantial evidence for the strong interfacial adhesion 53,62. Table 4. Mechanical properties of PLLA/LATC30 composites Samples PLA PCL PLACL PLACLS1 PLACLS3 PLACLS5 PLACLS7

Elastic Modulus (MPa) 3770 ± 3.4 469 ± 15 2008 ± 9.6 2857 ± 10.2 4477 ± 11.1 5644 ± 9.5 3543 ± 13.4

Tensile Elongation Strength at Break (MPa) (%) 66.2 ± 0.5 2.7 ± 1.4 14.4 ± 0.2 228 ± 8.1 26.08 ± 1.2 20.15 ± 0.7 28.49 ± 0.7 17.88 ± 0.5 30.09 ± 0.5 16.76 ± 0.6 31.01 ± 0.4 15.21 ± 0.5 24.51 ± 1.6 12.79 ± 0.9

Hardness (Shore D) 84 ± 0.7 29.3 ± 2.3 63.7 ± 1.2 67.8 ± 2.2 73.7 ± 1.3 76 ± 1 77.6 ± 0.9

Charpy Impact Strength (KJ/m2) 40.95 ± 2.6 No break 15.36 ± 2.9 17.29 ± 3 26.79 ± 2.6 34.04 ± 2.5 46.04 ± 1.4

Standard Deviation (n=5)

The hardness Shore D of the samples also reported in Table 4. Addition of elastic PCL to PLA has decreased the PLACL hardness comparing to the neat PLA 29. As can be observed, upon addition of the silk, composites hardness shows an increasing trend comparing to neat PLACL, which definitely is due to the interaction of polymer and silk and in higher contents the differences of the hardness values are very low. According to the obtained mechanical properties, it could be seen that PLACLS5 exhibits excellent mechanical properties alongside a ductile behavior making it a suitable candidate for a wide range of applications such as bone tissue engineering. Surface behavior and morphological studies Surface behavior of the biocomposites are obtained using contact angle measurement with water and diiodomethane (DIM) which have different natures and the results are reported in Table 5. Contact angle changes depending on the surface roughness, surface heterogeneity, chemical nature 24 ACS Paragon Plus Environment


of polymer surfaces, surface energy type of the solvent that is dropped on the surface and the crystallinity of the polymer 63. As can be observed the contact angle of the composites against both type of solvents shows a decreasing trend which could be due to the porous structure caused by the silk and increase of the water intake due to silk`s strong hydrophilic ability 19, therefore the composites show a hydrophilic behavior and these observations previously proved by in-vitro degradation tests. The surface energy is obtained based on the contact angles which shows an increasing trend. This increase also can be ascribed to the incorporation of hydrophilic silk in the composites, the porous structure of these composites and even presence of the silk on the surface of the composites and with an increase in silk content, the surface energy rises. Table 5. Contact angles and calculated surface energies of sample components and couple surface energies and sample wetting coefficients

Sample PLA PCL Silk PLACL PLACLS1 PLACLS3 PLACLS5 PLACLS7 Component Couple PLA/Silk PLA/PCL PCL/Silk 𝜔𝑃𝐿𝐴𝐶𝐿𝑆 𝑎 1 2

Contact Angle ( o ) Water Diiodomethane 72.5 55.5 77 62 57.3 46.2 88.1 44.2 78.3 42.3 61.2 39.5 58.7 37.6 55.5 34.3 1 𝛾12 (mN/m) 2.92 0.25 4.06 -4.58

Surface Energy (mN/m) 𝛾 𝛾d 𝛾p 34.11 20.95 13.16 30.13 18.33 11.80 44.50 20.99 23.51 38.47 36.85 1.62 40.45 32.86 7.59 43.95 26.51 17.44 45.62 26.56 19.06 47.97 27.06 20.91 2 𝛾12 (mN/m) 1.49 0.12 2.09 -4.79

Calculated using the harmonic mean equation Calculated using the geometric mean equation

It is generally known that hydrophobicity is inversely proportional to surface energy 64. Surface energy determined using Eq. 2 and 3. 𝛾d for deionized water and diiodomethane was reported to be 22.1 and 44.1 mN/m, respectively, while, 𝛾p for the these solvents was determined 50.7 and 6.7 mN/m

29,65.

After obtaining the surface energies of each component, the interfacial energies of 25 ACS Paragon Plus Environment

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Biomacromolecules

component couples based on harmonic and geometric mean equations are calculated and summarized in Table 5. The wetting coefficient is commonly utilized to anticipate the thermodynamic stable phase of additives in polymer blends. Based on the definition of 𝜔𝑃𝐿𝐴𝐶𝐿𝑆 ( 𝑎 = 𝜔𝑃𝐿𝐴𝐶𝐿𝑆 𝑎

𝛾𝑃𝐿𝐴/𝑆𝑖𝑙𝑘 ― 𝛾𝑃𝐶𝐿/𝑆𝑖𝑙𝑘 𝛾𝑃𝐿𝐴/𝑃𝐶𝐿

), if ωa>1 then the silk can only be found in PCL phase; if ωa

Preparation and Characterization of Composite Blends Based on

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