SCAFFOLDFOAMS BASED ON LOW MOLECULAR WEIGHT POLY(ε-CAPROLACTONE) OPENING A NEW DIMENSION

IN A TISSUE ENGINEERING

Amanda de C. Pereira, Daniela Sachs, Demétrio A. W. Soares, Élcio R. Barrak,

Álvaro A. A. de Queiroz

Centro de Estudos e Inovação em Materiais Biofuncionais Avançados, Universidade Federal de Itajubá,

Itajubá (MG), Brasil

E-mail:

Resumo.Synthetics polymers are being developed widely for tissue engineering use and these materials offer advantages with regard to avoid pathogenic contaminants and to be reproducibility in bigger scales. Poly(ε-caprolactone)(PCL)anditscopolymershavereceivedanincreasedattentionforapplicationsinregenerativemedicine. Our research group has developed a low molecular weight poly(ε-caprolactone)(LMWPCL) and we have blended it with PCL.Polymers biodegradation rate, pore structure and mechanical properties, such as crystallinity, play important roles for efficient clinical applications of the tissue scaffolds in medicine. In this context, this study aimed to evaluate the effect of molecular weight on pore size and distribution and crystallinity structure. So, we have modified PCL through blending with LMWPCL. The infrared analysis showed characteristics bands present in PCL and LMWPCL, and no extra peak or shift in band frequencies were observed in PCL/LMWPCL blends. For PCL/LMWPCL blends the crystallinity of LMWPCL component decreases as a function of increasing PCL concentration. By scanning electron microscopy examination of PCL/LMWPCL blends were observed characteristics microporous. TheexperimentalresultsofporositymercuryshowthathigherconcentrationsofLMWPCL produce higherporosityofthescaffold.Thedevelopment of three-dimensional scaffolds for tissue engineering needs to produce porous materialsthat allow cell attachment in its surface and then proliferation and differentiation of the cells. Like this,PCL/LMWPCL blends are able to produce scaffolds to be used in bone tissue engineering.

Key-words:Tissue engineering, Poly(ε-caprolactone), Scaffolds, Porosity, Cristallinity.

1.INTRODUCTION

Poly(ε-caprolactone)(PCL)anditscopolymershavereceivedanincreasedattentionforapplications as scaffolds in tissue engineering [Ng et al., 2001; Williamson et al., 2006; Savarino et al., 2007; Izquierdo et al., 2008]. PCL (the compound from which monocryl suture and the capronor is made) is FDA approved, and has a long history of safe use in humans [Pitt et al., 1980; Ory et al., 1983; Darney et al., 1989; Bezwada et al., 1995]. PCL is a biodegradable, biocompatible and semicrystalline aliphatic polymer having a very low glass transition temperature (-60ºC), slower degradation rate, melting temperature at about 58ºC - 60ºC and a high thermal stability. This has led to its application in the preparation of scaffolds for tissue engineering [Li et al., 2003; Williams et al., 2005].

Today it is well known that the biodegradation rate, pore structure and mechanical properties including polymer crystallinity and material strength play important roles for efficient clinical applications of the tissue scaffolds in medicine. An appropriate biodegradation rate is required to ensure the strength of the tissue scaffold is retained until the newly grown tissue has taken over the synthetic support [Engelberg et al., 1991; Ali et al., 1993]. The porosity and the pore structure of tissue scaffold can allow limited tissue ingrowth for tissue regeneration within or over a biological scaffold or matrix [Scott et al, 2005; Jones et al., 2006].

Recently,ourresearchergrouphasdevelopedauniquebioresorbablepolymerbasedinlowmolecularweightpoly(ε-caprolactone)(LMWPCL) [De Queiroz et al., 2002].ThekeyadvantagesofLMWPCLincludetheabilitytoallowthenaturalosteogenesisinbonedefectsandaperfectintegrationwithnativebone.Atsametime,theLMWPCLarealsoattractivebecausetheycanbeusedtofabricatescaffoldsintovariousshapeswithdesiredporemorphologicfeaturesconducivetotissuein-growth allowing use in tissue engineering.

Modification of PCL through blending with LMWPCL provides a potentially and relatively inexpensive and simple route to change and regulate properties of the biodegradable polymer offering opportunities to obtain cost-competitive scaffolds for tissue engineering modulating their specific property-ability to biodegrade.

The goal of this work is to study the effect of molecular weight of PCL on pore size and distribution and crystalline structure. Understanding the link between molecular weight of poly(-caprolactone) and porous formation will allow for better control over the properties of scaffolds based on PCL for tissue engineering.

2.MATERIALS AND METHODS

Polymers and blends preparation

LMWPCL wassynthesizedbyring-openingpolymerization ofε-caprolactonemonomercatalyzedbyiodinechargetransfercomplexasdescribedinourpreviouswork[De Queiroz et al., 2002].Reactiontimesvariedbetween10and24h.Themolecularweight of the LMWPCL evaluated by gel permeation chromatography (GPC) was 20,000g/mol.

TopreparetheLMWPCLscaffold,theaccuratelyweighedpolymerwasaddedintoaflaskand chloroform was added as solvent.Poly(-caprolactone) (PCL) of 80,000 g/mol molecular weight purchased from Sigma-Aldrich was used as received. PCL/LMWPCL blends were prepared using the phase separation process after a blend of the 0/100, 5/95, 10/90, 15/85, 20/80, 50/50, 80/100 and 100/0 weight percent PCL and LMWPCL, respectively were taken. Sufficient time was given for the polymers to dissolve (4h) in chloroform at room temperature (25 ºC) under constant magnetic stirring. Then the polymer solutions were cast uniformly onto Petri dish at room temperature (25 ºC) followed by annealing in an oven at 50 °C.

Fourier transform Infrared (FTIR)

The Fourier transform infrared-attenuated total reflectance spectroscopy (FTIR-ATR) spectra were recorded at room temperature (25 ºC) in a Perkin-Elmer Spectrum One Spectrometer in the 4000-600 cm-1 range.Each samples’ spectrum was recorded with a total of 16 scans.

Thermal analysis

The thermal behavior of PCL/LMWPCL blends was investigated using a differential scanning calorimeter (DSC 60, Shimadzu). Samples (3-5 mg) were sealed in aluminum pans and program-heated from room temperature (25 ºC) to 180 ºC at a rate of 10 ºC/min underaninert(N2)atmosphere andheldfor10mintodestroyanyresidualnucleibeforecoolingat10oC/min. The percentage crystallinity was estimated using a value of 139.5 J/g for the heat of fusion of fully crystalline PCL [Guo et al., 2001].

Mercury intrusion porosimetry

Mercury Intrusion Porosimetry (MIP) was performed using a Quantachrome Poremaster 33 (Quantachrome Instruments, Florida, USA). After driving off any moisture by vacuum over the PCL/LMWPCL membranes at 50°C for 4hours, mercury (Hg) was incrementally forced through the porous constructs using between 4–10MPa of pressure. Upon each incremental rise in pressure an equilibrium time of 10 seconds was implemented prior to taking readings of mean pore diameter, incremental and cumulative pore volume (mL/g) and pore area (m2/g) [Thompson et al, 1987].

X-ray diffraction (XRD)

ThecrystallinestructureofthescaffoldswasinvestigatedbyXRDanalysis.The XRD diffractometer used in this study was a Shimadzu model XRD 6000. Analyses were performed with monochromatic radiation of Cu-Ka (1.5406 Å) at a voltage of 40 kV and 40 mA, scan synchronized with the step of 0.05° in the range 2of 20-80° and 5 min exposure to 1 and 0.3° for the incident slit, and programmable divergent, respectively. The samples in powder form were deposited on a glass substrate.

Scanning electron microscopy (SEM)

The microstructures of PCL/LMWPCL blends were investigated using SEM images. The blends were sputtered with gold using Sputer Ion Coater (IC-50 Shimadzu) under a current of 8 mA under vacuum, to make them conductive material. Scanning was done using Shimadzu SS550 electronmicroscope.

3.RESULTS AND DISCUSSION

FTIR spectroscopy was used to investigate the PCL. The FTIR spectra of PCL/LMWPCL blends are shown in Fig. 1. The characteristic FTIR peaks of PCL and LMWPCL are observed at 2949 cm-1 (asymmetric CH2 stretching), 2865 cm-1 (symmetric CH2 stretching), 1727 cm-1(carbonyl stretching), 1293 cm-1 (C-O and C-C stretching in the crystalline phase), 1240 cm-1 (asymmetric COC stretching), 1190 cm-1 (OC-O stretching), 1170 cm-1 (symmetric COC stretching) and 1157 cm-1 (C-O and C-C stretching in the amorphous phase [Wang et al., 2002]. The PCL and LMWPCL FTIR absorption bands are observed in all PCL/LMWPCL blends and no extra peak or shift in band frequencies were observed, as a consequence of the lack of strong molecular interactions between PCL and LMWPCL.

Figure 1- FTIR spectra of PCL/LMWPCL blends: pure PCL (A), LMWPCL (B), PCL/LMWPCL 50/50 (C) and PCL/LMWPCL 20/80 (D).

PCL is a semi-crystalline polymer, and its final properties, such as strength, modulus, shape-memory effect and biodegradability depend, to a great extent, on the crystalline fraction, which is affected in turn by conditions of crystallization. Therefore, investigation of the crystallization kinetics of PCL is of considerable practical significance. In order to obtain a material with better physical properties, it is especially necessary to study the dynamic, non-isothermal crystallization process.

X-ray diffraction patterns of pure PCL and blends (Fig. 2) displayed their main peaks at 2equal to 21.2°, 21.8°, and 23.5° which are those typical of an orthorhombic crystalline unit cell [Hu et al., 1990]. The peak relative intensity was similar for all samples, evidencing a similar crystallinity degree for PCL/LMWPCL blends and pure PCL and LMWPCL. The less ordered amorphous regions in which the polymer chains are randomly arranged are thought to be the initial sites of hydrolysis.

Both the crystallization behavior and the structure of the crystallizable component in the crystalline/amorphous polymer blends should affect considerably the overall properties of the blends. In the previous studies, it was observed that if two components in the blends are through to be compatible in the amorphous state, as the blends are cooled from the melt, crystallization of the crystallizable component occurs, but the total degree of crystallinity of the blend decreases rapidly with increasing content of the amorphous component. A critical composition of crystallizable component, bellow which no crystallinity develops, is usually observed for several crystalline/amorphous polymer blends [Macknight et al., 1978].

Figure 2- XRD of PCL and the PCL/LMWPCL blends: pure PCL (A), LMWPCL (B), PCL/LMWPCL 50/50 (C) and PCL/LMWPCL 20/80 (D).

A plot of crystallinity versus weight percentage of PCL for PCL/LMWPCL is given in Fig. 3. For PCL/LMWPCL blends the crystallinity of LMWPCL component decreases as a function of increasing PCL concentration.According to the law mixtures, properties of blends are depending upon the fraction of components that made up the polymer blend. The relationship for PCL/LMWPCL is so often linear. This behavior is mainly caused by some compatibility factor between PCL and LMWPCL and also uniformity of size, shape and dispersion of PCL phase.

Figure 3- Relative crystallinity (%) measured for PCL/LMWPCL blends. The relative crystallinity was calculated from DSC data.

One of the major design concerns in the development of tissue engineering constructs is the structure of pores and the interconnectivity. A typical scaffold for tissue engineering of bone should be biocompatible along with the other desired properties such as biodegradability, porous structure and mechanical strength [Langer et al., 1993].

SEM examination of PCL/LMWPCL blends revealed characteristic microporous morphology with irregular pore shape and size in the range of 10-20 µm distributed in a lamellar or plate-shaped matrix (Fig. 4).

Figure 4- SEM of the PCL blends at LMWPCL concentration of 10% (w/w) (A) and 50% (w/w) (B).

Thepolymerconcentrationisanimportantparameterforthe porous scaffolddevelopment.Thedifferentpolymerconcentrations used in the study are 5-80wt%toseetheireffectonthescaffoldcharacteristics.TheexperimentalresultsofporositymercuryshowthathigherconcentrationsofLMWPCL produce higherporosityofthescaffold.Thescaffoldsresultedwereofhighlyporousstructuresintherangeof20-80% (wt) fortheLMWPCLathighconcentrationandthedensitywasfoundtodecrease(0.089-0.035g/cm3).

Mercury porosimetry and helium pycnometry were used to determine the pore characteristics of PCL/LWPCL membranes and the percent effective porosity are presented in Fig. 5. MIP data (Fig. 5) indicated that the PCL/LMWPCL blends possessed a bimodal pore distribution with peaks occurring in both the micro (<20μm) and macro (>50μm) range. In contrast the pore size distribution within PCL/LMWPCL 50/50 foam is distinctly mono-modal (Fig. 5) with the majority of pores (70.2%) ranged between 10 and 15 μm (Fig. 6).

Figure 5- Pore size distribution of PCL/LMWPCL foams determined via mercury porosimetry: PCL/LMWPCL 20/80 (A), PCL/LMWPCL 50/50 (B) and PCL/LMWPCL 80/20 (C).

Figure 6- Pore diameter distributions of pores found on PCL/LMWPCL blends at concentrations of 20/80 (w/w) (A) and 50/50 (w/w) (B).

To examine the molecular weight and viscosity influences on the porous formation, the intrinsic viscosity of the PCL/LMWPCL blends solutions at different blend compositions were determined and the results are showed at Fig. 7.

Figure 7- Intrinsic viscosities of the blends solutions where sp is the specific viscosity and c is the PCL/LMWPCL blend concentration (%w/w): 5/95 (A), 20/80 (B), 50/50 (C) and 80/20 (D). Extrapolation of the data to zero concentration yielded the intrinsic viscosity, [].

The viscosities of the blends showed a strength dependence on the PCL/LMWPCL solutions concentrations (Fig. 6). The values of [] for PCL/LMWPCL blends solutions are 2.7 (5/95), 3.8 (20/80), 3.55 (50/50) and 4.24 (80/20). Intrinsic viscosity measures the ratio of hydrodynamic volume to molecular weight. Thus, high concentration of LMWPCL on PCL/LMWPCL blends appears to generate more densely packed structures in solution, resulting in smaller hydrodynamic volumes compared to those blends of linear polymers with comparable molecular weights. Consequently, LMWPCL with minor hydrodynamic volume would naturally lead to a lower viscosity. In a shear field, the larger object dissipates more energy, resulting in a higher viscosity. Consequently, PCL have the larger hydrodynamic volume would naturally lead to a higher viscosity in detriment of the formation of pores in the PCL/LMWPCL membranes. Thus, the porous surface of PCL/LMWPCL foams appears to be due to the combination of both phase separation and or may be an issue of viscosity. The LMWPCL solution has a much lower viscosity than that of the PCL solution. The differences in viscosity made it difficult to determine whether changes in pore size and shape are due to molecular weight effects or differences in viscosity of the solutions. Research is currently in progress to investigate this issue.

4.CONCLUSIONS

Thedevelopmentof LMWPCLmaybenefitthedesignanddevelopmentofthree-dimensionaltemplatesorscaffoldsfortissue-engineeredproductstosupport,reinforceandinsomecasesorganizetheregeneratingtissue.Thesefunctionsrequireaporousscaffoldwithinterconnectedporosityanddesirablechemicalproperties.TheadvantageofLMWPCLoverPCListhattheycanbeeasilymass-producedandtheirproperties,inparticular,thedegradationrate,canbetailoredtosuitspecificapplicationsinmedicine.

Acknowledgments

The authors are gratefully to financial support of Finep, Fapemig and CNPq (Project: 501214/2011-9).

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