A versatile cell-friendly approach to produce PLA-based 3D micro-macro-porous blends for tissue engineering scaffolds

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A Versatile Cell-Friendly Approach to Produce PLA-Based 3D Micro-Macro-Porous Blends for Tissue Engineering Scaffolds Luciana Sartore , Stefano Pandini , Kamol Dey , Fabio Bignotti , Federica Chiellini PII: DOI: Reference:

S2589-1529(20)30032-6 https://doi.org/10.1016/j.mtla.2020.100615 MTLA 100615

To appear in:

Materialia

Received date: Accepted date:

25 November 2019 4 February 2020

Please cite this article as: Luciana Sartore , Stefano Pandini , Kamol Dey , Fabio Bignotti , Federica Chiellini , A Versatile Cell-Friendly Approach to Produce PLA-Based 3D Micro-Macro-Porous Blends for Tissue Engineering Scaffolds, Materialia (2020), doi: https://doi.org/10.1016/j.mtla.2020.100615

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A Versatile Cell-Friendly Approach to Produce PLA-Based 3D Micro-MacroPorous Blends for Tissue Engineering Scaffolds *

Luciana Sartorea, [email protected];[email protected], Stefano Pandinia, Kamol Deya,c, Fabio Bignottia and Federica Chiellinib

a

Department of Mechanical and Industrial Engineering, University of Brescia Via Branze 38 –

25133 Brescia Italy; b

c

Department of Chemistry and Industrial Chemistry, University of Pisa, Pisa Italy

Department of Applied Chemistry and Chemical Engineering, Faculty of Science, University of

Chittagong, Chittagong-4331, Bangladesh *

Corresponding author: Dipartimento di Ingegneria Meccanica e Industriale Università degli

Studi di Brescia Via Branze 38, 25133, Brescia (I) Tel. +39 030 3715786 Abstract. In this study we instituted an innovative, cost-effective, green and versatile methodology to produce a series of PLA-based open-pore porous blends with high porosity and interconnectivity as well physico-mechanical properties suitable for tissue engineering application. Parent poly-L-lactic acid (PLA) blend was prepared by melt-blending using crosslinked sodium polyacrylate particles as a porogen, commonly used as superabsorbent polymer (SAP). The obtained biphasic systems showed a regular distribution of SAP particles with diameters up to about 50 m and, most importantly, retained their superabsorbent ability within the thermoplastic PLA based matrices that facilitated swelling followed by leaching out from PLA based matrices in aqueous environment generating very high porosity. Very importantly, versatility of this developed methodology was judged by accommodating different polymers, such as, poly (3-hydroxybutyrate) (PHB) poly (ɛ-caprolactone) (PCL) or

poly (ethylene glycol) (PEG) or wood-cellulose microfiber (SP) to generate monophasic, biphasic, plasticized or reinforced blends, respectively, under identical benign condition. These blends were analyzed morphologically, thermally and mechanically to evaluate the degree of miscibility, thermal stability and mechanical property to apply as scaffolds in tissue engineering. Finally, all these scaffolds allowed good cell adhesion and proliferation during culture of mouse embryo fibroblasts cell line. Hence, this methodology of producing PLAbased polymeric system stunned with processability to accommodate other biocompatible polymers allows selectively modifying biomaterial properties for target application and appears very promising platform for several applications, particularly for scaffold production in tissue engineering. Keywords:

Poly-L-lactic

acid

(PLA),

poly

(ɛ-caprolactone)

(PCL),

poly

(3-

hydroxybutyrate) (PHB), micro-macro-porous blends, tissue engineering. 1. INTRODUCTION Tissue engineering aims at replacing or reconstructing fully functional substitute for damaged, diseased or lost tissues and organs. Biomaterials play a vital role in this field by acting as synthetic platforms referred as scaffolds or matrix, providing and maintaining a well-defined healthy microenvironment to encourage cells to enhance repair or generate new tissue. The challenging goals of tissue engineering have significantly boosted the development of biomaterial engineering over the last few decades [1-3]. Among the specific scaffold requirements, interconnected open porosity is a must have property that allows cell ingrowth and uniform cell distribution besides adequate vascularization to ensure enough transport of gases, nutrients, and regulatory factors for cell survivability [4-5]. Biodegradability is an added value, since it is often preferable that the scaffold is absorbed by the surrounding tissues at a

controllable rate that approximates the rate of tissue regeneration eventually creating space for new tissue growth under the culture conditions of interest, without the necessity of a surgical removal [6]. Numerous approaches such as, solvent casting, particle leaching, gas foaming, freeze-drying, thermal induced phase separation, electrospinning, rapid prototyping, micropatterning, and micromolding are utilized to produce porous structures [2, 5]. However, most of the techniques rely on the dissolution of polymeric phase into a specific solvent, which could be toxic to cells and might be intolerant to the environment. Melt blending by mechanical mixing of molten polymer mixture followed by particle leaching offers an eco-friendly approach to circumvent solvent-intolerant issues along with other advantages features like cost efficiency, easy controllability, rapid processability and customizability of the end-properties without altering the chemical fingerprint of the constituents [7]. Unfortunately, most of the melt blending technique employed today in biomedical field display very limited flexibility in terms of producing multiaspects blend under identical operating condition to meet various tissue-specific demand. Consequently, the paucity of a suitable blending technique pushes the researchers to focus on developing new blending strategies that allow tissue-specific customization of blends on-demand avoiding cumbersome additional processing requirements. So far, many different materials have been investigated for producing scaffolds using blending technique. Polymeric materials have shown a great affinity for cell transplantation and differentiation [8, 9] and their properties can be tuned by properly selecting the polymer from the wide variety available [10, 11]. Moreover, synthetic polymers represent the largest group of biodegradable polymers, they can be produced under controlled conditions, are often cheaper than biologic scaffolds, and many of them show physicochemical and mechanical properties comparable to those of biological tissues [11]. The most commonly used polymers in tissue

engineering are polyesters, such as poly(glycolic acid) (PGA), poly(ε-caprolactone) (PCL), poly(lactic acid) (PLA), and poly(lactic-co-glycolic acid) (PLGA) copolymers. These polymers are well known because of their high purity, convenient processing, availability with a wide range of molecular weight, inexpensiveness and good mechanical properties, along with biodegradability. Moreover, their degradation products can be resorbed through physiological metabolic pathways. They are already approved for human use in several forms and formulations worldwide such as resorbable sutures and fixation devices [12-15]. PLA, a bio-based polyester with high strength and modulus, degrades within the human body to form lactic acid, a naturally occurring chemical, which is easily removed from the body. PLA based scaffolds have already been used in applications as bone and cartilage tissue engineering, drug delivery and wound healing [16]. PLA is available as a fully crystalline or a fully amorphous form. PLLA, the semi-crystalline form of PLA, exhibits excellent biomimeticity and is the most biocompatible because it is a naturally occurring polymer [17]. PLLA has a relatively slow rate of degradation making it useful in engineering of tissues that require long-term high tensile strength [10]. In earlier studies, we developed a novel composite material based on PLA matrix and crosslinked particles of sodium polyacrylate, commonly used as superabsorbent polymer (SAP), to obtain superabsorbent thermoplastic products using melt blending [18, 19]. This system was proven to produce highly interconnected porous materials owing to the ability of SAP particles to swell and be leached in water. Consequently, the composite material may be considered as precursors for the safe preparation of highly macroporous PLA network with interconnected tunable porosity without the requirement of using organic solvents and chemical foaming agents which offers great potential for use in tissue engineering application. Though, this PLA-based scaffold was well proven as cell-friendly but suffered from poor cell colonization. To address this

constraints, we hypothesize to accommodate others polymers into PLA based blend and hence to modulate morphology and mechanics favorable for cell growth and colonization. Besides interconnected porosity, mechanical properties play a critical role in determining the in vitro and in vivo performance of scaffold, since scaffold’s mechanic alone can modulate different level of cellular functions as well as the healing and remodeling processes are greatly affected by the mechanic of the implanted scaffold [20, 21]. Often, the mechanical properties of a single polymer do not completely match those of the different tissues, but the properties of a composite or a blend may be programmatically varied by mixing different components in various ratios. Generally, PLA possesses high strength and stiffness, but it has low impact toughness and brittleness, which limit its wide-spread applications. Blending PLA with other polymers offers a convenient way to modify the material with desired properties or generate novel properties for a target application [7]. Poly(-caprolactone) (PCL) was the earliest polyester used in tissue-engineering due to its excellent biocompatibility, biodegradation, and bioresorption capability. Biodegradation of PCL occurs slower than that of PGA or PLA, making it the optimal polyester for development of longterm grafts like bone. Mechanically, PCL exhibits better flexibility than PLA, which can attribute improved ductility of PLA blend [22]. Moreover, PCL shows low glass transition and melting temperatures compared to those of PLA that could also enhance elasticity of the composites blend. Hence, PLA is blended with PCL to improve the ductility of the composite blend under identical condition. In this article, the previously developed simple method [18] is used to prepare a set of PLAbased composites blends to investigate the flexibility in tailoring the materials properties by incorporating a range of different polymers without perturbing the benign process conditions. We blended different polymers, such as, poly (3-hydroxybutyrate) (PHB) or poly (ɛ-caprolactone)

(PCL) or poly (ethylene glycol) (PEG) or wood-cellulose microfiber (SP) into parent PLA matrix along with SAP particles to attune final products for target applications. Finally, the effect of different materials composition i.e. degree of miscibility, plasticizing or fiber reinforcing, on physico-chemical properties, morphological and mechanical behavior as well as cell adhesion and viability were evaluated. 2. EXPERIMENTAL 2.1 Materials Poly-L-lactic acid (PLA) was purchased from Nature Works (Blair, NE) and had a nominal weight-average molecular weight (Mw) of 199,590 Da and the brand name 2002D. The material was dried at 70 °C under vacuum for 12 hrs before use. Cross-linked sodium polyacrylate was purchased from Evonik Industries AG (Essen, Germany) and had the brand name Produkt T 5066 F; the particles size was lower than 63μm and density is 0,7 g/cm3. It was dried at 60°C under vacuum for 12 hrs before use. Poly(-caprolactone) (PCL) with molecular weight Mn=10.000 Da and Poly(ethylene glycol) (PEG) with molecular weight Mn=600 Da were supplied by SigmaAldrich and used as received; wood-cellulose microfibers (SP), with a fiber length of 200 ÷ 300µm, were supplied by Gurit® (Zurich, Switzerland); the poly (3-hydroxybutyrate) (PHB) polymer was supplied by PHB Industrial SA, Serrana, SP, (Brazil) with average molecular weight Mw=425 kDa and polydispersity Mw/Mn=2.51. 2.2 Composite blends preparation Parent and composites blend with or without SAP particles were prepared by melt blending process using dried starting components through a discontinuous mixer (Brabender, Plastograph, Duisberg, Germany). The mixing treatment was done at 180 °C with a screw speed of 50 rpm for

a period of 6 min. The total polymer content inside the mixing chamber was 57 g. For all composite formulations, a 30 wt% SAP was used with respect to the total amount of matrix. Prior to the formation of sheets by means of a laboratory compression molding machine (Collin, P200E), all samples were recovered from the mixing chamber and dried in an oven under vacuum at 50 °C for 24 h. Small pieces of blend materials were first sandwiched between aluminum sheets followed by a compression molding machine using a specifically optimized temperature (180 °C), pressure (30 atm) and time program (15 min). To prepare composite blends, required amount of PLA and PCL/PHB/PEG/SP were blended along with 0% or 30% SAP particles. Table 1 reports composition of different formulations of parent PLA and PLA based composite blends. In case of composite blend the first number indicates the amount of SAP, while the last number indicates the amount of 2nd polymer in the blend. The symbol ‘W’ refers to the after water treatment (For example, PLA30PCL20W indicates the blend composed of PLA-80 wt%, PCL-20wt% subsequently added of SAP-30wt%, after water treatment). 2.3 Thermomechanical and Morphological analysis An electromechanic dynamometer (Instron Model 3366) was used to perform the tensile test. Elastic modulus (E), strength (b) and elongation at break (b) were determined on 0.2 mm thick and 10 mm wide strips, with a gauge length of 80 mm tested at a crosshead speed 2 mm/min. Prior to the tensile test, all specimen were dried at 55 °C for 4 hrs under vacuum to remove entrapped humidity in the composites. Thermal analysis was performed in nitrogen atmosphere with a differential scanning calorimeter (DSC Q100, TA Instruments) at a heating rate of 10 °C/min. The glass transition temperature (Tg), crystallization temperature (Tc), melting temperature (Tm) were recorded from

the second heating scan. The Tg is defined as the inflection point of the change in heat capacity versus temperature. Scanning electron microscopy (SEM) was performed using a LEO EVO 40 scanning electron microscope. The compression molded specimens were cryogenically fractured in transverse direction. Samples were mounted with carbon tape on aluminum stubs and then sputter coated with gold to make them conductive prior to SEM observation. 2.4 Swelling, density and porosity evaluation Swelling capacity was evaluated by a modification of tea-bag method [23] measuring the weight uptake after immersion in water as: Swelling capacity =

𝑊2 −𝑊1 𝑊1

100

Where W1 and W2 indicate the weight of dry and swollen specimen respectively. After water immersion and particles leaching, the density and porosity of samples were measured using liquid substitution method using ethanol as a displacing agent, described elsewhere [18, 24]. 2.5 Preparation and sterilization of the samples Starting from a 0.2 mm thick sheet for each material, about 80 disks with an average diameter of 1 cm were cut with a die. The disks with an average surface of 0.785 cm2 were arranged in 24 wells plate. Samples were sterilized under UV light for 30 minutes and covered with Dulbecco’s Phosphate Buffer Saline (DPBS) for 24 hours. Afterwards, samples were extensively washed with DPBS added with penicillin/streptomycin solution (1%), and subsequently pre-incubated for additional 3 hours with complete DMEM before cell seeding. 2.6 Cell culture To investigate the ability of parent PLA and PLA based blends compact/porous disk to support cell adhesion and viability, mouse embryo fibroblasts balb/3T3 clone A31 cell line

(CCL-163) was selected. Cells were purchased from American Type Culture Collection (ATCC) and propagated using Dulbecco’s Modified Eagle Medium (DMEM), supplemented with 4 mM of L-glutamine, 1% of penicillin: streptomycin solution (10,000 U/ml: 10 mg/ml), 10% of calf serum and antimycotic (complete DMEM). 2.7 Cell viability assay Samples were seeded with mouse embryo fibroblast balb/3T3 clone A31 cells (passage 73) at a concentration of 2 X 104 per well and incubated at 37°C in a 5% CO2 enriched atmosphere. Balb/3T3 cells cultured on tissue culture polystyrene (TCPS) were used as control. Cells were allowed to proliferate for 72 hours for the parent PLA blend disks and for 3, 7 and 10 days for the composites. The viability of balb/3T3, grown onto the tested polymer samples and also on the TCPS in the direct contact with the polymer disks was investigated by mean of WST-1 tetrazolium salt reagent (Roche). Briefly, cells were incubated for 4 hours with WST-1 reagent diluted 1:10, at 37°C and 5% CO2. Measurements of formazan dye absorbance were carried out with a microplate reader (Biorad) at 450 nm, using 655 nm as reference wavelength. In vitro biological tests are performed on triplicate and represented as mean ± standard deviation. Statistical difference was analyzed using one-way analysis of variance (ANOVA), and a p value <0.05 (*) and p value <0.001 (**) were considered significant. 2.8 Morphological investigations 2.8.1 Confocal laser scanning microscopy Morphology of balb/3T3 clone A31 cells cultured on tested samples was investigated by means of confocal laser scanning microscopy (CLSM) after 72 hours from seeding. Balb/3T3 cells cultured on 16 mm round glass cover slips were used as control. Cells were fixed with 3.8% paraformaldehyde for 1 hour in Phosphate Buffer Saline 0.01M pH 7.4 (PBS 1X) at room temperature and permeabilized with a PBS 1X/Triton X-100 solution (0.2%) for 10 min. After

blocking with 1% (w/v) bovine serum albumin (BSA) in PBS 1X for 30 min, cells were incubated with phalloidin-fluorescein isothiocyanate (FITC) labeled in 1% BSA solution at room temperature in the dark. After 1 hour, cells were incubated with 4’-6- diamidino-2phenylindole (DAPI) solution for 30 minutes. Following dyeing incubation, samples were extensively washed with PBS 1X and observed using confocal microscopy. A Nikon Eclipse TE2000 inverted microscope equipped with EZ-C1 confocal laser (Nikon, Japan) and Differential Interference Contrast (DIC) apparatus with 20X and 60X oil-immersion objectives were used to analyze the samples. A 405 nm laser diode (405 nm emission) and an Argon ion laser (488 nm emission) were used to excite DAPI and FITC fluorophores, respectively. Images were captured with Nikon EZ-C1 software with identical settings for each sample and further merged with Nikon ACT-2U Software. 2.8.2 Scanning Electron Microscopy (SEM) Morphological analysis of balb/3T3 clone A31 cells cultured on PLAPCL20 and PLA30PCL20W blends was carried out at days 3, 7 and 14 of culture also by SEM. After removal of the culture medium, each cell-cultured sample was rinsed twice with DPBS, and the cells were then fixed with 2% glutaraldehyde solution, which was diluted from a 25% glutaraldehyde solution (Sigma) with Phosphate Buffer Saline 0.01M pH 7.4 (PBS 1X), at 1.5 ml/well. After 1 hour of incubation, mouse embryo fibroblast cells/blends constructs were rinsed again with PBS and then treated with 1.5 ml/well of sodium cacodylate (0.1 M) pH 7.4 for approximately 1 minute. After cell fixation, the specimens were then dehydrated in ethanol solution of varying concentration (i.e. 10, 30, 50, 70, 90, and 100%, respectively) for 15 min at each concentration and finally dried in 100% of tetramethylsilane to remove any water traces. The fixed samples were mounted on a SEM stub, coated with gold at 15 mA for 20 minutes, and observed at different magnifications (300-2000X).

3

RESULTS AND DISCUSSION

3.1 Working Concept A plethora of materials and methods are used to fabricate porous scaffolds for biomedical application. While each method and material present distinct advantages and disadvantages, the rational selection of material(s) and technique must be warranted to meet the complex requirements of scaffolding substrate for the target tissue [5]. Despite of availability of existing vast panorama of materials and techniques, much work still can be done to satisfy increasing complexity and unmet demanding performances required by biomedical devices. Innovations in the material design and fabrication processes, indeed, are raising the possibility of production of implants with good performance. Blending being a quicker, easier and convenient physical modification technique without need of developing new polymers or copolymers has been widely exploited to tailor materials properties required for specific application. PLA is considered as a ‘material of choice’ for biomedical application thanks to its biocompatibility, biodegradability, mechanical strength, and processability. Our study aimed at fabricating a family of PLA based composites blends by combining other polymers which induced immiscibility/plasticity (biphasic system), miscibility/plasticity (monophasic system), molecular flexibility and reinforcing effects depending on the type of moieties used while maintaining the biodegradability of the both components. Three different biocompatible polymers, namely, PHB, PCL and PEG were blended with neat PLA to impart plasticizing phenomenon into final products while SP fibers were added as a reinforcing material. PLA based composites were prepared by melt-blending PLA, different biocompatible and biodegradable polymers and fibers, and crosslinked sodium polyacrylate particles (Table 1). According to our previous work, cross-linked particles of sodium polyacrylate, commonly used as superabsorbent polymer, retained their superabsorbent ability even if distributed in a PLA

thermoplastic matrix. In aqueous environments the particles swell and leave the matrix generating very high porosity. Figure 1 shows the mechanism of forming porous structure. It was demonstrated that a composite PLA having 30% SAP particles produced a macroporous network with interconnected porosity of about 60% void volume. This particles content was excellent both for obtained porosity and for mechanical performances preservation of the final porous PLA. For these reasons 30% particles content was selected and maintained constant for the preparation of all the PLA based composites blends. To selectively modify biomaterial properties and enhance their performances two PLA based blends were developed namely PLA30PCL20, PLA30PHB20, a plasticized PLA namely PLA30PEG and fiber reinforced PLA namely PLA30SP10; table 1 showed the composition of all produced composite systems. After blending, the resulting materials appeared visually homogeneous, and the components were well dispersed. 3.2 Morphology of the PLA based blends Figure 2a-d shows SEM micrographs of the parent PLA30 before and after water treatment. A homogeneous dispersion of SAP particles was observed in the parent PLA30 showing a biphasic system with a regular distribution of particles. The diameter of the SAP particles was found within a range of 20-50 µm in the PLA polymeric matrix. Generally speaking, a poor adhesion and low interaction between the SAP particles and the PLA matrix are observed as suggested by the discontinuous aspect of the surface between the matrix and the particles (Figure 2a). Most importantly, this polymeric biphasic system maintains excellent swelling property. The equilibrium absorbency capacity of PLA30 in water, evaluated by a modification of tea-bag method [23], was 3500 %, which indicated a water uptake about 35 times larger than its original

weight. This high level of water absorbency demonstrate that cross-linked SAP particles retain their intrinsic superabsorbent ability even though embedded in a thermoplastic polymeric matrix. SEM micrographs of the sample PLA30 after immersion in water for 15 days so to completely leach the SAP particles as demonstrated in a previous work [19] (Figure 2b-d) reveals a macroporous network material with pores, channels and interstices of different sizes well interconnected and distributed into the material. After water treatment, the particles swell possibly due to the low interaction with the matrix and leach out from PLA generating high porosity. In addition, the high magnification micrographs (Figure 2c-d) clearly show interconnected open porous structure, including the inner one, which is conducive to the infiltration of cells in the material. Elemental analysis done during SEM observation at different points of sample before water treatment confirms the identification of SAP particles (high content of Na level) in the blend (Figure 2a). On the other hand, elemental analysis shows increased percentage of C content and trace amount of Na content in agreement with hand-made materials, after water treatment of the sample, suggesting the successfully removal of SAP particles (Figure 2b). Density and porosity of pure PLA and of PLA based blends after swelling in water for 15 days were measured using liquid substitution method, and the results are shown in Table 2. As expected, reduced density and increased porosity were observed on the samples as a result of the leached particles. Parent PLA30W shows a porosity of about 60%. It is important to mention that after particles leaching, all samples maintained their shape integrity, and no significant deformations were observed during water treatment. Figure 3 shows the SEM images of the PLA based composite blends. The SEM micrographs of PLA30PCL20, in addition to the larger SAP particles with sharp edges, clearly shows the distribution of spherical PCL particle in the PLA matrix indicating the heterogeneous blend. PCL

particle sizes ranging from 5 to 10 µm are apparently impregnated inside the PLA matrix (Figure 3a). After water treatment, leaching out of SAP particles produced a biphasic PLA30PCL20W blend with well interconnected open porosity (Figure 3b). On the other hand, PLA30PHB20W and PLA30PEGW, incorporating PHB or PEG respectively into parent PLA matrix, display a monophasic porous blend without phase separation (Figure 3c-e). Additionally, the addition of SP fiber produced a randomly fiber distributed micro-macro-porous PLA30SP20W composite blend with protruded fiber diameter of 5-10 µm (Figure 3f). All composite blends exhibit a high porosity of about 60%, calculated by the liquid displacement method (Table 2). Most importantly, all PLA based blends show a well-developed heterogenous micro-macro-porous structure similar to the natural bone tissue, demonstrating the effectiveness of the leaching process irrespective of the chemical constituents of composite blends-a most welcome aspect from the manufacturing point of view. 3.3 Thermal properties of PLA based blends The thermal properties of PLA based blends were studied to evidence the interactions between polymer blends and SAP particles as well as to characterize the properties of the final obtained porous biomaterials. DSC analysis of SAP did not evidence transitions, while the glass transition temperature (Tg), crystallization peak temperature (Tc) and melting peak temperature (Tm) for parent PLA and PLA based blends both of compact (before water treatment) and porous (after water treatment) products, as evaluated on the second heating scan, are listed in Table 1. PLA presented a Tg at 60 °C which remained almost unchanged after introduction of SAP particles probably because the chain segment mobility of the PLA phase was not influenced by the introduction of SAP particles suggesting a reduced filler-matrix interaction. Conversely, while in PLA the second heating scan didn’t reveal any presence of the crystalline phase, in the

PLA-based composites cold crystallization and subsequent melting were found, because of nucleation effect promoted by SAP particles on PLA crystallization. Tg values are useful for a variety of purposes, for example, they tell us whether binary polymer blends are miscible, or compatible, or not miscible at all. All the blends show Tg values lower than that of neat PLA matrix. The blend PLAPHB20 presents two distinct Tg values at 0.7 °C and 58 °C correspondingly for PHB and PLA, while the PLA30PHB20 composite blend exhibits a single Tg value of 47 °C, by 13 °C lower than that of neat PLA (60 °C). This is the typical behavior of compatible blends with one distinct Tg value compared to starting pure polymers, suggesting the role of SAP that might promote the interaction between PLA and PHB increasing friction during melt mixing. The single glass transition temperature drops down even at a lower value of 40 °C for porous PLA30PHB20W blend after leaching out of SAP particles, indicating the full miscibility of the blend. Likewise, porous PLA30PEGW blend exhibits a significantly lower Tg value of 41 °C compared to that of neat PLA (60 °C) and displays full miscibility of the blend (Figure 4). This considerable reduced Tg value for porous PLA30PHB20W and PLA30PEGW blends might be attributed to plasticization effect of the PHB and PEG in the blend because of increased polymer chains mobility and SAP particles which probably increased the efficiency of melt mixing. Additionally, the reduced crystallization peak temperature (Tc) of the PLA30PHB20W (92 °C) and PLA30PEGW (90 °C) blends compared to that of PLA30 (129 °C) might be due to the disturbance of orderly arrangement of molecular chains that encourages chains segmental mobility reducing of the crystallization initiation temperature i.e. effective plasticizing effect of PHB and PEG in the blend (Table 1 and Figure 4). This miscibility concept of PLA30PHB20W and PLA30PEGW blends derived from DSC results is in well agreement with the SEM images (monophasic morphology).

On the other hand, PLA30PCL20W blend shows two distinct Tg values at -50 °C and 55 °C corresponding to the individual constituting components PCL and PLA, respectively, and suggesting the immiscibility of the blend, in good agreement with SEM biphasic morphology. Similar results are also obtained for PLAPCL blending by other researchers [26-28]. This blend also displays a double melting behavior due to the melting of PLA (154 °C) and the melting of PCL (56 °C). Similarly, PLA30SP10 shows a Tg value of 59 °C closed to that of pure PLA matrix probably because the chain segment mobility of the PLA phase was not influenced by the introduction of cellulose fibers. Generally, all porous PLA based blends present slightly reduced Tms compared to that of neat PLA and two melting peaks which might be ascribed to slow rates of crystallization and recrystallization [25]. 3.4 Mechanical properties of the PLA based blends Tensile mechanical test was performed to investigate the role of filler (i.e. polymeric crosslinked SAP particles), plasticizer (PEG), polymer blending (PHB, PCL) and reinforcing agent (SP fibers) on the overall mechanical behavior of PLA matrix. Young’s modulus (E), tensile strength (b) and elongation at break (b) of parent PLA and PLA based blends both before and after water treatment were presented in the Table 3. Figure 5 showed the representative tensile stress-strain curves for PLA-based composites blends compared to parent PLA composites before and after water treatment. As observed from Table 3 the mechanical properties are markedly modified by addition of SAP which induce a regular stiffening effect. For instance, elastic modulus increased to 4.3 GPa from 3.0 GPa by the introduction SAP particles into PLA matrix indicating the role of SAP as a stiffener particle. The presence of SAP did not significantly alter the inherent brittle behavior of PLA. However, water treatment reduced both elastic modulus and strength of PLA30W because of the porous nature (60% porosity) of

this blend generated by the leaching out of SAP from the matrix PLA during water treatment. Notable, this trend of reduction in stiffness and strength after water treatment is a general phenomenon for all composite blends owing to the introduction of voids (Figure 5). The high porosity of the material is probably accountable for the low elongation at break observed particularly for PLA30PEGW where a clear plasticizing effect was showed before water treatment (Figure 5). Porosity, as well as the presence of defect, generally decreases the elongation at break, while plasticization effect increases it. It is notable that, the effect of high porosity, which is reverse to the effect of plasticization from the mechanical point of view, eventually significantly nullifies the plasticization effect in final product, resulting into negligible increases in elongation at break. Among all PLA bassed blends after water treatment, the PLA30PCL20W showed the least elastic modulus and strength - suggesting the negligible interaction between PLA and PCL generating a biphasic system - which is in well agreement with SEM micrographs (Figure 3). In addition, before and after water treatment the SP fiber significantly reinforced both the stiffness and strength of matrix (Figure 5 and Table 3). 3.5 Biological evaluation of parent PLA based blends 3.5.1 Cell viability assay The evaluation of biomedical polymers’ biocompatibility represents a fundamental step in establishing and ensuring product applicability and safety. In the present study, balb/3T3 clone A31 mouse embryo fibroblasts were selected for a preliminary biological investigation of polymeric parent PLA based blend both in compact and porous disks starting from parent PLA with different thickness, namely, PLA 0.21 mm thickness (compact), PLA30W 0.17 mm thickness (porous) and PLA30W 0.32 mm thickness (porous).

The biocompatibility of all samples and the influence of the surface on cell behavior were assessed in a preliminary investigation on cell viability and adhesion. Cell viability was evaluated by WST-1 reagent, both for cells-seeded parent PLA disks and cells grown on TCPS. The test is based on the mitochondrial enzymatic conversion of the tetrazolium salt WST-1 into soluble formazan in metabolically active cells. Results highlighted very low values of viability for cell grown onto all the tested parent PLA disks (Figure 6a), which were in a range around 23% in respect to the control. However, it is worth noting a statistically significant difference in cell viability (p<0.05) observed between compact PLA 0.21 and porous PLA30W 0.32 samples. Figure 6b reports viability values of cells grown in contact with parent PLA disks, showing no toxic effects on cell viability in terms of chemicals released from the three typologies of disks. Moreover, an inverse correlation between cells grown on the samples and those on TCPS was found. In fact, higher number of viable cells resulted in lower number of cells on TCPS. Taken together, these preliminary results suggest that the investigated samples are non-toxic, but with poor capability to sustain an efficient cell adhesion and proliferation. In order to improve the ability of the investigated parent PLA disks to sustain a proper cell adhesion and proliferation, the surface features of the developed materials should be tuned to enhance cell-material interactions. Consequently, a family of PLA-based blends namely, PLAPHB20, PLASP10 and PLAPCL20 both in compact and porous structures were developed to improve the biological performance. 3.6 Biological evaluation of PLA based composite blends 3.6.1 Cell viability assay Three set of composite blends namely, PLAPHB20, PLASP10 and PLAPCL20 both in compact and porous structures were selected for assessing the viability and proliferation of mouse embryo fibroblasts balb/3T3 clone A31 cells. Quantitative evaluation of cell viability and

proliferation was performed by WST-1 reagent and monitored at days 3, 7 and 10 after seeding. The results are reported in Figure 6 (c-e) as cell proliferation (%) in respect to the control. The analysis of compact PLAPHB20 blend displayed a cell proliferation rate of approximately 20% compared to the control starting from day 3 (Figure 6c). However, it was evident a significant decrease of cell proliferation values at day 7 (p<0.05). As shown in Figure 6c, fibroblast cells reached cell proliferation values of about 10% at day 10. Cells cultured on porous PLA30PHB20W displayed a low value of proliferation in the range of 7% with respect to the control at day 3 of culture, with a significant increase of cell proliferation from day 7 (p<0.001). Significant higher values of cell proliferation were observed for porous PLA30PHB20W blends with respect to the compact PLAPHB20 samples at 7 and 10 day of culture (p<0.001). Likewise, at day 3 balb/3T3 cultured on compact PLASP10 and porous PLA30SP10W composite blends highlighted appreciable values of cell proliferation, in comparison to the cells cultured on TCPS, reaching a proliferation rate of about 16 % and 14% respectively (Figure 6d). Nevertheless, a statistically significant decrease of cell proliferation on compact PLASP10 blends was observed during the culturing period (p<0.05), with values of proliferation in the range of 7-10 % with respect to the control. On the contrary, fibroblast cells grown on porous PLA30SP10W displayed cell proliferation values not statistically different for all culturing period in comparison to the control. In this case also, significantly higher values of cell proliferation were observed for porous PLA30SP10W blend with respect to the compact PLASP10 blend at 7 and 10 day of culture (p<0.05) (Figure 6d). On the other hand, Figure 6e shows the balb/3T3 clone A31 cell proliferation (%) cultured on the compact PLAPCL20 and porous PLA30PCL20W composite blends in respect to the control TCPS at days 3, 7 and 14 after seeding. Results highlighted appreciable values of cell proliferation starting from day 3 of culture for both blend typologies in comparison to the cells

cultured on TCPS. Particularly, a statistically significant increase of cell proliferation on the compact PLAPCL20 blends was observed between 3 and 7 days (p<0.001) of culture, while similar value of cell proliferation, in the range of around 70% with respect to the control, was observed between 7 and 14 days of culture (Figure 6e). For the porous PLA30PCL20W blends, results highlighted a high cell proliferation rate of approximately 40% compared to the control starting from day 3. Moreover, as shown in Figure 6e, at day 7 and 14 of culture, fibroblast cells reached cell proliferation values of about 110% and 160 %, in respect to the control, respectively. This result, attributed to the ability of cells to adhere and proliferate also on the lower surface of the blend disk, was confirmed by the qualitative analysis carried out by CLSM. 3.5.2 Morphological investigations Observation of cell morphology provides direct information about the condition of cell attachment and viability. Therefore, a preliminary biological evaluation of cellular spreading and morphology for all the cell-cultured disks was carried out by CLSM technique. DAPI and phalloidin-FITC solution treatment were used to stain nucleic acids and cytoskeleton, respectively. Figure 7a displays images taken at different magnification, 20X (a-d) and 60X (eh) of balb/3T3 cultured on the three parent PLA disks with different thickness and on glass cover slips, as control. Microscopic observations revealed a low number of adherent cells on the surface of all samples, exhibiting a round morphology and an almost complete absence of spreading. Particularly, micrographs of PLA 0.21 mm sample (Figures 7a (a, e)) revealed the presence of cell clusters onto the surface of the disks generally arranged as cells aggregates. Furthermore, PLA30W 0.17 mm (Figures 7a (b, f)) and PLA30W 0.32 mm (Figures 7a (c, g)) showed a slightly higher number of adherent cells equally distributed on the surface of the disks but displaying a lack of spreading. Control cultures were well spread and have an elongated morphology, typical of fibroblasts (Figures 7a (d, h)). The low cell number and the absence of

cell spreading are in accordance with the quantitative results of cell viability obtained by using WST-1 assay. The observed results could be related to the surface characteristics of the parent PLA films in terms of wettability and surface energy. It appears clear that the process of cell spreading, typically mediated by the serum protein interaction with the surface of the films, is not favored, and therefore, cells results in low number and non-metabolically active. In order to improve the ability of the investigated parent PLA films to sustain a proper cell adhesion and spreading, the surface features of the developed materials should be tuned to enhance cellmaterial interactions. Consequently, a family of PLA-based blends were developed to improve the biological performance. 3.6.2 Morphological investigations Qualitative investigations of cell morphology and cytoskeleton organization of balb/3T3 clone A31 cells cultured on the compact/porous PLA based blends purposely developed to improve the biological performance were carried out by CLSM analysis at days 7 and 10 of culture. Cells were stained for F-actin and nuclei with FITC-phalloidin and DAPI, respectively. CLSM micrographs, taken at different magnification (20X and 60X), cells cultured on the compact/porous PLAPHB20 and compact/porous PLASP10 blends are presented in Figure 7 (be). As shown in Figure 7 (b, c) balb/3T3 cells cultured on compact PLAPHB20 and on porous PLA30PHB20W samples were able to adhere and proliferate on the surface of the blends. The cells grown on compact PLAPHB20 colonized very small areas of blends, while fibroblast covered larger areas but not all the available surface of porous PLA30PHB20W samples. However, the cells appeared well connected to each other with both fusiform and stellate shapes and numerous dendritic extensions from the cell membrane towards the samples surface. Moreover, higher magnification (60X) highlighted cell architecture showing consistent F-actin organization with great stress fibers stretched along the cytoplasm. On the other hand,

microscopic observations revealed the presence of adherent balb/3T3 cells on the surface of both compact PLASP10 and porous PLA30SP10W blends at day 7 and 10 of culture (Figure 7 d, e). However, only small areas of the surface localized on the border of the blends were covered by cells, in agreement with the quantitative results of cell viability obtained by using WST-1 assay. Nevertheless, the adherent cells were well spread and have an elongated morphology, typical of fibroblasts. The SEM micrographs of the surface of the compact PLAPCL20 and porous PLA30PCL20W blends seeded with balb/3T3 clone A31 cells after 3, 7 and 14 days of culture are reported in Figure 8(a-l). As shown in the SEM micrographs, 3T3 cells were able to colonize the blends surfaces (Figure 8 a-c, Figure 8 g-i), highlighting features indicative of good adhesion and spreading, including numerous filopodia and fiber-like processes that allowed the anchorage of the cells to the substrate (Figure 8 d-f, Figure 8 j-l). In particular, from day 3, cells grown onto porous PLA30PCL20W blend showed an improved multicellular coverage compared to cells cultured on compact PLAPCL20 blend. Moreover, from day 7 most of the pores, observed on the surface of the porous PLA30PCL20W blends, were covered by a continuous layer of cells (Figure 8 k). CLSM micrographs, taken at different magnification 10X, 20X and 60X, of balb/3T3 cultured at days 3, 7 and 14 on the compact PLAPCL20 and porous PLA30PCL20W blends are presented in Figure 9 (a-b). Images confirmed the presence of adherent 3T3 cells on the surface of the blends, starting from day 3 of culture up to day 14. A progressive increase of cells colonizing the surface of blends was evident and in agreement with the previously reported data of cell proliferation and scanning electron microscopy analysis. The cells appear well connected to each other with both fusiform and stellate shapes and numerous dendritic extensions from the cell membrane towards the samples surface. Moreover, higher magnification (60X) highlighted cell

architecture showing consistent F-actin organization with great stress fibers stretched along the cytoplasm. Moreover, as shown in Figure 10 (a-d), cells cultured on porous PLA30PCL20W blends were also able to adhere and proliferate on the lower surface of the blends starting from day 7 of cell seeding. The complete cellular colonization of the top blends surface and an initial cell adhesion and spreading on the lower surface nicely correlates with the higher values of cell proliferation compared to the controls obtained by mean of the biocompatibility assays. and confirms the excellent open interconnected porosity of the blends which is in well agreement with the SEM morphology (Figure 3b). Taken together, the biological investigations of all composite blends indicate that all samples are fully biocompatible and able to sustain cell adhesion and proliferation. In all cases, the porous composite blends exhibit increased cell viability and enhanced propensity of cell colonization. In fact, open interconnected porosity is a desired aspect for scaffold to provide cell ingrowth and survival and uniform cell distribution. Furthermore, the porous PLA30PCL20W blend clearly displayed a higher propensity to act as support for cell colonization, thanks to its biphasic structure that pushes cell movement throughout the sample, thus resulting an interesting material for the preparation of scaffolds for tissue engineering application. 4. CONCLUSIONS Highly micro-macro-porous PLA based blends with stable structural and good mechanical properties have been prepared starting from melt-processed PLA/SAP particles that lead to create tunable-porous three dimensional structure after water treatment owing to the leaching out of SAP thanks to retaining of its intrinsic swelling properties in the polymeric biphasic system without using any additives, catalysts and accelerators. The biological investigations of all micromacro-porous blends demonstrated biocompatibility, cell adhesion and proliferation. However, the obtained heterogenous porous structure encouraged improved cell viability and enhanced cell

colonization throughout the sample for all the three-dimensional systems compared to that of compact system. More importantly, this production approach allows to fabricate a diverse variety of composite blends with diverse morphology and mechanic that are better bio-mimetic for cell culture. This scaffold production technology seems very promising for many reasons: (i) it is suitable for accommodating several biocompatible thermoplastic materials, as for example PLA, PCL, PHB; (ii) the material filled with SAP particles, before the swelling phase, can be processed by technologies suitable for thermoplastic polymers, among which 3D printing; (iii) the new methodology provides a porous network in which porosity is tunable by proper use of superabsorbent particles; (iv) organic solvents and chemical foaming agents, necessary for the production of porous biomaterials, are not used, eliminating completely the residues remaining in the porous materials which may be harmful to adherent cells, protein growth factors or nearby tissues; and (v) data obtained for porous blends exhibits not only good physico-mechanical properties, but also good cell proliferation and colonization, particularly porous PLA30PCL20W, in well agreement with the requirements of scaffolds for bone regeneration.

ACKNOWLEDGMENTS The contribution of Gloria Spagnoli and Isabella Peroni in the experimental testing is gratefully acknowledged. REFERENCES 1. M.P. Lutolf, J.A. Hubbell, Synthetic biomaterials as instructive extracellular microenvironments for morphogenesis in tissue engineering, Nat. Biotechnol. 23 (1), (2005) 47-55. 2. K. Dey, S. Agnelli, F. Re, D. Russo, G. Lisignoli, C. Manferdini, S. Bernardi, E. Gabusi, L. Sartore, Rational design and development of anisotropic and mechanically strong gelatin-based stress relaxing hydrogels for osteogenic/chondrogenic differentiation, Macromol. Biosci. 19 (8), (2019) 1900099. 3. S. J. Lee, J. J. Yoo, A. Atala, "Biomaterials and tissue engineering." In Clinical Regenerative Medicine in Urology, Kim, Bup Wan Ed. Springer, Singapore, 2018, pp.1751. 4. H. Janik, M. Marzec, A review: Fabrication of porous polyurethane scaffolds, Mat. Sci. Eng. C 48, (2015) 586-591. 5. N. Annabi, J. W. Nichol, X. Zhong, C. Ji, S. Koshy, A. Khademhosseini, F. Dehghani, Controlling the porosity and microarchitecture of hydrogels for tissue engineering, Tissue Eng. Part B-Re. 16 (4), (2010) 371-383. 6. E. C. Novosel, C. Kleinhans, P. J. Kluger, Vascularization is the key challenge in tissue engineering, Adv. Drug Deliv. Rev. 63 (4-5), (2011) 300-311. 7. P. Saini, M. Arora, M.N.V. Ravi Kumar, Poly (lactic acid) blends in biomedical applications, Adv. Drug Deliv. Rev. 107, (2016) 47-59. 8. L. G. Griffith, M. A. Swartz, Capturing complex 3D tissue physiology in vitro, Nat. Rev. Mol. Cell Biol. 7 (3), (2006) 211-224. 9. F. Re, L. Sartore, V. Moulisova, M. Cantini, C. Almici, A. Bianchetti, C. Chinello, K. Dey, S. Agnelli, C. Manferdini, S. Bernardi, N. F. Lopomo, E. Sardini, E. Borsani, L. F. Rodella, F. Savoldi, C. Paganelli, P. Guizzi, G. Lisignoli, F. Magni, M. SalmeronSanchez, D. Russo, 3D gelatin-chitosan hybrid hydrogels combined with human platelet lysate highly support human mesenchymal stem cell proliferation and osteogenic differentiation, J. Tissue Eng. 10:2041731419845852 (2019) doi: 10.1177/2041731419845852. 10. L. Edgar, K. McNamara, T. Wong, R. Tamburrini, R. Katari, G. Orlando, Heterogeneity of scaffold biomaterials in tissue engineering, Mater. 9(5) (2016) 332-343. 11. P. Gunatillake, R. Mayadunne, R. Adhikari, Recent developments in biodegradable synthetic polymers, Biotechnol. Annu. Rev. 12, (2006) 301-347. 12. F. Rossi, M. Santoro, G. Perale, Polymeric scaffolds as stem cell carriers in bone repair, J Tissue Eng Regen Med. 9 (10), (2015) 1093-1119. 13. Haleh Bakht Khosh Hagh, Fahimeh Farshi Azhar, Reinforcing materials for polymeric tissue engineering scaffolds: A review, J. Biomed. Mater. Res. B 107 (5), (2019) 15601575.

14. T. Hoffman, A. Khademhosseini, R. Langer, Chasing the Paradigm: Clinical Translation of 25 Years of Tissue Engineering, Tissue Eng. Part A 25 (9-10), (2019) 679-687. 15. A. S. Mao, D. J. Mooney, Regenerative medicine: current therapies and future directions, Proc. Natl. Acad. Sci. USA, 112 (47), (2015) 14452-14459. 16. B. Dhandayuthapani, Y. Yoshida, T. Maekawa, D. S. Kumar, Polymeric scaffolds in tissue engineering application: A review, Int. J. Polym. Sci. 2011, (2011) 290602. http://dx.doi.org/10.1155/2011/290602. 17. K. Madhavan Nampoothiri, N.R. Nair, R.P. John, An overview of the recent developments in polylactide (PLA) research, Bioresour. Technol. 101(22), (2010) 84938501. 18. L. Sartore, S. Pandini, F. Baldi, F. Bignotti, L. Di Landro, Biocomposite based on poly(lactic acid) and superabsorbent sodium polyacrylate, J. Appl. Polymer Sci. 134 (48), (2017) 45655. 19. L. Sartore, N. Inverardi, S. Pandini, F. Bignotti, F. Chiellini, PLA/PCL-based foams as scaffolds for tissue engineering applications, Mater. Today Proc. 7, (2019) 410-417. 20. E. S. Schiffhauer, D. N. Robinson, Mechanochemical signaling directs cell-shape change, Biophys. J. 112(2), (2017) 207-214. 21. K. Dey, S. Agnelli, L. Sartore, Dynamic freedom: substrate stress relaxation stimulates cell responses, Biomater. Sci. 7(3), (2019), 836-842. 22. X. Dong, J. Zhang, L. Pang, J. Chen, M. Qi, S. You, N. Ren, An anisotropic threedimensional electrospun micro/nanofibrous hybrid PLA/PCL scaffold, RSC Adv. 9(17), (2019) 9838-9844. 23. M. J. Zohuriaan-Mehr, Z. Motazedi, K. Kabiri, A. Ershad-Langroudi, I. Allahdadi, Gum Arabic-acrylic superabsorbing hydrogel hybrids: studies on swelling rate and environmental responsiveness, J. Appl. Polym. Sci. 102, (2006) 5667-5674. 24. C. Kothapalli, M. Shaw, M. Wei, Biodegradable HA-PLA 3-D porous scaffolds: Effect of nano-sized filler content on scaffold properties, Acta Biomater. 1(6), (2005) 653-662. 25. M. Yasuniwa, S. Tsubakihara, Y. Sugimoto, C. Nakafuku,, Thermal analysis of the double‐ melting behavior of poly(L‐ lactic acid), J. Polym. Sci., Pol. Phys. 42, (2004) 25-32. 26. D. Cohn, A. H. Salomon, Designing biodegradable multiblock PCL/PLA thermoplastic elastomers, Biomaterials 26(15), (2005) 2297-2305. 27. L. Patrício, T. Domingos, M. Gloria, A. D'Amora, U. Coelho, P.J. Bártolo, Fabrication and characterisation of PCL and PCL/PLA scaffolds for tissue engineering, Rapid Prototyp. J., 20(2), (2014) 145-156. 28. R. Scaffaro, F. Lopresti, L. Botta, S. Rigogliuso, G. Ghersi, Integration of PCL and PLA in a monolithic porous scaffold for interface tissue engineering, J. Mech. Behav. Biomed. Mater. 63, (2016) 303-313.

Caption to Figures Figure 1: Novel production method of porous PLA-based materials for cells seeding and colonization.

Figure 2: SEM images of parent PLA30 blend (a) before water and (b, c, d) after water. The table refers to the C, O and Na content calculated by elemental analysis in the numbered portions of the blend before and after water treatment. Scale bars a: 100 µm, b: 200 µm, c: 20 µm and d: 10 µm. Figure 3: SEM images of PLA-based composites blends. (a) PLA30PCL20, (b) PLA30PCL20W, (c) PLA30PHB20, (d) PLA30PHB20W, (e) PLA30PEGW and (f) PLA30SP10W. From SEM image PLA30PCL20 is a biphasic blend. Figure 4: DSC traces (second heating scan) of porous PLA based blends. Figure 5: Representative tensile stress-strain curves for PLA-based composites blends before and after water treatment compared to parent PLA composites. Figure 6: Cell viability and proliferation of mouse embryo fibroblasts balb/3T3 clone A31 cultured on (a) parent PLA disks (b) below parent PLA disks on TCPS (c) PLAPHB20 and PLA30PHB20W (d) PLASP10 and PLA30SP10W and (e) PLAPCL20 and PLA30PCL20W blends evaluated by WST-1 assay.

Figure 7: CLSM micrographs of mouse embryo fibroblasts balb/3T3 clone A31 grown on (a) parent PLA 0.21 (a, e), PLA30W 0.17 (b, f), PLA30W 0.32 (c, g) blend disks and glass (d, h), (b) compact PLAPHB20, (c) porous PLA30PHB20W, (d) compact PLASP10, and (e) porous PLA30SP10W blends at 7 and 10 days. Scale bar represents 100 μm on 20X magnification and 30 μm on 60X magnification. Figure 8: SEM images of balb/3T3 clone A31 cells grown on (a-f) compact PLAPCL20 and (g-l) porous PLA30PCL20W blends at 3, 7 and 14 days. Figure 9: CLSM micrographs of mouse embryo fibroblasts balb/3T3 clone A31 grown on (a)

compact PLAPCL20 and (b) porous PLA30PCL20W blends at 3, 7 and 14 days. Scale bar represents 200 μm 10X magnification, 100 μm on 20X magnification and 30 μm on 60X magnification. Figure 10: CLSM micrographs of mouse embryo fibroblasts balb/3T3 clone A31 grown on lower surface of porous PLA30PCL20W blends at day 7 (a-b) and 14 (c-d).

Table 1. Composition, Glass Transition Temperature (Tg), Crystallization Temperature (Tc), Melting Temperature (Tm) for parent PLA and PLA based composite blends as evaluated on the second heating scan. Sample

Composition (%) PLA

Tg1

Tg2

SAP Polymer Fiber [°C] [°C]

Tm1

Tc Tm2a

[°C] [°C] [°C]

PLA

100

-

-

-

60

-

-

-

PLA30

68.7

31.3

-

-

59

-

PHB

-

-

100b

-

0.1

-

162

PLAPHB20

80

-

20b

-

0.7

58

128 151

PLA30PHB20

55

31

14b

-

47

107 153

PLA30PHB20W

80.0

-

20b

-

40

92

PCL

-

-

100c

-

-59

-

54

PLAPCL20

80

-

20c

-

-50

55

55

103

PLA30PCL20

54.8

31.5

13.7c

-

-50

55

57

105

PLA30PCL20W

80.0

-

20.0c

-

-50

55

56

101

PLA30SP10

59.9

30.1

-

10

59

112

PLA30SP10W

85.7

-

-

14.3

52

98

PLA30PEG

66.8

31.8

1.4d

-

47

100

PLA30PEGW

98.0

-

2.0d

-

41

90

129 154

149

144 154 147 156 144 154 148 156 140 151 135 146 132 147

Tg1: glass transition temperature of the polymeric phase blended with PLA; Tg2: glass transition temperature of PLA phase; Tm1: melting peak temperature of the polymeric phase blended with PLA; Tm2: melting peak temperature of the PLA phase; a : two melting peaks are found in some PLA polymer derivatives and they were attributed to slow rates of crystallization and recrystallization [25]. b Polymer used in the blend was PHB; c Polymer used in the blend was PCL; d Polymer used in the blend was PEGCOOH. W: indicates porous materials (i.e. samples after water treatment).

Table 2. Density and Porosity of PLA and PLA based blends evaluated by liquid substitution method [24] Sample

Density

Porosity

(g/cm3)

(%)

PLA

1.29

0.6

PLA30W

0.46

59.9

PLAPHB20W

1.20

0.2

PLA30PHB20W

0.46

61.1

PLAPCL20W

1.13

0.4

PLA30PCL20W

0.47

57.3

PLAPEGW

1.18

0.6

PLA30PEGW

0.46

62.2

PLASP10

1.12

0.3

PLA30SP10W

0.45

50.9

Table 3: Young’s modulus (E), tensile strength (b) and elongation at break (b) of parent PLA and PLA based composite blends before and after water treatment. Sample

Before water treatment

After water treatment

E

b

b

E

b

b

[GPa]

[MPa]

[%]

[GPa]

[MPa]

[%]

PLA

3.0±0.1

56±2

2.8±0.4

3.3±0.1

54±4

2.8±0.7

PLA30

4.3±0.1

31±3

1.1±0.1

1.3±0.1

18±1

1.8±0.2

PLAPCL20

2.4±0.5

32±1

3±1

2.6±0.03

35±2

3±1

PLA30PCL20

1.5±0.4

15±1

3±1

0.8±0.03

11±1

4±1

PLAPHB20

2.5±0.1

38±7

3.0±1

2.5±0.1

42±6

2.6±0.4

PLA30PHB20

2.6±0.5

24±1

1.4±0.2

1.2±0.1

13±1

1.5±0.2

PLA30SP10

5.3±0.1

45±5

1.0±0.2

1.5±0.1

20±1

2.1±0.3

PLA30PEG

3.5±0.2

22±3

1.6±0.4

1.2±0.1

14±1

1.9±0.2

Figure 1: Novel production method of porous PLA-based materials for cells seeding and colonization.

Figure 2: SEM images of parent PLA30 blend (a) before water and (b, c, d) after water. The table refers to the C, O and Na content calculated by elemental analysis in the numbered portions of the blend before and after water treatment. Scale bars a: 100 µm, b: 200 µm, c: 20 µm and d: 10 µm.

Figure 3: SEM images of PLA-based composites blends. (a) PLA30PCL20, (b) PLA30PCL20W, (c) PLA30PHB20, (d) PLA30PHB20W, (e) PLA30PEGW and (f) PLA30SP10W. From SEM image PLA30PCL20 is a biphasic blend.

Figure 4: DSC traces (second heating scan) of porous PLA based blends.

Figure 5: Representative tensile stress-strain curves for PLA-based composites blends before and after water treatment compared to parent PLA composites.

Figure 6: Cell viability and proliferation of mouse embryo fibroblasts balb/3T3 clone A31 cultured on (a) parent PLA disks (b) below parent PLA disks on TCPS (c) PLAPHB20 and PLA30PHB20W (d) PLASP10 and PLA30SP10W and (e) PLAPCL20 and PLA30PCL20W blends evaluated by WST-1 assay.

Figure 7: CLSM micrographs of mouse embryo fibroblasts balb/3T3 clone A31 grown on (a) parent PLA 0.21 (a, e), PLA30W 0.17 (b, f), PLA30W 0.32 (c, g) blend disks and glass (d, h), (b) compact PLAPHB20, (c) porous PLA30PHB20W, (d) compact PLASP10, and (e) porous PLA30SP10W blends at 7 and 10 days. Scale bar represents 100 μm on 20X magnification and 30 μm on 60X magnification.

Figure 8: SEM images of balb/3T3 clone A31 cells grown on (a-f) compact PLAPCL20 and (gl) porous PLA30PCL20W blends at 3, 7 and 14 days.

Figure 9: CLSM micrographs of mouse embryo fibroblasts balb/3T3 clone A31 grown on (a) compact PLAPCL20 and (b) porous PLA30PCL20W blends at 3, 7 and 14 days. Scale bar represents 200 μm 10X magnification, 100 μm on 20X magnification and 30 μm on 60X magnification.

Figure 10: CLSM micrographs of mouse embryo fibroblasts balb/3T3 clone A31 grown on lower surface of porous PLA30PCL20W blends at day 7 (a-b) and 14 (c-d).