polymers Article
Fabrication, Characterization, and Cytotoxicity of Thermoplastic Polyurethane/Poly(lactic acid) Material Using Human Adipose Derived Mesenchymal Stromal Stem Cells (hASCs) 3 and Krzysztof Marycz 2,4, * Anna Lis-Bartos 1,2 , Agnieszka Smieszek 2 , Kinga Franczyk ´ 1 2 3 4
*
AGH University of Science and Technology, Department of Biomaterials and Composites, Faculty of Materials Science and Science and Ceramics, Krakow 30-059, Poland;
[email protected] Department of Experimental Biology, Wroclaw University of Environmental and Life Sciences, Wroclaw 50-375, Poland;
[email protected] AGH University of Science and Technology, Faculty of Electrical Engineering, Automatics, Computer Science and Biomedical Engineering, Krakow 30-059, Poland;
[email protected] Faculty of Veterinary Medicine, Equine Clinic-Equine Surgery, Justus-Liebig-University, Gießen 35392, Germany Correspondence:
[email protected]; Tel.: +48-71-3205-201; Fax: +48-71-320-5854
Received: 25 July 2018; Accepted: 25 September 2018; Published: 28 September 2018
Abstract: Thermoplastic polyurethane (TPU) and poly(lactic acid) are types of biocompatible and degradable synthetic polymers required for biomedical applications. Physically blended (TPU+PLA) tissue engineering matrices were produced via solvent casting technique. The following types of polymer blend were prepared: (TPU+PLA) 7:3, (TPU+PLA) 6:4, (TPU+PLA) 4:6, and (TPU+PLA) 3:7. Various methods were employed to characterize the properties of these polymers: surface properties such as morphology (scanning electron microscopy), wettability (goniometry), and roughness (profilometric analysis). Analyses of hydrophilic and hydrophobic properties, thermogravimetric analysis (TGA), and differential scanning calorimetry (DSC) of the obtained polymer blends were conducted. Tensile tests demonstrated that the blends exhibited a wide range of mechanical properties. Cytotoxicity of polymers was tested using human multipotent stromal cells derived from adipose tissue (hASC). In vitro assays revealed that (TPU+PLA) 3:7 matrices were the most cytocompatible biomaterials. Cells cultured on (TPU+PLA) 3:7 had proper morphology, growth pattern, and were distinguished by increased proliferative and metabolic activity. Additionally, it appeared that (TPU+PLA) 3:7 biomaterials showed antiapoptotic properties. hASC cultured on these matrices had reduced expression of Bax-α and increased expression of Bcl-2. This study demonstrated the feasibility of producing a biocompatible scaffold form based on (TPU+PLA) blends that have potential to be applied in tissue engineering. Keywords: thermoplastic polyurethane; poly(lactic acid), polymer blends; stem cells; osteochondral tissue engineering
1. Introduction Tissue engineering (TE) and material science are valuable tool in regenerative medicine [1,2]. Until now, tissue engineering has been successfully used to obtain promising materials dedicated to the regeneration of various tissue, including soft and hard tissues [3,4]. Particular attention is paid to the development of polymer-based matrices, especially in terms of tailoring their chemical, physical, and biological properties, in order to obtain the most cytocompatible products [5–8]. Moreover,
Polymers 2018, 10, 1073; doi:10.3390/polym10101073
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polymers are important and attractive biomaterials due to high application potential, including scaffold design using three dimensional (3D) technology, which is fundamental in personalized medicine [9,10]. Obviously polymers used in the field of tissue engineering should be characterized in terms of their biocompatibility, biodegradability, but also immunomodulatory and biomechanical properties [11,12]. Both poly(lactic acid) (PLA) and thermoplastic polyurethane (TPU) are synthetic polymers commonly used in biomedical applications, however they differ in mechanical properties [5,13,14]. The former polymer has rigid mechanical properties, while the latter is characterized by flexible mechanical properties [6]. It is worth mentioning that appropriate mechanical properties of porous scaffolds determine various important aspects of tissue healing such as augmentation and filling of the defect, further promoting proper tissue repair [15]. Thus, the current TE perspectives are focused on manufacturing an ideal scaffold, characterized by mechanical features that would perfectly match the native tissue to be repaired [16]. Poly(lactic acid) (PLA)-based scaffolds have been widely used to promote bone and cartilage regeneration as well as to provide a substitute for blood vessels [7]. However, PLA scaffolds are characterized by brittle properties and relative high strength, which limits their applicability [5,17]. In turn, thermoplastic polyurethane (TPU) has been characterized as a polymer with a wide suitability in medical applications, not limited to the promotion of bone and cartilage regeneration [5,6,8]. For example TPU can be used to repair and reconstruct mechanically active soft tissues including blood vessels, cardiac walls and valves, or urinary bladders and skin [5]. TPU scaffolds are also characterized by high elongation, an attractive Young’s modulus, as well as excellent abrasion and tear resistance. Therefore, it seems reasonable to combine thermoplastic polyurethane with poly(lactic acid) (TPU+PLA) to create an attractive hybrid material that could have a wider spectrum of applicability in the field of regenerative medicine [5,8]. We decided to test TPU+PLA blend cytocompatibility using the model of human multipotent stromal cells originated from adipose tissue (hASCs). This aspect is important bearing in mind that current efforts in TE are also aimed on adapting TPU+PLA blend to obtain biomaterials suitable for multiple tissue applications. The multipotent character of those cells refers to their ability to differentiate, inter alia into bone and cartilage-forming cells [18–20]. Recently, adipose derived multipotent stromal cells have increasingly been used for orthopedic-based tissue engineering [21–23]. ASCs are for various reasons recognized as a powerful therapeutic tool in the field of regenerative medicine. ASCs are characterized by high proliferative and secretory activity [24,25]. The fact that ASCs are able to differentiate into osteoblasts, chondroblasts, and adipocytes, makes them very attractive in terms of broad range of potential clinical application [18,20]. More importantly, they exhibit an immunomodulatory ability, improving the regenerative capacity during wound healing [24]. Bearing in mind all aforementioned facts, hASCs are also commonly applied as a model for testing of biomaterials cytocompatibility [26–28]. It has been previously shown by our and other groups that cellular plasticity of hASCs is a feature that enables to perform valuable screening tests of various biomaterials, both natural and synthetic polymers, as well as metal-based implants [8,26–30]. The analysis of cytophysiological properties of hASCs such as morphology, proliferation, and metabolic activity in cultures with newly designed scaffolds is particularly important when potential autologous transplants are considered [31,32]. This study was aimed at the fabrication and characterization of (TPU+PLA) blends prepared by dissolving them in mixed organic solvents. The obtained (TPU+PLA) blends were used to produce films characterized by different Young’s moduli (E) in the MPa range and various chemical compositions. Generally, the following types of polymer blends were prepared (TPU+PLA) 7:3, (TPU+PLA) 6:4, (TPU+PLA) 4:6, and (TPU+PLA) 3:7. We decided to investigate biomaterials’ morphology, contact angle, and mechanical properties because surface and volume wettability of scaffolds intensively affects cell attachment and viability. We also decided to test the cytotoxicity of the obtained biomaterials using hASC model due to the fact that our goal was to obtain scaffolds for progenitor cells and matrices that could be potentially used in multiple tissue applications. Moreover, the proliferation of hASCs was studied as an effect of surface stiffness, which depends on the polymer blend composition.
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2. Materials and Methods Thermoplastic polyurethane—TPU (Ellastolan 1102A, PAN, Wroclaw, Poland) is a high flexible elastomers that provides elasticity to the designed blends. Its glass transition temperature (Tg ) is −50 ◦ C (temperature founded by DSC test). Poly(lactic acid)—PLA (PURASORB, Nederland) was chosen to improve the rigidity (physical stability) of the polymer blends. Its glass transition temperature is 60 to 63 ◦ C (based on completed DSC test). The N’N dimethylformamide (DMF) and dichloromethane (DCM) of analytical grade, as solvents were purchased from Sigma-Aldrich (Poland, Poznan). 2.1. Polymer Blends Fabrication Polymer films (cell culture matrices) were fabricated using a solvent casting method. Five percent thermoplastic polyurethane/poly(lactic acid) combinations were prepared by dissolving TPU and PLA in N’N dimethyloformamide and dichloromethane mixture. Processing conditions: (TPU+PLA) different composition (5% (w/w)) were dissolved in an 8:2 mixture of DMF and DCM by warming at ca. 35 ◦ C with magnetic stirring until the polymer solution became clear. This temperature was above the boiling point of DCM (39.6 ◦ C) and glass transition temperature of poly(lactic acid) (~60 ◦ C). Polyurethane and poly(lactic acid) were selected because of its known biocompatibility, easy of processing, and extensive use in the previous studies [26]. The polymers solution was sealed in glass bottle prior to use and used immediately after opening to ensure that minimal solvents were lost via evaporation. Polymer films were prepared by solvent casting onto glass Petri dish as previously described [26]. The following types of polymer blend were prepared (TPU+PLA) 7:3, (TPU+PLA) 6:4, (TPU+PLA) 4:6, and (TPU+PLA) 3:7. In comparison, (TPU+PLA) films were also fabricated by this technique but only DMF (with a high boiling point: 153 ◦ C) was used as a solvent with low evaporation rate. 2.2. Biomaterials Characterization 2.2.1. Scanning Electron Microscopy (SEM) The surface morphology and microstructure of the prepared polymer films was observed by scanning electron microscopy (SEM). SEM imaging was performed using Nova Nano SEM 200 microscope (FEI Europe Company, Hillsboro, OR, USA). The samples were fractured by a surgical blade. Prior to imaging scaffolds were made conductive with a thin layer of carbon by using a sputter-coater. Scanning electron microscopy was used for studying the surface morphology of determined by polymer blend composition. 2.2.2. Surface Wettability Goniometry was used to measure the surface contact angles of the various polymer blend formulations. This method provides information on surface hydrophilicity or hydrophilicity; important factors for in vitro tests. The surface wettability of prepared thin polymer films was measured by water contact angle analysis. Contact angle of the (TPU+PLA) films were measured on air atmosphere and at room temperature. The water contact angle of thin polymer samples was measured via sessile drop technique using a goniometer DSA 10 Mk2 (Kruss, Hamburg, Germany) equipped with internal image analysis software. Distilled water (purchase from Sigma Aldrich, Poznan, Poland) drops of the volume of 0.5 µL were put on each dry polymer films and the static contact angle was measured immediately. Contact angle was calculated by averaging the results of ten measurements (ten region in specimen). Wetting process was recorded using a digital camera.
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2.2.3. Surface Roughness Topography (surface roughness, as important feature for cell culture) of obtained polymer films were characterized by profilometry (Hommelwerke profilometer, Villingen-Schwenningen, Germany). The average roughness (Ra ) of polymer samples were determined. The various regions of each materials were examined. The presented results herein are the average of ten measurements (±standard deviation). 2.2.4. Mechanical Properties Evaluation The mechanical properties of the (TPU+PLA) different blends were determined by tensile test. Mechanical properties of polymer thin films were determined in tensile uniaxial mode with the use ZWICK universal testing machine model 1435, using the crosshead speed of 5 mm/min. Samples examined had the dimensions 60 mm × 5 mm. Depending on the polymer blend composition the thickness of the films varied from 50 µm to 0.1 mm. Young’s modulus (E) of the polymer blends was determined as the slope of the straight line stress–strain curve. Young’s modulus were obtained as the mean and standard deviation of ten measurements. 2.2.5. Thermal Degradation Test The thermal properties of obtained polymer blends and pure polymers were examined by thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC) The thermal degradation (thermostability) studies of the PLA, TPU, and (TPU+PLA) blends were carried out in NETZSCH analyzer, as well as the thermal properties of the blends measured by DSC. The samples (10 mg) were degraded under nitrogen atmosphere at the heating rate of 10 ◦ C/min. The samples were scanned from 20 to 600 ◦ C for TGA and DSC curve definition. 2.2.6. Sterilization Pure polymer and (TPU+PLA) blend films were cut into small disks (15 mm in diameter) with the aid of leather borer in order to locate the disks into 24 well tissue culture plates. All the samples were prewetted and sterilized in ethanol for 10 min, and then ethanol was evaporated. Dried disk was packed and sterilized by peroxide hydrogen cold plasma sterilization method. 2.3. Assessment of Biomaterials Cytocompatibility Using Model of Human Adipose Derived Mesenchymal Stromal Stem Cells (hASCs) 2.3.1. The Characteristics of Human Adipose-derived Stromal Cells (hASCs) Used for the Experiment The cells used for the current study were isolated using well-established method described previously in detail [26,33]. Subcutaneous adipose tissue was collected both from male and female subjects, age range 20 to 33 (n = 8, mean age 24 ± 1.5 years). All donors had given written consent prior to the procedure. The characteristic phenotype indicating on mesenchymal origin of hASCs was also determined using well established protocol and accordingly to the criteria established by International Society of Cellular Therapy [31,34]. The immunophenotyping was performed after third passage (p3) using protocol published previously [27]. The cells used for the experiment expressed the specific surface markers such as CD44, CD73, CD90, and CD105. Moreover hASCs used here did not expressed markers of hematopoietic origin i.e., CD34 and CD45. The cells underwent the osteogenic, chondrogenic, as well as adipogenic differentiation exhibiting their multipotent character. 2.3.2. The Metabolic Activity and Proliferation of hASCs Cultures on Biomaterials The cytocompatibility assay was performed in 24-well dishes covered with TPU+PLA films. For the test cells were inoculated at density equal 45,000 cells per well, suspended in a 0.5 mL of complete growth medium (CGM) consisting of Dulbecco’s Modified Eagle’s Medium with nutrient
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F-12 Ham supplemented with 10% of fetal bovine serum (FBS) and 1% antibiotic-antimycotic solution. Each culture was prepared at least in nine replicates. Cultures were propagated in aseptic conditions in CO2 incubator (37 ◦ C, 95% humidity and 5% CO2 ). The metabolic activity of hASCs was measured 120 h of cultures using resazurin-based assay (Alamar Blue dye, Sigma Aldrich, Poznan, Poland). The test was conducted accordingly to the protocol described in previously and in accordance with the manufacturer’s suggestions [26]. The metabolic activity of hASCs cultured on biomaterials was compared with the metabolic activity of cultures propagated on polystyrene (P/S) and expressed as metabolic factor (MF). The proliferation of cells was measured by detection of 5-bromo-2-deoxyuridine (BrdU) incorporation test. For this purpose after 120 h of cultures the cells were detached from the biomaterials surface and polystyrene (P/S) using TrypLE Express solution. Cells were then washed with Hanks’s Balanced Salt Solution (HBSS) containing 2% FBS, centrifuged, and resuspended at the total amount in a 96-well plates in six replications. Next, plates were cultured for 24 h and additionally treated with BrdU reagent and incubated overnight at 37 ◦ C in humidified atmosphere of 5% CO2 and 95% air. The BrdU assay was then performed using the general protocol, described in detail previously [35]. The reagents used for cell cultures and metabolic assay derived from Sigma Aldrich, Poznan, Poland. The BrdU assay as well as TrypLE Express solution were purchased from Abcam (Cambridge, UK) and Thermo Fisher Scientific (Waltham, MA, USA), respectively. 2.3.3. The Morphology of hASCs Cultures on Biomaterials The attachment of hASCs onto biomaterials surface, as well as the morphology and growth pattern of cultures were determined after 120 h of cells propagation. The microscopic evaluation of hASCs on biomaterials surface was performed using scanning electron microscope (SEM, Auriga 60, Zeiss, Oberkochen, Germany) and confocal microscope Cell Observer SD (Zeiss, Oberkochen, Germany) as described previously [36–38]. To prepare samples for observations the cultures were fixed with 4% paraformaldehyde. The specimens were either dehydrated in graded ethanol series (concentrations ranges from 50% to 100%, every 10%) for SEM analysis or stained with fluorescent dyes for confocal microscope observations. To visualize the nuclei and cytoskeleton the cultures were stained with diamidino-2-phenylindole (DAPI; 1:800) and atto-488-labeled phalloidin (1:800). The staining was performed using well established protocols [8,26,30]. All reagents used during this protocol derived from Sigma Aldrich, Poznan, Poland. 2.3.4. The Analysis of Antiapoptotic and Proapoptotic Bcl-2 Proteins After 120 h of culture the cells were harvested from the biomaterials and treated with TRI Reagent (Sigma Aldrich, Poznan, Poland). The total RNA isolation was performed using well-established protocol by Chomczynski and Sacchi [39]. Obtained RNA was diluted in DEPC-treated water, and its quantity and purity was determined using NanoDrop 8000 (ThermoFisher Scientific, Waltham, MA, USA). The genomic DNA (gDNA) traces were removed using prepared using PrecisionTMDNase kit (Primerdesign Ltd., Cambridge, UK). The cDNA was obtained from 0.5 µg of RNA using transcription Tetro cDNA Synthesis Kit (Bioline Reagents Ltd., London, UK). For qPCR the total volume of reaction mix (SensiFast SYBR & Fluorescein Kit; Bioline Reagents Ltd., London, UK) was 20 µL. The cDNA did not exceed 10% of the final PCR reaction volume. The specific primers used in concentration equal 400 nM derived from Sigma Aldrich (Poznan, Poland). The expression of B-cell lymphoma 2 (Bcl-2) was detected using the following primers forward 50 -ATCGCCCTGTGGATGACTGAG-3 and reverse 50 -CAGCCAGGAGAAATCAAACAGAGG-30 (129 bp). The sequences of the primers used for Bcl-2-associated X protein (Bax-α) amplification were as follows forward 50 -ACCAAGAAGCTGAGCGAGTGTC-30 and reverse 50 -ACAAAG ATGGTCACGGTCTGCC-30 (365 bp). The relative gene expression analysis (normalized quantity/Qn) was calculated in relation to a housekeeping gene, i.e., glyceraldehyde 3-phosphate dehydrogenase. The following primers were used forward 50 -GTCAGTGGTGGACCTGACCT-30 , reverse 50 -CACCACCCTGTTGCTGTAGC-30 (256 bp). The qPCR was performed using CFX Connect Real-Time
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PCR Detection System (Bio-Rad Polska Sp. z.o.o., Warszawa, Poland) using protocol precisely described elsewhere [40,41]. For the total Bax-α measurement cells were detached from biomaterials and lysed using ice-cold RIPA extraction buffer with the cocktail of protease and phosphatase inhibitors. The total protein in cell lysates was quantified using the Bicinchoninic Acid Kit Assay Kit (Sigma Polymers 2018, 10, xPEER FOR REVIEW PEER REVIEW Polymers 2018, 10, x FOR REVIEW 6 of 156 of 156 of 15 Polymers 2018, 10, x PEER FOR Aldrich, Poznan, Poland). The concentration of Bax-α was determined using sandwich ELISA derived from R&D Systems (Minneapolis, MN, USA). For each measurement samples with concentration of 30 sandwich ELISA derived from R&D Systems (Minneapolis, MN, For each measurement sandwich ELISA derived from R&D Systems (Minneapolis, MN,MN, USA). ForUSA). each measurement sandwich ELISA derived from R&D Systems (Minneapolis, USA). For each measurement µg/mL of total protein were used. The protocol of protein assays were conducted on abstract-treated samples with concentration 30 of total protein were The of protocol ofassays protein assays samples with concentration of 30ofμg/mL ofμg/mL total protein were used. Theused. protocol protein samples with concentration 30 of μg/mL of total protein were used. The protocol of protein assays cells according toon theabstract-treated manufacturer’s instructions. The readinstructions. oninstructions. a microplate reader were conducted on abstract-treated cells according to manufacturer’s thewas manufacturer’s instructions. The werewere conducted cells according to absorbance the manufacturer’s The The conducted on abstract-treated cells according to the Spectra MaxPlus (Molecular Devices, Syngen, Wroclaw, Poland) at 450 nm with wavelength correction absorbance wasa on read on a microplate reader Spectra MaxPlus (Molecular Devices, Syngen, Wroclaw, absorbance was was read on microplate reader Spectra MaxPlus (Molecular Devices, Syngen, Wroclaw, absorbance read a microplate reader Spectra MaxPlus (Molecular Devices, Syngen, Wroclaw, 450with nm with wavelength correction 540tested nm.tested All tested samples as standards well standards Poland) atPoland) 450 wavelength set toset540 nm. All samples as well standards at nm 450atwith nm wavelength correction toset 540to nm. All samples as well set toPoland) 540 nm. All tested samples ascorrection well standards were measured in three replicates. were measured in three replicates. werewere measured in three replicates. measured in three replicates.
2.4. Statistical Analysis 2.4. Statistical Analysis 2.4. Statistical Analysis 2.4. Statistical Analysis
The results obtained from biological assays are expressed as the mean values with standard The results obtained from biological are expressed the mean values with standard The The results obtained from biological assays areassays expressed as the mean values with standard results obtained from biological assays are expressed as theas mean values with standard deviation (SD). One-way analysis of variance and Dunnett’s post hoc analysis was done to determine deviation (SD). One-way analysis of variance and Dunnett’s post hoc analysis was done to determine deviation (SD). One-way analysis of variance and Dunnett’s post hoc analysis was done to determine deviation (SD). One-way analysis of variance and Dunnett’s post hoc analysis was done to determine statistical significance (p < 0.05). The data were analyzed using GraphPad Software (Prism 5.04, statistical significance (pThe < 0.05). The were analyzed using GraphPad Software (Prism statistical significance (p < (p 0.05). datadata weredata analyzed using GraphPad Software (Prism 5.04, statistical significance < 0.05). The were analyzed using GraphPad Software (Prism 5.04, 5.04, GraphPad Software, La Jolla, CA, USA). GraphPad Software, La Jolla, CA, USA). GraphPad Software, La Jolla, CA, USA). GraphPad Software, La Jolla, CA, USA).
3. Results
3. Results 3. Results 3. Results
3.1. Fabrication and Microstructure
3.1. Fabrication and Microstructure 3.1. Fabrication and Microstructure 3.1. Fabrication and Microstructure
Polyurethanes are widely existing synthetic biodegradable or nondegradable hydrophilic Polyurethanes areexisting widely existing synthetic biodegradable or nondegradable hydrophilic Polyurethanes are are widely synthetic biodegradable or nondegradable hydrophilic Polyurethanes widely existing synthetic biodegradable or nondegradable hydrophilic polymers. It can be used to composed with more hydrophilic poly(lactic acid) polymer blends to polymers. can becomposed used to composed more hydrophilic poly(lactic acid) polymer polymers. It can used to withwith morewith hydrophilic poly(lactic acid)acid) polymer blends to blends polymers. It be canIt be used to composed more hydrophilic poly(lactic polymer blends to to improve biocompatibility, degradability, hydrophilicity, and cell affinity of polymeric blend materials. improve biocompatibility, degradability, hydrophilicity, and cell affinity of polymeric improve biocompatibility, degradability, hydrophilicity, and cell affinity of polymeric blend improve biocompatibility, degradability, hydrophilicity, and cell affinity of polymeric blendblend A simple technique to fabricate of composition blends was solvent casting method. However, materials. A simple technique tocomposition fabricate acomposition various composition ofwas blends wascasting solvent casting materials. A simple technique toa various fabricate a various of blends solvent materials. A simple technique to fabricate a various of blends was solvent casting polyurethanes are generally insoluble in common organic solvent. Dichloromethane is usually used to method. However, polyurethanes are generally insoluble in common organic solvent. method. However, polyurethanes are are generally insoluble in common organic solvent. method. However, polyurethanes generally insoluble in common organic solvent. fabricate PLA films and other forms of for its good solubility with PLA polymers Dichloromethane isused usually used to PLA fabricate PLA films and other forms offamily scaffolds its and good Dichloromethane is usually to fabricate films and other forms of scaffolds for its good Dichloromethane is usually used to scaffolds fabricate PLA films and other forms of scaffolds for itsforgood solubility with PLA family polymers and itsevaporation fast evaporation rate. Some polyurethanes are solubility withwith PLAPLA family polymers and its evaporation rate. Some polyurethanes are soluble insoluble its fast evaporation rate. Some polyurethanes are soluble inrate. N’NSome dimethyloformamide with ainhighin solubility family polymers andfast its fast polyurethanes are soluble ◦ with N’N(T dimethyloformamide with a boiling high boiling (T B =°C). 153preparation °C).preparation For blends preparation of form different form N’NN’N dimethyloformamide apreparation high boiling point (TB point =(T 153 For of different dimethyloformamide with a high point B = °C). 153 For of form boiling point For of different form of (TPU+PLA) wedifferent use DMF mixed B = 153 C). ◦ of (TPU+PLA) blends we use DMF mixed with a DCM with a lowest boiling point (less than 40°). of (TPU+PLA) blends we use DMF mixed with a DCM with a lowest boiling point (less than 40°). In of (TPU+PLA) blends we use DMF mixed with a DCM with a lowest boiling point (less than 40°). In with a DCM with a lowest boiling point (less than 40 ). In this study, a mixed solvent of DCM and DMFIn this study, a mixed of DCM and DMF =was 8:2) was as asolvent suitable solvent for both PLA this study, awas mixed solvent ofsolvent DCM and DMF (v/v =(v/v 8:2) was tested asItatested suitable for both PLA this study, atested mixed of DCM and for DMF =(v/v 8:2) tested as abeen suitable solvent for both (v/v = 8:2) assolvent a suitable solvent both PLA and TPU. has found that TPU arePLA soluble and TPU. Itbeen has been found thatsoluble TPUsoluble are soluble insolvent organic solvent mixtures when the concentration and TPU. It has been found that TPU are in organic mixtures when the concentration and TPU. It has found that TPU are in organic solvent mixtures when the concentration in organic solvent mixtures when the concentration of DCM is 20% (v/v). Moreover the DMF and DCM is(v/v). 20% (v/v). Moreover the DMF and DCM mixture can runspeed the speed of solvent evaporation of DCM isof20% (v/v). Moreover the DMF and DCMDCM mixture can run speed of solvent evaporation of DCM is 20% Moreover the DMF and mixture can the run the of solvent evaporation DCM mixture can run the speed of solvent evaporation from polymer based materials. Subsequently, from polymer based materials. Subsequently, the more energetic evaporation of DCM might fromfrom polymer based materials. Subsequently, the more energetic evaporation of DCM might result polymer based materials. Subsequently, the more energetic evaporation of DCM might resultresult the more energetic evaporation of DCM might result in a better porous structure. The microstructure of inporous a better porous structure. The microstructure the blends polymer blends andsamples control samples in a in better structure. The The microstructure of the and control was a better porous structure. microstructure of polymer theofpolymer blends and control samples was was the polymer control samples was observed by1ain scanning electron microscopy. Figures in observed byand a electron scanning electron microscopy. Table 1 show examples the SEM observed by ablends scanning microscopy. Figures inFigures Table show examples of the images ofimages observed by a scanning electron microscopy. Figures in Table 1 show examples ofSEM theofSEM images of of Table 1 show examples of the SEM images of polymer films. A good integration of polyurethane and polymer films. A good integration of polyurethane and poly(lactic acid) can be found polymer films. A good integration of polyurethane and and poly(lactic acid)acid) phase canphase be in the polymer films. A good integration of polyurethane poly(lactic phase canfound be found in thein the poly(lactic acid)magnification. phase can be found inexamination the examination SEMofmagnification. examination offrom the SEM Microscopy of polymer the Microscopy polymer blend specimens all four SEMSEM magnification. Microscopy examination the polymer blend specimens fromfrom all four magnification. Microscopy of the blend specimens all polymer four blend specimens from alltheir four compositions confirm their surface roughness. compositions confirm their surface roughness. compositions confirm their surface roughness. compositions confirm surface roughness. Table 1. images SEM images offilms thinmade films made from PLA, TPU and (TPU+PLA) blends. Table 1.1.SEM images of thin films made fromfrom PLA,PLA, TPU TPU and blends. Table SEM images of thin TPU(TPU+PLA) and(TPU+PLA) (TPU+PLA) blends. Table 1. SEM of thinfilms made from PLA, and blends. TPU 100% (control) TPU TPU 100%100% (control) (control) TPU 100% (control)
(TPU+PLA) (TPU+PLA) 7:3 7:3 (TPU+PLA) 7:3 7:3 (TPU+PLA)
(TPU+PLA) (TPU+PLA) 6:4 6:4 6:4 (TPU+PLA) (TPU+PLA) 6:4
(TPU+PLA) (TPU+PLA) 4:6 (TPU+PLA) 4:6 4:6 4:6 (TPU+PLA)
(TPU+PLA) (TPU+PLA) (TPU+PLA) 3:7 3:7 (TPU+PLA) 3:7 3:7
PLA (control) PLA 100% (control) PLA100% 100%100% (control) PLA (control)
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Table 1. Cont.
3.2. Surface Wettability Surface Wettability 3.2.3.2. Surface Wettability The The surface hydrophilic or hydrophobic of polymer thinthin films matrices has ahas major on on on The surface hydrophilic or hydrophobic of polymer thin films matrices has ainfluence major influence surface hydrophilic or hydrophobic of polymer films matrices a major influence cell cell adhesion and and proliferation behavior. The The hydrophilicity of matrices has has an has influence on the cell adhesion and proliferation behavior. The hydrophilicity of matrices influence adhesion proliferation behavior. hydrophilicity of matrices an an influence on on the the surface energy, which might influence the serum proteins to adhere on the and and in and turn surface energy, which might influence serum proteins to adhere on matrices, in turn surface energy, which might influence the the serum proteins to adhere on matrices, the the matrices, in turn govern the biological response, suchsuch assuch cell adhesion, proliferations, and and differentiations. Table 2Table govern biological response, as cell adhesion, proliferations, and differentiations. govern the the biological response, as cell adhesion, proliferations, differentiations. Table 2 2 shows result ofresult contact angle measurement for pure TPU, PLA, and (TPU+PLA) films. Generally the the the shows of contact angle measurement pure TPU, PLA, and (TPU+PLA) films. Generally shows result of contact angle measurement for for pure TPU, PLA, and (TPU+PLA) films. Generally hydrophilicity of PLA films was higher than TPU and its and blends, proving that that thethat hydrophilicity of of of hydrophilicity of PLA films was higher than TPU its blends, proving hydrophilicity hydrophilicity of PLA films was higher than TPU and its blends, proving the the hydrophilicity polymer blends increased with increasing poly(lactic acid) concentration. The wettability of polymer blends increased with increasing poly(lactic acid) concentration. wettability polymer blends increased with increasing poly(lactic acid) concentration. TheThe wettability of of (TPU+PLA) polymer filmsfilms was found from 80° for PLA films tofilms 123° for123° TPU films. The significant (TPU+PLA) polymer films was found from PLA to TPU films. The significant (TPU+PLA) polymer was found from 80°80° for for PLA films to 123° for for TPU films. The significant differences in the angle values of TPU, and (TPU+PLA) blends were observed. Results differences in the contact angle values ofPLA, TPU, PLA, and (TPU+PLA) blends were observed. Results differences in contact the contact angle values of TPU, PLA, and (TPU+PLA) blends were observed. Results 3.2. Surface Wettability 3.2. Surface Wettability 3.2. Surface Wettability 3.2. Surface Wettability of our show that thethat incorporation of PLA in PLA TPU hasTPU major effect towards the hydrophilicity of our studies show incorporation of in major effect towards hydrophilicity of studies our studies show that the the incorporation of PLA in TPU hashas major effect towards the the hydrophilicity of (TPU+PLA) films, compared with the hydrophobic TPU films. ofsurface (TPU+PLA) films, compared with the hydrophobic TPU films. of (TPU+PLA) films, compared with the hydrophobic TPU films. The surface hydrophilic or hydrophobic ofthin polymer thin films matrices has a major influence on The hydrophilic or hydrophobic of polymer films matrices has ahas major on The surface hydrophilic or of thin films matrices has ainfluence major influence The surface hydrophilic orhydrophobic hydrophobic of polymer polymer thin films matrices a major influence on on cell adhesion and proliferation behavior. The hydrophilicity of has matrices has an influence cell adhesion adhesion and proliferation behavior. The hydrophilicity of an on cell adhesion proliferation behavior. hydrophilicity of matrices an influence on surface theon the and and proliferation behavior. TheThe hydrophilicity ofmatrices matrices hashas an influence influence on the the 3.3.surface Surface Roughness 3.3. Surface Roughness 3.3.energy, Surface Roughness surface energy, which might influence the serum to the and matrices, in the turn surface which might influence the serum proteins toproteins adhere onadhere the in turn energy, which might influence the serum proteins toon adhere on matrices, theonmatrices, and inand turn energy, which might influence the serum proteins to adhere the matrices, and in turn govern govern the biological response, such as cell adhesion, proliferations, and differentiations. Table govern the biological response, such as cell adhesion, proliferations, and differentiations. Table 2 govern the biological response, such as cell adhesion, proliferations, and differentiations. Table 2 Topography, among other chemical and physical factors such as substrate stiffness and polymer Topography, among other chemical and physical factors such as substrate stiffness and polymer Topography, among other chemical and physical factors such as substrate stiffness and polymer biological response, such as cell adhesion, proliferations, and differentiations. Table 2 shows result of 2 shows result contact angle for pure TPU, and (TPU+PLA) films. Generally the shows result of composition, contact angle measurement for pure PLA, and (TPU+PLA) films. Generally theof the shows result of contact measurement for pure TPU, PLA, and (TPU+PLA) films. Generally blend composition, isof known to have ahave great influence on PLA, cell behavior. Optimization blend isforknown to aTPU, great influence on cell behavior. Optimization blend composition, isangle known tomeasurement have a great influence on films. cell behavior. Optimization of of contact angle measurement pure TPU, PLA, and (TPU+PLA) Generally the hydrophilicity hydrophilicity of PLA films was higher than TPU and its blends, proving that the hydrophilicity hydrophilicity of PLA films was higher than TPU and its blends, proving that the hydrophilicity of hydrophilicity of PLA films was higher than TPU and its blends, proving that the hydrophilicity of topographical features, such as surface roughness, is the one of the key strategy to obtain the best topographical features, such as surface roughness, is the one ofhydrophilicity the strategy topolymer obtain bestof topographical features, such as surface is the one ofthe the keykey strategy toofobtain the the best of PLA films was higher than TPU and itsroughness, blends, proving that blends polymer blends increased with increasing poly(lactic acid) concentration. The wettability polymer blends increased with increasing poly(lactic acid) concentration. The wettability of polymer blends increased with increasing poly(lactic acid) concentration. The wettability of cellular scaffolds for various tissue applications. ThisThis work investigated the the influence of polymer cellular scaffolds various tissue applications. This work investigated the influence of polymerof cellular scaffolds for for various tissue applications. work investigated influence of polymer increased with increasing poly(lactic acid) concentration. The wettability of (TPU+PLA) polymer films (TPU+PLA) polymer films was found from 80° for PLA films to 123° for TPU films. The significant (TPU+PLA) polymer films was found from 80° for PLA films to 123° for TPU films. The significant (TPU+PLA) polymer films was found from 80° for PLA films to 123° for TPU films. The significant blend composition on surface roughness. The◦The average roughness (Ra) (R ofathe poly(lactic blend composition on surface roughness. The average roughness )the of the polyurethane, poly(lactic blend composition on surface roughness. average roughness )(R ofapolyurethane, polyurethane, poly(lactic ◦ for was found from 80 PLA films to 123 for TPU films. The significant differences in the contact differences in the contact angle values of PLA, and (TPU+PLA) blends were Results differences in the angle values of TPU, PLA, and (TPU+PLA) blends were observed. Results differences incontact the contact angle values ofin TPU, PLA, and blends observed. Results acid), and (TPU+PLA) blends are presented Table 2.Table The average roughness ofwere all blends acid), and (TPU+PLA) blends are presented in 2.(TPU+PLA) The average roughness of polymer all observed. polymer blends acid), and (TPU+PLA) blends are presented in TPU, Table 2. The average roughness ofpolymer all blends angle values of TPU, PLA, and (TPU+PLA) blends were observed. Results of our studies show that of our studies show that the incorporation of PLA in TPU has major effect towards the hydrophilicity of our studies show that the incorporation of PLA in TPU has major effect towards the hydrophilicity of our studies show that the incorporation of PLA in TPU has major effect towards the hydrophilicity waswas from 86from μm 7:3 to 102 for 3:7. As rule, for the with higher was 86 (TPU+PLA) μm for (TPU+PLA) 7:3μm to 102 μm for (TPU+PLA) a rule, for the blends with higher from 86 for μm for (TPU+PLA) 7:3 to 102 μm(TPU+PLA) for (TPU+PLA) 3:7.a3:7. As aAs rule, forblends the blends with higher the incorporation of PLA in TPU has major effect towards the hydrophilicity of (TPU+PLA) films, of (TPU+PLA) films, compared with the hydrophobic TPU films. ofconcentration (TPU+PLA) films, withroughness theroughness hydrophobic TPU films. of (TPU+PLA) films, compared with the TPU films. of PLA surface roughness is hydrophobic higher. Polymer blends withwith higher addition of elastic concentration of PLA surface is higher. Polymer blends with higher addition of elastic concentration ofcompared PLA surface is higher. Polymer blends higher addition of elastic compared with theflattered hydrophobic TPU TPUTPU areTPU more flattered surface, as well pure polyurethane films. are more flattered surface, asfilms. well as pure polyurethane films. are more surface, as well as pure polyurethane films. 3.3. Surface Roughness 3.3. Surface Roughness 3.3. Surface Roughness Table The surface properties ofofpolyurethane, poly(lactic acid), and their blends. Table 2.2.Table The properties of polyurethane, poly(lactic and their blends. 2.among The surface properties ofand polyurethane, poly(lactic acid), and their blends. Table 2. surface The surface properties poly(lactic acid), and their blends. Topography, other chemical physical factors such as substrate stiffness and polymer Topography, among other chemical and physical factors such asacid), substrate stiffness and polymer Topography, among other chemical andpolyurethane, physical factors such as substrate stiffness and polymer blend composition, is known to have a great influence on cell behavior. Optimization of blend composition, is known to have a great influence on cell behavior. Optimization of of blend composition, is known to have a great influence on cell behavior. Optimization TPU 100% (control) (TPU+PLA) 7:3 (TPU+PLA) 6:4 TPU 100% (control) (TPU+PLA) 7:3 (TPU+PLA) 6:4 TPU 100% (control) (TPU+PLA) 7:3 (TPU+PLA) 6:4 TPU 100% (control) (TPU+PLA) 7:3 (TPU+PLA) 6:4 topographical features, such as surface roughness, thestrategy keytostrategy to obtain the best topographical features, such as surface roughness, is the ofthe the key obtain the best topographical features, such as surface roughness, is one the isone ofone theofstrategy key to obtain the best cellular scaffolds fortissue various tissue applications. This work investigated the influence of polymer cellular scaffolds for various applications. ThisThis workwork investigated the influence of polymer cellular scaffolds for various tissue applications. investigated the influence of polymer blend composition on surface roughness. Theroughness average roughness a) of the polyurethane, poly(lactic blend composition on surface roughness. The average (Ra) of polyurethane, poly(lactic blend composition on surface roughness. The average roughness (Rathe ) of(R the polyurethane, poly(lactic and (TPU+PLA) are presented Table 2.average Theroughness average roughness of allblends polymer acid), andacid), (TPU+PLA) blends areblends presented in Table 2.inThe average of allofpolymer acid), and (TPU+PLA) blends are presented in Table 2. The roughness all polymer blendsblends was from 86 for (TPU+PLA) 7:3 102 for (TPU+PLA) 3:7. As the afor rule, the blends with higher was was fromfrom 86 μm for 7:3 to7:3 102 for (TPU+PLA) 3:7. As rule, for blends with higher 86 μm(TPU+PLA) forμm (TPU+PLA) toμm 102to μm forμm (TPU+PLA) 3:7.aAs a rule, thefor blends with higher concentration of surface PLA surface roughness is higher. Polymer blends with higher addition of elastic concentration of PLA surface roughness is higher. Polymer blends with higher addition of elastic concentration of PLA roughness is higher. Polymer blends with higher addition of elastic TPU are more flattered well as polyurethane pure polyurethane TPUTPU are more flattered surface, assurface, well as as pure polyurethane films. are more flattered surface, as well as pure films. films. ◦] ◦] ±±1.8 ] =θ118.9 ± 2.3 θθ==96.3 θ = 123.2 θ =θ 118.9 [°] 96.3 ± 1.3 θ±123.2 =1.8 123.2 1.8[◦[°] θ±=2.3 118.9 2.3 [°] θ = 96.3 1.3 [°] θ θ= = 123.2 ±[°] 1.8 [°] = 118.9 ± 2.3[± [°] θ =±96.3 ±[[°] 1.3± [°] Table 2. The surface properties of polyurethane, poly(lactic acid), and their blends. Table 2. The surface properties of polyurethane, poly(lactic acid), and their blends. Table 2. The surface properties of polyurethane, poly(lactic acid), and their blends. R a =R 72.1 ± 1.2 [µ m] R a = 86.1 ± 1.7 [µ m] R a = 83.1 ± 2.1 [µ m] R a = 72.1 ± 1.2 [µ m] R a = 86.1 ± 1.7 [µ m] R a = 83.1 ± 2.1 a = 72.1 ± 1.2 [µ m] R a = 86.1 ± 1.7 [µ m] R a = 83.1 ± 2.1 [µ Rax2018, = 72.1 ±x FOR 1.2 [µm] Ra = 86.1 ± 1.7 [µm] Ra = 83.1 ± 2.1 [µm] m] Polymers PEER REVIEW 8 of 15 Polymers 2018, 10, FOR PEER REVIEW 8[µ ofm] 15 Polymers 2018, 10, x 10, FOR PEER REVIEW 8 of 15 (TPU+PLA) 4:6 (TPU+PLA) 3:7 PLA 100% (control) (TPU+PLA) 4:6 (TPU+PLA) 3:7 PLA 100% (control) (TPU+PLA) 4:6 (TPU+PLA) 3:7 PLA 100% (control) TPU 100% (control) (TPU+PLA) (TPU+PLA) TPU TPU 100%100% (control) (TPU+PLA) 7:3 3:7 (TPU+PLA) 6:4 6:4 6:4 (control) (TPU+PLA) 7:3 7:3 (TPU+PLA) (TPU+PLA) 4:6 (TPU+PLA) PLA 100% (control)
◦] 87.7 ± ±1.8 θ1.8 123.2 ± [1.8 θ θ= =123.2 ±= θ87.7 1.8 [°] ==87.7 1.8 [°][°] ±θ1.8 [°] θ==θ123.2 87.7 ±±[°] 1.8 [°] a 97.4 =[µ 72.1 ±m] 1.2 RRa a==72.1 m] R aaR ==a±R 72.1 ± 1.2 aR =97.4 ±[µ 1.9 [µ[µ m]m] ±=1.2 1.9 m] R97.4 97.4 ±[µ 1.9 [µ m] ± 1.9 [µm] (TPU+PLA) (TPU+PLA) 4:6 4:6 4:6 (TPU+PLA)
◦] θθ86.9 = θ2.6 118.9 ± 2.3 θ θ= =118.9 ±±θ2.3 [°] 2.3[±[°] [°] =±=86.9 2.6 [°][°] [°] θ=86.9 =118.9 86.9 ±±2.6 2.6 R=1.6 a± =[µ 86.1 ±1.6 1.7 m] RRaaR ==a86.1 ± 1.7 m] R =R 86.1 ± [µ m] a± 102.5 ± 102.5 [µ m] R 102.5 ±1.7 1.6 [µ m][µ[µm] =aa =102.5 1.6 [µm] (TPU+PLA) (TPU+PLA) 3:7 3:7 3:7 (TPU+PLA)
◦] 79.9 1.1 θ =[°] 96.3 ± 1.3 θθθ==96.3 θθ ==±± 96.3 ± 1.3 = 79.9 1.1 [°][°] 79.9 ±θ1.3 1.1 79.9 ±[[°] 1.1±[°] [°] R=2.3 a2.3 =[µ 83.1 ±2.3 2.1 m] == 83.1 ± 2.1 m] R =R 83.1 ± [µ[µ m] a± 115.8 ± 115.8 [µ m] R aa = 115.8 ±2.1 2.3 m][µ[µm] RRRaaa= 115.8 ± [µm] PLA 100% (control) PLA PLA 100%100% (control) (control)
3.4. Thermal Properties of (TPU+PLA) Blends 3.4. 3.4. Thermal Properties of (TPU+PLA) Blends Thermal Properties of (TPU+PLA) Blends 3.3. Surface Roughness The TGA and DSC curves presented in Supplementary Figure S1the show the thermal degradation TheThe TGA and DSC curves presented in Supplementary Figure S1 show thermal degradation TGA and DSC curves presented in Supplementary Figure S1 show the thermal degradation
Topography, among other chemical and physical factors such as substrate stiffness and polymer behavior of the (TPU+PLA) blends and control samples heated to 600 °C aunder a nitrogen behavior of each theeach (TPU+PLA) blends andand control samples heated to 600 °C under nitrogen behavior of each the (TPU+PLA) blends control samples heated to 600 °C under a nitrogen blend composition, is known to have a great influence on cell behavior. Optimization of topographical atmosphere. TGA curves show similar profiles, as well as DSC curves, with thermal decomposition atmosphere. TGA curves show similar profiles, as well curves, withwith thermal decomposition atmosphere. TGA curves show similar profiles, as wellDSC as DSC curves, thermal decomposition features, such roughness, of°C. the keymaximum strategy tothermal obtain the bestofcellular opening atsurface 200 °C and extending toone 450 The decomposition of scaffolds polymer opening at 200 °C and extending to is 450 °C. The maximum thermal decomposition polymer opening atas200 °C and extending tothe 450 °C. The maximum thermal decomposition of polymer for various tissue applications. This work investigated the influence of polymer blend composition on samples was observed from 300 to (all 400 °C samples (all samples lost 50 percent of mass). samples waswas observed fromfrom 300 to 400 °C samples lost lost 50 percent of mass). samples observed 300 to 400 °C (all 50 percent of mass). 3.5. Tensile 3.5. 3.5. Tensile Test Tensile TestTest At this point, we themoduli, elastic moduli, which a role crucial role in cell culture. The substrate At this point, we test the test elastic which plays a plays crucial in cell culture. TheThe substrate At this point, we test the elastic moduli, which plays a crucial role in cell culture. substrate elastic moduli might regulate the adhesion, morphology, proliferation, or differentiation of cells. elastic moduli might regulate the adhesion, morphology, proliferation, or differentiation of cells. Four elastic moduli might regulate the adhesion, morphology, proliferation, or differentiation of cells. FourFour kinds of (TPU+PLA) blend films and control material samples were subjected to the measurement kinds of (TPU+PLA) blend films and control material samples were subjected to the measurement of kinds of (TPU+PLA) blend films and control material samples were subjected to the measurement of of
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surface roughness. The average roughness (Ra ) of the polyurethane, poly(lactic acid), and (TPU+PLA) blends are presented in Table 2. The average roughness of all polymer blends was from 86 µm for (TPU+PLA) 7:3 to 102 µm for (TPU+PLA) 3:7. As a rule, for the blends with higher concentration of PLA surface roughness is higher. Polymer blends with higher addition of elastic TPU are more flattered surface, as well as pure polyurethane films. 3.4. Thermal Properties of (TPU+PLA) Blends The TGA and DSC curves presented in Supplementary Figure S1 show the thermal degradation behavior of each the (TPU+PLA) blends and control samples heated to 600 ◦ C under a nitrogen atmosphere. TGA curves show similar profiles, as well as DSC curves, with thermal decomposition opening at 200 ◦ C and extending to 450 ◦ C. The maximum thermal decomposition of polymer samples was observed from 300 to 400 ◦ C (all samples lost 50 percent of mass). 3.5. Tensile Test At this point, we test the elastic moduli, which plays a crucial role in cell culture. The substrate elastic moduli might regulate the adhesion, morphology, proliferation, or differentiation of cells. Four kinds of (TPU+PLA) blend films and control material samples were subjected to the measurement of tensile tests, and Young moduli examination results are presented in Table 3. The strain–stress behavior of (TPU+PLA) films shows that PLA with TPU caused a reduction in mechanical stress and elasticity. The percent value of elongation of (TPU+PLA) films were higher compared to pure PLA indicating a good deformability and flexibility of obtained polymer blends. However, the percent elongation of (TPU+PLA) blends decreased significantly due higher PLA concentration in (TPU+PLA) blended polymeric films. Pure PLA films had a high tensile modulus (3.44 GPa) and exhibited an extremely low-elongation-at-break due to their inelastic nature. TPU film samples did not break during the tensile test even when the strain reached 500%. In summary, the ratios of 7:3 and 6:4 of (TPU+PLA) blend were therefore specifically chosen for the preparation of elastic scaffolds for cartilage zone and chondrocyte cell culture, with 3:7 and 4:6 of (TPU+PLA) chosen for more rigid scaffolds, for subchondralbone scaffolds, and osteoblast cell culture. All (TPU+PLA) compositions were selected for stem cell culture. Scaffolds have varying properties in different regions of the scaffolds for optimizing ingrowth of different tissues and cells. It is known that various topography, elasticity, and chemistry are suitable for ingrowth of different tissues. We carried out material characteristics and in vitro experiments evaluating the capability of (TPU+PLA) blends to modulate the behavior of stem cells for osteochondral tissue engineering. Table 3. Tensile Young’s moduli of thin films. Polymer Composition
Tensile Young’s Modulus for Thin Films [MPa]
TPU 100% (control)
29.6 ± 0.6
(TPU+PLA) 7:3
84.5 ± 0.5
(TPU+PLA) 6:4
162.4 ± 1.1
(TPU+PLA) 4:6
215.6 ± 1.9
(TPU+PLA) 3:7
237.0 ± 3.2
PLA 100% (control)
3.44 ± 2.5 [GPa]
3.6. The In Vitro Evaluation of Cytotoxicity of Obtained Biomaterial 3.6.1. Proliferation and Metabolic Activity of hASCs in Cultures with Obtained Biomaterials Both metabolic and proliferative activity were determined after 120 h of cell propagation in cultures with biomaterials (Figure 1a,b, respectively). The analysis of hASCs metabolism in cultures with (TPU+PLA) blends revealed that only (TPU+PLA) in ratio 7:3 may exert the cytotoxic effect
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toward hASCs lowering significantly the metabolic activity of the cells. Furthermore, the analysis of the proliferation status of hASCs showed that both (TPU+PLA) 7:3 and (TPU+PLA) 6:4 may inhibit the proliferative properties of hASCs. Bearing in mind both metabolic and proliferative potential of hASCs in cultures with (TPU+PLA) blends, the most cytocompatible matrices were characterized by lowered content of TPU, i.e., (TPU+PLA) 3:7 as well as (TPU+PLA) 4:6. Obtained results are in good agreement with the study of10,Mi et al. who developed the TPU/PLA scaffolds fabricated by microcellular Polymers 2018, x FOR PEER REVIEW 9 of injection 14 molding. In this study cultures of 3T3 fibroblast cell line exhibited slightly higher cell viabilities in the might be more associated the lowest water contactproliferative angle noted for (TPU+PLA) as well as for scaffolds with PLAwith content [6]. The increased activity might 3:7 be associated with the (TPU+PLA) 4:6 films, which was equal to 87.7 ± 1.8 and 86.9 ± 2.6, respectively. The obtained data lowest water contact angle noted for (TPU+PLA) 3:7 as well as for (TPU+PLA) 4:6 films, which was correlates with our previous research and that of others, where the increased proliferative activity of equal stromal to 87.7 ± 1.8 and 86.9 ± 2.6, respectively. The obtained data correlates with our previous research stem cells was related to water contact angle [8]. Moreover, the highest metabolic activity, and that others, whereonthe proliferative activity stromal cells was related to water thatofwas measured theincreased 5th day of experimental culture,ofwas foundstem in hASCs cultured onto contact angle [8].3:7,Moreover, metabolic that4:6. was measured on the 5th (TPU+PLA) (TPU+PLA)the 6:4,highest (TPU+PLA) 4:6, andactivity, (TPU+PLA) The results presented hereday of support previous that in polymer matrices (TPU+PLA) are not for hASCs6:4, and(TPU+PLA) may experimental culture, findings was found hASCsbland cultured onto (TPU+PLA) 3:7,toxic (TPU+PLA) serve as a scaffolds for progenitor cells [8,29]. This may shed a promising light for the further future 4:6, and (TPU+PLA) 4:6. The results presented here support previous findings that polymer bland application of (TPU+PLA) a wide and range biomedical for hard andcells soft [8,29]. matrices (TPU+PLA) are not material toxic forinhASCs may serve asapplications, a scaffoldsboth for progenitor tissue healing [7]. This may shed a promising light for the further future application of (TPU+PLA) material in a wide range biomedical applications, both for hard and soft tissue healing [7]. The proliferative activity factor
1.5
2.5 PF [arbitrary unit]
*** 1.0 0.5
***
1.5 1.0 0.5
(a)
4: 6
6: 4
3: 7
4: 6
6: 4
3: 7
0.0
7: 3
0.0
***
2.0
7: 3
MF [arbitrary unit]
The metabolic activity factor
(b)
Figure 1. Metabolic and proliferative activity of hASCs cultivated onto investigated (TPU+PLA) 1. Metabolic and proliferative factor activitywas of hASCs cultivated investigated (TPU+PLA) films. films.Figure The metabolic and proliferative determined in onto relation to the cultures propagated on The metabolic and proliferative factor was determined in relation to the cultures propagated polystyrene. The analysis was performed after 120 h of hASCs culture in obtained matrices. Anonasterisk Thesignificant analysis was performed h of hASCs culture in obtained matrices. An markspolystyrene. a statistically difference (***after p < 120 0.001). asterisk marks a statistically significant difference (*** p < 0.001).
3.6.2. The Morphology and Growth Pattern of hASCs Cultured on Obtained (TPU+PLA) Films 3.6.2. The Morphology and Growth Pattern of hASCs Cultured on Obtained (TPU+PLA) Films
The analysis of hASCs morphology and growth pattern in cultures with TPU+PLA films clearly The analysis of hASCs morphology and growth pattern in cultures with TPU+PLA films clearly indicated on the most cytocompatible blend (Figure 2). The proper morphology of hASCs, as well indicated on the most cytocompatible blend (Figure 2). The proper morphology of hASCs, as well as as growth pattern, with(TPU+PLA) (TPU+PLA) matrices. this culture growth pattern,was wasnoted noted in in culture culture with 3:7 3:7 matrices. The The cells cells in thisinculture were were spindle-shape or multipolar, which is typical morphotype for multipotent stromal cells of mesenchymal spindle-shape or multipolar, which is typical morphotype for multipotent stromal cells of origin.mesenchymal The analysis of growth pattern using confocal microscope revealed that TPU+PLA) origin. The analysis of performed growth pattern performed using confocal microscope revealed that TPU+PLA) 3:7 matrices cell-cell interactions. The hASC on (TPU+PLA) 3:7 dense 3:7 matrices promoted cell-cellpromoted interactions. The hASC cultured on cultured (TPU+PLA) 3:7 formed dense active layer of closely arranged cells. These were characterized bywell-developed cells with activeformed layer of closely arranged cells. These cultures were cultures characterized by cells with well-developed cytoskeleton and centrally located nuclei. Moreover hASCs, were distributed evenly cytoskeleton and centrally located nuclei. Moreover hASCs, were distributed evenly on the (TPU+PLA) on the (TPU+PLA) 3:7 matrices. The increased confluence of hASCs on (TPU+PLA) 3:7 matrices 3:7 matrices. The increased confluence of hASCs on (TPU+PLA) 3:7 matrices correlates with their high correlates with their high proliferative activity noted in cultures on those biomaterials. The proliferative activity noted in cultures on those(TPU+PLA) biomaterials. The of(TPU+PLA) cells propagated on morphology of cells propagated on remaining films, i.e.,morphology (TPU+PLA) 7:3, 6:4, remaining (TPU+PLA) films, i.e., (TPU+PLA) 7:3, (TPU+PLA) 6:4, and (TPU+PLA) 4:6 was typical and (TPU+PLA) 4:6 was typical for hASCs, and fibroblast-like cells were noted, despite that the cells for hASCs, fibroblast-like cells were noted, despite that the cells in these cultures did data not reveal in theseand cultures did not reveal tendency for the formation of intracellular connections. Obtained stands in good agreement with previous findings, showing that (TPU+PLA) blends with the higher tendency for the formation of intracellular connections. Obtained data stands in good agreement content of findings, PLA positively affectsthat the (TPU+PLA) adhesion and blends morphology progenitor cells, both adipose with previous showing withofthe higher content ofof PLA positively origin as well as derived from olfactory bulb [8,29,42]. affects the adhesion and morphology of progenitor cells, both of adipose origin as well as derived from olfactory bulb [8,29,42].
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Figure 2. The morphology of hASCs cultured ontoonto obtained (TPU+PLA) blends. The morphology of cells using scanning electronelectron microscope (SEM) (SEM) Figure 2. The morphology of hASCs cultured obtained (TPU+PLA) blends. The morphology ofwas cellsvisualized was visualized using scanning microscope and confocal microscope. The SEM images (revealed the adhesion rate of hASCs on tested (TPU+PLA) blends, and some biomimetic features of films). For confocal and confocal microscope. The SEM images (revealed the adhesion rate of hASCs on tested (TPU+PLA) blends, and some biomimetic features of films). For confocal observations, cells were stained with DAPI, to localize the nuclei (blue(blue dots)dots) and with phalloidin atto-488 (green, actin cytoskeleton). SEM microphotographs were were observations, cells were stained with DAPI, to localize the nuclei and with phalloidin atto-488 (green, actin cytoskeleton). SEM microphotographs captured under 1000× magnification (scale bar is 10 μm), while confocal observations were performed at magnification 40× (scale bar 200 μm). captured under 1000× magnification (scale bar is 10 µm), while confocal observations were performed at magnification 40× (scale bar 200 µm).
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3.6.3. Evaluation of Bax/Bcl-2 Ratio and Total Bax-α in hASCs Cultured on (TPU+PLA) Blends 3.6.3.The Evaluation Ratio and Total Bax-α in hASCs Cultured onof (TPU+PLA) Blends on analysis of of Bax/Bcl-2 the transcript levels indicated that the higher content PLA may influence increased mRNA level of transcript antiapoptotic Bcl2indicated gene (Figure PLA is described asmay less cytotoxic The analysis of the levels that 3a). the The higher content of PLA influence polymer than TPU, thus adequate content of TPU and PLA may improve biological activity of as blend on increased mRNA level of antiapoptotic Bcl2 gene (Figure 3a). The PLA is described less [6,7,13]. The high Bax/Bcl-2 ratio was noted in hASCs cultures propagated onto (TPU+PLA) 7:3 and cytotoxic polymer than TPU, thus adequate content of TPU and PLA may improve biological (TPU+PLA) 6:4. The increase mRNA level of Bax-α in those cultures reflected also on onto the activity of blend [6,7,13]. The high Bax/Bcl-2 ratio was noted in hASCs cultures propagated accumulation of Bax-α protein in cells, which was determined using the ELISA technique (Figure 3b). (TPU+PLA) 7:3 and (TPU+PLA) 6:4. The increase mRNA level of Bax-α in those cultures reflected The expression of proapoptotic Bax-αin incells, cultures on was biomaterials with the dominant of alsohigh on the accumulation of Bax-α protein which determined using the ELISAcontent technique TPU correlates with the results of proliferative activity of hASCs. Obtained data clearly indicate, that (Figure 3b). The high expression of proapoptotic Bax-α in cultures on biomaterials with the dominant the proper ratiowith maythe exert pro-proliferative andactivity antiapoptotic effectObtained toward multipotent content of (TPU+PLA) TPU correlates results of proliferative of hASCs. data clearly progenitor cells derived from adipose tissue. indicate, that the proper (TPU+PLA) ratio may exert pro-proliferative and antiapoptotic effect toward multipotent progenitor cells derived from adipose tissue.
total Bax-α measurment
1.0
*
15000
*
*
0.8
*
10000
pg/mL
0.6 0.4
5000
0.2 0.0
(a)
3
7
4
6
3:
6:
4:
4: 6
6: 4
3: 7
7: 3
0 7:
Qn ratio [Bax/Bcl-2]
Bax/Bcl-2 ratio
(b)
Figure 3. The analysis of Bax/Bcl-2 ratio (a) and total concentration of Bax-α (b). The transcripts levels Figure 3. The analysis of Bax/Bcl-2 (a) and total concentration Bax-α (b). The transcripts levels were determined using RT-qPCR ratio method, while total Bax-α was of evaluated using ELISA technique. were determined using method, while total Bax-α was evaluated using ELISA technique. An asterisk was used toRT-qPCR mark a statistically significant differences determined between tested groups An asterisk was used to mark a statistically significant differences determined between tested groups (* p < 0.05). (* p < 0.05).
4. Conclusions 4. Conclusions In this study we developed blend (TPU+PLA) films with a weight ratio of 7:3, 6:4, 4:6, and 3:7. The In results reported this studyblend demonstrated thatfilms the concentration PLAofin7:3, the6:4, polymer blends this study wein developed (TPU+PLA) with a weightofratio 4:6, and 3:7. wasresults strongly affected properties of polymer matrices. Mechanical tests confirmed very The reported in by thisthe study demonstrated that the concentration of PLA in the polymerthe blends highstrongly tensile range of the scaffolds fabricated by the solvent casting method, which can was affected by (TPU+PLA) the properties of polymer matrices. Mechanical tests confirmed the very potentially used tissue engineering in the many tissue applications. in vitro high tensile be range of in theosteochondral (TPU+PLA) scaffolds fabricated by solvent casting method, An which can proliferative metabolic assay using human adipose-derived stem cells (hASCs) potentially beand used in osteochondral tissue engineering in manymesenchymal tissue applications. An in vitro showed thatand (TPU+PLA) was using the most cytocompatible. Moderate cytocompatibility was also proliferative metabolic3:7 assay human adipose-derived mesenchymal stem cells (hASCs) showed the most cytocompatibility also observedthat for (TPU+PLA) (TPU+PLA) 3:7 4:6 was matrices. Bothcytocompatible. (TPU+PLA) 3:7 Moderate and (TPU+PLA) 4:6 can alsowas improve observed for (TPU+PLA) 4:6 matrices. Both of (TPU+PLA) 3:7Bcl-2 and and (TPU+PLA) 4:6proapoptotic can also improve hASCs viability by increasing the expression antiapoptotic decreasing Bax-α, hASCs by increasing thelevel. expression of antiapoptotic Bcl-2 and decreasing proapoptotic Baxboth atviability the mRNA and protein The obtained data clearly indicate, that our materials could be α, bothasatsufficient the mRNA and protein level. Thecells obtained data clearly indicate, ratio that our materials could used scaffolds for progenitor like hASCs. The different of TPU to PLA can be used as sufficient scaffolds for progenitor cells like hASCs.adhesion The different ratio of TPU pattern to PLA can significantly modulate proliferative and metabolic activity, as well as growth and significantly modulate proliferative metabolic activity, adhesion as well growth pattern and cell-cell contacts. Further research isand required to test whether our concept of as using different ratio of cell-cell contacts. research is required testsponges whetherorour concept of using different ratio of (TPU+PLA) filmsFurther in different physical forms,toi.e., fibrous matrices, might differentially (TPU+PLA) films in different physical forms, i.e., sponges or fibrous matrices, might differentially induce osteogenic and/or chondrogenic differentiation. induce osteogenic and/or chondrogenic differentiation. Supplementary Materials: The following are available online at http://www.mdpi.com/2073-4360/10/10/1073/ s1, Figure S1: TGA and DSC curves of different (TPU+PLA) blends: 7:3, 6:4, 4:6 and 3:7. Supplementary Materials: The following are available online at www.mdpi.com/link, Figure S1: TGA and DSC curves of different (TPU+PLA) blends: 7:3, 6:4, 4:6 and 3:7.
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Author Contributions: Conceptualization, A.L.-B., A.S. and K.M.; Methodology, A.L.-B., K.F. and A.S.; Software, A.L.-B. and A.S.; Validation A.L.-B. and A.S.; Formal Analysis, A.L.-B. and A.S.; Investigation, A.L.-B., K.F. and A.S.; Resources, A.L.-B. and K.M.; Data Curation, A.L.-B. and A.S.; Writing-Original Draft Preparation, A.L.-B., A.S. and K.M.; Writing-Review & Editing, A.L.-B., A.S. and K.M.; Visualization, A.L.-B., K.F. and A.S.; Supervision, K.M. Acknowledgments: Publication supported by Wroclaw Centre of Biotechnology, programme The Leading National Research Centre (KNOW) for years 2014–2018. The authors are grateful to PORT Polski O´srodek Rozwoju Technologii/Polish Center for Technology Development for ability to perform visualisation of cells morphology (SEM and epifluorescence images). Conflicts of Interest: The authors declare no conflicts of interest.
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