Synthesis, Characterization and Biocompatibility of Elastomeric Poly(L-co-D,L-lactic acid-co-Caprolactone)urethane for Biomedical Applications

Abstract

A versatile nature of polyurethanes allows for the modification of thermal, mechanical, and chemical properties, making them promising candidates for medical applications. This study focuses on the synthesis, characterization, and biocompatibility evaluation of a novel poly (L-co-D,L-co-lactic acid-co-Caprolactone)urethane (PLDLA-PCL-PU) material derived from L-lactide, D,L-lactide, polycaprolactone-diol (PCL-diol) and 1,6-hexamethylene diisocyanate (HDI). This research performs a meticulous two-stage synthesis process, ultimately leading to the formation of PLDLA-PCL-PU through polymerization of the prepolymer (PP) and HDI. The confirmation of successful synthesis and the characterization of PLDLA-PCL-PU was done by FTIR-ATR, and ¹H NMR. As evidenced by XRD, the amorphous nature of PLDLA reduces the degree of crystallinity in PLDLA-PCL-PU. Thermal decompositions, as well as the associated thermal events, were investigated using TGA and DSC. The biocompatibility of the material was evaluated using human mesenchymal stem cells. These assays reveal a notable enhancement in cell metabolic activity and proliferation when in contact with polyurethane membranes. By combining the properties of polyurethanes with the custom design of PLDLA-PCL-PU, this research strives to contribute with the advancement of biomaterials that promote tissue regeneration.

Keywords:
Polyurethane; Polycaprolactone; PLDLA; Biocompatibility; Biomaterials

1. Introduction

The increasing life expectancy of the global population requires progressive efforts in the advancement and development of therapeutic alternatives. In this context, the strategic elaboration and continuous improvement of biomaterials play a fundamental role. Among the available options, polymeric materials emerge as protagonists due to their inherent properties and design versatility. Elastomeric polyurethanes (PUs) have attracted significant attention in the biomedical area, boosting the formulation of biomaterials intended for hard and soft tissue regeneration¹.

The promising application of PUs in the medical field stems from their remarkable physical-chemical, mechanical, and structural attributes, enabling a wide spectrum of applications. They are extensively employed in the development of medical devices², implants³, drug delivery systems⁴, wound treatment⁵, scaffold structures in tissue engineering⁶, and other applications. Furthermore, its biocompatibility with the human organism, low cytotoxicity, resistance to enzymatic and bacterial hydrolysis, excellent hemocompatibility and probable absence of immunological reactions consolidate its prominence¹.

A significant additional advantage is the biological degradation capacity of these materials, even if gradual. This attribute resonates with the growing emphasis on sustainability and environmental safety. This approach has broadened the research and development focus of biodegradable and biologically active PUs, propelling them towards even more versatile and effective applications in the field of medicine¹.

The PUs are typically synthesized through the polyaddition reaction, involving polyols and isocyanates associated with catalysts, and additives resulting in the formation of urethane bonds (-NHCOO-)⁷. These polymers consist of alternating blocks of hard and soft segments. The polyols contribute to the soft segments, while the hard phase arises from the reaction between the diisocyanate and diols or the chain extenders. Consequently, the soft phase confers elastomeric properties to the PU, while the hard segment provides strength due to the hydrogen bonds present in the urethane bond⁸,⁹.

Polymers belonging to poly (α-hydroxy acids) are widely used in tissue engineering devices¹⁰. This class includes aliphatic polyesters, such as poly (L-lactic acid) (PLLA), poly(D,L-lactic acid) and polycaprolactone (PCL)¹¹,¹². PCL has frequently been used in PUs formulation, especially in the medical field, due to its flexibility, biocompatibility, and ability to hydrolyze into metabolizable products by the organism¹³. A series of studies addressing the use of PLA in the synthesis of PUs has been reported for the development of materials with shape memory performance¹⁴,¹⁵. Additionally, the chirality of PLA allows the formation of enantiomers that can be copolymerized and synthesized into a range of PU elastomers¹⁴. Pepponi et al.¹⁶ reported that the synthesis of PCL with PLLA results in obtaining a PU with shape memory behavior suitable for biomedical applications. According to the authors, PLLA acts as the fixed phase, responsible for promoting the shape memory behavior, while PCL contributes to the material's strength. However, studies addressing the development of PUs from L-lactide and D,L-lactide (PLDLA) are still scarce. Therefore, optimizing PU performance can be achieved through changes in the raw material, improvements in the production process, and exploration of novel synthetic routes, providing the PU with distinct physical-chemical and biological properties¹⁷.

In this context, the objective of this study was to synthesize and characterize physico-chemical and biologically the elastomeric polyurethane based on L-lactide and D,L-lactide (PLDLA) together with polycaprolactone diol (PCL-diol) called PDLA-PCL-PU. So, the PLDLA in the structure of synthesized PUs aims to improve its biocompatibility, and ability to hydrolyze into metabolizable products by the organism. In turn, the PCL has the function of giving more flexibility to the synthesized PU. So far, no studies have been identified in the scientific literature regarding the synthesis of PUs based on the PCL-diol with L-lactide and D,L-lactide. Therefore, this research aims to address this knowledge gap by exploring the potential of this material for advancements in the field of biomaterials, with a focus on future biomedical applications.

2. Materials and Methods

2.1. Materials

The L-lactide and D,L-lactide were purchased from PURAC. Polycaprolactone diol (PCL-diol) (M_n = 2000.0 g/mol), 1.2-dichloroethane (99.8%), and Tin(II) 2-ethylhexanoate (SnOct₂) were purchased from Sigma-Aldrich. The chloroform (99.8%) and methanol (99.8%) were purchased from Synth. The 1.6- hexamethylene diisocyanate (HDI) (98.0%) was purchased from Fluka. All reagents were used as received.

2.2. Methods

The polyurethanes (PLDLA-PCL-PU) were obtained through two steps. The first stage consisted of obtaining copolymer (CP), while the second stage consisted of obtaining the PLDLA-PCL-PU. The syntheses were based on a previous study of a PU based on PCL-PLLA¹⁵. The synthesis of PCL-PU was conducted as an experimental control.

2.2.1. Synthesis of copolymer

The copolymer (CP) was synthesized through a bulk polymerization reaction. PCL-diol (1.20x10^-2 mol) and monomers in a ratio 70/30 of L-lactide (2.9x10^-2 mol) and D,L-lactide (1.25x10^-2 mol) were utilized in this procedure. SnOct₂ was employed as the catalyst with a monomer/catalyst molar ratio of 5000. The reagents were added in a 250 mL two-neck flask. The flask was subjected in an oil bath at 150 °C, under a nitrogen (N₂) atmosphere and to magnetic stirring for 24 h. Subsequently, the CP was dissolved in chloroform and precipitated in methanol. After a 24 h period, the precipitated CP was separated from the supernatant, and the resulting product was dried at room temperature, followed by an additional 48 h in a vacuum chamber at room temperature. The molecular weight of CP was analyzed by Gel Permeation Chromatography (GPC). This information was important to calculate the molar ratio between CP and HDI to synthesize the PLDLA-PCL-PU in the next synthesis step.

2.2.2. Synthesis of PCL-PU

The molar ratio used between OH present in PCL-diol and NCO present in HDI was 1:8. PCL-diol (M_n = 2000.0 g/mol) was solubilized in 1.2-dichloroethane at a concentration of 33% w/v. HDI and the SnOct₂ catalyst were then added to the solution with a molar fraction between the monomer and the catalyst of 5000. The reagents were added to two-necked flasks under an N₂ atmosphere, heated to 70 °C and the reaction occurred for 6 h under magnetic stirring. The PCL-PU was dissolved in chloroform and precipitated in methanol. The polymer was dried under vacuum and at room temperature for 48 hours.

2.2.3. Synthesis of PLDLA-PCL-PU

The molar ratio of OH/NCO equivalent was 1:8 for synthesized PLDLA-PCL-PU. The CP (2.10x10^-4 mol) was solubilized in 1.2-dichloroethane at a concentration of 33% w/v. This solution was added to 2.02x10^-3 mol of HDI and SnOct₂ at a molar ratio of 5000 for copolymer/catalyst. The reagents were introduced into a two-necked flask under a N₂ atmosphere, heated by an oil bath at 70 °C, and stirred magnetically for 6 h. Subsequently, PLDLA-PCL-PU was dissolved in chloroform and precipitated in methanol. After 24 h, the precipitated PLDLA-PCL-PU was separated from the supernatant, and the obtained product was dried at room temperature in a vacuum chamber for 48 h. Figure 1 provides a schematic representation of the CP and PLDLA-PCL-PU synthesis process.

Figure 1
Schematic presentation of the synthesis of CP and PLDLA-PCL-PU.

2.3. Physicochemical characterization

2.3.1. Fourier transform infrared spectroscopy (FTIR-ATR)

The FTIR-ATR analysis was employed to confirm the chemical structure of both the PLDA, PCL-diol, CP, PCL-PU, and PLDLA-PCL-PU. This analysis was performed with the FTIR-ATR Spectrum 65 spectrometer (Perkin Elmer) with 32 scans, from 4000 to 500 cm^-1 and 4 cm^-1 resolution.

2.3.2. Hydrogen nuclear magnetic resonance spectroscopy (¹H NMR)

Analysis of the chemical structure of CP, PCL-PU, and PLDLA-PCL-PU was performed by ¹H NMR in Bruker Avance 500 Hz, using deuterated chloroform (CDCl₃) as solvent. Chemical shift values (δ) were reported in ppm.

2.3.3. X-ray diffraction (XRD)

Membranes of PCL-PU and PLDLA-PCL-PU obtained by casting were analyzed by XRD to verify the crystallinity and to determine the average crystallite size of the polymer. Wide-angle X-ray diffraction data was collected on an XRD-6100 SHIMADZU equipment. The diffractometer was used employing Cu Kα radiation (λ=1.5406 Å), generated at a voltage of 40 kV and current of 30 mA. Diffractogram was made by continuous scanning over the range of diffraction angle 2θ from 5° to 60° using a scan rate of 2°/min. The mean crystallite size was determined by the Scherrer formula (Equation 1)¹⁸:

t = $\frac{\left(K\lambda \right)}{Bcos\theta }$ (1)

Where t is the mean crystallite size, B is the broadening of the peak at half of the maximum peak, in radians. K is the shape factor with a typical value of 0.9, θ is the Bragg angle, λ is the wavelength of the incident wave of X-rays¹⁹.

The interplanar distances were determined by the Bragg’s law (Equation 2)

$d=\frac{n.\lambda }{2.sin\theta }$ (2)

Where n = is an integer (n=1) (diffraction order - reflections of order higher than 1 have low intensity, contributing essentially to the elevation of the baseline, making the usual and practical form of Bragg’s law n = 1)²⁰, λ is the wavelength of the incident wave of X-rays, and θ is the incident angle, and d is the interplanar distance.

The crystallinity value ($\chi$_c) was calculated mathematically using the ratio between the areas of the crystalline peaks (peaks area) and the total area under the XRD curve (total area)²¹. To calculate the areas, peak deconvolution was performed using a Gaussian function (Equation 3).

${\chi }_c=\frac{peaksarea}{totalarea}.100\%$ (3)

Where peaks area stands for the total crystalline area under the Gaussian curves and total area represents the total area given by the diffractogram in the 2θ range 7.5 to 37.5.

2.3.4. Thermogravimetric analysis (TGA)

The thermal stability of PCL-PU and PLDLA-PCL-PU was verified by TA Instruments equipment, model TGA55. The samples were heated from 25 to 500 °C in a dynamic nitrogen gas atmosphere with a flow rate of 40 mL/min, and a heating of 10 °C/min.

2.3.5. Differential scanning calorimetry (DSC)

The glass transition temperature (T_g) and melting temperature (T_m) of both CP, PCL-PU, and PLDLA-PCL-PU were evaluated using DSC. The DSC curves were obtained with a TA Instruments equipment, model Q20. The samples, approximately 5 mg, were deposited in a hermetically sealed pan (Tzero) and heated from 25 to 180 °C, followed by cooling to -80 °C, and then reheating to 250 °C. A heating and cooling rate of 10 °C/min with a 5 min isotherm was used during the analysis.

2.4. Biological characterization

2.4.1. Cell culture assays

The hMSC (human adipose derived mesenchymal stem cells) were acquired by BCRJ (Cell bank of Rio de Janeiro). The hMSC was seeded in the PLDLA-PCL-PU membranes (7 mm diameter x 0.5 mm height) at a concentration of 1x10⁴ cell/membrane and cultured in DMEM medium supplemented with 10% FBS and antibiotics, for 1 and 7 days. Before cell seeding, polyurethane samples were sterilized under UV light for one hour each side and conditioned for 4 h in pure DMEM at 37 °C in 5% CO₂.

2.4.2. Cell proliferation

The hMSC proliferation was evaluated by laser scanning confocal microscopy (LSCM) (microscope model TCS SP8 from Leica MicroSystems, Germany, supported with LAS X software). After each experimental period, PLDLA-PCL-PU membranes (Thermanox® coverslips from NUNC Inc., USA, were used as controls), were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton-X. Cell nuclei were labeled with Fluoroshield with DAPI (Sigma-Aldrich, St. Louis, MO, USA). The cytoskeleton was stained with Phalloidin-Alexa Fluor 647 (Thermo Fisher Scientific, Waltham, MA, USA). Laser lines of 405 nm and 638 nm in PMT mode were used to detect signals of DAPI (blue) and Alexa Fluor 647 (red), respectively. Cells densities were counted by nuclei identification using Image J software, while actin fibers alignment and spreading were evaluated as compared with controls.

2.4.3. Statistical analysis

The hMSC viability and proliferation assays were performed by one-way analysis of variance (ANOVA). Statistical differences between the groups were evaluated by the Tukey multi comparison parametric method. The P value < 0.01 was considered very significant. Data were expressed in sextuplicate and represented by means ± standard deviation (SD).

3. Results and Discussion

3.1. Fourier transform infrared spectroscopy (FTIR-ATR)

The FTIR-ATR spectra of PLDLA, PCL-diol, PCL-PU, copolymer (CP), isocyanate (HDI), and PLDLA-PCL-PU are shown in Figure 2.

Figure 2
FTIR-ATR spectra of PLDLA, PCL-diol, PCL-PU, CP, isocyanate (HDI), and PLDLA-PCL-PU.

The PLDLA sample exhibited the prominent peaks at 2998 and 2941 cm^-1, attributed to symmetric and asymmetric axial deformation CH₂ and CH₃, respectively. The C=O axial deformation of polyesters was indicated by a pronounced peak at 1750 cm^-1. Additionally, the peaks present at 1184 and 1084 cm^-1 were associated with C-C symmetrical axial stretching and =CO groups of polylactide²²,²³. The spectrum of PCL-diol displayed peaks at 2932 and 2859 cm^-1, which were assigned to the asymmetric stretching vibrations of CH₂ and CH₃, respectively²⁴. Two pronounced peaks in 1725 and 1165 cm^-1 indicate the stretching vibrations of the C=O and C-O bonds of the ester groups present in the soft domains of the PCL²⁵.

For comparison with the material resulting from polymerization (PLDLA-PCL-PU), the sample composed only of PCL-PU was analyzed. Thus, the FTIR spectrum of PCL-PU exhibited peaks characteristic of polyurethanes at 3334, 1725, and 1623 cm^-1 attributed to hydroxyls (-OH), stretching of the carbonyl group (C=O), and bending vibration of C=C, respectively²⁶. Furthermore, the HDI spectrum showed hydrocarbon peaks at 2932 and 2860 cm^-1, as well as at 2249 and 1350 cm^-1, associated with the N=C=O and asymmetric CH₂ stretching vibration of the isocyanate group²⁷,²⁸.

As observed in the CP and PCL-PU spectra, the PLDLA-PCL-PU spectrum showed that the polyurethane structure obtained maintained specific peaks for the carbonyl (1725 cm^-1) and methylene (2932 and 2859 cm^-1) groups. However, no specific peaks were observed around 3334 cm^-1, as evidenced in the PLC-PU spectrum. Furthermore, the PLDLA-PCL-PU spectrum also did not show the appearance of peaks associated with isocyanate (HDI) at 2250 cm^-1 (N=C=O). This fact suggests that the HDI was completely consumed during the copolymerization process, and urethane bonds were formed²⁹,³⁰. Additionally, compared to the CP spectrum, the synthesized material (PLDLA-PCL-PU) exhibited the appearance of a new peak in 1530 cm^-1, which was associated with amide vibration (NH) in polyurethanes, respectively²⁶,³¹-³³. The presence of these groups indicates the success of the synthesis through the reaction between the CP and isocyanate groups, resulting in the formation of PLDLA-PCL-PU¹⁶.

3.2. Hydrogen nuclear magnetic resonance spectroscopy (¹H NMR)

The ¹H NMR spectrum of copolymer (CP) is shown in Figure 3.

Figure 3
¹H NMR spectrum of copolymer (PP).

The ¹H NMR spectra corresponding to CP (Figure 3) exhibited proton signals present in PCL-diol and PLDLA. The signals found at 4.06 (c), 2.29 (f), 1.61 (d), and 1.35 (e) ppm were associated with the protons present in the methylene (CH₂) groups of the PCL-diol (O-CH₂-CH₂-CH₂- CH₂)³⁴. While the multiplet present at 5.11 – 5.24 ppm (a) and the quartet at 1.55 – 1.59 ppm (b) were attributed with protons present in the CH-O and CH₃ group of the PLDLA segments, respectively²³. The low intensity observed for CH-O protons (5.11 and 5.24 ppm) is due to the low PLDLA content relative to the PCL-diol in the CP. Furthermore, the signal identified in the region of 7.26 ppm is associated with the solvent (deuterated chloroform) used in the analysis³⁵.

For comparative purposes, the sample composed only of PCL-PU synthesis also had its ¹H NMR spectrum analyzed, as shown in Figure 4.

As observed in the CP spectrum (Figure 3), the ¹H NMR spectrum of the sample composed only of PCL-PU (Figure 4) showed the same signals (e, f, d, and e) associated with protons present in the PCL-diol structure. However, the appearance of a new signal at 3.15 ppm was attributed to methylene protons (-CH₂-NH-) adjacent to the urethane group formed during synthesis³⁶,³⁷.

Figure 4
¹H NMR spectrum of PCL-PU.

The ¹H NMR spectrum and chemical structure of PLDLA-PCL-PU are shown in Figure 5.

Figure 5
¹H NMR spectrum of PLDLA-PCL-PU.

In addition to presenting the main signals associated with protons linked to the chemical structure of PCL (c, f, d, and e) and PLDLA (a and b) in CP (Figure 3), the ¹H NMR spectrum of the synthesized PLDLA-PCL-PU (Figure 5) exhibited the appearance of a new signal at 3.1 ppm (g). As observed in the PCL-PU spectrum (Figure 5), the appearance of this new signal (3.1 ppm) indicates the presence of CH₂ protons linked to the urethane group evidenced in the attached chemical structure³⁸,³⁹. The appearance of these signals confirms the successful synthesis of PLDLA-PCL-PU, corroborating the FTIR analysis.

3.3. X-ray diffraction (XRD)

The X-ray diffraction (XRD) measurements of PCL-PU and PLDLA-PCL-PU were shown in Figure 6.

Figure 6
X-ray diffraction patterns of PCL-PU and PLDLA-PCL-PU.

Although the analysis was conducted within the angular range (2θ) of 5 to 60°, the relevant peaks associated with PCL-PU and PLDLA-PCL-PU samples were observed in 2θ between 19 to 36°. In the PCL-PU sample two small peaks at 21.4° and 23.6° on top of the amorphous halo were identified, corresponding to the (110) and (200) crystallographic planes of semi-crystalline nature of PCL⁴⁰,⁴¹. However, unlike pure PCL, PCL-PU showed fewer sharp peaks, indicating a decrease in the crystallites organization of PCL block in the synthesized PCL-PU when compared to pure PCL. According to the work of Dai et al.⁴², PU is an amorphous polymer. The diffractogram showed that the presence of PCL in the PU structure favored the organization of the PCL-PU chains, thereby making it a semi-crystalline polymer.

The diffractogram of PLDLA-PCL-PU showed phases that are related to PCL and PLDLA (Figure 6). As described by Zapata-Catzin et al.⁴³, and evident in the obtained diffractogram, the crystallites of PCL present in the PLDLA-PCL-PU are identified by the most intense peaks located at 21.0 and 23.2°. As seen in the PCL-PU, these peaks are corresponding to the (110) and (200) crystallographic planes of semi-crystalline nature of PCL. On the other hand, less prominent peaks emerged around 31.4, 34.1, and 36.0°³⁴,⁴³,⁴⁴. The presence of a shoulder between 18.5° and 20.5° corresponds to the amorphous phase of PLDLA⁴⁵. Thus, the XRD study reveals the coexistence and differentiation of the crystalline and amorphous phases of the PCL and PLDLA polymeric constituents in the PLDLA-PCL-PU. The size of the crystallites inherent to its structure and the distances between the atomic planes were determined using the Scherrer equation and Bragg’s law, respectively. The obtained values were listed in Table 1.

Table 1
Size of crystallites present in the PCL-PU and PLDLA-PCL-PU.

PLDLA-PCL-PU						PCL-PU
2Ɵ (°)	21.0	23.2	31.4	34.1	36.0	21.4	23.6
t (angstrom)	98.5	116.9	219.9	181.7	175.6	34.0	97.3
d (angstrom)	4.2	3.8	2.8	2.6	2.5	4.1	3.8
hkl	110	200	-	-	-	110	200

It is possible to observe in Table 1 that the crystallites formed in the PCL-PU are slightly smaller than those found in the PLDLA-PCL-PU. According to Selli et al.⁴⁶ the average crystallite size for pure PCL is 62 angstroms for the (110) crystallographic plane and 42 angstroms for the (200) crystallographic plane.

The crystallite size is important information for polymeric biomaterials, since the correlation between the degree of crystallinity and structural parameters can significantly influence the in vivo properties, exerting direct effects on the processes of resorption in the biological context. It happens because the amorphous phase is reabsorbed more rapidly than the crystalline phase, resulting in relevant implications for biomedical applications⁴⁷. Despite the presence of smaller crystallites in PCL-PU, its crystallinity value is higher than that of PLDLA-PCL-PU. In this case, the crystallinity percentages of PCL-PU and PLDLA-PCL-PU were 11.7% and 4.3%, respectively. Thus, it can be observed that the presence of PLDLA in the PU structure reduces the degree of crystallinity of the sample due to its amorphous nature⁴⁸. This reduction is caused by the steric hindrance generated by the D,L-lactide monomer in the PLDLA structure, which makes it difficult for the chains to come together, thereby reducing the organization of the PLDLA-PCL-PU chains. According to Baji et al.⁴⁹, pure PCL has a crystallinity of 45%. So, the crystallinity values of PLDLA-PCL-PU and PCL-PU are lower than those of pure PCL. This reduction in crystallinity could be due to the greater heterogeneity of the PCL-PU and PLDLA-PCL-PU chains, which have larger groups compared to the pure PCL chain. The larger groups create greater difficulty in organizing the polymer chains in PCL-PU and PLDLA-PCL-PU due to increased steric hindrance in these polymers.

Finally, although theory suggests that a shift of the peak to lower 2θ values indicates an increase in the interplanar distance⁵⁰, the 0.4° variation in the crystalline planes (110) and (220) of PCL-PU and PLDLA-PCL-PU caused virtually no change in the interplanar distances. This shows that the presence of PLDLA does not alter the periodic arrangement of atoms, meaning the distance between the atomic planes is not affected. Therefore, PLDLA only hinders the approach of the chains necessary to initiate the formation of crystallites, but it does not affect the structure of the final crystalline arrangement.

3.4 . Thermogravimetric analysis (TGA)

The results of thermogravimetric analysis of PCL-PU and PLDLA-PCL-PU are presented in Figure 7.

Figure 7
TGA and DTG curves of the (A) PCL-PU and (B) PLDLA-PCL-PU.

The determined derivative curves allowed for observing a three-stages decomposition process that might indicate the presence of micro-phase separation in synthesized PU. The presence of these stages can be attributed to structural variations within the synthesized material, as highlighted by Neto et al.⁵¹. The first stage presented slightly pronounced peaks (T_peak) around 250 °C (PCL-PU) and 240 °C (PLDLA-PCL-PU); it is related to the thermal degradation that begins with the thermal dissociation of urethane bonds (the weakest against the temperature)⁵². In this case, the temperature of the first stage is probably associated with the degradation of the hard segment of the polymer, where the urethane bonds, considered more susceptible, are broken²⁵,⁵². The second stage presented T_peak around 300 °C (PCL-PU) and 285 °C (PLDLA-PCL-PU); it is related to the thermal decomposition of soft segments that are present in synthesized PU (PCL part of chain - PCL-PU and PLDLA-PCL-PU samples)²⁵. In this case, the temperature of the second stage would be associated with two phenomena that can happen concomitantly. The first one is related to the decomposition of the soft segment, with a contribution from the hard segment (PCL-PU and PLDLA-PCL-PU samples). This analysis suggests the thermal decomposition of the ester groups of polyols (PCL) present in the PU samples⁸,⁵³. The second one is associated with the degradation of the PLDLA present on the PLDLA-PCL-PU structure⁵⁴. The third stage presented T_peak around 448 °C (PCL-PU) and 444 °C (PLDLA-PCL-PU); it is related to the complete degradation of isocyanate from 400 °C⁵⁵. The three-stage decomposition process temperatures were based on the T_peak of DTG curves of PCL-PU and PLDLA-PCL-PU samples. Therefore, the slightly higher values of T_onset and T_peak (first and second stages) of the PCL-PU when compared to the PLDLA-PCL-PU could be justified due to its greater crystallinity than PLDLA-PCL-PU, as shown in Table 2.

Table 2
Thermogravimetric analysis (TGA) and DTG results for synthesized PCL-PU and PLDLA-PCL-PU.

Sample	Events	Tonset (°C)	Tpeak (°C)	Weight loss (%)
PCL-PU	1	230	250	6
	2	290	300	84
	3	-	448	10
PLDLA-PCL-PU	1	226	240	4
	2	278	285	87
	3	-	444	9

3.5. Differential scanning calorimetry (DSC)

The thermal behavior of the CP, PCL-PU, and PLDLA-PCL-PU was investigated through the second heating ramp in the DSC analysis, as shown in Figure 8. The measurements were used to determine the glass transition temperature (T_g), melting temperature (T_m).

Figure 8
DSC curve of CP, PCL-PU, and PLDLA-PCL-PU.

The DSC analysis of the CP clearly showed the appearance of two distinct thermal events. The first event located at -50 °C was associated with the T_g of the polymer. According to the literature, pure PCL-diol has a T_g of approximately -69°C⁵⁶, while PLDLA has a T_g of 58°C²³. The copolymerization process of PCL-diol with L-lactide and D,L-lactide, in conjunction with the use of HDI, may have resulted in an increase in Tg (-50°C)¹⁴. The increase in hard segments may lead to a higher T_g due to the restriction of movement of the soft segments. On the other hand, the two endothermic events at 33 and 42 °C were attributed to the T_m of the PCL-diol present in the CP structure and the unreacted PCL-diol, respectively⁵⁶.

The DSC analysis of the PCL-PU showed the appearance of two distinct thermal events. The first event, located at -54 °C, was attributed to the T_g of the soft segments, while the second event, at 30 °C, was associated with the T_m of the crystal structure of PCL-diol that reacted with HDI in the PCL-PU.

Unlike what was observed for CP and PCL-PU, the DSC analysis of the PLDLA-PCL-PU sample exhibited three distinct thermal events. The first event, occurring at -50°C, was attributed to the Tg of the soft segments of PLDLA-PCL-PU, indicating its flexibility, similar to what was observed for CP. However, the second exothermic event observed at 0°C was attributed to the crystallization of PLDLA-PCL-PU due to the presence of soft PCL segments. This phenomenon may be associated with a more heterogeneous crystalline structure resulting from the hindered orientation of the PCL soft segments after reacting with HDI to generate urethane bonds in PLDLA-PCL-PU⁵⁷,⁵⁸. Furthermore, the cold crystallization⁵⁹ of the PLDLA segment present in the PLDLA-PCL-PU structure may have also occurred. Up to now, four different crystal forms (named α, β, γ, and ε forms) and one disordered crystal form δ have been identified⁶⁰. When crystallizing at low temperatures, the δ crystal form, with disordered helical chain conformation as well as disordered chain packing mode, is usually produced⁶¹. The third event located at 33 °C was attributed to the T_m of the crystallinity organization of PCL and PLDLA.

3.6. Cell proliferation

To evaluate the hMSC proliferation on the PLDLA-PCL-PU membranes, laser scanning confocal micrographs were obtained after 24 hours and 7 days of culture as shown in Figure 9.

Figure 9
LSCM images of hMSC adhered to coverslips (control) and PLDLA-PCL-PU after 24 hours and 7 days. The nuclei were stained with DAPI while the actin cytoskeleton was labeled with Phalloidin-Alexa Fluor 647.

After 24 hours in culture, controls presented a subconfluent homogeneous and spreaded pattern throughout the coverslip surface with fully assorted polygonal rich filopodia morphology, while after 7 days, the monolayer appearance indicated full occupation of the coverslip surface. Although adhered, striking morphological differences were noted in cells spread to PLDLA-PCL-PU as compared to controls. Changes in cell nuclei identified by distinct heterogeneous areas of euchromatin and heterochromatin, while decreased spreading and fewer filopodia indicated that cells presented a lower adhesion as compared to controls. Irregular shaped cells adhered in PLDLA-PCL-PU followed the material surface topography.

This difference in cellular morphology of PLDLA-PCL-PU can be explained by the adaptation of cells to the material's structure. Amokrane et al.⁶² evidenced that cells adhered to the PCL-based membrane exhibited delayed adaptation to the material after 7 days under culture, showing a more rounded morphology, with reduced spreading and decreased cell contact surface. The changes in spreading here observed were not evidenced after 7 days in culture as presented by these authors, which indicated that cells were able to adhere, spread and proliferate for long periods, this recovery pattern reinforces that the cell adapted to the materials surface. Furthermore, as evidenced in the literature, PLDLA, PCL, and PU polymers are materials that do not exhibit cytotoxicity and cellular death behavior; instead, they are widely used for biomedical applications due to their individual characteristics²⁹,⁶³,⁶⁴.

The induction of cell migration increases chromatin compaction⁶⁵. Although no specific markers for chromatin were present, the DAPI dye revealed heterogeneous labeling that indicates chromatin decondensation in cells adhered to PLDLA-PCL-PU after 24 hours, while the homogeneous nuclei labeling in control is an indicative that cell behavior as migration and adhesion were well established. Since mobility and adhesion are related⁶⁶, early cell interaction to the material was affected. As the cell cycle is influenced by cell adhesion, the significant lower absorbance identified between groups after 24 hours in culture. Such changes were not maintained after 7 days, which showed a complete recovery of cells' cytoskeleton and nuclei morphology despite a slight decrease in cell quantity although considered not significant as observed in Figure 10. Although images were digitally contrast/brightness filtered, a discrete cyan background observed in PLDLA-PCL-PU images of LSCM at Figure 9 was attributed to a slight auto fluorescence detected by the laser 405, used to identify the DAPI labeling. Such fluorescent properties were already observed elsewhere in other polyurethane based materials⁶⁷.

Figure 10
Quantitative analysis of total cell counts after 24 hours and 7 days in culture on control and PLDLA-PCL-PU. Six random chosen areas under culture were digitally imaged and the total cell number of each image was counted. Values are expressed by means ± standard deviation. Statistical differences between groups are shown by **p < 0.01.

According to Asalpour et al.⁶⁸, the increase in adhesion and proliferation of cells in scaffolds based on polyurethane and polycaprolactone was shown from the 4^th day of cultivation and were continuous over time. Likewise, it was observed by Kemppi et al.⁶⁹ that PLDLA-based structures were able to maintain cell viability and proliferation for 14 days in culture, indicating the biocompatibility property of the material. Furthermore, polyurethane presents a very promising wound dressing property, showing its angiogenic potential and collagen deposition⁷⁰.

It is well known that L-lactide, DL-lactide and PCL-diol based materials have strong biocompatibility properties and are routinely used in the medical field to support cell growth⁷¹-⁷³. In addition, polyurethane also shows the ability to act in several medical areas, as well as to act as an antimicrobial dressing⁷⁴, in orthopedics, being able to improve osteogenesis⁷⁵ and in the cardiovascular area, the which contributes to the construction of heart valves⁷⁶. Polyurethane based materials synthesized with HDI highly showed cell biocompatibility as compared to other different diisocyanates synthesis⁷⁷. Therefore, the overall set of assays indicated that PLDLA-PCL-PU is highly biocompatible, despite early cell adaptation to the material's surface delayed cell adhesion. Thus, the polyurethane here presented owns the requirements for future applications in tissue engineering.

4. Conclusions

Confirmation of the newly synthesized PLDLA-PCL-PU was achieved through FTIR and ¹H NMR analyses. The PLDLA-PCL-PU presented a semicrystalline nature structure by XRD. The thermal analysis (TGA) revealed the thermal stability of PLDLA-PCL-PU up to 210 °C. Beyond this limit, the progressive increase in temperature begins the polyurethane degradation process. The DSC results showed T_g values, which characterized PLDLA-PCL-PU as a flexible polymer even under environmental conditions. Furthermore, the synthesized material showed biocompatibility in viability and proliferation assays with hMSCs after 7 days of culture. These results highlight the potential of PLDLA-PCL-PU as a biomaterial for future biomedical applications.

5. Acknowledgements

The authors thanks for the use permission by PPGBMA of the Laser Scanning Confocal Microscopy granted by Pró-Equipamentos (CAPES: 3420/2013-17, 2610/2014-90). The authors also acknowledge the FAPESP (Fundação de Amparo à Pesquisa do Estado de São Paulo) (Process no. 2023/17083-8).

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Publication Dates

• Publication in this collection
  14 Oct 2024
• Date of issue
  2024

History

• Received
  02 May 2024
• Reviewed
  26 Aug 2024
• Accepted
  16 Sept 2024