Mechanical Properties of Membranes of Poly ( Lco-DL-lactic acid ) with Poly ( caprolactone triol ) and Study In Vivo

1Department of Physiological Sciences, Faculty of Medical Sciences, Pontifical Catholic University of São Paulo (PUC-SP), 18030-095 Sorocaba, SP, Brazil 2University Center SOCIESC of Santa Catarina (UNISOCIESC), 89206-001 Joinville, SC, Brazil 3Department of Materials Engineering, Faculty of Mechanical Engineering, State University of Campinas (UNICAMP), 13083-970 Campinas, SP, Brazil 4Departamento de Ciências Fisiológicas, Centro de Ciências Médicas e Biológicas, Pontif́ıcia Universidade Católica de São Paulo (CCMB/PUC-SP), Praça Dr. José Ermı́rio de Moraes 290, Jardim Vergueiro, CP 1570, 18030-095 Sorocaba, SP, Brazil


Introduction
The biomaterials are meant to be implanted in the human body as constituents of devices that are designed to perform certain biological functions by substituting or repairing different tissues such as bone, cartilage or ligaments, and tendons and even by guiding bone repair when necessary.For this reason, polymers need to be biocompatible and bioresorbable in order to minimize adverse reactions in the organism [1].The increasing therapeutic use of polymers as biomaterial implants makes it essential to evaluate the behavior of subcutaneous implants in vivo.This is generally done in rats, which provide an ideal model for analyzing the rate of neovascularization, the formation of collagen types I and III, and the consolidation of the conjunctive capsule around the polymer [2].
Poly(L-lactide) is characterized by a high content of crystalline, high strength particles, and prolonged period of degradation.Poly(Dl lactide) is in contrast characterized by lower strength and quicker rates of degradation.Combining both poly(L-lactide) and poly(D,L lactide) results in a single amorphous copolymer that has a structure that combines the best characteristics of poly(L-lactic acid) and poly(DL-lactic acid), that is, the mechanical properties of the former and the shorter degradation time of the latter; these properties have made PLDLA a compound of great relevance in the controlled release of drugs and the treatment of bone fractures [3].In addition to these applications, PLDLA serves as 2 International Journal of Polymer Science substrates for regenerative medicine applications with special focus on skin tissue engineering [4].Despite these advantages, biomaterials containing poly(L-co-D,L lactic acid) are generally extremely dense, that is, devoid of pores and very rigid, and have a high modulus of elasticity, low elongation, and are hydrophobic; these characteristics limit the use of this polymer in a variety of medical applications, particularly when tissue/implant interactions are an important factor.
Many studies have shown that one way to improve the elongation characteristics, reduce the hydrophobicity of the polymer, and facilitate its interaction with tissue is to incorporate a low molecular weight polymer that can act as a plasticizer in polymeric matrices [5].Polycaprolactone triol, a low molecular weight aliphatic polyester obtained by ring opening of -caprolactone to yield three OH groups in the monomer molecule of the polymer chain terminal, has been used as a plasticizer.
The aim of this work was to study the thermal, mechanical, morphological, and biological properties of PLDLA membranes containing different concentrations of poly(caprolactone triol) (PCL-T) as a plasticizer and to assess their potential usefulness in vivo.

Membranes.
PLDLA/PCL-T membranes were prepared by the solvent casting technique, in blends of 100/0, 90/10, 70/30, and 50/50% (mass/mass), respectively, PLDLA/PCL-T in dichloromethane (Merck KgaA, Darmstadt, Germany), at a concentration of 10% (m⋅vol −1 ).After complete dissolution of the copolymer, the solution was poured onto a glass plate and placed in a glass container that had been saturated with the solvent.After solvent evaporation, the membranes were dried under vacuum for 8 h and stored in a desiccator under vacuum until characterized.

Dynamic Mechanical Analysis (DMA).
DMA was done with a model 242 dynamic mechanical analyzer (Netzsch).The samples were subjected to deformation (amplitude: 240 m) at a constant frequency of 1.00 Hz over a temperature range of −100 ∘ C to 200 ∘ C and a heating rate of 5 ∘ C/min, using a traction system.

Differential Scanning Calorimetry (DSC).
Calorimetric measurements were done with a DSC 2920 calorimeter (TA-Instruments) in which 7-10 mg samples sealed in aluminum pans were heated from 25 ∘ C to 200 ∘ C at a rate of 10 ∘ C/min and maintained at the final temperature for 5 min prior to cooling to 25 ∘ C at the same rate.The samples were maintained at 25 ∘ C for 5 min and then reheated.All tests were done under a nitrogen atmosphere.

Fourier
Transform Infrared (FTIR) Spectroscopy.The quality of the PLDLA/PCL-T blends was assessed by FTIR spectroscopy.The IR measurements were done on Perkin-Elmer Spectrum One Perkin-Elmer 983G infrared spectrophotometer, using the KBr pellet technique, over the frequency range of 400-4000 cm −1 , with a total of 256 scans.

Surface Contact
Angle of the Membrane.The contact angles were measured with a contact angle goniometer (Ramé-Hart 100-00) and drop shape analysis software (RHI 2001 Imaging Software) at room temperature.A drop of deionized water (0.2 mL) was placed on the sample and the images of the water on the sample were recorded using a digital camera at 0.5 second intervals.From the angle measured between the droplet baseline and the tangent at the water/air boundary, a contact angle was calculated as the average of the measured left and right contact angles.For statistical analysis, three contact angle measurements were obtained.
Contact angle measurements were also used to evaluate the surface energy of the samples.This was done by measuring the angle () to a polar liquid (water) and nonpolar liquid (diiodomethane) using the method harmonic [7].

Tensile Strength
Test.PLDLA/PCL-T membranes containing polymer blends of 100/0, 90/10, 70/30, and 50/50 (m/m) were cut into strips 13.01 ± 0.45 mm wide and approximately 0,20 ± 0.45 mm thick and the tensile strength was determined with a model 810 Testar II apparatus (Instron) at 50 mm⋅min −1 and a load capacity of 1 kN.The samples were tested five times at a fixed humidity (50%) and temperature (25 ± 2 ∘ C).The rats were anesthetized with a combination of ketamine and xylazine (50 mg/kg and 10 mg/kg, i.p., resp.).Subsequently, two lateral incisions approximately 1 cm long were made in the dorsal cervical region.A membrane disc 6 mm in diameter was inserted into the left incision while the right incision served as the control (sham operated, no membrane inserted) (Figure 1).One implant was placed in each rat.The incisions were then sutured with nylon 5-0 suture.

Studies In
At each time interval after implantation, the rats were anesthetized with ketamine 10% (100 mg/Kg) and xylazine 2% (6 mg/Kg) and the tissues surrounding the implant were excised.

Histological Analysis.
The material surrounding the tissues was washed in saline solution and immediately fixed in 10% paraformaldehyde.The specimens were dehydrated in an increasing alcohol series, embedded in a paraffin (Histosec, Merck) and sectioned (4 m) with a high-precision microtome (Leica RM 2245).The sections were subsequently mounted and stained with hematoxylin and eosin (H&E) and with Masson's trichrome (for 30-and 60-day intervals).The stained sections were examined with a Nikon Eclipse E800 light microscope and images were captured using a Nikon FDX 35 digital camera attached to the microscope.

Mechanical and Physicochemical
Properties.DMA was done on membranes containing PLDLA/PCL-T blends of 100/0, 90/10, 70/30, and 50/50.The -relaxation associated with the glass transition (  ) of the amorphous phases of PLDLA and PCL-T was determined from curves of loss of modulus versus temperature (  versus ), as described in ASTMD 4065-2001.The   versus  curves showed the presence of two glass transitions (  ) typical of the presence of PLDLA and PCL-T (Figure 2(a)).The   values for PLDLA decreased significantly in membranes containing 10% and 30% PCL-T (Table 1), indicating that the components were partially miscible.These findings agreed with those of Meier et al. ( 2004) [8] who used PCL-T as a plasticizer in cellulose acetate films and observed that the   of cellulose acetate decreased as the PCL-T content increased.
The 50/50 blend (Figure 2(a)) showed behavior indicative of immiscibility (phase separation) of the components and this was confirmed by the   values, which were very close to those of pure polymers; this immiscibility probably reflected the presence of excess plasticizer.Experiments with PLLA/PEG blends found that a plasticizer concentration > 20% caused phase separation [9].
Figure 2(a) shows that there was secondary -type relaxation in PLDLA/PCL-T blend 100/0 (X) caused by the mobility of the methyl group linked to its main chain.This relaxation disappeared in the presence of PCL-T, probably because of the steric hindrance caused by PCL-T or overlap in the melting temperature of PCL-T.

Calculation of the Interaction Parameter (𝜒 12
) for PLDLA/ PCL-T Blends.The interaction parameters of the polymer blends were calculated based on the   obtained by DMA.According to the Flory-Huggins theory, for a system to show complete miscibility, the interaction parameter ( 12 ) should be <0.5; in this case only the   [10].The  12 for PLDLA/PCL-T blends was calculated using the Fox equation as follows: where  Based on the content of PLDLA and PCL-T, (2) [11] was used to obtain the interaction parameter of the blend ( 12 ), the values of which are shown in Table 2: where  1 = degree of polymerization of PLDLA = 1780.67and  2 = degree of polymerization of PCL-T = 2.63.Table 2 shows that the addition of 10%, 30%, and 50% PCL-T to the blends resulted in  12 values of 0.6, 0.5, and 0.8, respectively.The decrease in  12 for the 70/30 blend may be directly related to the higher concentration of polar components (plasticizer) that interact with the ester groups of PLDLA.The 50/50 blend had the highest  12 compared to the other blends and was again indicative of the immiscibility of the system.
There was no notable difference in the   values for the first and second heating of membranes containing pure PLDLA (100/0 blend); there was also no melting transition phase in these curves, indicating that the material was amorphous, in case the membranes containing PCL-T has been a fall in the value of   compared to pure PLDLA, which  is a consequence of the increased flexibility of the material.The membranes of the blends 70/30 and 50/50 showed similar behavior in their curves.The   and crystallization temperature of PCL-T were well defined.

Fourier Transform Infrared (FTIR) Spectroscopy.
The FITR spectra in Figure 4 show all the characteristic groups of the copolymer poly(L-D,L-lactide).The spectrum for the PLDLA membrane showed absorption bands (, cm −1 ) at 2997-2965 (CH 2 , CH 3 ), 1747 (C=O), 1360-1450 (CH 3 ), 1266 (C-O bond in COO), and 750 (CH) characteristic of this material [12].PCL-T showed bands similar to those of PLDLA that differed only in the axial asymmetrical deformation of fading in the 2995 cm −1 OH stretch and the appearance of a peak in the terminal region of the polymer chains (around 3467 cm −1 ).The FTIR spectra for PLDLA/PCL-T blends revealed a shift in the OH stretch in blends 90/10 and 70/30 at a wavelength of 3500 cm −1 with respect to pure PCL-T (3467 cm −1 ); this shift can be assigned to links (H-bridges) between PLDLA and PCL-T.The 50/50 blend showed a band shift in the OH stretch to a shorter wavelength and this change was probably associated with excess plasticizer that could result in competition between the polymer/linked plasticizer, plasticizer/plasticizer.

Contact Angles.
Measurement of the contact angle between water and a film surface is one of the easiest ways to characterize the hydrophilicity/hydrophobicity of a film.Good hydrophilicity is beneficial for biocompatibility and flow of nutritional liquid in vivo [13].Cells adhere to and spread easily on substrates that have moderate hydrophilicity compared to very hydrophilic or hydrophobic substrates [14].For the present analysis, the values (limits) for defining hydrophilic/hydrophobic interactions were a contact angle of 65 ∘ and a water adhesion tension () of 30 dynes⋅cm −1 , as described by Lim et al. (2004) [15].
The water adhesion tension was calculated as where  = water surface tension (72.8 dynes⋅cm −1 ) and  = contact angle [15].Surfaces with contact angles < 65 ∘ and high values of  were defined as hydrophilic, whereas surfaces high contact angles and low values of , that is, low wettability, were considered to be hydrophobic.Table 4 shows the contact angles, surface energy, and water adhesion tension for PLDLA/PCL-T membranes.The values of  were calculated according to (3).
Based on the values shown in Table 4, the surface of PLDLA/PCL-T 100/0 membranes was hydrophobic and the addition of PCL-T made this more hydrophilic, as shown by the decrease in the contact angle.This effect was expected because of the presence of polar groups in PCL-T that increase the interaction with water, increasing the surface energy and water adhesion tension.Figure 5 shows the wettability of the membrane surface of different blends of PLDLA/PCL-T with deionized water; as can be seen, wettability increased proportionally to the amount of PCL-T, in agreement with the results in Table 4.
International Journal of Polymer Science  3.6.Tensile Strength Tests.Table 5 and Figure 6 show the relationship between tensile properties and the PCL-T content of PLDLA/PCL-T membranes (100/0, 90/10, 70/30, and 50/50 blends).PLDLA showed the typical characteristics of a vitreous polymer, with a modulus of elasticity of 1564 ± 141 MPa, resistance to traction ( max ) of 21 ± 5.5 MPa, and deformation at rupture of 8.7 ± 1.2% (Table 5).The addition of PCL-T significantly reduced the modulus of elasticity () (Table 5), in agreement with the findings of Meier et al. ( 2004) [8] in their study of the effects of PCL-T on cellulose acetate.The presence of PCL-T significantly altered the deformation at rupture, indicating an increase in flexibility (Table 5, Figure 6); the 90/10 blend showed the greatest deformation at rupture, with a value of 21.4 ± 6% compared to pure PLDLA, which had a value of 8.7 ± 1.2%.The higher value of the 50/50 blend reflected the ability of the plasticizer to dissipate energy at fracture interfaces, thereby delaying rupture of the membrane [9].With regard to the 70/30 blend, one possible explanation for the unexpectedly higher modulus values obtained for this membrane compared to the 90/10 membrane could be the proximity between the temperature  The values are the mean ± SD of 5 determinations.In all cases, the inflammatory infiltrate in the controls was markedly less than that at the implanted sites (Figure 7(c)).At three days after implantation, sites implanted with the 100/0 blend showed an area delimited by organized fibrin deposition, with congested vessels and the presence of mononuclear cells, but no giant multinucleated cells.Sites implanted with the 90/10 blend also showed the fibrin "capsule, " but it was not as organized and there were frequently macrophages around the implant, in addition to congestion and edema.The control sites of both groups had a similar appearance, that is, granulated tissues with a chronic inflammatory infiltrate containing macrophages, plasmocytes, and mast cells.
At 15 days after implantation with the 100/0 blend, there was a well-defined capsule delimited by dense collagen and an internal surface containing a few mononuclear cells International Journal of Polymer Science ).All control sites showed complete healing with no significant alterations.At 60 days after implantation, sites implanted with the 100/0 blend showed thick fibrosis around the capsule and practically no inflammatory infiltrate except for a small number of mononuclear cells in the interstitium (Figure 9).Sites implanted with the 90/10 blend showed a similar capsular organization to that seen with the 100/0 blend but with a discrete chronic inflammatory infiltrate; there were no giant multinucleated cells.The control sites showed no marked alterations.In contrast to our findings, previous studies of PLLA membranes implanted subcutaneously in rats reporting the presence of giant multinucleated cells up to 180 days after the implantation reported that poly(DL-lactic acid) without plasticizer caused a chronic inflammatory response involving giant cells, macrophages with phagocytosed material, and fibroblast proliferation [5,16].Macrophages have an important role in tissue repair through their ability to produce growth factors and mediators responsible for fibroblast proliferation and collagen synthesis [17].

Staining with Masson's
Trichrome.The histological alterations observed with specific staining (Masson's trichrome) confirmed those observed with general staining (HE).In the case of the 100/0 blend, the capsule was practically devoid of an inflammatory infiltrate, while, in the case of the 90/10 blend, the collagen was looser and there was granulous tissue containing macrophages.
At 60 days after implantation, for both of the blends tested, there was well-formed, dense collagen that was practically devoid of an inflammatory infiltrate.No giant multinucleated cells were observed at any of the sites.For the 100/0 blend, the capsule was well organized with mature collagen and no inflammatory infiltrate (Figure 9(a)), while for the 90/10 blend the collagen was more loosely organized and there were few mononuclear cells (Figure 9(b)).
Although this in vivo analysis was only a preliminary investigation, our results indicate that PLDLA membranes containing PCL-T are potentially useful, particularly since these polymers are nonallergic, nontoxic, and flexible; the latter characteristic in particular facilitates the handling of these membranes during surgical procedures.

Conclusions
In this study, we have shown that the addition of PCL-T to PLDLA membranes markedly affected the mechanical, thermal, morphological, physicochemical, and biological properties of the resulting membrane, especially increasing the flexibility of PLDLA.The addition of PCL-T at concentrations of 10% and 30% resulted in partially miscible systems while the addition of 50% resulted in immiscibility, probably because of the excess of plasticizer.
The analysis of PLDLA/PCL-T membranes in vivo indicated satisfactory polymer/tissue interaction.By 60 days after implantation, both 100/0 and 90/10 blends resulted in dense collagen deposition that was more loosely organized with the latter blend, probably because of the slower degradation.
These results indicate that the use of PLDLA PCL-T membranes in medical devices could be advantageous, particularly where there is no need for long-term implants, for example, in supporting cell cultures, in dressing skin ulcers, and in guided regeneration in periodontics.

Figure 1 :
Figure 1: Surgical implantation of PLDLA/PCL-T membrane.A membrane disc was implanted in the incision on the left while the incision on the right served as the control.

Figure 6 :
Figure 6: Tension versus deformation curves for PLDLA/PCL-T membranes.Standard deviation bars have been omitted for clarity.

Figure 7 :
Figure 7: Photographs of sites one day after implantation of PLDLA/PCL-T membranes.(a) Site implanted with the 100/0 blend showing the formation of a fibrous capsule ( * ) around the membrane (M) and an infiltrate of polymorphonuclear cells, (b) site implanted with the 90/10 blend showing a similar result to (a), and (c) control site showing an infiltrate of polymorphonuclear cells.Staining: hematoxylin and eosin (H&E).Magnification: 100x.

Figure 8 :Figure 9 :
Figure 8: Photographs of sites 15 days after implantation of PLDLA/PCL-T membranes.(a) Site implanted with the 100/0 blend showing the formation of a capsule delimited by dense collagen (yellow arrow), (b) site implanted with the 90/10 blend showing individualized capsules and the presence of giant cells (green arrow) and blood vessels (red arrow).Staining: HE.Magnification: 100x.

Table 1 :
The   temperatures for PLDLA/PCL-T membranes determined by DMA based on the loss of modulus (  ).

Table 4 :
Contact angle, surface energy, and water adhesion tension for membranes containing PLDLA and PLDLA/PCL-T blends.

Table 5 :
Maximum tension, tension at rupture, deformation at rupture, and modulus of elasticity () obtained in the tensile strength tests of PLDLA/PCL-T membranes.