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collagen blends
journal of the mechanical behavior of biomedical materials 103 (2020) 103577
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Thermal and mechanical behavior of ultra-high molecular weight polyethylene/collagen blends ^nica Rufino Senra, Maria de Fa �tima Vieira Marques * Mo Instituto de Macromoleculas Eloisa Mano, IMA-UFRJ, Universidade Federal do Rio de Janeiro, Cidade Universit� aria, Av. Hor� acio Macedo, 2.030. Centro de Tecnologia. Bloco J, Rio de Janeiro, RJ, 21941-598, Brazil
A R T I C L E I N F O
A B S T R A C T
Keywords: UHMWPE Collagen Polymer blend Orthopedic applications
Bone defects or diseases significantly affect quality of life, thus the development of materials with improved performance that can be used as bone substitutes is increasingly studied. As an alternative, ultra-high molecular weight polyethylene (UHMWPE) has been employed for orthopedic applications since it combines high wear resistance, high impact resistance and low friction coefficient. However, it is a bioinert material and difficult to process. In the present work, the addition of collagen (hydrolyzed or type II), one of the constituents of natural bone, to UHMWPE was studied aiming to improve its processability and possibly its biocompatibility. The blends were prepared by compression and twin-screw extrusion. The results show that addition of higher amounts of both collagens to UHMWPE reduced the degree of crystallinity. However, crystallization and melting tempera tures were not affected. The thermogravimetric analysis exhibited two thermal events correlated to the degra dation of collagens (Tmax~300 � C) and of UHMWPE (Tmax~480 � C), corroborating the FTIR analysis that presented bands corresponding to these materials. The extrusion process promoted a better dispersion of the collagens, especially the hydrolyzed one. In addition, the obtained materials presented better mechanical properties when extruded. Torque reduction during extrusion showed that hydrolyzed collagen aid processing, even more than collagen due to its smaller molecular weight.
1. Introduction Millions of people worldwide suffer from bone disorders, bone fractures/injuries and diverse musculoskeletal problems that are usually treated by drug therapies or surgeries, which generally include partial or total replacement of the diseased tissue (Ferreira et al., 2012). Ultra-high molecular weight polyethylene (UHMWPE) is a material widely studied for prosthesis manufacturing. Mechanical properties similar to natural bone, high wear and impact resistance, low friction coefficient and biocompatibility turns this polymer a suitable material for bone replacement (Mirsalehi et al., 2016; Sattari et al., 2014). For this reason, it is broadly used in parts of hip, knee and shoulder prosthesis. UHMWPE has a very high molecular weight, which provides to this material excellent characteristics such as high impact resistance, low friction coefficient and high wear resistance. However, their high mo lecular weight causes an increase in the entanglements between the polymer chains, reducing their mobility. The high viscosity of UHMWPE makes it very hard to be processed and also the dispersion of fillers in
this matrix is inhibited (Zhang and Liang, 2018). Due to the difficulties presented, it is a challenge to process UHMWPE and, therefore, many studies have been looking for ways to facilitate its processing since conventional injection and extrusion cannot be applied owing to the high viscosity in the melt. As a conse quence, compression molding and RAM extrusion are the two widely manufacturing processes used to produce UHMWPE artifacts (Kurtz, 2015). The compression molding was the first manufacturing process used to process UHMWPE powder, having been originated in Germany in 1950. It is a discontinuous process based on material sintering without limiting molecular weight and melt viscosity. This manufacturing method is still being used by two companies, Orthoplastics and Medi TECH, who produce compression mould sheets of GUR 1020 (MM~3.5 � 106 g/mol) and GUR 1050 (MM~5.5-6.0 � 106 g/mol) (Baena et al., 2015). The RAM extrusion was developed in the United States in 1970 and can be considered as a continuous process of compression and sintering at a temperature close to the UHMWPE melting. Unlike conventional
* Corresponding author. E-mail address: [email protected] (M.F. Vieira Marques). https://doi.org/10.1016/j.jmbbm.2019.103577 Received 9 August 2019; Received in revised form 2 October 2019; Accepted 29 November 2019 Available online 2 December 2019 1751-6161/© 2019 Elsevier Ltd. All rights reserved.
M. Rufino Senra and M.F. Vieira Marques
Journal of the Mechanical Behavior of Biomedical Materials 103 (2020) 103577
Fig. 1. FTIR spectra (a) pure HC and COL, (b) UHMWPE/HC pressed, (c) UHMWPE/HC extruded, (d) UHMWPE/COL pressed and (e) UHMWPE/COL extruded.
extrusion where material is carried by a screw, RAM extrusion is a plug flow process, in other words, in batch without shear, where the partic ulate material is fed to an elongated die while a piston compatible with the die cavity travels a round trip in the feed zone. In this path occurs the powder compaction and the heat transfer to the polymer, taking place the sintering. Soon after, the extruded goes through the cooling (Kurtz, 2015). In this type of process plates, tubes and rods can be produced being made UHMWPE products with good surface quality. However,
residual stresses may occur in the bulk of the material, thereby the extrusion products must undergo a high temperature annealing treat ment to remove these residual stresses. This procedure can increase the crystallinity of the polymer, which is useful for maintaining the excellent mechanical performances of UHMWPE. However, some drawbacks are associated with this type of processing, such as fluctuations in product quality, long plasticization cycle, slow extrusion rate and high power consumption (Zhang and Liang, 2018). 2
M. Rufino Senra and M.F. Vieira Marques
Journal of the Mechanical Behavior of Biomedical Materials 103 (2020) 103577
ability of bone to withstand mechanical forces and fractures depends not only on the amount of bone tissue (mineralization), but also on its quality (collagen structure organization) (Daneault et al., 2015). There are 28 types of collagens, which have as a common structural feature the presence of a triple helix chains formed by three polypeptide, called alpha chains, which may be identical or different (Mayne and Burgeson, 1987). Collagen derivatives, such as gelatin and hydrolyzed collagen, are widely used in the food, cosmetics and pharmaceutical industries, as well as in tissue engineering. Gelatin is obtained by ther mal denaturation of collagen, which promotes the separation of collagen
Table 1 Band assignments for HC, COL and UHMWPE blends. Bands Assignment
ν (cm 1)
Assignment
HC and COL
3406 3078 1651 1542 1242 561
Stretching N–H of Amide A Stretching C–H of Amide B Stretching C– –O of Amide I Stretching C–N and bending N–H of Amide II Stretching C–N and bending N–H of Amide III Out of plane bending N–H
UHMWPE blends
3340 2916 2849 1651 1542 1472 1462 1245 1153 731 719
Stretching C–O–C Stretching CH2 antisymmetric Stretching CH2 symmetric Stretching C– –O of Amide I Stretching C–N e bending N–H of Amide II In plane bending CH2 In plane bending CH2 Stretching C–O–C Stretching C–O–C In plane bending CH2 In plane bending CH2
Table 2 Degree of crystallinity of neat UHMWPE and UHMWPE blends.
Bone can be considered a natural nanocomposite formed of collagen fiber networks impregnated with calcium phosphate, mainly by hy droxyapatite (HA) nanocrystals (Jiang and Liu, 2016). In bone, collagen plays an important role in force transmission and maintenance of tissue structure and determines the amount of mineral deposition. Thus, the
�
Sample
Xc (%)
Sample
Xc (%)
UHMWPE� 1% HC� 2% HC� 5% HC� 10% HC�
48.2 50.7 48.1 46.8 43.9
UHMWPEΔ 1% HCΔ 2% HCΔ 5% HCΔ 10% HCΔ
47.1 50.0 47.3 43.8 42.8
1%COL� 2%COL� 5%COL� 10%COL�
47.0 47.3 42.7 42.3
1%COLΔ 2%COLΔ 5%COLΔ 10%COLΔ
44.9 43.7 44.3 39.2
pressed material; Δ extruded material.
Fig. 2. XRD profiles of pure UHMWPE and processed UHMWPE blends. 3
M. Rufino Senra and M.F. Vieira Marques
Journal of the Mechanical Behavior of Biomedical Materials 103 (2020) 103577
2. Materials and methods 2.1. Materials The UHMWPE (UTEC 3040), with average molecular weight of 3.0.106 gmol 1, was purchased from Braskem SA. Collagen type II was donated by the Health Science Center of Rio de Janeiro Federal Uni versity (UFRJ), obtained by direct extraction of the extracellular matrix of bovine cartilage. The extraction was carried out for 24 h in acidic aqueous solution of pH ¼ 3 containing 5% DMSO and acetic acid. After extraction, the collagen was washed with deionized water until neutral pH and then was lyophilized for 76 h. Hydrolyzed collagen was acquired from Amazonas Rio Norte Produtos Naturais Ltda and vitamin E, a �rios farmac^euticos. natural antioxidant, was provided by Ach�e laborato 2.2. UHMWPE processing conditions Fig. 3. Thermal degradation profiles of pure HC and COL.
UHMWPE blends with 1, 2, 5 and 10 wt% of HC or COL with 0.8 wt% of vitamin E were prepared by twin-screw extrusion and press molding. Before both processing methods, the components of the blend were firstly mixed in an analytical mill (A11, IKA) for 2 min. UHMWPE with 0.8 wt% of vitamin E was also processed with the same conditions (extrusion and press molding) for comparison.
chains in the triple helix through the destruction of crosslinks. In order to obtain hydrolyzed collagen, gelatin undergoes enzymatic hydrolysis and collagen chains are broken down into small peptides (Daneault et al., 2015). Mineralization is a process of bone formation promoted by osteo blasts that create an environment for the concentration of calcium and phosphate. Osteoblasts secrete type I collagen, in addition to many noncollagenous proteins and collagen serves as a template to initiate and propagate mineralization (Ferreira et al., 2012). So, the addition of collagen to biomaterials is a strategy to improve osseointegration. Some works already have reported that collagen on the materials surface improved cell adhesion and proliferation (Cheng and Teoh, 2004; Müller et al., 2005; Peng et al., 2001; Yu et al., 2014). This can be attributed by a biospecific interaction that works between the cell and the collagen surface (Tamada and Ikada, 1994). In this work, hydrolyzed and type II collagens were used because they are inexpensive compared to type I collagen, whose use is widely reported in the literature. Type II collagen is more easily extracted from bovine cartilage compared to type I collagen extracted from bone. This occurs because the organic bone matrix is more complex and removing all the protein residues makes the process extensive. Moreover, the demineralization process (removal of HA) can lead to rupture of collagen chains. In the literature, several methods for obtaining low density poly ethylene (LDPE) film with collagen are found (Castiello et al., 2009; Dascǎlu et al., 2005; Haroun, 2010; Puccini et al., 2017, 2015). How � et al., 2008) or high ever, the blend of collagen with UHMWPE (Bo�ckova density polyethylene (HDPE) (Kinoshita et al., 1993; Matsumura et al., 2000; Tamada and Ikada, 1994) is not very well studied. Moreover, in these works the materials were not processed by extrusion or compres sion, the polyethylenes (PE) had their surface modified. Briefly, the PE was chemically modified on their surface and peroxide groups were introduced, then they were grafted with carbonyl monomers, in which the acrylic acid is widely used. After that, PE and collagen react and the carbonyl groups of the grafted chain binds to the nitrogen of the collagen amide groups. For this reason, this is the first work that processes UHMWPE with collagen. The aim of introducing collagen to UHMWPE was first improve its processability and possibly improve its biocom patibility with bone tissue. The purpose of the work is to prepare blends of UHMWPE with hy drolyzed collagen (HC) and collagen type II (COL) by twin-screw extrusion and by compression, the most common manufacturing pro cess of UHMWPE pieces and verifies the plasticizer effect of the collagens.
2.2.1. Twin-screw extrusion conditions For processing the blends, a Haake Minilab Rheomex (CTW5) from Thermo Scientific was used, with counter-rotating twin-screws and automatic bypass for circulation/extrusion. The extruder was operated at 200 � C with an initial rotation speed of 20 rpm. After the torque remained constant (around 4 min), rotation speed was raised to 60 rpm until the end of the process (total extrusion time: 12 min). 2.2.2. Press molding conditions The compressed materials were produced by pressing the premixed materials at 90 MPa for 10 min and at constant temperature of 200 � C. It was prepared films and specimens for the mechanical and thermal analyses. 2.3. Fourier-transform infrared spectroscopy (FTIR) FTIR spectra of collagens were recorded using Nicolet-Magna 760 spectrophotometer with KBr pellets in the wavenumber range between 400 and 4000 cm-1. For the polymeric samples FTIR spectra were recorded using IRAffinity-1 spectrophotometer in the wavenumber range between 650 and 4000 cm-1. The polymeric films were analyzed in the ATR (Attenuated total reflection) mode and the spectra were normalized for comparison. 2.4. X-ray diffraction X-ray diffraction analysis was performed using a Rigaku XRD diffractometer at room temperature, using Cu Kα (0.154 nm) radiation. The scanning was performed in the Bragg angle (2θ) range of 2–60� , with a scanning rate of 0.05� /min. Samples were analyzed as films. UHMWPE is a semicrystalline polymer with a most common ortho rhombic unit cell. The orthorhombic form is characterized by the crys tallographic planes (100) and (200) at 2θ ffi 21� e 23� , respectively (Fang et al., 2007; Joo et al., 2000). To study the effect of the addition of collagens to the polymeric matrix the degree of crystallinity of the blends was obtained by the equation 1, through the areas of crystalline (Sc) and amorphous (Sa) peaks. Xc ¼
4
Sc x100 Sc þ Sa
M. Rufino Senra and M.F. Vieira Marques
Journal of the Mechanical Behavior of Biomedical Materials 103 (2020) 103577
Fig. 4. TG and DTG curves for pure UHMWPE and for blends.
5
M. Rufino Senra and M.F. Vieira Marques
Journal of the Mechanical Behavior of Biomedical Materials 103 (2020) 103577
Table 3 Thermal degradation properties for UHMWPE/HC blends. Sample
�
1st thermal event
Table 6 Dynamic-mechanical properties of pure UHMWPE and UHMWPE blends.
2nd thermal event
Tonset (� C)
Tmax (� C)
Weight (%)
Tonset (� C)
Tmax (� C)
Weight (%)
UHMWPE� 1% HC� 2% HC� 5% HC� 10%HC�
– 251 238 246 245
– 298 303 297 305
– 0.9 1.6 4.7 7.2
447 454 462 444 455
469 472 480 465 473
99.7 98.6 98.2 94.0 90.2
UHMWPEΔ 1% HCΔ 2% HCΔ 5% HCΔ 10% HCΔ
– 256 255 247 253
– 310 303 321 319
– 1.2 2.1 4.1 5.1
469 471 457 460 469
490 489 476 479 487
99.6 98.3 96.8 94.8 92.5
pressed material; Δ extruded material.
Table 4 Thermal degradation properties for UHMWPE/COL blends. Sample
�
1st thermal event
2nd thermal event
Tonset (� C)
Tmax (� C)
Weight (%)
Tonset (� C)
Tmax (� C)
Weight (%)
UHMWPE� 1% COL� 2% COL� 5% COL� 10% COL�
– 248 254 261 270
– 298 295 302 298
– 1.5 2.4 3.4 7.0
447 460 456 463 460
469 479 476 480 478
99.7 97.5 96.9 95.3 89.8
UHMWPEΔ 1% COLΔ 2% COLΔ 5% COLΔ 10% COLΔ
– 262 267 272 267
– 299 300 303 300
– 1.6 2.2 4.4 5.2
469 460 462 459 461
490 478 480 479 479
99.6 98.0 97.4 94.3 92.2
�
UHMWPE UHMWPEΔ
�
Tc (oC)
Tm (oC)
119 118
132 133
Sample
Tc (oC)
Tm (oC)
Sample
Tc (oC)
Tm (oC)
1% HC 2% HC� 5% HC� 10% HC�
119 119 120 120
131 131 132 131
1% HC 2% HCΔ 5% HCΔ 10% HCΔ
120 119 119 119
132 132 132 132
1% COL� 2% COL� 5% COL� 10% COL�
120 120 119 120
131 131 132 131
1% COLΔ 2% COLΔ 5% COLΔ 10% COLΔ
119 119 119 119
132 133 132 133
�
E00 25 � C (MPa)
Tβ (oC)
UHMWPE� UHMWPEΔ
713,9 989,5
58.7 79.2
103.9 105.1
109.4 107.7
18.2 25.3
44.8 44.8
1% HC� 2% HC� 5% HC� 10% HC�
721.5 582.4 569.2 858.2
51.3 49.9 43.0 67.7
104.1 112.6 104.4 106.2
107.0 108.8 108,6 109.7
21.1 22.4 15.2 22.7
45.6 40.2 50.6 42.9
1% HCΔ 2% HCΔ 5% HCΔ 10% HCΔ
921.7 785.4 927.4 717.9
74.6 68.7 69.4 53.8
104.5 110.2 106.9 105.4
107.0 113.9 110.1 108,9
23.8 22.4 20.1 22.0
43.9 40.5 49.3 46.0
1% COL� 2% COL� 5% COL� 10% COL�
632.1 716.3 635.3 761.8
55.6 61.7 57.0 66.1
108.5 108.3 108.7 109.2
111.5 111.5 111.2 113.0
18.8 20.7 20.7 22.2
40.7 41.2 42.4 40.8
1% COLΔ 2% COLΔ 5% COLΔ 10% COLΔ
940.7 929.4 913.4 921.1
83.3 80.1 78.2 81.9
109.8 108.3 108.85 109.78
112.7 112.6 111.7 113.7
23.9 22.4 24.0 24.7
42.1 42.1 41.8 41.5
Tα (oC)
pressed material; Δ extruded material.
2.7. Dynamic-mechanical thermal analysis (DMA) DMA was carried out on a TA Instruments Q 800 equipment. The storage modulus (E0 ), loss modulus (E00 ) and tan δ values were found under a sinusoidal applied strain over a range of temperatures from 140 to 120 � C at a heating rate of a 3 � C/min and an operating fre quency of 1 Hz. The glass transition temperature (Tg) was also deter mined from the tan δ curve as a function of temperature. Specimens were obtained with approximate dimensions of 13.3 � 6.08 � 0.60 mm from the materials processed by press molding or extrusion.
Table 5 Thermal characteristics obtained from DSC curves. �
E0 25 � C (MPa)
verify the thermal transitions of the processed samples. The samples (4–5 mg) were hermetically sealed in an aluminum sample holder. The tests were conducted in a nitrogen atmosphere with flow of 50 mL/min. In the first run the material was heated from 0 � C to 200 � C with a heating rate of 10 � C/min to eliminate thermal history. After, the sample was cooled to 0 � C at the same rate, and the crystallization temperature (Tc) was determined. Thereafter, the sample was heated again at 10 � C/ min to 200 � C and the melting temperature (Tm) was determined.
pressed material; Δ extruded material.
Sample
Tg, tan δ (� C)
Tγ (oC)
Sample
Δ
2.8. Mechanical tensile testing The mechanical tensile test was carried out according to the inter national standard ASTM D638 (Standard Test Method for Tensile Properties of Plastic) on a universal testing machine Emic model L 3000 with a load cell of 30 KN and a speed of 10 mm/min. In the present work, the modulus was calculated by linear regression using the stress-strain curve points up to 0.5% deformation. Since the yield strength at the stress-strain curve was not possible to detect, the offset yield strength at 10% was calculated. Five specimens were tested for each sample and the results presented refer to the average values calculated.
pressed material; Δ extruded material.
2.5. Thermogravimetric analysis (TGA) Thermal analysis was conducted in a TA Instruments Q500 analyzer and was used to investigate the collagen influence on UHMWPE degradation rate. Samples of 10 to 15 mg were heated from 25 up to 700 � C (polymeric samples) and up to 900 � C (pure collagens) under nitrogen atmosphere at a flow rate of 60 mL/min. All the experiments were conducted at 10 � C/min and TG/DTG curves were recorded. The tem peratures of the start of degradation (Tonset) and maximum degradation rate (Tmax) were determined, as well as the weight loss.
3. Results and discussion The FTIR spectra of pure hydrolyzed collagen (HC) and collagen type II (COL) and their blends with UHMWPE are shown in Fig. 1. The presence of a band at 1651 cm 1 in HC and COL spectra corresponds to a – O stretching of amide I; at 1542 cm 1 there is a band that is attrib C– uted to a N–H bending and to a C–N stretching vibrations of amide II, while the band at 1242 cm 1 is typical of amide III due to stretching and bending vibrations of C–N and N–H bonds, respectively (Ficai et al., 2013). It can also be noticed a band at 561 cm 1 which refers to the out
2.6. Differential scanning calorimetry (DSC) This analysis was performed on a TA Instruments model Q1000 to 6
M. Rufino Senra and M.F. Vieira Marques
Journal of the Mechanical Behavior of Biomedical Materials 103 (2020) 103577
Fig. 5. Storage modulus (E0 ) of processed neat UHMWPE and UHMWPE/HC or COL blends.
of plane bending of N–H; at 3406 cm 1 is found the collagen amide A band, associated with the frequency of a stretching deformation of –NH and a band at 3078 cm 1 related to amide B due to stretching vibrations � ska and Sionkowska, 1996; Wang of C–H (Camacho et al., 2001; Kamin et al., 2009). Through the spectra of the blends it is possible to identify bands related to the vibrational modes of methylene and also bands at 1651 and 1542 cm 1 due to deformations of amide I and II from collagens. They confirm that the spectrum of the blends resulted in overlapping of the pure components typical peaks showing that no reaction took place between the polyethylene and the collagens, as reported by Castiello et al. (2009) that prepared films of LDPE with HC. In the HC blends, the collagen bands intensity increased with the increment of HC contents. However, in UHMWPE/COL blends the intensity of these bands was lower and this may be because the dispersion of COL into the matrix may have been hampered as COL is a triple helix with a higher molecular weight than HC. Bands at 1245 and 1153 cm 1 were also detected in some pressed and extruded materials referring to the UHMWPE oxidation. It is well known that UHMWPE is susceptible to oxidative degradation when subjected to high temperatures and pressures, showing the importance of adding an antioxidant during the polymer processing. These bands can be attributed to the vibrations of C–O–C groups as reported by Rocha et al. (2009) and by Tretinnikov et al. (1998). The wavenumbers and assignment of these bands are reported in Table 1.
Fig. 2 shows X-ray diffraction profiles of pure UHMWPE and their blends with HC and COL processed by compression and extrusion. The most intense peaks obtained correspond to the orthorhombic unit cell of polyethylene. This phase is characterized by the crystallographic planes (100) and (200) at 2θ ¼ 21� e 23� , respectively (Fang et al., 2007; Joo et al., 2000). It is also evident a peak less intense at 2θ ¼ 36.5� that correspond to the crystallographic plane (020) (Baker and Windle, 2001; Jaggi et al., 2015; McDaniel et al., 2015; Zheng et al., 2002). In addition, it is also possible to note a reflection at 2θ ffi 19� related to a change in the orthorhombic structure of the polymer matrix to a monoclinic structure. The monoclinic structure is a metastable phase present in smaller quantity and was possible to detect this phase because some crystals in the orthorhombic form when subjected to pressure, temper ature and/or shear can be partially transformed in the monoclinic structure (Kurelec et al., 2000). Table 2 resumes the degree of crystallinity (Xc) obtained for the blends and for the processed neat UHMWPE. In general, increasing the amount of both collagens led to a decrease in the Xc and the reduction was more highlighted for the blends with COL, since their higher mo lecular weight can hinder the polymeric material reorganize. Appar ently, samples with 1 wt% HC obtained by compression and extrusion present Xc slightly higher than pure UHMWPE, which could indicate a plasticization of the polymer, which facilitated its crystallization. Be sides, the blends with COL, specially the extruded ones, presented lower Xc. 7
M. Rufino Senra and M.F. Vieira Marques
Journal of the Mechanical Behavior of Biomedical Materials 103 (2020) 103577
Fig. 6. Loss modulus (E00 ) of processed neat UHMWPE and UHMWPE/HC or COL blends.
TGA was conducted in order to evaluate the thermal stability of UHMWPE samples when HC and COL were incorporated. The ther mogravimetric curves are shown in Fig. 3 for the pure collagens and in Fig. 4 for the prepared blends. Fig. 3 shows that the thermal stability of COL is higher than for the HC, as expected, due to the higher molecular weight of COL. Also, that HC is more hygroscopic than COL. This is indicated by the first thermal event that is assigned to the loss of water adsorbed onto the material. The water loss is approximately of 9.7% for HC and of 6.8% for the COL. The TG and DTG curves of neat UHMWPE showed thermal stability of the polymer up to 447 � C when pressed and up to 469 � C when extruded. The higher thermal stability for the extruded material can be attributed to the polymer chain orientation that occurs during processing. In the curves related to the blends, two thermal events were detected: the first is related to the collagen degradation and the second correspond to the polymeric matrix degradation. Table 3 and Table 4 report the values of the initial degradation temperature (Tonset) and temperature of maximum degradation rate (Tmax) for both events. It can be easily remarked that the weight losses of all blends, in the first thermogravimetric event, are lower than the theoretical values in higher concentrations. This indicates that protein fraction incorporated in the polymer matrix improves by far its thermal resistance. This was also reported by Castiello et al. (2009), Dascǎlu et al. (2005) and Puccini et al. (2015) when they prepared blends of LDPE with HC and it was imputed to the reciprocal influence of the components.
Regarding the results for the composition of 5 and 10 wt% of HC in either processing, it can be noted that there is a distinction between the two types of processing used. Since the extrusion provides a more inti mate mixture of the materials it may have led to a higher thermal resistance of the HC as well as the. UHMWWPE matrix, although the matrix has no affinity to the collagen. The increased on the Tmax value for the first degradation event in these compositions may indicate that UHMWPE is protecting the decomposition of HC into a more dispersed mixture, in the case of the materials obtained by extrusion. Indeed, in both thermal events, through the values of Tonset and Tmax, in general, for all HC blends, these temperatures are higher when the processing method is extrusion. The pressed and extruded materials with COL showed a similar thermal stability as evidenced by Tonset and Tmax values for the two degradation events observed in the blends. It can be inferred that the dispersion of COL in the matrix was harmed because of their high mo lecular weight and as a consequence the ispersion in the blend was not efficient resulting in a heterogeneous sample. Therefore, the polymeric matrix protected less the COL against thermal degradation. Results of second heating run, as well of cooling run of neat UHMWPE and the blends with HC and COL are presented in Table 5. The blends with the two types of collagen in the evaluated concentrations and in both manufacturing processes insignificantly changed crystalli zation and melting temperatures of the material. This indicates that the addition of collagen practically did not affect this thermal property of 8
M. Rufino Senra and M.F. Vieira Marques
Journal of the Mechanical Behavior of Biomedical Materials 103 (2020) 103577
Fig. 7. tan δ of processed neat UHMWPE and UHMWPE/HC or COL blends.
the matrix. DMA is a technique used to study the viscoelastic properties of the materials. The analysis has as a main objective to relate the macroscopic properties, such as thermo-mechanical properties, with molecular re laxations associated by conformational changes and microscopic de formations generated from molecular rearrangements due to change in the temperature (Lorandi et al., 2016). This test provides the storage modulus (E0 ) that reflects the stiffness of the material; the loss modulus (E00 ) that indicates the energy dissipated by the material and the tan δ (E’/E00 ) that was used to determine the glass transition temperature (Tg) of the samples. Table 6 resumes the dynamic-mechanical properties of pure UHMWPE as well the blends with HC and COL for both processing methods. Fig. 5 presents the storage modulus in function of temperature that show a decrease in E0 with an increase in the temperature for all curves. This happens because the increment in the temperature provides more energy to the system, allowing a higher mobility to the polymeric chains and as a consequence E0 decreases. Comparing the extruded pure UHMWPE with the pressed one, the first presented higher E0 , indicating again that the extrusion of the material promoted an alignment of the chains, which generates an increase of the material stiffness. It is ex pected that collagen acts as a plasticizer due to its lower molecular weight, generating a reduction in E0 values. This was observed, in gen eral, for all UHMWPE/collagen blends. The exception was for the pressed blends with 1 and 10 wt% of HC and 10 wt% COL that showed a higher E’. This can be attributed to an inefficient mixing process,
providing a poor dispersion, which can cause errors in the measurement due to heterogeneity in the sample. As it will be seen ahead, in the mechanical test these values do not match with the ones found in the DMA analysis. Comparing the E’ obtained for the pressed and extruded HC blends, the extruded ones presented higher storage modulus due to the alignment of the chains. The same occurred for the blends with COL. Comparing the processing methods, in general, the blends with COL showed higher values than the blends with HC showing an increase of material stiffness when COL was added. This indicates that COL has a lower plasticizing effect due to its higher molecular weight. The E00 curves for all blends were presented in Fig. 6 allowing study α, β and γ relaxations of UHMWPE, as presented in Table 6. The γ relaxa tion is related to the movement of chains in the amorphous phase associated with the glass transition. The β relaxation, which was less intense, corresponds to the amorphous-crystalline interface and is assigned to interlamellar grain boundary phenomena associated with orientational and distortionary dispersions of non-crystalline materials between oriented lamellae (Gøuchel and Ulrich, 2009), and its intensity decreases as crystallinity increases being more pronounced in low den sity polyethylenes (Sirotkin and Brooks, 2001). Lastly, α relaxation is related to the crystalline phase and is due to the motion of the amor phous chains segments within the crystalline lamellae and Tα increases as the lamellar thickness increases (Sirotkin and Brooks, 2001). In the prepared blends, E00 tended to decreases, showing a lower molecular relaxation at room temperature, which indicates orientation of the crystalline phase, especially for the extruded blends. 9
M. Rufino Senra and M.F. Vieira Marques
Journal of the Mechanical Behavior of Biomedical Materials 103 (2020) 103577
Fig. 8. Mechanical properties of pure UHMWPE and UHMWPE blends.
The glass transition temperature (Tg) of a polymer depends on the mobility of the polymer chains. When a plasticizer is added to a polymer, it acts increasing free volume and decreasing interactions between polymer chains, which results in greater mobility at low temperatures, decreasing Tg. As can be seen from the analysis of tan δ curves (Fig. 7) and on the Tg values presented in Table 6, Tg in general was reduced with the addition of both collagens. Analyzing the tan δ curve, there is a peak at elevated temperatures (~100 � C). This peak can be associated
with phenomena such as intracrystalline relaxations (connected with α relaxations) and sliding of the entanglement chains within the crystal line blocks of PE (Alexandre et al., 2002). Mechanical properties of the blends are presented in Fig. 8. The elastic modulus, in general, reduced slightly for the prepared blends. This behavior is related to the decrease of the UHMWPE crystallinity by effect of HC and COL introduction and it was also reported by Castiello et al. (2009) and Haroun (2010) that prepared LDPE/HC blends. The 10
M. Rufino Senra and M.F. Vieira Marques
Journal of the Mechanical Behavior of Biomedical Materials 103 (2020) 103577
extruded, except for the 10 wt% concentration of collagens. Further more, torque analysis as a function of time of extrusion showed that collagen acted as a plasticizer due to torque reduction in the region where it is stabilized. Therefore, the results achieved in this research show that is possible to process UHMWPE with collagen and the mate rials obtained could improve UHMWPE biocompatibility with a poten tial to be used in orthopedic applications. Declaration of competing interest We have no conflict of interest to declare. Acknowledgements The authors acknowledge FAPERJ, CAPES and CNPq for financial support. References
Fig. 9. Curve of torque x time for pure UHMWPE and for the blends with 2% of HC and COL acquired during extrusion.
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elongation at break tended to decrease with the addition of collagen and the decrease was more pronounced with 10 wt% of HC or COL. It is worth to mention that Dascǎlu et al. (2005) reported that elongation at break continuously decreased with increase of HC content in LDPE/HC blends and the compatibilizing agents used do not excessively influence this property. So, we obtained a material with the same behavior without using a compatibilizing agent. It is worth to mention that the elongation at break is related to the tenacity of the material and it was the mechanical property most affected by adding HC and COL in the matrix. This is an important property considering the suggested appli cation, since it predicts the capacity in absorbing energy before fracture. Addition up to 5% of both collagens did not promote a sharp change in this property. The tensile strength at break for the pressed materials showed a more pronounced reduction by the addition of 10 wt% of HC and 5 and 10 wt% of COL, indicating that HC was better dispersed in the polymeric matrix than COL due to the lower molecular weight of HC. In blends containing 10 wt% HC obtained by compression molding, there is a greater standard deviation in the determination of tensile strength at break, yield strength and elongation at break compared with the extruded material, showing that there is a better dispersion using the latter method. In general, the extruded materials exhibit higher tensile strength at break and elongation at break. This behavior confirms once again that the extrusion process improves the dispersion of the bio component in the UHMWPE matrix. Therefore, it is possible to produce a UHMWPE-based material that is potentially more biocompatible, with good mechanical properties as the matrix, which could be applied in bone prostheses. Fig. 9 shows Torque-Time curve obtained during extrusion for pure UHMWPE and for the blends with the addition of 2 wt% HC or COL. The results showed a reduction on the torque, mainly in the region where it is stabilized, confirming that the collagens acted as plasticizer in the blend, helping in the processing, particularly HC, due to higher torque reduction. 4. Conclusions UHMWPE is widely used in biomedical applications and through the methodology employed, blends of UHMWPE with HC and COL by compression and extrusion were successfully obtained. The analyzes performed indicate that the extrusion allowed a more intimate mixture of components. Although a decrease in the mechanical properties could be expected because of the poor adhesion between UHMWPE and collagen, the obtained results showed that the addition of both collagens positively affected these properties, mainly when the materials were 11
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Journal of the Mechanical Behavior of Biomedical Materials 103 (2020) 103577 Puccini, M., Seggiani, M., Vitolo, S., 2015. Polyethylene and hydrolyzed collagen blend films produced by blown extrusion. Chem. Eng. Trans. 43, 1705–1710. https://doi. org/10.3303/CET1543285. Puccini, M., Stefanelli, E., Seggiani, M., Balestri, E., Vitolo, S., 2017. Biodegradability of polyethylene/hydrolyzed collagen blends in terrestrial and marine environmental conditions. J. Renew. Mater. 5, 117–123. https://doi.org/10.7569/ jrm.2017.634138. Rocha, M., Mansur, A., Mansur, H., 2009. Characterization and accelerated ageing of UHMWPE used in orthopedic prosthesis by peroxide. Materials 2, 562–576. https:// doi.org/10.3390/ma2020562. Sattari, M., Naimi-Jamal, M.R., Khavandi, A., 2014. Interphase evaluation and nanomechanical responses of UHMWPE/SCF/nano- SiO2 hybrid composites. Polym. Test. 38, 26–34. https://doi.org/10.1016/j.polymertesting.2014.06.006. Sirotkin, R.O., Brooks, N.W., 2001. The dynamic mechanical relaxation behaviour of polyethylene copolymers cast from solution. Polymer 42, 9801–9808. https://doi. org/10.1016/S0032-3861(01)00535-3. Tamada, Y., Ikada, Y., 1994. Fibroblast growth on polymer surfaces and biosynthesis of collagen. J. Biomed. Mater. Res. 28, 783–789. https://doi.org/10.1002/ jbm.820280705. Tretinnikov, O.N., Ogata, S., Ikada, Y., 1998. Surface crosslinking of polyethylene by electron beam irradiation in air. Polymer 39, 6115–6120. https://doi.org/10.1016/ S0032-3861(98)00075-5. Wang, Xiaoliang, Wang, Xiaomin, Tan, Y., Zhang, B., Gu, Z., Li, X., 2009. Synthesis and evaluation of collagen-chitosan- hydroxyapatite nanocomposites for bone grafting. J. Biomed. Mater. Res. A 89, 1079–1087. https://doi.org/10.1002/jbm.a.32087. Yu, X., Walsh, J., Wei, M., 2014. Covalent immobilization of collagen on titanium through polydopamine coating to improve cellular performances of MC3T3-E1 cells. RSC Adv. 4, 7185–7192. https://doi.org/10.1039/c3ra44137g. Zhang, H., Liang, Y., 2018. Extrusion processing of ultra-high molecular weight polyethylene extrusion processing of ultra-high molecular weight polyethylene. In: Qamar, S.Z. (Ed.), Extrusion of Metals, Polymers and Food Products. InTech, London. https://doi.org/10.5772/intechopen.72212. Zheng, L., Waddon, A.J., Farris, R.J., Coughlin, E.B., 2002. X-ray characterizations of polyethylene polyhedral oligomeric silsesquioxane copolymers. Macromolecules 35, 2375–2379. https://doi.org/10.1021/ma011855e.
Kurelec, L., Rastogi, S., Meier, R.J., Lemstra, P.J., 2000. Chain mobility in polymer systems: on the borderline between solid and melt. 3. Phase transformations in nascent ultrahigh molecular weight polyethylene reactor powder at elevated pressure as revealed by in situ Raman spectroscopy. Macromolecules 33, 5593–5601. https://doi.org/10.1021/ma9911187. Kurtz, S.M., 2015. UHMWPE Biomaterials Handbook: Ultra-high Molecular Weight Polyethylene in Total Joint Replacement and Medical Devices. Lorandi, N.P., Odila, M., Cioffi þþ, H., Ornaghi, H.þ, 2016. An� alise din^ amico-mec^ anica de Materiais comp� ositos polim�ericos dynamic mechanical analysis (DMA) of polymeric composite materials. Sci. Cum Ind. 4, 48–60. https://doi.org/10.18226/ 23185279.v4iss1p48. Matsumura, K., Hyon, S.H., Nakajima, N., Peng, C., Tsutsumi, S., 2000. Surface modification of poly(ethylene-co-vinyl alcohol) (EVA). Part I. Introduction of carboxyl groups and immobilization of collagen. J. Biomed. Mater. Res. 50, 512–517. https://doi.org/10.1002/(SICI)1097-4636(20000615)50:4<512::AIDJBM6>3.0.CO;2-G. Mayne, R., Burgeson, R.E., 1987. Structure and Function of Collagen Types. Academic Press, Cambridge. McDaniel, P.B., Deitzel, J.M., Gillespie, J.W., 2015. Structural hierarchy and surface morphology of highly drawn ultra high molecular weight polyethylene fibers studied by atomic force microscopy and wide angle X-ray diffraction. Polymer 69, 148–158. https://doi.org/10.1016/j.polymer.2015.05.010. Mirsalehi, S.A., Sattari, M., Khavandi, A., Mirdamadi, S., Naimi-Jamal, M.R., 2016. Tensile and biocompatibility properties of synthesized nano-hydroxyapatite reinforced ultrahigh molecular weight polyethylene nanocomposite. J. Compos. Mater. 50, 1725–1737. https://doi.org/10.1177/0021998315595711. Müller, R., Abke, J., Schnell, E., Macionczyk, F., Gbureck, U., Mehrl, R., Ruszczak, Z., Kujat, R., Englert, C., Nerlich, M., Angele, P., 2005. Surface engineering of stainless steel materials by covalent collagen immobilization to improve implant biocompatibility. Biomaterials 26, 6962–6972. https://doi.org/10.1016/j. biomaterials.2005.05.013. Peng, C., Tsutsumi, S., Matsumura, K., Nakajima, N., Hyon, S.H., 2001. Morphologic study and syntheses of type I collagen and fibronectin of human periodontal ligament cells cultured on poly(ethylene-co-vinyl alcohol) (EVA) with collagen immobilization. J. Biomed. Mater. Res. 54, 241–246. https://doi.org/10.1002/10974636(200102)54:2<241::AID-JBM11>3.0.CO;2-P.
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Journal of the Mechanical Behavior of Biomedical Materials journal homepage: http://www.elsevier.com/locate/jmbbm
Thermal and mechanical behavior of ultra-high molecular weight polyethylene/collagen blends ^nica Rufino Senra, Maria de Fa �tima Vieira Marques * Mo Instituto de Macromoleculas Eloisa Mano, IMA-UFRJ, Universidade Federal do Rio de Janeiro, Cidade Universit� aria, Av. Hor� acio Macedo, 2.030. Centro de Tecnologia. Bloco J, Rio de Janeiro, RJ, 21941-598, Brazil
A R T I C L E I N F O
A B S T R A C T
Keywords: UHMWPE Collagen Polymer blend Orthopedic applications
Bone defects or diseases significantly affect quality of life, thus the development of materials with improved performance that can be used as bone substitutes is increasingly studied. As an alternative, ultra-high molecular weight polyethylene (UHMWPE) has been employed for orthopedic applications since it combines high wear resistance, high impact resistance and low friction coefficient. However, it is a bioinert material and difficult to process. In the present work, the addition of collagen (hydrolyzed or type II), one of the constituents of natural bone, to UHMWPE was studied aiming to improve its processability and possibly its biocompatibility. The blends were prepared by compression and twin-screw extrusion. The results show that addition of higher amounts of both collagens to UHMWPE reduced the degree of crystallinity. However, crystallization and melting tempera tures were not affected. The thermogravimetric analysis exhibited two thermal events correlated to the degra dation of collagens (Tmax~300 � C) and of UHMWPE (Tmax~480 � C), corroborating the FTIR analysis that presented bands corresponding to these materials. The extrusion process promoted a better dispersion of the collagens, especially the hydrolyzed one. In addition, the obtained materials presented better mechanical properties when extruded. Torque reduction during extrusion showed that hydrolyzed collagen aid processing, even more than collagen due to its smaller molecular weight.
1. Introduction Millions of people worldwide suffer from bone disorders, bone fractures/injuries and diverse musculoskeletal problems that are usually treated by drug therapies or surgeries, which generally include partial or total replacement of the diseased tissue (Ferreira et al., 2012). Ultra-high molecular weight polyethylene (UHMWPE) is a material widely studied for prosthesis manufacturing. Mechanical properties similar to natural bone, high wear and impact resistance, low friction coefficient and biocompatibility turns this polymer a suitable material for bone replacement (Mirsalehi et al., 2016; Sattari et al., 2014). For this reason, it is broadly used in parts of hip, knee and shoulder prosthesis. UHMWPE has a very high molecular weight, which provides to this material excellent characteristics such as high impact resistance, low friction coefficient and high wear resistance. However, their high mo lecular weight causes an increase in the entanglements between the polymer chains, reducing their mobility. The high viscosity of UHMWPE makes it very hard to be processed and also the dispersion of fillers in
this matrix is inhibited (Zhang and Liang, 2018). Due to the difficulties presented, it is a challenge to process UHMWPE and, therefore, many studies have been looking for ways to facilitate its processing since conventional injection and extrusion cannot be applied owing to the high viscosity in the melt. As a conse quence, compression molding and RAM extrusion are the two widely manufacturing processes used to produce UHMWPE artifacts (Kurtz, 2015). The compression molding was the first manufacturing process used to process UHMWPE powder, having been originated in Germany in 1950. It is a discontinuous process based on material sintering without limiting molecular weight and melt viscosity. This manufacturing method is still being used by two companies, Orthoplastics and Medi TECH, who produce compression mould sheets of GUR 1020 (MM~3.5 � 106 g/mol) and GUR 1050 (MM~5.5-6.0 � 106 g/mol) (Baena et al., 2015). The RAM extrusion was developed in the United States in 1970 and can be considered as a continuous process of compression and sintering at a temperature close to the UHMWPE melting. Unlike conventional
* Corresponding author. E-mail address: [email protected] (M.F. Vieira Marques). https://doi.org/10.1016/j.jmbbm.2019.103577 Received 9 August 2019; Received in revised form 2 October 2019; Accepted 29 November 2019 Available online 2 December 2019 1751-6161/© 2019 Elsevier Ltd. All rights reserved.
M. Rufino Senra and M.F. Vieira Marques
Journal of the Mechanical Behavior of Biomedical Materials 103 (2020) 103577
Fig. 1. FTIR spectra (a) pure HC and COL, (b) UHMWPE/HC pressed, (c) UHMWPE/HC extruded, (d) UHMWPE/COL pressed and (e) UHMWPE/COL extruded.
extrusion where material is carried by a screw, RAM extrusion is a plug flow process, in other words, in batch without shear, where the partic ulate material is fed to an elongated die while a piston compatible with the die cavity travels a round trip in the feed zone. In this path occurs the powder compaction and the heat transfer to the polymer, taking place the sintering. Soon after, the extruded goes through the cooling (Kurtz, 2015). In this type of process plates, tubes and rods can be produced being made UHMWPE products with good surface quality. However,
residual stresses may occur in the bulk of the material, thereby the extrusion products must undergo a high temperature annealing treat ment to remove these residual stresses. This procedure can increase the crystallinity of the polymer, which is useful for maintaining the excellent mechanical performances of UHMWPE. However, some drawbacks are associated with this type of processing, such as fluctuations in product quality, long plasticization cycle, slow extrusion rate and high power consumption (Zhang and Liang, 2018). 2
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Journal of the Mechanical Behavior of Biomedical Materials 103 (2020) 103577
ability of bone to withstand mechanical forces and fractures depends not only on the amount of bone tissue (mineralization), but also on its quality (collagen structure organization) (Daneault et al., 2015). There are 28 types of collagens, which have as a common structural feature the presence of a triple helix chains formed by three polypeptide, called alpha chains, which may be identical or different (Mayne and Burgeson, 1987). Collagen derivatives, such as gelatin and hydrolyzed collagen, are widely used in the food, cosmetics and pharmaceutical industries, as well as in tissue engineering. Gelatin is obtained by ther mal denaturation of collagen, which promotes the separation of collagen
Table 1 Band assignments for HC, COL and UHMWPE blends. Bands Assignment
ν (cm 1)
Assignment
HC and COL
3406 3078 1651 1542 1242 561
Stretching N–H of Amide A Stretching C–H of Amide B Stretching C– –O of Amide I Stretching C–N and bending N–H of Amide II Stretching C–N and bending N–H of Amide III Out of plane bending N–H
UHMWPE blends
3340 2916 2849 1651 1542 1472 1462 1245 1153 731 719
Stretching C–O–C Stretching CH2 antisymmetric Stretching CH2 symmetric Stretching C– –O of Amide I Stretching C–N e bending N–H of Amide II In plane bending CH2 In plane bending CH2 Stretching C–O–C Stretching C–O–C In plane bending CH2 In plane bending CH2
Table 2 Degree of crystallinity of neat UHMWPE and UHMWPE blends.
Bone can be considered a natural nanocomposite formed of collagen fiber networks impregnated with calcium phosphate, mainly by hy droxyapatite (HA) nanocrystals (Jiang and Liu, 2016). In bone, collagen plays an important role in force transmission and maintenance of tissue structure and determines the amount of mineral deposition. Thus, the
�
Sample
Xc (%)
Sample
Xc (%)
UHMWPE� 1% HC� 2% HC� 5% HC� 10% HC�
48.2 50.7 48.1 46.8 43.9
UHMWPEΔ 1% HCΔ 2% HCΔ 5% HCΔ 10% HCΔ
47.1 50.0 47.3 43.8 42.8
1%COL� 2%COL� 5%COL� 10%COL�
47.0 47.3 42.7 42.3
1%COLΔ 2%COLΔ 5%COLΔ 10%COLΔ
44.9 43.7 44.3 39.2
pressed material; Δ extruded material.
Fig. 2. XRD profiles of pure UHMWPE and processed UHMWPE blends. 3
M. Rufino Senra and M.F. Vieira Marques
Journal of the Mechanical Behavior of Biomedical Materials 103 (2020) 103577
2. Materials and methods 2.1. Materials The UHMWPE (UTEC 3040), with average molecular weight of 3.0.106 gmol 1, was purchased from Braskem SA. Collagen type II was donated by the Health Science Center of Rio de Janeiro Federal Uni versity (UFRJ), obtained by direct extraction of the extracellular matrix of bovine cartilage. The extraction was carried out for 24 h in acidic aqueous solution of pH ¼ 3 containing 5% DMSO and acetic acid. After extraction, the collagen was washed with deionized water until neutral pH and then was lyophilized for 76 h. Hydrolyzed collagen was acquired from Amazonas Rio Norte Produtos Naturais Ltda and vitamin E, a �rios farmac^euticos. natural antioxidant, was provided by Ach�e laborato 2.2. UHMWPE processing conditions Fig. 3. Thermal degradation profiles of pure HC and COL.
UHMWPE blends with 1, 2, 5 and 10 wt% of HC or COL with 0.8 wt% of vitamin E were prepared by twin-screw extrusion and press molding. Before both processing methods, the components of the blend were firstly mixed in an analytical mill (A11, IKA) for 2 min. UHMWPE with 0.8 wt% of vitamin E was also processed with the same conditions (extrusion and press molding) for comparison.
chains in the triple helix through the destruction of crosslinks. In order to obtain hydrolyzed collagen, gelatin undergoes enzymatic hydrolysis and collagen chains are broken down into small peptides (Daneault et al., 2015). Mineralization is a process of bone formation promoted by osteo blasts that create an environment for the concentration of calcium and phosphate. Osteoblasts secrete type I collagen, in addition to many noncollagenous proteins and collagen serves as a template to initiate and propagate mineralization (Ferreira et al., 2012). So, the addition of collagen to biomaterials is a strategy to improve osseointegration. Some works already have reported that collagen on the materials surface improved cell adhesion and proliferation (Cheng and Teoh, 2004; Müller et al., 2005; Peng et al., 2001; Yu et al., 2014). This can be attributed by a biospecific interaction that works between the cell and the collagen surface (Tamada and Ikada, 1994). In this work, hydrolyzed and type II collagens were used because they are inexpensive compared to type I collagen, whose use is widely reported in the literature. Type II collagen is more easily extracted from bovine cartilage compared to type I collagen extracted from bone. This occurs because the organic bone matrix is more complex and removing all the protein residues makes the process extensive. Moreover, the demineralization process (removal of HA) can lead to rupture of collagen chains. In the literature, several methods for obtaining low density poly ethylene (LDPE) film with collagen are found (Castiello et al., 2009; Dascǎlu et al., 2005; Haroun, 2010; Puccini et al., 2017, 2015). How � et al., 2008) or high ever, the blend of collagen with UHMWPE (Bo�ckova density polyethylene (HDPE) (Kinoshita et al., 1993; Matsumura et al., 2000; Tamada and Ikada, 1994) is not very well studied. Moreover, in these works the materials were not processed by extrusion or compres sion, the polyethylenes (PE) had their surface modified. Briefly, the PE was chemically modified on their surface and peroxide groups were introduced, then they were grafted with carbonyl monomers, in which the acrylic acid is widely used. After that, PE and collagen react and the carbonyl groups of the grafted chain binds to the nitrogen of the collagen amide groups. For this reason, this is the first work that processes UHMWPE with collagen. The aim of introducing collagen to UHMWPE was first improve its processability and possibly improve its biocom patibility with bone tissue. The purpose of the work is to prepare blends of UHMWPE with hy drolyzed collagen (HC) and collagen type II (COL) by twin-screw extrusion and by compression, the most common manufacturing pro cess of UHMWPE pieces and verifies the plasticizer effect of the collagens.
2.2.1. Twin-screw extrusion conditions For processing the blends, a Haake Minilab Rheomex (CTW5) from Thermo Scientific was used, with counter-rotating twin-screws and automatic bypass for circulation/extrusion. The extruder was operated at 200 � C with an initial rotation speed of 20 rpm. After the torque remained constant (around 4 min), rotation speed was raised to 60 rpm until the end of the process (total extrusion time: 12 min). 2.2.2. Press molding conditions The compressed materials were produced by pressing the premixed materials at 90 MPa for 10 min and at constant temperature of 200 � C. It was prepared films and specimens for the mechanical and thermal analyses. 2.3. Fourier-transform infrared spectroscopy (FTIR) FTIR spectra of collagens were recorded using Nicolet-Magna 760 spectrophotometer with KBr pellets in the wavenumber range between 400 and 4000 cm-1. For the polymeric samples FTIR spectra were recorded using IRAffinity-1 spectrophotometer in the wavenumber range between 650 and 4000 cm-1. The polymeric films were analyzed in the ATR (Attenuated total reflection) mode and the spectra were normalized for comparison. 2.4. X-ray diffraction X-ray diffraction analysis was performed using a Rigaku XRD diffractometer at room temperature, using Cu Kα (0.154 nm) radiation. The scanning was performed in the Bragg angle (2θ) range of 2–60� , with a scanning rate of 0.05� /min. Samples were analyzed as films. UHMWPE is a semicrystalline polymer with a most common ortho rhombic unit cell. The orthorhombic form is characterized by the crys tallographic planes (100) and (200) at 2θ ffi 21� e 23� , respectively (Fang et al., 2007; Joo et al., 2000). To study the effect of the addition of collagens to the polymeric matrix the degree of crystallinity of the blends was obtained by the equation 1, through the areas of crystalline (Sc) and amorphous (Sa) peaks. Xc ¼
4
Sc x100 Sc þ Sa
M. Rufino Senra and M.F. Vieira Marques
Journal of the Mechanical Behavior of Biomedical Materials 103 (2020) 103577
Fig. 4. TG and DTG curves for pure UHMWPE and for blends.
5
M. Rufino Senra and M.F. Vieira Marques
Journal of the Mechanical Behavior of Biomedical Materials 103 (2020) 103577
Table 3 Thermal degradation properties for UHMWPE/HC blends. Sample
�
1st thermal event
Table 6 Dynamic-mechanical properties of pure UHMWPE and UHMWPE blends.
2nd thermal event
Tonset (� C)
Tmax (� C)
Weight (%)
Tonset (� C)
Tmax (� C)
Weight (%)
UHMWPE� 1% HC� 2% HC� 5% HC� 10%HC�
– 251 238 246 245
– 298 303 297 305
– 0.9 1.6 4.7 7.2
447 454 462 444 455
469 472 480 465 473
99.7 98.6 98.2 94.0 90.2
UHMWPEΔ 1% HCΔ 2% HCΔ 5% HCΔ 10% HCΔ
– 256 255 247 253
– 310 303 321 319
– 1.2 2.1 4.1 5.1
469 471 457 460 469
490 489 476 479 487
99.6 98.3 96.8 94.8 92.5
pressed material; Δ extruded material.
Table 4 Thermal degradation properties for UHMWPE/COL blends. Sample
�
1st thermal event
2nd thermal event
Tonset (� C)
Tmax (� C)
Weight (%)
Tonset (� C)
Tmax (� C)
Weight (%)
UHMWPE� 1% COL� 2% COL� 5% COL� 10% COL�
– 248 254 261 270
– 298 295 302 298
– 1.5 2.4 3.4 7.0
447 460 456 463 460
469 479 476 480 478
99.7 97.5 96.9 95.3 89.8
UHMWPEΔ 1% COLΔ 2% COLΔ 5% COLΔ 10% COLΔ
– 262 267 272 267
– 299 300 303 300
– 1.6 2.2 4.4 5.2
469 460 462 459 461
490 478 480 479 479
99.6 98.0 97.4 94.3 92.2
�
UHMWPE UHMWPEΔ
�
Tc (oC)
Tm (oC)
119 118
132 133
Sample
Tc (oC)
Tm (oC)
Sample
Tc (oC)
Tm (oC)
1% HC 2% HC� 5% HC� 10% HC�
119 119 120 120
131 131 132 131
1% HC 2% HCΔ 5% HCΔ 10% HCΔ
120 119 119 119
132 132 132 132
1% COL� 2% COL� 5% COL� 10% COL�
120 120 119 120
131 131 132 131
1% COLΔ 2% COLΔ 5% COLΔ 10% COLΔ
119 119 119 119
132 133 132 133
�
E00 25 � C (MPa)
Tβ (oC)
UHMWPE� UHMWPEΔ
713,9 989,5
58.7 79.2
103.9 105.1
109.4 107.7
18.2 25.3
44.8 44.8
1% HC� 2% HC� 5% HC� 10% HC�
721.5 582.4 569.2 858.2
51.3 49.9 43.0 67.7
104.1 112.6 104.4 106.2
107.0 108.8 108,6 109.7
21.1 22.4 15.2 22.7
45.6 40.2 50.6 42.9
1% HCΔ 2% HCΔ 5% HCΔ 10% HCΔ
921.7 785.4 927.4 717.9
74.6 68.7 69.4 53.8
104.5 110.2 106.9 105.4
107.0 113.9 110.1 108,9
23.8 22.4 20.1 22.0
43.9 40.5 49.3 46.0
1% COL� 2% COL� 5% COL� 10% COL�
632.1 716.3 635.3 761.8
55.6 61.7 57.0 66.1
108.5 108.3 108.7 109.2
111.5 111.5 111.2 113.0
18.8 20.7 20.7 22.2
40.7 41.2 42.4 40.8
1% COLΔ 2% COLΔ 5% COLΔ 10% COLΔ
940.7 929.4 913.4 921.1
83.3 80.1 78.2 81.9
109.8 108.3 108.85 109.78
112.7 112.6 111.7 113.7
23.9 22.4 24.0 24.7
42.1 42.1 41.8 41.5
Tα (oC)
pressed material; Δ extruded material.
2.7. Dynamic-mechanical thermal analysis (DMA) DMA was carried out on a TA Instruments Q 800 equipment. The storage modulus (E0 ), loss modulus (E00 ) and tan δ values were found under a sinusoidal applied strain over a range of temperatures from 140 to 120 � C at a heating rate of a 3 � C/min and an operating fre quency of 1 Hz. The glass transition temperature (Tg) was also deter mined from the tan δ curve as a function of temperature. Specimens were obtained with approximate dimensions of 13.3 � 6.08 � 0.60 mm from the materials processed by press molding or extrusion.
Table 5 Thermal characteristics obtained from DSC curves. �
E0 25 � C (MPa)
verify the thermal transitions of the processed samples. The samples (4–5 mg) were hermetically sealed in an aluminum sample holder. The tests were conducted in a nitrogen atmosphere with flow of 50 mL/min. In the first run the material was heated from 0 � C to 200 � C with a heating rate of 10 � C/min to eliminate thermal history. After, the sample was cooled to 0 � C at the same rate, and the crystallization temperature (Tc) was determined. Thereafter, the sample was heated again at 10 � C/ min to 200 � C and the melting temperature (Tm) was determined.
pressed material; Δ extruded material.
Sample
Tg, tan δ (� C)
Tγ (oC)
Sample
Δ
2.8. Mechanical tensile testing The mechanical tensile test was carried out according to the inter national standard ASTM D638 (Standard Test Method for Tensile Properties of Plastic) on a universal testing machine Emic model L 3000 with a load cell of 30 KN and a speed of 10 mm/min. In the present work, the modulus was calculated by linear regression using the stress-strain curve points up to 0.5% deformation. Since the yield strength at the stress-strain curve was not possible to detect, the offset yield strength at 10% was calculated. Five specimens were tested for each sample and the results presented refer to the average values calculated.
pressed material; Δ extruded material.
2.5. Thermogravimetric analysis (TGA) Thermal analysis was conducted in a TA Instruments Q500 analyzer and was used to investigate the collagen influence on UHMWPE degradation rate. Samples of 10 to 15 mg were heated from 25 up to 700 � C (polymeric samples) and up to 900 � C (pure collagens) under nitrogen atmosphere at a flow rate of 60 mL/min. All the experiments were conducted at 10 � C/min and TG/DTG curves were recorded. The tem peratures of the start of degradation (Tonset) and maximum degradation rate (Tmax) were determined, as well as the weight loss.
3. Results and discussion The FTIR spectra of pure hydrolyzed collagen (HC) and collagen type II (COL) and their blends with UHMWPE are shown in Fig. 1. The presence of a band at 1651 cm 1 in HC and COL spectra corresponds to a – O stretching of amide I; at 1542 cm 1 there is a band that is attrib C– uted to a N–H bending and to a C–N stretching vibrations of amide II, while the band at 1242 cm 1 is typical of amide III due to stretching and bending vibrations of C–N and N–H bonds, respectively (Ficai et al., 2013). It can also be noticed a band at 561 cm 1 which refers to the out
2.6. Differential scanning calorimetry (DSC) This analysis was performed on a TA Instruments model Q1000 to 6
M. Rufino Senra and M.F. Vieira Marques
Journal of the Mechanical Behavior of Biomedical Materials 103 (2020) 103577
Fig. 5. Storage modulus (E0 ) of processed neat UHMWPE and UHMWPE/HC or COL blends.
of plane bending of N–H; at 3406 cm 1 is found the collagen amide A band, associated with the frequency of a stretching deformation of –NH and a band at 3078 cm 1 related to amide B due to stretching vibrations � ska and Sionkowska, 1996; Wang of C–H (Camacho et al., 2001; Kamin et al., 2009). Through the spectra of the blends it is possible to identify bands related to the vibrational modes of methylene and also bands at 1651 and 1542 cm 1 due to deformations of amide I and II from collagens. They confirm that the spectrum of the blends resulted in overlapping of the pure components typical peaks showing that no reaction took place between the polyethylene and the collagens, as reported by Castiello et al. (2009) that prepared films of LDPE with HC. In the HC blends, the collagen bands intensity increased with the increment of HC contents. However, in UHMWPE/COL blends the intensity of these bands was lower and this may be because the dispersion of COL into the matrix may have been hampered as COL is a triple helix with a higher molecular weight than HC. Bands at 1245 and 1153 cm 1 were also detected in some pressed and extruded materials referring to the UHMWPE oxidation. It is well known that UHMWPE is susceptible to oxidative degradation when subjected to high temperatures and pressures, showing the importance of adding an antioxidant during the polymer processing. These bands can be attributed to the vibrations of C–O–C groups as reported by Rocha et al. (2009) and by Tretinnikov et al. (1998). The wavenumbers and assignment of these bands are reported in Table 1.
Fig. 2 shows X-ray diffraction profiles of pure UHMWPE and their blends with HC and COL processed by compression and extrusion. The most intense peaks obtained correspond to the orthorhombic unit cell of polyethylene. This phase is characterized by the crystallographic planes (100) and (200) at 2θ ¼ 21� e 23� , respectively (Fang et al., 2007; Joo et al., 2000). It is also evident a peak less intense at 2θ ¼ 36.5� that correspond to the crystallographic plane (020) (Baker and Windle, 2001; Jaggi et al., 2015; McDaniel et al., 2015; Zheng et al., 2002). In addition, it is also possible to note a reflection at 2θ ffi 19� related to a change in the orthorhombic structure of the polymer matrix to a monoclinic structure. The monoclinic structure is a metastable phase present in smaller quantity and was possible to detect this phase because some crystals in the orthorhombic form when subjected to pressure, temper ature and/or shear can be partially transformed in the monoclinic structure (Kurelec et al., 2000). Table 2 resumes the degree of crystallinity (Xc) obtained for the blends and for the processed neat UHMWPE. In general, increasing the amount of both collagens led to a decrease in the Xc and the reduction was more highlighted for the blends with COL, since their higher mo lecular weight can hinder the polymeric material reorganize. Appar ently, samples with 1 wt% HC obtained by compression and extrusion present Xc slightly higher than pure UHMWPE, which could indicate a plasticization of the polymer, which facilitated its crystallization. Be sides, the blends with COL, specially the extruded ones, presented lower Xc. 7
M. Rufino Senra and M.F. Vieira Marques
Journal of the Mechanical Behavior of Biomedical Materials 103 (2020) 103577
Fig. 6. Loss modulus (E00 ) of processed neat UHMWPE and UHMWPE/HC or COL blends.
TGA was conducted in order to evaluate the thermal stability of UHMWPE samples when HC and COL were incorporated. The ther mogravimetric curves are shown in Fig. 3 for the pure collagens and in Fig. 4 for the prepared blends. Fig. 3 shows that the thermal stability of COL is higher than for the HC, as expected, due to the higher molecular weight of COL. Also, that HC is more hygroscopic than COL. This is indicated by the first thermal event that is assigned to the loss of water adsorbed onto the material. The water loss is approximately of 9.7% for HC and of 6.8% for the COL. The TG and DTG curves of neat UHMWPE showed thermal stability of the polymer up to 447 � C when pressed and up to 469 � C when extruded. The higher thermal stability for the extruded material can be attributed to the polymer chain orientation that occurs during processing. In the curves related to the blends, two thermal events were detected: the first is related to the collagen degradation and the second correspond to the polymeric matrix degradation. Table 3 and Table 4 report the values of the initial degradation temperature (Tonset) and temperature of maximum degradation rate (Tmax) for both events. It can be easily remarked that the weight losses of all blends, in the first thermogravimetric event, are lower than the theoretical values in higher concentrations. This indicates that protein fraction incorporated in the polymer matrix improves by far its thermal resistance. This was also reported by Castiello et al. (2009), Dascǎlu et al. (2005) and Puccini et al. (2015) when they prepared blends of LDPE with HC and it was imputed to the reciprocal influence of the components.
Regarding the results for the composition of 5 and 10 wt% of HC in either processing, it can be noted that there is a distinction between the two types of processing used. Since the extrusion provides a more inti mate mixture of the materials it may have led to a higher thermal resistance of the HC as well as the. UHMWWPE matrix, although the matrix has no affinity to the collagen. The increased on the Tmax value for the first degradation event in these compositions may indicate that UHMWPE is protecting the decomposition of HC into a more dispersed mixture, in the case of the materials obtained by extrusion. Indeed, in both thermal events, through the values of Tonset and Tmax, in general, for all HC blends, these temperatures are higher when the processing method is extrusion. The pressed and extruded materials with COL showed a similar thermal stability as evidenced by Tonset and Tmax values for the two degradation events observed in the blends. It can be inferred that the dispersion of COL in the matrix was harmed because of their high mo lecular weight and as a consequence the ispersion in the blend was not efficient resulting in a heterogeneous sample. Therefore, the polymeric matrix protected less the COL against thermal degradation. Results of second heating run, as well of cooling run of neat UHMWPE and the blends with HC and COL are presented in Table 5. The blends with the two types of collagen in the evaluated concentrations and in both manufacturing processes insignificantly changed crystalli zation and melting temperatures of the material. This indicates that the addition of collagen practically did not affect this thermal property of 8
M. Rufino Senra and M.F. Vieira Marques
Journal of the Mechanical Behavior of Biomedical Materials 103 (2020) 103577
Fig. 7. tan δ of processed neat UHMWPE and UHMWPE/HC or COL blends.
the matrix. DMA is a technique used to study the viscoelastic properties of the materials. The analysis has as a main objective to relate the macroscopic properties, such as thermo-mechanical properties, with molecular re laxations associated by conformational changes and microscopic de formations generated from molecular rearrangements due to change in the temperature (Lorandi et al., 2016). This test provides the storage modulus (E0 ) that reflects the stiffness of the material; the loss modulus (E00 ) that indicates the energy dissipated by the material and the tan δ (E’/E00 ) that was used to determine the glass transition temperature (Tg) of the samples. Table 6 resumes the dynamic-mechanical properties of pure UHMWPE as well the blends with HC and COL for both processing methods. Fig. 5 presents the storage modulus in function of temperature that show a decrease in E0 with an increase in the temperature for all curves. This happens because the increment in the temperature provides more energy to the system, allowing a higher mobility to the polymeric chains and as a consequence E0 decreases. Comparing the extruded pure UHMWPE with the pressed one, the first presented higher E0 , indicating again that the extrusion of the material promoted an alignment of the chains, which generates an increase of the material stiffness. It is ex pected that collagen acts as a plasticizer due to its lower molecular weight, generating a reduction in E0 values. This was observed, in gen eral, for all UHMWPE/collagen blends. The exception was for the pressed blends with 1 and 10 wt% of HC and 10 wt% COL that showed a higher E’. This can be attributed to an inefficient mixing process,
providing a poor dispersion, which can cause errors in the measurement due to heterogeneity in the sample. As it will be seen ahead, in the mechanical test these values do not match with the ones found in the DMA analysis. Comparing the E’ obtained for the pressed and extruded HC blends, the extruded ones presented higher storage modulus due to the alignment of the chains. The same occurred for the blends with COL. Comparing the processing methods, in general, the blends with COL showed higher values than the blends with HC showing an increase of material stiffness when COL was added. This indicates that COL has a lower plasticizing effect due to its higher molecular weight. The E00 curves for all blends were presented in Fig. 6 allowing study α, β and γ relaxations of UHMWPE, as presented in Table 6. The γ relaxa tion is related to the movement of chains in the amorphous phase associated with the glass transition. The β relaxation, which was less intense, corresponds to the amorphous-crystalline interface and is assigned to interlamellar grain boundary phenomena associated with orientational and distortionary dispersions of non-crystalline materials between oriented lamellae (Gøuchel and Ulrich, 2009), and its intensity decreases as crystallinity increases being more pronounced in low den sity polyethylenes (Sirotkin and Brooks, 2001). Lastly, α relaxation is related to the crystalline phase and is due to the motion of the amor phous chains segments within the crystalline lamellae and Tα increases as the lamellar thickness increases (Sirotkin and Brooks, 2001). In the prepared blends, E00 tended to decreases, showing a lower molecular relaxation at room temperature, which indicates orientation of the crystalline phase, especially for the extruded blends. 9
M. Rufino Senra and M.F. Vieira Marques
Journal of the Mechanical Behavior of Biomedical Materials 103 (2020) 103577
Fig. 8. Mechanical properties of pure UHMWPE and UHMWPE blends.
The glass transition temperature (Tg) of a polymer depends on the mobility of the polymer chains. When a plasticizer is added to a polymer, it acts increasing free volume and decreasing interactions between polymer chains, which results in greater mobility at low temperatures, decreasing Tg. As can be seen from the analysis of tan δ curves (Fig. 7) and on the Tg values presented in Table 6, Tg in general was reduced with the addition of both collagens. Analyzing the tan δ curve, there is a peak at elevated temperatures (~100 � C). This peak can be associated
with phenomena such as intracrystalline relaxations (connected with α relaxations) and sliding of the entanglement chains within the crystal line blocks of PE (Alexandre et al., 2002). Mechanical properties of the blends are presented in Fig. 8. The elastic modulus, in general, reduced slightly for the prepared blends. This behavior is related to the decrease of the UHMWPE crystallinity by effect of HC and COL introduction and it was also reported by Castiello et al. (2009) and Haroun (2010) that prepared LDPE/HC blends. The 10
M. Rufino Senra and M.F. Vieira Marques
Journal of the Mechanical Behavior of Biomedical Materials 103 (2020) 103577
extruded, except for the 10 wt% concentration of collagens. Further more, torque analysis as a function of time of extrusion showed that collagen acted as a plasticizer due to torque reduction in the region where it is stabilized. Therefore, the results achieved in this research show that is possible to process UHMWPE with collagen and the mate rials obtained could improve UHMWPE biocompatibility with a poten tial to be used in orthopedic applications. Declaration of competing interest We have no conflict of interest to declare. Acknowledgements The authors acknowledge FAPERJ, CAPES and CNPq for financial support. References
Fig. 9. Curve of torque x time for pure UHMWPE and for the blends with 2% of HC and COL acquired during extrusion.
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elongation at break tended to decrease with the addition of collagen and the decrease was more pronounced with 10 wt% of HC or COL. It is worth to mention that Dascǎlu et al. (2005) reported that elongation at break continuously decreased with increase of HC content in LDPE/HC blends and the compatibilizing agents used do not excessively influence this property. So, we obtained a material with the same behavior without using a compatibilizing agent. It is worth to mention that the elongation at break is related to the tenacity of the material and it was the mechanical property most affected by adding HC and COL in the matrix. This is an important property considering the suggested appli cation, since it predicts the capacity in absorbing energy before fracture. Addition up to 5% of both collagens did not promote a sharp change in this property. The tensile strength at break for the pressed materials showed a more pronounced reduction by the addition of 10 wt% of HC and 5 and 10 wt% of COL, indicating that HC was better dispersed in the polymeric matrix than COL due to the lower molecular weight of HC. In blends containing 10 wt% HC obtained by compression molding, there is a greater standard deviation in the determination of tensile strength at break, yield strength and elongation at break compared with the extruded material, showing that there is a better dispersion using the latter method. In general, the extruded materials exhibit higher tensile strength at break and elongation at break. This behavior confirms once again that the extrusion process improves the dispersion of the bio component in the UHMWPE matrix. Therefore, it is possible to produce a UHMWPE-based material that is potentially more biocompatible, with good mechanical properties as the matrix, which could be applied in bone prostheses. Fig. 9 shows Torque-Time curve obtained during extrusion for pure UHMWPE and for the blends with the addition of 2 wt% HC or COL. The results showed a reduction on the torque, mainly in the region where it is stabilized, confirming that the collagens acted as plasticizer in the blend, helping in the processing, particularly HC, due to higher torque reduction. 4. Conclusions UHMWPE is widely used in biomedical applications and through the methodology employed, blends of UHMWPE with HC and COL by compression and extrusion were successfully obtained. The analyzes performed indicate that the extrusion allowed a more intimate mixture of components. Although a decrease in the mechanical properties could be expected because of the poor adhesion between UHMWPE and collagen, the obtained results showed that the addition of both collagens positively affected these properties, mainly when the materials were 11
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