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New semi-IPN scaffolds based on HEMA and collagen modified with itaconic anhydride
Materials Letters 67 (2012) 95–98
Contents lists available at SciVerse ScienceDirect
Materials Letters journal homepage: www.elsevier.com/locate/matlet
New semi-IPN scaffolds based on HEMA and collagen modified with itaconic anhydride S. Potorac a,⁎, M. Popa a, L. Verestiuc b, D. Le Cerf c a b c
“Gheorghe Asachi” Technical University, Faculty of Chemical Engineering and Environmental Protection, 73 Prof. dr. docent Dimitrie Mangeron Street, 700050 Iasi, Romania “Gr. T. Popa” University of Medicine and Pharmacy, Faculty of Medical Bioengineering, 16 Universitatii Street, 700115, Iasi, Romania Université de Rouen UMR 6270 & FR 3038 CNRS, Polymères, Biopolymères, Surfaces, F-76821 Mont Saint Aignan, France
a r t i c l e
i n f o
Article history: Received 1 May 2011 Accepted 7 September 2011 Available online 16 September 2011 Keywords: Collagen 2-Hydroxyethyl methacrylate Tissue engineering Biomaterials Polymers
a b s t r a c t Porous semi-IPN scaffolds were synthesized via free radical copolymerization of collagen modified with itaconic anhydride and 2-hydroxyethyl methacrylate, using ammonium persulfate and N,N,N′,N′-tetramethylethylenediamine as redox initiator system, with the aim of overcoming the high enzymatic degradation rate of native collagen scaffolds. The chemical modification of collagen was confirmed by 1H NMR and FTIR spectroscopy. The physico-chemical properties of the resulted matrices were investigated by elemental analysis, SEM, enzymatic degradation and water retention studies. The synthesized scaffolds, obtained through an innovative process, have fine microstructures, controlled water retention degree and enhanced stability against enzymatic digestion compared to native collagen. This may broaden the use of collagenbased scaffolds in tissue engineering, particularly for wound dressings. © 2011 Elsevier B.V. All rights reserved.
1. Introduction Collagen is a major constituent of skin dermis and a vital molecule for normal wound healing process [1]. Due to its excellent biocompatibility and biodegradability, the use of collagen in biomedical applications has been rapidly growing and widely expanding to bioengineering areas [2]. However, unprocessed collagen is often limited by its rapid degradability and low mechanical strength which prevents full exploitation of its potential in vivo. Multiple methods have been tested aiming to improve the properties of the collagenbased medical devices. Physical [3] and [4] chemical crosslinking, blending with natural [5] or synthetic [6] polymers, incorporating exogenous compounds [7] and preparation of three-dimensional semior full-interpenetrated networks (IPN) [8] may offer new and improved applications for collagen-based biomaterials especially in drug-delivery and tissue engineering [9,10]. Several chemical agents (glutaraldehyde) [11] or physical methods (dehydrothermal crosslinking) have been used to obtain crosslinked collagen scaffolds, but they can be cytotoxic or can compromise biochemical features of natural collagen [12,13]. Crosslinking of collagen materials without incorporation of any chemical reagent, by using carbodiimide compounds has also been reported [14, 15]. To achieve the purpose of obtaining a semi-IPN scaffold, that combines the physical characteristics of a neutral hydrophilic monomer and the bioactive features of collagen, we have designed and synthesized a modified collagen carrying mono-itaconate amide groups ⁎ Corresponding author. E-mail address: [email protected] (S. Potorac). 0167-577X/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.matlet.2011.09.039
that can be copolymerized with 2-hydroxyethyl methacrylate, without the involving of any chemical crosslinking agent. The influence of protein to monomer ratio and concentration of the initiator on the structure and characteristics of the formed semi-IPNs were analyzed and reported in this study. 2. Materials and methods 2.1. Materials Acid-soluble collagen (C), type I + III, 1% in H2O2 solution, was supplied by Lohmann & Rauscher GmbH, Germany. 2-Hydroxyethyl methacrylate (HEMA, Merck Co.) was purified by passing it through an inhibitor removal column. Ammonium persulfate (APS, SigmaAldrich) was purified by recrystallization from a mixture of water/ methanol (1/2, v/v). Itaconic anhydride (ITA, Merck Co.), collagenase (Clostridium histolyticum, EC 3.4.24.3, US Biological, USA), N,N,N′,N′tetramethylethylenediamine and dimethyl sulfoxide (TEMED and DMSO, Sigma-Aldrich) were used as received. 2.2. Preparation of itaconic anhydride modified collagen (CITA) and copolymerization with HEMA In order to provide reactive sites on collagen macromolecules that can be copolymerized with HEMA, the collagen solution 200 mL (1%, wt/v) was adjusted to pH=8.0 by adding 1% NaOH solution. Then, a solution of ITA (C:ITA, 1:3 g g−1) in DMSO (20 mL) was gradually added and the pH was readjusted and maintained constantly at 8.0 during reaction, by adding 1 N NaOH solution. The reaction mass was stirred for 24 h at
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Table 1 Terminology, composition and degradation degree of the synthesized scaffolds. Terminology
Collagen CITA16H2 CITA30H1.3 CITA30H2 CITA30H2.7 CITA44H2
Composition CITA, %
HEMA, %
APS, %
– 16 30 30 30 44
– 84 70 70 70 56
– 2.0 1.3 2.0 2.7 2.0
Degradation degree, % 100 ± 2.0 11.6 ± 1.2 16.4 ± 0.4 18.0 ± 0.3 17.6 ± 0.2 17.8 ± 0.3
room temperature and then the product was purified by dialysis against deionized water for 3 days. The water was changed 3 times daily. With the aim of studying the composition influence on scaffolds characteristics, specific weight ratios of CITA/HEMA (g g−1) were used. To complete the crosslinking reaction, the initiator system was added and the mixture was stirred vigorously. APS (5 wt.% in deionized water) was used in molar ratio with the monomer (% mol:mol) and TEMED:APS ratio was always constant (mol:mol). The mixture was rapidly poured into polyethylene molds and allowed to react for 24 h at room temperature. The obtained scaffolds (CITAH) were washed in deionized water for 3 days and freeze-dried (−62 °C and 0.03 mbar). Various formulations were prepared: CITA/HEMA ratio was ranged from 1/5.3 to 1/1.2 and APS concentration from 1.3% to 2.7% (Table 1). 2.3. Characterization methods A Bruker Avance DRX 400 spectrometer operating at 400 MHz was used to confirm the chemical modification of collagen through 1H
NMR spectroscopy. HCl solution in D2O (pH ~ 1.5) was used as solvent and the resulting 1H spectra were calibrated to TMS at 0.0 ppm. FT-IR spectra were obtained using a Nicolet Avatar 360 FT-IR Spectrometer with the range of 4000 cm −1 to 675 cm −1 in the ATR mode: mono reflection device, using a diamond crystal at room temperature (incidence angle 45°). The Kjeldahl method was used for quantitative determination of nitrogen [16] and the measured nitrogen was related to the amount of protein in the composition of the matrices (100 mg of each sample were analyzed). The cross-section morphology of the studied materials was examined using a SEM Tescan-Vega microscope. Water uptake studies were performed in PBS (pH = 7.2) at 37 °C and the equation used was Water uptake; WUð%Þ ¼ ½ðwt wo Þ=wo 100 where wt is the weight at time point t and wo is the initial sample weight (ca. 20 mg). Triplicate measurements were performed for each sample. The in vitro degradation was analyzed by photometric nynhidrine method. The samples (50 mg) were incubated in 30 mL PBS (0.01 M) pH = 7.2, containing 0.01% collagenase in a shaking water bath, at 37 °C over a period of 72 h. 1 mL of sample solution was mixed with 5 mL nynhidrine reagent [17] and heated for 20 min at boiling temperature. The solution was then cooled and mixed with 25 mL water/2-propanol (1/1, v/v). Optical absorbance of the solution was measured at 570 nm using a UV–VIS spectrophotometer, (Shimadzu UV-1700 PharmaSpec, Japan). The degradation degree was calculated from the calibration curve of collagen hydrolizate and the equation Degradation degree; DDð%Þ ¼ ðN=NC Þ 100
Fig. 1. 1H NMR spectra of C and CITA (I) and FT-IR spectra of ITA, C and CITA (II).
S. Potorac et al. / Materials Letters 67 (2012) 95–98
where, DD is the degradation degree, N and NC are the concentrations of the α-NH2 groups (mmol/mL ×10−10) for CITAH matrices, respectively for pure C. 3. Results and discussion 1
H NMR spectra of pure C and CITA can be seen in Fig. 1. Specifically characteristic peaks from amide group (8.3 ppm), methylene protons next to carboxylic acid (5.9–6.3 ppm) and methylene next to carbonyl (2.7 ppm), were seen in the CITA spectrum (Fig. 1.II). Chemical modification of C has also been investigated by FT-IR technique (Fig. 1). The spectrum corresponding to unmodified collagen (Fig. 1.b) presents characteristic absorption peaks of amide groups at 3280 cm −1 (amide A, NH stretching), 2924 cm −1 (amide B, CH stretching), 1640 cm −1 (amide I, C_O stretching), 1550 cm −1 (amide II, NH bending) and 1230 cm −1 (amide III, NH bending). The spectrum of CITA reveals characteristic peaks to both C and ITA (Fig. 1.c). Peak frequencies of amide I and amide II (1639, 1547 cm − 1) are shifted to lower values in comparison to the frequencies for pure C (1640, 1550 cm − 1) due to hydrogen bonding from ITA. The absorption band at 1724 cm −1 is attributed to C_O stretching from ester linkage. In addition, the C\N stretching (O_C\NH) appears at 1446 cm −1 and the 690 cm −1 is for the weak band N\H out of plane bending. The 1157 cm −1 band is from C\O stretching and the band at 1072 cm −1 is probably due to the S_O groups of
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the residual DMSO solvent. These results are indicative of successful grafting of ITA to collagen. Collagen chains are crosslinked with pHEMA bridges and interpenetrated with linear pHEMA chains, thus the obtained products have a semi-IPN character. Collagen content was determined based on total amount of nitrogen found in the final composition of the synthesized matrices. The collagen content is increasing with initial content of CITA. Water retention degree is also strongly dependent on the collagen content of the CITAH matrices (Fig. 2). With increasing concentrations of initiator, the quantity of HEMA involved in the composition of the semi-IPNs increases; at the same time, cross-linking density is higher. Water retention tended to increase with increasing concentration of APS, reached an optimum value (4.86 × 100% for CITA30H2) and then decreased (4.62 × 100% for CITA30H2.7). Porosity and texture of the freeze-dried samples were examined and the obtained images are presented in Fig. 3. SEM micrograph of pure C (Fig. 3.I) revealed a non-uniform morphology, while CITA (Fig. 3.II) shows an interconnected porous cross-section. SEM examination confirms the open and interconnecting porosity of the CITA30H2 scaffold (Fig. 3.III, IV) the pores being smaller, as pHEMA bridges stabilize the three-dimensional network and increase the scaffold homogeneity. To simulate the physiological human body conditions, we used collagenase to determine the degradation behavior of native collagen as well as of the obtained scaffolds. We observed that the unmodified
Fig. 2. Collagen content, water uptake (I) and degradation degree (II) of the obtained semi-IPNs.
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Fig. 3. SEM micrographs of pure C (I), CITA (II) and CITA30H2 scaffold (III — 500 μm, IV — 100 μm).
collagen was fully digested after approximately 48 h. CITAH hydrogels are not fully degradable and showed greatly enhanced stability due to their crosslinked structure. When APS concentrations are higher than 2.0 mol% of monomer, enzymatic digestion decreases due to higher crosslinking density of the scaffolds (Table 1). In addition, a high dependency of the degradation degree on the collagen concentration was observed, as we expected.
4. Conclusion In this work, new semi-IPN scaffolds based on itaconic anhydride modified collagen and HEMA were prepared through a radical copolymerization-crosslinking method and characterized. The elemental analysis showed that the amount of collagen in the obtained matrices increases particularly with the increasing quantity of collagen in the initial composition. The synthesized scaffolds are able to retain water at physiological pH and temperature, the water uptake and degradation rate being strongly dependent by the final composition of the semi-IPNs. Increasing concentration of redox initiator (N2.0 mol% of monomer) leads to a higher crosslinking density, consecutively decreasing water retention and restricting the enzymatic digestion of the matrices. These advantages indicate the synthesized scaffolds as excellent candidates for applications in tissue engineering.
Acknowledgment This paper was realized with the support of BRAIN “Doctoral scholarships as an investment in intelligence” project, financed by the European Social Fund and Romanian Government. References [1] Greenhalgh DG. Wound Healing. In: Herndon D, editor. Total wound care. 3 rd ed. Saunders Elsevier. Inc; 2007. p. 578–95. [2] Lee C-H, Singla A, Lee Y. Int J Pharm 2001;221:1–22. [3] Usha R, Rajaram A, Ramasami T. J Photoch Photobio B 2009;97:34–9. [4] Kanth SV, Ramaraj A, Rao JR, Nair BU. Process Biochem 2009;44:869–74. [5] Skopinska-Wisniewska J, Sionkowska A, Kaminska A, Kaznica A, Jachimiak R, Drewa T. Appl Surf Sci 2009;255:8286–92. [6] Jose MV, Thomas V, Dean DR, Nyairo E. Polymer 2009;50:3778–85. [7] Geiger M, Li RH, Friess W. Adv Drug Deliv Rev 2003;55:1613–9. [8] Suri S, Schmidt CE. Acta Biomater 2009;5:2385–97. [9] Chen JP, Lee WL. Appl Surf Sci 2008;255:412–5. [10] Duan X, McLaughlin C. J Biomater 2007;28(1):78–88. [11] Veríssimo DM, Leitão RFC, Ribeiro RA, Figueiró SD, Sombra ASB, Góes JC, Brito GAC. Acta Biomater 2010;6:4011–8. [12] Gupta B, Hilborn J, Plummer C, Bisson I, Frey P. J Appl Polym Sci 2002;85:1874–80. [13] Wess TJ, Orgel JP. Therm Acta 2000;365:119–28. [14] Harley BA, Leung JH, Silva ECCM, Gibson LJ. Acta Biomater 2007;3:463–74. [15] Nam K, Kimura T, Kishida A. J Biomater 2007;28:1–8. [16] Lynch JM, Barbano DM. J AOAC Int 1999;82:1389–98. [17] Moore S, Stein WH. J Biol Chem 1948;176:367–88.
Contents lists available at SciVerse ScienceDirect
Materials Letters journal homepage: www.elsevier.com/locate/matlet
New semi-IPN scaffolds based on HEMA and collagen modified with itaconic anhydride S. Potorac a,⁎, M. Popa a, L. Verestiuc b, D. Le Cerf c a b c
“Gheorghe Asachi” Technical University, Faculty of Chemical Engineering and Environmental Protection, 73 Prof. dr. docent Dimitrie Mangeron Street, 700050 Iasi, Romania “Gr. T. Popa” University of Medicine and Pharmacy, Faculty of Medical Bioengineering, 16 Universitatii Street, 700115, Iasi, Romania Université de Rouen UMR 6270 & FR 3038 CNRS, Polymères, Biopolymères, Surfaces, F-76821 Mont Saint Aignan, France
a r t i c l e
i n f o
Article history: Received 1 May 2011 Accepted 7 September 2011 Available online 16 September 2011 Keywords: Collagen 2-Hydroxyethyl methacrylate Tissue engineering Biomaterials Polymers
a b s t r a c t Porous semi-IPN scaffolds were synthesized via free radical copolymerization of collagen modified with itaconic anhydride and 2-hydroxyethyl methacrylate, using ammonium persulfate and N,N,N′,N′-tetramethylethylenediamine as redox initiator system, with the aim of overcoming the high enzymatic degradation rate of native collagen scaffolds. The chemical modification of collagen was confirmed by 1H NMR and FTIR spectroscopy. The physico-chemical properties of the resulted matrices were investigated by elemental analysis, SEM, enzymatic degradation and water retention studies. The synthesized scaffolds, obtained through an innovative process, have fine microstructures, controlled water retention degree and enhanced stability against enzymatic digestion compared to native collagen. This may broaden the use of collagenbased scaffolds in tissue engineering, particularly for wound dressings. © 2011 Elsevier B.V. All rights reserved.
1. Introduction Collagen is a major constituent of skin dermis and a vital molecule for normal wound healing process [1]. Due to its excellent biocompatibility and biodegradability, the use of collagen in biomedical applications has been rapidly growing and widely expanding to bioengineering areas [2]. However, unprocessed collagen is often limited by its rapid degradability and low mechanical strength which prevents full exploitation of its potential in vivo. Multiple methods have been tested aiming to improve the properties of the collagenbased medical devices. Physical [3] and [4] chemical crosslinking, blending with natural [5] or synthetic [6] polymers, incorporating exogenous compounds [7] and preparation of three-dimensional semior full-interpenetrated networks (IPN) [8] may offer new and improved applications for collagen-based biomaterials especially in drug-delivery and tissue engineering [9,10]. Several chemical agents (glutaraldehyde) [11] or physical methods (dehydrothermal crosslinking) have been used to obtain crosslinked collagen scaffolds, but they can be cytotoxic or can compromise biochemical features of natural collagen [12,13]. Crosslinking of collagen materials without incorporation of any chemical reagent, by using carbodiimide compounds has also been reported [14, 15]. To achieve the purpose of obtaining a semi-IPN scaffold, that combines the physical characteristics of a neutral hydrophilic monomer and the bioactive features of collagen, we have designed and synthesized a modified collagen carrying mono-itaconate amide groups ⁎ Corresponding author. E-mail address: [email protected] (S. Potorac). 0167-577X/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.matlet.2011.09.039
that can be copolymerized with 2-hydroxyethyl methacrylate, without the involving of any chemical crosslinking agent. The influence of protein to monomer ratio and concentration of the initiator on the structure and characteristics of the formed semi-IPNs were analyzed and reported in this study. 2. Materials and methods 2.1. Materials Acid-soluble collagen (C), type I + III, 1% in H2O2 solution, was supplied by Lohmann & Rauscher GmbH, Germany. 2-Hydroxyethyl methacrylate (HEMA, Merck Co.) was purified by passing it through an inhibitor removal column. Ammonium persulfate (APS, SigmaAldrich) was purified by recrystallization from a mixture of water/ methanol (1/2, v/v). Itaconic anhydride (ITA, Merck Co.), collagenase (Clostridium histolyticum, EC 3.4.24.3, US Biological, USA), N,N,N′,N′tetramethylethylenediamine and dimethyl sulfoxide (TEMED and DMSO, Sigma-Aldrich) were used as received. 2.2. Preparation of itaconic anhydride modified collagen (CITA) and copolymerization with HEMA In order to provide reactive sites on collagen macromolecules that can be copolymerized with HEMA, the collagen solution 200 mL (1%, wt/v) was adjusted to pH=8.0 by adding 1% NaOH solution. Then, a solution of ITA (C:ITA, 1:3 g g−1) in DMSO (20 mL) was gradually added and the pH was readjusted and maintained constantly at 8.0 during reaction, by adding 1 N NaOH solution. The reaction mass was stirred for 24 h at
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S. Potorac et al. / Materials Letters 67 (2012) 95–98
Table 1 Terminology, composition and degradation degree of the synthesized scaffolds. Terminology
Collagen CITA16H2 CITA30H1.3 CITA30H2 CITA30H2.7 CITA44H2
Composition CITA, %
HEMA, %
APS, %
– 16 30 30 30 44
– 84 70 70 70 56
– 2.0 1.3 2.0 2.7 2.0
Degradation degree, % 100 ± 2.0 11.6 ± 1.2 16.4 ± 0.4 18.0 ± 0.3 17.6 ± 0.2 17.8 ± 0.3
room temperature and then the product was purified by dialysis against deionized water for 3 days. The water was changed 3 times daily. With the aim of studying the composition influence on scaffolds characteristics, specific weight ratios of CITA/HEMA (g g−1) were used. To complete the crosslinking reaction, the initiator system was added and the mixture was stirred vigorously. APS (5 wt.% in deionized water) was used in molar ratio with the monomer (% mol:mol) and TEMED:APS ratio was always constant (mol:mol). The mixture was rapidly poured into polyethylene molds and allowed to react for 24 h at room temperature. The obtained scaffolds (CITAH) were washed in deionized water for 3 days and freeze-dried (−62 °C and 0.03 mbar). Various formulations were prepared: CITA/HEMA ratio was ranged from 1/5.3 to 1/1.2 and APS concentration from 1.3% to 2.7% (Table 1). 2.3. Characterization methods A Bruker Avance DRX 400 spectrometer operating at 400 MHz was used to confirm the chemical modification of collagen through 1H
NMR spectroscopy. HCl solution in D2O (pH ~ 1.5) was used as solvent and the resulting 1H spectra were calibrated to TMS at 0.0 ppm. FT-IR spectra were obtained using a Nicolet Avatar 360 FT-IR Spectrometer with the range of 4000 cm −1 to 675 cm −1 in the ATR mode: mono reflection device, using a diamond crystal at room temperature (incidence angle 45°). The Kjeldahl method was used for quantitative determination of nitrogen [16] and the measured nitrogen was related to the amount of protein in the composition of the matrices (100 mg of each sample were analyzed). The cross-section morphology of the studied materials was examined using a SEM Tescan-Vega microscope. Water uptake studies were performed in PBS (pH = 7.2) at 37 °C and the equation used was Water uptake; WUð%Þ ¼ ½ðwt wo Þ=wo 100 where wt is the weight at time point t and wo is the initial sample weight (ca. 20 mg). Triplicate measurements were performed for each sample. The in vitro degradation was analyzed by photometric nynhidrine method. The samples (50 mg) were incubated in 30 mL PBS (0.01 M) pH = 7.2, containing 0.01% collagenase in a shaking water bath, at 37 °C over a period of 72 h. 1 mL of sample solution was mixed with 5 mL nynhidrine reagent [17] and heated for 20 min at boiling temperature. The solution was then cooled and mixed with 25 mL water/2-propanol (1/1, v/v). Optical absorbance of the solution was measured at 570 nm using a UV–VIS spectrophotometer, (Shimadzu UV-1700 PharmaSpec, Japan). The degradation degree was calculated from the calibration curve of collagen hydrolizate and the equation Degradation degree; DDð%Þ ¼ ðN=NC Þ 100
Fig. 1. 1H NMR spectra of C and CITA (I) and FT-IR spectra of ITA, C and CITA (II).
S. Potorac et al. / Materials Letters 67 (2012) 95–98
where, DD is the degradation degree, N and NC are the concentrations of the α-NH2 groups (mmol/mL ×10−10) for CITAH matrices, respectively for pure C. 3. Results and discussion 1
H NMR spectra of pure C and CITA can be seen in Fig. 1. Specifically characteristic peaks from amide group (8.3 ppm), methylene protons next to carboxylic acid (5.9–6.3 ppm) and methylene next to carbonyl (2.7 ppm), were seen in the CITA spectrum (Fig. 1.II). Chemical modification of C has also been investigated by FT-IR technique (Fig. 1). The spectrum corresponding to unmodified collagen (Fig. 1.b) presents characteristic absorption peaks of amide groups at 3280 cm −1 (amide A, NH stretching), 2924 cm −1 (amide B, CH stretching), 1640 cm −1 (amide I, C_O stretching), 1550 cm −1 (amide II, NH bending) and 1230 cm −1 (amide III, NH bending). The spectrum of CITA reveals characteristic peaks to both C and ITA (Fig. 1.c). Peak frequencies of amide I and amide II (1639, 1547 cm − 1) are shifted to lower values in comparison to the frequencies for pure C (1640, 1550 cm − 1) due to hydrogen bonding from ITA. The absorption band at 1724 cm −1 is attributed to C_O stretching from ester linkage. In addition, the C\N stretching (O_C\NH) appears at 1446 cm −1 and the 690 cm −1 is for the weak band N\H out of plane bending. The 1157 cm −1 band is from C\O stretching and the band at 1072 cm −1 is probably due to the S_O groups of
97
the residual DMSO solvent. These results are indicative of successful grafting of ITA to collagen. Collagen chains are crosslinked with pHEMA bridges and interpenetrated with linear pHEMA chains, thus the obtained products have a semi-IPN character. Collagen content was determined based on total amount of nitrogen found in the final composition of the synthesized matrices. The collagen content is increasing with initial content of CITA. Water retention degree is also strongly dependent on the collagen content of the CITAH matrices (Fig. 2). With increasing concentrations of initiator, the quantity of HEMA involved in the composition of the semi-IPNs increases; at the same time, cross-linking density is higher. Water retention tended to increase with increasing concentration of APS, reached an optimum value (4.86 × 100% for CITA30H2) and then decreased (4.62 × 100% for CITA30H2.7). Porosity and texture of the freeze-dried samples were examined and the obtained images are presented in Fig. 3. SEM micrograph of pure C (Fig. 3.I) revealed a non-uniform morphology, while CITA (Fig. 3.II) shows an interconnected porous cross-section. SEM examination confirms the open and interconnecting porosity of the CITA30H2 scaffold (Fig. 3.III, IV) the pores being smaller, as pHEMA bridges stabilize the three-dimensional network and increase the scaffold homogeneity. To simulate the physiological human body conditions, we used collagenase to determine the degradation behavior of native collagen as well as of the obtained scaffolds. We observed that the unmodified
Fig. 2. Collagen content, water uptake (I) and degradation degree (II) of the obtained semi-IPNs.
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Fig. 3. SEM micrographs of pure C (I), CITA (II) and CITA30H2 scaffold (III — 500 μm, IV — 100 μm).
collagen was fully digested after approximately 48 h. CITAH hydrogels are not fully degradable and showed greatly enhanced stability due to their crosslinked structure. When APS concentrations are higher than 2.0 mol% of monomer, enzymatic digestion decreases due to higher crosslinking density of the scaffolds (Table 1). In addition, a high dependency of the degradation degree on the collagen concentration was observed, as we expected.
4. Conclusion In this work, new semi-IPN scaffolds based on itaconic anhydride modified collagen and HEMA were prepared through a radical copolymerization-crosslinking method and characterized. The elemental analysis showed that the amount of collagen in the obtained matrices increases particularly with the increasing quantity of collagen in the initial composition. The synthesized scaffolds are able to retain water at physiological pH and temperature, the water uptake and degradation rate being strongly dependent by the final composition of the semi-IPNs. Increasing concentration of redox initiator (N2.0 mol% of monomer) leads to a higher crosslinking density, consecutively decreasing water retention and restricting the enzymatic digestion of the matrices. These advantages indicate the synthesized scaffolds as excellent candidates for applications in tissue engineering.
Acknowledgment This paper was realized with the support of BRAIN “Doctoral scholarships as an investment in intelligence” project, financed by the European Social Fund and Romanian Government. References [1] Greenhalgh DG. Wound Healing. In: Herndon D, editor. Total wound care. 3 rd ed. Saunders Elsevier. Inc; 2007. p. 578–95. [2] Lee C-H, Singla A, Lee Y. Int J Pharm 2001;221:1–22. [3] Usha R, Rajaram A, Ramasami T. J Photoch Photobio B 2009;97:34–9. [4] Kanth SV, Ramaraj A, Rao JR, Nair BU. Process Biochem 2009;44:869–74. [5] Skopinska-Wisniewska J, Sionkowska A, Kaminska A, Kaznica A, Jachimiak R, Drewa T. Appl Surf Sci 2009;255:8286–92. [6] Jose MV, Thomas V, Dean DR, Nyairo E. Polymer 2009;50:3778–85. [7] Geiger M, Li RH, Friess W. Adv Drug Deliv Rev 2003;55:1613–9. [8] Suri S, Schmidt CE. Acta Biomater 2009;5:2385–97. [9] Chen JP, Lee WL. Appl Surf Sci 2008;255:412–5. [10] Duan X, McLaughlin C. J Biomater 2007;28(1):78–88. [11] Veríssimo DM, Leitão RFC, Ribeiro RA, Figueiró SD, Sombra ASB, Góes JC, Brito GAC. Acta Biomater 2010;6:4011–8. [12] Gupta B, Hilborn J, Plummer C, Bisson I, Frey P. J Appl Polym Sci 2002;85:1874–80. [13] Wess TJ, Orgel JP. Therm Acta 2000;365:119–28. [14] Harley BA, Leung JH, Silva ECCM, Gibson LJ. Acta Biomater 2007;3:463–74. [15] Nam K, Kimura T, Kishida A. J Biomater 2007;28:1–8. [16] Lynch JM, Barbano DM. J AOAC Int 1999;82:1389–98. [17] Moore S, Stein WH. J Biol Chem 1948;176:367–88.