ORIGINAL ARTICLE
J Appl Biomater Funct Mater 2014 ; 12 (3): 183 - 192 DOI: 10.5301/jabfm.5000187
Development of semi- and grafted interpenetrating polymer networks based on poly(ethylene glycol) diacrylate and collagen Marta Madaghiele, Francesco Marotta, Christian Demitri, Francesco Montagna, Alfonso Maffezzoli, Alessandro Sannino Department of Engineering for Innovation, University of Salento, Lecce - Italy
ABSTRACT Purpose: The objective of this work was to develop composite hydrogels based on poly(ethylene glycol) diacrylate (PEGDA) and collagen (Coll), potentially useful for biomedical applications. Methods: Semi-interpenetrating polymer networks (semi-IPNs) were obtained by photo-stabilizing aqueous solutions of PEGDA and acrylic acid (AA), in the presence of collagen. Further grafting of the collagen macromolecules to the PEGDA/ poly(AA) network was achieved by means of a carbodiimide-mediated crosslinking reaction. The resulting hydrogels were characterized in terms of swelling capability, collagen content and mechanical properties. Results and conclusions: The grafting procedure was found to significantly improve the mechanical stability of the IPN hydrogels, due to the establishment of covalent bonding between the PEGDA/poly(AA) and the collagen networks. The suitability of the composite hydrogels to be processed by means of stereolithography (SLA) was also investigated, toward creating biomimetic constructs with complex shapes, which might be useful either as platforms for tissue engineering applications or as tissue mimicking phantoms. Key words: Collagen, Hydrogel, Interpenetrating polymer networks Accepted: March 4, 2013
INTRODUCTION Interpenetrating polymer networks (IPNs) are composite systems made up of 2 (or more) independently crosslinked networks, at least one of which is crosslinked in the presence of the other. The intimate entanglements between the 2 polymer networks are well known to enhance the mechanical properties of the single networks, and/or to form a new material showing the key functional properties of both components. IPN hydrogels are particularly attractive for biomedical applications, especially for the creation of biomimetic platforms for tissue engineering, which usually combine the typical advantages of synthetic polymers (i.e., designable and tunable properties) with the intrinsic bioactivity of naturally derived biomaterials (e.g., cell-interactive domains and enzymatic degradability). In particular, polymers derived from the extracellular matrix (ECM) are ideal candidates for the development of semi-IPNs, which differ from IPNs in the fact that one network only is crosslinked (e.g., the synthetic polymer), and formed around uncrosslinked chains of a second polymer (e.g., the ECM macromolecules).
Poly(ethylene glycol) (PEG)–based hydrogels have been widely investigated in the last decade in both in vitro and in vivo applications, especially as tissue engineering templates, due to the resistance of PEG to protein adsorption, which makes it a nonfouling material for the design of scaffolds with specific bioactivity (1-11). Hydrogels are usually obtained by the photocrosslinking of acrylated PEG derivatives in aqueous solution, which is particularly advantageous for the design of injectable hydrogel formulations (12-14), as well as for the possible use of rapid prototyping techniques (e.g. stereolithography [SLA]) and other photopatterning approaches for the design of devices with complex or customized structures (15-18). Bioactive PEG-based hydrogels can be obtained by copolymerizing PEG derivatives with short peptide sequences and/or ECM components, which have been previously modified (e.g., acrylated or methacrylated) to be incorporated into the hydrogel network during the crosslinking reaction (1-9). Alternatively, biomimetic semi-IPNs can be synthesized by crosslinking the PEG derivatives in the presence of unmodified ECM molecules, such as hyaluronic acid (19, 20). In this case, in addition to inducing a given cell– material interaction, the ECM component acts as a spacer
© 2014 Società Italiana Biomateriali - eISSN 2280-8000
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Poly(ethylene glycol) and collagen IPNs
in the PEG network, thus modifying the microstructure of the network itself. The aim of this work was to investigate the creation of biomimetic semi-IPNs and grafted IPNs based on PEG derivatives and collagen (Coll), potentially useful for biomedical applications. Semi-IPNs were synthesized starting from aqueous mixtures of poly(ethylene glycol) diacrylate (PEGDA), acrylic acid (AA) and collagen as bioactive molecule. The effect of collagen on the PEGDA/ poly(AA) network formed in a photocrosslinking reaction was assessed in terms of swelling behavior and mechanical properties. The use of SLA for the production of hydrogels with complex shapes was also investigated. Further grafting of the collagen macromolecules to the PEGDA/ poly(AA) network was explored by using a carbodiimidemediated crosslinking reaction (21-23).
TABLE I - HYDROGEL FORMULATIONS: PEGDA (10% w/v), AA (0.2% v/v), DAROCUR 1173 (0.3% v/v) WERE USED FOR ALL SAMPLES Formulation code
Collagen loading (% w/v)
EDAC treatment (Yes/No)
PEG
0
No
PEG-X
0
Yes
Coll(0.9)/PEG
0.9
No
Coll(0.9)/PEG-X
0.9
Yes
Coll(1.8)/PEG
1.8
No
Coll(1.8)/PEG-X
1.8
Yes
AA = acrylic acid; Coll = collagen; EDAC = 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide; PEG = poly(ethylene glycol); PEGDA = poly(ethylene glycol) diacrylate.
MATERIALS AND METHODS All chemicals used in this study were purchased from Sigma-Aldrich (Milan, Italy), unless otherwise stated, and used as received. Hydrogel synthesis To develop composite hydrogels based on PEGDA and Coll, a 2-step process was employed which involved: (a) the synthesis of semi-IPNs by photostabilizing aqueous solutions of PEGDA (molecular weight 700 Da) and AA, in the presence of collagen; (b) the grafting of collagen to the PEGDA/poly(AA) network by means of a carbodiimide (EDAC)–mediated crosslinking reaction. Semi-IPNs An aqueous suspension of Type I collagen from calf skin (3% w/v, Semed S collagen; Kensey Nash Corporation) was prepared. PEGDA, AA and an acrylate-soluble photoinitiator (Darocur 1173; Basf) were dissolved in distilled water. A given volume of collagen suspension was then added to the above solution and thoroughly mixed, in order to obtain a homogeneous polymer mixture with fixed PEGDA and AA amounts and variable collagen concentrations, according to the following: PEGDA (10% w/v), AA (0.2% v/v), Darocur 1173 (0.3% v/v) and collagen at a concentration of either 0.9% or 1.8% w/v. Control solutions devoid of collagen were also prepared. The concentrations of PEGDA, AA and Darocur 1173 were chosen based on a previous protocol (10), which was slightly modified to reduce the AA amount. With the concentrations used in this study, the molar ratio between PEGDA and AA was 5:1. Hydrogel samples were then obtained by photostabilizing 1.5 mL of the polymer mixture in a 35-mm Petri dish by exposure to UV light (365 nm) 184
for 10 minutes. The average distance between the sample surface and the UV lamp was 3.5 cm. The resulting semi-IPNs were either subjected to a further grafting procedure, as described in the following, or directly washed in excess distilled water (50 mL) overnight, to remove unreacted chemicals. A synoptic overview of all the hydrogel formulations prepared in this work (semi-IPNs, grafted IPNs and control samples) is given in Table I. The crosslinking and the characterization of hydrogels based on PEGDA only (i.e., not containing AA) was not investigated, because it has already been reported in the literature (10, 24). The formation of a 3-dimensional hydrogel network for the different formulations tested was itself representative of the reactivity of PEGDA, which was also confirmed by FT-Raman spectra (Fig. 1). From semi-IPNs to grafted IPNs To induce a chemical grafting of collagen to the PEGDA/poly(AA) network, the semi-IPNs described above were subjected to a crosslinking procedure activated by exposure to water-soluble 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDAC). EDAC is known to induce the formation of peptide linkages (22, 23), which, in this study, may form between the amine groups of collagen and the carboxylic groups brought by either the PEGDA/ poly(AA) network or the collagen itself (Fig. 2). Hydrogel samples were subjected to the grafting treatment by placing them in a water solution containing EDAC (14 mM) and N-hydroxysuccinimide (NHS, 5.5 mM), the latter working as a catalyst to improve the rate and the efficacy of the crosslinking reaction (22, 23). NHS is indeed well known to convert the intermediate O-acylisourea group, created between a carboxylic group and EDAC, into an NHS-activated carboxylic group, which is much
© 2014 Società Italiana Biomateriali - eISSN 2280-8000
Madaghiele et al
water (50 mL) overnight, to remove unreacted chemicals and/or by-products. Assessment of collagen content
Fig. 1 - FT-Raman spectra of pure poly(ethylene glycol) diacrylate (PEGDA), before and after UV exposure (365 nm, 1 minute) in the presence of 3% w/w of Darocur 1173. Spectra were acquired averaging on 64 scans and using a laser (1064 nm) power of 390 mW. The characteristic peaks of the acrylate moieties of PEGDA, highlighted in the upper curve, either disappear or are significantly reduced following UV exposure. Highlighted peaks: 1 (3,109 cm-1) and 2 (3,041 cm-1), both ascribable to =CH2; 3 (1,728 cm-1) related to C=O; 4 (1,641 cm-1) related to C=C; 5 (1,413 cm-1), ascribable to CH2=CH.
To assess the amount of collagen retained in swollen IPNs, as well as the physical and/or chemical interaction between the collagen and the PEGDA/poly(AA) network in both semi-IPNs and grafted IPNs, an aliquot (1 mL) of the distilled water used to wash the samples (50 mL) was analyzed to detect any protein traces, by means of the bicinchoninic acid (BCA) protein quantification assay. BCA is known to form a purple-blue complex with Cu+1 in alkaline environments, where Cu+1 is obtained from the reduction of Cu+2 achieved in the presence of a protein and proportional to the amount of protein present (25). The BCA assay used in this study had a linear sensitivity range of 200-1,000 μg/mL of protein. Following the manufacturer’s protocol, the content of collagen released in the washing solution was measured by reading the absorbance of the purple-blue complex at 562 nm, against a standard curve. Measurements were run in triplicate. The amount of collagen released from each sample was then expressed as the percentage of the theoretical protein loading used for the hydrogel synthesis. Such a loading, calculated from the volume of the starting polymer mixture (1.5 mL) and the collagen concentration, was equal to 13.5 mg and 27 mg for Coll(0.9)/PEG and Coll(1.8)/PEG samples, respectively. Swelling measurements The hydrogel swelling capability in distilled water was assessed by measuring the mass swelling ratio, defined as follows:
Fig. 2 - Synthesis of composite PEGDA/Coll hydrogels: a) Schematic representation of semi-IPNs based on PEGDA/poly(AA) and collagen; b) Subsequent grafting reaction of collagen to the PEGDA/poly(AA) network induced by EDAC. AA = acrylic acid; Coll = collagen; EDAC = 1-ethyl3-(3-dimethylaminopropyl)carbodiimide; IPN = interpenetrating polymer network; PEGDA = poly(ethylene glycol) diacrylate.
more reactive toward free amine groups and less sensitive to hydrolysis (23). The needed volume of the EDAC/NHS solution for each sample was calculated by considering a molar ratio of 5:1 between the EDAC and the total amount of carboxylic groups (Coll + AA) theoretically present in each sample (23). The crosslinking reaction was run for 3 hours, after which the samples were soaked in distilled
SR =
Msw − Md [1] Md
where Msw is the mass of the swollen hydrogel sample and Md the mass of the dried sample. For the swelling measurements, each hydrogel type described above was synthesized in triplicate, and from each sample three 6-mm-diameter disks were punched out. After washing in distilled water overnight, the weight of the disks was measured to determine Msw. Before weighing, the hydrogel samples were gently blotted with soft paper to remove excess water from their surface. The samples were then air-dried at room temperature under a chemical hood for 24-48 hours, and weighed again to determine Md. Both Msw and Md values, estimated as described above, did account for the release of any unreacted chemicals and/or collagen from the hydrogel network during the washing, and thus were considered as representative of the true water holding capacity of the samples.
© 2014 Società Italiana Biomateriali - eISSN 2280-8000
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Poly(ethylene glycol) and collagen IPNs
To verify whether the hydrogel formulations were sensitive to ionic strength variations, the swelling ratio of the hydrogels was also assessed in water solutions at different NaCl concentrations (10–4, 10–3, 10–2, 10–1 and 1 M) (26). Results were averaged over 3 independent measurements. Uniaxial compression tests After swelling in distilled water for 24 hours, semiIPN and grafted IPN samples were subjected to uniaxial compression at 20°C by means of a parallel plate rheometer (ARES; Scientific Rheometric), as described previously (27). Such tests were carried out both to assess the mechanical properties of the samples and to verify if the theory of rubber elasticity could be applied to them to evaluate their crosslink density. Indeed, in the case of a perfect rubber-like polymer network subjected to small uniaxial deformations (i.e., for negligible volume changes either in compression or elongation), the following equation derived by Flory (28) might be used to estimate the elastically effective degree of crosslinking: 1 σ = RT ρxeV21s/ 3 α − 2 α
[2]
where σ is the uniaxial stress, R is the universal gas constant, T is the absolute temperature, ρxe is the crosslink density, V2s is the polymer volume fraction in the swollen state, i.e., the inverse of the volume swelling ratio, and α=L/Li is the deformation ratio, with L the actual thickness of the deformed sample and Li the initial thickness of the swollen sample (α>1 for elongation and α
J Appl Biomater Funct Mater 2014 ; 12 (3): 183 - 192 DOI: 10.5301/jabfm.5000187
Development of semi- and grafted interpenetrating polymer networks based on poly(ethylene glycol) diacrylate and collagen Marta Madaghiele, Francesco Marotta, Christian Demitri, Francesco Montagna, Alfonso Maffezzoli, Alessandro Sannino Department of Engineering for Innovation, University of Salento, Lecce - Italy
ABSTRACT Purpose: The objective of this work was to develop composite hydrogels based on poly(ethylene glycol) diacrylate (PEGDA) and collagen (Coll), potentially useful for biomedical applications. Methods: Semi-interpenetrating polymer networks (semi-IPNs) were obtained by photo-stabilizing aqueous solutions of PEGDA and acrylic acid (AA), in the presence of collagen. Further grafting of the collagen macromolecules to the PEGDA/ poly(AA) network was achieved by means of a carbodiimide-mediated crosslinking reaction. The resulting hydrogels were characterized in terms of swelling capability, collagen content and mechanical properties. Results and conclusions: The grafting procedure was found to significantly improve the mechanical stability of the IPN hydrogels, due to the establishment of covalent bonding between the PEGDA/poly(AA) and the collagen networks. The suitability of the composite hydrogels to be processed by means of stereolithography (SLA) was also investigated, toward creating biomimetic constructs with complex shapes, which might be useful either as platforms for tissue engineering applications or as tissue mimicking phantoms. Key words: Collagen, Hydrogel, Interpenetrating polymer networks Accepted: March 4, 2013
INTRODUCTION Interpenetrating polymer networks (IPNs) are composite systems made up of 2 (or more) independently crosslinked networks, at least one of which is crosslinked in the presence of the other. The intimate entanglements between the 2 polymer networks are well known to enhance the mechanical properties of the single networks, and/or to form a new material showing the key functional properties of both components. IPN hydrogels are particularly attractive for biomedical applications, especially for the creation of biomimetic platforms for tissue engineering, which usually combine the typical advantages of synthetic polymers (i.e., designable and tunable properties) with the intrinsic bioactivity of naturally derived biomaterials (e.g., cell-interactive domains and enzymatic degradability). In particular, polymers derived from the extracellular matrix (ECM) are ideal candidates for the development of semi-IPNs, which differ from IPNs in the fact that one network only is crosslinked (e.g., the synthetic polymer), and formed around uncrosslinked chains of a second polymer (e.g., the ECM macromolecules).
Poly(ethylene glycol) (PEG)–based hydrogels have been widely investigated in the last decade in both in vitro and in vivo applications, especially as tissue engineering templates, due to the resistance of PEG to protein adsorption, which makes it a nonfouling material for the design of scaffolds with specific bioactivity (1-11). Hydrogels are usually obtained by the photocrosslinking of acrylated PEG derivatives in aqueous solution, which is particularly advantageous for the design of injectable hydrogel formulations (12-14), as well as for the possible use of rapid prototyping techniques (e.g. stereolithography [SLA]) and other photopatterning approaches for the design of devices with complex or customized structures (15-18). Bioactive PEG-based hydrogels can be obtained by copolymerizing PEG derivatives with short peptide sequences and/or ECM components, which have been previously modified (e.g., acrylated or methacrylated) to be incorporated into the hydrogel network during the crosslinking reaction (1-9). Alternatively, biomimetic semi-IPNs can be synthesized by crosslinking the PEG derivatives in the presence of unmodified ECM molecules, such as hyaluronic acid (19, 20). In this case, in addition to inducing a given cell– material interaction, the ECM component acts as a spacer
© 2014 Società Italiana Biomateriali - eISSN 2280-8000
183
Poly(ethylene glycol) and collagen IPNs
in the PEG network, thus modifying the microstructure of the network itself. The aim of this work was to investigate the creation of biomimetic semi-IPNs and grafted IPNs based on PEG derivatives and collagen (Coll), potentially useful for biomedical applications. Semi-IPNs were synthesized starting from aqueous mixtures of poly(ethylene glycol) diacrylate (PEGDA), acrylic acid (AA) and collagen as bioactive molecule. The effect of collagen on the PEGDA/ poly(AA) network formed in a photocrosslinking reaction was assessed in terms of swelling behavior and mechanical properties. The use of SLA for the production of hydrogels with complex shapes was also investigated. Further grafting of the collagen macromolecules to the PEGDA/ poly(AA) network was explored by using a carbodiimidemediated crosslinking reaction (21-23).
TABLE I - HYDROGEL FORMULATIONS: PEGDA (10% w/v), AA (0.2% v/v), DAROCUR 1173 (0.3% v/v) WERE USED FOR ALL SAMPLES Formulation code
Collagen loading (% w/v)
EDAC treatment (Yes/No)
PEG
0
No
PEG-X
0
Yes
Coll(0.9)/PEG
0.9
No
Coll(0.9)/PEG-X
0.9
Yes
Coll(1.8)/PEG
1.8
No
Coll(1.8)/PEG-X
1.8
Yes
AA = acrylic acid; Coll = collagen; EDAC = 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide; PEG = poly(ethylene glycol); PEGDA = poly(ethylene glycol) diacrylate.
MATERIALS AND METHODS All chemicals used in this study were purchased from Sigma-Aldrich (Milan, Italy), unless otherwise stated, and used as received. Hydrogel synthesis To develop composite hydrogels based on PEGDA and Coll, a 2-step process was employed which involved: (a) the synthesis of semi-IPNs by photostabilizing aqueous solutions of PEGDA (molecular weight 700 Da) and AA, in the presence of collagen; (b) the grafting of collagen to the PEGDA/poly(AA) network by means of a carbodiimide (EDAC)–mediated crosslinking reaction. Semi-IPNs An aqueous suspension of Type I collagen from calf skin (3% w/v, Semed S collagen; Kensey Nash Corporation) was prepared. PEGDA, AA and an acrylate-soluble photoinitiator (Darocur 1173; Basf) were dissolved in distilled water. A given volume of collagen suspension was then added to the above solution and thoroughly mixed, in order to obtain a homogeneous polymer mixture with fixed PEGDA and AA amounts and variable collagen concentrations, according to the following: PEGDA (10% w/v), AA (0.2% v/v), Darocur 1173 (0.3% v/v) and collagen at a concentration of either 0.9% or 1.8% w/v. Control solutions devoid of collagen were also prepared. The concentrations of PEGDA, AA and Darocur 1173 were chosen based on a previous protocol (10), which was slightly modified to reduce the AA amount. With the concentrations used in this study, the molar ratio between PEGDA and AA was 5:1. Hydrogel samples were then obtained by photostabilizing 1.5 mL of the polymer mixture in a 35-mm Petri dish by exposure to UV light (365 nm) 184
for 10 minutes. The average distance between the sample surface and the UV lamp was 3.5 cm. The resulting semi-IPNs were either subjected to a further grafting procedure, as described in the following, or directly washed in excess distilled water (50 mL) overnight, to remove unreacted chemicals. A synoptic overview of all the hydrogel formulations prepared in this work (semi-IPNs, grafted IPNs and control samples) is given in Table I. The crosslinking and the characterization of hydrogels based on PEGDA only (i.e., not containing AA) was not investigated, because it has already been reported in the literature (10, 24). The formation of a 3-dimensional hydrogel network for the different formulations tested was itself representative of the reactivity of PEGDA, which was also confirmed by FT-Raman spectra (Fig. 1). From semi-IPNs to grafted IPNs To induce a chemical grafting of collagen to the PEGDA/poly(AA) network, the semi-IPNs described above were subjected to a crosslinking procedure activated by exposure to water-soluble 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDAC). EDAC is known to induce the formation of peptide linkages (22, 23), which, in this study, may form between the amine groups of collagen and the carboxylic groups brought by either the PEGDA/ poly(AA) network or the collagen itself (Fig. 2). Hydrogel samples were subjected to the grafting treatment by placing them in a water solution containing EDAC (14 mM) and N-hydroxysuccinimide (NHS, 5.5 mM), the latter working as a catalyst to improve the rate and the efficacy of the crosslinking reaction (22, 23). NHS is indeed well known to convert the intermediate O-acylisourea group, created between a carboxylic group and EDAC, into an NHS-activated carboxylic group, which is much
© 2014 Società Italiana Biomateriali - eISSN 2280-8000
Madaghiele et al
water (50 mL) overnight, to remove unreacted chemicals and/or by-products. Assessment of collagen content
Fig. 1 - FT-Raman spectra of pure poly(ethylene glycol) diacrylate (PEGDA), before and after UV exposure (365 nm, 1 minute) in the presence of 3% w/w of Darocur 1173. Spectra were acquired averaging on 64 scans and using a laser (1064 nm) power of 390 mW. The characteristic peaks of the acrylate moieties of PEGDA, highlighted in the upper curve, either disappear or are significantly reduced following UV exposure. Highlighted peaks: 1 (3,109 cm-1) and 2 (3,041 cm-1), both ascribable to =CH2; 3 (1,728 cm-1) related to C=O; 4 (1,641 cm-1) related to C=C; 5 (1,413 cm-1), ascribable to CH2=CH.
To assess the amount of collagen retained in swollen IPNs, as well as the physical and/or chemical interaction between the collagen and the PEGDA/poly(AA) network in both semi-IPNs and grafted IPNs, an aliquot (1 mL) of the distilled water used to wash the samples (50 mL) was analyzed to detect any protein traces, by means of the bicinchoninic acid (BCA) protein quantification assay. BCA is known to form a purple-blue complex with Cu+1 in alkaline environments, where Cu+1 is obtained from the reduction of Cu+2 achieved in the presence of a protein and proportional to the amount of protein present (25). The BCA assay used in this study had a linear sensitivity range of 200-1,000 μg/mL of protein. Following the manufacturer’s protocol, the content of collagen released in the washing solution was measured by reading the absorbance of the purple-blue complex at 562 nm, against a standard curve. Measurements were run in triplicate. The amount of collagen released from each sample was then expressed as the percentage of the theoretical protein loading used for the hydrogel synthesis. Such a loading, calculated from the volume of the starting polymer mixture (1.5 mL) and the collagen concentration, was equal to 13.5 mg and 27 mg for Coll(0.9)/PEG and Coll(1.8)/PEG samples, respectively. Swelling measurements The hydrogel swelling capability in distilled water was assessed by measuring the mass swelling ratio, defined as follows:
Fig. 2 - Synthesis of composite PEGDA/Coll hydrogels: a) Schematic representation of semi-IPNs based on PEGDA/poly(AA) and collagen; b) Subsequent grafting reaction of collagen to the PEGDA/poly(AA) network induced by EDAC. AA = acrylic acid; Coll = collagen; EDAC = 1-ethyl3-(3-dimethylaminopropyl)carbodiimide; IPN = interpenetrating polymer network; PEGDA = poly(ethylene glycol) diacrylate.
more reactive toward free amine groups and less sensitive to hydrolysis (23). The needed volume of the EDAC/NHS solution for each sample was calculated by considering a molar ratio of 5:1 between the EDAC and the total amount of carboxylic groups (Coll + AA) theoretically present in each sample (23). The crosslinking reaction was run for 3 hours, after which the samples were soaked in distilled
SR =
Msw − Md [1] Md
where Msw is the mass of the swollen hydrogel sample and Md the mass of the dried sample. For the swelling measurements, each hydrogel type described above was synthesized in triplicate, and from each sample three 6-mm-diameter disks were punched out. After washing in distilled water overnight, the weight of the disks was measured to determine Msw. Before weighing, the hydrogel samples were gently blotted with soft paper to remove excess water from their surface. The samples were then air-dried at room temperature under a chemical hood for 24-48 hours, and weighed again to determine Md. Both Msw and Md values, estimated as described above, did account for the release of any unreacted chemicals and/or collagen from the hydrogel network during the washing, and thus were considered as representative of the true water holding capacity of the samples.
© 2014 Società Italiana Biomateriali - eISSN 2280-8000
185
Poly(ethylene glycol) and collagen IPNs
To verify whether the hydrogel formulations were sensitive to ionic strength variations, the swelling ratio of the hydrogels was also assessed in water solutions at different NaCl concentrations (10–4, 10–3, 10–2, 10–1 and 1 M) (26). Results were averaged over 3 independent measurements. Uniaxial compression tests After swelling in distilled water for 24 hours, semiIPN and grafted IPN samples were subjected to uniaxial compression at 20°C by means of a parallel plate rheometer (ARES; Scientific Rheometric), as described previously (27). Such tests were carried out both to assess the mechanical properties of the samples and to verify if the theory of rubber elasticity could be applied to them to evaluate their crosslink density. Indeed, in the case of a perfect rubber-like polymer network subjected to small uniaxial deformations (i.e., for negligible volume changes either in compression or elongation), the following equation derived by Flory (28) might be used to estimate the elastically effective degree of crosslinking: 1 σ = RT ρxeV21s/ 3 α − 2 α
[2]
where σ is the uniaxial stress, R is the universal gas constant, T is the absolute temperature, ρxe is the crosslink density, V2s is the polymer volume fraction in the swollen state, i.e., the inverse of the volume swelling ratio, and α=L/Li is the deformation ratio, with L the actual thickness of the deformed sample and Li the initial thickness of the swollen sample (α>1 for elongation and α
