Subscriber access provided by Brown University Library
Article
A Novel Strategy for Softening the Gelatin-Bioactive Glass Hybrids Ali Negahi Shirazi, Ali Fathi, Francia Garces Suarez, Yiwei Wang, Peter K. Maitz, and Fariba Dehghani ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.5b09006 • Publication Date (Web): 04 Jan 2016 Downloaded from http://pubs.acs.org on January 6, 2016
Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
ACS Applied Materials & Interfaces is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.
Page 1 of 36
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
ACS Applied Materials & Interfaces
A Novel Strategy for Softening the GelatinBioactive Glass Hybrids Ali Negahi Shirazi1, Ali Fathi1, Francia Garces Suarez2, Yiwei Wang2, Peter K. Maitz2,3 and Fariba Dehghani1* 1
School of Chemical & Biomolecular Engineering, the University of Sydney, 2006, Australia
2
Burns Research and Reconstructive Surgery ANZAC Research Institute, Concord Repatriation
General Hospital, 2139, Australia 3
Burn Injury and Reconstructive Surgery, Concord Repatriation General Hospital, 2139,
Australia Keywords: organic-inorganic hybrids, photo-crosslinking, sol-gel, bioactive glasses, tissue engineering
Abstract
The brittle structure of polymer-bioactive glass hybrids is a hurdle for their biomedical applications. To address this issue here, we developed a novel method to cease the overcondensation of bioactive glass by a polymer crosslinking. Here, an organosilane-functionalized gelatin methacrylate (GelMA) is covalently bonded to a bioactive glass during the sol-gel process, and the condensation of silica networks is controlled by photo-crosslinking of GelMA. The physicochemical properties and mechanical strength of these hybrids are tunable by the
ACS Paragon Plus Environment
1
ACS Applied Materials & Interfaces
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Page 2 of 36
incorporation of secondary crosslinking agents. These hydrogels display elastic properties with ultimate compression strain above 0.2 mm.mm-1 and tunable compressive modulus in the range of 42 kPa to 530 kPa. In addition, these hydrogels are bioactive as they promoted the alkaline phosphatase activity of bone progenitor cells. They are also well-tolerated in mice subcutaneous model. Therefore, our method is efficient for the prevention of over-condensation and allows preparation of soft bioactive hydrogels from organic-inorganic matrices, suitable for soft and hard tissue regeneration.
1.
Introduction
Bioactive glass (BG) is a class of biomaterials with unique bioactivity1, osteoconductivity2 and osteoinductivity3. Various studies confirmed the favorable properties of BG for bone regeneration4, dental applications5, ophthalmology6, the regeneration of nerve7, and for the remodeling of soft tissues8. However, BG is only clinically used for hard tissue regeneration due to its intrinsic brittle properties and inferior water uptake behavior. Several studies proposed to fabricate organic-inorganic hybrids by chemical conjugation of BG with a variety of polymers to tackle these issues and to prepare structures with tunable physicochemical properties9,10. In the fabrication of organic-inorganic hybrids, a polymer is first functionalized with an organosilane compound11 and it is subsequently chemically bonded with an inorganic precursor to form a hybrid network structure12. By acquiring the sol-gel method, natural polymers including gelatin9,10, chitosan13,14 and poly(γ-glutamic acid)15,16 as well as synthetic polymers such as poly(methyl methacrylate)17 were covalently bonded to silica precursors and formed organic-inorganic hybrids for biomedical application. A monolith structure is commonly formed by complete condensation of inorganic compounds through prolonged drying and aging of these
ACS Paragon Plus Environment
2
Page 3 of 36
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
ACS Applied Materials & Interfaces
hybrids18. Some of polymer-bioactive glass hybrids from previous attempts displayed brittle twodimensional structures that also had limited water uptake properties15-17. To this end, we proposed a novel technique to control the condensation of silica network during the sol-gel method to fabricate a homogenous network of gelatin and BG. It was hypothesized that the crosslinking reaction of a polymer that chemically bonded to a BG network tune the degree of silica condensation and thus can potentially reduce the brittleness of bioactive hybrid structure. To assess this hypothesis, photo-crosslinkable gelatin (GelMA) was functionalized with glycidoxypropyl-methyldiethoxysilane (GPTMS) prior to hybrid formation. The physicochemical properties of these hybrids were tuned by optimizing the concentration of organic and inorganic compounds to assure the applicability of these constructs for a broad range of biomedical applications. The precise mechanism of organosilation was determined to assure the secondary polymer crosslinking did not have a predominant impact on the hybrid formation. To confirm the instructive effect of BG within the structure of hybrids on their biological and structural properties series of in vitro osteoblast cell and in vivo subcutaneous studies were conducted. 2.
Results and Discussion
The aim of this study was to fabricate bioactive 3D elastic organic-inorganic structures. The preliminary results showed the over condensation of BG, even at very low concentrations (e.g. 0.5 mg.mg-1) in different polymers solutions led to the formation of brittle monoliths. In this study, the feasibility of controlling the condensation level of silica networks by crosslinking of the organic polymer chain was investigated. Photo-crosslinkable gelatin (GelMA) was used as the organic segment. In addition, GelMA and silica networks were chemically bonded to acquire
ACS Paragon Plus Environment
3
ACS Applied Materials & Interfaces
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Page 4 of 36
uniform structures. To this end, GelMA was initially functionalized with GPTMS to add the capacity of bonding of this polymer to silica networks. The functionalization reaction was carried out in PBS solution at pH 7.4. However, care must be taken in the functionalization of polymers with this hetero-bifunctional agent as GPTMS undergoes competitive reactions between epoxy-ring hydrolysis and self-condensation of activated silanol groups of GPTMS. Temperature and the reaction time are the important processing parameters to control the GPTMS-functionalization reaction19. By increasing the temperature, the selectivity of the reaction leans toward the self-condensation of silanol groups in GPTMS, which is unfavorable20. The functionalisation reaction was, therefore, conducted at 40 °C to reduce the risk of self-condensation of silanol group and also to avoid physical crosslinking of GelMA. The reaction time between GelMA and GPTMS was optimized at 40 °C and neutral pH. Our preliminary results showed that less than 14 h was not adequate for this reaction and above this period, GPTMS was condensed. This result was in agreement with a previous study by Gabrielli et al. who confirmed GPTMS condensation after 16 h in neutral pH21. Therefore, GelMA functionalization with GPTMS was set at 40 °C for 14 h to prevent fast solidification of FnGelMA solution. It is important to verify the mechanism of the functionalization reaction of GelMA with GPTMS as it can have an impact on the biological activity of this protein. Glycidoxy functional group of GPTMS can form a covalent bond with nucleophilic functional groups, either carboxylic acid9,10 or amine22 groups in amino acid residues of gelatin23. Colorimetric ninhydrin assay was therefore used to quantify the amine groups in Fn-GelMA hydrogels for determining the mechanism of
ACS Paragon Plus Environment
4
Page 5 of 36
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
ACS Applied Materials & Interfaces
functionalization reaction, as depicted in Figure1-a. The results show that the amount of free amine functional group in GelMA was 15%, which was in agreement with 1H-NMR results24. However, the fraction of amine functional groups in GelMA was decreased to 13% following the functionalization with GPTMS (p
Article
A Novel Strategy for Softening the Gelatin-Bioactive Glass Hybrids Ali Negahi Shirazi, Ali Fathi, Francia Garces Suarez, Yiwei Wang, Peter K. Maitz, and Fariba Dehghani ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.5b09006 • Publication Date (Web): 04 Jan 2016 Downloaded from http://pubs.acs.org on January 6, 2016
Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
ACS Applied Materials & Interfaces is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.
Page 1 of 36
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
ACS Applied Materials & Interfaces
A Novel Strategy for Softening the GelatinBioactive Glass Hybrids Ali Negahi Shirazi1, Ali Fathi1, Francia Garces Suarez2, Yiwei Wang2, Peter K. Maitz2,3 and Fariba Dehghani1* 1
School of Chemical & Biomolecular Engineering, the University of Sydney, 2006, Australia
2
Burns Research and Reconstructive Surgery ANZAC Research Institute, Concord Repatriation
General Hospital, 2139, Australia 3
Burn Injury and Reconstructive Surgery, Concord Repatriation General Hospital, 2139,
Australia Keywords: organic-inorganic hybrids, photo-crosslinking, sol-gel, bioactive glasses, tissue engineering
Abstract
The brittle structure of polymer-bioactive glass hybrids is a hurdle for their biomedical applications. To address this issue here, we developed a novel method to cease the overcondensation of bioactive glass by a polymer crosslinking. Here, an organosilane-functionalized gelatin methacrylate (GelMA) is covalently bonded to a bioactive glass during the sol-gel process, and the condensation of silica networks is controlled by photo-crosslinking of GelMA. The physicochemical properties and mechanical strength of these hybrids are tunable by the
ACS Paragon Plus Environment
1
ACS Applied Materials & Interfaces
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Page 2 of 36
incorporation of secondary crosslinking agents. These hydrogels display elastic properties with ultimate compression strain above 0.2 mm.mm-1 and tunable compressive modulus in the range of 42 kPa to 530 kPa. In addition, these hydrogels are bioactive as they promoted the alkaline phosphatase activity of bone progenitor cells. They are also well-tolerated in mice subcutaneous model. Therefore, our method is efficient for the prevention of over-condensation and allows preparation of soft bioactive hydrogels from organic-inorganic matrices, suitable for soft and hard tissue regeneration.
1.
Introduction
Bioactive glass (BG) is a class of biomaterials with unique bioactivity1, osteoconductivity2 and osteoinductivity3. Various studies confirmed the favorable properties of BG for bone regeneration4, dental applications5, ophthalmology6, the regeneration of nerve7, and for the remodeling of soft tissues8. However, BG is only clinically used for hard tissue regeneration due to its intrinsic brittle properties and inferior water uptake behavior. Several studies proposed to fabricate organic-inorganic hybrids by chemical conjugation of BG with a variety of polymers to tackle these issues and to prepare structures with tunable physicochemical properties9,10. In the fabrication of organic-inorganic hybrids, a polymer is first functionalized with an organosilane compound11 and it is subsequently chemically bonded with an inorganic precursor to form a hybrid network structure12. By acquiring the sol-gel method, natural polymers including gelatin9,10, chitosan13,14 and poly(γ-glutamic acid)15,16 as well as synthetic polymers such as poly(methyl methacrylate)17 were covalently bonded to silica precursors and formed organic-inorganic hybrids for biomedical application. A monolith structure is commonly formed by complete condensation of inorganic compounds through prolonged drying and aging of these
ACS Paragon Plus Environment
2
Page 3 of 36
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
ACS Applied Materials & Interfaces
hybrids18. Some of polymer-bioactive glass hybrids from previous attempts displayed brittle twodimensional structures that also had limited water uptake properties15-17. To this end, we proposed a novel technique to control the condensation of silica network during the sol-gel method to fabricate a homogenous network of gelatin and BG. It was hypothesized that the crosslinking reaction of a polymer that chemically bonded to a BG network tune the degree of silica condensation and thus can potentially reduce the brittleness of bioactive hybrid structure. To assess this hypothesis, photo-crosslinkable gelatin (GelMA) was functionalized with glycidoxypropyl-methyldiethoxysilane (GPTMS) prior to hybrid formation. The physicochemical properties of these hybrids were tuned by optimizing the concentration of organic and inorganic compounds to assure the applicability of these constructs for a broad range of biomedical applications. The precise mechanism of organosilation was determined to assure the secondary polymer crosslinking did not have a predominant impact on the hybrid formation. To confirm the instructive effect of BG within the structure of hybrids on their biological and structural properties series of in vitro osteoblast cell and in vivo subcutaneous studies were conducted. 2.
Results and Discussion
The aim of this study was to fabricate bioactive 3D elastic organic-inorganic structures. The preliminary results showed the over condensation of BG, even at very low concentrations (e.g. 0.5 mg.mg-1) in different polymers solutions led to the formation of brittle monoliths. In this study, the feasibility of controlling the condensation level of silica networks by crosslinking of the organic polymer chain was investigated. Photo-crosslinkable gelatin (GelMA) was used as the organic segment. In addition, GelMA and silica networks were chemically bonded to acquire
ACS Paragon Plus Environment
3
ACS Applied Materials & Interfaces
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Page 4 of 36
uniform structures. To this end, GelMA was initially functionalized with GPTMS to add the capacity of bonding of this polymer to silica networks. The functionalization reaction was carried out in PBS solution at pH 7.4. However, care must be taken in the functionalization of polymers with this hetero-bifunctional agent as GPTMS undergoes competitive reactions between epoxy-ring hydrolysis and self-condensation of activated silanol groups of GPTMS. Temperature and the reaction time are the important processing parameters to control the GPTMS-functionalization reaction19. By increasing the temperature, the selectivity of the reaction leans toward the self-condensation of silanol groups in GPTMS, which is unfavorable20. The functionalisation reaction was, therefore, conducted at 40 °C to reduce the risk of self-condensation of silanol group and also to avoid physical crosslinking of GelMA. The reaction time between GelMA and GPTMS was optimized at 40 °C and neutral pH. Our preliminary results showed that less than 14 h was not adequate for this reaction and above this period, GPTMS was condensed. This result was in agreement with a previous study by Gabrielli et al. who confirmed GPTMS condensation after 16 h in neutral pH21. Therefore, GelMA functionalization with GPTMS was set at 40 °C for 14 h to prevent fast solidification of FnGelMA solution. It is important to verify the mechanism of the functionalization reaction of GelMA with GPTMS as it can have an impact on the biological activity of this protein. Glycidoxy functional group of GPTMS can form a covalent bond with nucleophilic functional groups, either carboxylic acid9,10 or amine22 groups in amino acid residues of gelatin23. Colorimetric ninhydrin assay was therefore used to quantify the amine groups in Fn-GelMA hydrogels for determining the mechanism of
ACS Paragon Plus Environment
4
Page 5 of 36
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
ACS Applied Materials & Interfaces
functionalization reaction, as depicted in Figure1-a. The results show that the amount of free amine functional group in GelMA was 15%, which was in agreement with 1H-NMR results24. However, the fraction of amine functional groups in GelMA was decreased to 13% following the functionalization with GPTMS (p
