Degradation prediction model and stem cell growth of gelatin-PEG composite hydrogel Nan Zhou,
1,2
Chang Liu,
1,4
Shijie Lv,
3
Dongsheng Sun,
1
Qinglong Qiao,
1
Rui
Zhang, 5 Yang Liu, 1 Jing Xiao, 2 Guangwei Sun, 1 1
Scientific Research Center for Translational Medicine, Dalian Institute of Chemical
Physics, Chinese Academy of Sciences, Dalian, 116023, China 2
Department of Oral Pathology, College of Stomatology, Dalian Medical University,
Dalian, 116044, China 3
Dalian Maternity & Child Healthcare Hospital, Dalian, 116033, China
4
Dalian Municipal Central Hospital, Dalian, 116033, China
5
Department of Stomatology, First Affiliated Hospital, Dalian Medical University,
Dalian, 116023, China
E-mail addresses: [email protected] (Nan Zhou), [email protected] (Chang Liu), [email protected] (Shijie Lv), [email protected] (Dongsheng Sun), [email protected] (Qinglong Qiao), [email protected] (Rui Zhang), [email protected]
(Yang
Liu),
[email protected]
(Jing
Xiao),
[email protected] (Guangwei Sun)
This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process which may lead to differences between this version and the Version of Record. Please cite this article as an ‘Accepted Article’, doi: 10.1002/jbm.a.35847 This article is protected by copyright. All rights reserved.
Journal of Biomedical Materials Research: Part A
Correspondence to: Y. Liu; e-mail: [email protected] and G. Sun; e-mail: [email protected]
*Corresponding author: Dr. Yang Liu. Dalian Institute of Chemical Physics, Chinese Academy of Sciences, 457 Zhongshan Road, Dalian 116023, China. Tel. /fax: +86411-82463027. E-mail address: [email protected]
*Corresponding author: Dr. Guangwei Sun. Dalian Institute of Chemical Physics, Chinese Academy of Sciences, 457 Zhongshan Road, Dalian 116023, China. Tel. /fax: +86-411-82463027. E-mail address: [email protected]
2
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Page 3 of 38
Journal of Biomedical Materials Research: Part A
Abstract Gelatin hydrogel has great potential in regenerative medicine. The degradation of gelatin hydrogel is important to control the release profile of encapsulated biomolecules and regulate in vivo tissue repair process. As a plasticizer, PEG can significantly improve the mechanical property of gelatin hydrogel. However, how preparation parameters affect the degradation rate of gelatin-PEG composite hydrogel is still not clear. In this study, the significant effect factor, glutaraldehyde (GA) concentration, was confirmed by means of Plackett-Burman method. Then a mathematical model was built to predict the degradation rate of composite hydrogels under different preparation conditions using the response surface method (RSM), which was helpful to prepare the certain composite hydrogel with desired degradation rate. In addition, it was found that gelatin-PEG composite hydrogel surface well supported the adhension and growth of human mesenchymal stem cells (MSCs). Moreover, PEG concentration not only could adjust hydrogel degradation more subtly, but also might increase the cross-linking degree and affect the cell migration. Therefore, these results would be useful to optimize the preparation of gelatin-PEG composite hydrogel for drug delivery or tissue engineering. Key words: gelatin-PEG hydrogel, degradation model, response surface methodology, stem cells 3
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Journal of Biomedical Materials Research: Part A
INTRODUCTION Gelatin is derived from collagen by the partial hydrolysis, and it is a useful natural polymer that has great potential in pharmaceutical and medical applications due to its biodegradability and biocompatibility.1,2 Gelatin can be covalently cross-linked by cross-linking agents, such as glutaraldehyde (GA) and carbodiimide, to form gelatin hydrogel with better stability.2-4 The covalently cross-linked gelatin hydrogel has a three-dimensional network structure and can be used as a carrier for the controlled release of bioactive molecules, including growth factors and drugs,5-10 by the polyion complexation.2 In addition, gelatin hydrogel is also used as a biodegradable cell delivery matrice for cell transplantation, which can accelerate in vivo tissue repair.1113
For gelatin hydrogel, the degradation rate is very important, because it not only
controls the release profile of encapsulated biomolecules, but also regulates in vivo tissue repair process.2 It is known that plasticizers can improve the physicochemical properties of biomaterial scaffolds. Polyethylene glycol (PEG), one of widespread used plasticizers, has good biocompatibility as well as low cytotoxicity and immunogenicity, which makes it widely used as the modifier of biomaterial scaffolds.10,14-15 It is reported that PEG can interact with gelatin molecule by the strong inter/intra-molecular hydrogen bond,10 which exhibits better plasticizing influence on the mechanical property of 4
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Page 4 of 38
Page 5 of 38
Journal of Biomedical Materials Research: Part A
gelatin scaffolds.16 However, how preparation parameters affect the degradation rate of gelatin-PEG composite hydrogel is still not clear. In this work, we prepared gelatin-PEG composite hydrogel, and ascertained the significant effect factor that affected the degradation rate of composite hydrogel by means of Plackett-Burman method. And then a mathematical model was built to predict the degradation rate of composite hydrogel using the response surface method (RSM). This model would be helpful to prepare the certain composite hydrogel with desired degradation rate. In addition, gelatin-PEG composite hydrogel carrying stem cells might be helpful to repair damaged tissues, such as periodontal tissue regeneration. So the adhension and growth of human umbilical cord Wharton's jellyderived mesenchymal stem cells (WJ MSCs) on the surface of composite hydrogel was also evaluated compared to the conventional gelatin hydrogel without PEG. This study would be benefit to optimize the preparation of gelatin-PEG composite hydrogel for drug delivery or tissue engineering.
MATERIALS AND METHODS
Materials
5
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Journal of Biomedical Materials Research: Part A
Gelatin (type B, Gel Strength = 225 g Bloom), glycine, trypsin, calcein AM and ethidiumhomodimer-1 (ED-1) were purchased from Sigma (St. Louis, MO, USA). Collagenase I was purchased from Gibco (BRL Co. Ltd., Eugene, OR, USA). Glutaraldehyde (GA) and PEG (molecular weight 6 kDa) was procured from Damao Chemical Reagent Factory (Tianjin, China) and Sinopharm Chemical Reagent Co. Ltd. (Shanghai, China),
respectively. Trinitro-benzene-sulfonic acid (TNBS) was
purchased from Ark Pharm (Libertyville, IL, USA). Preparation of gelatin-PEG composite hydrogels Gelatin-PEG composite hydrogels were prepared according to the procedures reported previously with some modification.10,17,18 In brief, gelatin and PEG were dissolved in water, respectively. And these two solutions were mixed together. Then GA was added into the mixed solution to generate gelatin-PEG composite hydrogels via the cross-linking at 4 °C for 18 h. After that, the hydrogels were immersed into 100 mM glycine aqueous solution for 1 h at 37 °C to block the residual aldehyde groups of GA, and then they were washed three times with double-distilled water. The final concentration ranges of gelatin, PEG and GA inside hydrogels were 3-9 wt %, 03 wt % and 0.1-0.3 wt %. In vitro enzymatic degradation of composite hydrogels
6
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Page 6 of 38
Page 7 of 38
Journal of Biomedical Materials Research: Part A
Gelatin-PEG hydrogels were immersed into PBS containing 15 µg/ml collagenase I at 37 °C for 24 h.7 And they were washed 3 times with deionized water and dried to constant weight in a vacuum oven (DZF 6050, Longyue Instrument Equipment Co., LTD., shanghai, China) at 35 °C. Then the dry weight of dried hydrogels were measured accurately. The degradation rate (Y) of composite hydrogels was calculated using the following equation: Y = (W1 − W2 ) /W1
(1)
W1, dry weight of hydrogels before degradation; W2, dry weight of hydrogels after degradation.
Plackett-Burman screening and response surface methodology Firstly, significant factor that influenced the degradation of gelatin-PEG composite hydrogel was screened using Plackett-Burman method.19 Gelatin concentration (X1), GA concentration (X2) and PEG concentration (X3) were chosen as three effect factors, and the degradation rate of gelatin-PEG composite hydrogel after enzymatic degradation for 24 h as the response value (Y). Table Ⅰ showed the design of Plackett-Burman screening experiment. Then RSM was used to investigate the effect of hydrogel formulation variables (X1, X2, X3) on the dependent value (Y). Box-Behnken design consisting of 17 experiments was employed for RSM (Table Ⅱ).19 A mathematical model of the degradation rate of 7
John Wiley & Sons, Inc. This article is protected by copyright. All rights reserved.
Journal of Biomedical Materials Research: Part A
gelatin-PEG composite hydrogel was built according to the experiment results. ANOVA was used to evaluate the mathematical model. In addition, three random checkpoints were used to check the reliability of the mathematical model. The degradation rates obtained from experiments were compared with the responses calculated by the mathematical model.
WJ MSC culture on the hydrogel surface Primary WJ MSCs were donated by Zhongyuan Union Stem Cell Bioengineering Corporation (Tianjin, China). WJ MSCs were cultured in the special culture medium that was also kindly gifted by this company. The medium was changed every three days and cells were subcultured when reaching 90% confluence.20 WJ MSCs (Passage 5-7) were cultured on the surface of gelatin-PEG composite hydrogels with different formulation (104 cells/cm2). After incubating for 24 h, nonadherent cells were removed by washing with culture medium. The morphology of adherent cells was observed under a microscope (Eclipse TE2000-U, Nikon, Tokyo, Japan). Then the adherent cells were harvested using trypsin and the cell numbers on different hydrogel surfaces were statistically analyzed.
Live/Dead staining Live/Dead staining of adherent cells on different hydrogel surfaces was performed on 24 h using the previously reported method with some modification.21 Cells were 8
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Page 8 of 38
Page 9 of 38
Journal of Biomedical Materials Research: Part A
incubated with live/dead staining working solution composed of 2 µM calcein AM and 4 µM ED-1 at 37 °C for 30 min. Then cells were observed with confocal laser scanning microscopy (CLSM, LeicaSP2, Heidelberger, Germany). Cell distribution on the hydrogel surface or inside hydrogels on Day 6 was observed by living cell station (Revolution WD, Andor Technology, Belfast, Northern Ireland) using Live/Dead staining.
F-actin staining F-actin staining of adherent cells on different hydrogel surfaces was performed at 24 h using the previously reported method.22 Cells were fixed with 4 wt % paraformaldehyde in phosphate-buffered saline (PBS, pH 7.2) for 30 min and rinsed with PBS. The samples were treated with Alexa Flour® 488 phalloidin (50 µg/ml) and incubated overnight at 4 °C. Then samples were rinsed with PBS and observed using the CLSM.
Microstructure analysis Gelatin hydrogels were freeze-dried and then the microstructures of cross-sections were observed by SEM (JSM-7800F, JEOL, Akishima, Tokyo, Japan).
Cross-linking degree determination The cross-linking degree was evaluated as the difference between the chemicallydetermined number of free amines groups before and after crosslinking, relative to the 9
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Journal of Biomedical Materials Research: Part A
initial free amine content. Uncrosslinked amines groups were assayed according to the reported method. 23 In brief, 5.5 mg of freeze-dried gelatin hydrogel was mixed with 0.5 ml of 4% NaHCO3 and 0.5 ml of 0.5% TNBS, and heated at 40 °C for 4 h. 1.5 ml of 6M HCl was added and the mixture was autoclaved for 1 h at 120 °C. The hydrolysate was diluted with 2.5 ml of water, then extracted with ethyl ether. Aqueous phase was heated for 15 min in a hot water bath, cooled to room temperature, and diluted again with 7.5 ml of water. The absorbance was measured at 346 nm against a blank. Uncrosslinked gelatin hydrogel without PEG was used as control to determine the initial free amine content.
Statistical analysis Data were presented as the means ± standard deviation (SD). Plackett-Burman and Box-Behnken design were performed and statistically analyzed by minitab and Design Expert software, respectively. One-way analysis of variance (ANOVA) was performed for multiple comparisons. Differences were considered significant for *p F
R2
Adj R2
Adeq. Prec.
C.V. (%)
29.43
< 0.0001
0.9742
0.9411
17.183
25.72
Adj. R2, adjusted R2; Adeq Prec., Adequate precision; C.V., coefficient of variation. Significant influence (p< 0.05).
27
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Journal of Biomedical Materials Research: Part A
TABLE Ⅳ. Summary of Variance Analysis for Regression Equation Factor
Y F-Value
p-Value
X1
3.73
0.0949
X2
168.14
1,2
Chang Liu,
1,4
Shijie Lv,
3
Dongsheng Sun,
1
Qinglong Qiao,
1
Rui
Zhang, 5 Yang Liu, 1 Jing Xiao, 2 Guangwei Sun, 1 1
Scientific Research Center for Translational Medicine, Dalian Institute of Chemical
Physics, Chinese Academy of Sciences, Dalian, 116023, China 2
Department of Oral Pathology, College of Stomatology, Dalian Medical University,
Dalian, 116044, China 3
Dalian Maternity & Child Healthcare Hospital, Dalian, 116033, China
4
Dalian Municipal Central Hospital, Dalian, 116033, China
5
Department of Stomatology, First Affiliated Hospital, Dalian Medical University,
Dalian, 116023, China
E-mail addresses: [email protected] (Nan Zhou), [email protected] (Chang Liu), [email protected] (Shijie Lv), [email protected] (Dongsheng Sun), [email protected] (Qinglong Qiao), [email protected] (Rui Zhang), [email protected]
(Yang
Liu),
[email protected]
(Jing
Xiao),
[email protected] (Guangwei Sun)
This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process which may lead to differences between this version and the Version of Record. Please cite this article as an ‘Accepted Article’, doi: 10.1002/jbm.a.35847 This article is protected by copyright. All rights reserved.
Journal of Biomedical Materials Research: Part A
Correspondence to: Y. Liu; e-mail: [email protected] and G. Sun; e-mail: [email protected]
*Corresponding author: Dr. Yang Liu. Dalian Institute of Chemical Physics, Chinese Academy of Sciences, 457 Zhongshan Road, Dalian 116023, China. Tel. /fax: +86411-82463027. E-mail address: [email protected]
*Corresponding author: Dr. Guangwei Sun. Dalian Institute of Chemical Physics, Chinese Academy of Sciences, 457 Zhongshan Road, Dalian 116023, China. Tel. /fax: +86-411-82463027. E-mail address: [email protected]
2
John Wiley & Sons, Inc. This article is protected by copyright. All rights reserved.
Page 2 of 38
Page 3 of 38
Journal of Biomedical Materials Research: Part A
Abstract Gelatin hydrogel has great potential in regenerative medicine. The degradation of gelatin hydrogel is important to control the release profile of encapsulated biomolecules and regulate in vivo tissue repair process. As a plasticizer, PEG can significantly improve the mechanical property of gelatin hydrogel. However, how preparation parameters affect the degradation rate of gelatin-PEG composite hydrogel is still not clear. In this study, the significant effect factor, glutaraldehyde (GA) concentration, was confirmed by means of Plackett-Burman method. Then a mathematical model was built to predict the degradation rate of composite hydrogels under different preparation conditions using the response surface method (RSM), which was helpful to prepare the certain composite hydrogel with desired degradation rate. In addition, it was found that gelatin-PEG composite hydrogel surface well supported the adhension and growth of human mesenchymal stem cells (MSCs). Moreover, PEG concentration not only could adjust hydrogel degradation more subtly, but also might increase the cross-linking degree and affect the cell migration. Therefore, these results would be useful to optimize the preparation of gelatin-PEG composite hydrogel for drug delivery or tissue engineering. Key words: gelatin-PEG hydrogel, degradation model, response surface methodology, stem cells 3
John Wiley & Sons, Inc. This article is protected by copyright. All rights reserved.
Journal of Biomedical Materials Research: Part A
INTRODUCTION Gelatin is derived from collagen by the partial hydrolysis, and it is a useful natural polymer that has great potential in pharmaceutical and medical applications due to its biodegradability and biocompatibility.1,2 Gelatin can be covalently cross-linked by cross-linking agents, such as glutaraldehyde (GA) and carbodiimide, to form gelatin hydrogel with better stability.2-4 The covalently cross-linked gelatin hydrogel has a three-dimensional network structure and can be used as a carrier for the controlled release of bioactive molecules, including growth factors and drugs,5-10 by the polyion complexation.2 In addition, gelatin hydrogel is also used as a biodegradable cell delivery matrice for cell transplantation, which can accelerate in vivo tissue repair.1113
For gelatin hydrogel, the degradation rate is very important, because it not only
controls the release profile of encapsulated biomolecules, but also regulates in vivo tissue repair process.2 It is known that plasticizers can improve the physicochemical properties of biomaterial scaffolds. Polyethylene glycol (PEG), one of widespread used plasticizers, has good biocompatibility as well as low cytotoxicity and immunogenicity, which makes it widely used as the modifier of biomaterial scaffolds.10,14-15 It is reported that PEG can interact with gelatin molecule by the strong inter/intra-molecular hydrogen bond,10 which exhibits better plasticizing influence on the mechanical property of 4
John Wiley & Sons, Inc. This article is protected by copyright. All rights reserved.
Page 4 of 38
Page 5 of 38
Journal of Biomedical Materials Research: Part A
gelatin scaffolds.16 However, how preparation parameters affect the degradation rate of gelatin-PEG composite hydrogel is still not clear. In this work, we prepared gelatin-PEG composite hydrogel, and ascertained the significant effect factor that affected the degradation rate of composite hydrogel by means of Plackett-Burman method. And then a mathematical model was built to predict the degradation rate of composite hydrogel using the response surface method (RSM). This model would be helpful to prepare the certain composite hydrogel with desired degradation rate. In addition, gelatin-PEG composite hydrogel carrying stem cells might be helpful to repair damaged tissues, such as periodontal tissue regeneration. So the adhension and growth of human umbilical cord Wharton's jellyderived mesenchymal stem cells (WJ MSCs) on the surface of composite hydrogel was also evaluated compared to the conventional gelatin hydrogel without PEG. This study would be benefit to optimize the preparation of gelatin-PEG composite hydrogel for drug delivery or tissue engineering.
MATERIALS AND METHODS
Materials
5
John Wiley & Sons, Inc. This article is protected by copyright. All rights reserved.
Journal of Biomedical Materials Research: Part A
Gelatin (type B, Gel Strength = 225 g Bloom), glycine, trypsin, calcein AM and ethidiumhomodimer-1 (ED-1) were purchased from Sigma (St. Louis, MO, USA). Collagenase I was purchased from Gibco (BRL Co. Ltd., Eugene, OR, USA). Glutaraldehyde (GA) and PEG (molecular weight 6 kDa) was procured from Damao Chemical Reagent Factory (Tianjin, China) and Sinopharm Chemical Reagent Co. Ltd. (Shanghai, China),
respectively. Trinitro-benzene-sulfonic acid (TNBS) was
purchased from Ark Pharm (Libertyville, IL, USA). Preparation of gelatin-PEG composite hydrogels Gelatin-PEG composite hydrogels were prepared according to the procedures reported previously with some modification.10,17,18 In brief, gelatin and PEG were dissolved in water, respectively. And these two solutions were mixed together. Then GA was added into the mixed solution to generate gelatin-PEG composite hydrogels via the cross-linking at 4 °C for 18 h. After that, the hydrogels were immersed into 100 mM glycine aqueous solution for 1 h at 37 °C to block the residual aldehyde groups of GA, and then they were washed three times with double-distilled water. The final concentration ranges of gelatin, PEG and GA inside hydrogels were 3-9 wt %, 03 wt % and 0.1-0.3 wt %. In vitro enzymatic degradation of composite hydrogels
6
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Page 6 of 38
Page 7 of 38
Journal of Biomedical Materials Research: Part A
Gelatin-PEG hydrogels were immersed into PBS containing 15 µg/ml collagenase I at 37 °C for 24 h.7 And they were washed 3 times with deionized water and dried to constant weight in a vacuum oven (DZF 6050, Longyue Instrument Equipment Co., LTD., shanghai, China) at 35 °C. Then the dry weight of dried hydrogels were measured accurately. The degradation rate (Y) of composite hydrogels was calculated using the following equation: Y = (W1 − W2 ) /W1
(1)
W1, dry weight of hydrogels before degradation; W2, dry weight of hydrogels after degradation.
Plackett-Burman screening and response surface methodology Firstly, significant factor that influenced the degradation of gelatin-PEG composite hydrogel was screened using Plackett-Burman method.19 Gelatin concentration (X1), GA concentration (X2) and PEG concentration (X3) were chosen as three effect factors, and the degradation rate of gelatin-PEG composite hydrogel after enzymatic degradation for 24 h as the response value (Y). Table Ⅰ showed the design of Plackett-Burman screening experiment. Then RSM was used to investigate the effect of hydrogel formulation variables (X1, X2, X3) on the dependent value (Y). Box-Behnken design consisting of 17 experiments was employed for RSM (Table Ⅱ).19 A mathematical model of the degradation rate of 7
John Wiley & Sons, Inc. This article is protected by copyright. All rights reserved.
Journal of Biomedical Materials Research: Part A
gelatin-PEG composite hydrogel was built according to the experiment results. ANOVA was used to evaluate the mathematical model. In addition, three random checkpoints were used to check the reliability of the mathematical model. The degradation rates obtained from experiments were compared with the responses calculated by the mathematical model.
WJ MSC culture on the hydrogel surface Primary WJ MSCs were donated by Zhongyuan Union Stem Cell Bioengineering Corporation (Tianjin, China). WJ MSCs were cultured in the special culture medium that was also kindly gifted by this company. The medium was changed every three days and cells were subcultured when reaching 90% confluence.20 WJ MSCs (Passage 5-7) were cultured on the surface of gelatin-PEG composite hydrogels with different formulation (104 cells/cm2). After incubating for 24 h, nonadherent cells were removed by washing with culture medium. The morphology of adherent cells was observed under a microscope (Eclipse TE2000-U, Nikon, Tokyo, Japan). Then the adherent cells were harvested using trypsin and the cell numbers on different hydrogel surfaces were statistically analyzed.
Live/Dead staining Live/Dead staining of adherent cells on different hydrogel surfaces was performed on 24 h using the previously reported method with some modification.21 Cells were 8
John Wiley & Sons, Inc. This article is protected by copyright. All rights reserved.
Page 8 of 38
Page 9 of 38
Journal of Biomedical Materials Research: Part A
incubated with live/dead staining working solution composed of 2 µM calcein AM and 4 µM ED-1 at 37 °C for 30 min. Then cells were observed with confocal laser scanning microscopy (CLSM, LeicaSP2, Heidelberger, Germany). Cell distribution on the hydrogel surface or inside hydrogels on Day 6 was observed by living cell station (Revolution WD, Andor Technology, Belfast, Northern Ireland) using Live/Dead staining.
F-actin staining F-actin staining of adherent cells on different hydrogel surfaces was performed at 24 h using the previously reported method.22 Cells were fixed with 4 wt % paraformaldehyde in phosphate-buffered saline (PBS, pH 7.2) for 30 min and rinsed with PBS. The samples were treated with Alexa Flour® 488 phalloidin (50 µg/ml) and incubated overnight at 4 °C. Then samples were rinsed with PBS and observed using the CLSM.
Microstructure analysis Gelatin hydrogels were freeze-dried and then the microstructures of cross-sections were observed by SEM (JSM-7800F, JEOL, Akishima, Tokyo, Japan).
Cross-linking degree determination The cross-linking degree was evaluated as the difference between the chemicallydetermined number of free amines groups before and after crosslinking, relative to the 9
John Wiley & Sons, Inc. This article is protected by copyright. All rights reserved.
Journal of Biomedical Materials Research: Part A
initial free amine content. Uncrosslinked amines groups were assayed according to the reported method. 23 In brief, 5.5 mg of freeze-dried gelatin hydrogel was mixed with 0.5 ml of 4% NaHCO3 and 0.5 ml of 0.5% TNBS, and heated at 40 °C for 4 h. 1.5 ml of 6M HCl was added and the mixture was autoclaved for 1 h at 120 °C. The hydrolysate was diluted with 2.5 ml of water, then extracted with ethyl ether. Aqueous phase was heated for 15 min in a hot water bath, cooled to room temperature, and diluted again with 7.5 ml of water. The absorbance was measured at 346 nm against a blank. Uncrosslinked gelatin hydrogel without PEG was used as control to determine the initial free amine content.
Statistical analysis Data were presented as the means ± standard deviation (SD). Plackett-Burman and Box-Behnken design were performed and statistically analyzed by minitab and Design Expert software, respectively. One-way analysis of variance (ANOVA) was performed for multiple comparisons. Differences were considered significant for *p F
R2
Adj R2
Adeq. Prec.
C.V. (%)
29.43
< 0.0001
0.9742
0.9411
17.183
25.72
Adj. R2, adjusted R2; Adeq Prec., Adequate precision; C.V., coefficient of variation. Significant influence (p< 0.05).
27
John Wiley & Sons, Inc. This article is protected by copyright. All rights reserved.
Journal of Biomedical Materials Research: Part A
TABLE Ⅳ. Summary of Variance Analysis for Regression Equation Factor
Y F-Value
p-Value
X1
3.73
0.0949
X2
168.14
