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In-vitro and in-vivo evaluation of hemostatic potential of decellularized ECM hydrogels
Accepted Manuscript In-vitro and in-vivo evaluation of hemostatic potential of decellularized ECM hydrogels Reiza D. Ventura, Andrew R. Padalhin, Byong Taek Lee PII: DOI: Reference:
S0167-577X(18)31203-5 https://doi.org/10.1016/j.matlet.2018.08.013 MLBLUE 24724
To appear in:
Materials Letters
Received Date: Revised Date: Accepted Date:
31 May 2018 4 July 2018 5 August 2018
Please cite this article as: R.D. Ventura, A.R. Padalhin, B.T. Lee, In-vitro and in-vivo evaluation of hemostatic potential of decellularized ECM hydrogels, Materials Letters (2018), doi: https://doi.org/10.1016/j.matlet. 2018.08.013
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
In-vitro and in-vivo evaluation of hemostatic potential of decellularized ECM hydrogels Reiza D. Ventura1, Andrew R. Padalhin2 and Byong Taek Lee1,2 1
Department of Regenerative Medicine, College of Medicine, Soonchunhyang University, Cheonan, South Korea 2 Institute of Tissue Regeneration, College of Medicine, Soonchunhyang University, Cheonan, South Korea Soonchunhyang University 366-1, Ssangyoung-dong, Cheonan City, Chungnam 330-090, Korea Tel.: +82-41-570-2427, Fax: +82-41-577-2415 [email protected] (Byong-Taek Lee)
Abstract
Extracellular matrix (ECM) hydrogels with a porous microstructure and good swelling behavior were fabricated via lyophilization and cross-linked with 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide (EDC). Platelet adhesion studies revealed that platelets easily adhered to hydrogels while coagulation studies showed rapid blood aggregate formation. In-vitro cytotoxicity studies showed ECM hydrogels are biocompatible. In-vivo studies using rat tail bleeding and rabbit arterial injury showed ECM hydrogels' ability in hemostasis. ECM hydrogels formed a physical barrier and activated platelets that resulted in rapid thrombin formation during in-vivo application. The present study revealed ECM hydrogels could be a potential biomaterial for hemostat application.
Keywords: Extracellular matrix, hydrogel, hemostat, porcine dermal
Introduction Traumatic injuries during combat are the most common cause of preventable death in civilian trauma centers [1]. Limited time to control a life-threatening hemorrhage, delayed access, transportation to care centers and severity of wound are the challenges of wound management in battlefield [2]. Thus, there is need for an inexpensive, easy and safe to use hemostatic agents and dressings that are capable to stop severe bleeding and to increase the survival rate of injured victims [3]. Extracellular matrix (ECM) is an exellent biomaterial due to its inherent physical and chemical properties. It is a complex structure that composes of collagen, glycoproteins and glycosaminoglycan [4]. ECM affects various cellular functions and intracellular signaling activated by cell adhesion molecules. When processed properly with the aid of decellularization, ECM can be fabricated into different forms and using and can be applied in different tissue engineering such as cell delivery, soft and hard tissue regeneration [5-12]. The aim of this study is to evaluate the hemostatic property of ECM hydrogel. Decellularized hydrogels with different concentrations were fabricated using lyophilization method and crosslinked using 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide (EDC) solution. Materials and Methods Decellularization of porcine skin The porcine skin was cut into small pieces, exposed to trypsin solution and decellularized using sodium dodecyl sulfate followed by Triton X-100. Samples were washed with distilled water and lyophilized, ground and stored in -20°C until further use. Evaluation of Decellularization Efficiency
To determine the decellularization efficiency, DNA content was quantified before and after decellularization. DNA content of native and decellularized ECM samples was extracted and analyzed using a nanophotometer (Implen GmbH). Collagen content was analyzed using a Sirius Red Total Detection kit (Chondrex, USA) based on manufacturer’s protocol. Sulfated glycosaminoglycan content was quantified using Blyscan glycosaminoglycan kit (Biocolor, UK) based on the manufacturer’s protocol. Elastin content was quantified using Blyscan Elastin kit (Biocolor, UK) based on the manufacturer’s protocol. Fabrication of ECM hydrogel Decellularized ECM powder was solubilized in pepsin buffer for 48 h to obtain a homogenous solution. pH of the homogenized solution was neutralized using 1M NaOH. Homogenized and neutralized ECM solution was diluted using PBS to obtain 10% (HECM10), 15% (HECM15) and 20% (HECM20) concentration (w/v). ECM solution was placed in a plastic mould (5mm by 5 mm) and cross-linked with EDC/NHS (50mmol:20mmol) solution for 24 h. Samples were then freezed and lyophilized in freeze dryer for 48 hours to remove the ice formed within the hydrogel resulting to a porous structure. Characterization of ECM hydrogels Crosslinked hydrogels were analyzed by Fourier transform infrared (FTIR) analysis with uncrosslinked hydrogel as control. Morphology was evaluated using scanning electron microscope and porosity and pore size distribution was analyzed using Image J based on SEM images. For swelling rate, dried samples were weighed (Wi) and incubated in PBS at 37°C at different time points. Final weight (Wf) were recorded after hydrogel blotted with filter paper to remove excess water after specified time. Swelling rate (%) were calculated as: |Wi-Wf|/Wi x 100.
For degradation study, initial dry weight (Wi) of the hydrogels were obtained and incubated in PBS solution at 37°C. Final weight (Wf) of lyophilized hydrogels (1, 3, 5 and 7 days) were recorded. Degradation (%) was calculated as: |Wi-Wf|/Wi x 100. Hemostatic properties of ECM hydrogel For platelet adhesion, platelet-rich plasma (PRP) was added to samples and incubated at different time points (0, 2.5, 5 and 10 mins). Samples were wash with PBS to remove nonadherent PRP and fixed with 4% paraformaldehyde dried prior to SEM observation. For blood coagulation studies, ACD blood was added to cover the samples, 0.2M CaCl2 was added and incubated at 37°C for 0, 2.5, 5 and 10 min. DI water was added and centrifuge. The supernatant was decanted in a new tube and DI water was added. For control, 0.5 ml ACD whole blood was transferred in a tube with DI water. The absorbance of the solutions were read at 540 nm using Elisa plate reader. Blood clotting index were computed as: BCI (%)= (Absorbancesample/Absorbancecontrol)x100 In-vitro study Cytotoxicity studies were carried out based on ISO 10993-5 protocol with modification. Hydrogel extracts were prepared by incubating the HECM hydrogels (10% w/v) in media for 1 and 7 days at 37°C with 5% CO2. L929 cells were seeded into 24-well cell culture plate at a concentration of 6x104 cells per well and incubated for 1 day in a humidified incubator. After one day, culture media was replaced with 100% hydrogel extracts. Adherent L929 cells viability was assessed by MTT assay based on manufacuturer’s protocol. In-vivo Studies Two different in-vivo model was used determine the hemostatic ability of HECM hydrogels. Animals (Samtaco, Korea) were housed in a temperature controlled environment. Food and
water were supplied according to the guidelines of the Animal Care Center at Soonchunhyang University, Chungcheongnam-do, South Korea. 12 Sprague Dawley rats were divided into three groups: control, HECM15, and HECM20. The tail was cut at 50% length using surgical scissors. HECM hydrogels were applied with minimal pressure. Hemostatic time was recorded until no blood came out from the tail. For control, the cut was not treated and allowed bleeding until the bleeding stopped. For leg arterial bleeding, 12 New Zealand white rabbits rabbits were divided into three groups. Leg artery was cut using a surgical blade, allowed bleeding for 1 min and applied ECM hydrogels while for the control, blood was allowed to flow until stopped. Hemostatic time was recorded with triplicates conducted for each group. Results and Discussion DNA quantification result showed significantly removal of DNA content in decellularized ECM compared with the native ECM. Collagen, elastin and sGAG content of decellularized ECM were preserved after decellularization (Figure 1). SEM micrographs showed porous with pore interconnectivity in uncross-linked ECM hydrogels. After crosslinking with EDC/NHS solution, HECM hydrogels have enhanced porosity and pore interconnectivity with increased surface area of pore walls as ECM concentration increased (Figure 2). Hydrogels had a swelling rate of 93-95% after 3 hours exposure to PBS. It absorbed PBS readily without drastic changes in dimension. HECM10 degraded faster while no significant difference in degradation rate was observed in HECM15 and HECM20 after 7 days in PBS. Uncross-linked HECM hydrogels were too weak and easily disintegrated upon exposure to DI water making it unsuitable as a hemostat agent (Figure 3).
Hemostatic Properties Platelets easily adhered in all of the HECM hydrogels after 10 minutes with more platelets in HECM20. Increased ECM concentrations resulted to increased platelet adhesion which may be due to higher amount of proteins that resulted in efficient platelet adhesion. Blood Cotting Index (BCI) determined the hemostatic capacity of HECM hydrogels. It was used to measure the uncoagulated blood in the solution after sample exposure. HECM20 has the lowest BCI values which indicated highest hemostatic properties among the samples (Figure 4). In- vitro Studies Cytotoxicity studies revealed that hydrogel extracts did not contain toxic properties upon exposure to L929 cells after 24 hours. Results indicated that all HECM hydrogels are biocompatible (Figure 5A). In-vivo studies HECM10 was excluded in in-vivo study because it was too weak based on the degradation study. Hemostatic bleeding time of HECM15 and HECM20 were not significantly different but significantly different from the control in both bleeding models. Blood absorption of HECM15 was better even with the presence of blood aggregate in contrast with HECM20. This may be due to HECM15 higher %porosity with larger pore size compared with HECM20. HECM20 contained higher ECM proteins that rapidly activated platelets and formed blood aggregates that harder to absorbed by smaller pore size of HECM20 (Figure 5B-D). HECM hydrogel could be a better alternative for gelatin hydrogel because it contain large amount of collagen that aid in indirect thrombin formation, responsible in complete activation of platelets and highly absorbable [13, 14]. In contrast, gelatin hydrogels traps the platelet within
the
pores and form an insoluble fibrin clot that controls the bleeding [15]. Also, gelatin
hydrogels are brittle at a concentration greater than 5% and have low absorption potential [16]. Conclusion HECM hydrogels cross-linked with EDC/NHS solution had porous structure with interconnected porosity with a good swelling and degradation rate. Platelet adhesion were enhanced and hemostatic properties were increased as the concentration of ECM in hydrogel increased. HECM hydrogels in non-lethal and lethal bleeding significantly decreased the bleeding time compared with control. ECM hydrogels could serve a potential and promising biomaterial for hemostat application. Acknowledgement This study was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT and future planning (No.2015R1A2A1A10053272) and partially supported by Soonchunhyang University. Reference 1.
2. 3.
4.
5. 6.
Sambasivan, C.N., et al., A highly porous silica and chitosan-based hemostatic dressing is superior in controlling hemorrhage in a severe groin injury model in swine. The American Journal of Surgery, 2009. 197(5): p. 576-580. Granville-Chapman, J., N. Jacobs, and M. Midwinter, Pre-hospital haemostatic dressings: a systematic review. Injury, 2011. 42(5): p. 447-459. Ward, K.R., et al., Comparison of a new hemostatic agent to current combat hemostatic agents in a swine model of lethal extremity arterial hemorrhage. Journal of Trauma and Acute Care Surgery, 2007. 63(2): p. 276-284. Kim, E.J., et al., Injectable and thermosensitive soluble extracellular matrix and methylcellulose hydrogels for stem cell delivery in skin wounds. Biomacromolecules, 2015. 17(1): p. 4-11. Badylak, S., T. Gilbert, and J. Myers-Irvin, The extracellular matrix as a biologic scaffold for tissue engineering. Science, 1993. 260: p. 920-6. Wolf, M.T., et al., A hydrogel derived from decellularized dermal extracellular matrix. Biomaterials, 2012. 33(29): p. 7028-38.
7. 8.
9. 10.
11. 12. 13. 14. 15.
16.
Ventura, R.D., et al., Bone Regeneration Using Hydroxyapatite Sponge Scaffolds with In Vivo Deposited Extracellular Matrix. Tissue Eng Part A, 2015. 21(21-22): p. 2649-61. Hoshiba, T., et al., Decellularized extracellular matrix as an in vitro model to study the comprehensive roles of the ECM in stem cell differentiation. Stem cells international, 2016. 2016. Wolf, M.T., et al., A hydrogel derived from decellularized dermal extracellular matrix. Biomaterials, 2012. 33(29): p. 7028-7038. Damien, E., et al., A preliminary study on the enhancement of the osteointegration of a novel synthetic hydroxyapatite scaffold in vivo. Journal of Biomedical Materials Research Part A, 2003. 66(2): p. 241-246. Rozario, T. and D.W. DeSimone, The extracellular matrix in development and morphogenesis: a dynamic view. Developmental biology, 2010. 341(1): p. 126-140. Bonnans, C., J. Chou, and Z. Werb, Remodelling the extracellular matrix in development and disease. Nature reviews Molecular cell biology, 2014. 15(12): p. 786. Bergmeier, W. and R.O. Hynes, Extracellular matrix proteins in hemostasis and thrombosis. Cold Spring Harb Perspect Biol, 2012. 4(2). Farndale, R.W., et al., The role of collagen in thrombosis and hemostasis. J Thromb Haemost, 2004. 2(4): p. 561-73. Kobatake, K., K. Mita, and M. Kato, Effect on hemostasis of an absorbable hemostatic gelatin sponge after transrectal prostate needle biopsy. International braz j urol, 2015. 41(2): p. 337-343. Wu, X., et al., Preparation of aligned porous gelatin scaffolds by unidirectional freezedrying method. Acta Biomater, 2010. 6(3): p. 1167-77.
Figures
Figure 1. Determination of decellularization efficiency. DNA quantification (A) of native and decellularized ECM showing significant decreased of cellular components after decellularization. Collagen, Elastin and sGAG contents in decellularized ECM were preserved after decellularization (B).
Figure 2. Macro photographs of HECM10, HECM15 and HECM20 with corresponding SEM micrographs (A) and FTIR spectra of uncrosslinked and crosslinked HECM hydrogels (B).
Figure 3. Porosity (A), pore size (B) was evaluated using Image J. Swelling rate (C) and degradation studies (D) of HECM10, HECM15 and HECM20 after 3 hours and 7 days respectively.
Figure 4. Evaluation of Hemostatic properties of HECM hydrogels. Platelet adhesion (A) and Blood clotting studies (B) after 10 minutes of exposure to citrated blood.
Figure 5. Cytotoxicity studies of L929 cells after exposure to 1 and 7 days hydrogel extract for 24 hours (A). Significant reduction in HECM hydrogels’ hemostatic time in rat tail bleeding (A) and rabbit artery bleeding (B) were observed compared with the control. In vivo pictures of HECM15 and HECM20 applied to rat tail bleeding and rabbit artery bleeding (D).
HIGHLIGHTS
HECM hydrogels are biocompatible and do not contained toxic byproducts Increased platelet adhesion as ECM concentration in hydrogels increased Hemostatic properties increased as ECM concentration in hydrogels increased ECM proteins were attributed to good hemostatic properties of HECM hydrogels HECM hydrogels decreased the bleeding time when applied in-vivo
S0167-577X(18)31203-5 https://doi.org/10.1016/j.matlet.2018.08.013 MLBLUE 24724
To appear in:
Materials Letters
Received Date: Revised Date: Accepted Date:
31 May 2018 4 July 2018 5 August 2018
Please cite this article as: R.D. Ventura, A.R. Padalhin, B.T. Lee, In-vitro and in-vivo evaluation of hemostatic potential of decellularized ECM hydrogels, Materials Letters (2018), doi: https://doi.org/10.1016/j.matlet. 2018.08.013
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
In-vitro and in-vivo evaluation of hemostatic potential of decellularized ECM hydrogels Reiza D. Ventura1, Andrew R. Padalhin2 and Byong Taek Lee1,2 1
Department of Regenerative Medicine, College of Medicine, Soonchunhyang University, Cheonan, South Korea 2 Institute of Tissue Regeneration, College of Medicine, Soonchunhyang University, Cheonan, South Korea Soonchunhyang University 366-1, Ssangyoung-dong, Cheonan City, Chungnam 330-090, Korea Tel.: +82-41-570-2427, Fax: +82-41-577-2415 [email protected] (Byong-Taek Lee)
Abstract
Extracellular matrix (ECM) hydrogels with a porous microstructure and good swelling behavior were fabricated via lyophilization and cross-linked with 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide (EDC). Platelet adhesion studies revealed that platelets easily adhered to hydrogels while coagulation studies showed rapid blood aggregate formation. In-vitro cytotoxicity studies showed ECM hydrogels are biocompatible. In-vivo studies using rat tail bleeding and rabbit arterial injury showed ECM hydrogels' ability in hemostasis. ECM hydrogels formed a physical barrier and activated platelets that resulted in rapid thrombin formation during in-vivo application. The present study revealed ECM hydrogels could be a potential biomaterial for hemostat application.
Keywords: Extracellular matrix, hydrogel, hemostat, porcine dermal
Introduction Traumatic injuries during combat are the most common cause of preventable death in civilian trauma centers [1]. Limited time to control a life-threatening hemorrhage, delayed access, transportation to care centers and severity of wound are the challenges of wound management in battlefield [2]. Thus, there is need for an inexpensive, easy and safe to use hemostatic agents and dressings that are capable to stop severe bleeding and to increase the survival rate of injured victims [3]. Extracellular matrix (ECM) is an exellent biomaterial due to its inherent physical and chemical properties. It is a complex structure that composes of collagen, glycoproteins and glycosaminoglycan [4]. ECM affects various cellular functions and intracellular signaling activated by cell adhesion molecules. When processed properly with the aid of decellularization, ECM can be fabricated into different forms and using and can be applied in different tissue engineering such as cell delivery, soft and hard tissue regeneration [5-12]. The aim of this study is to evaluate the hemostatic property of ECM hydrogel. Decellularized hydrogels with different concentrations were fabricated using lyophilization method and crosslinked using 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide (EDC) solution. Materials and Methods Decellularization of porcine skin The porcine skin was cut into small pieces, exposed to trypsin solution and decellularized using sodium dodecyl sulfate followed by Triton X-100. Samples were washed with distilled water and lyophilized, ground and stored in -20°C until further use. Evaluation of Decellularization Efficiency
To determine the decellularization efficiency, DNA content was quantified before and after decellularization. DNA content of native and decellularized ECM samples was extracted and analyzed using a nanophotometer (Implen GmbH). Collagen content was analyzed using a Sirius Red Total Detection kit (Chondrex, USA) based on manufacturer’s protocol. Sulfated glycosaminoglycan content was quantified using Blyscan glycosaminoglycan kit (Biocolor, UK) based on the manufacturer’s protocol. Elastin content was quantified using Blyscan Elastin kit (Biocolor, UK) based on the manufacturer’s protocol. Fabrication of ECM hydrogel Decellularized ECM powder was solubilized in pepsin buffer for 48 h to obtain a homogenous solution. pH of the homogenized solution was neutralized using 1M NaOH. Homogenized and neutralized ECM solution was diluted using PBS to obtain 10% (HECM10), 15% (HECM15) and 20% (HECM20) concentration (w/v). ECM solution was placed in a plastic mould (5mm by 5 mm) and cross-linked with EDC/NHS (50mmol:20mmol) solution for 24 h. Samples were then freezed and lyophilized in freeze dryer for 48 hours to remove the ice formed within the hydrogel resulting to a porous structure. Characterization of ECM hydrogels Crosslinked hydrogels were analyzed by Fourier transform infrared (FTIR) analysis with uncrosslinked hydrogel as control. Morphology was evaluated using scanning electron microscope and porosity and pore size distribution was analyzed using Image J based on SEM images. For swelling rate, dried samples were weighed (Wi) and incubated in PBS at 37°C at different time points. Final weight (Wf) were recorded after hydrogel blotted with filter paper to remove excess water after specified time. Swelling rate (%) were calculated as: |Wi-Wf|/Wi x 100.
For degradation study, initial dry weight (Wi) of the hydrogels were obtained and incubated in PBS solution at 37°C. Final weight (Wf) of lyophilized hydrogels (1, 3, 5 and 7 days) were recorded. Degradation (%) was calculated as: |Wi-Wf|/Wi x 100. Hemostatic properties of ECM hydrogel For platelet adhesion, platelet-rich plasma (PRP) was added to samples and incubated at different time points (0, 2.5, 5 and 10 mins). Samples were wash with PBS to remove nonadherent PRP and fixed with 4% paraformaldehyde dried prior to SEM observation. For blood coagulation studies, ACD blood was added to cover the samples, 0.2M CaCl2 was added and incubated at 37°C for 0, 2.5, 5 and 10 min. DI water was added and centrifuge. The supernatant was decanted in a new tube and DI water was added. For control, 0.5 ml ACD whole blood was transferred in a tube with DI water. The absorbance of the solutions were read at 540 nm using Elisa plate reader. Blood clotting index were computed as: BCI (%)= (Absorbancesample/Absorbancecontrol)x100 In-vitro study Cytotoxicity studies were carried out based on ISO 10993-5 protocol with modification. Hydrogel extracts were prepared by incubating the HECM hydrogels (10% w/v) in media for 1 and 7 days at 37°C with 5% CO2. L929 cells were seeded into 24-well cell culture plate at a concentration of 6x104 cells per well and incubated for 1 day in a humidified incubator. After one day, culture media was replaced with 100% hydrogel extracts. Adherent L929 cells viability was assessed by MTT assay based on manufacuturer’s protocol. In-vivo Studies Two different in-vivo model was used determine the hemostatic ability of HECM hydrogels. Animals (Samtaco, Korea) were housed in a temperature controlled environment. Food and
water were supplied according to the guidelines of the Animal Care Center at Soonchunhyang University, Chungcheongnam-do, South Korea. 12 Sprague Dawley rats were divided into three groups: control, HECM15, and HECM20. The tail was cut at 50% length using surgical scissors. HECM hydrogels were applied with minimal pressure. Hemostatic time was recorded until no blood came out from the tail. For control, the cut was not treated and allowed bleeding until the bleeding stopped. For leg arterial bleeding, 12 New Zealand white rabbits rabbits were divided into three groups. Leg artery was cut using a surgical blade, allowed bleeding for 1 min and applied ECM hydrogels while for the control, blood was allowed to flow until stopped. Hemostatic time was recorded with triplicates conducted for each group. Results and Discussion DNA quantification result showed significantly removal of DNA content in decellularized ECM compared with the native ECM. Collagen, elastin and sGAG content of decellularized ECM were preserved after decellularization (Figure 1). SEM micrographs showed porous with pore interconnectivity in uncross-linked ECM hydrogels. After crosslinking with EDC/NHS solution, HECM hydrogels have enhanced porosity and pore interconnectivity with increased surface area of pore walls as ECM concentration increased (Figure 2). Hydrogels had a swelling rate of 93-95% after 3 hours exposure to PBS. It absorbed PBS readily without drastic changes in dimension. HECM10 degraded faster while no significant difference in degradation rate was observed in HECM15 and HECM20 after 7 days in PBS. Uncross-linked HECM hydrogels were too weak and easily disintegrated upon exposure to DI water making it unsuitable as a hemostat agent (Figure 3).
Hemostatic Properties Platelets easily adhered in all of the HECM hydrogels after 10 minutes with more platelets in HECM20. Increased ECM concentrations resulted to increased platelet adhesion which may be due to higher amount of proteins that resulted in efficient platelet adhesion. Blood Cotting Index (BCI) determined the hemostatic capacity of HECM hydrogels. It was used to measure the uncoagulated blood in the solution after sample exposure. HECM20 has the lowest BCI values which indicated highest hemostatic properties among the samples (Figure 4). In- vitro Studies Cytotoxicity studies revealed that hydrogel extracts did not contain toxic properties upon exposure to L929 cells after 24 hours. Results indicated that all HECM hydrogels are biocompatible (Figure 5A). In-vivo studies HECM10 was excluded in in-vivo study because it was too weak based on the degradation study. Hemostatic bleeding time of HECM15 and HECM20 were not significantly different but significantly different from the control in both bleeding models. Blood absorption of HECM15 was better even with the presence of blood aggregate in contrast with HECM20. This may be due to HECM15 higher %porosity with larger pore size compared with HECM20. HECM20 contained higher ECM proteins that rapidly activated platelets and formed blood aggregates that harder to absorbed by smaller pore size of HECM20 (Figure 5B-D). HECM hydrogel could be a better alternative for gelatin hydrogel because it contain large amount of collagen that aid in indirect thrombin formation, responsible in complete activation of platelets and highly absorbable [13, 14]. In contrast, gelatin hydrogels traps the platelet within
the
pores and form an insoluble fibrin clot that controls the bleeding [15]. Also, gelatin
hydrogels are brittle at a concentration greater than 5% and have low absorption potential [16]. Conclusion HECM hydrogels cross-linked with EDC/NHS solution had porous structure with interconnected porosity with a good swelling and degradation rate. Platelet adhesion were enhanced and hemostatic properties were increased as the concentration of ECM in hydrogel increased. HECM hydrogels in non-lethal and lethal bleeding significantly decreased the bleeding time compared with control. ECM hydrogels could serve a potential and promising biomaterial for hemostat application. Acknowledgement This study was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT and future planning (No.2015R1A2A1A10053272) and partially supported by Soonchunhyang University. Reference 1.
2. 3.
4.
5. 6.
Sambasivan, C.N., et al., A highly porous silica and chitosan-based hemostatic dressing is superior in controlling hemorrhage in a severe groin injury model in swine. The American Journal of Surgery, 2009. 197(5): p. 576-580. Granville-Chapman, J., N. Jacobs, and M. Midwinter, Pre-hospital haemostatic dressings: a systematic review. Injury, 2011. 42(5): p. 447-459. Ward, K.R., et al., Comparison of a new hemostatic agent to current combat hemostatic agents in a swine model of lethal extremity arterial hemorrhage. Journal of Trauma and Acute Care Surgery, 2007. 63(2): p. 276-284. Kim, E.J., et al., Injectable and thermosensitive soluble extracellular matrix and methylcellulose hydrogels for stem cell delivery in skin wounds. Biomacromolecules, 2015. 17(1): p. 4-11. Badylak, S., T. Gilbert, and J. Myers-Irvin, The extracellular matrix as a biologic scaffold for tissue engineering. Science, 1993. 260: p. 920-6. Wolf, M.T., et al., A hydrogel derived from decellularized dermal extracellular matrix. Biomaterials, 2012. 33(29): p. 7028-38.
7. 8.
9. 10.
11. 12. 13. 14. 15.
16.
Ventura, R.D., et al., Bone Regeneration Using Hydroxyapatite Sponge Scaffolds with In Vivo Deposited Extracellular Matrix. Tissue Eng Part A, 2015. 21(21-22): p. 2649-61. Hoshiba, T., et al., Decellularized extracellular matrix as an in vitro model to study the comprehensive roles of the ECM in stem cell differentiation. Stem cells international, 2016. 2016. Wolf, M.T., et al., A hydrogel derived from decellularized dermal extracellular matrix. Biomaterials, 2012. 33(29): p. 7028-7038. Damien, E., et al., A preliminary study on the enhancement of the osteointegration of a novel synthetic hydroxyapatite scaffold in vivo. Journal of Biomedical Materials Research Part A, 2003. 66(2): p. 241-246. Rozario, T. and D.W. DeSimone, The extracellular matrix in development and morphogenesis: a dynamic view. Developmental biology, 2010. 341(1): p. 126-140. Bonnans, C., J. Chou, and Z. Werb, Remodelling the extracellular matrix in development and disease. Nature reviews Molecular cell biology, 2014. 15(12): p. 786. Bergmeier, W. and R.O. Hynes, Extracellular matrix proteins in hemostasis and thrombosis. Cold Spring Harb Perspect Biol, 2012. 4(2). Farndale, R.W., et al., The role of collagen in thrombosis and hemostasis. J Thromb Haemost, 2004. 2(4): p. 561-73. Kobatake, K., K. Mita, and M. Kato, Effect on hemostasis of an absorbable hemostatic gelatin sponge after transrectal prostate needle biopsy. International braz j urol, 2015. 41(2): p. 337-343. Wu, X., et al., Preparation of aligned porous gelatin scaffolds by unidirectional freezedrying method. Acta Biomater, 2010. 6(3): p. 1167-77.
Figures
Figure 1. Determination of decellularization efficiency. DNA quantification (A) of native and decellularized ECM showing significant decreased of cellular components after decellularization. Collagen, Elastin and sGAG contents in decellularized ECM were preserved after decellularization (B).
Figure 2. Macro photographs of HECM10, HECM15 and HECM20 with corresponding SEM micrographs (A) and FTIR spectra of uncrosslinked and crosslinked HECM hydrogels (B).
Figure 3. Porosity (A), pore size (B) was evaluated using Image J. Swelling rate (C) and degradation studies (D) of HECM10, HECM15 and HECM20 after 3 hours and 7 days respectively.
Figure 4. Evaluation of Hemostatic properties of HECM hydrogels. Platelet adhesion (A) and Blood clotting studies (B) after 10 minutes of exposure to citrated blood.
Figure 5. Cytotoxicity studies of L929 cells after exposure to 1 and 7 days hydrogel extract for 24 hours (A). Significant reduction in HECM hydrogels’ hemostatic time in rat tail bleeding (A) and rabbit artery bleeding (B) were observed compared with the control. In vivo pictures of HECM15 and HECM20 applied to rat tail bleeding and rabbit artery bleeding (D).
HIGHLIGHTS
HECM hydrogels are biocompatible and do not contained toxic byproducts Increased platelet adhesion as ECM concentration in hydrogels increased Hemostatic properties increased as ECM concentration in hydrogels increased ECM proteins were attributed to good hemostatic properties of HECM hydrogels HECM hydrogels decreased the bleeding time when applied in-vivo