Abstract
Statement of Purpose: Tissue-engineered heart valves (TEHVs) have the potential to fuse with the native tissue and facilitate growth and remodelling; however, this is dependent on having a suitable scaffold material that can undergo the mechanical strain experienced during the cardiac cycle. Fibrin and collagen show particular promise as TEHV scaffolds due to their biocompatibility. Although weak mechanical properties have limited their use to date, crosslinking treatments can be used to increase the mechanical properties of these collagen-based biomaterials by forming bonds between collagen molecules. Common crosslinking treatments for collagen-glycosaminoglycan (CG) scaffolds include dehydrothermal (DHT) and 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide (EDAC). The overall aim of this study was to investigate the effect of these crosslinking treatments on the mechanical properties of fibrin infused CG scaffolds (FCG) and compare the results to mechanical properties of native valves. Additionally, the effect of CG and FCG scaffold material on human umbilical cord blood endothelial progenitor cell’s (hUCBEPCs) viability and proliferation was investigated.
Methods: CG scaffolds were fabricated using a freeze drying process with a final freezing temperature of -10 ⁰C. A CG suspension was produced with microfibrillar, type 1 collagen in a solution of acetic acid and glycosaminoglycan (GAG) (chondroitin sulphate) at a final suspension of 0.75 wt% collagen and 0.044 wt% chondroitin-6-sulfate. Scaffolds were cross-linked with DHT, EDAC, or DHT/EDAC. After crosslinking, tris-buffered saline, 50 mM CaCl2, 40 IU/ml thrombin and 10 mg/ml of fibrinogen were drop-loaded onto the CG scaffold to fabricate FCG scaffolds (Brougham C. Acta Biomater. 2015; 26:205-214). Uniaxial tensile tests were performed using a 5 N load cell at a strain rate of 10% per minute. The burst strength was measured using a burst pressure rig. EPCs were isolated from human umbilical cord blood and seeded onto CG and FCG scaffold crosslinked with DHT/EDAC for 1-14 days. To observe the response of hUCBEPCs in CG and FCG scaffolds, their metabolic activity was measured using AlamarBlue and cell-mediated contraction was determined by measuring the scaffold dimensional stability.
Results: Tensile testing showed that crosslinking significantly increased the tensile strength and elastic modulus, although EDAC significantly decreased the elongation of FCG scaffolds (Figure 1). Uniaxial tensile testing also showed that the elastic modulus of CG and FCG cross-linked with EDAC (4.6 kPa and 5.5 kPa) and DHT/EDAC (5.0 and 5.4 kPa) was in line with that seen in adolescent pulmonary valve leaflets (4.5 kPa (Geemen DV. PLOS ONE. 2016;11)). Burst pressure testing also showed that the crosslinked scaffolds had a significantly higher burst strength than non-crosslinked scaffolds and all scaffolds could withstand pressure higher than 200 mmHg before failure, indicating that they can withstand the pressure seen in-vivo. When the metabolic activity of cells embedded in scaffolds was measured, there was significantly greater metabolic activity after 7 and 14 days, suggesting that the hUCBEPCs can proliferate in the CG and FCG scaffolds. However, the most interesting finding was that at day 14 cells seeded in the FCG scaffolds had greater metabolic activity than when seeded in CG scaffolds (Figure 2A). After 14 days, the CG and FCG scaffolds showed no significant change in diameter indicating their dimensional stability (Figure 2B).
Conclusions: This research determined that crosslinking both CG and FCG scaffolds with DHT/EDAC yielded scaffolds with superior mechanical properties. DHT/EDAC crosslinking increased the strength of the FCG scaffold while not compromising its elasticity. The most interesting finding was that hUCBEPCs had a greater metabolic activity when embedded in FCG scaffolds than in CG scaffolds after 14 days. This indicates that FCG scaffolds crosslinked with DHT/EDAC provided an environment that allows for greater cell viability and proliferation. These initial results form part of a bigger project, identifying critical parameters for developing successful TEHVs.
Methods: CG scaffolds were fabricated using a freeze drying process with a final freezing temperature of -10 ⁰C. A CG suspension was produced with microfibrillar, type 1 collagen in a solution of acetic acid and glycosaminoglycan (GAG) (chondroitin sulphate) at a final suspension of 0.75 wt% collagen and 0.044 wt% chondroitin-6-sulfate. Scaffolds were cross-linked with DHT, EDAC, or DHT/EDAC. After crosslinking, tris-buffered saline, 50 mM CaCl2, 40 IU/ml thrombin and 10 mg/ml of fibrinogen were drop-loaded onto the CG scaffold to fabricate FCG scaffolds (Brougham C. Acta Biomater. 2015; 26:205-214). Uniaxial tensile tests were performed using a 5 N load cell at a strain rate of 10% per minute. The burst strength was measured using a burst pressure rig. EPCs were isolated from human umbilical cord blood and seeded onto CG and FCG scaffold crosslinked with DHT/EDAC for 1-14 days. To observe the response of hUCBEPCs in CG and FCG scaffolds, their metabolic activity was measured using AlamarBlue and cell-mediated contraction was determined by measuring the scaffold dimensional stability.
Results: Tensile testing showed that crosslinking significantly increased the tensile strength and elastic modulus, although EDAC significantly decreased the elongation of FCG scaffolds (Figure 1). Uniaxial tensile testing also showed that the elastic modulus of CG and FCG cross-linked with EDAC (4.6 kPa and 5.5 kPa) and DHT/EDAC (5.0 and 5.4 kPa) was in line with that seen in adolescent pulmonary valve leaflets (4.5 kPa (Geemen DV. PLOS ONE. 2016;11)). Burst pressure testing also showed that the crosslinked scaffolds had a significantly higher burst strength than non-crosslinked scaffolds and all scaffolds could withstand pressure higher than 200 mmHg before failure, indicating that they can withstand the pressure seen in-vivo. When the metabolic activity of cells embedded in scaffolds was measured, there was significantly greater metabolic activity after 7 and 14 days, suggesting that the hUCBEPCs can proliferate in the CG and FCG scaffolds. However, the most interesting finding was that at day 14 cells seeded in the FCG scaffolds had greater metabolic activity than when seeded in CG scaffolds (Figure 2A). After 14 days, the CG and FCG scaffolds showed no significant change in diameter indicating their dimensional stability (Figure 2B).
Conclusions: This research determined that crosslinking both CG and FCG scaffolds with DHT/EDAC yielded scaffolds with superior mechanical properties. DHT/EDAC crosslinking increased the strength of the FCG scaffold while not compromising its elasticity. The most interesting finding was that hUCBEPCs had a greater metabolic activity when embedded in FCG scaffolds than in CG scaffolds after 14 days. This indicates that FCG scaffolds crosslinked with DHT/EDAC provided an environment that allows for greater cell viability and proliferation. These initial results form part of a bigger project, identifying critical parameters for developing successful TEHVs.
| Original language | English (Ireland) |
|---|---|
| Pages | 884 |
| Number of pages | 1 |
| Publication status | Published - 19 Apr 2023 |
| Event | Society for Biomaterials annual meeting 2023 - Sheraton San Diego Hotel & Marina, San Diego, United States Duration: 19 Apr 2023 → 22 Apr 2023 https://2023.biomaterials.org/ |
Conference
| Conference | Society for Biomaterials annual meeting 2023 |
|---|---|
| Country/Territory | United States |
| City | San Diego |
| Period | 19/04/23 → 22/04/23 |
| Internet address |