Hydrogel Engineering and Conditioned Media Present Two Basic Tools in Cardiovascular Regenerative Medicine

Objectives: Regenerative Medicine is a promising field in cardiovascular research. Here, we show that hydrogels, known for their use in myocardial infarction research, and conditioned media (CM) present two basic tools. A new hydrogel engineering approach was investigated to determine whether degradation of a gel and invasion of cells can be controlled. Growth agents are essentials in regenerative research but allogenic sources risk immunogenic reactions. This may be circumvented by the use of CM; its proliferative effect was confirmed.

Methods: Eight-arm PEG hydrogels polymerized with either a peptide generally permissive for matrix metalloproteinases (pMMP1) or a hydrolytically degradable PEG monomer (hdPEG) were investigated. These cross-linkers were used in the following ratios: 100% pMMP1, 75% pMMP1 + 25% hdPEG, 50% pMMP1 + 50% hdPEG, 25% pMMP1 + 75% hdPEG, 100% hdPEG. Rheology studies and a spheroid assay were conducted to investigate biomechanical properties and cell invasion. A 14-day in vivo experiment (rat subcutaneous implant) was performed with these hydrogels to determine tissue ingrowth in a preclinical setting. To gain CM, human dermal fibroblasts were incubated for 3 days in DMEM without any supplements under hypoxic/normoxic conditions. After harvesting the CM, it was transferred to human fibrosarcoma cells and its proliferative capacity was compared with that of DMEM containing none or 5% FBS with a proliferation assay.

Result: With hdPEG presenting ≥50% of the cross-linkers incorporated into a gel, the stiffness decreases while swelling ratio (Q) increases significantly (e.g., 100% hdPEG, day 1/7: Q = 20/40, p < 0.001). With higher ratios of pMMP1, cell sprouting from the spheroids is promoted. Gels assembled through 100% pMMP1/75% pMMP1 + 25% hdPEG allow initial sprouting (sprout length, 24 hours: 62.16 µm/89.48 µm, respectively) and a significant increase of sprout length (p < 0.001 in both cases, n = 9) after another 24 hours. Surprisingly, hdPEG, as a cross-linker, leads to less sprouting in vitro, whereas tissue ingrowth in vivo is supported. CM improves cell growth (p = 0.032), but less than FBS does (p < 0.0001).

Conclusion: The degree of invasion into the various hydrogels was found to be linked to the relative proportion of peptide/hdPEG. Also, we could show that immunologically inert and very accessible PEG monomers can be used to control tissue invasion and degradation of hydrogels. CM enhances cell growth, suggesting a high potential as a regenerative growth medium without the risks that allogenic serum alternatives present.

Publication History

Article published online:
19 February 2021