Tailorable Cell Culture Platforms from Enzymatically Cross-Linked Multifunctional Poly(ethylene glycol)-Based Hydrogels

Abstract

As stem-cell-based therapies rapidly advance toward clinical applications, there is a need for cheap, easily manufactured, injectable gels that can be tailored to carry stem cells and impart function to such cells. Herein we describe a process for making hydrogels composed of hydroxyphenyl propionic acid (HPA) conjugated, branched poly(ethylene glycol) (PEG) via an enzyme mediated, oxidative cross-linking method. Functionalization of the branched PEG with HPA at varying degrees of substitution was confirmed via attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR) and (1)H NMR. The versatility of this hydrogel system was exemplified through variations in the degree of HPA substitution, polymer concentration, and the concentration of cross-linking reagents (horseradish peroxidase and H(2)O(2)), which resulted in a range of mechanical properties and gelation kinetics for these gels. Cross-linking of the PEG-HPA conjugate with a recombinantly produced Fibronectin fragment (Type III domains 7-10) encouraged attachment and spreading of human mesenchymal stem cells (hMSCs) when assessed in both two-dimensional and three-dimensional formats. Interestingly, when encapsulated in both nonfunctionalized and functionalized cross-linked PEG-HPA gels, MSCs showed good viability over all time periods assessed. With tunable gelation kinetics and mechanical properties, these hydrogels provide a flexible in vitro cell culture platform that will likely have significant utility in tissue engineering as an injectable delivery platform for cells to sites of tissue damage.

Enzymatically Cross-Linked Hyperbranched Polyglycerol Hydrogels as Scaffolds for Living Cells

“…The HRP-catalyzed oxidative cross-linking successfully took place in the presence of H 2 O 2 for both hPG-HPA and hPG-TA (SI Figure S8). These results, along with others on HRP-crosslinked linear polymers from the literature, 13,22,24,25 clearly show that HRP-catalyzed oxidative cross-linking is suitable for dendritic polymers regardless of the polymer's architecture.…”

“…22,24,25 Therefore, we also initially applied their grafting to hPG for HRP crosslinking. hPG-HPA (compound 1 in Table 1) was synthesized with an amide coupling reaction between hPG-amine and HPA (Materials and Methods, SI Figures S1 and S2), while hPG-TA Table 1) was obtained by coupling TA with hPG-succinic acid in two steps (Supporting Information).…”

“…This is in agreement with the literature that G′ is not affected above a certain HRP activity level. 25,43,44 The mechanical strength of hydrogels, on the other hand, strongly depends on the H 2 O 2 concentration (from 2.5 to 12.5 mM) with G′ and G″ changing over 3 orders of magnitude ( Figure 1f). However, for the concentration higher than 12.5 mM H 2 O 2 , G′ and G″ reached a plateau, thus apparently achieving the maximum gel strength.…”

Introduction to In Situ Forming Hydrogels for Biomedical Applications

“…1 There has been an increased interest in the enzymatically crosslinked hydrogels that shows few side reactions since their high specificity for substrates. Horseradish peroxidase (HRP)/hydrogen peroxide (H 2 O 2 ), transglutaminase (TG), phosphatase (PP), tyrosinase, or thermolysin catalyzed crosslinking provides in situ hydrogel formation of hydroxyphenyl propionic acid (HPA) functionalized 8-arm PEG [42], thiol functionalized poly(glycidol) [43], Tetronic-tyramine (Tet-TA)/gelatin-HPA (GFPA) [44], dextran-tyramine (Dex-TA) [45], alginate-g-pyrrole [46], or protein polymers containing either lysine or glutamine [47]. These enzymatic crosslinks provide fast gelation.…”

Effects of Synthetic Biomacromolecule Addition on the Flow Behavior of Concentrated Mesenchymal Cell Suspensions

“…3,4,6 In earlier work, 1 This is the case for one of the most commonly used synthetic biomacromolecules, polyethylene glycol (PEG). These molecules are obviously not naturally present in the body, however they have received significant attention for the development of new tissue engineering scaffolds 11 for two main reasons. Firstly, this polymer is relatively biologically inert and is well tolerated in vivo.…”