Assistant Professor Clarkson University Potsdam, New York, United States
Introduction:: Osteoarthritis is among the most common degenerative joint diseases, affecting more than 32 million people in the US. Cartilage degradation is considered irreversible due to the avascular and nearly acellular structure of cartilage. One potential therapy for cartilage regeneration is using human mesenchymal stem cells (hMSCs) injected into the damaged joint due to the chondrogenic potential of these cells. However, hMSCs have low survival rates due to shear forces during injection, which hinders their therapeutic use. To address this issue, the hMSCs may be encapsulated within an injectable hydrogel, which acts as a supportive delivery vehicle. Hydrogels are water-swollen polymers that are biocompatible and mimetic of the native, extracellular matrix-dense cartilage microenvironment. Two major components of the cartilage extracellular matrix are hyaluronic acid (HA) and collagen. However, as natural polymers, these form hydrogels with low mechanical properties and thereby need to be combined with synthetic polymers and/or crosslinked with chemical crosslinking in order to achieve hydrogels with sufficient physical properties [1-3]. However, synthetic polymers lack integrin binding domains, and chemical crosslinking methods typically involve cytotoxic materials (i.e., photoinitiators, catalysts, and enzymes) and gelation occurs in harsh chemical conditions [4]. We propose to use a novel approach, bioorthogonal Diels-Alder click chemistry which is a benign process occurring under cell culture conditions, to covalently crosslink HA with type I collagen, investigating the effects of changing HA molecular weight on the chondrogenic differentiation of the hMSCs.
Materials and Methods:: We functionalized two different molecular weights (MW) of de-salted sodium hyaluronate (low MW: Mn ~66 kDa; LMW and high MW: Mn ~1 MDa; HMW) with a furan group [5-7]. Briefly, we dissolved HA in 2-morpholinoethane sulfonic acid (MES) buffer and added 1:1 Molar ratio 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4 methylmorpholinium chloride (DMTMM) to activate HA. Then, we added a 1:1 Molar ratio furfurylamine:HA repeat unit, and let the solution stir for 24 h at room temperature (RT), followed by dialysis against 18.2 MΩ water and lyophilization. Similarly, we furan-functionalized type I collagen according to a previously reported protocol for gelatin with some modifications [8]. Briefly, we added furfuryl glycidyl ether (FGE) to collagen following the adjustment of pH to 10 using NaOH for 24 h at RT under dark and inert conditions. Following incubation, we adjusted the pH to 7 using HCl, dialyzed against 18.2 MΩ water for 48 h, and lyophilized. Proton nuclear magnetic resonance (1H NMR, Bruker Advance 400 MHz) spectroscopy and a ninhydrin assay were used to evaluate furan conjugation in HA and collagen, respectively. Bone marrow-derived hMSCs were expanded, suspended at 250,000 cells/mL, and mixed with furan-modified HA and furan-modified type I collagen in the MES buffer. Following the suspension, a two-arm, dimaleimide poly(ethylene glycol) (mal-PEG-mal) crosslinker (MW = 2000 Da) was added to form a hydrogel at 37 ◦C and 5% CO2. Pellets at 250,000 cells/mL were made as controls.
Results, Conclusions, and Discussions:: We successfully conjugated both HA and type I collagen to furan functional groups, achieving 36.5% and 27.8% conjugation efficiencies as evaluated using 1H NMR and a ninhydrin assay, respectively. Following conjugation, we fabricated different hydrogels (i.e., LMW HA-PEG, HMW HA-PEG, LMW HA-PEG-collagen, HMW HA-PEG-collagen in MES buffer or hMSC growth media with or without encapsulated hMSCs) according to the previously described procedure (Figure 1A). hMSCs were encapsulated throughout the hydrogels (Figure 1B), though hydrogels without collagen had notably decreased cell adhesion. In addition, HMW HA hydrogels (with and without collagen) gelled within two hours, while LMW HA hydrogels (with and without collagen) gelled overnight. Dynamic mechanical analysis characterization showed that the addition of the collagen to LMW HA resulted in a hydrogel with a higher stiffness compared to the LMW HA-PEG hydrogel alone, likely due to an increase in covalent crosslinking via Diels-Alder click chemistry (Figure 1C). Moreover, the LMW HA-PEG-collagen hydrogel exhibited the highest stiffness among the hydrogels. We hypothesize that the stiffness of these hydrogels, tunable depending on the hydrogel composition, will affect the chondrogenic differentiation of encapsulated hMSCs. Current work is focused on characterizing the physical properties of the hydrogels in MES buffer and hMSC growth media with and without hMSCs and evaluating hMSCs chondrogenesis within the hydrogels. In particular, we are interested in investigating the effects of HA MW and concentrations on hMSC chondrogenesis.
Figure 1. Hyaluronic acid (HA)/ type I collagen hydrogels. (A) Photos of i) 1% w/v LMW HA-PEG hydrogel in MES buffer, ii) 1% w/v HMW HA-PEG hydrogel in MES buffer, iii) 1% w/v LMW HA-PEG-0.5% w/v collagen hydrogel in MES buffer, iv) 1% w/v HMW HA-PEG-0.5% w/v collagen hydrogel in MES buffer, v) 1% w/v LMW HA-PEG hydrogel in hMSC growth media, and vi) hMSC-loaded 1% w/v HMW HA-PEG hydrogel in hMSC growth media, (B) Phase contrast image of hMSCs loaded within a 1% w/v HMW HA-PEG hydrogel, showing distribution of the cells throughout the hydrogel. Scale bar = 200 µm, and (C) Stiffness values for hydrogels with different components (A i-iv n=2) showing stiffnesses as a function of the polymer composition.
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