(598e) Achieving Cartilage-Mimetic Properties in a Human Mesenchymal Stem Cell-Laden Hydrogel Using Diels-Alder Click Chemistry

Cartilage is an avascular, mostly acellular tissue that is susceptible to irreversible injury and degradation from diseases such as osteoarthritis. Injection of human mesenchymal stem cells (hMSCs) into the damaged joint has garnered significant attention as a potential therapy for cartilage regeneration due to their chondrogenic differentiation potential. In order to protect hMSCs from death during injection and subsequent clearance from the joint, researchers are currently investigating the incorporation of these cells within hydrogel scaffolds prior to injection. Hydrogels are widely used in cartilage tissue engineering due to their inherent biocompatibility, ability to support the viability of encapsulated cells, and high water content, mimicking the native extracellular matrix of cartilage. In addition, hydrogels may be developed from extracellular matrix and cartilage-derived polysaccharides and proteins, such as hyaluronic acid (HA) and collagen. Despite this, hydrogels fabricated from these natural polymers are limited by low mechanical properties, resulting in incomplete regeneration of cartilage from encapsulated hMSCs [1]. Synthetic polymers and covalent crosslinking approaches may be used to enhance the mechanical properties of hydrogels [2,3]. However, these approaches require the use of cytotoxic photoinitiators, crosslinkers, catalysts, and enzymes, and gelation occurs in harsh chemical conditions [4]. We propose to use a novel, benign crosslinking method wherein bioorthogonal Diels-Alder click chemistry is used to covalently crosslink HA with type I collagen. In this presentation, we explore the effects of changing HA molecular weight, HA and collagen density, and crosslinker density on the properties of the hydrogel, with the goal of achieving articular cartilage-mimetic mechanical properties in hydrogels fabricated using these natural polymers.

Materials and Methods:

Two different molecular weights (MW) of de-salted sodium hyaluronate (low MW: M_n ~66 kDa; LMW and high MW: M_n ~1 MDa; HMW) was functionalized with a furan group based on previous reports [5-7]. Briefly, HA was dissolved in 2-morpholinoethane sulfonic acid (MES) buffer and activated using 1:1 Molar ratio 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM). 1:1 Molar ratio furfurylamine:HA repeat unit was added dropwise, and the solution was stirred for 24 h at room temperature (RT), followed by dialysis against 18.2 MΩ water and lyophilization. Similarly, collagen type I from calf skin (M_n ~1 MDa) was functionalized with a furan group according to a previously reported protocol for gelatin with some modifications [8]. Briefly, collagen was dissolved in 0.5 M acetic acid, and furfuryl glycidyl ether (FGE) was added dropwise following the adjustment of pH to 10 using NaOH. The solution was stirred for 24 h at RT under dark and inert conditions. After the reaction was complete, pH was adjusted to 7 using HCl. Next, the final solution was dialyzed against 18.2 MΩ water for 48 h and lyophilized. Hydrogels were fabricated by mixing furan-modified HA and furan-modified type I collagen with a two-arm, dimaleimide poly(ethylene glycol) (mal-PEG-mal) crosslinker (Mw = 2000 Da) in MES buffer at concentrations ranging from 0-10% w/v. The mal-PEG-mal was added to the HA/type I collagen solution at varying furan:maleimide ratios to form the crosslinked hydrogel. Proton nuclear magnetic resonance (¹H NMR, Bruker Advance 400 MHz) spectroscopy was performed to confirm furan modification. Hydrogel stiffness was measured using dynamic mechanical analysis (DMA 850, TA instruments) under unconfined compression mode at 37 °C.

Results:

Different hydrogels (i.e., LMW HA-PEG, HMW HA-PEG, LMW HA-PEG-collagen, HMW HA-PEG-collagen) were made following the previously described procedure (Figure 1A). HMW HA hydrogels with and without collagen gelled within two hours, whereas LMW HA hydrogels gelled overnight. DMA analysis (Figure 1B) demonstrated that LMW HA-PEG-collagen had the highest stiffness among other hydrogel groups. Interestingly, the combination of LMW HA and collagen resulted in a hydrogel with a higher compressive modulus compared to the LMW HA hydrogel alone, potentially due to an increase in covalent crosslinking via Diels-Alder click chemistry as opposed to high levels of physical crosslinking (polymer entanglement) for HMW HA with the collagen. In these preliminary studies, the stiffnesses of the hydrogels are lower than previously reported values in the literature for 1% HA hydrogels. This may be due to a myriad of reasons, including low functionalization with furan (¹H-NMR yielded a conjugation efficiency of 36.46% for HA-furan) and low Molar ratios between the furan and maleimide. By tuning the crosslinker density, we can improve the stiffness values.

Figure 1. Hyaluronic acid (HA)/ type I collagen hydrogels formed via Diels-Alder click chemistry. (A) Gelation photos of i) 1% w/v LMW HA-PEG hydrogel, ii) 1% w/v HMW HA-PEG hydrogel, iii) 1% w/v LMW HA-PEG-0.5% w/v collagen hydrogel, and iv) 1% w/v HMW HA-PEG-0.5% w/v collagen hydrogel. (B) Compressive modulus for the different hydrogel compositions, n=2.

Conclusions:

In conclusion, we were able to successfully fabricate HA/type I collagen hydrogels using bioorthogonal Diels-Alder click chemistry. The combination of LMW HA and collagen had the greatest compressive modulus likely due to increased covalent crosslinking between furan and maleimide. Future studies will focus on investigating the effects of increasing crosslinker density and polymer density on hydrogel stiffness, as well as the integration of hMSCs with the hydrogels and gelation in cell culture media.

References:

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[2] Antich C. Acta Biomaterialia 2020;106:114-123

[3] Yu F. Polymer Chemistry 2014;5(17):5116-5123

[4] Hu X. Frontiers in Chemistry 2019;7:477

[5] Nimmo C. M. Biomacromolecules 2011;12(3):824-830

[6] Yu F. Carbohydrate polymers. 2013;97(1):188-195

[7] Yang Y. Acta Biomaterialia 2021;128:163-174

[8] García-Astrainet C. RSC Advances 2014;4(67):35578-35587.