Poster P2 - PEG-Alginate Hybrid Double-Network Cryogels with Tunable Degradation Rates for Cartilage Tissue Engineering

Introduction: Articular cartilage has limited self-healing capacity and any joint trauma or “wear-and tear” due to overuse can lead to widespread cartilage erosion and the development of painful degenerative osteoarthritis (OA). Clinically used cell therapies for articular cartilage repair have achieved partial clinical success and involve direct implantation of either autologous chondrocytes (ACI) or mesenchymal stem cells (MSCs) with or without a support matrix. There has been a growing interest in using double network (DN) hydrogel as support matrix for cartilage tissue engineering due to their enhanced toughness compared to conventional hydrogels. Inspite of the high toughness, the non-porous and non-degradable characteristic of the DN hydrogel limits their application for tissue engineering. Several studies emphasize the need for macroporosity and degradability to facilitate cell infiltration and extracellular matrix deposition in cartilage constructs. Thus, a critical need remains for robust 3D macroporous and biodegradable DN hydrogels that can promote cell infiltration and accelerate deposition of high-quality cartilage.
To address this need, in this study we have developed biodegradable, macroporous hybrid DN cryogel by integrating two distinct crosslinked networks: 8-arm polyethylene glycol (PEG) acrylate and sulfated alginate. Cryogels are gel matrices that are synthesized through cryogelation of gel precursors at subzero temperatures leading to formation of interconnected macroporous structures. Furthermore, we demonstrate that by judicious choice of structurally similar crosslinkers we can customize the degradation kinetics of the resulting hybrid DN cryogels.

Materials and

Methods: Cryogels were synthesized through simultaneous crosslinking of two polymeric networks. Briefly, 8-arm PEG-acrylate (8-arm PEGAc, Jenkem Technology) and alginate (GMB, Manugel, FMC Biopolymer) solutions were mixed and the crosslinker mix composed of respective crosslinkers for each of the network was added. The gel precursor solution was mixed quickly and placed in a cryostat bath maintained at -20°C for at least 18h. Crosslinking in alginate is facilitated using CaCO3 and glucono-delta-lactone (GDL) pair. The 8-arm PEGAc network is crosslinked using dithiol crosslinker via Michael addition click reaction. Three different dithiol crosslinkers dithiothreitol (DTT), (S)-2-aminobutane-1,4-dithio (DTBA) and ethylene glycol dimethacrylate (EGBMA, Sigma-Aldrich)) were used. Scanning electron microscopy (SEM) was used to analyze pore size. Mechanical analysis of the cryogels was done using compression tester. Degradation studies were conducted by incubating dried and pre-weighed cryogel in complete media and measuring change in weight until complete degradation. Swelling kinetics was determined by incubating dried cryogels in buffer and monitoring the wet weight at regular intervals. For release studies 30 ng each of TGF-β1 and IGF-1 are loaded onto cryogels and the concentration of the growth factors in releasates collected at regular intervals is determined using respective ELISAs.
Mouse D1 MSCs (ATCC) were seeded in hybrid DN cryogels in presence and absence of chondrogenic media. After day 7,14 and 21 of culture, MSC chondrogenesis was assessed by quantitative real time polymerase chain reaction (qPCR) and collagen II immunostaining. Cells seeded in tissue culture plastic on day 1 were used as controls.

Results, Conclusions, and Discussions: Hybrid DN cryogel of 8-arm PEGAc-alginate were synthesized by gelation of the precursor polymers under subzero conditions. The 8-arm PEG alginate hybrid DN cryogels were found to have macroporous interconnected structure as indicated by SEM imaging. The hybrid DN cryogels swelled rapidly within 2 min of placing in buffer and reached equilibrium within 30 min. The degradation rate of the hybrid DN cryogels could be varied between 10-40 days depending upon the dithiol crosslinker used for the PEG network (Figure 1 lower panel). Hybrid DN cryogels exhibited a compressive modulus of 54.9 ±10.2 – 388 ±51.9 kPa depending upon the dithiol crosslinker used for the PEG network. The hybrid DN cryogels showed greater than 90% loading efficiency and sustained release of TGF-β1 and IGF-1 for upto 7days. Mouse D1 MSCs could be successfully cultured in the cryogels for 21 days. The D1 cells cultured in hybrid DN cryogels in absence or presence of chondrogenic media showed signs of early chondrogenesis by day 7. The pro-chondrogenic gene markers such as collagen 2, SOX-9, and aggrecan were found to be upregulated. While the hypertrophy markers collagen 1 and RUNX 2 were downregulated. Further, sustained expression of collagen 2 was observed by immunostaining D1 cells cultured in 3D cryogel until day 21.

PEG-alginate hybrid DN macroporous cryogels were successfully synthesized using radical free chemistry. The rapid swelling ability of these cryogels indicates their interconnected macroporous nature and efficient transport characteristics. Macroporous nature of the cryogels is further confirmed by SEM analysis. The ability to control the degradation rate in these cryogels by use of different dithiol crosslinkers provides a unique and facile handle to modulate their degradation properties as needed. Furthermore, the cryogels can retain and sustainably release growth factors which can potentially accelerate chondrogenesis. Lastly, the PEG-alginate DN cryogels synthesized in this study support the differentiation of mouse MSCs into chondrocytes both in the absence and presence of exogenously added growth factors. Thus, our study introduces a novel method for producing macroporous hybrid DN cryogels with customizable degradation rates, offering promising biocompatible scaffolds for cartilage tissue engineering.

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