Using chondroitin sulfate to improve the viability and biosynthesis of chondrocytes encapsulated in interpenetrating network (IPN) hydrogels of agarose and poly(ethylene glycol) diacrylate

We recently introduced agarose-poly(ethylene glycol) diacrylate (PEGDA) interpenetrating network (IPN) hydrogels to cartilage tissue engineering that were able to encapsulate viable cells and provide a significant improvement in mechanical performance relative to its two constituent hydrogels. The goal of the current study was to develop a novel synthesis protocol to incorporate methacrylated chondroitin sulfate (MCS) into the IPN design hypothesized to improve cell viability and biosynthesis. The IPN was formed by encapsulating porcine chondrocytes in agarose, soaking the construct in a solution of 1:10 MCS:PEGDA, which was then photopolymerized to form a copolymer network as the second network. The IPN with incorporated CS (CS-IPN) (~0.5 wt%) resulted in a 4- to 5-fold increase in the compressive elastic modulus relative to either the PEGDA or agarose gels. After 6 weeks of in vitro culture, more than 50% of the encapsulated chondrocytes remained viable within the CS-modified IPN, in contrast to 35% viability observed in the unmodified. At week 6, the CS-IPN had significantly higher normalized GAG contents (347 ± 34 µg/µg) than unmodified IPNs (158 ± 27 µg/µg, P < 0.05). Overall, the approach of incorporating biopolymers such as CS from native tissue may provide favorable micro-environment and beneficial signals to cells to enhance their overall performance in IPNs

Hard Tissue Engineering

Peer reviewe

The bioactivity of agarose-PEGDA interpenetrating network hydrogels with covalently immobilized RGD peptides and physically entrapped aggrecan

Our previous reports of interpenetrating networks (IPNs) have demonstrated drastic improvements in mechanical performance relative to individual constituent networks while maintaining viability of encapsulated cells. The current study investigated whether covalent linkage of RGD to the poly(ethylene glycol) diacrylate (PEGDA) network could improve upon cell viability and performance of agarose-PEGDA IPNs compared to unmodified IPNs (control) and to IPNs with different concentrations of physically entrapped aggrecan, providing a point of comparison to previous work. The inclusion of RGD or aggrecan generally did not adversely affect mechanical performance, and significantly improved chondrocyte viability and performance. Although both 4 and 100 μ g/mL of aggrecan improved cell viability, only 100 μ g/mL aggrecan was clearly beneficial to improving biosynthesis, whereas 100 μg/mL of RGD was beneficial to both chondrocyte viability and biosynthesis. Interestingly, clustering of cells within the IPNs with RGD and the higher aggrecan concentration were observed, likely indicating cell migration and/or preferred regional proliferation. This clustering resulted in a clearly visible enhancement of matrix production compared to the other IPNs. With this cell migration, we also observed significant cell proliferation and matrix synthesis beyond the periphery of the IPN, which could have important implications in facilitating integration with surrounding cartilage in vivo. With RGD and aggrecan (at its higher concentration) providing substantial and comparable improvements in cell performance, RGD would be the recommended bioactive signal for this particular IPN formulation and cell source given the significant cost savings and potentially more straightforward regulatory pathway in commercialization

Recellularization of decellularized heart valves: Progress toward the tissue-engineered heart valve

The tissue-engineered heart valve portends a new era in the field of valve replacement. Decellularized heart valves are of great interest as a scaffold for the tissue-engineered heart valve due to their naturally bioactive composition, clinical relevance as a stand-alone implant, and partial recellularization in vivo. However, a significant challenge remains in realizing the tissue-engineered heart valve: assuring consistent recellularization of the entire valve leaflets by phenotypically appropriate cells. Many creative strategies have pursued complete biological valve recellularization; however, identifying the optimal recellularization method, including in situ or in vitro recellularization and chemical and/or mechanical conditioning, has proven difficult. Furthermore, while many studies have focused on individual parameters for increasing valve interstitial recellularization, a general understanding of the interacting dynamics is likely necessary to achieve success. Therefore, the purpose of this review is to explore and compare the various processing strategies used for the decellularization and subsequent recellularization of tissue-engineered heart valves

Mimicking the Extracellular Matrix: Tuning the Mechanical Properties of Chondroitin Sulfate Hydrogels by Copolymerization with Oligo(ethylene glycol) Diacrylates

ABSTRACTChondroitin sulfate (CS) is one of the major glycosaminoglycans (GAGs) present in the connective tissue extracellular matrix (ECM) and is responsible for the regulation of cellular activities as well as providing mechanical support for the surrounding tissue. Due to presence of CS in the natural tissues including cartilage, hydrogels of CS and other GAGs have been widely used in cartilage regeneration. Due to their polyelectrolyte nature, GAG-based hydrogels are brittle and require modifications to overcome the weak mechanical properties. In this work, we showed copolymerization of methacrylated chondroitin sulfate with oligo(ethylene glycol)s improved the crosslink density of the gels from 2 to 20 times depending on the methacrylation degree of CS and length of the crosslinking monomer. Copolymerization of CS with oligo(ethylene glycol) acrylates is a method to design hydrogels with tunable swelling and mechanical properties.</jats:p

Decellularized cartilage may be a chondroinductive material for osteochondral tissue engineering.

Extracellular matrix (ECM)-based materials are attractive for regenerative medicine in their ability to potentially aid in stem cell recruitment, infiltration, and differentiation without added biological factors. In musculoskeletal tissue engineering, demineralized bone matrix is widely used, but recently cartilage matrix has been attracting attention as a potentially chondroinductive material. The aim of this study was thus to establish a chemical decellularization method for use with articular cartilage to quantify removal of cells and analyze the cartilage biochemical content at various stages during the decellularization process, which included a physically devitalization step. To study the cellular response to the cartilage matrix, rat bone marrow-derived mesenchymal stem cells (rBMSCs) were cultured in cell pellets containing cells only (control), chondrogenic differentiation medium (TGF-β), chemically decellularized cartilage particles (DCC), or physically devitalized cartilage particles (DVC). The chemical decellularization process removed the vast majority of DNA and about half of the glycosaminoglycans (GAG) within the matrix, but had no significant effect on the amount of hydroxyproline. Most notably, the DCC group significantly outperformed TGF-β in chondroinduction of rBMSCs, with collagen II gene expression an order of magnitude or more higher. While DVC did not exhibit a chondrogenic response to the extent that DCC did, DVC had a greater down regulation of collagen I, collagen X and Runx2. A new protocol has been introduced for cartilage devitalization and decellularization in the current study, with evidence of chondroinductivity. Such bioactivity along with providing the 'raw material' building blocks of regenerating cartilage may suggest a promising role for DCC in biomaterials that rely on recruiting endogenous cell recruitment and differentiation for cartilage regeneration