Even though articular cartilage is known for its durability and ability to resist high stresses and strains, once injured it has a very poor intrinsic capacity to heal itself. Unlike most tissue, cartilage is composed of one cell type, chondrocytes, and an extracellular matrix (ECM) which is avascular and lacks a neuronal supply. Damaged cartilage progressively deteriorates, often ending in joint destruction and even complete loss of function. Surgical treatments available today are microfracture, drilling, abrasion chondroplasty, mosaicplasty and autologous chondrocyte implantation (ACI). The concept behind microfracture, drilling, and abrasion chondroplasty is to form a connection between the defect and its subchondral bone by inducing blood clot formation. Although new cartilage is formed, it is inferior in its composition and biomechanical properties when compared to native articular cartilage [1
]. Relief from pain is what justifies these procedures to be used as standard surgical intervention strategies for repairing damaged cartilage. ACI is a newer surgical intervention strategy in which chondrocytes are harvested arthroscopically, expanded in vitro
and implanted in the debrided area, where they are secured in place by a periosteal flap [2
]. In order to reduce the amount of tissue hypertrophy associated with ACI, a second generation of ACI techniques was developed in which scaffolds were used instead of periosteum to secure the cells into the defect area [2
]. These improved results have led to an explosion of interest in developing an ideal scaffold for the next generation of ACI [4
]. In general, each of these surgical interventions works by creating a functional tissue that reduces some of the pain associated with damaged cartilage. However, they each are associated with their own specific shortcomings that include donor site morbidity, extensive surgical procedures, cost, host pain and availability of cells.
In theory, cartilage tissue engineering combines a scaffold, cells and growth factors to create a tissue mimicking native healthy cartilage biochemically and structurally. This discipline, however, is still in its infancy. There is no consensus on an optimal scaffold, best cell type or combination of growth factors. Moreover, since there are numerous diseased and damaged cartilage states, an individualistic approach to cartilage repair may be required, using different combinations for optimal repair of each state of damage or disease. With the lack of intrinsic repair modalities, repairing damaged cartilage with engineered cartilage tissue constructs is one possible solution.
In the present study, oligo(poly(ethylene glycol) fumarate) (OPF), a derivate of poly(ethylene glycol) (PEG) was used to create hydrogels with different charges. PEG itself has been under investigation in cartilage tissue engineering for years [5
]. For example, it has been shown that the cross-linking density of PEG has an effect on chondrocyte morphology [7
], and loading has an effect on chondrocyte metabolism [6
]. In 2001, Jo et al. were able to synthesize OPF by connecting PEG and fumaric acid through ester bonds [8
]. OPF was chosen in this study because it has a high degree of swelling in aqueous environments (>95%) and other properties which mimic native cartilage [9
]. Cross-linking density, water content, modulus and surface tension can be modified in this hydrogel in order to optimize cell survival, proliferation and extracellular matrix secretion [10
]. Also, it has been previously shown that OPF is biodegradable, biocompatible and degraded through hydrolysis of the ester bonds [9
Recent research efforts have been directed towards the effect of incorporating charged molecules into PEG-based hydrogels [11
]. When chondroitin was incorporated into PEG-hydrogels there was a positive effect versus pure chondroitin sulfate gels [11
]. Total collagen content and collagen type II gene expression increased, but the aggrecan content remained unchanged. In 2010, the same group demonstrated an increase of collagen and proteoglycan content of bovine chondrocytes in charged PEG hydrogels under dynamic loading conditions [12
]. However, the effect of incorporating charge into OPF hydrogels has yet to be determined. From our laboratory, Dadsetan et al. recently demonstrated that neuron attachment and differentiation of dorsal root ganglia improved, and neurite extension was significantly greater when the neurons were cultured on OPF hydrogels with small charged monomers [13
]. Although OPF had been shown to be a promising candidate for cartilage tissue engineering [9
], the impact of charge in the OPF hydrogels on chondrocyte behavior is still unknown. Since previous studies using PEG hydrogels with incorporated charge demonstrated a positive effect in other cell lineages and chondrocytes in particular, it seems logical to deduce that charge might also affect the cartilage tissue quality when incorporated into OPF hydrogels.
Aggrecan is the most abundant protein expressed by chondrocytes. These huge molecules have a high anionic charge from the numerous branches of charged anionic sulfate (SO3−
) and carboxyl (COO−
) that they contain. Given that fixed charge density of the ECM plays a key role in maintaining healthy cartilage [12
], the charge status of engineered cartilage matrix is also likely to have an effect on the regenerated cartilage tissue.
In this study, we compared the effects of negative and positive fixed charge on chondrocyte behavior in vitro to test the hypothesis that engineered cartilage incorporating negatively charged molecules in the matrix would more closely resemble the structure and function of native cartilage than engineered cartilage with positively charged matrix. Small negatively charged molecules of sodium methacrylate (SMA) were copolymerized with the OPF hydrogel to produce a negatively charged hydrogel. [2-(methacryloyloxy) ethyl]-trimethylammonium chloride (MAETAC), which is a positively charged monomer, was copolymerized with the OPF for comparison of the charge effects. The resulting polymers were characterized by assessing the swelling ratio, zeta potential, ion conductivity and surface composition. After hydrogel characterization, chondrocytes were seeded on top of the hydrogels and stained for viability and collagen type II. For protein expression, the normalized GAG production was assessed. These results were then compared with those of the neutrally charged hydrogels.