Hyaluronic Acid Enhances the Mechanical Properties of Tissue-Engineered Cartilage Constructs

Abstract

There is a need for materials that are well suited for cartilage tissue engineering. Hydrogels have emerged as promising biomaterials for cartilage repair, since, like cartilage, they have high water content, and they allow cells to be encapsulated within the material in a genuinely three-dimensional microenvironment. In this study, we investigated the mechanical properties of tissue-engineered cartilage constructs using in vitro culture models incorporating human chondrocytes from osteoarthritis patients. We evaluated hydrogels formed from mixtures of photocrosslinkable gelatin-methacrylamide (Gel-MA) and varying concentrations (0–2%) of hyaluronic acid methacrylate (HA-MA). Initially, only small differences in the stiffness of each hydrogel existed. After 4 weeks of culture, and to a greater extent 8 weeks of culture, HA-MA had striking and concentration dependent impact on the changes in mechanical properties. For example, the initial compressive moduli of cell-laden constructs with 0 and 1% HA-MA were 29 and 41 kPa, respectively. After 8 weeks of culture, the moduli of these constructs had increased to 66 and 147 kPa respectively, representing a net improvement of 69 kPa for gels with 1% HA-MA. Similarly the equilibrium modulus, dynamic modulus, failure strength and failure strain were all improved in constructs containing HA-MA. Differences in mechanical properties did not correlate with glycosaminoglycan content, which did not vary greatly between groups, yet there were clear differences in aggrecan intensity and distribution as assessed using immunostaining. Based on the functional development with time in culture using human chondrocytes, mixtures of Gel-MA and HA-MA are promising candidates for cartilage tissue-engineering applications.

Hydrogels and bioreactors for cartilage research and functional tissue engineering

“…Consistent with our previous studies [162], the Young's moduli of free swelling constructs increased from ~ 40 kPa to ~ 80 kPa over 28 days in both free swelling and loaded cultures (Figure 4.6B). Accumulation of GAGs in the constructs was not affected by loading (Figure 4.6C, D).…”

“…However, stronger intra-and pericellular staining was observed in loaded constructs ( Figure 4.7B, F). As previously reported [161,162], strong collagen I immunoreactivity was observed at the outer surface of cell-hydrogel constructs ( induced by developing muscles contribute to the development of articular cartilage [340]. In later life, mechanical loading represents a key factor for tissue maintenance, and if excessive, can lead to the development of pathological conditions such as osteoarthritis.…”

“…To yield tissue-specific 3D models, methacryloyl-substituted ECM components may be co-polymerized with GelMA. [316], which is very similar to the 10 % typically used in our experiments [162]. Although it is not specifically outlined in the following protocol, this principle…”

“…Thus, the development of novel strategies involving extended in vitro pre-culture periods with the application of physiological stimuli may improve clinical outcomes in future. In such approaches, isolated autologous cells produce ECM in vitro, leading to biological implants with superior biochemical composition and mechanical functionality [161][162][163]. Additionally, these ex vivo tissue constructs can also be utilised to study chondrocyte biology as they mimic the native tissue environment superior to traditional cell culture approaches [164].…”

“…However, this method appears more tedious than the use of PDMS rings and requires large quantities of sterile hydrogel material which in turn could lead to increased material costs and preparation times. The bioreactor system is capable of controlling oxygen levels within the culture chamber using a commercial hypoxia system; however, the effects of hypoxia were not investigated in this study as we aimed to examine the reproducibility of tissue engineering using GelMA-HAMA as scaffold for human chondrocytes [161,162], as well as the effects of uni-and biaxial mechanical stimulation in this system. Nevertheless, future experiments will involve the examination of chondrocyte response to loading under various atmospheric oxygen concentrations.…”