Introduction: Since the development of the first generation of cell-based therapies, autologous chondrocyte implantation (ACI) and mesenchymal stem cell transplantation have been used in clinical trials for joint repair[1]. However, a major challenge is the difficulty of the surgical procedure to deliver cells to the site of injury. In ACI, for example, surgeons utilize a periosteal flap to secure the cell-implant, and a high cell density to make up for hypertrophy[2]. During the culture, dedifferentiation and loss of phenotype constitute an added complexity. In this study, we introduce a novel injectable system that (i) serves as a safe vehicle for controlled cell delivery, (ii) possesses a degree of adhesiveness to confine cell to the defect site at the time of implantation, and (iii) exhibits a tunable mechanical environment to drive the commitment of the cells toward the desired lineage. This system must (i) be injectable, (ii) adhere to the chondral interface, and (iii) sustain cell release over time without compromising the level of repair.
Materials and Methods: The proposed hydrogel system consisted of a crosslinked and a non-crosslinked domain. The proportion of the former impacts the mechanical environment in which the cells reside, the injectability of the system and the degree of adhesiveness, while the latter determines cell release rate. A 3% alginate/polyethylene glycol solution (1:3) was prepared in PBS at 60°C overnight followed by internal gelation during vortex with 1% calcium solution at room temperature. Porcine chondrocytes were mixed at a density of 1×106 cell/mL with the gel, and injected in a 3 mm circular defect model in a porcine patella in vitro. External gelation took place in a bath of 5% calcium solution immediately after the injection. Samples were incubated at 37°C with PPRF-MSC6 medium[3], in 5% CO2 until the completion of the delivery phase.
Results and Discussion: The results showed that the gel demonstrated a viscous flow behavior at the time of injection to effectively penetrate throughout the cylindrical defect models and fully embed the implantation site, then demonstrated solid-like properties in situ post external gelation with adequate mechanical stability to not flow from the defect vicinity (Fig 1).
Also, the gel was able to completely fill the focal defect model, fuse with the surrounding cartilage tissue (Fig 2a), and seal the implantation site flush to the articular surface (Fig 2). There was no need for additional fixation such as patches since the sealant demonstrated ample resistance to peeling off from the superficial layer. Figure 3 shows how the cells were released at a controlled rate due to the spontaneous discharging of the non-crosslinked domain which resulted in the concomitant breakdown of the matrix by day 7. As the release rate increased with time, the gel strength decreased and cell migration accelerated (Fig 3).
Conclusion: This research introduced an injectable sealant, which also served as a cell delivery vehicle and an adhesive to treat chondral defects of different size and shape. By design, it represented a multifunctional mimicry of the chondrocytes niche in cartilage since it encompassed cells in a three-dimensional matrix, under a hypoxic condition and with a relatively similar mechanical environment to that of native cartilage. From a surgical viewpoint, the system can be cured in situ within seconds and demonstrated a reliable and a cost-effective additional step that can benefit various cell-based therapies.
Alberta Innovates: Health Solutions; Natural Sciences and Engineering Research Council of Canada (NSERC)
References:
[1] Caplan, A.I., Review: mesenchymal stem cells: cell-based reconstructive therapy in orthopedics. Tissue Eng, 2005. 11(7-8): p. 1198-211.
[2] Brittberg, M., Autologous chondrocyte implantation—technique and long-term follow-up. Injury, 2008. 39(1): p. 40-49.
[3] Jung, S., et al., Human mesenchymal stem cell culture: Rapid and efficient isolation and expansion in a defined serum-free medium. Journal of Tissue Engineering and Regenerative Medicine, 2012. 6(5): p. 391-403.