Introduction:: Osteoarthritis (OA), a painful joint disease manifested by cartilage damage, affects 654 million people worldwide. Currently, there is no cure for OA; available medicines only alleviate the disease symptoms (e.g. pain and inflammation) while surgical approaches including the use of replacement autograft or allograft cartilage suffers from the problems of donor-site morbidity, infection, and especially, limited tissue supply.
Piezoelectric materials with an exciting ability to self-convert mechanical deformation into electricity, are an appealing platform for creating self-powered electrical stimulators. Prior research has shown that piezoelectric scaffolds made of poly (l-lactic acid) (PLLA) promote bone and cartilage healing (1). However, surgical interventions are required to deliver those scaffolds to the wound. The implantation could cause complications such as damage to other healthy tissues, infection, inflammation, and recovery time. In this study, we present a novel injectable and biodegradable piezoelectric hydrogel with ultrasound (US) activation to provide a minimally invasive approach to OA treatment (Fig. a-1). The piezoelectric hydrogel can be injected into the joints and self-produce localized electrical charges under US activation to promote cartilage healing. In vitro study showed that culturing adipose-derived stem cells inside piezoelectric hydrogel under US treatment induced a 9.4-fold increase of COL2A1, 10.6-fold of ACAN gene expression, higher than the control groups without piezoelectric effect or US activation. Especially in, on rabbits’ model, we found significant regeneration of hyaline cartilage and subchondral bone within the defects with strong mechanical properties for the animals receiving our piezoelectric hydrogel after 2 months of US activation.
Materials and Methods:: Material fabrication:
Piezoelectric fibers were fabricated using the electrospinning method. The piezoelectric fibers were then sectioned into individual fibers with 25um lengths by Cryostat. Then we integrated the chopped fibers with collagen hydrogel to create injectable piezo hydrogel. PDLLA, non-piezoelectric fibers were implemented as a control.
Material characterization:
DSC, XRD, SEM, and rheometer techniques were used for material characterization and injection ability.
Cartilage regeneration hypothesis testing:
In Vitro: To study the effect of piezoelectric charges on chondrogenesis, ADSCs were seeded with piezo, non-piezo, and control hydrogel. Ultrasound was used to activate the electric charges in the piezo hydrogel. After 2 weeks, the cells were collected, rt-qPCR, collagen II staining Alcian blue staining, and IF staining of type II collagen were performed.
In Vivo: The rabbit osteochondral defect model was utilized to evaluate cartilage healing in vivo. Gross view, histology Safranin O/, and fast green staining were carried out. Nanoindentation was employed to test new form cartilage mechanical properties.
Results, Conclusions, and Discussions:: Material characterization: Fig. a,2-5 demonstrates that we have successfully fabricated the hydrogel and the nanofibers were distributed homogeneously inside the collagen matrix. We loaded the hydrogel into a 1ml - G29 insulin syringe needle and eventually injected the hydrogel out showing this hydrogel can be injected into the body. We also confirmed that the two important parameters of PLLA including β-form crystal structure and the crystallinity did not change after sectioning (Fig. a,6-7). Fig. a-8 shows the representative output voltage waveforms of piezo (PLLA hydrogel) and non-piezo (PDLLA hydrogel). The piezo generates a clear signal with consistent intervals and peak magnitude. Meanwhile, NonPiezo’s waveform has a smaller amplitude and irregularity with random peaks.
In vitro: Data shows that piezo hydrogel with US activation triggered the most COL2A1, and ACAN gene expression compared to other groups (Fig. b,1-2). This data is consistent with collagen type II staining; the piezo + US group produced much more collagen II in the matrix compared to other groups (Fig. b-3).
In vivo: From the collected knees, we observed less cartilage and subchondral-bone tissue in the defects of control and sham groups compared to the experimental group of Piezo + US (Fig. c,1-4). The experimental group has a better macroscopic appearance with the new tissue which was integrated well with the defect border and had a higher degree of defect repair which more resembled the surrounding native host tissues. Noticeably, in the histology data the newly formed cartilage tissues in the piezo + US had a clear shape of chondrocyte structure and cell distribution. For mechanical properties, Fig. c-5 displayed the load-displacement curve of the 2-month piezo + US group which gets closer to that of the native healthy cartilage which indicates the mechanical property of newly formed cartilage in piezo +US has significantly improved compared to other groups.
Conclusion: We report the first injectable, biodegradable piezoelectric hydrogel that promotes cartilage regeneration. The fabricated hydrogel activated by the external US can induce chondrogenesis both in vitro and in vivo. This hydrogel could be a minimally invasive alternative treatment, and easy to administer for OA patients.
Acknowledgements (Optional): : This project was funded NIH.
Special thanks GE Fellowship for providing financial support to graduate student Tra Vinikoor during 2022-2023
References (Optional): : Liu, Yang, Godwin Dzidotor, Thinh T. Le, Tra Vinikoor, Kristin Morgan, Eli J. Curry, Ritopa Das et al. "Exercise-induced piezoelectric stimulation for cartilage regeneration in rabbits." Science translational medicine 14, no. 627 (2022): eabi7282.
