(J-375) Application of Cartilage acellularized matrix based on IEDDA click chemistry as a therapeutic rhinoplasty filler

Introduction:: The demand for non-surgical cosmetic procedures, such as filler injections, increases because of their short procedure time and fast recovery. Despite the proven safety of commercially available hyaluronic acid (HA) fillers, concerns about potential side effects remain. In addition, fillers used primarily for volume maintenance only last for a short period of time, highlighting the need for safe, long-lasting, and tissue-integrable functional fillers. Extracellular matrix (ECM)-based fillers are gaining traction. ECM exhibits a low inflammatory response, good biocompatibility, and inherent biodegradability, has the potential to promote cell adhesion, regulate cellular responses, and tissue integration. However, conventional ECM-based hydrogels have limitation such as crosslinker’s cytotoxicity problem and non-injectability, making them unfit for use as fillers. An injectable hydrogel was developed using cartilage acellular matrix (CAM) and HA with IEDDA click chemistry and utilized to deliver cell. This approach aims to develop long-lasting functional replacements by promoting tissue integration with the host tissue and increasing cell viability and replacing conventional fillers.



Materials and Methods:: Preparation of hydrogels

Collagen, glycosaminoglycans (GAGs) contents were measured to analyze the preservation of essential properties of CAM and toxicity assays were performed. The CAM-HA hydrogel was developed using norbornene (Nb)-tetrazine (Tz) click chemistry, which resulted in a highly porous structure and injectable hydrogel. The synthesis of CAM-Nb and HA-Tz was confirmed by Fourier transform infrared spectroscopy (FT-IR). The CAM-HA hydrogels were compared with CAM hydrogels cross-linked by EDC/NHS and HA hydrogels cross-linked by 1,4-Butanediol diglycidyl ether (BDDE).

Characterization of CAM-HA hydrogel

The functional properties of the hydrogel were evaluated through rheological characterization, swelling test, injectability analysis, and toxicity analysis.

Formation of cartilage tissue in vivo

To confirm that the CAM-HA hydrogel can be maintained subcutaneously for a long time, hydrogels were monitored and tracked using a fluorescence in vivo imaging system with nude mice as models. Additionally, experiments were also conducted using subcutaneous injection in nude mice to demonstrate the superior cartilage formation of the injectable hydrogel in vivo. 4 weeks after injection, chondrogenesis was observed by strength analysis, collagen, GAGs formation assays, real-time qPCR, histological analysis, and IHC.



Results, Conclusions, and Discussions:: Results
CAM was found to effectively remove tissue components/cells, nuclei, and other genetic material. However, preserved collagen and GAGs content compared to native cartilage. The FT-IR results showed no significant change between the CAM and CAM-Nb peaks, confirming that the synthesis was successful. The CAM-HA hydrogel displayed the ability to gel in a short time with an increased storage modulus starting at 6 minutes. The CAM-HA hydrogel could be injected with the same force as the HA hydrogel, compared to the CAM hydrogel, which was difficult to inject. The CAM-HA hydrogels exhibited higher mechanical strength compared to HA hydrogels and CAM hydrogels. The swelling tests revealed a lower swelling rate than the HA hydrogel, indicating higher mechanical strength and stability, with minimal displacement problems during injection. The toxicity analysis also showed good results Moreover, we found that under the subcutaneous of nude mice, CAM-HA hydrogels remained their fluorescence intensity longer than HA hydrogels, suggesting that CAM-HA hydrogels are less degradable in vivo and can be maintained for a longer period. In vivo experiments indicated more GAGs and collagen formation and faster chondrogenesis when using CAM-HA compared to HA hydrogel..

Conclusions
In conclusion, we could fabricate a CAM-HA hydrogel with excellent mechanical strength and biocompatibility. Using CAM-HA hydrogels, we were able to deliver cell that was superior to HA hydrogel in chondrogenesis. This hydrogel can be used as a functional filler for injectable cartilage tissue delivery and maintained in the long term, providing a safe and effective alternative to traditional fillers. These findings demonstrate the authors' development of advanced ECM fillers to meet the growing demand for non-surgical aesthetic procedures.


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