Poster BB2 - Effect of copper- and zinc-doped bioactive glass biopolymeric scaffolds for bone tissue engineering applications utilizing RUNX-2 and osteocalcin reporters: an in vitro study

Introduction:

Introduction:
Bone grafts are by far the most utilized solid organ transplantation worldwide. The bone graft market, which includes various types of grafts such as autografts, allografts, and synthetic grafts, was valued at USD 4.46 billion in 2022 and is expected to reach USD 6.69 billion by 2030. Lack of donor graft tissue is a perennial problem that could be alleviated using tissue-engineered material. Bone tissue engineering (BTE) involves a multifaceted and dynamic process that focuses on developing methods and techniques to regenerate bone and enhance its function within the body. This approach is designed to mitigate the complications and obstacles associated with alternative treatments like allografts or autografts. Within BTE, 3D scaffolds play a pivotal role by acting as an external extracellular matrix (ECM) for cell attachment and promoting the formation of bone tissue within the body. Our study employed a unique methodology. We used engineered MSCs to non-invasively report on bone differentiation events in real-time. This allowed us to test the effectiveness of incorporating bioactive bioglass that delivers copper or zinc into biopolymeric scaffolds. Bioactive glass has been increasingly utilized in tissue engineering because of its exceptional biocompatibility, appropriate mechanical properties, and controllable biodegradation. Further, bioglass can be adapted to deliver biologically active substances. Copper and zinc are essential trace elements involved in many physiological activities and also bone tissue engineering applications.

Materials and

Methods: Materials and

Methods:
Our research utilized a novel method to create composite porous tissue engineering scaffolds. These scaffolds consisted of biopolymers like gelatin and alginate of optimized concentration, along with borosilicate bioactive glass nanoparticles (of base composition in wt%: 37.5 SiO2, 22.6 CaO, 5.9 Na2O, 4.0 P2O5, 12.0 K2O, 5.5 MgO, 12.5 B2O3) zinc and copper bioglass formulations. The scaffolds were crosslinked with calcium chloride and then lyophilized. We conducted comprehensive physico-chemical characterization studies of the scaffolds and in vitro experiments using hMSCs engineered to express luciferase under the control of RUNX-2 and Osteocalcin promoters. First, the experiments were performed only with the cultured reporter cells in osteogenic differentiation media (specific amounts of ascorbic acid, dexamethasone and β-glycerol phosphate) and a standard medium. Later, experiments were conducted to evaluate the bone-forming ability on scaffolds with and without bioactive glass based on the release of luciferase in luminescence units.

Results, Conclusions, and Discussions: Results
Our study showed that RUNX-2 reporter cells treated with osteogenic medium had significantly higher expression of reporter genes compared to those treated with normal medium on day 7. This expression decreased by day 10 in both types of medium (see Fig 1). We used WST-8 assay to test the compatibility of bioactive glass with hMSCs on day 3 and day 7. The results showed that when the concentration of bioactive glass was below 5% (m/v), it did not compromise cell viability and improved degradation rates. The functionalized scaffolds enhanced osteogenic differentiation by activating the RUNX2 and osteocalcin reporters. Importantly, the presence of Zn++ and Cu++ ions significantly improved the maintenance of the osteogenic phenotype, as indicated by the sustained activity of the RUNX2 and osteocalcin reporters, compared to scaffolds without bioactive glass. The most significant effect on the activation of both reporters was observed with the bioactive glass formulations doped with Cu.

Conclusion:
Cells grown on porous biopolymeric scaffolds functionalized with Cu++ and Zn++ ion-dispersing bioactive glass showed enhanced and sustained osteogenic activity based on the activation of the Runx2 and Osteocalcin reporters. These novel scaffold formulations have significant applications as a bone graft substitute and/or for tissue engineering applications.

Discussion:
The use of noninvasive/non-destructive molecular reporters in these experiments demonstrated the utility of these sensors to monitor critical tissue engineering outcomes and their potential utility for feedback control of the engineering process. They will thus help improve the manufacturing of bone graft substitutes by tissue engineering. Our findings further suggest that the incorporation of bioactive glass into tissue engineering scaffolds can enhance the osteogenic potential of cells, paving the way for the development of more effective bone graft substitutes and tissue engineering strategies.

Acknowledgements (Optional): This work was funded in part by a grant from NIH/NIBIB EB021911 (Welter)