Cell-mediated mineralization of synthetic polyethylene glycol hydrogels to create bone tissue engineering constructs

Abstract

Event Abstract Back to Event Cell-mediated mineralization of synthetic polyethylene glycol hydrogels to create bone tissue engineering constructs Çiğdem Demirkaya1, Evita Willems2, Yoke C. Chai3, 4, Frank P. Luyten3, 4 and Jennifer Patterson2, 3 1 Ege University, Department of Bioengineering, Türkiye 2 KU Leuven, Department of Materials Engineering, Belgium 3 KU Leuven, Prometheus, Division of Skeletal Tissue Engineering, Belgium 4 KU Leuven, Tissue Engineering Laboratory, Skeletal Biology and Engineering Research Center, Belgium Introduction: A key principle of tissue engineering is that the appropriate combination of a biomaterial scaffold, cells, and biological signals can be used to create a living implant, a tissue engineering construct, that will help a damaged tissue to regenerate its lost function[1]. To make a bone tissue engineering construct, we chose to combine: an enzymatically degradable, synthetic hydrogel scaffold based on the Michael-type addition reaction between end-functionalized, multi-arm polyethylene glycol (PEG) and protease-sensitive peptides[2]; human periosteum-derived cells (hPDCs), a progenitor cell type with mesenchymal stem cell characteristics[3]; and in vitro culture with calcium (Ca) and phosphate (P) supplementation in the medium, which has been shown to promote hPDC proliferation, collagen deposition, and mineralization[4]. Interestingly, culture of these hydrogels in the Ca- and P-rich medium led to the deposition of mineral inside the scaffold only when cells were encaspulated in the hydrogels, suggesting a cell-mediated mineratization process. In this study, we examined the behavior of hPDCs throughout the culture period and characterized the mineral that was formed. Materials and Methods: hPDCs were encapsulated in degradable PEG hydrogels that were modified with RGD. The hydrogels, with and without cells, were cultured in growth medium that was or was not supplemented with Ca and P for between 21 and 42 days. In some cases, a two-step procedure was followed where culture in growth medium was followed by Ca- and P-supplementation. Constructs were analyzed to measure cell proliferation (Presto blue), morphology (phalloidin/DAPI), viability (Live/Dead), and differentiation (alkaline phosphatase). To characterize the deposited mineral, nanofocus computed tomography (nanoCT) was used to examine the distribution and volume of mineral, X-ray diffraction (XRD) was used to identify the type of mineral, and a dissolution study was performed at 37°C. Results and Discussion: Ca- and P-supplementation led to the precipitation of a layer of mineral on the surface of the hydrogels, both with and without cells. Intriguigingly, nanoCT analysis (Fig. 1) revealed a punctate, radio-opaque phase inside the hydrogels, but only for samples that contained cells and were incubated in Ca- and P-supplemented medium. Quantitatively, this mineral volume was significantly higher in samples with cells than those without cells. In addition, XRD spectra showed peaks that were consistent with the presence of hydroxyapatite. Moreover, a dissolution study showed that release of Ca and P reached a plateau after 10 days for the cell-free samples whereas hydrogels with cells continued to release Ca and P for over 21 days. Importantly, cells within the hydrogels not only remained viable over the entire culture period, as indicated by a Live/Dead assay (Fig. 2), but they also proliferated, as seen by an increase in Presto blue readings. Additionally, the cells adopted a spread morphology, which was expected given the use of a protease-sensitive and RGD-functionalized PEG hydrogel. Conclusion: We have demonstrated that synthetic hydrogels based on PEG can not only support the proliferation of hPDCs, an osteoprogenitor cell, but can promote cell-mediated mineralization when cultured in medium supplemented with Ca and P. These hydrogels are particularly exciting for tissue engineering because they can be degraded and cleared during the tissue regeneration process. This work is part of Prometheus, the R&D Division of Skeletal Tissue Engineering of the KU Leuven: http://www.kuleuven.be/prometheus.; This work was supported by Research Programme G.0982.11 of the Research Foundation - Flanders (FWO) and the special research fund of the KU Leuven, grant number CREA/13/017.; Yoke Chin Chai was supported by a postdoctoral mandate (1.5.172.13N-Interdisc.) from the Research Foundation - Flanders (FWO).; We acknowledge Abhijith Kudva for assistance with cell culture and Yujing He for assistance with data analysis.