(M1330-04-22) Controlling the Release of Copper from 3D Printed Calcium Phosphate Scaffolds for Bone Regeneration

Purpose: Bone grafts have found widespread use in the treatment of non-union fractures due to their ability to temporarily establish mechanical stability in the defect site as well as their ability to engender the growth of new bone tissue. The formation of hydroxyapatite (HA), a prominent mineral present in the bone from calcium phosphate cements (CPCs) when in contact with water has shown promising potential to be used as bone replacement materials. In regions of damage, these CPC cements possess the ability to provide mechanical integrity to the bone during repair. Further, 3D bioprinting provides the capability to curate the graft to the defect site allowing for optimal osteointegration. However, angiogenesis is a key aspect for successful repair to the defect site for optimal osteointegration of the graft. Copper is a mineral well investigated for its role in promoting angiogenesis which is essential for tissue growth or repair. The release of copper from the scaffolds in a sustained manner can provide long term bone regeneration affects. In this study copper was utilized in two ways: 1. The calcium phosphate scaffolds were directly seeded with copper and 2. The copper was loaded into poly lactic co glycolic acid (PLGA) microparticles which were later loaded into the CPC scaffolds. The present study ultimately investigates the ability of controlled delivery of copper ions using 3D printed copper loaded calcium phosphate scaffolds.

Methods: Copper nanoparticles and PLGA copper microparticles were prepared and loaded into the calcium phosphate cements. The cements were then printed using a bioprinter and set by exposure to humidity. The copper loaded CPC scaffolds were then tested for its porosity using scanning electron microscopy. Further the interactions of copper with the calcium phosphate were analyzed using Fourier transform infrared microscopy. Mechanical test characterizations were conducted using a texture analyzer. In vitro release was conducted in DPBS at 37˚C over a period of 2 weeks. The samples were further analyzed for copper release using inductively coupled plasma mass spectrometry. Finally, cell viability studies were conducted on the copper loaded scaffolds using mesenchymal stem cells for a period of two weeks using calcium phosphate scaffolds without copper loading as the control.

Results: First we prepared copper loaded calcium phosphate scaffolds from which we got the pore diameter to be and the strut diameter to be (Figure 1.). Further, copper loaded PLGA microparticles were successfully prepared. Further the copper loaded scaffolds as well as the PLGA loaded scaffolds were successfully prepared and printed. Further SEM images showed varied porosities in the scaffolds (Figure 2.). FTIR studies showed no significant interaction between copper, PLGA and calcium phosphate scaffolds (Figure 3.). The in vitro release studies displayed the potential of these scaffolds to release copper over sustained periods.

Conclusion: From the results it was clear that calcium phosphate cements can be used for the delivery of copper to the defect site to promote angiogenesis. This approach to provide a sustained release scaffolds for bone regeneration could have future applications to treat bone cancer patients.

References: 1. Finney, L., Vogt, S., Fukai, T., Glesne, D., 2009. Copper and Angiogenesis: Unravelling a Relationship Key to Cancer Progression. Clin. Exp. Pharmacol. Physiol. 36, 88–94. https://doi.org/10.1111/j.1440-1681.2008.04969.x
2. Ginebra, M.-P., Canal, C., Espanol, M., Pastorino, D., Montufar, E.B., 2012. Calcium phosphate cements as drug delivery materials. Adv. Drug Deliv. Rev. 64, 1090–1110. https://doi.org/10.1016/j.addr.2012.01.008
3. Ginebra, M.P., Traykova, T., Planell, J.A., 2006. Calcium phosphate cements as bone drug delivery systems: A review. J. Controlled Release 113, 102–110. https://doi.org/10.1016/j.jconrel.2006.04.007
4. Mroczek-Sosnowska, N., Sawosz, E., Vadalasetty, K.P., Łukasiewicz, M., Niemiec, J., Wierzbicki, M., Kutwin, M., Jaworski, S., Chwalibog, A., 2015. Nanoparticles of Copper Stimulate Angiogenesis at Systemic and Molecular Level. Int. J. Mol. Sci. 16, 4838–4849. https://doi.org/10.3390/ijms16034838


Figure 1. Optical images of 3D printed calcium phosphate and copper loaded calcium phosphate scaffolds with uniform pore size and strut diameter


Figure 2. SEM images of the printed scaffolds. a) Calcium phosphate cement before cell seeding b) Copper loaded calcium phosphate cement scaffold c) Calcium phosphate scaffold after 24h incubation in serum media d) Copper loaded calcium phosphate scaffold after 24h incubation in serum media


Figure 3. FTIR spectra showing characteristic bands of calcium phosphate and copper loaded calcium phosphate scaffolds