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The biological and physical performance of high strength dicalcium phosphate cement in physiologically relevant models
Uppsala University, Disciplinary Domain of Science and Technology, Technology, Department of Engineering Sciences, Applied Materials Sciences. Uppsala University. (Materials in Medicine)ORCID iD: 0000-0001-7004-2853
2017 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The chemical properties of calcium phosphate cements (CPCs) are very similar to the mineral phase of bone. CPCs are, consequently, very effective substrates (scaffolds) for tissue engineering; bone and stem cells attach readily, and can proliferate and differentiate to form new bone tissue. Unlike other CPCs that may remain largely unchanged in the body for years, such as hydroxyapatite, dicalcium phosphates are remodelled by the body and rapidly converted to new bone. Unfortunately, the dicalcium phosphates are also typically too weak to support load bearing in the human body. Our laboratory has recently developed a novel, high strength brushite CPC, (hsCPC), which can reach 10-50 fold higher failure strength than many commercially available CPCs. The aim of this thesis was to investigate the physical, chemical and biological performance of hsCPCs in physiologically relevant model of drug release, load bearing, osteoconductivity, and as a scaffold for bone tissue engineering.

Multiple CPCs were compared in a model of screw augmentation to determine whether the physical properties of the cement, such as bulk strength and porosity, affected orthopedic screw holding strength. In an in vitro model of bone regeneration stem cells were grown on macroporous scaffolds that were fabricated from hsCPC. Drug releasing scaffolds were fabricated to examine whether the low porosity of hsCPC impeded drug release during a 4 week incubation period. The biological activity of an incorporated drug, Rebamipide, was examined after acute and chronic incubation periods. In the drug release study it was noted that the biological response to hsCPC was significantly better than tissue culture grade polystyrene, even in groups without drug. The mechanism underlying this biological response was further investigated by testing the effect of pyrophosphate, a common cement additive, on bone cell proliferation and differentiation. This thesis concludes that a high strength cement can produce significant improvement in screw augmentation strength, if there is sufficient cortical bone near the augmentation site. The hsCPC is also cytocompatible, and can support bone and stem cell proliferation and differentiation. hsCPC scaffolds stimulated osteogenic gene expression comparable to native bone scaffolds. hsCPC scaffolds are also capable of delivering drug for up to 4 weeks, in vitro. Finally, a cement additive, pyrophosphate, stimulated differentiation, but not proliferation of bone cells.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2017. , p. 78
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 1502
Keywords [en]
biomaterial, bioceramic, osteoinduction, calcium phosphate, cement, osteoblast, pyrophosphate, Rebamipide, drug delivery, screw augmentation
National Category
Medical Materials
Research subject
Engineering Science with specialization in Materials Science
URN: urn:nbn:se:uu:diva-319495ISBN: 978-91-554-9885-6 (print)OAI:, id: diva2:1087377
Public defence
2017-06-02, Å2001, Ångströmlaboratoriet, Lägerhyddsvägen 1, Uppsala, 09:15 (English)
Swedish Research Council, GA 621-2011-3399EU, FP7, Seventh Framework Programme, 262948Available from: 2017-05-05 Created: 2017-04-06 Last updated: 2017-08-09Bibliographically approved
List of papers
1. Rebamipide Delivered by Brushite Cement Enhances Osteoblast and Macrophage Proliferation
Open this publication in new window or tab >>Rebamipide Delivered by Brushite Cement Enhances Osteoblast and Macrophage Proliferation
2015 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 10, no 5Article in journal (Refereed) Published
Abstract [en]

Many of the bioactive agents capable of stimulating osseous regeneration, such as bone morphogenetic protein-2 (BMP-2) or prostaglandin E2 (PGE2), are limited by rapid degradation, a short bioactive half-life at the target site in vivo, or are prohibitively expensive to obtain in large quantities. Rebamipide, an amino acid modified hydroxylquinoline, can alter the expression of key mediators of bone anabolism, cyclo-oxygenase 2 (COX-2), BMP-2 and vascular endothelial growth factor (VEGF), in diverse cell types such as mucosal and endothelial cells or chondrocytes. The present study investigates whether Rebamipide enhances proliferation and differentiation of osteoblasts when delivered from brushite cement. The reactive oxygen species (ROS) quenching ability of Rebampide was tested in macrophages as a measure of bioactivity following drug release incubation times, up to 14 days. Rebamipide release from brushite occurrs via non-fickian diffusion, with a rapid linear release of 9.70%+/- 0.37% of drug per day for the first 5 days, and an average of 0.5%-1% per day thereafter for 30 days. Rebamipide slows the initial and final cement setting time by up to 3 and 1 minute, respectively, but does not significantly reduce the mechanical strength below 4% (weight percentage). Pre-osteoblast proliferation increases by 24% upon exposure to 0.4uM Rebamipide, and by up to 73% when Rebamipide is delivered via brushite cement. Low doses of Rebamipide do not adversely affect peak alkaline phosphatase activity in differentiating pre-osteoblasts. Rebamipide weakly stimulates proliferation in macrophages at low concentrations (118 +/- 7.4% at 1uM), and quenches ROS by 40-60%. This is the first investigation of Rebamipide in osteoblasts.

National Category
Other Engineering and Technologies
urn:nbn:se:uu:diva-257022 (URN)10.1371/journal.pone.0128324 (DOI)000355319400083 ()26023912 (PubMedID)
Available from: 2015-06-29 Created: 2015-06-29 Last updated: 2018-02-08Bibliographically approved
2. Fabrication of rnacroporous cement scaffolds using PEG particles: In vitro evaluation with induced pluripotent stem cell-derived mesenchymal progenitors
Open this publication in new window or tab >>Fabrication of rnacroporous cement scaffolds using PEG particles: In vitro evaluation with induced pluripotent stem cell-derived mesenchymal progenitors
Show others...
2016 (English)In: Materials science & engineering. C, biomimetic materials, sensors and systems, ISSN 0928-4931, E-ISSN 1873-0191, Vol. 69, p. 640-652Article in journal (Refereed) Published
Abstract [en]

Calcium phosphate cements (CPCs) have been extensively used in reconstructive dentistry and orthopedics, but it is only recently that CPCs have been combined with stem cells to engineer biological substitutes with enhanced healing potential. In the present study, macroporous CPC scaffolds with defined composition were fabricated using an easily reproduced synthesis method, with minimal fabrication and processing steps. Scaffold pore size and porosity, essential for cell infiltration and tissue ingrowth, were tuned by varying the content and size of polyethylene glycol (PEG) particles, resulting in 9 groups with different architectural features. The scaffolds were characterized for chemical composition, porosity and mechanical properties, then tested in vitro with human mesenchymal progenitors derived from induced pluripotent stem cells (iPSC-MPs). Biomimetic decellularized bone scaffolds were used as reference material in this study. Our manufacturing process resulted in the formation of macroporous monetite scaffolds with no residual traces of PEG. The size and content of PEG particles was found to affect scaffold porosity, and thus mechanical properties. Irrespective of pore size and porosity, the CPC scaffolds fabricated in this study supported adhesion and viability of human iPSC-MPs similarly to decellularized bone scaffolds. However, the architectural features of the scaffolds were found to affect the expression of bone specific genes, suggesting that specific scaffold groups could be more suitable to direct human iPSC-MPs in vitro toward an osteoblastic phenotype. Our simplistic fabrication method allows rapid, inexpensive and reproducible construction of macroporous CPC scaffolds with tunable architecture for potential use in dental and orthopedic applications.

Bioinaterial scaffold, Calcium phosphate cement, Macroporosity, Mesenchymal progenitors, Induced pluripotent stem cells, Tissue engineering
National Category
Materials Engineering
urn:nbn:se:uu:diva-306232 (URN)10.1016/j.msec.2016.06.075 (DOI)000383930900076 ()27612757 (PubMedID)
EU, FP7, Seventh Framework Programme, 262948
Available from: 2016-10-27 Created: 2016-10-26 Last updated: 2018-02-08Bibliographically approved
3. Pyrophosphate Stimulates Differentiation, Matrix Gene Expression and Alkaline Phosphatase Activity in Osteoblasts
Open this publication in new window or tab >>Pyrophosphate Stimulates Differentiation, Matrix Gene Expression and Alkaline Phosphatase Activity in Osteoblasts
Show others...
2016 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 11, no 10, article id e0163530Article in journal (Refereed) Published
Abstract [en]

Pyrophosphate is a potent mitogen, capable of stimulating proliferation in multiple cell types, and a critical participant in bone mineralization. Pyrophosphate can also affect the resorption rate and bioactivity of orthopedic ceramics. The present study investigated whether calcium pyrophosphate affected proliferation, differentiation and gene expression in early (MC3T3 pre-osteoblast) and late stage (SAOS-2 osteosarcoma) osteoblasts. Pyrophosphate stimulated peak alkaline phosphatase activity by 50% and 150% at 100 mu M and 0.1 mu M in MC3T3, and by 40% in SAOS-2. The expression of differentiation markers collagen 1 (COL1), alkaline phosphatase (ALP), osteopontin (OPN), and osteocalcin (OCN) were increased by an average of 1.5, 2, 2 and 3 fold, by high concentrations of sodium pyrophosphate (100 mu M) after 7 days of exposure in MC3T3. COX-2 and ANK expression did not differ significantly from controls in either treatment group. Though both high and low concentrations of pyrophosphate stimulate ALP activity, only high concentrations (100 mu M) stimulated osteogenic gene expression. Pyrophosphate did not affect proliferation in either cell type. The results of this study confirm that chronic exposure to pyrophosphate exerts a physiological effect upon osteoblast differentiation and ALP activity, specifically by stimulating osteoblast differentiation markers and extracellular matrix gene expression.

National Category
Biomaterials Science Engineering and Technology
urn:nbn:se:uu:diva-307554 (URN)10.1371/journal.pone.0163530 (DOI)000385696900011 ()27701417 (PubMedID)
Available from: 2016-11-17 Created: 2016-11-17 Last updated: 2018-02-08Bibliographically approved
4. Influence of cement compressive strength and porosity on augmentation performance in a model of orthopedic screw pull-out
Open this publication in new window or tab >>Influence of cement compressive strength and porosity on augmentation performance in a model of orthopedic screw pull-out
Show others...
2018 (English)In: Journal of The Mechanical Behavior of Biomedical Materials, ISSN 1751-6161, E-ISSN 1878-0180, Vol. 77, p. 624-633Article in journal (Refereed) Published
Abstract [en]

Disease and injuries that affect the skeletal system may require surgical intervention and internal fixation, i.e. orthopedic plate and screw insertion, to stabilize the injury and facilitate tissue repair. If the surrounding bone quality is poor the screws may migrate, or the bone may fail, resulting in screw pull-out. Though numerous studies have shown that cement augmentation of the interface between bone and implant can increase screw holding strength in bone, the physical properties of cement that influence pull-out force have not been investigated. The present study sought to determine how the physical properties of calcium phosphate cements (CPCs), and the strength of the biological or synthetic material surrounding the augmented screw, affected the corresponding orthopedic screw pull-out force in urethane foam models of healthy and osteoporotic bone (Sawbones). In the simplest model, where only the bond strength between screw thread and cement (without Sawbone) was tested, the correlation between pull-out force and cement compressive strength (R2 = 0.79) was weaker than correlation with total cement porosity (R2 = 0.89). In open pore Sawbone that mimics “healthy” cancellous bone density the stronger cements produced higher pull-out force (50-60% increase). Higher strength, lower porosity, cements also produced higher pull-out forces (50-190% increase) in Sawbones with cortical fixation if the failure strength of the cortical material was similar to (bovine tibial bone), or greater than (metal shell), actual cortical bone. This result is of particular clinical relevance where fixation with a metal plate implant is indicated, as the nearby metal can simulate a thicker cortical shell and, thereby, increase the pull-out force of screws augmented with stronger cements. The improvement in pull-out force was apparent even at low augmentation volumes of 0.5 ml (50% increase), which suggest that in clinical situations where augmentation volume is limited the stronger, lower porosity CPCs may still produce a significant improvement in screw holding strength. When correlations of all the tested models were compared both cement porosity and compressive strength accurately predicted pull-out force (R2=1.00, R2=0.808), though prediction accuracy depended upon the strength of the material surrounding the Sawbone. The correlations strength was low for bone with no, or weak, cortical fixation. Higher strength and lower porosity CPCs also produced greater pull-out force (1-1.5 kN) than commercial CPC (0.2-0.5kN), but lower pull-out force than PMMA (2-3 kN). The results of this study suggest that the likelihood of screw fixation failure may be reduced by selecting calcium phosphate cements with lower porosity and higher bulk strength, in patients with healthy bone mineral density and/or sufficient cortical thickness. This is of particular clinical relevance when fixation with metal plates is indicated, or where the augmentation volume is limited.

Place, publisher, year, edition, pages
Elsevier, 2018
Screw pull-out, Orthopedic screw augmentation, Calcium phosphate cement, Bioceramic, Bone biomechanics, Sawbones, Cortical fixation
National Category
Medical Materials Ceramics Applied Mechanics Biomaterials Science
Research subject
Engineering Science with specialization in Materials Science
urn:nbn:se:uu:diva-320157 (URN)10.1016/j.jmbbm.2017.10.016 (DOI)000418309500073 ()29100205 (PubMedID)
Swedish Research Council, 621–2011-3399EU, FP7, Seventh Framework Programme, 262948
Available from: 2017-04-16 Created: 2017-04-16 Last updated: 2018-02-16Bibliographically approved

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