Biofunctionalization and biological evaluation of newly designed biomimetic organic/inorganic multilayer composites as bone replacement materials
Final Report Abstract
As outlined in the initial proposal the idea underlying this project is to prefabricate a novel calcium phosphate polyelectrolyte multilayer composite similar to the matrix produced by bone, in order to reduce the cellular energy contribution to bone growth. The subsequent loading of the novel artificial matrix with bone growth factors would thus enhance bone growth as well as the osteoconductive potential of biomaterials. The general goal of the joint project was therefore focussed on the development of procedures for the biofunctionalization with BMP-2 of the new materials based on the in situ formation of calcium phosphates (crystalline or amorphous) within a structured organic matrix of polyelectrolytes (CaP-PE). To achieve this general goal four major aims were outlined: 1. State of the art preparation and further development of a calcium phosphate polyelectrolyte multilayer composite on titanium as a matrix for biofunctionalization with BMP-2 for the stimulation of bone regeneration. 2. Development of techniques for the functional inclusion of bioactive BMP-2 into the CaP-PE layer either by immobilization or coprecipitation procedures. 3. Development of a controlled release technology for BMP-2 from the artificial matrix allowing defined half-lives of release in the range of weeks or months. 4. Cell biological in vitro evaluation in special consideration of stimulated cell-matrix-material interactions and the induction of biomineralization. The work schedule was therefore subdivided into Seven Tasks, which are the basis of this report. It can be clearly stated that all tasks were fulfilled by the investigators. A significant part of these tasks has been published in two peer reviewed journals and in seven extended reviewed abstracts. The following goals were reached: To 1: The state of the art preparation and development of PEMs was significantly improved by the introduction of the reflectometric interference spectroscopy (RIfS) for the analysis of PEM film growth. In addition it was shown that the highly charged Ca-binding protein phosvitin destabilizes PEMs and therefore has to be utilized carefully. PEMs containing amorphous calcium phosphates were tested for rhBMP-binding for the first time. Force spectroscopic measurements revealed that PEMs containing chondroitin sulphate instead of polyglutamic acid are very soft with a very low Young's modulus and low cell adhesion capacity. To 2: The front line of international research was reached by analyzing the binding of rhBMP-2 to PEMs by isotope labelling of rhBMP-2 with 125I. It could be demonstrated for the first time that rhBMP-2 binding increases exponentially with the number of PEM double layers. Contrary to expectations the inclusion of amorphous calcium phosphates in the PEMs only had a negligible influence on rhBMP-2 binding. In addition the initial kinetics of rhBMP-2 adsorption were analyzed by evanescent wave technology for the first time, demonstrating a double exponential binding reaction with a mean half –life of only ~ 50 s. To 3: Utilizing the isotope labelling procedure the controlled release technology could be demonstrated as a new standard. For the first time it was shown that the release of rhBMP-2 is a double exponential function with a half-life for the burst phase (15% of total release) of t1/2 ~ 1.5.-2.3 hours and a half-life for the sustained release phase (85% of total release) of t1/2 ~ 34-38 days. To 4: Cell biological in vitro evaluation of MC3T3-E1 cells was performed by both groups yielding similar results and indicating a low cell adhesion capacity on the evaluated PEMs. Cross-linking experiments with the employed condroitin sulphate containing PEMS showed a reduced rhBMP-2 binding capacity which excluded this technology in the present project. rhBMP-2 immobilized within PEMS was shown to be highly active. The relative expression of ALP, osteocalcin and osteonectin was 10 to 200 fold overexpressed by the rhBMP-2 containing PEMs as compared to the untreated control cells. Conclusion: Very important successful steps toward achieving the general goal of the joint project in the development of procedures for the biofunctionalization of PEMs with BMP-2 were taken. These internationally relevant results clearly warrant an extension and conclusion of the project in order to perform animal experiments and to complete the experiments.
Publications
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(2010). Cell biological investigations on BMP-2 modified polyelectrolyte multilayer films. Biomaterialien, 11 (S1), 108
Grohmann, S., Eisenhuth, S., Rothe, H., Zurlinden, K., Jennissen, H. P., Füredi-Milhofer, H., & Liefeith, H.
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(2010). Dependence of rhBMP-2 binding on the number of layer pairs in polyelectrolyte multilayers. Biomaterialien, 11 (S1), 62
Madenci, S., Laub, M., & Jennissen, H. P.
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(2010). Immobilization of rhBMP-2 on glass surfaces: Evanescent wave kinetics and binding. Biomaterialien, 11, 142-143
Meißner, M., Zurlinden, K., Laub, M., Fischer, H., Lindner, K., Koczur, K., Kirsten, A., Oliviera, C., Seifert, G., Gemming, S., Müller-Mai, C., & Jennissen, H. P.
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(2010). In vivo comparison of osteoinduction by glycosylated and non-glycosylated rhBMP-2. Biomaterialien, 11 (S1), 102
Zurlinden, K., Laub, M., Moser, D., Spassova, E., Ewers, R., & Jennissen, H. P.
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(2010). On the definition of biomaterials. Biomaterialien, 11 (S1), 110
Jennissen, H. P.
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(2010). Polyelectrolyte multilayers on glass as a model system for the immobilization of rhBMP-2. Biomaterialien, 11 (S1), 103
Lange, M., Madenci, S., Piontek, J., Laub, M., Grohmann, S., Liefeith, H., Füredi-Milhofer, H., & Jennissen, H. P.
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(2010). Real-time kinetics of the binding of rhBMP-2 to polyelectrolyte multilayers on quartz glass. Biomaterialien, 11 (S1), 63
Meißner, M., Madenci, S., & Jennissen, H. P.
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(2011). Biomimetic assembly of polyelectrolyte multilayers containing phosvitin monitored with reflectometric interference spectroscopy. Biointerphases., 6, 54-62
Grohmann, S., Rothe, H., Eisenhuth, S., Hoffmann, C., & Liefeith, K.
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(2011). Colloidal force spectroscopy and cell biological investigations on biomimetic polyelectrolyte multilayer coatings composed of chondroitin sulfate and heparin. Biomacromolecules, 12, 1987-1997
Grohmann, S., Rothe, H., Frant, M., & Liefeith, K.
