Combination of pro-angiogenic and bone cell attracting coatings on titanium implants

摘要

Introduction: The aim of this study is to combine the trace elements copper, zinc and strontium with inorganic surface modification techniques already successful in bone applications. The chemotactic signals from released Cu ions are intended to provoke enhanced vascularization (demonstrated in vivo for amounts on the ng scale) while released Zn ions recruit circulating bone forming cells to the site of surgery. The incorporation of strontium is intended for shifting the balance between bone forming and bone degrading cells in bone tissue. Calcium phosphate phases (CPP) are utilized as a carrier for the selected trace elements in order to (ⅰ) provide osteoconductive surfaces and (ⅱ) allow fine tuning of immobilized amounts and release behavior of the trace elements. Experimental Methods: Depositions of brushite onto c.p.Ti discs were performed by means of electrochemically assisted deposition (ECAD) using electrolytes composed of Ca(NO_3)_2 and NH_4H_2PO_4 with additives of copper, zinc or strontium nitrate in different concentrations. The obtained coatings were characterized by (ⅰ) scanning electron microscopy (SEM) combined with energy dispersive X-ray spectroscopy (EDX), (ⅱ) chemical analysis of calcium, phosphate and trace element content. Ion release from coated surfaces was analyzed after incubation in simulated body fluid with or without additional serum proteins. The pro-angiogenic stimulation of human umbilical vein endothelial cells (HUVECs) by copper ions was studied with a proliferation and a 3D angiogenic assay. Results and Discussion: The deposition of the trace elements into brushite layers depended mainly on the concentration of the respective ions in the electrolyte as well as on total charge density and reached values of up to ～50 μg/cm~2 for Zn and Sr and ～13 μg/cm~2 for Cu. For brushite depositions in presence of Cu or Zn discrete phases of brushite plates decorated by Cu and Zn species were observed by SEM and EDX-mapping. This was in contrast to co-depositions within hydroxyapatite (HAp) layers, where Cu was solely co-located with Ca and P within the HAp needles. While Cu did not affect the morphology of the brushite plates, the presence of Zn delayed the brushite crystallization and led to shorter but thicker plates. For strontium some HAp like needles appeared at the edges of the brushite plates which could be assigned to HAp with Sr occupying the Ca places in the crystal lattice. Zn was delivered with a small initial burst followed by a slow steady release irrespective of the presence of proteins. Contrarily, 65 - 85% (depending on initial amount) of the immobilized Cu was delivered in presence of proteins within 24 h. Further, the pro-angiogenic impact of Cu ions could be verified in vitro dependend on the applied ion concentration. Conclusion: It was shown that copper, zinc and strontium can be co-deposited together with calcium phosphate onto metallic implant materials by means of electrochemically assisted deposition with only minor changes in general structure and amount of the deposited calcium phosphate. The relative amounts of the respective trace elements to calcium are tunable in a wide range. Current investigations focus on improving retention of copper within the coatings by including other components like chitosan, and on cell culture studies regarding the regulation of osteoclasts for different strontium contents. The applicability of several combinations of the trace ions is subject of ongoing experiments.