Human Osteoblast-Derived Extracellular Matrix with High Homology to Bone Proteome Is Osteopromotive

Efficient osteogenic differentiation of mesenchymal stromal cells (MSCs) is crucial to accelerate bone formation. In this context, the use of extracellular matrix (ECM) as natural 3D framework mimicking in vivo tissue architecture is of interest. The aim of this study was to generate a devitalized human osteogenic MSC-derived ECM and to investigate its impact on MSC osteogenic differentiation to improve MSC properties in bone regeneration. The devitalized ECM significantly enhanced MSC adhesion and proliferation. Osteogenic differentiation and mineralization of MSCs on the ECM were quicker than in standard conditions. The presence of ECM promoted in vivo bone formation by MSCs in a mouse model of ectopic calcification. We analyzed the ECM composition by mass spectrometry, detecting 846 proteins. Of these, 473 proteins were shared with the human bone proteome we previously described, demonstrating high homology to an in vivo microenvironment. Bioinformatic analysis of the 846 proteins showed involvement in adhesion and osteogenic differentiation, confirming the ECM composition as key modulator of MSC behavior. In addition to known ECM components, proteomic analysis revealed novel ECM functions, which could improve culture conditions. In sum

Multifunctional implants Prevention is better than cure

Millions of people around the globe receive orthopedic implants every year. These implants help people to regain their mobility and contribute tremendously to improve the quality of life. However, a significant number of patients suffer from complications, such as implant associated infections (IAI) and aseptic loosening. The number of orthopedic implants is expected to increase due to an aging and increasingly obese population. As a result, the number of complications will rise too. In addition, the treatment of IAI is complicated by the development of antibiotic resistant bacteria. The focus of researchers has, therefore, shifted more and more towards the prevention of complications. In the words of Desiderius Erasmus: “Prevention is better than cure.”Biomaterials & Tissue Biomechanic

Antibacterial Titanium Implants Biofunctionalized by Plasma Electrolytic Oxidation with Silver, Zinc, and Copper: A Systematic Review

Patients receiving orthopedic implants are at risk of implant-associated infections (IAI). A growing number of antibiotic-resistant bacteria threaten to hamper the treatment of IAI. The focus has, therefore, shifted towards the development of implants with intrinsic antibacterial activity to prevent the occurrence of infection. The use of Ag, Cu, and Zn has gained momentum as these elements display strong antibacterial behavior and target a wide spectrum of bacteria. In order to incorporate these elements into the surface of titanium-based bone implants, plasma electrolytic oxidation (PEO) has been widely investigated as a single-step process that can biofunctionalize these (highly porous) implant surfaces. Here, we present a systematic review of the studies published between 2009 until 2020 on the biomaterial properties, antibacterial behavior, and biocompatibility of titanium implants biofunctionalized by PEO using Ag, Cu, and Zn. We observed that 100% of surfaces bearing Ag (Ag-surfaces), 93% of surfaces bearing Cu (Cu-surfaces), 73% of surfaces bearing Zn (Zn-surfaces), and 100% of surfaces combining Ag, Cu, and Zn resulted in a significant (i.e., >50%) reduction of bacterial load, while 13% of Ag-surfaces, 10% of Cu-surfaces, and none of Zn or combined Ag, Cu, and Zn surfaces reported cytotoxicity against osteoblasts, stem cells, and immune cells. A majority of the studies investigated the antibacterial activity against S. aureus. Important areas for future research include the biofunctionalization of additively manufactured porous implants and surfaces combining Ag, Cu, and Zn. Furthermore, the antibacterial activity of such implants should be determined in assays focused on prevention, rather than the treatment of IAIs. These implants should be tested using appropriate in vivo bone infection models capable of assessing whether titanium implants biofunctionalized by PEO with Ag, Cu, and Zn can contribute to protect patients against IAI.Biomaterials & Tissue Biomechanic

Emergent collective organization of bone cells in complex curvature fields

Individual cells and multicellular systems respond to cell-scale curvatures in their environments, guiding migration, orientation, and tissue formation. However, it remains largely unclear how cells collectively explore and pattern complex landscapes with curvature gradients across the Euclidean and non-Euclidean spectra. Here, we show that mathematically designed substrates with controlled curvature variations induce multicellular spatiotemporal organization of preosteoblasts. We quantify curvature-induced patterning and find that cells generally prefer regions with at least one negative principal curvature. However, we also show that the developing tissue can eventually cover unfavorably curved territories, can bridge large portions of the substrates, and is often characterized by collectively aligned stress fibers. We demonstrate that this is partly regulated by cellular contractility and extracellular matrix development, underscoring the mechanical nature of curvature guidance. Our findings offer a geometric perspective on cell-environment interactions that could be harnessed in tissue engineering and regenerative medicine applications.</p

Inorganic Agents for Enhanced Angiogenesis of Orthopedic Biomaterials

Aseptic loosening of a permanent prosthesis remains one of the most common reasons for bone implant failure. To improve the fixation between implant and bone tissue as well as enhance blood vessel formation, bioactive agents are incorporated into the surface of the biomaterial. This study reviews and compares five bioactive elements (copper, magnesium, silicon, strontium, and zinc) with respect to their effect on the angiogenic behavior of endothelial cells (ECs) when incorporated on the surface of biomaterials. Moreover, it provides an overview of the state-of-the-art methodologies used for the in vitro assessment of the angiogenic properties of these elements. Two databases are searched using keywords containing ECs and copper, magnesium, silicon, strontium, and zinc. After applying the defined inclusion and exclusion criteria, 59 articles are retained for the final assessment. An overview of the angiogenic properties of five bioactive elements and the methods used for assessment of their in vitro angiogenic potential is presented. The findings show that silicon and strontium can effectively enhance osseointegration through the simultaneous promotion of both angiogenesis and osteogenesis. Therefore, their integration onto the surface of biomaterials can ultimately decrease the incidence of implant failure due to aseptic loosening.Biomaterials & Tissue Biomechanic

Data on the surface morphology of additively manufactured Ti-6Al-4V implants during processing by plasma electrolytic oxidation

textabstractAdditively manufactured Ti-6Al-4V implants were biofunctionalized using plasma electrolytic oxidation. At various time points during this process scanning electron microscopy imaging was performed to analyze the surface morphology (van Hengel et al., 2017) [1]. This data shows the changes in surface morphology during plasma electrolytic oxidation. Data presented in this article are related to the research article “Selective laser melting porous metallic implants with immobilized silver nanoparticles kill and prevent biofilm formation by methicillin-resistant Staphylococcus aureus” (van Hengel et al., 2017) [1]

The effects of plasma electrolytically oxidized layers containing Sr and Ca on the osteogenic behavior of selective laser melted Ti6Al4V porous implants

Surface biofunctionalization is frequently applied to enhance the functionality and longevity of orthopedic implants. Here, we investigated the osteogenic effects of additively manufactured porous Ti6Al4V implants whose surfaces were biofunctionalized using plasma electrolytic oxidation (PEO) in Ca/P-based electrolytes with or without strontium. Various levels of Sr and Ca were incorporated in the oxide layers by using different current densities and oxidation times. Increasing the current density and oxidation time resulted in thicker titanium oxide layers and enhanced the release of Ca2+ and Sr2+. Biofunctionalization with strontium resulted in enhanced pore density, a thinner TiO2 layer, four-fold reduced release of Ca2+, and mainly anatase phases as compared to implants biofunctionalized in electrolytes containing solely Ca/P species under otherwise similar conditions. Different current densities and oxidation times significantly increased the osteogenic differentiation of MC3T3-E1 cells on implants biofunctionalized with strontium, when the PEO treatment was performed with a current density of 20 A/dm2 for 5 and 10 min as well as for a current density of 40 A/dm2 for 5 min. Therefore, addition of Sr in the PEO electrolyte and control of the PEO processing parameters represent a promising way to optimize the surface morphology and osteogenic activity of future porous AM implants.Biomaterials & Tissue Biomechanic

Osteogenic and antibacterial surfaces on additively manufactured porous Ti-6Al-4V implants: Combining silver nanoparticles with hydrothermally synthesized HA nanocrystals

The recently developed additively manufacturing techniques have enabled the fabrication of porous biomaterials that mimic the characteristics of the native bone, thereby avoiding stress shielding and facilitating bony ingrowth. However, aseptic loosening and bacterial infection, as the leading causes of implant failure, need to be further addressed through surface biofunctionalization. Here, we used a combination of (1) plasma electrolytic oxidation (PEO) using Ca-, P-, and silver nanoparticle-rich electrolytes and (2) post-PEO hydrothermal treatments (HT) to furnish additively manufactured Ti-6Al-4V porous implants with a multi-functional surface. The applied HT led to the formation of hydroxyapatite (HA) nanocrystals throughout the oxide layer. This process was controlled by the supersaturation of Ca2+ and PO43− during the hydrothermal process. Initially, the high local supersaturation resulted in homogenous nucleation of spindle-like nanocrystals throughout the surface. As the process continued, the depletion of reactant ions in the outermost surface layer led to a remarkable decrease in the supersaturation degrees. High aspect-ratio nanorods and hexagonal nanopillars were, therefore, created. The unique hierarchical structure of the microporous PEO layer (pore size &lt; 3 μm) and spindle-like HA nanocrystals (&lt;150 nm) on the surface of macro-porous additively manufactured Ti-6Al-4V implants provided a favorable substrate for the anchorage of cytoplasmic extensions assisting cell attachment and migration on the surface. The results of our in vitro assays clearly showed the important benefits of the HT and the spindle-like HA nanocrystals including a significantly stronger and much more sustained antibacterial activity, significantly higher levels of pre-osteoblasts metabolic activity, and significantly higher levels of alkaline phosphatase activity as compared to similar PEO-treated implants lacking the HT.</p

Functionality-packed additively manufactured porous titanium implants

The holy grail of orthopedic implant design is to ward off both aseptic and septic loosening for long enough that the implant outlives the patient. Questing this holy grail is feasible only if orthopedic biomaterials possess a long list of functionalities that enable them to discharge the onerous task of permanently replacing the native bone tissue. Here, we present a rationally designed and additive manufacturing (AM) topologically ordered porous metallic biomaterial that is made from Ti-6Al-4V using selective laser melting and packs most (if not all) of the required functionalities into a single implant. In addition to presenting a fully interconnected porous structure and form-freedom that enables realization of patient-specific implants, the biomaterials developed here were biofunctionalized using plasma electrolytic oxidation to locally release both osteogenic (i.e. strontium) and antibacterial (i.e. silver ions) agents. The same single-step biofunctionalization process also incorporated hydroxyapatite into the surface of the implants. Our measurements verified the continued release of both types of active agents up to 28 days. Assessment of the antibacterial activity in vitro and in an ex vivo murine model demonstrated extraordinarily high levels of bactericidal effects against a highly virulent and multidrug-resistant Staphylococcus aureus strain (i.e. USA300) with total eradication of both planktonic and adherent bacteria. This strong antibacterial behavior was combined with a significantly enhanced osteogenic behavior, as evidenced by significantly higher levels of alkaline phosphatase (ALP) activity compared with non-biofunctionalized implants. Finally, we discovered synergistic antibacterial behavior between strontium and silver ions, meaning that 4–32 folds lower concentrations of silver ions were required to achieve growth inhibition and total killing of bacteria. The functionality-packed biomaterial presented here demonstrates a unique combination of functionalities that make it an advanced prototype of future orthopedic biomaterials where implants will outlive patients.</p

Self-defending additively manufactured bone implants bearing silver and copper nanoparticles

Effective preventive measures against implant-associated infection (IAI) are desperately needed. Therefore, the development of self-defending implants with intrinsic antibacterial properties has gained significant momentum. Biomaterials biofunctionalized with silver (Ag) have resulted in effective antibacterial biomaterials, yet regularly induce cytotoxicity. In this study, the use of both Ag and copper (Cu) nanoparticles (NPs) on TiO2 surfaces was investigated to generate antibacterial and osteoconductive biomaterials. Hence, additively manufactured Ti-6Al-4V volume-porous implants were biofunctionalized with plasma electrolytic oxidation (PEO) through the incorporation of varying ratios of Ag and/or Cu NPs in the TiO2 layer covering the implant surface. For all experimental groups, the surface morphology, chemical composition, ion release profile, generation of reactive ion species, antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA) in vitro and ex vivo, as well as the response of pre-osteoblastic MC3T3-E1 cells in metabolic activity and differentiation assays were determined. PEO biofunctionalization resulted in rough and highly porous surfaces that released Ag and Cu ions for 28 days and generated hydroxyl as well as methyl radicals. A strong synergistic bactericidal behavior between Ag and Cu ions was detected, which allowed to decrease the concentration of Ag ions by 10-fold, while maintaining the same level of antibacterial activity. Antibacterial agar diffusion and quantitative assays indicated strong antibacterial activity in vitro for the implants containing Ag and Ag/Cu, while no antibacterial activity was observed for implants bearing only Cu NPs. Moreover, the biofunctionalized implants with ratios of up to 75% Ag and 25% Cu NP totally eradicated all bacteria in an ex vivo model using murine femora. Meanwhile, the biofunctionalized implants did not show any signs of cytotoxicity, while implants bearing only Cu NPs improved the metabolic activity after 7 and 11 days. The biomaterials developed here, therefore, exploit the synergistic behavior of Ag and Cu to simultaneously offer strong antibacterial behavior while fully mitigating the cytotoxicity of Ag against mammalian cells.Biomaterials & Tissue Biomechanic