Despite being the most widely used biomaterials in orthopedic surgery, metallic implants do not induce new bone growth because they are bioinert. Surface biofunctionalization of implants with immunomodulatory mediators is a recent approach to promote osteogenic factors that facilitate bone regeneration. Liposomes (Lip) can be used as a low-cost, efficient and simple immunomodulator to stimulate immune cells in favor of bone regeneration. Even though liposomal coating systems have been reported previously, their main disadvantage is their limited ability to preserve liposome integrity after drying. In order to address this issue, we developed a hybrid system in which liposomes could be embedded in a polymeric hydrogel namely gelatin methacryloyl (GelMA). Specifically, we have developed a novel versatile coating strategy using electrospray technology to coat implants with GelMA/Liposome without using adhesive intermediate layer. The two differently charged Lip (i.e., anionic and cationic) were blended with GelMA and coated via electrospray technology on the bone-implant surfaces. The results showed that the developed coating withstood mechanical stress during surgical replacement, and Lip inside GelMA coating stayed intact in different storage conditions for a minimum of 4 weeks. Surprisingly, bare Lip, either cationic or anionic, improved the osteogenesis of human Mesenchymal Stem Cells (MSCs) by inducing pro-inflammatory cytokines, even at a low dosage of Lip released from the GelMA coating. More importantly, we showed that the inflammatory response could be fine-tuned by selecting the Lip concentration, Lip/hydrogel ratio, and coating thickness to determine the timing of the release such that we can accommodate different clinical needs. These promising results pave the way to use these Lip coatings to load different types of therapeutic cargo for bone-implant applications.@en

Cationic host defense peptides (HDPs) are a promising alternative to antibiotics in the fight against Staphylococcus aureus infections. In this study, we investigated the antibacterial and immunomodulatory properties of three HDPs namely IDR-1018, CATH-2, and LL-37. Although all three HDPs significantly inhibited LPS-induced activation of human macrophages, only CATH-2 prevented S. aureus growth. When applied to different infection models focused on intracellularly surviving bacteria, only IDR-1018 showed a consistent reduction in macrophage bacterial uptake. However, this observation did not correlate with an increase in killing the efficiency of intracellular S. aureus. Here, we conclude that despite the promising antibacterial and anti-inflammatory properties of the selected HDPs, macrophages’ intrinsic antibacterial functions were not improved. Future studies should either focus on combining different HDPs or using them synergistically with other antibacterial agents to improve immune cells’ efficacy against S. aureus pathogenesis.

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Additively manufactured (AM) porous metallic biomaterials, in general, and AM porous titanium, in particular, have recently emerged as promising candidates for bone substitution. The porous design of such materials allows for mimicking the elastic mechanical properties of native bone tissue and showed to be effective in improving bone regeneration. It is, however, not clear what role the other mechanical properties of the bulk material such as ductility play in the performance of such biomaterials. In this study, we compared the bone tissue regeneration performance of AM porous biomaterials made from the commonly used titanium alloy Ti6Al4V-ELI with that of commercially pure titanium (CP-Ti). CP-Ti was selected because of its high ductility as compared to Ti6Al4V-ELI. Critical-sized (6 mm diameter) femoral defects in rats were treated with implants made from both Ti6Al4V-ELI and CP-Ti. Bone regeneration was assessed up to 11 weeks using micro-CT scanning. The regenerated bone volume was assessed ex vivo followed by histology and biomechanical testing to assess osseointegration of the implants. The bony defects treated withAMCP-Ti implants generally showed higher volumes of regenerated bone as compared to those treated with AM Ti6Al4V-ELI. The torsional strength of the two titanium groups were similar however, and both considerably lower than those measured for intact bony tissue. These findings show the importance of material type and ductility of the bulk material in the ability for bone tissue regeneration of AM porous biomaterials.

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Additive manufacturing has facilitated fabrication of complex and patient-specific metallic meta-biomaterials that offer an unprecedented collection of mechanical, mass transport, and biological properties as well as a fully interconnected porous structure. However, applying meta-biomaterials for addressing unmet clinical needs in orthopedic surgery requires additional surface functionalities that should be induced through tailor-made coatings. Here, we developed multi-functional layer-by-layer coatings to simultaneously prevent implant-associated infections and stimulate bone tissue regeneration. We applied multiple layers of gelatin- and chitosan-based coatings containing either bone morphogenetic protein (BMP)-2 or vancomycin on the surface of selective laser melted porous structures made from commercial pure Titanium (CP Ti) and designed using a triply periodic minimal surface (i.e., sheet gyroid). The additive manufacturing process resulted in a porous structure and met the the design values comparatively. X-ray photoelectron spectroscopy spectra confirmed the presence and composition of the coating layers. The release profiles showed a continued release of both vancomycin and BMP-2 for 2–3 weeks. Furthermore, the developed meta-biomaterials exhibited a very strong antibacterial behavior with up to 8 orders of magnitude reduction in both planktonic and implant-adherent bacteria and no signs of biofilm formation. The osteogenic differentiation of mesenchymal stem cells was enhanced, as shown by two-fold increase in the alkaline phosphatase activity and up to four-fold increase in the mineralization of all experimental groups containing BMP-2. Eight-week subcutaneous implantation in vivo showed no signs of a foreign body response, while connective tissue ingrowth was promoted by the layer-by-layer coating. These results unequivocally confirm the superior multi-functional performance of the developed biomaterials.

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Application of bio-functionalized coatings on bone implantable devices is a promising approach to direct rapid bone-implant integration. Plasma polymer (PP) films have become increasingly popular as platforms for surface bio-functionalization of implantable devices. However, the production of a reactive, yet stable PP film represents a technological challenge; as achieving a balance between the film's stability and functional group density is not trivial. Here we report the development of highly reactive and stable radical-functionalized PP films, using a combination of plasma polymerization and plasma immersion ion implantation. We provide new insights into the role of energetic ion bombardment on the growth mechanisms of plasma polymers by measuring the hydrogen content of PP structures using elastic recoil detection analysis. Nano-indentation and nano-scratch tests, as well as stability studies in simulated body fluid show a strong correlation between the degree of energetic ion bombardment and physico-chemical stability of the coatings. The potential of such ion-treated PP films to fabricate biofunctionalized implants that promote the functionality of primary osteoprogenitor cells is confirmed by studying cellular interactions after covalent attachment of fibronectin or bone morphogenetic protein (BMP)-2. We found that covalent attachment of fibronectin improved adhesion, spreading and proliferation of primary osteoblasts; whereas covalent attachment of BMP-2 enhanced the osteocalcin expression in bone-marrow isolated mesenchymal stem cells (MSC). These results present great promise for the fabrication of a new class of robust, biologically-functionalized interfaces for the surface engineering of biomaterials, particularly implants that need to be overgrown with bone-producing cells and thereby become firmly attached to host tissue.

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Application of bio-functionalized coatings on bone implantable devices is a promising approach to direct rapid bone-implant integration. Plasma polymer (PP) films have become increasingly popular as platforms for surface bio-functionalization of implantable devices. However, the production of a reactive, yet stable PP film represents a technological challenge; as achieving a balance between the film's stability and functional group density is not trivial. Here we report the development of highly reactive and stable radical-functionalized PP films, using a combination of plasma polymerization and plasma immersion ion implantation. We provide new insights into the role of energetic ion bombardment on the growth mechanisms of plasma polymers by measuring the hydrogen content of PP structures using elastic recoil detection analysis. Nano-indentation and nano-scratch tests, as well as stability studies in simulated body fluid show a strong correlation between the degree of energetic ion bombardment and physico-chemical stability of the coatings. The potential of such ion-treated PP films to fabricate biofunctionalized implants that promote the functionality of primary osteoprogenitor cells is confirmed by studying cellular interactions after covalent attachment of fibronectin or bone morphogenetic protein (BMP)-2. We found that covalent attachment of fibronectin improved adhesion, spreading and proliferation of primary osteoblasts; whereas covalent attachment of BMP-2 enhanced the osteocalcin expression in bone-marrow isolated mesenchymal stem cells (MSC). These results present great promise for the fabrication of a new class of robust, biologically-functionalized interfaces for the surface engineering of biomaterials, particularly implants that need to be overgrown with bone-producing cells and thereby become firmly attached to host tissue.

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Nanopillar arrays that are bactericidal but not cytotoxic against the host cells could be used in implantable medical devices to prevent implant-associated infections. It is, however, unclear what heights, widths, interspacing, and shape should be used for the nanopillars to achieve the desired antibacterial effects while not hampering the integration of the device in the body. Here, we present an in-silico approach based on finite element modeling of the interactions between Staphylococcus aureus and nanopatterns on the one hand and osteoblasts and nanopatterns on the other hand to find the best design parameters. We found that while the height of the nanopillars seems to have little impact on the bactericidal behavior, shorter widths and larger interspacings substantially increase the bactericidal effects. The same combination of parameters could, however, also cause cytotoxicity. Our results suggest that a specific combination of height (120 nm), width (50 nm), and interspacing (300 nm) offers the bactericidal effects without cytotoxicity.

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Magnesium and its alloys have the intrinsic capability of degrading over time in vivo without leaving toxic degradation products. They are therefore suitable for use as biodegradable scaffolds that are replaced by the regenerated tissues. One of the main concerns for such applications, particularly in load-bearing areas, is the sufficient mechanical integrity of the scaffold before sufficient volumes of de novo tissue is generated. In the majority of the previous studies on the effects of biodegradation on the mechanical properties of porous biomaterials, the change in the elastic modulus has been studied. In this study, variations in the static and fatigue mechanical behavior of porous structures made of two different Mg alloys (AZ63 and M2) over different dissolution times ( 6, 12, and 24 h) have been investigated. The results showed an increase in the mechanical properties obtained from stress-strain curve (elastic modulus, yield stress, plateau stress, and energy absorption) after 6-12 h and a sharp decrease after 24 h. The initial increase in the mechanical properties may be attributed to the accumulation of corrosion products in the pores of the porous structure before degradation has considerably proceeded. The effects of mineral deposition was more pronounced for the elastic modulus as compared to other mechanical properties. That may be due to insufficient integration of the deposited particles in the structure of the magnesium alloys. While the bonding of the parts being combined in a composite-like material is of great importance in determining its yield stress, the effects of bonding strength of both parts is much lower in determining the elastic modulus. The results of the current study also showed that the dissolution rates of the studied Mg alloys were too high for direct use in human body.

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Prevention and treatment of biomaterial-associated infections (BAI) are imperative requirements for the effective and long-lasting function of orthopedic implants. Surface-functionalization of these materials with antibacterial agents, such as antibiotics, nanoparticles and peptides, is a promising approach to combat BAI. The well-known silver nanoparticles (AgNPs) in particular, although benefiting from strong and broad-range antibacterial efficiency, have been frequently associated with mammalian cell toxicity when physically adsorbed on biomaterials. The majority of irreversible immobilization techniques employed to fabricate AgNP-functionalized surfaces are based on wet-chemistry methods. However, these methods are typically substrate-dependent, complex, and time-consuming. Here we present a simple and dry strategy for the development of polymeric coatings used as platforms for the direct, linker-free covalent attachment of AgNPs onto solid surfaces using ion-assisted plasma polymerization. The resulting coating not only exhibits long-term antibiofilm efficiency against adherent Staphylococcus aureus (S. aureus), but also enhances osteoblast adhesion and proliferation. High resolution X-ray photoelectron spectroscopy (XPS), before and after sodium dodecyl sulfate (SDS) washing, confirms covalent bonding. The development of such silver-functionalized surfaces through a simple, plasma-based process holds great promise for the fabrication of implantable devices with improved tissue-implant integration and reduced biomaterial associated infections.@en

Additively manufactured porous titanium implants, in addition to preserving the excellent biocompatible properties of titanium, have very small stiffness values comparable to those of natural bones. Although usually loaded in compression, biomedical implants can also be under tensional, shear, and bending loads which leads to crack initiation and propagation in their critical points. In this study, the static and fatigue crack propagation in additively manufactured porous biomaterials with porosities between 66% and 84% is investigated using compact-tension (CT) samples. The samples were made using selective laser melting from Ti-6Al-4V and were loaded in tension (in static study) and tension-tension (in fatigue study) loadings. The results showed that displacement accumulation diagram obtained for different CT samples under cyclic loading had several similarities with the corresponding diagrams obtained for cylindrical samples under compression-compression cyclic loadings (in particular, it showed a two-stage behavior). For a load level equaling 50% of the yield load, both the CT specimens studied here and the cylindrical samples we had tested under compression-compression cyclic loading elsewhere exhibited similar fatigue lives of around 10⁴ cycles. The test results also showed that for the same load level of 0.5 F_y, the lower density porous structures demonstrate relatively longer lives than the higher-density ones. This is because the high bending stresses in high-density porous structures gives rise to local Mode-I crack opening in the rough external surface of the struts which leads to quicker formation and propagation of the cracks. Under both the static and cyclic loading, all the samples showed crack pathways which were not parallel to but made 45^° angles with respect to the notch direction. This is due to the fact that in the rhombic dodecahedron unit cell, the weakest struts are located in 45^° direction with respect to the notch direction.

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Implant-associated infections are notoriously difficult to treat and may even result in amputation and death. The first few days after surgery are the most critical time to prevent those infections, preferably through full eradication of the micro-organisms entering the body perioperatively. That is particularly important for patients with a compromised immune system such as orthopedic oncology patients, as they are at higher risk for infection and complications. Full eradication of bacteria is, especially in a biofilm, extremely challenging due to the toxicity barrier that prevents delivery of high doses of antibacterial agents. This study aimed to use the potential synergistic effects of multiple antibacterial agents to prevent the use of toxic levels of these agents and achieve full eradication of planktonic and adherent bacteria. Silver ions and vancomycin were therefore simultaneously delivered from additively manufactured highly porous titanium implants with an extremely high surface area incorporating a bactericidal coating made from chitosan and gelatin applied by electrophoretic deposition (EPD). The presence of the chitosan/gelatin (Ch+Gel) coating, Ag, and vancomycin (Vanco) was confirmed by X-ray photoelectron spectroscopy (XPS) and Fourier transform infrared spectroscopy (FTIR). The release of vancomycin and silver ions continued for at least 21 days as measured by inductively coupled plasma (ICP) and UV-spectroscopy. Antibacterial behavior against Staphylococcus aureus, both planktonic and in biofilm, was evaluated for up to 21 days. The Ch+Gel coating showed some bactericidal behavior on its own, while the loaded hydrogels (Ch+Gel+Ag and Ch+Gel+Vanco) achieved full eradication of both planktonic and adherent bacteria without causing significant levels of toxicity. Combining silver and vancomycin improved the release profiles of both agents and revealed a synergistic behavior that further increased the bactericidal effects.

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Enhanced bone tissue regeneration and improved osseointegration are among the most important goals in design of multifunctional orthopedic biomaterials. In this study, we used additive manufacturing (selective laser melting) to develop multifunctional porous nitinol that combines superelasticity with a rationally designed microarchitecture and biofunctionalized surface. The rational design based on triply periodic minimal surfaces aimed to properly adjust the pore size, increase the surface area (thereby amplifying the effects of surface biofunctionalization), and resemble the curvature characteristics of trabecular bone. The surface of additively manufactured (AM) porous nitinol was biofunctionalized using polydopamine-immobilized rhBMP2 for better control of the release kinetics. The actual morphological properties of porous nitinol measured by microcomputed tomography (e.g., open/close porosity, and surface area) closely matched the design values. The superelasticity originated from the austenite phase formed in the nitinol porous structure at room temperature. Polydopamine and rhBMP2 signature peaks were confirmed by X-ray photoelectron spectroscopy and Fourier transform infrared spectroscopy tests. The release of rhBMP2 continued until 28 days. The early time and long-term release profiles were found to be adjustable independent of each other. In vitro cell culture showed improved cell attachment, cell proliferation, cell morphology (spreading, spindle-like shape), and cell coverage as well as elevated levels of ALP activity and increased calcium content for biofunctionalized surfaces as compared to as-manufactured specimens. The demonstrated functionalities of porous nitinol could be used as a basis for deployable orthopedic implants with rationally designed microarchitectures that maximize bone tissue regeneration performance by release of biomolecules with adjustable and well-controlled release profiles.@en

Enhanced bone tissue regeneration and improved osseointegration are among the most important goals in design of multifunctional orthopedic biomaterials. In this study, we used additive manufacturing (selective laser melting) to develop multifunctional porous nitinol that combines superelasticity with a rationally designed microarchitecture and biofunctionalized surface. The rational design based on triply periodic minimal surfaces aimed to properly adjust the pore size, increase the surface area (thereby amplifying the effects of surface biofunctionalization), and resemble the curvature characteristics of trabecular bone. The surface of additively manufactured (AM) porous nitinol was biofunctionalized using polydopamine-immobilized rhBMP2 for better control of the release kinetics. The actual morphological properties of porous nitinol measured by microcomputed tomography (e.g., open/close porosity, and surface area) closely matched the design values. The superelasticity originated from the austenite phase formed in the nitinol porous structure at room temperature. Polydopamine and rhBMP2 signature peaks were confirmed by X-ray photoelectron spectroscopy and Fourier transform infrared spectroscopy tests. The release of rhBMP2 continued until 28 days. The early time and long-term release profiles were found to be adjustable independent of each other. In vitro cell culture showed improved cell attachment, cell proliferation, cell morphology (spreading, spindle-like shape), and cell coverage as well as elevated levels of ALP activity and increased calcium content for biofunctionalized surfaces as compared to as-manufactured specimens. The demonstrated functionalities of porous nitinol could be used as a basis for deployable orthopedic implants with rationally designed microarchitectures that maximize bone tissue regeneration performance by release of biomolecules with adjustable and well-controlled release profiles.@en

he effects of substrate composition on oxide layer properties following plasma electrolytic oxidation under similar conditions have been evaluated for α-cpTi, α/β-Ti6Al7Nb, β-Ti35Zr10Nb and β-Ti45Nb alloys. All oxidised surfaces revealed enhanced wettability, surface free energy and roughness relative to the non-oxidised surfaces. Nevertheless, the resultant oxides differed with respect to average pore size, pores density, layer chemistry and phase composition. The β-titanium alloys developed oxides with a larger average pore size and lower pore density relative to the α-cpTi and α/β-Ti6Al7Nb substrates. Anatase dominated the oxide layer formed on α-cpTi and β-Ti45Nb alloys, a mixture of anatase and rutile was present on the oxidised α/β-Ti6Al7Nb surface, whereas Ti2ZrO6 was the only phase detected on the oxidised surface of the β-Ti35Zr10Nb alloy.@en