Porous titanium scaffolds modified with Zeolitic Imidazolate Framework (ZIF-8) with enhanced osteogenic activity for the prevention of implant-associated infections.

In this study, zeolitic imidazolate framework 8 (ZIF-8) was coated on porous Ti6Al4V scaffolds, either bare or previously modified using hydroxyapatite (HA) or HA and gelatin (HAgel), via a growing single-step method in aqueous media using two contact times at 6 h and 24 h. The coated scaffolds termed ZIF-8@Ti, ZIF-8@HA/Ti, and ZIF-8@HAgel/Ti were characterized via scanning electron microscopy (SEM), powder X-ray diffraction (PXRD), attenuated total reflectance-Fourier transform infrared (ATR-FTIR), and molecular plasma-atomic emission spectroscopy (MP-AES). In order to assess the cell proliferation rate, the cytocompatibility of the scaffolds was evaluated in primary osteoblasts (hOBs) using alamarBlue assay, while the osteoconductivity was analyzed in hOBs using a real-time approach, evaluating the expression of secreted phosphoprotein 1 (SPP1). Osteopontin, which is the protein encoded by this gene, represents the major non-collagenous bone protein that binds tightly to HA. The scaffolds were shown to be non-cytotoxic based on hOB proliferation at all time points of analysis (24 h and 72 h). In hOB cultures, the scaffolds induced the upregulation of SPP1 with different fold changes. Some selected scaffolds were assayed in vitro for their antibacterial potential against Staphylococcus epidermidis; the scaffolds coated with ZIF-8 crystals, regardless of the presence of HA and gelatin, strongly inhibited bacterial adhesion to the materials and reduced bacterial proliferation in the culture medium, demonstrating the suitable release of ZIF-8 in a bioactive form. These experiments suggest that the innovative scaffolds, tested herein, provide a good microenvironment for hOB adhesion, viability, and osteoconduction with effective prevention of S. epidermidis adhesion.

Evaluating Osteogenic Cell Differentiation Efficacy in the Presence of Polylactide Samples With Varied Compositions for Bone Grafting: In Vitro Study.

In oral implantology, surgeons often confront the need to improve alveolar bone quality and volume before implantation in patients with bone defects. Whereas guided bone regeneration with titanium meshes is a clinical gold standard for bone augmentation, mesh removal pre-implantation presents a drawback. This study explores biodegradable scaffolds as an alternative. The research investigates the impact of various compositions of customized bone-grafting scaffolds on proliferation and osteogenic differentiation processes in vitro. Plates (10 × 10 × 0.5 mm) were fabricated from polylactide (PLA), PLA with 15% hydroxyapatite nanoparticles (PLA/HA), and polylactide with glycolic acid copolymers (PLGA 60:40 and 85:15). Gingival fibroblasts assessed the influence of experimental samples on proliferation and osteogenic differentiation in a low-glucose medium. Osteogenic differentiation was induced, and alizarin red staining measured extracellular matrix calcification via spectrophotometry. Active proliferation of gingival fibroblasts occurred along scaffold edges during cultivation. Although cells proliferated with experimental samples, rates were lower than control cells. PLA/HA showed higher alizarin red staining intensity, indicating enhanced matrix calcification. Experimental samples (PLA, PLA/HA, PLGA 85:15, PLGA 60:40) supported cell proliferation at lower rates than control. PLA/HA demonstrated increased matrix calcification. Biodegradable membranes were nontoxic, suggesting potential for bone augmentation.

3D Printing and Surface Engineering of Ti6Al4V Scaffolds for Enhanced Osseointegration in an In Vitro Study.

Ti6Al4V superalloy is recognized as a good candidate for bone implants owing to its biocompatibility, corrosion resistance, and high strength-to-weight ratio. While dense metal implants are associated with stress shielding issues due to the difference in densities, stiffness, and modulus of elasticity compared to bone tissues, the surface of the implant/scaffold should mimic the properties of the bone of interest to assure a good integration with a strong interface. In this study, we investigated the additive manufacturing of porous Ti6Al4V scaffolds and coating modification for enhanced osteoconduction using osteoblast cells. The results showed the successful fabrication of porous Ti6Al4V scaffolds with adequate strength. Additionally, the surface treatment with NaOH and Dopamine Hydrochloride (DOPA) promoted the formation of Dopamine Hydrochloride (DOPA) coating with an optimized coating process, providing an environment that supports higher cell viability and growth compared to the uncoated Ti6Al4V scaffolds, as demonstrated by the higher proliferation ratios observed from day 1 to day 29. These findings bring valuable insights into the surface modification of 3D-printed scaffolds for improved osteoconduction through the coating process in solutions.

Preparation of Cu2+/TA/HAP composite coating with anti-bacterial and osteogenic potential on 3D-printed porous Ti alloy scaffolds for orthopedic applications.

Because of stress shielding effects, traditional titanium (Ti) alloy scaffolds have a high elastic modulus, which might promote looseness and bone disintegration surrounding the implant, increasing the likelihood of a second surgery. In contrast, 3D-printed porous Ti alloy scaffolds can reduce the scaffold weight while enhancing biocompatibility. Further, these scaffolds' porous nature allows bone tissue ingrowth as well as strong pore connectivity, which can improve nutrient absorption. Nevertheless, bare Ti alloy implants may fail because of inadequate bone integration; hence, adding a coating on the implant surface is an effective technique for improving implant stability. In this study, a composite coating comprising hydroxyapatite (HAP), chitosan (CS), tannic acid (TA) and copper ions (Cu2+) (Cu2+/TA/HAP composite coating) was prepared on the surface of 3D printed porous Ti alloy scaffolds using electrophoretic deposition. Using the standard plate count method, Live/Dead bacteria staining assay, FITC Phalloidin and 4',6-diamidino-2-phenylindole staining assay, and live/dead staining of cells we determined that the composite coating has better antibacterial properties and cytocompatibility as well as lower cytotoxicity. The Alkaline Phosphatase assay revealed that the coating results showed good osteogenesis potential. Overall, the composite coatings produced in this investigation give new potential for the application of Ti alloys in clinics.

Human dental pulp stem cell-derived exosomes decorated titanium scaffolds for promoting bone regeneration.

Exosomes, nanoscale extracellular vesicles crucial for intercellular communication, hold great promise as a therapeutic avenue in cell-free tissue regeneration. In this study, we identified and utilized exosomes to adorn anodized titanium scaffolds, inducing osteogenic differentiation in human dental pulp stem cells (hDPSCs). The osteogenesis of hDPSCs was stimulated by exosomes derived from hDPSCs that underwent various periods of osteogenic differentiation. After purification, these exosomes were loaded onto anodized titanium scaffolds. Notably, the scaffolds loaded with exosomes deriving from osteogenic differentiated hDPSCs demonstrated superior bone tissue regeneration compared to those loaded with exosomes deriving from hDPSCs within 10-week. RNA-sequencing analysis shed light on the underlying mechanism, revealing that the osteogenic exosomes carried specific cargo, which is due to upregulated miRNAs (Hsa-miR-29c-5p, Hsa-miR-378a-5p, Hsa-miR-10b-5p and Hsa-miR-9-3p) associated with osteogenesis. And down-regulated anti-osteogenic miRNA (Hsa-miR-31-3p, Hsa-miR-221-3p, Hsa-miR-183-5p and Hsa-miR-503-5p). In conclusion, the identification and utilization of exosomes derived from osteogenic differentiated stem cells offer a novel and promising strategy for achieving cell-free bone regeneration.

Numerical design of open-porous titanium scaffolds for Powder Bed Fusion using Laser Beam (PBF-LB).

The paper concerns the numerical design of novel three-dimensional titanium scaffolds with complex open-porous structures and desired mechanical properties for the Powder Bed Fusion using Laser Beam (PBF-LB). The 60 structures with a broad range of porosity (38-78%), strut diameters (0.70-1.15 mm), and coefficients of pore volume variation, CV(Vp), 0.35-5.35, were designed using the Laguerre-Voronoi tessellations (LVT). Their Young's moduli and Poisson's ratios were calculated using Finite Element Model (FEM) simulations. The experimental verification was performed on the representative designs additively manufactured (AM) from commercially pure titanium (CP Ti) which, after chemical polishing, were subjected to uniaxial compression tests. Scanning Electron Microscopy (SEM) observations and microtomography (µ-CT) confirmed the removal of the support structures and unmelted powder particles. PBF-LB structures after chemical polishing were in close agreement with the CAD models' dimensions having 4-12% more volume. The computational and experimental results show that elastic properties were predicted in very close agreement for the low CV(Vp), and with even 30-40% discrepancies for CV(Vp) higher than 4.0, mainly due to PBF-LB scaffold architecture drawbacks rather than CAD inaccuracy. Our research demonstrates the possibility of designing the open-porous scaffolds with pore volume diversity and tuning their elastic properties for biomedical applications.

Structural and antibacterial studies of novel ZnO and ZnxMn(1-x)O nanostructured titanium scaffolds for biomedical applications.

In the biomedical field, the demand for the development of broad-spectrum biomaterials able to inhibit bacterial growth is constantly increasing. Chronic infections represent the most serious and devastating complication related to the use of biomaterials. This is particularly relevant in the orthopaedic field, where infections can lead to implant loosening, arthrodesis, amputations and sometimes death. Antibiotics are the conventional approach for implanted-associated infections, but they have the limitation of increasing antibiotic resistance, a critical worldwide healthcare issue. In this context, the development of anti-infective biomaterials and infection-resistant surfaces can be considered the more effective strategy to prevent the implant colonisation and biofilm formation by bacteria, so reducing the occurrence of implant-associated infections. In the last years, inorganic nanostructures have become extremely appealing for chemical modifications or coatings of Ti surfaces, since they do not generate antibiotic resistance issues and are featured by superior stability, durability, and full compatibility with the sterilization process. In this work, we present a simple, rapid, and cheap chemical nanofunctionalization of titanium (Ti) scaffolds with colloidal ZnO and Mn-doped ZnO nanoparticles (NPs), prepared by a sol-gel method, exhibiting antibacterial activity. ZnO NPs and ZnxMn(1-x)O NPs formation with a size around 10-20nm and band gap values of 3.42 eV and 3.38 eV, respectively, have been displayed by characterization studies. UV-Vis, fluorescence, and Raman investigation suggested that Mn ions acting as dopants in the ZnO lattice. Ti scaffolds have been functionalized through dip coating, obtaining ZnO@Ti and ZnxMn(1-x)O@Ti biomaterials characterized by a continuous nanostructured film. ZnO@Ti and ZnxMn(1-x)O@Ti displayed an enhanced antibacterial activity against both Gram-positive Staphylococcus aureus (S. aureus) and Gram-negative Pseudomonas aeruginosa (P. aeruginosa) bacterial strains, compared to NPs in solution with better performance of ZnxMn(1-x)O@Ti respect to ZnO@Ti. Notably, it has been observed that ZnxMn(1-x)O@Ti scaffolds reach a complete eradication for S. aureus and 90 % of reduction for P. aeruginosa. This can be attributed to Zn2+ and Mn2+ metal ions release (as observed by ICP MS experiments) that is also maintained over time (72 h). To the best of our knowledge, this is the first study reported in the literature describing ZnO and Mn-doped ZnO NPs nanofunctionalized Ti scaffolds with improved antibacterial performance, paving the way for the realization of new hybrid implantable devices through a low-cost process, compatible with the biotechnological industrial chain method.

Plasma Spray vs. Electrochemical Deposition: Toward a Better Osteogenic Effect of Hydroxyapatite Coatings on 3D-Printed Titanium Scaffolds.

Surface modification of three-dimensional (3D)-printed titanium (Ti) scaffolds with hydroxyapatite (HA) has been a research hotspot in biomedical engineering. However, unlike HA coatings on a plain surface, 3D-printed Ti scaffolds have inherent porous structures that influence the characteristics of HA coatings and osteointegration. In the present study, HA coatings were successfully fabricated on 3D-printed Ti scaffolds using plasma spray and electrochemical deposition, named plasma sprayed HA (PSHA) and electrochemically deposited HA (EDHA), respectively. Compared to EDHA scaffolds, HA coatings on PSHA scaffolds were smooth and continuous. In vitro cell studies confirmed that PSHA scaffolds have better potential to promote bone mesenchymal stem cell adhesion, proliferation, and osteogenic differentiation than EDHA scaffolds in the early and late stages. Moreover, in vivo studies showed that PSHA scaffolds were endowed with superior bone repair capacity. Although the EDHA technology is simpler and more controllable, its limitation due to the crystalline and HA structures needs to be improved in the future. Thus, we believe that plasma spray is a better choice for fabricating HA coatings on implanted scaffolds, which may become a promising method for treating bone defects.

Bone Conduction Capacity of Highly Porous 3D-Printed Titanium Scaffolds Based on Different Pore Designs.

In porous titanium scaffolds manufactured via 3D printing, the differences in bone formation according to pore design and implantation period were studied. Titanium scaffolds with three types of different pore structures (Octadense, Gyroid, and Dode) were fabricated via 3D printing using the selective laser melting method. Mechanical properties of scaffolds were investigated. Prepared specimens were inserted into both femurs of nine rabbits and their clinical characteristics were observed. Three animals were sacrificed at the 2nd, 4th, and 6th weeks, and the differences in bone formation were radiologically and histologically analyzed. The percentage of new bone and surface density in the pore structure were observed to be approximately 25% and 8 mm2/mm3, respectively. There was no difference in the amount of newly formed bone according to the pore design at 2, 4, and 6 weeks. In addition, no differences in the amount of newly formed bone were observed with increasing time within the same pore design for all three designs. During the 6-week observation period, the proportion of new bones in the 3D-printed titanium scaffold was approximately 25%. Differences in bone formation according to the pore design or implantation period were not observed.

A Magnetic Iron Oxide/Polydopamine Coating Can Improve Osteogenesis of 3D-Printed Porous Titanium Scaffolds with a Static Magnetic Field by Upregulating the TGFß-Smads Pathway.

3D-printed porous titanium-aluminum-vanadium (Ti6Al4V, pTi) scaffolds offer surgeons a good option for the reconstruction of large bone defects, especially at the load-bearing sites. However, poor osteogenesis limits its application in clinic. In this study, a new magnetic coating is successfully fabricated by codepositing of Fe3 O4 nanoparticles and polydopamine (PDA) on the surface of 3D-printed pTi scaffolds, which enhances cell attachment, proliferation, and osteogenic differentiation of hBMSCs in vitro and new bone formation of rabbit femoral bone defects in vivo with/without a static magnetic field (SMF). Furthermore, through proteomic analysis, the enhanced osteogenic effect of the magnetic Fe3 O4 /PDA coating with the SMF is found to be related to upregulate the TGFß-Smads signaling pathway. Therefore, this work provides a simple protocol to improve the osteogenesis of 3D-printed porous pTi scaffolds, which will help their application in clinic.

Mechanobiologically optimization of a 3D titanium-mesh implant for mandibular large defect: A simulated study.

Recently, a novel 3D titanium-mesh scaffold with bone grafting material has been proposed to reconstruct the large defect of mandible. However, how to design and optimize the 3D scaffolds of mandible is still unclear. Therefore, the aim of this study was to investigate the optimization of 3D scaffolds for mandibular defect. Both the biomechanical behavior and mechanobiological property of scaffolds were considered in this study. Four configurations (regular hexahedron, cuboctahedron, regular dodecahedron, and diamond) and three strut diameters (0.2 mm, 0.5 mm and 0.8 mm) were divided into 12 groups. By employing Finite Element Analysis and bone "Mechnostat" theory, the optimal unit cell was selected from 12 scaffolds. Then, the original implant for mandible defects was designed with the optimal unit cell, and the final implant was optimized to promote osteogenesis and avoid mechanical failure under bi-lateral chewing bite (200N) and maximum force (worse-case) bite (800 N). The results illustrated a strong correlation between the configurations and the load transmission capacity, while mechanical failure highly depended on strut size and architecture. Regular dodecahedron with a strut diameter of 0.8 mm provided a good load transfer to bone tissue while resisting the mechanical failure. Ultimately, the optimized implant was constructed with regular dodecahedron unit cell, and the strut diameters of scaffold gradually varied according to the biomechanical analysis. The computational results indicated that the optimized implant can provide an excellent mechanical environment for bone regeneration, thus achieving a long-term stability and occlusal reconstruction with dental implant. This study is expected to provide a scientific basis for the design and optimization of 3D mesh scaffolds to reconstruct a mandibular functionally and aesthetically.

Direct ink writing of porous titanium (Ti6Al4V) lattice structures.

Ti6Al4V components, for biomedical and aerospace sectors, are receiving a great interest especially after the advent of additive manufacturing technologies. The most used techniques are Selective Laser Sintering (SLS), Selective Laser Melting (SLM) and Electron Beam Melting (EBM). In the current research, we developed 3D-printed Ti6Al4V scaffolds by Direct Ink Writing (DIW) technology. Appropriate ink formulations, based on water-titanium powder suspensions, were achieved by controlling the rheological properties of the developed inks. After printing process, and drying, the printed components were sintered at 1400 °C under high vacuum for 3 h. Highly porous titanium scaffolds (with porosity up to 65 vol%) were produced and different geometries were printed. The influence of the porosity on the morphology, compression strength and biocompatibility of the scaffolds was investigated.

The influence of chemical polishing of titanium scaffolds on their mechanical strength and in-vitro cell response.

Selective Laser Melting (SLM) is a powder-bed-based additive manufacturing method, using a laser beam, which can be used to produce metallic scaffolds for bone regeneration. However, this process also has a few disadvantages. One of its drawbacks is the necessity of post-processing in order to improve the surface finish. Another drawback lies in the removal of unmelted powder particles from the build. In this study, the influence of chemical polishing of SLM fabricated titanium scaffolds on their mechanical strength and in vitro cellular response was investigated. Scaffolds with bimodal pore size (200 µm core and 500 µm shell) were fabricated by SLM from commercially pure titanium powder and then chemically treated in HF/HNO3 solutions to remove unmelted powder particles. The cell viability and mechanical strength were compared between as-made and chemically-treated scaffolds. The chemical treatment was successful in the removal of unmelted powder particles from the titanium scaffold. The Young's modulus of the fabricated cellular structures was of 42.7 and 13.3 GPa for as-made and chemically-treated scaffolds respectively. These values are very similar to the Young's modulus of living human bone. Chemical treatment did not affect negatively cell proliferation and differentiation. Additionally, the chemically-treated scaffolds had a twofold increase in colonization of osteoblast cells migrating out of multicellular spheroids. Furthermore, X-ray computed microtomography confirmed that chemically-treated scaffolds met the dimensions originally set in the CAD models. Therefore, chemical-treatment can be used as a tool to cancel the discrepancies between the designed and fabricated objects, thus enabling fabrication of finer structures with regular struts and high resolution.

In vitro evaluation of marrow clot enrichment on microstructure decoration, cell delivery and proliferation of porous titanium scaffolds by selective laser melting three-dimensional printing.

Titanium alloy is a clinically approved material for bone substitution. Although three-dimensional printing (3DP) fabrication technique can build up porous Ti scaffolds with the designed shape and microstructure, the biomechanical performance of 3DP Ti scaffolds still need to be improved to increase the reliability of osseointegration capacity. To address this issue, rabbit bone marrow clot (MC) is used to modify 3DP Ti scaffolds by stem cell delivery and microenvironment decoration inside the pores of these scaffolds. Moreover, 3DP Ti scaffolds were built up using selective laser melting, and 3DP MC-Ti scaffolds were constructed through the enrichment of MC with Ti scaffolds in vitro. Results demonstrated that the obtained 3DP Ti scaffolds in current study has an average modulus of elasticity (ME) at 1294.48 MPa with average yield strength of 33.154 MPa. For MC-Ti scaffolds, MC enrichment obstructs the pores of 3DP scaffolds due to the large amount of fibrin and erythrocytes and leads to a decrease in ratio of live cells at 1-week culture. Cell proliferation and osteogenic differentiation performance of MC-Ti scaffolds were promoted with porous recanalization in the later 3 weeks. After 2 weeks in vitro culture, fivefold of cell number in MC-Ti scaffolds were observed than bone marrow-derived mesenchymal stem cell-seeded Ti scaffolds. Compared to Ti scaffolds, fourfold of deoxyribonucleic acid content, type I collagen-α1, osteocalcin, and alkaline phosphatase expression in MC-Ti scaffolds were observed after 4 weeks in vitro culture. Results suggested that the combination with MC is a highly efficient method that improves the biological performance of Ti scaffolds. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 2245-2253, 2018.

Biomechanical stability of novel mechanically adapted open-porous titanium scaffolds in metatarsal bone defects of sheep.

Open-porous titanium scaffolds for large segmental bone defects offer advantages like early weight-bearing and limited risk of implant failure. The objective of this experimental study was to determine the biomechanical behavior of novel open-porous titanium scaffolds with mechanical-adapted properties in vivo. Two types of the custom-made, open-porous scaffolds made of Ti6Al4V (Young's modulus: 6-8 GPa and different pore sizes) were implanted into a 20 mm segmental defect in the mid-diaphysis of the metatarsus of sheep, and were stabilized with an osteosynthesis plate. After 12 and 24 weeks postoperatively, torsional testing was performed on the implanted bone and compared to the contralateral non-treated side. Maximum torque, maximum angle, torsional stiffness, fracture energy, shear modulus and shear stress were investigated. Furthermore, bone mineral density (BMD) of the newly formed bone was determined. Mechanical loading capabilities for both scaffolds were similar and about 50% after 12 weeks (e.g., max. torque of approximately 20 Nm). A further increase after 24 weeks was found for most of the investigated parameters. Results for torsional stiffness and shear modulus as well as bone formation depended on the type of scaffold. Increased BMD after 24 weeks was found for one scaffold type but remained constant for the other one. The present data showed the capability of mechanically adapted open-porous titanium scaffolds to function as bone scaffolds for large segmental defects and the influence of the scaffold's stiffness. A further increase in the biomechanical stability can be assumed for longer observation periods of greater than six months.