Evaluation of Osseointegration of Titanium Alloyed Implants Modified by Plasma Polymerization

By means of plasma polymerization, positively charged, nanometre-thin coatings can be applied to implant surfaces. The aim of the present study was to quantify the adhesion of human bone cells in vitro and to evaluate the bone ongrowth in vivo, on titanium surfaces modified by plasma polymer coatings. Different implant surface configurations were examined: titanium alloy (Ti6Al4V) coated with plasma-polymerized allylamine (PPAAm) and plasma-polymerized ethylenediamine (PPEDA) versus uncoated. Shear stress on human osteoblast-like MG-63 cells was investigated in vitro using a spinning disc device. Furthermore, bone-to-implant contact (BIC) was evaluated in vivo. Custom-made conical titanium implants were inserted at the medial tibia of female Sprague-Dawley rats. After a follow-up of six weeks, the BIC was determined by means of histomorphometry. The quantification of cell adhesion showed a significantly higher shear stress for MG-63 cells on PPAAm and PPEDA compared to uncoated Ti6Al4V. Uncoated titanium alloyed implants showed the lowest BIC (40.4%). Implants with PPAAm coating revealed a clear but not significant increase of the BIC (58.5%) and implants with PPEDA a significantly increased BIC (63.7%). In conclusion, plasma polymer coatings demonstrate enhanced cell adhesion and bone ongrowth compared to uncoated titanium surfaces.     

Superior Osteo-Inductive and Osteo-Conductive Properties of Trabecular Titanium vs. PEEK Scaffolds on Human Mesenchymal Stem Cells: A Proof of Concept for the Use of Fusion Cages

Fusion cages composed of titanium and its alloys are emerging as valuable alternative to standard polyetheretherketone (PEEK) ones routinely used in cervical and lumbar spine surgery. Aim of this study was to evaluate osteo-inductive and osteo-conductive ability of an innovative trabecular titanium (T-Ti) scaffold on human mesenchymal stem cells (hMSCs), in both absence and presence of biochemical osteogenic stimuli. Same abilities were assessed on PEEK and standard 2D plastic surface, the latter meant as gold-standard for in vitro differentiation studies. hMSCs adhered and colonized both T-Ti and PEEK scaffolds. In absence of osteogenic factors, T-Ti triggered osteogenic induction of MSCs, as demonstrated by alkaline phosphatase activity and calcium deposition increments, while PEEK and standard 2D did not. Addition of osteogenic stimuli reinforced osteogenic differentiation of hMSCs cultured on T-Ti in a significantly higher manner with respect to standard 2D plastic culture surfaces, whereas PEEK almost completely abolished the process. T-Ti driven differentiation towards osteoblasts was confirmed by gene and marker expression analyses, even in absence of osteogenic stimuli. These results clearly indicate superior in vitro osteo-inductive and osteo-conductive capacity of T-Ti compared to PEEK, and make ground for further studies supporting the use of T-Ti cages to improve bone fusion.     

Characterization of Osteogenesis and Chondrogenesis of Human Decellularized Allogeneic Bone with Mesenchymal Stem Cells Derived from Bone Marrow,Adipose Tissue,and Wharton’s Jelly

Allogeneic bone grafts are a promising material for bone implantation due to reduced operative trauma, reduced blood loss, and no donor-site morbidity. Although human decellularized allogeneic bone (hDCB) can be used to fill bone defects, the research of revitalizing hDCB blocks with human mesenchymal stem cells (hMSCs) for osteochondral regeneration is missing. The hMSCs derived from bone marrow, adipose tissue, and Wharton’s jelly (BMMSCs, ADMSCs, and UMSCs, respectively) are potential candidates for bone regeneration. This study characterized the potential of hDCB as a scaffold for osteogenesis and chondrogenesis of BMMSCs, ADMSCs, and UMSCs. The pore sizes and mechanical strength of hDCB were characterized. Cell survival and adhesion of hMSCs were investigated using MTT assay and F-actin staining. Alizarin Red S and Safranin O staining were conducted to demonstrate calcium deposition and proteoglycan production of hMSCs after osteogenic and chondrogenic differentiation, respectively. A RT-qPCR was performed to analyze the expression levels of osteogenic and chondrogenic markers in hMSCs. Results indicated that BMMSCs and ADMSCs exhibited higher osteogenic potential than UMSCs. Furthermore, ADMSCs and UMSCs had higher chondrogenic potential than BMMSCs. This study demonstrated that chondrogenic ADMSCs- or UMSCs-seeded hDCB might be potential osteochondral constructs for osteochondral regeneration.     

Polymer Functionalization and Thin Film Deposition by Remote Cold Nitrogen Plasma Process

Modification of polymer surfaces by cold plasma processes is attracting a growing interest. Especially, the use of plasma gases such as N₂, O₂ or air, which are cheap and environmentally safe, is very attractive from an industrial point of view. The lifetime of atomic oxygen being very short, it is very difficult to operate with air or oxygen plasma when wide plasma volume is required. However, it is possible to obtain cold remote nitrogen plasma reaching volume of several m³ due to the long lifetime of atomic nitrogen because of a re-dissociation mechanism. Moreover, the temperature being close to the ambient makes this plasma very attractive for functionalization and/or coating of polymer surfaces. Several applications of this plasma process are presented in this paper. The incorporation of new chemical functions during the treatment of polymers leads to an increase of their adhesion properties. Several industrial applications (painting, sticking, bonding, foaming and thermo-covering) are presented. The ability of this remote plasma to decompose, to polymerize, or to react with a volatile chemical component is described through three examples involving polymer substrates: (i) the deposition of metallic films; (ii) the synthesis of a nitride film combining hardness and elastic behavior; (iii) the synthesis of an organosilicon film showing interesting barrier properties. Adhesion aspects are investigated for all these examples.     

Improvement of paint adhesion to a polypropylene bumper by plasma treatment

Improvement of the paint adhesion to a polypropylene (PP) bumper has been investigated without using a primer by treating the bumper surface with O₂, H₂O, and acetylene plasmas. All the plasma treatments resulted in an increase of the adhesion strength in dry conditions. The adhesion strength could be increased up to a value comparable to that obtained by applying a primer. The treated surfaces were quite stable for 7 days in air. After exposure to wet conditions, however, the adhesion strengths for both O₂ and H₂O plasma-treated samples decreased significantly, while the adhesion strength for the acetylene plasma-treated sample did not change much.     

Role of Hydrogen Peroxide in Selective OH Group Functionalization of Polypropylene Surfaces using Underwater Capillary Discharge

Plasma chemical methods are well suited for introducing functional groups to the surfaces of chemically inert polymers such as polyolefins. However, a broad variety of functional groups are often formed. Unfortunately, for further chemical processing such as grafting of molecules for advanced applications a highly dense monotype functionalized polyolefin surface is needed. Therefore, the main task was to develop a selective surface functionalization process, which formed preferably only a single type of functional groups at the surface in high concentration. Amongst the novel plasma methods, the underwater plasma process (UWP) is one of most attractive options to solve the problem of monotype functionalization. Such plasma is an efficient source of ions, electrons, UV-radiation, high-frequency shock waves, radicals such as hydroxyl radical, and reactive neutral molecules such as hydrogen peroxide. In contrast to established gas phase glow discharge processes, the water phase limits the particle and radiation energies and thus the energy input into the polymer. By virtue of the liquid water environment, which moderates highly energetic plasma species, extensive oxidation, degradation, cross-linking and radical formation on the polymer are more limited as compared to gas plasma exposure. The variety of plasma produced species in the water phase is also much smaller because of the limited reaction possibilities of the plasma with water. The possibility to admix a broad variety of chemical additives makes underwater plasma even more attractive. Hydrogen peroxide and the catalyst (Fe-ZSM5) should influence and increase the equilibrium concentration of OH radicals in the underwater plasma process. It was found that these radicals played a very important role in OH functionalization of polyolefin surfaces. Hydrogen peroxide was identified to be the most prominent precursor for OH group formation in the UWP. The catalyst would affect the steady state of OH radical formation and its reaction with the substrate surface and thus accelerates the functionalization process.     

Antiosteoporotic Nanohydroxyapatite Zoledronate Scaffold Seeded with Bone Marrow Mesenchymal Stromal Cells for Bone Regeneration: A 3D In Vitro Model

Background: Bisphosphonates are widely employed drugs for the treatment of pathologies with high bone resorption, such as osteoporosis, and display a great affinity for calcium ions and apatitic substrates. Here, we aimed to investigate the potentiality of zoledronate functionalized hydroxyapatite nanocrystals (HAZOL) to promote bone regeneration by stimulating adhesion, viability, metabolic activity and osteogenic commitment of human bone marrow derived mesenchymal stromal cells (hMSCs). Methods: we adopted an advanced three-dimensional (3D) in vitro fracture healing model to study porous scaffolds: hMSCs were seeded onto the scaffolds that, after three days, were cut in halves and unseeded scaffolds were placed between the two halves. Scaffold characterization by X-ray diffraction, transmission and scanning electron microscopy analyses and cell morphology, viability, osteogenic differentiation and extracellular matrix deposition were evaluated after 3, 7 and 10 days of culture. Results: Electron microscopy showed a porous and interconnected structure and a uniform cell layer spread onto scaffolds. Scaffolds were able to support cell growth and cells progressively colonized the whole inserts in absence of cytotoxic effects. Osteogenic commitment and gene expression of hMSCs were enhanced with higher expressions of ALPL, COL1A1, BGLAP, RUNX2 and Osterix genes. Conclusion: Although some limitations affect the present study (e.g., the lack of longer experimental times, of mechanical stimulus or pathological microenvironment), the obtained results with the adopted experimental setup suggested that zoledronate functionalized scaffolds (GHAZOL) might sustain not only cell proliferation, but positively influence osteogenic differentiation and activity if employed in bone fracture healing.     

Collagen-Based Osteogenic Nanocoating of Microrough Titanium Surfaces

The aim of the present study was to develop a collagen/heparin-based multilayer coating on titanium surfaces for retarded release of recombinant human bone morphogenic protein 2 (rhBMP2) to enhance the osteogenic activity of implant surfaces. Polyelectrolyte multilayer (PEM) coatings were constructed on sandblasted/acid-etched surfaces of titanium discs using heparin and collagen. PEM films of ten double layers were produced and overlayed with 200 µL of a rhBMP2 solution containing 15 µg rhBMP2. Subsequently, cross-linking of heparin molecules was performed using EDC/NHS chemistry to immobilize the incorporated rhBMP2. Release characteristics for 3 weeks, induction of Alkaline Phosphatase (ALP) in C2C12 cells and proliferation of human mesenchymal stem cells (hMSCs) were evaluated to analyze the osteogenic capacity of the surface. The coating incorporated 10.5 µg rhBMP2 on average per disc and did not change the surface morphology. The release profile showed a delivery of 14.5% of the incorporated growth factor during the first 24 h with a decline towards the end of the observation period with a total release of 31.3%. Cross-linking reduced the release with an almost complete suppression at 100% cross-linking. Alkaline Phosphatase was significantly increased on day 1 and day 21, indicating that the growth factor bound in the coating remains active and available after 3 weeks. Proliferation of hMSCs was significantly enhanced by the non-cross-linked PEM coating. Nanocoating using collagen/heparin-based PEMs can incorporate clinically relevant amounts of rhBMP2 on titanium surfaces with a retarded release and a sustained enhancement of osteogenic activity without changing the surface morphology.     

Characterization of plasma-polymerized allyl alcohol polymers and copolymers with styrene

Pulsed plasma was used to initiate chemical copolymerization of allyl alcohol and styrene. In this way the concentration of OH groups at the surface of the copolymer layer could be adjusted from 0 (styrene homopolymerization) to 31 OH groups/100 C atoms (allyl alcohol homopolymerization). The copolymerization kinetics correspond to those of a pure chemical (radical) copolymerization. Thus, the variation of OH group density is only possible in a small range of comonomers ratio, otherwise styrene homopolymerization dominates.     

Gas-phase surface functionalization of multi-walled carbon nanotubes with vacuum UV photo-oxidation

For bulk processing of carbon nanotubes, an important first step in adhesion to the nanotubes is often liquid-phase functionalization through chemical oxidation with acids (e.g., nitric and sulfuric), peroxides and/or potassium permanganate. In comparison, gas-phase photo-oxidation can be an alternative to introduce oxygenated functional groups on the surfaces of carbon nanotubes without the generation of liquid waste. In the present study, vacuum UV photo-oxidation of multi-walled carbon nanotube (MWNT) paper was investigated downstream from an Ar microwave plasma. X-ray Photoelectron Spectroscopy (XPS) was used to detect the carbon- and oxygen-containing functional groups in the top 2–5 nm of the sample's surface. The current results are compared to previous investigations using MWNT powder and single-walled carbon nanotube (SWNT) paper showing decreased levels of oxidation in MWNT samples.     

3D Printed SiOC(N) Ceramic Scaffolds for Bone Tissue Regeneration: Improved Osteogenic Differentiation of Human Bone Marrow-Derived Mesenchymal Stem Cells

Bone tissue engineering has developed significantly in recent years as there has been increasing demand for bone substitutes due to trauma, cancer, arthritis, and infections. The scaffolds for bone regeneration need to be mechanically stable and have a 3D architecture with interconnected pores. With the advances in additive manufacturing technology, these requirements can be fulfilled by 3D printing scaffolds with controlled geometry and porosity using a low-cost multistep process. The scaffolds, however, must also be bioactive to promote the environment for the cells to regenerate into bone tissue. To determine if a low-cost 3D printing method for bespoke SiOC(N) porous structures can regenerate bone, these structures were tested for osteointegration potential by using human mesenchymal stem cells (hMSCs). This includes checking the general biocompatibilities under the osteogenic differentiation environment (cell proliferation and metabolism). Moreover, cell morphology was observed by confocal microscopy, and gene expressions on typical osteogenic markers at different stages for bone formation were determined by real-time PCR. The results of the study showed the pore size of the scaffolds had a significant impact on differentiation. A certain range of pore size could stimulate osteogenic differentiation, thus promoting bone regrowth and regeneration.     

Polypropylene surface functionalization with chitosan

Chitosan coatings on oxygen-plasma pre-treated polypropylene (PP) surfaces were formed to improve their wettability, dyeing behavior and reactivity without altering material bulk properties. XPS, electrokinetic potential and contact angle measurements as well as dye uptake tests were carried out for surface characterization of modified PP, evaluation of chitosan coatings stability, and the effects of temperature and pH on coatings formation. About 20–30% of the total amount of chitosan immobilized on PP was found to be covalently bonded to the plasma pre-treated surface through the heat induced reactions with oxygen-containing functional groups at T > 80°C that corresponded to 47% of surface coverage. Subsequent cross-linking reaction with epichlorohydrin proved to be an efficient way to reduce the susceptibility of chitosan coatings to acidic hydrolysis.     

Plasma surface modification of poly(phenylene sulfide) films for copper metallization

Poly(phenylene sulfide) (PPS) films were modified by Ar, O₂, N₂ and NH₃ plasmas in order to improve their adhesion to copper metal. All four plasmas modified the PPS film surfaces, but the NH₃ plasma modification was the most effective in improving adhesion. The NH₃ plasma modification brought about large changes in the surface topography and chemical composition of the PPS film surfaces. The peel strength for the Cu/plasma-modified PPS film systems increased linearly with increasing surface roughness, R _a or R _rms, of the PPS film. The plasma modification also led to considerable changes in the chemical composition of the PPS film surfaces. A large fraction of phenylene units and a small fraction of sulfide groups in the PPS film surfaces were oxidized during the plasma modification process. Nitrogen functional groups also were formed on the PPS film surfaces. The NH₃ plasma modification formed S—H groups on the PPS film surfaces by reduction of S—C groups in the PPS film. Not only the mechanical interlocking effect but also the interaction of the S—H groups with the copper metal may contribute to the adhesion of the Cu/PPS film systems.     

Different surface treatments to improve the adhesion of polypropylene

Injection-molded samples of polypropylene were exposed to oxygen plasma and SACO (SAndblasting and COating) treatments. The pretreated surfaces were successively adhesively bonded or lacquered. The adhesion strength and failure mode of these specimens were examined. The surfaces obtained after treatments were characterized by electron spectroscopy for chemical analysis (ESCA), contact angle measurements, and scanning electron microscopy (SEM). Both microroughness and chemical modification of the surface led to an increase in adhesion by up to a factor of 10. The stability of the surface changes generated during the plasma and SACO pretreatments was observed by different kinds of aging experiments in air and water. The aging of SACO-treated surfaces led to no significant change on the surface. In the case of plasma-treated surfaces, hydrophobic recovery during aging in air reduced the polarity of the surface layer. During aging in water, no hydrophobic recovery on the surface was observed.     

Thermal imidization of poly(amic acid) on Si(100) surface modified by plasma polymerization of glycidyl methacrylate

The plasma polymerization of glycidyl methacrylate (GMA) on pristine and Ar plasma-pretreated Si(100) surfaces was carried out. The epoxide functional groups of the plasma-polymerized GMA (pp-GMA) could be preserved, to a large extent, through the control of the glow discharge parameters, such as the radio-frequency (RF) power, carrier gas flow rate, system pressure, and monomer temperature. The pp-GMA film was used as an adhesion promotion layer for the Si substrate. The polyimide (PI)/pp-GMA-Si laminates, formed by thermal imidization of the poly(amic acid) (PAA) precursor poly(pyromellitic dianhydride-co-4,4′-oxydianiline) (PMDA-ODA) on the pp-GMA-deposited Si surface (the pp-GMA-Si surface), exhibited a 180°-peel adhesion strength as high as 9.0 N/cm. This value was much higher than the negligible adhesion strength for the PI/Si laminates obtained from thermal imidization of the PAA precursor on both the pristine and the argon plasma-pretreated Si(100) surfaces. The high adhesion strength of the PI/pp-GMA-Si laminates was attributed to the synergistic effect of coupling the curing of epoxide functional groups in the pp-GMA layer with the imidization process of the PAA, and the fact that the plasma-deposited GMA chains were covalently tethered onto the Si(100) surface. The chemical composition and structure of the deposited films were characterized, respectively, by X-ray photoelectron spectroscopy (XPS) and Fourier transform infrared (FTIR) spectroscopy, while the surface morphology of the deposited films was characterized by atomic force microscopy (AFM).     

Surface characterization of polymers modified by keV and MeV ion beams

The present work deals with two different surface modification techniques for altering the surface properties of polymers: plasma treatment and ion implantation. Polymer foils were exposed in an inductively-coupled r.f. (13.56 MHz) plasma system with and without applying a negative high voltage pulse to the sample stage. The influence of low pressure plasmas of oxygen, nitrogen, or argon on the chemical composition, topography, and wettability of polymer surfaces was studied in detail. Etch rates of poly(ethylene terephthalate) for different plasma parameters were monitored. The polymer surface was also modified by a high energy ion beam process. Polyimide films were implanted with different ion species such as Ar⁺, N⁺, C⁺, He⁺, and O⁺ at doses from 1 × 10¹⁵ to 1 × 10¹⁷ ion/cm². Ion energy was varied from 10 to 60 keV for the plasma source ion implantation (PSII) experiment. Polyimide samples were also implanted with 1 MeV hydrogen, carbon, and oxygen ions at a dose of 1 × 10¹⁴ ion/cm². Depending on the ion energy, dose, and ion species, the surface resistivity of the film was reduced by several orders of magnitude. A study on the plasma-treated and ion beam-treated polymer surfaces was performed using TOF-SIMS, XPS, SEM, AFM, and water contact angle measurements.     

Enhanced adhesion of silica for epoxy molding compounds (EMCs) by plasma polymer coatings

Silica for epoxy molding compounds (EMCs) was coated via plasma polymerization using an RF plasma (13.56 MHz) as a function of the plasma power, gas pressure, and treatment time. The monomers utilized for the plasma polymer coatings were 1,3-diaminopropane, allylamine, pyrrole, 1,2-epoxy-5-hexene, allyl mercaptan, and allyl alcohol. The EMC samples were prepared from biphenyl epoxy resin, phenol novolac, triphenyl phosphine, and plasma polymer-coated silica, and the loading of silica was controlled to 60 wt%. The EMC samples were cured at 175°C for 4 h and subjected to T_g, CTE, and water absorption measurements. The adhesion of silica to epoxy resin was evaluated by measuring the flexural strength of EMC samples and the fracture surfaces were analyzed by SEM. Plasma polymer coatings were also characterized by FT-IR and coating thickness measurements. The plasma polymer coating of silica with 1,3-diaminopropane and allylamine enhanced the flexural strength of EMC samples (167 and 165 MPa), compared with the control sample (140 MPa), and exhibited a higher T_g, a lower CTE, and lower water absorption. The enhanced properties with 1,3-diaminopropane and allylamine plasma polymer coatings can be attributed to the amine functional groups in the plasma polymer coatings.     

Investigation of Apatite Deposition onto Charged Surfaces in Aqueous Solutions Using a Quartz-Crystal Microbalance

Hydroxyapatite (HAp) deposition onto positively charged surfaces (i.e., self-assembled monolayers (SAMs) terminated with NH₂ head groups) and negatively charged surfaces (i.e., OH-SAMs (weak) and COOH-SAMs (strong)) soaked at 50°C in aqueous supersaturated solutions (1.5 SBF, pH 7.0–7.6; SBF = simulated body fluid) was investigated using a quartz-crystal microbalance. The results revealed that the solution conditions greatly influenced the formation of HAp on the charged surfaces. In a stable supersaturated solution of simulated body fluid (1.5 SBF, pH <7.4), more strongly negative surfaces had a more powerful induction capability for the heterogeneous nucleation of HAp (COOH > OH), whereas nucleation was obviously prohibited on a positive surface (NH₂-SAM). On the other hand, after the calcium phosphate particles had nucleated homogeneously in an unstable soaking solution (1.5 SBF, pH ≧7.4), adhesion of the HAp microparticles to the NH₂-SAM was observed. A two-step interaction is conceivable to describe the formation of HAp on the positive NH₂-SAM: At the first stage, electrostatic interaction dominates the adhesion of HAp microparticles; at the second stage, hydrogen bonds possibly form between the HAp microparticles and the amino head groups of the NH₂-SAM, for a firm bonding with the substrate, and the microparticles grow progressively into a thin film. The electrophoretic behaviors of the HAp microparticles confirmed this hypothesis.     

Adhesion and differentiation of human mesenchymal stem cells on plasma-functionalized graphenes with different feeding gases

Surface functionalisation of graphene layer has been investigated to improve the efficiencies in bio-applications such as stem cell differentiation or protein sensing. Plasma treatment can induce various functionalities on graphene surface simply by changing feeding gas. In this study, the surface chemistry of graphene is modified by plasma treatment using three different feeding gases of H₂, N₂, and O₂. The effects of differential functionalisation are examined by the adsorption of fibronectin and the adhesion, proliferation, and differentiation of human mesenchymal stem cells. Fibronection adsorption is enhanced on the N₂ plasma-treated graphene, but it is not clear on the others. hMSCs adhesion is broadened on all of the plasma-treated graphene substrates, especially on the N₂ plasma-treated one. H₂ plasma treatment enhances cell proliferation and osteogenic differentiation, and N₂ and O₂ plasma treatments show a tendency of heterogeneous differentiation to osteoblasts and myoblasts. Different responses of stem cell adhesion, proliferation, and differentiation depending on the plasma-treated graphene surface indicate the importance of highly specific surface functionality in stem cell engineering.