ICMCTF2013 Session D1-1: Surface Functionalization, Drug Delivery, and Anti-microbial Coatings

Various coatings are evaluated for biocompatibility and antibacterial property. These coatings for evaluations are such as TiN, DLC and thin film metallic glasses (TFMGs). TiN and DLC are commonly known as the hard coatings, while TFMGs have a great potential owing to their unique properties, such as high strength, high hardness, smooth surface, thermal plastic forming and antibacterial properties. In this study, Zr-based TFMGs and conventional hard coatings were deposited by magnetron sputtering. The effects of these coatings on microstructures, surface roughness, amorphization of TFMG, mechanical, antibacterial properties and cell adhesion behavior are explored, and the results will be presented in this talk.

Biomaterials are used clinically to restore form and function to patients. When biomaterials are implanted into the body, they are first coated with proteins from blood, aiding in initial stem cell attachment to the material surface. In this way, stem cells can differentiate in cells that induce healing and tissue formation. In this presentation I will focus mainly in biomaterials implanted in bone. In this application, stem cells attached to the implant surface differentiate into bone, forming a direct contact between bone and implant termed osseointegration. In dental and orthopaedic implants, osseointegration is crucial to maintain implant stability. However, if the stem cells that attach do not form bone, the implant will fail, requiring additional procedures. Therefore, it is important to design biomaterial surfaces to control stem cell fate once they are implanted. Previous studies have shown that material surface characteristics on which cells are grown influence their growth, development and differentiation, but these studies commonly use exogenous growth factors, hormones, and synthetic molecules. In this presentation, I will discuss the effect of biomaterial properties such as surface roughness, chemistry, and energy on stem cell fate and to explore the signaling pathways involved in osteoblastic differentiation of stem cells in response to these surface properties. Designing “smart” materials that control cellular activities or cell fate would translate to more efficient regenerative medicine and the abolishment of exogenous growth factors that can produce adverse effects.

It has been recognized that bacteria have affinities for different proteins that can be present in saliva. The aim of this study was to determine the effect of the adsorption of two salivary proteins and its effect on the adhesion of two bacterial strains on different microestructured titanium (Ti) samples with different surface properties. To evaluate the protein adsorption on the surfaces, Alpha-amylase, Cystatin-S and Histatin 5, were incubated during 2 hours on PT [pre-treatment (Ra<0.2µm)], A [acid-etched (Ra<0.8 µm)], and SLA [sand-blasted/acid-etched (Ra=4 µm)] Ti surfaces. The amount of proteins absorbed on each surface was measured by ELISA assay. To assess the bacterial adhesion, two oral microorganisms: Streptococcus gordonii and Porphyromonas gingivalis were incubatedindividually on the Ti surfaces previously coated with Alpha-amylase, Cystatin-S or Histatin 5. After incubation time, bacteria were detached by sonication and the number of colony forming units (CFUs) was counted by direct observation. In addition, the surfaces were observed by fluorescence microscopy to determine the surface coverage of bacteria using the LIVE/DEAD^® BacLightTM (Invitrogen) kit. Significant differences were determined using ANOVA test. Results show that Alpha-amylase was detected on PT, A and SLA surfaces (41.1 ng/mL, 41.1 ng/mL and 52.03 ng/mL, respectively) while Cystatin-S was adsorbed on the same surfaces at 27.2 ng/mL, 20.3 ng/mL and 25.1 ng/mL, respectively), finally Histatin 5 was detected on higher quantities on the PT, A and SLA surfaces (107.1 ng/mL, 100.8 ng/mL and 103.7 ng/mL, respectively). In general, higher bacterial counts were detected on the SLA surfaces. P. gingivalis was detected in higher counts comparing with S. gordonii in all substrates tested regardless previous protein adsorption. Alpha-amylase decreased the adhesion of S. gordonii while Histatin 5 decreased the adhesion of P. gingivalis. The protein adsorption was influenced by the physical properties of each substrate while the bacterial adhesion was influenced mainly by the surface topography but also by the previous adsorption of the proteins that were tested.

In this work, we deposited amorphous and crystalline titanium oxide (TiO₂) films to study the effect of atomic ordering, and micro-nano roughness on the cell adhesion, proliferation and differentiation. The films were deposited from a metallic Ti target using a magnetron sputtering method, a reactive Ar/O₂ atmosphere (80:20) and 200 W RF-power. Amorphous films were deposited at room temperature and crystalline films at substrate temperatures of 250^oC. The films were deposited on pure Ti grade 2 (cp-Ti) substrates with 2 different micro-roughness; pre-treated PT [Ra=0.4 mm] and sand blasted-acid etched, SLA [Ra=3.2 mm]). Moreover, at the nanometric level, it was observed that the amorphous-crystalline structure induce also modifications in the nano-roughness. The films were characterized by X-ray diffraction, which confirmed the crystallinity, and X-ray photoelectron spectroscopy to obtain the composition, which was similar for the amorphous and crystalline phases. Images of the surface topography were obtained by both atomic force microscopy and secondary electron microscopy. To characterize the biological response, human mesenchymal stem cells (HMSC) were plated at 10000 cells/cm² on uncoated PT and SLA cp-Ti substrates, as well as on TiO2-coated substrates; amorphous TiO₂/PT (aPT), crystalline TiO₂/PT (cPT), amorphous TiO₂/SLA (aSLA) and crystalline TiO₂/SLA (cSLA). Tissue culture polystyrene (TCPS) was used as a control. Cells were cultured in Mesenchymal Stem Cell Growth Media (Lonza) at 37C, and media was changed every other day. Cells were cultured for 7 days. After the incubation period, cells were harvested for cell number and alkaline phosphatase specific activity. Meanwhile, the conditioned media was analyzed for osteocalcin. HMSC grown on SLA and cSLA surfaces decreased cell number and increased alkaline phosphatase activity in comparison to TCPS, PT, or cPT. HMSC grown on aPT and aSLA showed lower cell number in comparison to TCPS, but higher cell number when compared to PT, SLA, cPT, or cSLA. Osteocalcin levels were higher on SLA and cSLA compared to TCPS, PT, or cPT. However, amorphous modifications on PT and SLA had higher osteocalcin levels when compared to control group, or the crystalline modification. The results show that HMSC differentiation is sensitive to surface micro- and nano-features, as well as to the surface atomic ordering of the TiO₂ films.

Acknowledgements: CONACYT 152995

Surface properties influence adsorption of adhesive proteins and subsequent cell adhesion on orthopaedic implants and greatly affect its biocompatibility. The role dielectric permittivity of the implant coating in the protein adsorption is poorly understood. We calculated the contribution of electrostatic interactions to the free energy of binding between protein and dielectric surface as a function of its dielectric permittivity using non local electrostatic approach [1]. We show that the unfavorable desolvation effects expected in classical consideration are considerably reduced. The formation of complex of protein with the dielectric implant surface is assisted by electrostatic interaction if coatings have a moderate dielectric constant. This compensation for the unfavorable desolvation effects is due to the presence of an interfacial solvent layer with low dielectric constant. We show that the electrostatic binding energy of protein with the dielectric implant surface depends non-monotonically on the dielectric constant of the latter. It is strongest for a medium with the dielectric permittivity of ~4. Thus, ceramic implant coating of reduced dielectric permittivity may be beneficial to strengthen the electrostatic binding of the adhesive proteins to the implant. Because biocompatibility of CoCrMo and Ti implant can be modifies by nanocrystalline coating therefore we performed a comparative analyze of the electrostatic interactions of protein with typical ceramics coatings TiO2 (anatase and rutile), ZrO2, Ta2O5 and glass. The results correlate with the experimental findings of adhered cells counting determined by Alamar Blue Assay on respective substrates fabricated by ion-beam assisted deposition.[2]

1. A.Rubinstein, R. F. Sabirianov, W. N. Mei, F. Namavar and A. Khoynezhad. Effect of the ordered interfacial water layer in protein complex formation: a non-local electrostatic approach. Phys. Rev. E, 82, 021915(2010)

2. J. D. Jackson and J. G. Sharp, H. Haider, K. L. Garvin and F. Namavar, Preliminary analysis of attachment, survival, and growth of bone marrow stromal cells on nanocrystalline hard ceramic coatings, Ceramics, Cells and Tissues: Materials for Scaffolding of Biologically Engineered Systems. Interfaces and Interactions on a Nanoscale ed A Ravaglioli and A Krajewski (Rome: Consiglio Nazionale Delle Ricerche) p 109