Maciej Domanski

The influence of nanoscale grooved substrates on osteoblast behavior and extracellular matrix deposition

To fight bone diseases characterized by poor bone quality like osteoporosis and osteoarthritis, as well as in reconstructive surgery, there is a need for a new generation of implantable biomaterials. It is envisioned that implant surfaces can be improved by mimicking the natural extracellular matrix of bone tissue, which is highly a organized nano-composite. In this study we aimed to get a better understanding of osteoblast response to nanometric grooved substrates varying in height, width and spacing. A throughput screening biochip was created using electron beam lithography. Subsequently, uniform large-scale nanogrooved substrates were created using laser interference lithography and reactive ion etching. Results showed that osteoblasts were responsive to nanopatterns down to 75 nm in width and 33nm in depth. SEM and TEM studies showed that an osteoblast-driven calcium phosphate (CaP) mineralization was observed to follow the surface pattern dimensions. Strikingly, aligned mineralization was found on even smaller nanopatterns of 50 nm in width and 17 nm in depth. A single cell based approach for real time PCR demonstrated that osteoblast-specific gene expression was increased on nanopatterns relative to a smooth control. The results indicate that nanogrooves can be a very promising tool to direct the bone response at the interface between an implant and the bone tissue.

The interaction between nanoscale surface features and mechanical loading and its effect on osteoblast-like cells behavior

Osteoblasts respond to mechanical stimulation by changing morphology, gene expression and matrix mineralization. Introducing surface topography on biomaterials, independently of mechanical loading, has been reported to give similar effects. In the current study, using a nanotextured surface, and mechanical loading, we aimed to develop a multi-factorial model in which both parameters interact. Mechanical stimulation to osteoblast-like cells was applied by longitudinal stretch in parallel direction to the nanotexture (300 nm wide and 60 nm deep grooves), with frequency of 1 Hz and stretch magnitude varying from 1% to 8%. Scanning electron microscopy showed that osteoblast-like cells subjected to mechanical loading oriented perpendicularly to the stretch direction. When cultured on nanotextured surfaces, cells aligned parallel to the texture. However, the parallel cell direction to the nanotextured surface was lost and turned to perpendicular when parallel stretch to the nanotexture, greater than 3% was applied to the cells. This phenomenon could not be achieved when a texture with micro-sized dimensions was used. Moreover, a significant synergistic effect on upregulation of fibronectin and Cfba was observed when dual stimulation was used. These findings can lead to a development of new biomimetic materials that can guide morphogenesis in tissue repair and bone remodeling.

In vitro and in vivo evaluation of the inflammatory response to nanoscale grooved substrates

The immune response to an implanted biomaterial is orchestrated by macrophages. In this study various nanogrooved patterns were created by using laser interference lithography and reactive ion etching. The created nanogrooves mimic the natural extracellular matrix environment. Macrophage cell culture demonstrated that interleukin 1β and TNF-α cytokine production were upregulated on nanogrooved substrates. In vivo subcutaneous implantation in a validated mouse cage model for 14 days demonstrated that nanogrooves enhanced and guided cell adhesion, and few multinucleated cells were formed. In agreement with the in vitro results, cytokine production was found to be nanogroove dependent, as interleukin 1β, TNF-α, TGF-β and osteopontin became upregulated. The results indicate that biomaterial surface texturing, especially at the nanometric scale, can be used to control macrophage activation to induce a wound healing response, rather than a profound inflammatory response. From the Clinical Editor: The authors investigate various nano-grooved patterns that mimic the natural extracellular matrix environment and demonstrate (both in macrophage cultures and in vivo) that interleukin 1β and TNF-α cytokine production is dependent upon surface texturing at the nanometric scale. They propose that modified surfaces may trigger macrophage activation to promote a wound healing response.

The effect of nanometric surface texture on bone contact to titanium implants in rabbit tibia

Designing biomaterial surfaces to control the reaction of the surrounding tissue is still considered to be a primary issue, which needs to be addressed systematically. Although numerous in vitro studies have described different nano-metrically textured substrates capable to influence bone cellular response, in vivo studies validating this phenomenon have not been reported. In this study, nano-grooved silicon stamps were produced by laser interference lithography (LIL) and reactive ion etching (RIE) and were subsequently transferred onto the surface of 5 mm diameter Titanium (Ti) discs by nanoimprint lithography (NIL). Patterns with pitches of 1000 nm (500 nm ridge and groove, 150 nm depth), 300 nm (150 nm ridge and groove, 120 nm depth; as well as a 1:3 ratio of 75 nm ridge and 225 nm groove, 120 nm depth) and 150 nm (75 nm ridge and groove, 30 nm depth) were created. These samples were implanted in a rabbit tibia cortical bone. Histological evaluation and histomorphometric measurements were performed, comparing each sample to conventional grit-blasted/acid-etched (GAE) titanium controls. Results showed a significantly higher bone-to-implant contact at 4 weeks for the 300 nm (1:3) specimens, compared to GAE (p = 0.006). At 8 weeks, there was overall more bone contact compared to 4 weeks. However, no significant differences between the nano-textured samples and the GAE occurred. Further studies will need to address biomechanical testing and the use of trabecular bone models.

Submicron-patterning of bulk titanium by nanoimprint lithography and reactive ion etching

Nanopatterns on titanium may enhance endosseous implant biofunctionality. To enable biological studies to prove this hypothesis, we developed a scalable method of fabricating nanogrooved titanium substrates. We defined nanogrooves by nanoimprint lithography (NIL) and a subsequent pattern transfer to the surface of ASTM grade 2 bulk titanium applying a soft-mask for chlorine-based reactive ion etching (RIE). With respect to direct write lithographic techniques the method introduced here is fast and capable of delivering uniformly patterned areas of at least 4 cm(2). A dedicated silicon nanostamp process has been designed to generate the required thickness of the soft-mask for the NIL-RIE pattern transfer. Stamps with pitch sizes from 1000 nm down to 300 nm were fabricated using laser interference lithography (LIL) and deep cryogenic silicon RIE. Although silicon nanomachining was proven to produce smaller pitch sizes of 200 nm and 150 nm respectively, successful pattern transfer to titanium was only possible down to a pitch of 300 nm. Hence, the smallest nanogrooves have a width of 140 nm. An x-ray photoelectron spectroscopy study showed that only very few contaminations arise from the fabrication process and a cytotoxicity assay on the nanopatterned surfaces confirmed that the obtained nanogrooved titanium specimens are suitable for in vivo studies in implantology research.