The influence of microgrooved surfaces on the behavior and celluar function of osteoblasts

Since 1945 Weiss Paul described the phenomenon ‘contact guidance’ which means the cell elongates along the direction of the groove and migrates guided by the grooves. Cell could sense the surface topography where it lies and react to these surface cues. Many researches have devoted themselves to reveal the potential mechanisms. The interaction is mainly mediated by the cytoskeleton, the focal adhesions and the extracellular matrix (ECM). But how would the groove dimensions affect the cellular behavior is still obscure. Nowadays, micro fabrication techniques such as electron beam lithography have been applied to the production of microtextured surfaces. They are relatively fast and cheap, and could fabricate microgrooves of reasonable size. Thus, they have been widely utilized to generate (micro-) nano-topographical surfaces or scaffolds for in vitro cell research. According to the report of P. CLARK, the response of cells to micro-grooved surfaces is cell typedependent, so the focus of this review is on the osteoblast(s) reaction to micro-grooved surfaces. Correspondence to: Yongyue Wang, DDS, Ph.D, Professor, Department of Oral Implantology, West China Hospital of Stomatology, Sichuan University, #14, 3rd Section, Renmin Nan Road Chengdu, Sichuan 610041, China, Tel.:86-2885503579; Fax: 86-28-85582167; E-mail: westchinawangyy@163.com


Introduction
A century ago, in 1911 Harrison depicted that cells cultured on spider's webs grew along the fibers [1]. Later on, in 1945 Weiss P initially named the phenomenon 'contact guidance': a tendency of cells to align, grow, or migrate along the grooves [2]. Cell can 'sense' the surface topography and then take reaction to these surface cues. The interaction between substrates and cells is achieved through the effort of the cytoskeleton, the extracellular matrix (ECM) [3][4][5] and the focal adhesions [6].
In terms of the defined (micro-) nano-topographical surface, they are usually produced by the micromachining technology: lithographic patterning (photolithography, electron beam lithography, colloidal lithography), galvanoformung abformung process LIGA, focused ion beam-chemical vapor deposition FIB-CVD and so on. Some of these techniques such as electron beam lithography (EBL) have been developed for creating well-defined patterns with feature sizes <10 nm [7]. Recent years, femtosecond laser patterning has obtained a position in the microgrooves‵ machining [8,9]. These techniques promoted the development of biomaterials and tissue engineering greatly.
As the response of cells to micro-grooved surfaces is cell typedependent [10], the react of osteoblasts to the micro-machined surface might be different from other cell types. This review is based on the gathered information about the defined microgrooves, ranging from nanometers to microns, role on the osteoblasts, aiming at finding out the interaction between these ultrafine arrays and the bone-forming cells.

Micro/Nanofabrication technologies
A variety of methods such as Femtosecond laser microtexturing can be applied to the fabrication of microtextured surface. These nano/ micro patterning techniques were early used in the semiconductor and microelectronics industries [11], later they were increasingly applied in biology, medicine, and biomedical engineering fields [12]. Researchers [13,14] use these techniques to manufacture materials, attempting to get a value that is optimal for the growth of cells. These technologies both have their adaptations as well as limitations, also they have got developments. Hence it is hard to define the best tech in this field [15].

Microgrooved surface influences cellular behavior
Cell adhesive to the grafting materials, more importantly, they are in reciprocity with them. Different surface materials and topographies may induce distinct cell morphology, proliferation, and gene expression [16]. Cells can "sense" substrate elasticity [17,18] as long as its surface patterns in the scope of 10 nm to 100 mm [19,20]. Different dimensions are thought to play varied roles in cellular behavior [10,21]. The average size of the osteoblasts is 20-30μm. When the dimensions of grooves/ridges are reduced to the sizes of the cells and less, topographic effects on cell orientation become more prominent [22]. As will be discussed below, a majority of results focused on groove width of the micro-or-nanoscaled surface, some reports show that ridge width is more important in conducting the cellular behavior, while maybe the groove depth is the leading factor inducing cellular activities.
Groove/ridge topographies are important modulators of both cellular adhesion and osteospecific function and that groove width is vital in determining cellular response [23]. Certain groove width guides the cell to align along the direction [8,9,24,25]. The change of the width affects celluar shape [26], attachment [27], cellular proliferation [28] as well as bone forming ability [25,26]. Form these opinions and Table 1 and 2, we can infer that substrates with the microgroove width of 1-5μm, particularly 2μm seems to be optical for the biological behavior of osteoblasts. On 2μm-wide-grooves the cellular adhesion [29], proliferation [28], osteogenic differentiation [28,30] as well as calcification [28]. Also，these nanophase material increased adhesions of osteoblasts compared with the conventional materials [31]. Depicted in table 2, almost all of these dimensions guide the cells to align along the long axis of micropatterns. Some nano-dimensions display an osteogenic influencing function [25,30,32].
Those who focused on the effect of ridge part had some limited findings. Alexey Klymov et al. designed the substrates with ridge to groove ratios of 1:1, 1:3 and 3:1. He demonstrated that nano-grooved patterns with the ridge to groove ratio of 1:3 showed cell repelling, meanwhile grooves with the ridge to groove ratio of 3:1 partially showed cell attraction during cellular selective migration [33]. Apart from that the ridge width clearly enhanced differentiation of MSCs towards specific lineages [30]. Tests on other kinds of cells, say fibroblasts, found that ridge width is the main parameter affecting cell alignment (alignment being inversely proportional to ridge width) [34].
Actually there is no defined item about the influence of groove depth on the osteoblast. From the information Azeem A reported, 306nm and 2046nm promoted osteoblasts alignment parallel to underlined topography. Besides this size showed its osteogenesis ability [32]. Kenichi Matsuzaka observed that on a 0.5μm deep and 10μm wide grooved surface, the cell descends into the groove, on a 1.5μm deep and 1μm wide grooved surface, cells attach to the ridges only. Nowhere, differences were observed between specimens with different groove depths. Instead Kenichi Matsuzaka attributed this phenomenon to the width of the ridge merely [27].
In vivo studies on effect of the surface micromachining to the osseointegration also take for the positive side. The laser micromachining technology enhances bone [24] and soft-tissue integration and controls the local microstructural geometry of attached bone [35]. The organized pattern of the microgrooved surfaces is capable of resulting in transverse collagen fiber microenvironment reaction to the load, being positive to promote and to maintain the bone remodeling; in addition, blood vessels and bone cells are able to penetrate microgrooved surfaces [36]. What's more, micromachined implants enhances primary and secondary implant stability, preserves crestal bone levels [36,37].

Conclusions and outlook
With the acceptance of 'contact guidance' theory, many defined patterns were made by various micro/nano technoloies, prompting the study of different dimensions to the cellular behavior. The limited collected data in the table 1 and 2 showed that the groove width is the most influencing factor affecting the osteoblasts. On the micropatterned substrates, osteoblasts adhere and elongate along the long axis of the microgrooves. Improper width of microgrooves may lead to adhesion down growth. On certain groove width cell density, proliferation and osteogenic ability show an improvement. The differentiation also can be affected by the nanotopography. However, the reports based on the virtues of the ridge width and the depth of the array still needs further exploration. Moreover, we can do a further step research on the effect ROS, silicon from 1 to 20 --width less than 10 μm induced the alignment of osteoblasts, increase osteogenic proteins  of the wettability and inclination of ridge. Soluble biochemical cues, dynamic control and regulation of topographical features, as well as cell co-culture systems, have all been declared to act in synergy with physical cues in regulating stem cell fate [38]. When we design a test, multi-factors should be taken into consideration. In conclusion, critical dimensions do play a part in regulating celluar behavior. However, it is a pity that we have not completely revealed the mystery of micronanotopographical on the osteoblasts. In addition, which dimension of microarrays is optimal for the adhesion, proliferation, differentiation, and osteogenisis is still under research. We can use the obtained data as a guide and reference for the study in the future. Besides, these results could be helpful in the design and fabrication of implants and biomaterials. Rat MSCs, Silicone rubber 300 (600 pitch) 1 μm(pitch 2 μm) ∼150, 500 perpendicular the substrates when parallel stretch to the nanotexture greater than 3% applied (Klymov et al. 2015) [33] Rat MSCs, Silicon, polystyrene 10 -1000 ridge to groove ratios of 1:1, 1:3 and 3:1 --All sizes of squares showed strong cell-repelling capacity. 3:1 partially showed cell attraction Table 2. The influence of nanoscale microgrooves on osteoblasts′ function.