Tissues in the body have micro-architecture which help to maintain tissue homeostasis. Some examples include blood vessels (internal structure), rete ridges in skin and limbal epithelial crypts in the cornea (both surface structures). Therefore, to properly mimic native tissue, such architectural characteristics should not be overlooked and be incorporated into the engineered construct.
Surface anisotropy has been shown to be important in controlling cell alignment and proliferation and differentiation [244-246]. Cells align themselves parallel to grooves present on the surface of substrates [247,248]. The dimensions of the micro-texture were shown to affect cell responses. Both the repeat distance
between grooves and the depths of the grooves have been found to affect cell alignment, with the latter having the greater influence [249]. Baby hamster kidney (BHK) cells showed greater degrees of alignment with increasing groove depths over the range of 100 – 400 µm. Similarly, fibroblasts aligned more in the direction of the grooves the deeper the grooves were [250].
The nucleus of a cell has been found to be segregated into regions of differing degrees of gene activities by nuceloskeletal intermediate filaments A, B1, B2 and C (which make up the nuclear lamina) [251-254]. The nuclear lamina provides both support to the nucleus and a physical connection to the cytoskeleton. Therefore, changes in cell shape can affect nuclear organisation and thus gene expression. Using micro-grooves, hTERT BJ-1 fibroblasts were found to reposition chromosomes resulting in significant changes in gene expression levels [255]. This suggests that external mechanical forces affect subcellular organisation which in turn affects downstream gene expression pathways.
Topography also has implications in stem cell niches. The epidermis of skin is made up of five layers, one of which – the basal layer, contains stem cells. Within this population of stem cells there are two sub-divisions of stem cells located either at the top or bottom of rete ridges [256-258]. The rete ridges in ageing skin were found to be flattened and stem cell markers have been shown to be downregulated [259]. This implicates the peaks and troughs of the rete ridges in providing specialised microenvironments for the maintenance of skin stem cells. Limbal stem cells of the eye reside in micro-scale crypts where it is conducive for the
maintenance of their plasticity [260]. Stem cells migrate from these limbal epithelial crypts (LECs) towards the centre of the cornea during wound healing or corneal regeneration. Therefore, micro-topograpical patterning is necessary in engineered tissue to capture the natural behaviour and responses of cells in their local microenvironment.
Micro-topographical patterning has been performed using wet etching, micro- contact printing, photolithography, hot embossing, microfluidic patterning, polishing grinding, abrasion, plasma spraying, acid etching and grit-blasting [261]. However, these techniques are viable only for synthetic, usually hard materials such as glass and metals. Soft lithography is an approach that has been used for fabricating micro-structures on organic materials. Replica moulding, solvent- assisted micro-moulding, microcontact printing, micro-moulding in capillaries (MIMIC), phase-shifting edge lithography, decal transfer lithography and nanoskiving are examples of soft lithography techniques [262]. Soft lithography has been used to pattern silk films [263], collagen films [264-266], gelatin and collagen- glycosaminoglycan films [267], collagen gels [268-270], calcium alginate hydrogels [271], methacrylated hyaluronic acid (MeHA) and poly(ethylene glycol) diacrylate (PEGDA) [272]. These generally involve the use of photolithography to generate a pattern template usually using polydimethylsiloxane (PDMS). The template is then either pushed into pre-polymerised solution or the pre-polymer is cast over the template. Separation of the two yields a gel with topography moulded into a surface.
Micro-patterned collagen films were prepared by pouring collagen solution over a patterned PDMS template and air-dried overnight [266]. These films required chemical cross-linking for stabilisation. Similarly, micro-patterned ‘basal lamina analogs’ were fabricated by coating a PDMS template with either gelatin or collagen-GAG solutions. These were air-dried overnight, peeled off the template and cross-linked by thermal dehydration under vacuum [272]. Tang et al. used microtransfer moulding as well as MIMIC to pattern collagen gels into hexagonal or rectangular shapes while Yeh et al. showed that micro-scale square prisms, discs or strings could be produced from MeHA or PEGDA using soft lithography and photo- crosslinking.
However, soft lithography for micro-patterning membranes creates useful materials only for investigations on the effects of 2D topography on cell behaviour and not 3D. The use of hydrogels instead of films with soft lithography does have the added functionality of the third dimension, but in most cases, micro-patterning has been performed to create discrete micro-gels (of different shapes and sizes), rather than gels with micro-topography. Furthermore, the hydrogels used were highly hydrated, with densities much lower than that of native tissues.
Micro-patterning of hydrogels has also been done by casting a hydrogel around a removable pattern template [273]. Chrobak et al. cast collagen gels around a 120 µm-diameter needle [268]. Pulling out of the needle left a ~120 µm-diameter channel within the gel. Golden and Tien encapsulated a pre-formed pattern made from gelatin within collagen, fibrinogen or Matrigel [274]. Raising the temperature
of the set-up to 37°C resulted in the melting of the gelatin pattern, which was then washed out to leave a hollow pattern in the shape of the original gelatin mould. Dissolution of conical phosphate-based glass fibres cast within compressed collagen constructs have been shown to form micro-channels in a directional and temporal manner [275,276]. 3D-printing has been used to fabricate carbohydrate glass lattices which were then placed within pre-polymer solutions of PEGDA/acrylate- PEG-RGDS, fibrin, Matrigel, agarose or alginate [277]. Exposure of the lattice-gel composite to complete medium for 10 min led to the dissolution of the carbohydrate-glass, leaving behind an interconnected tubular network within the hydrogels.