5.7.  Tissue Engineering and Scaffold Structure and Materials
Tissue engineering can perhaps be best defined as the use of a combination of cells, engineering materials, and suitable biochemical factors to improve or replace biological functions [412]. The advanced medicine indicates an interdisciplinary field that applies the principles of engineering and life sciences toward the development of biological substitutes that restore, maintain, or improve tissue function for understanding the principles of tissue growth, and applying this to produce functional replacement tissue for clinical use [412]. The term “regenerative medicine” is often used synonymously with “tissue engineering”, although those involved in regenerative medicine place more emphasis on the use of stem cells to produce tissues [412]. Tissue engineering in vitro and in vivo involves the interaction of cells with a material surface. The nature of the surface can directly influence cellular response, ultimately affecting the rate and quality of new tissue formation. Initial events at the surface include the orientated adsorption of molecules from the surrounding fluid, creating a conditioned interface to which the cell responds. The gross morphology, as well as the micro-topography and chemistry of the surface, determine which molecules can adsorb and how cells will attach and align themselves. The local attachments made of the cells with their substrate determine cell shape, which, when transduced via the cytoskeleton to the nucleus, result in expression of specific phenotypes. Osteoblasts and chondrocytes are sensitive to subtle differences in surface roughness and surface chemistry. Boyan et al. [413] investigated the chondrocyte response to TiO2 of differing crystallinities, and showed that cells can discriminate between surfaces at this level as well. Cellular response also depends on the local environmental and state of maturation of the responding cells. It was mentioned that optimizing surface structure for site-specific tissue engineering is one option; modifying surfaces with biological means is another biological engineering [413].
One major determination of the suitability of various engineering materials for use in biological settings is the relative strength of adhesion obtained between those materials and their contacting viable phases [414]. Maximal adhesive strength and immobility are desired for orthopedic and dental implants. For example, while minimal bio-adhesion is critical to preventing unwanted thrombus formation in cardiovascular devices, plaque buildup on dental prostheses, and bacterial fouling [414]. Attention should be directed to adhesive phenomena in the oral environment, examining new surface conditioning methods for the prevention of micro-organism deposits, as well as the promotion of excellent tissue bonding to implanted prosthetic devices. Other bio-adhesive phenomena considered included those important to the safe and effective function of new cardiovascular devices [414].
Scaffold material has a two-fold function: artificial extracellular matrices (ECM) and as a spacer keeping a certain open space [415]. Furthermore, scaffold material has to be dissolved completely into the living body after auto-cell is regenerated with artificial extracellular matrices [415]. There are several important biodegradable and/or biofunctional scaffold architectures, structures and materials. They include blended-polymer scaffolds, collagen-based scaffolds, and composite scaffolds of polyhydroxybutyrate-polyhydroxyvalerate with bioactive wollastonite (CaSiO3) [416]. Using an ink-injection technique [417], a thin film (with thickness of about 0.1mm) of calcium phosphate and binding agent is injected onto the substrate to build 3-D bony-like structures [418]. Lee et al. [419] employed three-dimensional printing (3DP) technology to fabricate porous scaffolds by inkjet printing liquid binder droplets. Direct 3DP, where the final scaffold materials are utilized during the actual 3DP process, imposes several limitations on the final scaffold structure. An indirect 3DP protocol was developed, where molds are printed and the final materials are cast into the mold cavity to overcome the limitations of the direct technique. Results of SEM observations revealed highly open, well interconnected, uniform pore architecture (about 100–150 μm) [419]. Scaffold materials for bone tissue engineering often are supplemented with bone morphogenetic proteins (BMPs). Walboomers et al. [420] investigated a bovine extracellular matrix product containing native BMPs. Hollow cylindrical implants were made from titanium fiber mesh, and were implanted subcutaneously into the back of Wistar rats. It was reported that (i) after eight weeks, in two out of six loaded specimens, newly-formed bone and bone marrow-like tissues could be observed, and (ii) after 12 weeks, this had increased to five out of six loaded samples. It was, therefore, concluded that the bovine extracellular matrix product loaded in a titanium fiber mesh tube showed bone-inducing properties [420].
Electrospinning [421] has recently emerged as a leading technique for generating biomimetic scaffolds made of synthetic and natural polymers for tissue engineering applications. Li et al. [422] compared collagen, gelatin (denatured collagen), solubilized alpha-elastin, and recombinant human tropoelastin as biopolymeric materials for fabricating tissue engineered scaffolds by electrospinning. It was reported that (i) the average diameter of gelatin and collagen fibers could be scaled down to 200–500 nm without any beads, while the alpha-elastin and tropoelastin fibers were several microns in width, and (ii) cell culture studies confirmed that the electrospun engineered protein scaffolds support attachment and growth of human embryonic palatal mesenchymal cells [422]. For fabricating meshes of collagen and/or elastin by means of electrospinning from aqueous solutions, Buttafoco et al. [423] added polyethylene oxide and NaCl to spin continuous and homogeneous fibers. It was reported that (i) upon crosslinking, polyethylene oxide and NaCl were fully leached out, and (ii) smooth muscle cells grew as a confluent layer on top of the crosslinked meshes after 14 days of culture [423].
Surface properties of scaffolds play an important role in cell adhesion and growth. Biodegradable poly(α-hydroxy acids) have been widely used as scaffolding materials for tissue engineering; however, the lack of functional groups is a limitation. Liu et al. [424] mentioned in their studies that gelatin was successfully immobilized onto the surface of poly(α-hydroxy acids) films and porous scaffolds by an entrapment process. It was found that (i) the amount of entrapped gelatin increased with the ratio of dioxane/water in the solvent mixture used, (ii) chemical crosslinking after physical entrapment considerably increased the amount of retained gelatin on the surface of poly(α-hydroxy acids), (iii) osteoblasts were cultured on these films and scaffolds, (iv) cell numbers on the surface-modified films and scaffolds were significantly higher than those on controls 4 h and 1 day after cell seeding, (v) the osteoblasts showed higher proliferation on surface-modified scaffolds than on the control during 4 weeks of in vitro cultivation, and (vi) more collagen fibers and other cell secretions were deposited on the surface-modified scaffolds than on the control scaffolds [424].
There are still unique scaffold systems developed, such as the collagen-carbon nanotubes composite matrices [425], chitosan-based hyaluronan hybrid polymer fibers system [426], bioactive porous CaSiO3 scaffold structure [427], or a three-dimensional porous scaffold composed of biodegradable polyesters [428].