In Vitro Studies

In the bone tissue engineering applications, biomimetic nanofibrous scaffolds derived from natural biopolymers require good mechanical and biological performances including biomineralization. Pangon et al. demonstrated the utility of chitin

Scanning electron microscope images of representative bio-nanocomposite fibers before

Fig. 7.4 Scanning electron microscope images of representative bio-nanocomposite fibers before (top) and after (bottom) immersion in deionized water for 4 h, showing electrospun CS/PVA nanofibrous membranes containing CTWK with different content (a) as-spun and (b)GA-crosslinked CS/PVA/CTWK-2 nanofibers for (c) 8 wt% (CS/PVA/CTWK-1), (d) 17 wt% (CS/PVA/CTWK-2), and (e) 26 wt% (CS/PVA/CTWK-3) (Adapted with permission from Ref. [89]. Copyright 2016 Elsevier Ltd)

whisker (CTWK) to promote mechanical properties of chitosan/poly(vinyl alcohol) (CS/PVA) nanofibers and to provide osteoblast cell growth with hydroxyapatite (HA) mineralization [89]. In their study, electrospun CS/PVA nanofibrous membranes containing CTWK can be easily gained (Fig. 7.4) by using diacid as a solvent. The dimension stability of nanofibrous CS/PVA/CTWK bio-nanocomposite can be further controlled by exposing to glutaraldehyde vapor.

The nanofibrous membranes obtain the mineralization of HA in concentrated simulated body fluid resulting in an improvement of Young’s modulus and tensile strength. The CTWK combined with HA in bio-nanocomposite is a key to promoting osteoblast cell adhesion and proliferation. The present work, for the first time, demonstrates the use of CTWKs for bio-nanocomposite fibers of chitosan and its hydroxyapatite with the function of bio-mineralization in osteoblast cell culture. Figure 7.5 was the scanning electron microscope images of the MC3T3-E1 osteoblast cells adhered on different bionanocomposite fibers. These hydroxyapatite- hybridized CS/PVA/CTWK nanocompositefibers (CS/PVA/CTWK-HA) offer a great potential for bone tissue engineering applications. In their present study, chi- tosan/CTWK nanocomposites in the form of HA-hybridized nanofibers were developed for bone tissue engineering scaffolds. The well-defined CS/PVA/CTWK nanocomposite fibers were electrospun from CS/PVA blend containing CTWK for 8 wt%, 17 wt%, and up to 26 wt% in succinic acid solution. The fiber stability was improved remarkably, after cross-linked using glutaraldehyde vapor. When subjected to 10x simulated body fluid solution for 2 h, the cross-linked nanofibers were randomly mineralized with Ca-deficient HA. The thermal gravimetric analysis results indicated that such mineralization process resulted in HA deposition for 17.6-20.2 wt%, independent of CTWK content. The addition of CTWK could

Scanning electron microscope images of the MC3T3-E1 osteoblast cells adheredon

Fig. 7.5 Scanning electron microscope images of the MC3T3-E1 osteoblast cells adheredon (a) control, (b) GA-crosslinked CS/PVA, (c) CS/PVA-HA, (d) CS/PVA/CTWK-1-HA, and (e) CS/ PVA/CTWK-2-HA scaffolds after 7 days of culture, showing that the CTWK combined with HA in bio-nanocomposite can promoteosteoblast cell adhesion (Adapted with permission from Ref. [89]. Copyright 2016 Elsevier Ltd)

change mechanical properties of the bio-nano-composite fibers containing their HA-hybridized forms, while have less impact on their biocompatibility and biodegradability. The increase of CTWK contentled to a improving the Young’s modulus of the bio-nano-composite fibers, and particularly, of the nanofibers with HA hybridization. The studies on MTT assessment showed that, firstly, the HA-hybridized bio-nano-composite scaffolds had no toxic effect on osteoblast cells, and secondly, the cell viability and ability to proliferate could be improved by increasing CTWK as reinforcement for chitosan nanofibrous scaffolds. It was found that the CS/PVA/CTWK-2-HA hybrid nanofibers with good mechanical and biological properties containing biomineralization are promising scaffolds for bone tissue engineering applications.

Over the last two decades, biodegradable polymers, such as poly(lactic acid) (PLA), poly(glycolic acid) (PGA) and PCL, have emerged as a class of biomaterials of growing interest for application in surgery, drug delivery, and tissue engineering. They can be used in sutures for wound healing, devices for bone fracture internal fixation, carriers for delivery of bioactive molecules, scaffolds for the regeneration of tissues or organs. Li et al. prepared the porous three-dimensional PLLA scaffolds reinforced by the chitin fibers (PLLA/CF) with and without the link [90], and evaluated the properties in vitro [91]. To get an appropriate content of the chitin fibers in the PLLA/CF, they measured pH value of the phosphate buffered saline lixivium of the PLLA/CF with different content of the chitin fibers. Then, they compared the cell proliferation, attachment, alkaline phosphatase level, total protein per unit cell, and osteopontin, osteonectin, and osteocalcin gene expression of human osteoblastlike cells cultured on the PLLA/CF with or without the link, and PLLA scaffold. The results showed that the link treatment had no significant effect on the pH value of the lixivium of the scaffolds. Moreover, 30% volume content might be a suitable content of the chitin fibers in PLLA/CF scaffold. During the lixiviation time of 16 weeks, the PLLA/CF scaffold was significantly better for the attachment, proliferation, differentiation, and mineralization of the osteoblast than PLLA. Similarly, Guarino et al. developed the composite scaffolds through combining two or more types of biodegradable polymers with selected properties, which meet all the mechanical and physiological demands of the host tissue [92]. A new type of composite material for bone regeneration, consisting of a porous PCL matrix enhanced by continuous poly(L-lactic acid) (PLLA) fibers, was processed by the synergistic use of filament winding and phase inversion/salt leaching technique (Fig. 7.6). The chemistry and the degrees of micro- and macro-porosity gained by such techniques decide the structural and biological performances of the composite. Considering the degradation properties, the treatment with NaOH solution (pH 12) aroused the most significant changes over 5 weeks. These just related to the more hydrophilic PLLA component, which experienced significant structural rearrangements, decrease of polymeric chain length and increase of crystallinity. Furthermore, encouraging results are showed in cellular studies in the case of human osteoblasts and marrow stromal cells: the latter were found more active on the composite, due either to a slightly higher number of cells or to a higher activity of the cells compared to the former.

The stiffening effect on mechanical models used to measure the efficient of stiffening in different mineralization methods on nanofiber polymer scaffolds and compared strictly examine the mechanic nanofiber PLGA scaffolds with gradients in the mineral. Lipner et al. determined the local mechanical properties and the mineral volume fractions, and developed a mathematical model to compare the experimental results of the optical bounds [93]. It is found that the two mineralization methods all stiffened the scaffolds, but a varied substantially of magnitudes,

Fig. 7.6 Scanning electron microscope analysis of fiber-reinforced composite scaffolds: (a) bimodal pore size distribution and (b) organization of PLLA fibers inside porous PCL matrix. The microporosity with small pore sizes ranging 1-10 pm and a macroporosity with 100-400 pm pore size (Adapted with permission from Ref. [92]. Copyright 2008 Elsevier Ltd)

with modified simulated body fluid (m10SBF) showing a more efficient effect than simulated body fluid (10SBF). When compared to the composite bounds we developed, the stiffening by mineralization achieved using 10SBF proved to be weaker than the lowest possible stiffening predicted by homogenization theory, which indicates that mineral linked weakly to the scaffold. In contrast, mineralization-using m10SBF achieved stiffening that was hardly an order of magnitude greater than10SBF. This stiffening was near to that predicted by the Hashin-Shtrikman upper homogenization bound, which suggests that the new method might fit the tendon to bone tissue engineering.

Complexation with other more rigid polymeric constructs, such as electrospun fiber meshes, can be used to provide a template for tissue regeneration and improve structural rigidity [94, 95]. The traditional role of the scaffold as simply a template for tissue formation has evolved and the new generation of scaffolds are increasingly used as delivery vehicles for therapeutic molecules such as drugs, proteins, and genes that initiate biological events leading to the regeneration of tissue [96]. Hoppe et al. found ions can also be classified as therapeutics [97, 98]. One method of delivery is the release of Si and Caions from bioactive glasses, which are defined as inorganic surface-active bioceramics. Moreover, previous studies indicated that

Scanning electron microscopy micrograph of the fibers before heating

Fig. 7.7 Scanning electron microscopy micrograph of the fibers before heating. (a). 45S5 submicron BG fibers, (b) 45S5 sub-micron BG fibers containing Cu (Adapted with permission from Ref. [101]. Copyright 2016 Elsevier Ltd)

direct mixing of Cu ions with bioactive materials is a feasible way to improve angiogenesis [99, 100]. Sharifi et al. prepared Cu-containing composite scaffolds reinforced by bioglass (BG) fibers and investigate their effects on properties of scaffolds for bone tissue engineering [101]. In that research, three types of scaffolds were fabricated, including gelatin-collagen hydrogel, gelatin-collagen composite containing submicron 45S5 (in wt%: 45% SiO2, 24.5% Na2O, 24.4% CaO and 6% P2O5) BG fibers, and Cu-doped composites. Sub-micron BG fibers were fabricated by combination of sol-gel and electrospinning processes, the fibers were then mixed with hydrogel matrix containing gelatin and collagen, freeze dried, followed by genipin cross-linking to fabricate final composite scaffolds (Fig. 7.7). Growth and viability of human osteoblast-like cell line SaOS-2 were investigated on these biomimetic scaffolds. Cellular biocompatibility assays illustrated that scaffolds containing Cu ion in the BG structure had more viability and osteoblast growth in comparison with other scaffolds. The scaffolds containing Cu ion in the 45S5 BG network showed better biocompatibility compared to the hydrogel and composite scaffold containing the 45S5 BG. Cu ion increased the growth of osteoblasts on composite scaffold containing sub-micron fibers BG, compared to hydrogel without sub-micron fibers BG. The results showed that the prepared scaffolds, especially Cu-doped BG fibers containing scaffold isnon-cytotoxic and its surface is ideal for osteoblast attachment, growth, viability, and bone regeneration.

Except for those above, ploymer fiber and BG fiber, collagen fiber has been simultaneously studied widely. A series of highly porous scaffolds derived from type I collagen fibers and the polysaccharide have been developed by a controlled freeze-drying process [102-104]. These scaffolds with an optimized composition to facilitate osteogenesis have been shown to promote bone repair in vivo in the case of minimally loaded calvarial defects [105]. The emphasis of this study was to incorporate cobalt bioactive glass into CG scaffolds that have been developed and optimized for bone tissue regeneration to improve the mechanical and structural properties of the CG scaffold, and vitally importantly, enhance the initial angiogenic step vital for bone regeneration. Specifically, the aims were to evaluate the effect of the bioactive glass on the pore structure, including porosity, compressive module and biological activity of the resultant composites, by inspecting their ability to induce an angiogenic and osteogenic response from cells. When Combined novel hypoxia-mimicking cobalt bioactive glasses with CG scaffolds, bone were repaired. As the results demonstrated, these scaffolds with collagen fibers could create a microenvironment that stimulates both angiogenesis and vascularization through the release of cobalt, a known hypoxia mimic, as well as supporting osteogenesis, which benefit from the addition of osteoinductive bioactive glass particles. Moreover, Liu et al. produced hydrogel scaffolds reinforced by collagen fibers in vitro, in the aspect of improving adhesion and differentiation of bone cells, most hydrogels fail to be independently used in load bearing sites for their less mechanical integrity [106]. Hydrogels, networked and hydrophilic polymers, are typically formed via cross-linking or chain entanglement, absorbing significant amounts of water [107-109]. Therefore, there has been great interest in preparing composites on basis of hydrogels. The first approach is to compound hydrogel materials with other biomaterials, such as hyaluronate composites and collagen fiber. The composites reinforced by fiber are capable of providing the enhanced mechanical properties. Moreover, the composites performed greater osteoconductivity compared to either material independently, indicating the satisfactory synergy of the components.

The anterior cruciate ligament (ACL) is the ligament of the knee injured most commonly as the number of the patients diagnosed with ACL disruptions is more than 200,000 every year [110-112]. The ACL consisting of elastin, water, cells, proteoglycans, and collagens (typesI, III, and V), is a highly organized, dense, cable-liketissue [113]. Moreover, it is vital to normal stability and kinematics as the key intra-articular ligament of the knee [114]. To repair the knee damage created by ACL disruptions, various materials and structures have been studied for their use in tissue engineered ligament replacements. It also has been investigated that polydiox- anone (PDS) can be used for scaffold construction as a promising material [115]. In addition, Dunn et al. have also studied the use of type I collagen fibers in ACL scaffolds [116]. Furthermore, compositing some more complex fibers in tissue- engineered structures can resolve section of these possible problems. A recent tissue-engineered structure using twisted silk fibers conforms two different tissue engineering methods to achieve the regeneration of damaged or lost tissue: using isolated cells that have been expanded in vitro and using a three-dimensional, porous matrix. The matrix composited by silk performs hierarchical structure, and is non-toxic in proliferation tests using bone marrow stromal cells. Wounding bundles of silk fibers into strands, which will be wound into cords can form the matrix. The twisted fiber architecture promotes the similarity to ACL in scaffold mechanical properties. The scaffolds also showed the three phase mechanical behavior observed in ligament and tendon, a linear region (high stress per unit strain), and consecutively, a toe region (low stress per unit strain), which is important for the protection of damage deriving from creep and fatigue. Moreover, in other studies, Altman et al. coated the surface with RGD sequences (Arg-Gly-Asp), having improved the ability to elicit new tissue growth and the biocompatibility of this matrix [117]. The presence of the RGD sequences on the silk fiber surfaces also increased the production of extracellular matrix by bone marrow stromal cells creating the possibility of quicker and more complete tissue regeneration.

More recently, Laurencin and his partners have put forward a tissue-engineered method, which is based on a degradable, cell seeded, 3D braided PLLA fibers scaffold [118]. The scaffold used 3D braiding techniques to product a new scaffold with integrated pores, controlled pore size and mechanical properties compared to natural ACL and resistance to wear and rupture. It is very important to control the size and distribution of pores in the scaffold. Because it enhances bone cell tissue and proliferation in growth. The braided scaffold with hierarchical structure is similar to the ligament. The braided scaffold is consists of fibers in the same diameter to collagen fibers. Then the fibers are integrated into bundles and wound through the thickness of the scaffold. The braids include three regions: ligament region, femoral tunnel attachment site, and tibial tunnel attachment site. The intra-articular zone has a lower-angle fiber orientation and the femoral tunnel attachment sites have a high- angle fiber. The different orientations of fiber lead to diversification about pore size between different regions. The three regions of the scaffold comprise pore sizes within these listed ranges to facilitate capillary supply and tissue (ligament and bone) in growth. The smaller pore size (and higher braiding angle) at the insertion sites improve resistance to wear in bone tunnels and enhance the quality of anchorage within bone tunnels via the integration of bone tissue. Pore size as well as overall scaffold porosity is able to change the response to the implant of cells. The pore interconnectivity stretching via an implant expands the overall surface area of cell attachment, which can simultaneously reinforce the regenerative properties of the implant via permitting tissue growth into the internal matrix.

In addition, bone-anterior cruciate ligament-bone (B-ACL-B) grafts were studied by incorporating surfactants lauryl sulfate (SDS), Triton X-100, and/or an organic solvent (tributyl phosphate (TnBP)) [119]. The study showed that the mechanical and biochemical properties of B-ACL-B grafts satisfy the further investigation of the repair B-ACL-B defect (Fig. 7.8). The further study was to prepare the scaffolds reinforced by fibers to repair the B-ACL-B damage.

Besides the fibers, nanotubes are also used extensively for bone repair in vitro. Karla et al. compared the osteogenic cell behavior on titania nanotubes surfaces with carbon-coated titania nanotubes surfaces, including osteoblast and mesenchymal stem cell interactions with the different surface chemistries [120]. Their experiments showed that nanotubes had good influence on the alkaline phosphatase activity of osteoblast cells and promoted osteogenic differentiation. The carbon- coated titania nanotubes had higher levels of osteo-differentiation. Similarly, Wan et al. prepared a three-dimensional network-structured silica nanotubes scaffold, and did cell studies using a mouse fibroblast cell line and a human osteoblast-like cell line [121]. The three-dimensional network structured silica nanotubes scaffold benefited for the attachment, spreading, and proliferation of cells and showed excellent biocompatibility. Thus, silica nanotubes are promising used in bone tissue engineering and regeneration.

(a) Potted B-ACL-B sample mounted onto servo-hydraulic tensile testing apparatus

Fig. 7.8 (a) Potted B-ACL-B sample mounted onto servo-hydraulic tensile testing apparatus. (b) The schematic diagram showing the details of the gripping of the B-ACL-B samples. This set-up was suit for the tensile properties of ACLs loaded along the axis of the Ligament independent of the angle of knee flexion and engaging the greatest amount of ligament fibers (Adapted with permission from Ref. [119]. Copyright 2005 Elsevier Ltd)

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