# Recombinant Proteins-Based Strategies in Bone Tissue Engineering
## 9 Recombinant Spider Silk  
Spider silk is made of protein fibers spun by spiders. Spiders use their silk to make webs or other structures, which function as sticky nets to catch other animals, or as nests or cocoons to protect their offspring or to wrap up prey. The properties of native spider silk vary within and across species due to the presence of different genes containing conserved repetitive core domains encoding a variety of silk proteins. Spider silk is biocompatible, strong and elastic, and hence an attractive biomaterial [87; 88]. The most used recombinant spider silk proteins are based on sequences from Nephila clavipes or Araneus diadematus [89]. These proteins can form various types of scaffolds--fibers, films, 3D-foams, hydrogels, tubes, and microcapsules--because they can self-assemble. Two independent groups have made great progress in bone tissue engineering based on the structure of these silk proteins. [PERSON] et al. [90] generated two recombinant proteins from the consensus sequence of the major component of dragline silk from Nephila clavipes, including RGD cell-binding domains. The proteins produced were processed into films and fibers. It was observed that these scaffolds increased the differentiation of human marrow stromal cells into osteoblastic progeny in the presence of osteogenic stimulants. In addition, [PERSON] et al. [53] generated biomimetic calcium phosphate coatings on recombinant spider silk fiber, showing that they supported the attachment and growth of hMSCs. In 2011, an interesting recombinant chimeric protein of spider silk (the consensus repeat for the Nephila clavipes spider dragline protein) and bone sialoprotein (BSP) was designed. The clone carrying the DNA sequence coding for BSP was inserted in the vector pET30L (Novagen, San Diego, CA, USA) carrying the silk block copolymer. After the expression and purification of the chimeric protein, a film chimeric scaffold was obtained that had more elastic modulus than the film of spider silk alone without the BSP. This protein retained mineralization potential and sustained human mesenchymal stem cell proliferation and differentiation into the osteogenic lineage [91]. An interesting contribution was made more recently, using a scaffold produced from a recombinant spider silk, based on the consensus motif of the repetitive core domain of one of the major ampullate silk fibroins of the garden cross spider, _A. diadematus_ fibroin 4 [92]. This recombinant protein contains sixteen repeats of the polypeptide module C (GSSAAAAAAAASGPGYGPENQGPSGPGYGPGGP amino acid sequence), referred to hereafter as eADF4(C16). It was found to display multiple carboxylic acid moieties capable of binding calcium ions, which would favor the mineralization process. In vitro studies with MSCs seeded on these scaffolds showed a significant increase in ALP activity, as well as its potential in bone tissue engineering. Moreover, a scaffold was produced from cloning a chimera protein containing the spider silk inspired domain (SGRGGLGGQG AGAAAAAGGA QGGYGGGLGSQGT)15--serving as an organic scaffold to control material stability and to allow multiple modes of processing--and the HA binding domain VTKHLNQISQSY (VTK), providing control of osteogenesis [93]. These scaffolds increased differentiation of bone marrow-derived hMSCs into osteoblastic progeny. Recently, the spider silk proteome has provided the structural characterization of the entire structure of _Nephila clavipes_ flagelliform spidroin [94]. The long sequence of this flagelliform silk protein presents 45 hydroxylated proline residues, each located in the GPGX motif, which may contribute to explain the mechanoelastic property of these fibers. This is a very interesting property to be present in scaffolds to be applied in bone tissue engineering. Knowing this sequence could be applied to the construction of recombinant chimeric proteins with this property. A recent study by [PERSON] et al.