# Recombinant Proteins-Based Strategies in Bone Tissue Engineering
## 10 Future Perspectives  
The development of new scaffolds using recent advances in molecular biology undoubtedly provides novel strategies in tissue engineering [97]. In particular, scaffolds with recombinant proteins for bone tissue engineering application have broad prospects and wide frontiers to explore. Scaffolds based on human recombinant R-spondin (RSPO) proteins are a good example in this respect. RSPO comprises a family of secreted proteins with important roles in cell proliferation and differentiation [98]. RSPOs display characteristic domains which are conserved among vertebrates, such as: (1) a thrombospondin\(1\) repeat domain; (2) a cysteine-rich furin-like domain; (3) a basic amino acid-rich domain of variable length in the C-terminal region; and (4) a hydrophilic signal peptide sequence [99]. They are broadly expressed during skeletal development, and modulate osteoblasts and bone formation by inducing the canonical WNT/\(\beta\)-catenin pathway [100]. A stable platform for the production of recombinant hRSPO1 from HEK293 cells was generated, leading to the production of a purified, fully characterized bioactive protein product for bone tissue engineering applications [101]. From the perspective of recombinant protein strategies, new technologies have emerged that are still in their infancy but have a high potential in bone regenerative medicine. Particularly noteworthy in this regard, a very recent report from [PERSON] et al. [102] has exploited the fact that BMP2 is known to induce the expression of its antagonist noggin that self-restricts its bioactivity. Therefore, they proposed that a CRISPR-based system providing noggin inhibition concurrently to BMP2 overexpression might improve bone healing. In fact, this complex system was found to stimulate osteogenic differentiation of adipose-derived stem cells in vitro, and promoted the mineralization and repair of a calcaneal bone lesion in vivo. Indeed, bone tissue engineering using molecular biology strategies and recombinant protein production is advancing rapidly. The advent of recombinant DNA strategies as well as the development of genomic strategies that allow the design of oligomeric sequences coding for proteins of interest represent great improvements for recombinant protein production. In addition, production of chimeric proteins would provide alternatives for the development of multifunctional scaffolds to promote osteogenesis at the site of bone injury. This is particularly important to repair non-union fractures and to avoid double surgeries required by autologous bone replacement [103; 104; 105]. Novel protocols applying tissue engineering strategies based on recombinant proteins for bone tissue repair in the clinical practice are in constant development, with very encouraging results [106; 107; 108; 109]. Current strategies in this regard intend to promote a close relationship between basic researchers who develop recombinant scaffolds and physicians who face the different types of problems associated with bone pathology [110; 111; 112]. In this way, knowledge of the metabolic pathways supporting bone health, such as those shown in Figure 1, facilitates the development of recombinant scaffolds to meet specific needs, without undesirable side effects, for each type of bone lesion. All of the above indicates that bone tissue repair based on recombinant molecular biology strategies has a promising future in bone regenerative medicine. However, use of recombinant chimeric proteins for use in medical practice requires further exploration of the physiological mechanisms of bone repair in order to generate bone tissue repair biomolecules that possess the appropriate structure to be delivered at the site of injury. By this regard, researchers have been increasingly focusing on recombinant materials that can be produced with full reproducibility, and can be designed according to specific needs to form scaffolds.