# Recombinant Proteins-Based Strategies in Bone Tissue Engineering
## 6 Collagen-Based Recombinants  
Currently, collagen used in tissue engineering applications are derived from animal tissues, which raises concerns related to the quality, purity, and predictability of their performance, as well as the risk of transmission of infectious agents and precipitating immune reactions. Therefore, in recent years, the interest of collagen in tissue engineering focused on the development of recombinant sources of human collagen that provide a predictable and chemically defined source of purified human collagen free of animal components [53]. The advantage found in this type of scaffold is that, in addition to being perfectly biocompatible, it has the ideal chemical and mechanical properties to replace the extracellular matrix of damaged tissue, such as structure, strength, mechanical integrity, and porosity, that allows cellular infiltration and nutrient diffusion required for osteoinduction and osteodifferentiation. Mechanical properties are a critical factor for cell adhesion, growth, and stem cell differentiation [54]. In 2004, [PERSON] et al. [41] used recombinant human type I collagen (RCP) as a scaffold. Its production was achieved by cross-linking followed by lyophilization, forming a 3D porous structured scaffold with extensive surface areas, compared to those observed in type I collagen of animal origin, to allow cell proliferation, in vitro and in vivo. In vitro activity was analyzed in cell cultures for three months, considering osteogenic differentiation and mineralization. In the in vivo studies, using a cranial lesion in rats, the biocompatibility of the scaffold with mild and insignificant inflammation was demonstrated. Thereafter, [PERSON] et al. [55] performed in vitro and in vivo studies, with an interesting scaffold produced with a human recombinant type 1 collagen nanohydroxapatite-poly (lactic acid) composite. It was tested in vivo in rabbit radial defects that healed 24 weeks after surgery. These studies demonstrate that scaffolds with this recombinant collagen are similarly efficient as those containing collagen of animal origin, but avoid potential hazards (e.g., infection by viruses). [PERSON] et al. [54], in 2017, investigated the properties of recombinant type I collagen-based scaffolds by comparing three cross-linking procedures, using dehydrothermal, hexamethylene diisocyanate or genipin, to evaluate whether the chemical properties of the scaffolds could vary depending on the treatment. The mechanical properties were not significantly altered. The chemistry of the scaffolds was changed, affecting properties such as water uptake, and initial adhesion of human mesenchymal stem cells (MSCs); genipin crosslinking supported the lowest MSC adhesion. In vitro analysis using hMSC cultures showed that, regardless of the cross-linking method, the scaffolds can support osteoblast differentiation and mineralization. In another study, three types of RCP scaffolds enriched in arginine-glycine-aspartate sequence were evaluated: one scaffold with non-mineralized RCP, another with mineralized RCP, and the third one with mineralized RCP in the presence of magnesium, all three with 3D isotropic porous structure and produced by lyophilization [35]. In vitro studies using MSC cultures revealed that the latter scaffold induced cell growth and increased gene and protein expression of osteogenic markers, best mimicking bone mineral composition, thus the most promising candidate for future clinical applications. In 2017, [PERSON] et al. [56] produced a new microcarrier type constituted of a recombinant collagen I peptide This microcarrier allows the GMP-compliant upscaling of scaffold and cell production, as needed for cell delivery in a clinical scenario. [PERSON] et al. [57] developed collagen and elastin-like polypeptide (ELP)-based bone regenerative hydrogels loaded with recombinant human bone morphogenetic protein-2 (rhBMP-2) with mechanical properties suitable for osteogenesis.