# Recombinant Proteins-Based Strategies in Bone Tissue Engineering
## 5 Recombinant Human BMPs in Bone Tissue Engineering  
Among the first human recombinant proteins selected for use in bone tissue engineering were BMPs, in particular BMP-2 [41]. Recombinant human BMP-2 (rhBMP-2) was approved by the Food and Drug Administration (FDA) and used clinically. The mechanism of action of the two most studied BMPs, BMP-2 and BMP-9, is shown in Figure 1.  
Though rhBMP-2 shows a well-documented ability to induce bone formation, it has a very short half-life, requiring clinicians to use supraphysiological doses, and even its uncontrolled release in vivo could lead to ectopic bone formation, inflammation, and increased cancer risk [42]. This reinforces the idea that this recombinant protein must be applied through tissue engineering strategies, namely attached to matrices that slowly releases it in situ. The  
Figure 1: A brief summary of BMP-2 and BMP-9 signaling pathways, and their relation to the Wnt canonical pathway. _ECM_, extracellular matrix; _CM_, cell membrane; _NM_, nuclear membrane; _BMP-2_, bone morphogenetic protein 2; _BMP-4_, bone morphogenetic protein 4; _BMP-9_, bone morphogenetic protein 9; _BMPr2_, bone morphogenetic protein 2 receptor; _BMPr1_, bone morphogenetic protein 1 receptor; _miRR_, microRNA; _miRR-24_, microRNA 27a; _miRR20u_, microRNA 20a; _Wnt3A_, Wnt family member 3A; _Wnt5a/b_, Wnt family member 5a and 5b; _DKK-1_, Dickkopf-related protein 1; _LRP5_, low-density lipoprotein receptor-related protein 5; _YAP_, yes-associated protein; _TAZ_, transcriptional co-activator with PDZ-binding motif; _APC_, activated protein C; _GSK_, glycogen synthase kinase-3; _CKI_, casein kinase 1; _TEADs_, transcriptional enhancer activator domains; _Runz2_, run-related transcription factor 2; _Sp7_/Oxx, transcription factor Sp7 or Osterix; _TCF1/LEFI_, specific T-cell factor/factor 1 transcriptional factor; _MMP13_, matrix metallopeptidase 13; _RANK-L_, receptor activator for nuclear factor xB ligand; _OCN_, osteoclastin; _OPN_, osteepontin; _ALP_, alkaline phosphatase; _OPG_, osteeprotegerin; _Co1I_, type 1 collagen; _IGFBP-4_, insulin-like growth factor binding-protein 4.  
scaffolds act in these situations, not only as structures promoting tissue neoformation, but also as favoring the release of recombinant proteins at the site where they are implanted. A novel rhBMP-2-atelocollagen composite scaffold implanted in an experimental bone defect in rats induced intense new bone formation activity as demonstrated by the expression of bone formation markers, alkaline phosphatase (ALP), osteoclastcin (OCN), osteepontin, and bone sialoprotein, detected by reverse transcription-polymerase chain reaction and immunohistochemistry [43]. In addition, other research groups proposed to attach rhBMP-2 to polimeric lactic scaffolds (PLLA), with encouraging preliminary results [44]. More recently, a polydopamine (pDA)-assisted BMP-2-derived peptide (named as P24) was developed by a surface modification strategy for attachment to a scaffold consisting of nano-hydroxyapatite (nHA)/recombinant human-like collagen (RHLC)/poly (lactic acid)(PLA), to improve its osteogenic capacity [45]. The immobilization efficiency and release kinetics of P24, and in vitro osteoinductive activity of the nHA/RHLC/PLA-pDA-P24 scaffold were examined in rat-derived mesenchymal stem cells (rMSCs) in vitro, showing increased ALP activity and _mRNA_ expression of bone specific markers, compared with non-P24-loaded nHA/RHLC/PLA scaffold. Moreover, in vivo, nHA/RHLC/PLA-pDA-P24 scaffolds significantly enhanced bone regeneration in a rat critic-sized calvarial defect [45]. Furthermore, a pioneering study has recently compared the osteogenic potential of rhBMP-2 and rhBMP-9 [46]. In vitro, undifferentiated mouse ST2 stromal bone marrow cells were seeded onto bovine-derived natural bone mineral (NBM) particles used as scaffolds for the different BMPs. Neither rhBMP2 nor rhBMP9 influenced cell attachment to the scaffold particles, but both BMPs stimulated cell proliferation.