This study confirmed the existence of BMP-4, 6, 7, and 9 in IMs using human samples, and demonstrated that these BMPs serve as osteoinductive factors in the treatment of patients with bone defects using the Masquelet technique. This finding helps understand the mechanism by which IM promotes bone regeneration and repair in the treatment of bone defects using the Masquelet technique. Therefore, the presence of these BMPs as osteoinductive factors in IM may lead surgeons to omit the addition of exogenous BMPs to bone grafts. The application of BMPs to surgery is expensive, and use of IM in the Masquelet technique can be considered as an alternative approach to supply BMPs at a low cost.
BMPs constitute the largest subdivision of the transforming growth factor-β (TGF-β) family of ligands, with nearly 30 distinct human proteins bearing the name [17, 18]. Among the various BMPs, we selected four for this study, the reasons for which are enumerated below.
BMP-4 belongs to the same subgroup of bone-inducing BMPs as BMP-2, based on the homology of their amino acid sequences. BMP-2 is known to be the most representative osteoinductive factor and is widely used clinically to treat bone fracture or nonunion [22, 31, 32]. BMP-4 has been detected along with BMP-2 in the area of endochondral ossification, particularly in the matrix between the newly formed osteoid in human fracture callus . The expression of BMP-4 and Noggin, a major BMP antagonist in tissues, is highlighted in the newly formed callus tissue, thereby confirming the central role of BMP signaling in bone fracture repair .
BMP-7 has been clinically applied to treat nonunion of fractures. In a randomized controlled trial, the efficacy of recombinant human BMP-7 (rhBMP-7) in tibial nonunion of 124 patients who received either autologous bone grafting or a device containing rhBMP-7 was tested . The bone healing rate was found to be inferior in the rhBMP-7 treated group, albeit not statistically significant, and the bone healing capacity of rhBMP-7 was assessed to be comparable to that of autologous bone grafting. The Food and Drug Administration (FDA) and European Medicines Agency (EMA) approved rhBMP-7 as a “humanitarian use device” for tibial nonunion. In addition, rhBMP-7 has been used off-label for various indications, including nonunion of the scaphoid, humerus, and clavicle [33–36].
BMP-6 is a paralog of BMP-7 with 87% similarity in their amino acid sequences. BMP-6 is more potent in promoting osteoblast differentiation in vitro and inducing bone regeneration in vivo when compared with its closely related BMP-7 paralog. This is explained by the reversible binding of BMP-6 to Noggin, and unlike BMP-7, BMP-6 may dissociate from Noggin and escape Noggin inhibition . A novel rhBMP-6 containing osteogenic device aimed to accelerate bone regeneration has been developed and is being tested in clinical trials [18, 38, 39].
BMP-9 is a recent discovery in the BMP family. BMP-9 is resistant to Noggin, thus facilitating a more robust cellular differentiation of osteoprogenitor cells into preosteoblasts and osteoblasts . It was reported that BMP-9 stimulated callus formation in osteoporotic rats during fracture healing . Besides its osteogenic activity, BMP-9 exerts a broad range of biological functions, including stem cell differentiation, angiogenesis, neurogenesis, tumorigenesis, and metabolism . It is expected that BMP-9 will be a promising alternative to clinically available BMPs.
We revealed the existence of BMP-4, 6, 7, and 9 in all human IM samples. In addition to immunolocalization of BMP-4, 6, 7, and 9 in blood vessels and fibroblasts, they were also observed within the bone inside the IM in one sample, in which osteogenesis inside the IM was observed. This histological finding of osteogenesis inside the IM was found in one sample out of six. Other investigators have also reported this finding in limited samples [14, 28]. We consider BMP-4, 6, 7, and 9 existing inside the IM and contributing to osteoinduction as an important finding.
The Masquelet technique is frequently applied to the lower extremity rather than the upper extremity. The tibia and femur are long bones of the lower extremities and frequently become subject to bone defects. The volume of the surrounding soft tissue is greater in the femur than in the tibia, and can be considered to reflect vascularity, thereby potentially affecting the formation and properties of IM. Therefore, we included both tibia and femur cases in this study. The results of our study revealed that IMs harvested from both tibia and femur cases expressed BMP-4, 6, 7, and 9.
The strength of this study was the use of human IM samples harvested from patients with bone defects treated using the Masquelet technique. In addition, we revealed the existence of BMPs that have not been proven at the protein or the mRNA level. The limitation of this study is that specimens were harvested from a limited number of patients. Moreover, this was a retrospective study conducted at a single institution, and could therefore be susceptible to selection bias and limited generalizability.