Recombinant Human Bone Morphogenetic Protein 6 Delivered Within Autologous Blood Coagulum Restores Critical Size Segmental Defects of Ulna in Rabbits

Abstract

BMP2 and BMP7, which use bovine Achilles tendon–derived absorbable collagen sponge and bovine bone collagen as scaffold, respectively, have been approved as bone graft substitutes for orthopedic and dental indications. Here, we describe an osteoinductive autologous bone graft substitute (ABGS) that contains recombinant human BMP6 (rhBMP6) dispersed within autologous blood coagulum (ABC) scaffold. The ABGS is created as an injectable or implantable coagulum gel with rhBMP6 binding tightly to plasma proteins within fibrin meshwork, as examined by dot‐blot assays, and is released slowly as an intact protein over 6 to 8 days, as assessed by ELISA. The biological activity of ABGS was examined in vivo in rats (Rattus norvegicus) and rabbits (Oryctolagus cuniculus). In a rat subcutaneous implant assay, ABGS induced endochondral bone formation, as observed by histology and micro‐CT analyses. In the rabbit ulna segmental defect model, a reproducible and robust bone formation with complete bridging and restoration of the defect was observed, which is dose dependent, as determined by radiographs, micro‐CT, and histological analyses. In ABGS, ABC scaffold provides a permissive environment for bone induction and contributes to the use of lower doses of rhBMP6 compared with BMP7 in bovine bone collagen as scaffold. The newly formed bone undergoes remodeling and establishes cortices uniformly that is restricted to implant site by bridging with host bone. In summary, ABC carrier containing rhBMP6 may serve as an osteoinductive autologous bone graft substitute for several orthopedic applications that include delayed and nonunion fractures, anterior and posterior lumbar interbody fusion, trauma, and nonunions associated with neurofibromatosis type I. © 2018 American Society for Bone and Mineral Research.

Autologous blood coagulum is a physiological carrier for BMP6 to induce new bone formation and promote posterolateral lumbar spine fusion in rabbits

“…ABC was chosen as a physiological carrier as it suppresses foreign body response, promotes tight rhBMP6 binding with plasma proteins within the fibrin meshwork, allows a sustained release of rhBMP6, and avoids generation of antibodies to rhBMP6, thus providing a permissive environment for endochondral bone differentiation. In our previous work (Grgurevic et al, ), we have demonstrated that the use of ABC as a carrier reduced the immune response when compared with the use of bovine collagen, as shown by reduced neutrophil accumulation and myeloperoxidase activity in rat subcutaneous implants. The ALLO particles are added and distributed uniformly across the ABC to provide adequate biomechanical and biocompatible good handling properties.…”

“…As rhBMP6 is mainly bound to plasma proteins in ABGS (Grgurevic et al, ), the calculated cumulative total release measured in vitro over 10 days from ABGS was only 3–5% of total rhBMP6 dose (Figure c). Addition of ALLO in different particle sizes seemed to slightly attribute to more cumulative release.…”

“…BMP6 was chosen as a preferred BMP as its binding to Noggin, a natural BMP antagonist present in abundance in bone, is lesser than other BMPs (Klineberg et al, ; Song et al, ; Vukicevic et al, ). BMP6 also binds to most of the Types I and II BMP receptors and exhibits a high specific alkaline phosphatase activity in osteoblastic cell cultures (Grgurevic et al, ; Song et al, ), hence permitting the use of lower doses as compared with BMP2 or BMP7. ABC was chosen as a physiological carrier as it suppresses foreign body response, promotes tight rhBMP6 binding with plasma proteins within the fibrin meshwork, allows a sustained release of rhBMP6, and avoids generation of antibodies to rhBMP6, thus providing a permissive environment for endochondral bone differentiation.…”

“…ABGS without ALLO was produced by dispersing rhBMP6 into autologous blood, which then allowed to form coagulum with defined structure and rheological properties as determined by stiffness, elasticity, and strain, testing as previously described (Grgurevic et al, ). Blood samples were collected from rabbit marginal ear veins into tubes without any anticoagulant substance, supplemented with 0.1 ml of 50‐mM CaCl 2 solution in a volume of 2.5 ml, 2 hr before surgery, and lyophilized rhBMP6 (Genera Research, Croatia) was dissolved in water for injection (c = 2.5 mg/ml) and mixed with blood in an appropriate volume and then left in room temperature to coagulate.…”

Recombinant Human BMP6 Applied Within Autologous Blood Coagulum Accelerates Bone Healing: Randomized Controlled Trial in High Tibial Osteotomy Patients

“…The optimal effective dose (100 μg/mL) was chosen for a single administration based on several preclinical studies using small and large animal models for fracture healing and posterolateral lumbar fusion. (6,7,9) Since we employed autologous carrier in animals as well as in human patients instead of xenogeneic animal-derived collagen as a carrier, the effective dose is translatable from preclinical models to human trials. Avoidance of animal-derived collagen helped to minimize the inflammatory response at the local implant site and the systemic immune responses, and thus prevented edema, erythema, and tenderness.…”