Synthetic ceramic macroporous blocks as a scaffold in ectopic bone formation induced by recombinant human bone morphogenetic protein 6 within autologous blood coagulum in rats.

PURPOSE: We have recently developed an autologous bone graft substitute (ABGS) containing recombinant human bone morphogenetic protein 6 (rhBMP6) in autologous blood coagulum (ABC) that induces new bone formation in vivo. In order to improve biomechanical properties of the implant, compression resistant matrix (CRM) consisting of synthetic ceramics in the form of macroporous cylinders was added to the ABGS and we evaluated the biomechanical properties and the quantity and quality of bone formation following subcutaneous implantation in rats. METHODS: ABGS implants containing rhBMP6 in ABC with cylindrical ceramic blocks were implanted subcutaneously (n = 6 per time point) in the axillary region of Sprague-Dawley rats and removed at specified time points (7, 14, 21, 35, and 50 days). The quantity and quality of newly formed bone were analyzed by microCT, histology, and histomorphometric analyses. Biomechanical properties of ABGS formulations were determined by employing the cut test. RESULTS: MicroCT analyses revealed that ABGS implants induced formation of new bone within ceramic blocks. Histological analysis revealed that on day seven following implantation, the endochondral ossification occupied the peripheral part of implants. On days 14 and 21, newly formed bone was present both around the ceramic block and through the pores inside the block. On both days 35 and 50, cortical bone encircled the ceramic block while inside the block, bone covered the ceramic surface surrounding the pores. Within the osseous circles, there were few trabeculae and bone marrow containing adipocytes. ABGS containing cylindrical ceramic blocks were more rigid and had significantly increased stiffness compared with implants containing ceramic particles as CRM. CONCLUSION: We demonstrated that macroporous ceramic blocks in a form of cylinders are promising CRMs with good handling and enhanced biomechanical properties, supporting bone formation with ABGS containing rhBMP6 within autologous blood coagulum. Hence, ABGS containing ceramic blocks should be tested in preclinical models including diaphyseal segmental defects and non-unions in larger animals.

Biology of bone morphogenetic protein in bone repair and regeneration: A role for autologous blood coagulum as carrier.

BMPs were purified from demineralized bone matrix based on their ability to induce new bone in vivo and they represent a large member of the TGF-ß superfamily of proteins. BMPs serve as morphogenic signals for mesenchymal stem cell migration, proliferation and subsequently differentiation into cartilage and bone during embryonic development. A BMP when implanted with a collagenous carrier in a rat subcutaneous site is capable of inducing new bone by mimicking the cellular events of embryonic bone formation. Based on this biological principle, BMP2 and BMP7 containing collagenous matrix as carrier have been developed as bone graft substitutes for spine fusion and long bone fractures. Here, we describe a novel autologous bone graft substitute that contains BMP6 delivered within an autologous blood coagulum as carrier and summarize the biology of osteogenic BMPs in the context of bone repair and regeneration specifically the critical role that carrier plays to support osteogenesis.

Evaluation of synthetic ceramics as compression resistant matrix to promote osteogenesis of autologous blood coagulum containing recombinant human bone morphogenetic protein 6 in rabbit posterolateral lumbar fusion model.

Posterolateral lumbar fusion (PLF) is a commonly performed surgical procedure for the treatment of pathological conditions of the lumbosacral spine. In the present study, we evaluated an autologous bone graft substitute (ABGS) containing rhBMP6 in autologous blood coagulum (ABC) and synthetic ceramics used as compression resistant matrix (CRM) in the rabbit PLF model. In the pilot PLF rabbit experiment, we tested four different CRMs (BCP 500-1700 µm, BCP 1700-2500 µm and two different TCP in the form of slabs) which were selected based on achieving uniform ABC distribution. Next, ABGS implants composed of 2.5 mL ABC with 0.5 g ceramic particles (TCP or BCP (TCP/HA 80/20) of particle size 500-1700 µm) and 125 µg rhBMP6 (added to blood or lyophilized on ceramics) were placed bilaterally between transverse processes of the lumbar vertebrae (L5-L6) following exposition and decortication in 12 New Zealand White Rabbits observed for 7 weeks following surgery. Spinal fusion outcome was analysed by µCT, palpatory segmental mobility testing and selected specimens were either tested biomechanically (three-point bending test) and/or processed histologically. The total fusion success rate was 90.9% by both µCT analyses and by palpatory segmental mobility testing. The volume of newly formed bone between experimental groups with TCP or BCP ceramics and the different method of rhBMP6 application was comparable. The newly formed bone and ceramic particles integrated with the transverse processes on histological sections resulting in superior biomechanical properties. The results were retrospectively found superior to allograft devitalized mineralized bone as a CRM as reported previously in rabbit PLF. Overall, this novel ABGS containing rhBMP6, ABC and the specific 500-1700 µm synthetic ceramic particles supported new bone formation for the first time and successfully promoted posterolateral lumbar fusion in rabbits.

A novel autologous bone graft substitute comprised of rhBMP6 blood coagulum as carrier tested in a randomized and controlled Phase I trial in patients with distal radial fractures.

Bone morphogenetic proteins (BMPs) are known to induce new bone formation in vivo but treating trabecular bone defects with a BMP based therapeutic remains controversial. Here, we evaluated the safety and efficacy of a novel Autologous Bone Graft Substitute (ABGS) comprised of recombinant human BMP6 (rhBMP6) dispersed within an autologous blood coagulum (ABC) as a physiological natural carrier in patients with a closed distal radial fracture (DRF). We enrolled 32 patients in a randomized, standard of care (SoC) and placebo (PBO) controlled, double-blinded Phase I First in Human (FiH) clinical trial. ABGS was prepared from peripheral blood as 250 µg rhBMP6/mL ABC or PBO (1 mL ABC containing excipients only) and was administered dorsally via a syringe injection into the fracture site following closed fracture fixation with 3 Kirschner wires. Patients carried an immobilization for 5 weeks and were followed-up for 0 to 26 weeks by clinical examination, safety, serial radiographic analyses and CT. During the 13 weeks follow-up and at 26 weeks post study there were no serious adverse reactions recorded. The results showed that there were no detectable anti-rhBMP6 antibodies in the blood of any of the 32 patients at 13- and 26-weeks following treatment. Pharmacokinetic analyses of plasma from patients treated with ABGS showed no detectable rhBMP6 at any time point within the first 24 h following administration. The CT image and radiographic analyses score from patients treated with AGBS showed significantly accelerated bone healing as compared to PBO and SoC at 5 and 9 weeks (with high effect sizes and P = 0.027), while at week 13 all patients had similar healing outcomes. In conclusion, we show that intraosseous administration of ABGS (250 µg rhBMP6/mL ABC) into the distal radial fracture site demonstrated a good tolerability with no serious adverse reactions as well as early accelerated trabecular bone healing as compared to control PBO and SoC patients.

Discovery of bone morphogenetic proteins - A historical perspective.

Bone morphogenetic proteins (BMPs) were purified from demineralized bone matrix by their ability to induce new bone formation in vivo. BMPs represent a large sub-family of proteins structurally related to TGF-beta and activins. Two BMP bone graft substitutes, BMP2 (InFuse®) and BMP7 (OP1®) have been developed as products for the repair of long bone non-union fractures and lumbar spinal fusion in humans. The approval of BMP2 and BMP7 based products for use in the clinic supports that the signals responsible for bone formation at ectopic sites can form a basis as therapeutics for bone repair and regeneration. This article describes a historical perspective of the discovery BMPs.

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

Bone morphogenetic proteins (BMPs) are potent osteogenic proteins that induce new bone formation in vivo. However, their effect on bone healing in the trabecular bone surfaces remains challenging. We evaluated the safety and efficacy of recombinant human BMP6 (rhBMP6) applied within an autologous blood coagulum (ABC) in a surgically created wedge defect of the proximal tibia in patients undergoing high tibial osteotomy (HTO) for varus deformity and medial osteoarthritis of the knee. We enrolled 20 HTO patients in a randomized, placebo-controlled, double-blinded phase I/II clinical trial. RhBMP6/ABC (1.0 mg/10 mL ABC prepared from peripheral blood) or placebo (10 mL ABC containing excipients) was administered into the tibial wedge defects. Patients were followed for 0 to 24 months by clinical examination (safety) and computed tomography (CT) and serial radiographic analyses (efficacy). The results show that there were no detectable anti-rhBMP6 antibodies in the blood of any of the 20 patients at 14 weeks after implantation. During the 24 months of follow-up, there were no serious adverse reactions recorded. The CT scans from defects of patients treated with rhBMP6/ABC showed an accelerated bone healing compared with placebo at 9 weeks (47.8 ± 24.1 versus 22.2 ± 12.3 mg/cm3 ; p = 0.008) and at 14 weeks (89.7 ± 29.1 versus 53.6 ± 21.9 mg/cm3 ; p = 0.006) follow-up. Radiographic analyses at weeks 6 and 24 and months 12 and 24 suggested the advanced bone formation and remodeling in rhBMP6/ABC-treated patients. In conclusion, we show that rhBMP6/ABC at a dose of 100 µg/mL accelerated bone healing in patients undergoing HTO without serious adverse events and with a good tolerability compared with placebo alone. Overall, for the first time, a BMP-based osteogenic implant was examined against a placebo for bone healing efficacy in the trabecular bone surface, using an objective bone mineral density measurement system. © 2020 The Authors. Journal of Bone and Mineral Research published by American Society for Bone and Mineral Research.

Autologous blood coagulum containing rhBMP6 induces new bone formation to promote anterior lumbar interbody fusion (ALIF) and posterolateral lumbar fusion (PLF) of spine in sheep.

In the present study, we evaluated an autologous bone graft substitute (ABGS) composed of recombinant human BMP6 (rhBMP6) dispersed within autologous blood coagulum (ABC) used as a physiological carrier for new bone formation in spine fusion sheep models. The application of ABGS included cervical cage for use in the anterior lumbar interbody fusion (ALIF), while for the posterolateral lumbar fusion (PLF) sheep model allograft devitalized bone particles (ALLO) were applied with and without use of instrumentation. In the ALIF model, ABGS (rhBMP6/ABC/cage) implants fused significantly when placed in between the L4-L5 vertebrae as compared to control (ABC/cage) which appears to have a fibrocartilaginous gap, as examined by histology and micro CT analysis at 16 weeks following surgery. In the PLF model, ABGS implants with or without ALLO showed a complete fusion when placed ectopically in the gutter bilaterally between two decorticated L4-L5 transverse processes at a success rate of 88% without instrumentation and at 80% with instrumentation; however the bone volume was 50% lower in the instrumentation group than without, as examined by histology, radiographs, micro CT analyses and biomechanical testing at 27 weeks following surgery. The newly formed bone was uniform within ABGS implants resulting in a biomechanically competent and histologically qualified fusion with an optimum dose in the range of 100 µg rhBMP6 per mL ABC, while in the implants that contained ALLO, the mineralized bone particles were substituted by the newly formed remodeling bone via creeping substitution. These findings demonstrate for the first time that ABGS (rhBMP6/ABC) without and with ALLO particles induced a robust bone formation with a successful fusion in sheep models of ALIF and PLF, and that autologous blood coagulum (ABC) can serve as a preferred physiological native carrier to induce new bone at low doses of rhBMP6 and to achieve a successful spinal fusion.

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

In the present study, we describe autologous blood coagulum (ABC) as a physiological carrier for BMP6 to induce new bone formation. Recombinant human BMP6 (rhBMP6), dispersed within ABC and formed as an autologous bone graft substitute (ABGS), was evaluated either with or without allograft bone particles (ALLO) in rat subcutaneous implants and in a posterolateral lumbar fusion (PLF) model in rabbits. ABGS induced endochondral bone differentiation in rat subcutaneous implants. Coating ALLO by ABC significantly decreased the formation of multinucleated foreign body giant cells (FBGCs) in implants, as compared with ALLO alone. However, addition of rhBMP6 to ABC/ALLO induced a robust endochondral bone formation with little or no FBGCs in the implant. In rabbit PLF model, ABGS induced new bone formation uniformly within the implant resulting in a complete fusion when placed between two lumbar transverse processes in the posterolateral gutter with an optimum dose of 100-µg rhBMP6 per ml of ABC. ABGS containing ALLO also resulted in a fusion where the ALLO was replaced by the newly formed bone via creeping substitution. Our findings demonstrate for the first time that rhBMP6, with ABC as a carrier, induced a robust bone formation with a complete spinal fusion in a rabbit PLF model. RhBMP6 was effective at low doses with ABC serving as a physiological substratum providing a permissive environment by protecting against foreign body reaction elicited by ALLO.

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

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.

Excessive transforming growth factor-ß signaling is a common mechanism in osteogenesis imperfecta.

Osteogenesis imperfecta (OI) is a heritable disorder, in both a dominant and recessive manner, of connective tissue characterized by brittle bones, fractures and extraskeletal manifestations. How structural mutations of type I collagen (dominant OI) or of its post-translational modification machinery (recessive OI) can cause abnormal quality and quantity of bone is poorly understood. Notably, the clinical overlap between dominant and recessive forms of OI suggests common molecular pathomechanisms. Here, we show that excessive transforming growth factor-ß (TGF-ß) signaling is a mechanism of OI in both recessive (Crtap(-/-)) and dominant (Col1a2(tm1.1Mcbr)) OI mouse models. In the skeleton, we find higher expression of TGF-ß target genes, higher ratio of phosphorylated Smad2 to total Smad2 protein and higher in vivo Smad2 reporter activity. Moreover, the type I collagen of Crtap(-/-) mice shows reduced binding to the small leucine-rich proteoglycan decorin, a known regulator of TGF-ß activity. Anti-TGF-ß treatment using the neutralizing antibody 1D11 corrects the bone phenotype in both forms of OI and improves the lung abnormalities in Crtap(-/-) mice. Hence, altered TGF-ß matrix-cell signaling is a primary mechanism in the pathogenesis of OI and could be a promising target for the treatment of OI.

Sevelamer restores bone volume and improves bone microarchitecture and strength in aged ovariectomized rats.

Sevelamer hydrochloride, a noncalcium phosphate binder, has been shown to reduce coronary artery and aortic calcification, and to improve trabecular bone mineral density in hemodialysis patients with chronic kidney disease. Here, we examined whether sevelamer given orally for 12 wk with normal food could restore bone volume (BV) and strength in aged ovariectomized (OVX) rats starting at 4 wk after OVX. Dual-energy x-ray absorptiometry, microcomputerized tomography, and bone histomorphometry analyses showed that OVX animals receiving sevelamer had increased trabecular BV (51%), trabecular number (43%), trabecular thickness (9%), cortical thickness (16%), mineral apposition rate (103%), bone formation rate (25%), and enhanced cortical and trabecular bone mechanical strength as compared with OVX rats. Sevelamer decreased collagen C telopeptide, increased osteocalcin levels, and decreased phosphate and magnesium levels without affecting calcium levels in the blood. Although sevelamer was not absorbed systemically, it stimulated osteoblast differentiation in BM-derived mesenchymal stem cell cultures, as evaluated by alkaline phosphatase positive colony-forming units, and inhibited recombinant human soluble receptor activator of nuclear factor-kappaB ligand-induced osteoclast differentiation, as evaluated by tartrate-resistant acid phosphatase positive cells in bone mineral-hematopoietic stem cell cultures. Surface enhanced laser desorption/ionization time-of-flight mass spectrometry analysis revealed that 69 proteins were differently expressed after OVX, of which 30% (20 of 69) were reversed to sham activity after sevelamer intake. PTH, fibroblast growth factor-23, and cytokine profile in serum were not significantly changed. Together, these results suggest that sevelamer in food increases the BV and improves biomechanical properties of bone in OVX rats.

Newly reported roles of thyroid-stimulating hormone and follicle-stimulating hormone in bone remodelling.

Thyroid-stimulating hormone (TSH) and follicle-stimulating hormone (FSH) have both been recently implicated in bone remodelling. Clinical evidence, as well as data from TSH receptor and thyroid hormone receptor knockout mice, suggest that TSH has a direct effect on skeletal homeostasis, although some data are conflicting. Recently, the exogenous administration of TSH has been shown to positively impact bone in oophrectomised rats. These data, along with their potential implications for the treatment of severe osteoporosis, are discussed.

Thyroid-stimulating hormone restores bone volume, microarchitecture, and strength in aged ovariectomized rats.

UNLABELLED: We show the systemic administration of low levels of TSH increases bone volume and improves bone microarchitecture and strength in aged OVX rats. TSH's actions are mediated by its inhibitory effects on RANKL-induced osteoclast formation and bone resorption coupled with stimulatory effects on osteoblast differentiation and bone formation, suggesting TSH directly affects bone remodeling in vivo. INTRODUCTION: Thyroid-stimulating hormone (TSH) receptor haploinsufficient mice with normal circulating thyroid hormone levels have reduced bone mass, suggesting that TSH directly affects bone remodeling. We examined whether systemic TSH administration restored bone volume in aged ovariectomized (OVX) rats and influenced osteoclast formation and osteoblast differentiation in vitro. MATERIALS AND METHODS: Sprague-Dawley rats were OVX at 6 months, and TSH therapy was started immediately after surgery (prevention mode; n = 80) or 7 mo later (restoration mode; n = 152). Hind limbs and lumbar spine BMD was measured at 2- or 4-wk intervals in vivo and ex vivo on termination at 8-16 wk. Long bones were subjected to microCT, histomorphometric, and biomechanical analyses. The direct effect of TSH was examined in osteoclast and osteoblast progenitor cultures and established rat osteosarcoma-derived osteoblastic cells. Data were analyzed by ANOVA Dunnett test. RESULTS: In the prevention mode, low doses (0.1 and 0.3 microg) of native rat TSH prevented the progressive bone loss, and importantly, did not increase serum triiodothyroxine (T3) and thyroxine (T4) levels in aged OVX rats. In restoration mode, animals receiving 0.1 and 0.3 microg TSH had increased BMD (10-11%), trabecular bone volume (100-130%), trabecular number (25-40%), trabecular thickness (45-60%), cortical thickness (5-16%), mineral apposition and bone formation rate (200-300%), and enhanced mechanical strength of the femur (51-60%) compared with control OVX rats. In vitro studies suggest that TSH's action is mediated by its inhibitory effects on RANKL-induced osteoclast formation, as shown in hematopoietic stem cells cultivated from TSH-treated OVX rats. TSH also stimulates osteoblast differentiation, as shown by effects on alkaline phosphatase activity, osteocalcin expression, and mineralization rate. CONCLUSIONS: These results show for the first time that systemically administered TSH prevents bone loss and restores bone mass in aged OVX rats through both antiresorptive and anabolic effects on bone remodeling.

Bone morphogenetic protein signaling in articular chondrocyte differentiation.

Articular chondrocytes progressively undergo dedifferentiation into a spindle-shaped mesenchymal cellular phenotype in monolayers. Chondrocyte dedifferentiation is stimulated by retinoic acid. On the other hand, bone morphogenic proteins (BMPs) stimulate differentiation of chondrocytes. We examined the mechanism of effects of BMP in chondrocyte differentiation with use of a recombinant adenovirus vector system. Constitutively active forms of BMP type I receptors (BMPR-IA and BMPR-IB) and those of activin receptor-like kinase (ALK)-1 and ALK-2 maintained differentiation of chondrocytes in the presence of retinoic acid. The BMP receptor-regulated signaling substrates, Smad1/5, weakly induced chondrocyte differentiation; the effects of Smad1/5 were enhanced by BMP-7 treatment. Inhibitory Smad, Smad6, blocked increase of expression of chondrocyte markers by BMP-7 in a dose-dependent manner. SB202190, a p38 mitogen-activated protein kinase inhibitor, inhibited this effect of BMP-7; however, since SB202190 suppressed phosphorylation of Smad1/5, this may be due to blockade of BMP receptor activation. These results together strongly suggest that induction of chondrocyte differentiation by BMP-7 is regulated by Smad pathways.

Treatment of a murine model of high-turnover renal osteodystrophy by exogenous BMP-7.

BACKGROUND: The secondary hyperparathyroidism of chronic kidney disease (CKD) produces a high turnover osteodystrophy that is associated with peritrabecular fibrosis. The nature of the cells involved in the development of peritrabecular fibrosis may represent osteoprogenitors expressing a fibroblastic phenotype that are retarded from progressing through osteoblast differentiation. METHODS: To test the hypothesis that osteoblast differentiation is retarded in secondary hyperparathyroidism due to CKD producing bone marrow fibrosis, we administered bone morphogenetic protein 7 (BMP-7), a physiologic regulator of osteoblast regulation, to C57BL6 mice that had CKD produced by electrocautery of one kidney followed by contralateral nephrectomy two weeks later. Following the second surgical procedure, a subgroup of mice received daily intraperitoneal injections of BMP-7 (10 microg/kg). Three to six weeks later, the animals were sacrificed, blood was obtained for measurements of blood urea nitrogen (BUN) and parathyroid hormone (PTH) levels, and the femora and tibiae were processed for histomorphometric analysis. RESULTS: The animals had significant renal insufficiency with BUN values of 77.79 +/- 22.68 mg/dL, and the level of renal impairment between the CKD untreated mice and the CKD mice treated with BMP-7 was the same in the two groups. PTH levels averaged 81.13 +/- 51.36 and 75.4 +/- 43.61 pg/mL in the CKD and BMP-7 treated groups, respectively. The animals with CKD developed significant peritrabecular fibrosis. In addition, there was an increase in osteoblast surface and osteoid accumulation as well as increased activation frequency and increased osteoclast surface consistent with high turnover renal osteodystrophy. Treatment with BMP-7 eliminated peritrabecular fibrosis, increased osteoblast number, osteoblast surface, mineralizing surface and single labeled surface. There was also a significant decrease in the eroded surface induced by treatment with BMP-7. CONCLUSIONS: These findings indicate that BMP-7 treatment in the setting of high turnover renal osteodystrophy prevents the development of peritrabecular fibrosis, affects the osteoblast phenotype and mineralizing surfaces, and decreases bone resorption. This is compatible with a role of osteoblast differentiation in the pathophysiology of osteitis fibrosa.