We investigated bone induction by bone morphogenetic protein in primates, comparing it with that seen in rodents. Twelve Millipore diffusion chambers containing 5 mg of semipurified bone morphogenetic protein were implanted into the dorsal muscles of 12 young, adult crab-eating monkeys (Macaca fascicularis) and were retrieved six weeks later. In six of nine unbroken chambers, new bone with haematopoietic marrow had been induced on the host-side surface of the filter. The incidence of trans-filter bone induction in the monkeys was almost equal to that observed in mice, and the new bone yield was approximately half as much as in mice. Our results show that bone morphogenetic protein can induce new bone formation in primates within six weeks, and support the hope that it will be useful as a substitute for bone graft in man

Heterotopic ossification occurring after the use of commercially available bone morphogenetic proteins has not been widely reported. We describe four cases of heterotopic ossification in patients treated with either recombinant bone morphogenetic protein 2 or recombinant bone morphogenetic protein 7. We found that while some patients were asymptomatic, heterotopic ossification which had occurred around a joint often required operative excision with good results

We describe a 13-year-old boy with atrophic tibial pseudarthrosis associated with neurofibromatosis who had undergone nine unsuccessful operations. Eventually, union was obtained by the use of bone morphogenetic protein 7 in conjunction with intramedullary stabilisation and autologous bone graft

In dogs, resection of a length of the ulna equal to twice the diameter of the mid-shaft leaves a defect which consistently fails to unite. In response to an implant of 100 mg of bovine bone morphogenetic protein (BMP), the defect becomes filled by callus consisting of fibrocartilage, cartilage and woven bone within four weeks. The cartilage is resorbed and replaced by new bone in four to eight weeks. Woven bone is then resorbed, colonised by bone marrow cells and remodelled into lamellar bone. Union of the defect is produced by 12 weeks. Control defects filled with autogeneic cortical bone chips unite after the same period. In regeneration induced by bone morphogenetic protein (BMP) and in repair enhanced by bone graft, union depends upon the proliferation of cells within and around the bone ends. Our working hypothesis is that BMP induces the differentiation of perivascular connective tissue cells into chondroblasts and osteoprogenitor cells and thereby augments the process of bone regeneration from the cells already present in the endosteum and periosteum

In this work, we combined tissue engineering and gene therapy technologies to develop a therapeutic platform for bone regeneration. We have developed photothermal fibrin-based hydrogels that incorporate degradable CuS nanoparticles (CuSNP) which transduce incident near-infrared (NIR) light into heat. A heat-activated and rapamycin-dependent transgene expression system was incorporated into mesenchymal stem cells to conditionally control the production of bone morphogenetic protein 2 (BMP-2). Genetically engineered cells were entrapped in the photothermal hydrogels. In the presence of rapamycin, photoinduced mild hyperthermia induced the release of BMP-2 from the NIR responsive cell constructs. Transcriptome analysis of BMP-2 expressing cells showed a signature of induced genes related to stem cell proliferation and angiogenesis. We next generated 4 mm diameter calvarial defects in the left parietal bone of immunocompetent mice. The defects were filled with NIR-responsive hydrogels entrapping cells that expressed BMP-2 under the control of the gene circuit. After one and eight days, rapamycin was administered intraperitoneally followed by irradiation with an NIR laser. Ten weeks after implantation, the animals were euthanized and samples from the bone defect zone were processed for histological analysis using Masson's trichrome staining and for immunohistochemistry analyses using specific CD31 and CD105 antibodies. Samples from mice that were only administered rapamycin or vehicle or that were only NIR-irradiated showed the persistence of fibrous tissue bridging the defect. In animals that were treated with rapamycin, NIR irradiation of implants resulted in the formation of new mineralized tissue with a high degree of vascularization, thus indicating the therapeutic potential of the approach. Acknowledgements: This research was supported by grants RTI2018-095159-B-I00 and PID2021-126325OB-I00 (MCIN/AEI/10.13039/501100011033 and “ERDF A way of making Europe”), by grant P2022/BMD- 7406 (Regional Government of Madrid). M.A.L-J. is the recipient of predoctoral fellowship PRE2019-090430 (MCIN/AEI/10.13039/501100011033)

Objectives. MicroRNAs (miRNAs) have been reported as key regulators of bone formation, signalling, and repair. Fracture healing is a proliferative physiological process where the body facilitates the repair of a bone fracture. The aim of our study was to explore the effects of microRNA-186 (miR-186) on fracture healing through the bone morphogenetic protein (BMP) signalling pathway by binding to Smad family member 6 (SMAD6) in a mouse model of femoral fracture. Methods. Microarray analysis was adopted to identify the regulatory miR of SMAD6. 3D micro-CT was performed to assess the bone volume (BV), bone volume fraction (BVF, BV/TV), and bone mineral density (BMD), followed by a biomechanical test for maximum load, maximum radial degrees, elastic radial degrees, and rigidity of the femur. The positive expression of SMAD6 in fracture tissues was measured. Moreover, the miR-186 level, messenger RNA (mRNA) level, and protein levels of SMAD6, BMP-2, and BMP-7 were examined. Results. MicroRNA-186 was predicted to regulate SMAD6. Furthermore, SMAD6 was verified as a target gene of miR-186. Overexpressed miR-186 and SMAD6 silencing resulted in increased callus formation, BMD and BV/TV, as well as maximum load, maximum radial degrees, elastic radial degrees, and rigidity of the femur. In addition, the mRNA and protein levels of SMAD6 were decreased, while BMP-2 and BMP-7 levels were elevated in response to upregulated miR-186 and SMAD6 silencing. Conclusion. In conclusion, the study indicated that miR-186 could activate the BMP signalling pathway to promote fracture healing by inhibiting SMAD6 in a mouse model of femoral fracture. Cite this article: Bone Joint Res 2019;8:550–562

Background. Bone Morphogenetic Protein (BMP) has been used in clinical practice to stimulate fracture healing and spinal arthrodesis. Difficulty in localising and maintaining BMP at the target site has resulted in the use of large doses of BMP, and has been associated with significant adverse effects. We have previously shown clay hydrogels can bind growth factors for localised efficacy. We hypothesised that localisation of BMP within clay gels would reduce the dose required to mediate bone formation. Methods. 2×10-4mg and 1×10-5 mg BMP were mixed in Laponite and applied to collagen sponge. 3 sponges containing high dose, and 3 containing low dose BMP were implanted subcutaneously in a mouse. This process was repeated in 8 mice, for controls, alginate hydrogel was used in a further 8 mice, and 1 mouse received 6 blank collagen scaffolds. Micro Computed Tomography was used to assess bone formation fortnightly; at 8 weeks the mice were culled and underwent histological analysis. Results. Mean Bone Volume formed within collagen per μg BMP was significantly greater with Laponite and low dose BMP compared to Alginate and Laponite with high dose BMP (p<0.0001). No bone formation was observed with Alginate and low dose BMP. Conclusions. We have demonstrated that Laponite is able to reduce, by several orders, the effective dose of BMP required to mediate ectopic bone formation compared to current gold standard methods of BMP delivery. Clinical translation of this finding offers, potentially, great significance to orthopaedic surgery. Level of Evidence. In vivo study. Approval. Our study received ethical approval complied with Home Office licensing. Acknowledgments. Funded by grants from EU(FP7) Biodesign, Rosetrees Trust, BBSRC and EPSRC

Congenital pseudarthrosis of the tibia is an uncommon manifestation of neurofibromatosis type 1 (NF1), but one that remains difficult to treat due to anabolic deficiency and catabolic excess. Bone grafting and more recently recombinant human bone morphogenetic proteins (rhBMPs) have been identified as pro-anabolic stimuli with the potential to improve the outcome after surgery. As an additional pharmaceutical intervention, we describe the combined use of rhBMP-2 and the bisphosphonate zoledronic acid in a mouse model of NF1-deficient fracture repair. Fractures were generated in the distal tibiae of neurofibromatosis type 1-deficient (Nf1. +/−. ) mice and control mice. Fractures were open and featured periosteal stripping. All mice received 10 μg rhBMP-2 delivered in a carboxymethylcellulose carrier around the fracture as an anabolic stimulus. Bisphosphonate-treated mice also received five doses of 0.02 mg/kg zoledronic acid given by intraperitoneal injection. When only rhBMP but no zoledronic acid was used to promote repair, 75% of fractures in Nf1. +/−. mice remained ununited at three weeks compared with 7% of controls (p < 0.001). Systemic post-operative administration of zoledronic acid halved the rate of ununited fractures to 37.5% (p < 0.07). These data support the concept that preventing bone loss in combination with anabolic stimulation may improve the outcome following surgical treatment for children with congenital pseudarthoris of the tibia and NF1

Ossification of the ligamentum flavum and secondary spinal-cord compression were produced experimentally in mice by implanting bone morphogenetic protein (BMP) in the lumbar extradural space. The ligamentum flavum became hypertrophied and ossified, and protruded into the spinal canal. The thickness of the ossified ligament increased gradually with time, leading to compression and deformation of the spinal cord which showed various degrees of degeneration. Demyelination occurred in the posterior and lateral white columns and neuronal loss or chromatolysis in the grey matter. The pathological findings in the experimental animals closely resemble those found in the human disease and suggest that BMP may be a causative factor of ossification of the ligamentum flavum in man. This experimental model may be useful for the study of myelopathy caused by gradual spinal-cord compression

There is a growing interest in the development of tissue engineering (TE) therapies to repair damaged bone. Among the scaffolds for TE applications, injectable hydrogels have demonstrated great potential as three-dimensional cell cultures in bone TE, owing to their high water content, porous structure that allows cell transplantation and proliferation, similarity to the natural extracellular matrix and ability to match irregular defects. We investigated whether fibrin-based hydrogels capable of transducing near infrared (NIR) energy into heat can be employed to lead bone repair. Hollow gold nanoparticles with a plasmon surface band absorption at ∼750 nm, a NIR wavelength within the so called “tissue optical window”, were used as fillers in injectable fibrin-based hydrogels. These composites were loaded with genetically-modified cells harbouring a heat-activated and rapamycin-dependent gene circuit to regulate transgenic expression of the reporter gene firefly luciferase (fLuc). NIR-responsive cell constructs were injected to fill a 4 mm diameter critical-sized defect (CSD) in the parietal bone of mouse calvaria. NIR-irradiation in the presence of rapamycin triggered a pattern of fLuc activity that faithfully matched the illuminated area of the implanted hydrogel. Having shown that this platform can control the expression of a transgene product, we tested its effectiveness on regulating the secretion of transgenic bone morphogenetic protein 2 (BMP-2) from NIR-responsive hydrogels implanted in CSD. The spatiotemporal pattern of transgenic BMP-2 secretion induced by NIR-irradiation in the presence of rapamycin significantly stimulated bone regeneration from the edge of osteotomy in the CSD practiced, validating the therapeutic approach

Introduction: Periosteum is a tissue with pluripotential mesenchymal cells (MSCs). During fracture repair several growth factors are released from periosteum, including bone morphogenetic proteins (BMPs), which induce the differentiation of bone marrow stromal cells towards the osteoblastic lineage, therefore increasing the pool of mature bone forming cells and enhance the differentiated function of osteoblasts. The purpose of our study is to evaluate the expression of periosteal BMPs mRNA from fracture samples, collected within 24 hours of fracture and to compare it with BMPs expression from periosteal samples of normal (non-fractured) bones. Materials and Methods: Periosteum samples were collected from 25 patients with recent fracture (during the past 24 hours) (age: 12–80) and 25 individuals without fracture (age: 10–73). BMPs (BMP2, BMP4, BMP6) mRNA levels were analysed by Real Time RT-PCR by using the Light Cycler machine and PBGD as a housekeeping gene. Results: BMP2 mRNA levels were significantly higher (p< 0.05) in normal samples (median:12.15) than in fracture (median:4.39). BMP6 and BMP4 mRNA expression followed similar pattern to that of BMP2 but in significant lower levels. In normal samples, BMP4 mRNA median levels were 1.99, while in fracture samples the levels were significantly lower (median:0.35), (p< 0.05). BMP6 mRNA levels were also higher in normal samples (median:2.21) than in fractures (median:1.87) (p> 0.05). Furthermore, the decrease of BMPs mRNA levels in fracture samples was higher for BMP4 followed by BMP2 and BMP6. Discussion: Our results indicate high BMP2 mRNA levels expressed from periosteal cells. In recent fractures there is a significant reduction of BMP2 compared to normal samples; however, the expression of BMP2 remains more elevated in comparison to the other BMPs highlighting the potential role of BMP2 at the initiation of healing process of fractures. BMP6 and BMP4 expression was similar among normal periosteal cells while levels of BMP6 were higher than BMP4 in fracture periosteal cells. The suppression of BMP6 expression was minimum and less significant than BMP2 and BMP4 suppression indicating the potential role of BMP6 at the early stages of MSCs differentiation in periosteum. On the other hand, BMP4 remains in low levels in any confrontation and seems that plays a minor role in early healing process of fracture. BMPs are considered to play central role in fracture response and bone remodelling but further investigation has to be done as much in their correlation and toward other growth factors as in their expression levels during bone fracture repair process

Recombinant Bone Morphogenetic Protein 7 (OP-1) has been available in the UK since 2001, but there has been little published data on its use in the upper limb. In our institution OP-1 has been used in the management of 23 upper limb patients between 2001–2005, including 10 humeral non-unions. We believe this represents one of the largest single-unit cohorts of humeral fractures treated with OP-1. We reviewed the 10 humeral patients, who were all tertiary referrals with established non-unions. Two had been treated non-operatively before referral. The remaining eight had undergone a mean of 2.1 operations before OP-1 was used, with autologous bone grafting used in the majority of cases. Surgery occurred at a mean of 70.5 months following initial fracture. Seven patients underwent revision of the fracture fixation, and autologous bone graft was used with the OP-1 in 8 cases. Clinical union was established in 8 patients (80%) within a mean of 7.4 months. Radiological union was achieved in 8 patients (80%) within a mean of 9.1 months. No complications or adverse effects from the use of OP-1 were encountered. Both cases which failed to unite had a definite history of deep infection prior to index surgery, despite initial treatment with a staged revision procedure before OP-1 use. This study shows that OP-1 can be used successfully in the treatment of recalcitrant non-unions of the humerus following failure of traditional fracture management methods

Background: Bone morphogenetic proteins (BMPs) induce new bone in patients with bone defects and at extraskeletal sites in animals. Standard treatment for symptomatic scaphoid non-unions is bone graft with or without internal fixation by a screw or wires. We tested the ability of human recombinant osteogenic protein-1 (OP-1, BMP-7) with compressed autologous or allogeneic bone graft to accelerate the healing of scaphoid non-union. Study Design: Randomized and controlled pilot study in 17 patients with a scaphoid nonunion. Methods: Patients were randomly assigned to one of three groups: (1) Autologous iliac graft (n=6), (2) Autologous iliac graft + OP-1 (n=6) and (3) Allogeneic iliac graft + OP-1 (n=5). Radiographic, scintigraphic and clinical outcomes were assessed throughout the follow-up period of 24 months. Results: OP-1 improved the performance of both autologous and allogeneic bone implants. Three dimensional helical CT scans and scintigraphy showed that the pre-existing sclerotic bone within proximal scaphoid poles was mainly replaced in OP-1 treated patients with well vascularized new bone. Addition of OP-1 to allogeneic bone implant equalized the clinical outcome with the autologous graft procedure and enabled circumventing the second donor graft harvest procedure resulting in less blood loss, shorter anesthesia and no pain at the donor side. Conclusion: This is the first evidence that a recombinant human BMP accelerates scaphoid bone non-union repair and resorption of sclerotic bone in this specific microenvironment. Clinical Relevance: OP-1 might be successfully used in healing of scaphoid non-union

Statement of purpose. To determine the outcome of the use of Bone Morphogenetic Protein 7 (BMP7) as a replacement for bone graft in a limb reconstruction unit. Methods. Retrospective case note and imaging review was performed on a cohort of 71 consecutive patients from October 2009 to October 2012 in whom BMP7 was used to achieve union. The patients were identified from a pharmacy database. Factors analysed included the perceived indication, location in the skeleton, age, comorbidities, type of procedure (non-union, fusion, docking site etc), complications and need for revision surgery. Results. BMP7 was used in 71 patients with a median age of 63 (mean 58, range 17–86). The majority of patients were in the seventh decade. 62 were non-unions, eight were for fusion of docking site and one for fusion of an osteotomy. Circular external fixation was used in 20, IM nails in four, and plate fixation in 47. Indications for using BMP7 as opposed to bone graft included advanced age, immunosuppressive comorbidities or medication and obesity. In 65 cases bony union was achieved after the index procedure and one patient needed further surgery. There were five failures: two patients died before union and three failed to heal with the index intervention, leading to 74 procedures in 71 patients. BMP7 was used in several anatomic sites: femur (n=29), humerus (n=15), tibia (n=18) and in fusion of a joint (n=9). Overall success rate for achieving union was 93%. Local inflammation lasting a mean of three weeks was seen in six cases, and five patients developed heterotopic ossification, of which one was symptomatic. Conclusion. BMP 7 can be used as a replacement for autologous bone graft with predictable success in situations where bone graft harvest may be deemed unsuitable either because of local or systemic factors

Objectives. Fracture non-union poses a significant challenge to treating orthopaedic surgeons. These patients often require multiple surgical procedures. The incidence of complications after Autologous Bone Graft (ABG) harvesting has been reported up to 44%. These complications include persistent severe donor site pain, infection, heterotopic ossification and antalgic gait. We retrospectively compared the use of BMP-7 alone in long bone fracture Non-union, with patients in whom BMP-7 was used in combination with the Autologous Bone Graft (ABG). Material and Methods. The databases of our dedicated Limb Reconstruction Unit were searched for patient with three common long bone fractures Non-unions (Tibia, Femur and Humerus). The patients who had intra-operative use of Bone Morphogenetic Protein (BMP-7) alone and in combination with ABG were evaluated. 53 Patients had combined use of ABG and BMP-7, and 65 patients had BMP-7 alone. Results. In the ABG and BMP-7 group, the union rate for femoral (n=18) Non-unions was 83%, for humeral (n=16) Non-unions 81%, and for tibia (n=19) Non-unions it was 47%. In the BMP-7 alone group, 83% of the femoral (n=12) Non-unions, 87% of the humeral (n=16) and 56% of the tibial (n=37) Non-unions healed. The common risk factors for Non-union were comparable in both the groups and included location and nature (open vs closed) of fracture, infection, smoking and NSAIDs use. The average time to union in ABG+BMP-7 group was 8.1 months (range 3-30 months) and in BMP-7 alone group it was 7.2 months (range 3-24 months). Conclusion. Autologous Bone Grafting has a pivotal role in limb reconstruction surgery but its indication should be carefully evaluated in view of considerable morbidity associated with graft donor site. Our study did not show any significant difference in the union rates of common long bone fracture Non-unions treated with BMP-7 alone or with a combination of Autologous Bone Graft and BMP-7

Cahill et. al. published a large review of the use of BMP in spinal fusions. They reviewed the nationwide inpatient database, which represents approximately 25% of use U.S. Community Hospitals from the years 2002 to 2006. This included over 300,000 fusion type procedures. They noted increased complications with the use of anterior cervical procedures specifically increased complications with increased dysphasia and wound complications.

Due to these concerns, the Food and Drug Administration released last year a public health notification about the potential life threatening complications related to the use of BMP in anterior cervical spine fusions. Joseph & Rampersaud noticed a 20% incidence of heterotopic ossification in patients undergoing this procedure versus only 8% for patients who had undergone fusions without BMP.

Wong et. al. published a report on five cases of neurologic injury that relate to the use of BMP and the formation of heterotopic bone. Again, the suggestion of a barrier or closure defect was brought up and this may help minimise the risks; however, further work is noted. Multiple authors have noted a phenomenon of osteolysis occurring around graft fusion sites for the use of BMP. McCullen et. al. evaluated that 32 levels in 26 patients who had undergone a TLIF procedure. It is unclear the carcinogenic and tetraogenic effects of the use of BMP in the spine and also, the effects of repeat exposures on BMP has yet to be addressed. Finally, the long term cost and benefits of the use of BMP on the health care system has yet to be fully addressed.

So in conclusion, BMP2 is effective in producing fusions especially in challenging environments, deformity, smoking and infection. However, the complications continue to be a concern especially with regards to interbody fusions as well as in the cervical spine.

The feasibility of bone transport with bone substitute and the factors which are essential for a successful bone transport are unknown. We studied six groups of 12 Japanese white rabbits. Groups A to D received cylindrical autologous bone segments and groups E and F hydroxyapatite prostheses. The periosteum was preserved in group A so that its segments had a blood supply, cells, proteins and scaffold. Group B had no blood supply. Group C had proteins and scaffold and group D had only scaffold. Group E received hydroxyapatite loaded with recombinant human bone morphogenetic protein-2 and group F had hydroxyapatite alone.

Distraction osteogenesis occurred in groups A to C and E which had osteo-conductive transport segments loaded with osteo-inductive proteins. We conclude that scaffold and proteins are essential for successful bone transport, and that bone substitute can be used to regenerate bone.

Objectives

There is increasing application of bone morphogenetic proteins (BMPs) owing to their role in promoting fracture healing and bone fusion. However, an optimal delivery system has yet to be identified. The aims of this study were to synthesise bioactive BMP-2, combine it with a novel α-tricalcium phosphate/poly(D,L-lactide-co-glycolide) (α-TCP/PLGA) nanocomposite and study its release from the composite.

Aims: Previous work at this institution has demonstrated that perfusion beneath circumferential negative pressure wound therapy (NPWT) is decreased, which conflicts with most studies on NPWT and perfusion. This study investigates perfusion beneath non-circumferential NPWT in humans and also discusses the potential methodology flaw common to all previous research in this field.

Methods: Tests were conducted on both lower limbs of six volunteers (N=12). Volunteers were sequentially randomised into two groups, which would receive different suction pressures (−400 mmHg and −125 mmHg). A doughnut shaped NPWT dressing was placed over the shin of each leg. The central hole allowed for measurement of the transcutaneous partial pressure of oxygen (tcpO2), an indirect measure of perfusion. Readings were taken every five minutes throughout the experiment. After acquiring readings for 15 minutes to establish a baseline, suction was switched on and readings were taken for another 15 minutes. Suction was then disconnected and readings were taken for 15 minutes. Suction was then reapplied and the sequence was repeated. Data were analysed using the Wilcoxon and Mann-Whitney tests.

Results: On applying suction pressures of −400 mmHg, there was a significant reduction of the tcpO2 (mean reduction 7.35 mmHg, SD 7.4, p< 0.0005). At −125 mmHg, there was also a significant reduction of the tcpO2 (mean reduction 5.10 mmHg, SD 7.4, p< 0.0005). Although there was a tendency for greater reductions in the −400 mmHg group this was not significantly different to the −125 mmHg group (p=0.07).

Conclusion: NPWT reduces tissue perfusion, with higher suction pressure resulting in greater reductions in perfusion. Studies on perfusion using laser Doppler, which report findings contrary to these, may be flawed due to the measuring technique of this device. The compressive forces of NPWT are likely to result in false increased readings on application of suction when using the laser Doppler.

This represents a paradigm shift in our understanding of NWPT and that these dressings should be used with caution on tissues with compromised perfusion.