Bone Morphogenetic Proteins

Biologic alternatives to bone graft can be classified into cell based or molecule based. Bone Morphogenetic Proteins (BMPs) are a group of molecules that work by inducing the mesenchymal stem cells to differentiate into bone forming cell lines that form new bone. BMPs are involved in many physiological and pathological processes such as inflammatory response, bone formation and resorption, growth signaling pathways, oncogenesis and immune response. The purity, local effects, systemic effects, immunogenicity, and biocompatibility influence the safety and efficacy of BMP as a bone graft substitute. The two commercially available forms; rhBMP-2 and rhBMP-7 result in endochondral ossification.

Introduction

Formation of bone (ossification) occurs by both intramembranous ossification and endochondral ossification. In intramembranous ossification, the primitive mesenchymal cells are first transformed to osteoprogenitor cells and then into osteoblasts which lays down osteoid. Intramembranous ossification is seen in the skull, mandible, and the clavicle. Endochondral ossification is seen in the long bones; here the primitive mesenchymal cells transform into chondroblasts which lays down cartilage, the cartilage matures then degenerates. Degenerated cartilage is invaded by blood vessels and also by osteoblasts which forms bone.
The cellular events of both endochondral and intramembranous ossification involve the mesenchymal stem cells (MSCs). MSCs may be bone marrow derived or periosteum derived. MSCs are pluripotent progenitors that can differentiate into osteoblast, chondroblast and other connective tissue cell lines. Differentiation of MSCs is regulated by signaling pathways and molecules such as bone morphogenetic proteins (BMP), Wnt, Notch, Hedgehog, and Fibroblast growth factor (FGF).
The cellular and molecular events that govern the bone formation during development and fracture healing are similar. The process of fracture healing is similar to endochondral ossification. Healing of fracture needs appropriate cellular environment, adequate growth factors, sufficient bone matrix and mechanical stability. In some situations, the process of fracture healing may fail, leading to nonunion or delayed union. Such conditions as well as traumatic bone loss and spinal fusion surgery need stimulation of the process of bone formation. This can be achieved by biophysical methods such as ultrasound or biological interventions such as bone graft, bone marrow or biologically active molecules. Autogenous bone graft is capable of stimulation of bone formation by the process of osteogenesis, osteoconduction and osteoinduction. Osteogenesis is the direct formation of bone by the living osteoblasts in the graft. Osteoconduction is the ability to promote bone growth by allowing bone formation on its surface. Osteoinduction is the ability to induce the cells of recipient area to form new bone. Autogenous iliac crest bone graft (AICBG) is considered the gold standard for stimulation of bone formation in the treatment of bone defects and nonunions. However, limited availability of bone graft, morbidity of graft harvest, and the variable success rate of union highlights the need for a better option.
One of the solutions to avoid the problems of autologous bone graft is the use of the parathyroid hormone (PTH), hypoxia-inducible factor 1α (HIF-1α), modulators of the Wnt signalling pathway and the BMPs as a bone graft substitutes as they are capable of osteoinduction. It was hoped that equal or better results can be achieved without the morbidity of graft harvest.

History

In 1965, Marshal Urist demonstrated the ability of crude bone extracts to induce new bone in an ectopic site. He coined the term “bone morphogenetic protein” or “osteogenic protein” for the active ingredient of this extract. Later investigators identified and purified a variety of proteins capable of osteoinduction from these extracts. BMP became commercially available in 2002. In 2004, US FDA approved the use of rhBMP-2 in single level anterior lumbar interbody fusion.

Structure

BMPs constitute the largest part of the TGF-ß super family which also comprises activins and inhibins. The TGF-ß superfamily is comprised of growth factors and differentiation factors which have homology in their primary amino acid sequence. BMPs are highly conserved across animal species. The BMP-7 of humans and mouse share a 98% similarity in their amino acid sequence. Currently there are 20 known BMPs.
In the bone, BMPs are secreted by osteoprogenitor cells, osteoblasts and platelets. BMPs are synthesized as precursor proteins, which contains a hydrophobic leader sequence and propeptides. The biologically active portion is located at the carboxy terminal of the precursor molecule. Active BMP is released by proteolytic removal of the signal peptide and pro-peptide. The bioactive portions of all BMPs contain seven cysteine amino acid residues at positions similar to other members of the TGF superfamily. They form dimeric molecules with subunits. Each subunit has three intrachain disulfide bonds. The subunits are held together by a fourth bond.
The BMPs can be broadly classified into several subfamilies. Both BMP-2 and BMP-4 have 80% amino acid sequence homology form the first subfamily. The second group consisting of BMP-5, -6, -7 and -8’has 78% amino acid sequence homology. BMP-9 and BMP-10 form the next group. The BMP-3 and BMP-13 inhibits bone formation. BMP-1 is a metalloproteinase and is not a member of the superfamily.

Functions

The actions of BMPs depend on the target cell type, the maturation phase of target cells, local concentration of BMPs and other biological signals. BMPs are mitogens (growth factors) that stimulate the multiplication of connective tissue cells as well as morphogens (differentiation factors) that transform connective tissue cells into osteoprogenitor cells. They serve in signal pathways that influence cell division, matrix synthesis and tissue differentiation. They play a pivotal role in embryonic development. They play a role in the specification of positional information in the embryo. They regulate the growth, differentiation, chemotaxis and apoptosis of various cell types, including epithelial, mesenchymal, haemopoietic and neuronal cells. BMPs induce bone through intramembranous ossification or endochondral ossification. They induce osteoclasts as well leading to bone resorption.
BMP receptors are transmembranous cell surface receptors. The receptors are made up of Type I and Type II serine/threonine kinase proteins. BMPs bind as dimmers to the receptors forming an oligomeric complex. On binding of the ligand to the receptors, the Type II receptor kinase phosphorylates the Type I receptor. The Type I receptor phosphorylates the intracellular effector proteins, the receptor-regulated Smads (RSmads), Smad1, Smad5 and Smad8. Once phosphorylated, the Smads 1, 5, and 8 bind to co-Smads and Smad 4 and translocate into the cell nucleus and results in the activation of transcriptional factors for the BMP response genes. The transcription factor Cbfa1/Runx2 is considered as a key intersection of many signal pathways regulating osteoblast differentiation.
The local effects of BMP is regulated by a number of extra cellular and intracellular antagonists. Known extracellular antagonists are noggin, chordin, twisted gastrulation (Tsg), gremlin, follistatin and BMPER. These are cystine knot-containing proteins that form complexes with BMPs to prevent them from binding to their receptors. BMP-3 through a TGF-β/activin pathway in its bone formation. Known intracellular antagonists are Smad6, Smad7, Smad8b, Smurf1 and Smurf2. They either interfere with the activation of R-Smads or facilitate their degradation.

Pharmacology

Bone morphogenetic protein is a water-soluble, low-molecular weight protein which diffuses easily in the body fluids. If administered alone, the protein will lost rapidly due to diffusion or irrigation. Hence BMP is administered in a carrier to have a prolonged localized effect at the bone healing site. The BMP carriers can be broadly classified into inorganic salts, naturally occurring polymeric substances, synthetic polymers, and composites of synthetic and naturally occurring polymers.
Collagen is the most commonly used carrier, and Type I collagen is preferred. It may be sourced from bone or tendons. BMP binds tightly to collagen extracted from bone and less tightly to collagen extracted from tendons. Compression of the collagen carrier leads to rapid diffusion of BMPs; hence it should be protected by a cage. If collagen extracted from bone is used, it need not be protected by cage as it tightly binds to BMP.
BMP has widespread biological effects; many unknown or not clearly understood. Hence there are several long-term concerns about the use of BMPs in humans. The effects of BMP on the embryo are not fully known; hence it should not be used during pregnancy. Even though BMP is a human protein, it may induce an allergic response. The development of antibodies against the rhBMPs or the bovine collagen carrier is the most common adverse effect.
Some spontaneously evolving osteosarcomas contain high levels of BMPs and hence the cancer risk is a concern. BMP is a morphogen as well as a mitogen hence risk of cancer is possible.
Uncontrollable bone growth into the spinal canal or intervertebral foramen is a known complication of clinical use of BMP. Revision surgery with excision of heterotopic ossification in the spinal canal and/or intervertebral foramen is a difficult task with unpredictable results.
BMPs may have clinical uses other than osteogenesis. BMP is believed to be a protective to the brain. It has been shown to reduce the size of the infarct in an ischemic rat model. In patients with chronic renal disease levels of BMP are lower and it may have a place in the management of these patients.

Bone Morphogenetic Protein (BMP) Controversy

BMPs were available for use from 2002 onwards. From 2002-04 many industry sponsored clinical trials were published in reputed journals comparing it with autologous iliac crest graft. Not a single adverse effect due to BMP was reported in 13 industry sponsored clinical trials involving 780 patients. The estimated risk of rhBMP-2 use as per these clinical trials could be calculated to be less than 0.5% with 99% certainty which was 40 times lower than common analgesics and antibiotics. These studies reported a high morbidity rate of 40% to 60% with iliac crest graft harvest. These publications lead to tremendous increase in the clinical use of BMP. In United States of America, rhBMP-2 was used in more than 50% of primary anterior lumbar interbody fusion (ALIF), 43% of posterior lumbar interbody fusion (PLIF) and 30% of posterolateral fusion (PLF) by 2007.
However, several independent studies started reporting serious complications with rhBMP-2 use started appearing from 2006 onwards. Reported adverse event rates were from 20% to 70%. These complications included increased postoperative pain and radiculitis, increased infection rates, increased postoperative swelling, increased risk of spinal canal heterotopic ossification and increased risk of malignancy.
US Justice Department and the US senate committee investigated the rhBMP-2 manufacturer, Medtronic Inc. (Memphis, TN, USA) regarding the off-label use of rhBMP-2. The Yale University Open Data Access Project (YODA Project) did a review by 2 independent research teams into the original statistical data of the industry sponsored studies and they found appalling findings. The reported median known financial association between the authors of the industry supported clinical trials and Medtronic Inc. was found to be approximately $12,000,000–$16,000,000 per study (range, $560,000–$23,500,000). They found that adverse events were 50-70 times more common in the BMP group either not reported or not highlighted in the industry sponsored trials.
Patients given BMPs had greater incidence of back and leg pain in the early postoperative period possibly due to proinflammatory effects of BMP. The rate of epidural hematoma and wound complications with rhBMP-2 was 5 times higher when compared to AICBG. Osteolysis, subsidence of implants, and adverse neurologic and urologic events were more commonly seen with rhBMP-2 use. Loss of stability, collapse of the disc space by up to 50%, and large osteolytic lesions extending up to 50% of the vertebral height were found. Rate of retrograde ejaculation in patients treated with ALIF and rhBMP-2 was 5-6 times higher than control group. Bladder retention was twice more common with rhBMP-2. Deep infections were 2-5 times more common with rhBMP-2. Bone overgrowth into the spinal canal or intervertebral foramen in the rhBMP-2 group after PLIF was over 70% compared to 12% in the control group. Postoperative radiculitis and osteolysis was found in 20% to 70% of cases after PLIF. US FDA found ‘‘notably increased cancer rates with the use of high dose rhBMP -2 for posterolateral fusion. After anterior cervical fusion using rhBMP-2; high rates of wound problems, soft tissue swelling, airway compromise, graft subsidence, and end-plate erosion were reported.
The outcome of this misadventure is that stringent measures have now come into force regarding efficacy and safety profile of new medical devices and interventions and stringent disclosure of financial relationship of authors and industry have been enforced.
In spite of this debacle it remains a fact that BMPs are an important discovery that have clinical applications. The ideal molecule or molecules, their dose, timing and indications as well as contraindications need further refinement.

Further Reading

Safety and Effectiveness of Recombinant Human Bone Morphogenetic Protein-2 for Spinal Fusion. A Meta-analysis of Individual- Participant Data. Mark C. Simmonds,Jennifer V.E. Brown, Morag K. Heirs, Julian P. T. Higgins, Richard J. Mannion, Mark A. Rodgers and Lesley A. Stewart. Ann Intern Med. 2013;158:877-889.
Carragee EJ, Hurwitz EL, Weiner BK. A critical review of recombinant human bone morphogenetic protein-2 trials in spinal surgery: emerging safety concerns and lessons learned. SpineJ.2011;11:471-91. [PMID:21729796]
Brown JV, Heirs MK, Higgins JP, Mannion RJ, Rogers MA, Seneschall CC, et al. Systematic review and meta-analysis of the safety and efficacy of recombinant human bone morphogenetic protein-2 (rhBMP-2) for spinal fusion. Report to the YODA Project. Available from http://medicine.yale.edu/core/projects/yodap/index.aspx
Thawani JP, Wang AC,Than KD, Lin CY, LaMarca F, Park P. Bone morphogenetic proteins and cancer : review of the literature. Neurosurgery. 2010; 66:233-46. [PMID:20042986]
Boden SD, Zdeblick TA, Sandhu HS, Heim SE. The use of rhBMP-2 in interbody fusion cages. Definitive evidence of osteoinduction in humans: a preliminary report. Spine 2000;25:376–81.
Boden SD, Kang J, Sandhu H, Heller JG. Use of recombinant human bone morphogenetic protein-2 to achieve posterolateral lumbar spine fusion in humans: a prospective, randomized clinical pilot trial: 2002 Volvo Award in clinical studies. Spine 2002;27:2662–73.
Suzanne N. Lissenberg-Thunnissen, David J.J. deGorter, Cornelis F.M. Sier, Inger B. Schipper. Use and efficacy of bone morphogenetic proteins in fracture healing. International Orthopaedics (SICOT) (2011)35:1271–1280

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