A Review of the Clinical Side Effects of Bone Morphogenetic Protein-2

Abstract

Bone morphogenetic protein-2 (BMP-2) is currently the only Food and Drug Administration (FDA)-approved osteoinductive growth factor used as a bone graft substitute. However, with increasing clinical use of BMP-2, a growing and well-documented side effect profile has emerged. This includes postoperative inflammation and associated adverse effects, ectopic bone formation, osteoclast-mediated bone resorption, and inappropriate adipogenesis. Several large-scale studies have confirmed the relative frequency of adverse events associated with the clinical use of BMP-2, including life-threatening cervical spine swelling. In fact, the FDA has issued a warning of the potential life-threatening complications of BMP-2. This review summarizes the known adverse effects of BMP-2, including controversial areas such as tumorigenesis. Next, select animal models that replicate BMP-2's adverse clinical effects are discussed. Finally, potential molecules to mitigate the adverse effects of BMP-2 are reviewed. In summary, BMP-2 is a potent osteoinductive cytokine that has indeed revolutionized the bone graft substitute market; however, it simultaneously has accrued a worrisome side effect profile. Better understanding of these adverse effects among both translational scientists and clinicians will help determine the most appropriate and safe use of BMP-2 in the clinical setting.

Introduction

Perhaps no single protein has revolutionized the study of bone biology from bench to bedside as has bone morphogenetic protein-2 (BMP-2). In 1965, Marshall R. Urist discovered a substance in the extracellular bone matrix that had the ability in induce osteogenesis when implanted in extraskeletal soft tissue.¹ BMP-2 is a member of the transforming growth factor beta (TGF-β) superfamily, originally cloned by Wozney et al.² and is among more than 20 human BMPs described.³ Although many BMPs have osteogenic properties,⁴ recombinant human (rh)BMP-2 was the first and only to be introduced as a bone graft substitute.

First introduced in the United States in 2002 as approved by Food and Drug Administration (FDA) for single-level anterior lumbar interbody fusion (ALIF) within a specific threaded titanium tapered cage,^5,6 BMP-2 was then approved for tibial nonunions as an alternative to autograft in 2004, and for oral maxillofacial reconstructions in 2007.^7,8 In the United States alone, during the time period of 2002 to 2006, the use of BMP-2 dramatically increased from 0.7% to 24.9% of all spine fusion procedures performed, including 36.6% of all spine fusion revisions.⁹ By 2007, use of BMP-2 rose to greater than 50% of all primary ALIF, and off-label use of BMP-2 rose correspondingly. For example, BMP-2 was used in 43% of posterior lumbar interbody fusion (PLIF)/transforaminal lumbar interbody fusion (TLIF), 30% in posterolateral lumbar fusion (PLF), and 20% of all anterior cervical discectomy and fusion (ACDF) procedures.¹⁰ Indeed, in many instances, BMP-2 has been championed as a much needed clinical alternative to autograft bone. For example, a retrospective analysis of combined data from seven clinical trials demonstrated that BMP-2 is more effective than autogenous bone graft for radiographic spinal fusion in patients with single-level degenerative disc disease.¹¹ Compared to use of autograft alone, use of BMP-2 was correlated with reduction in both duration of operation time (25 min) and length of hospital stay (0.75 days).¹¹ Likewise, the efficacy of BMP-2 use has been elucidated in the treatment of open tibial fractures. The rate of secondary interventions is reduced 1.55-fold with BMP-2, from 27.1% in control patients to 17.5% in rhBMP-2 patients.¹² Furthermore, treatment failure rate of the control group was significantly higher than that of the rhBMP-2 treatment group (34.3% vs. 21.4%).¹²

The exponential rise in BMP-2 use has been associated with an increasing side effect profile. Despite preliminary human trials heralding adverse events with BMP-2 use in lumbar fusion,^13,14 initial industry-sponsored trials examining the use of BMP-2 showed no significant side effects in various common surgical procedures, including ALIF,^5,6,13,15 PLIF,¹⁶ACDF,^17,18 and PLF.^14,19–21 Remarkably, the combined reported risk from these initial industry-sponsored trials of the early 2000s accumulated to less than 0.5%.²²

After FDA approval, BMP-2 was believed to be “near-perfect” in achieving bone induction without harmful adverse events. However, subsequent independent assessments of the original industry-sponsored publications and independent assessment of original FDA data revealed that BMP-2-associated complications were not nominal and infrequent, but rather ranged from 20% to 70% of cases, and were potentially life threatening because of significant cervical swelling and corresponding dyspnea and dysphagia, requiring intubation.²² Systematic reviews through investigations from the FDA reports, Yale University of Open Data Access Project, and Medtronic internal reports examined the safety and efficacy of BMP-2. These reviews concluded that there was evidence of reporting bias in the initial industry-sponsored publications and no clear advantages of BMP-2 use as compared with iliac crest bone graft (ICBG).^23,24 Moreover, adverse events associated with BMP-2 were not only frequent but were also occasionally catastrophic especially in anterior cervical spine fusions.²⁴ These included cervical and soft tissue swelling, airway compromise, and need for reoperation. These new insights prompted the FDA to issue a Public Health Notification about BMP-2 use in 2008.²⁵

The current review seeks to summarize the known clinical adverse effects of BMP-2 by presenting the relevant cellular effects of BMP-2, reviewing animal models previously shown to replicate these adverse events, and highlighting preclinical attempts to augment BMP-2 to improve its safety profile. Table 1 provides a summary of complications associated with BMP-2.

Table 1.

The Adverse Effects of BMP-2 in Clinical and Preclinical Models

Clinical side effects associated with BMP-2
Side effect	Main findings	References
Inflammatory complications	Cervical spine swelling	65,69,145–148
	Seroma formation	65,66,149–154
Radiculopathy	Nerve root compression/postoperative radiculitis	48,55,67,152,155–158
Ectopic bone	Neuroforaminal and soft tissue ectopic bone and bone cyst formation	48,158–163
Osteoclast activation, osteolysis, and subsidence	Vertebral and nonvertebral bone resorption	41,48,55,60,61,156,164–170
	Graft subsidence	41,60,61,166,171
Urogenital events	Retrograde ejaculation	76,172–175
	Bladder retention	172,175
Wound complications	Hematoma	9,79,80,146,152,176,177
	Wound dehiscence	9,13,79,152,176
	Infection	9,48,79,80,151,152,16

Preclinical side effects with BMP-2 (in vitro and in vivo)
Side effect	Main findings	References
Induction of inflammation	Increase in IL-1β, IL-6, IL-10, IL-17, IL-18, TNF-α	102,105,123,178–180
Osteoclast activation	Potentiation of nuclear factor-kβ ligand and induction of RANKL	104,181–184
	Upregulation of PPARγ pathway and repression of Wnt signaling pathways	185
Induction of adipogenesis and bone cyst formation	Dose-dependent induction of PPARγ expression	64,102,178,186–188

BMP, bone morphogenetic protein-2; IL, interleukin; PPARγ, peroxisome proliferator-activated receptor gamma; RANKL, receptor activator of nuclear kappa-B ligand; TNF-α, tumor necrosis factor-α.

Review of BMP-2 Signaling

Canonical BMP signaling transduction begins when BMPs bind to BMP receptor types I and II whose signal is regulated by the phosphorylation of receptor-regulated R-Smad (typically Smad 1, 5, and 8).²⁶ Phosphorylated R-Smads perpetuate the signal by forming a heterodimer complex with Co-Smad (typically Smad 4) to translocate into the nucleus to direct transcriptional response.^27,28 Among other factors, BMP-2 induces the master osteogenic transcription factor runt-related transcription factor 2 (Runx2) in osteoblasts to induce osteogenic programming^29–31 (see Chen et al.³² for a more complete review of BMP-2 signaling).

However, the cellular effects of BMP-2 a far more pleiotropic than this would suggest. Indeed, among many contexts, BMP-2 activates peroxisome proliferator-activated receptor gamma (PPARγ) signaling,³³ leading to adipogenesis differentiation and fat formation.³⁴ In addition, BMP-2 induces expression of numerous inflammatory cytokines and chemokines including interleukins (ILs) and tumor necrosis factor (TNF)-α.³⁵ Moreover, BMP-2 is well known to activate osteoclast through receptor activator of nuclear kappa-B ligand (RANKL).³⁶ With disparate effects on adipogenesis, inflammation, and osteoclast activation, it is understandable that BMP-2 has been found to elicit a range of clinically relevant side effects.

Clinical Side Effects Associated with BMP-2 Use

BMP-2 has marked species-specific concentration requirements for osteogenesis,^37,38 whereby the BMP-2 concentration necessary for inducing consistent bone formation is substantially higher in nonhuman primates (0.75–2.0 mg/mL) than in rodents (0.02–0.4 mg/mL)^37,38 (reviewed in Boden et al.¹⁴). The current FDA-approved 1.5 mg/mL BMP-2 concentration for human use was determined through nonhuman primate efficacy testing.^37,39 The dose-dependent efficacy in humans has been observed in studies of fracture healing, where the median time to fracture healing was reduced with 1.50 mg/mL but not 0.75 mg/mL BMP-2 (median time to healing was 184, 187, and 145 days in the control, 0.75 mg/mL, and 1.50 mg/mL BMP-2 groups, respectively).⁴⁰

As effective bone healing in humans requires high amounts of proteins, the incidence of side effects is concomitantly increased. In fact, excessive concentrations of BMP-2 may be the most important contributory factor to the majority of adverse events, and increasing doses of BMP-2 do not necessarily result in higher fusion rates in spine procedures and long bone nonunions.^41,42 Various clinical trials are aimed toward elucidating the optimal dosage of BMP-2.⁴³ Currently, a phase-I clinical trial is evaluating a single percutaneous injection at varying doses of rhBMP-2 in patients with closed diaphyseal fractures.⁴⁴ Another clinical study examined the dose efficacy of rhBMP-2 in patients requiring extraction socket augmentation and found statistically significant differences between the 0.75 and 1.50 mg/mL groups. At 25% extraction socket length, 25% of implant positions demonstrated adequate bone formation in the 0.75 mg/mL group compared with 56.25% in the 1.50 mg/mL group.⁴⁵ Likewise, a feasibility sinus floor augmentation study showed that although 0.43 mg/mL rhBMP-2 was effective at inducing bone formation, 1.5 mg/mL rhBMP-2 was identified as the most effective concentration for inducing de novo bone.^46,47

Ectopic bone formation with BMP-2

The most recognized adverse event related to BMP-2 use is ectopic bone formation, associated with BMP-2 leakage outside the implant site. Ectopic bone formation is estimated to occur at a rate of nearly six times more than control patients when BMP-2 is added into spinal canal/foramen; CT scan evaluation showed ectopic bone formation in 70.1% of patients who were administered rhBMP-2 compared with 12.9% of patients who were not administered rhBMP-2.²² Leakage can occur into the epidural space and lead to root compression of nerves by ectopic bone impingement, and the incidence rate of postoperative radiculitis was 14.0% with rhBMP-2 treatment (in comparison with 3.0% in control groups).⁴⁸ Biologically, the occurrence of ectopic bone formation with BMP-2 is not surprising, as multiple nonosteoblastic cells undergo osteogenic programming when exposed to BMP-2, including myoblasts,⁴⁹ adipocytes,⁵⁰ and fibroblasts⁵¹ among others.

Ectopic bone formation presumably could occur because of premature leakage of BMP-2 during manual manipulation of the absorbable collagen sponge into the cage^52,53 while preparing for insertion during an interbody fusion. While comparing the overall sizes in different segments of the spinal column, because of the narrower dimensions of the cervical spine, any leakage during graft placement of the BMP-soaked sponge would be in proximity to the spinal canal or foramina than the larger diameter of the other sections of the spine, ultimately leading to increased susceptibility to cord compression.⁵⁴ In summary, the implantation of BMP-2, delivered on the absorbable collagen sponge, increases the potential of BMP-2 leakage, resulting in subsequent neural impingement, secondary to ectopic bone formation. Of note, efforts have been made to determine mechanisms to reduce ectopic bone formation with BMP-2 on a collagen sponge carrier. Recent emphasis centers on the exact placement of the sponge. For example, placement of the rhBMP-2 sponge anterior to and within the interbody cage rather than in the posterior half of the interbody space minimizes the risk of ectopic bone formation in the spinal canal and intervertebral foramen.⁵⁵ In addition, the manufacturer of rhBMP-2 recommends a few cautions in the manipulation of the rhBMP-2/sponge product to minimized BMP-2 extravasation. According to the package insert and instructions for preparation of the Medtronic rhBMP-2 product, it is crucial to avoid “use of irrigation or suction near the implanted device” and “excessive squeezing of the wetted sponge.”⁵⁶

Osteoclast activation, osteolysis, and subsidence with BMP-2

Osteolysis and subsidence are serious adverse events of spinal surgeries that are also observed at an increased rate with BMP-2 treatment. As previously noted, application of BMP-2 is well known to enhance osteoclastic activity in a cancellous bone environment.⁵⁷ Three studies have examined the rates of subsidence and related complications in spinal procedures with BMP-2.^5,58,59 Postoperative radiological depiction of the surgical area discovered collapse of the disc space, large osteolytic cystic lesions, implant displacement or loosening, and subsidence all in increased frequency in the BMP-2 groups as compared with the control group in a randomized controled trial (RCT) of ALIF procedures.^5,58,59 Occurrence rates of these events summed to 9.6% in groups with BMP-2 use, nearly a 1.5-fold increase when compared with ICBG control group especially during the immediate postsurgical period. In another study, McClellan et al. found a 68% and 74% incidence of vertebral bone resorption and graft subsidence, respectively, in patients treated with rhBMP-2.⁶⁰ Similarly, Vaidya et al. found an 82% rate of lumbar end plate resorption correlated with use of rhBMP-2.⁶¹ In summary, in keeping with the known osteoclastogenic effects of BMP-2, a significant increase in osteolysis and subsidence is observed with BMP-2 use.

Bone cyst formation with BMP-2

The eventual fate of mesenchymal stem cells (MSCs) is hypothesized to be controlled by the antagonistic balance between RUNX2 and PPARγ.⁶² PPARγ promotes adipogenesis upon activation while suppressing osteogenesis by downregulating the expression and transactivation ability of Runx2.³³ Although there is no clinical summary of bone cyst formation at the patient level, it has been previously reported in in vivo preclinical studies.⁶³ Although bone volume is significantly increased with the use of BMP-2, the quality of bone mineral density has been reported to be poor. Previous studies have demonstrated an increased spacing in trabecular bone formed in proximity to BMP-2 implantation, with interspersed lipid deposition, resulting in cystic bone formation.^63,64 Thus, despite the robust proosteogenic effect BMP-2 elicits, the proadipogenic effects on MSCs decrease the overall quality of bone formed.

Inflammatory complications with BMP-2

Previous studies have also noted a significant host of side effects associated with induction of local inflammation secondary to BMP-2 implantation. These range from benign seroma formations to life-threatening effects, such as cervical spine swelling. As issued in 2008, the FDA released a black box warning for BMP-2 use owing to the recognized risk of cervical spine swelling and death.²⁵ Each inflammatory side effect of BMP-2 will be discussed sequentially hereunder.

Seroma formation is a common side effect of BMP-2, encountered most commonly in the first week postoperatively, as demonstrated by several case studies.^65,66 Similarly, Rihn et al. found that lumbar seroma occurred in 1.2% of rhBMP-2 patients compared with 0% of control patients.⁴⁸ Frequently seroma formations are subclinical until impingement on nearby organs by mass effect, causing pain or dysesthesias.⁵⁴ For example, Robin et al. described a postoperative seroma formation with BMP-2 use in the cervical region that led to bilateral paresthesia of the upper extremities until subsequent drainage.⁶⁶ In fact, investigators have found elevated levels of inflammatory cytokines in BMP-2-induced seromas, including ∼3000-, 5000-, and 34-fold levels of IL-6, IL-8, and TNF-α, respectively.⁶⁶

Similarly, the incidence of radiculitis has been reported to be increased among BMP-2-treated patients. The incidence of radiculitis in the context of BMP-2 treatment is highest among PLIF and TLIF procedures.⁶⁷ Rihn et al. reported rates of radiculitis among BMP-2-treated patients as high as 14.0% in comparison with control groups (3.0%).⁴⁸ These clinical findings were independent of nerve root compression from ectopic bone formation and appear to be mediated by BMP-2-induced inflammation of the nerve root.⁶⁸ However, not all studies have reported a significant difference in leg pain scores between rhBMP-2 and control patient groups. Interestingly, the 2010 FDA Executive Summary of the Dimar et al. trial²⁰ found that postoperative leg pain scores in both the ICBG control and rhBMP-2 matrix groups improved in an almost identical manner over all time intervals. In addition, Boden et al. showed that patients in both the BMP-2 and autograft control groups reported similar degrees of leg pain in the early postoperative period, as indicated by their Oswestry Disability Index scores of 13.0 and 12.0, respectively.¹⁴

Most worrisome, however, is the well-described increased incidence in cervical spine swelling—a potentially life-threatening side effect of BMP-2, resulting in a FDA black box warning.²⁵ Inflammation of the cervical spine was first described as a side effect by Smucker et al. in 2006, and since six fatalities have been reported.⁶⁹ Overall, the FDA received 38 reports of complications because of “off-label” use of BMP-2 in anterior cervical surgery in 2008. Patients having swelling of the neck and throat tissues experienced compression of the airway dysphagia, and difficulty breathing or speaking.⁷⁰ In an attempt to define appropriate use of BMP-2, in 2014 the North American Spine Society released coverage policy recommendations stating: “Based on the available evidence, BMP[-2] is indicated as an adjunct to spinal fusion in cases in which other alternatives are either not available or are not likely to lead to effective fusion.”⁷¹ Furthermore, BMP-2 is not indicated in the following scenarios: “Routine anterior and posterior cervical fusion procedures, single level posterior/posterolateral fusion in healthy adults, and routine pediatric spine fusion procedures.”⁷¹ To the authors' knowledge, there have not been reports of major complications of BMP-2 use in cervical spinal surgeries since this time, which most likely reflects change in practices to avoid BMP-2 in this anatomic region.

Urogenital events

BMP-2 has also been observed to elicit urogenital complications in numerous clinical reports, including retrograde ejaculation and persistent bladder retention. Retrograde ejaculation after anterior lumbar surgery occurs at a rate less than 1%;^72–74 however, the adverse event of retrograde ejaculation associated with the use of BMP-2 with the LT-cage was reassessed by the FDA Summary of Safety and Effectiveness Data⁵⁹ using the same cohort and found to be significantly increased in the group receiving BMP-2.⁷⁵ This was confirmed by an RCT that observed higher rates of retrograde ejaculations in male patients who received BMP-2 than those who received an ICBG. The rate of retrograde ejaculation after anterior lumbar surgery with the use of BMP-2 increases the frequency of retrograde ejaculation by at least sixfold, depending on the surgical procedure specifics.^76,77 Specifically, Carragee et al. found a 7.2% rate of retrograde ejaculation in the rhBMP-2 groups (as compared with 0.6% rate among the control group).⁷⁶

Bladder retention is also a concern with any invasive procedure, whether it be from anatomical obstruction (lysis of the ureter) or damage to the nervous system (obliteration of the parasympathetic system). Early bladder retention was studied by the FDA Summary of Safety and Effectiveness Data and found to be twice as common in patients receiving BMP-2. The frequency of early bladder retention in the control group saw a rate of 3.6%, whereas in those receiving BMP-2, a rate of 7.9% was observed; an increase that proved to be statistically significant. Thus, increased incidence of both retrograde ejaculation and persistent bladder retention has been observed in multiple independent reports related to BMP-2 use.

Wound complications with BMP-2

Wound complications entail a variety of adverse events including epidural hematoma, wound dehiscence, postoperative fever, and hemorrhage, to name a few. A reassessment of the pilot study of Boden et al. found wound complication rates to be 10% in the BMP-2 group, significantly higher than the control group without BMP-2 use in PLF.⁷⁸ Likewise, Chan et al. found significantly higher wound complication rates among the rhBMP-2 treatment group (31%) than among the control group (18%).⁷⁹ Further analysis of complications in posterior fusion surgeries illustrates a fivefold increase (2.1% vs. 0.4%) in wound complications associated with BMP-2 use during a posterior approach.⁸⁰

Infections, both local and systemic, are known complications of all invasive procedures. Early wound infections (less than 6 weeks postoperatively) were equivalent in BMP-2 and ICBG groups in lumbar fusion surgeries (9.4%); however, an increase was observed in delayed infections (>6 weeks postoperatively), whereby BMP-2 led to a threefold increase (4.2% in BMP-2 group vs. 1.4% in ICBG group).⁵⁹ Moreover, deep wound infections were found at a fivefold greater rate (2.1% vs. 0.4%) in patients receiving BMP-2 in anterior/posterior surgeries.⁸⁰ The mechanisms behind increased rates of infection with BMP-2 are not fully understood.

Risk of tumor formation with BMP-2

The role of BMP-2 in carcinogenesis is a controversial topic, for which the linkage (if any truly exists) is tenuous. However, it is clear that deviation from normal physiologic expression of BMP-2 is associated with multiple tumor types, involving diverse organs including prostate, breast, oral mucosa, pleura, and bone.^81–85 Although variation by organ system is seen, BMP-2 is generally upregulated in diverse tumors and is associated with tumor cell proliferation, invasion, and at times a poor clinical prognosis. The basic biologic importance of BMPs in diverse cancers raises potential concerns about its clinical use.⁸⁶ Data from a multicenter, RCT of patients undergoing posterolateral fusions with BMP-2 did show a higher incidence of carcinoma among patients with BMP-2 treatment than those with autograft control.⁸⁷ Of note, multiple investigators have studied the link between BMP-2 and the incidence of cancer in large-scale studies and have not been able to replicate these findings.^88–91 Of note, most studies are limited by long-term follow-up. Perhaps the most comprehensive study was recently published by Kelly et al. who examined the records of 467,916 Medicare patients undergoing spinal arthrodesis with or without BMP-2.⁹² Their study that had an average of 2.9-year follow-up period showed that BMP-2 exposure actually reduced the risk of developing cancer (relative risk: 0.938; 95% CI: 0.913–0.964). Thus, the largest clinical studies to date have not been able to find a link between BMP-2 application and carcinogenesis.

Nevertheless, basic biologic studies have shown upregulated BMP ligand expression in breast carcinoma,^82,93 lung,⁹⁴ pancreatic,⁹⁵ and oral squamous cell carcinoma (SCC),⁹⁶ to name a few. BMP-2 increases proliferation within multiple carcinoma types, including breast,^97,98 lung,⁹⁴ and some pancreatic tumor cell lines.⁹⁵ Moreover, BMP-2 has been shown to increase invasiveness of prostate,⁹⁹ lung,¹⁰⁰ oral SCC,⁹⁶ and breast carcinoma,⁹⁷ potentially by activating Tenascin-W¹⁰¹ and increasing CCL5 expression.⁹⁶ Moreover, aberrant BMP signaling has been proposed to be of biologic importance in bone metastases of breast⁸² and prostate carcinoma.⁹⁹ Thus, the oncologic concerns of clinical BMP-2 use are largely rooted in the basic biology of BMP signaling, with clinical studies predominantly not able to find a cause for concern.

Bone-Related Side Effects with BMP-2 Use in Animal Studies

Induction of inflammation

In association with BMP-2 use, in vitro, studies have shown increased concentrations of numerous cytokines and chemokines including IL-1α, IL-1β, IL-6, IL-10, IL-18, TNF-α, macrophage inflammatory protein 1 α (MIP-1 α), and monocyte chemotactic protein 1 (MCP-1) in animals treated with BMP-2. Supra-physiologic levels of BMP-2 can alter the balance between proinflammatory and anti-inflammatory cytokines tipping it in favor of inflammation.

Recent studies by Zara et al. have extended these findings, showing that high-dose BMP-2 induces inflammation in an orthotopic model that results in structurally abnormal bone formation in vivo.¹⁰² Here, BMP-2 (4 mg/mL) was applied to rats in a femoral-only model, assessing inflammation at early timepoints (3–14 days).¹⁰² Compared with control-treated rats, BMP-2-treated animals exhibited significant local inflammation, including increased presence of monomorphonuclear and polymorphonuclear cells and calcitonin receptor (CTR)-positive osteoclast-like cells.¹⁰² When extended to a femoral bone defect model, a similar induction of inflammation was noted along with a dose-dependent induction of structurally abnormal bone cysts. Of note, the time course of inflammation in these rodent experiments mimics that of published clinical reports of BMP-2-induced cervical swelling.⁶¹

Interestingly, Yamazaki et al. also determined that TNF-α represses BMP-2 signaling by interfering with the DNA binding of Smads through the activation of nuclear factor-kβ.¹⁰³ TNF-α inhibits both osteoblast differentiation and bone formation, as pretreatment with TNF-α abolished BMP-2-induced ALP and Id1-luciferase activity.¹⁰³ Thus, the increased levels of TNF-α associated with BMP-2 use result in not only induction of inflammation but also an unwanted inhibition of osteoblastogenesis.

Interferon-γ is one of the major proinflammatory cytokines that leads to significant inflammatory changes by elevating TNF-α expression, which is one of the principle cytokines that activates the inflammatory cascade. Acting as chemokines, IL-8 and MCP-1 recruit neutrophils and monocytes to their location and trigger the firm adhesion of monocytes to the local vascular endothelium.

Osteoclast activation

Multiple lines of evidence suggest that BMP-2 induces osteoclastic activity both in vitro and in vivo, through direct and indirect mechanisms. BMP-2 directly stimulates osteoclastogenesis by potentiating receptor activator of nuclear factor-kβ ligand. In addition, BMP-2 enhances RANKL-induced osteoclast differentiation.¹⁰⁴ In addition, numerous inflammatory cytokines induced by BMP-2 secondarily induce osteoclastic activity, including IL-6.¹⁰⁵ Indirectly, BMP-2 affects osteoclast activity by upregulation of the PPARγ pathway and repression of the Wnt signaling pathway. Also, this increases osteoclast quantity by upregulating c-fos expression.

In recent novel studies, Tasca et al. determined that expression of Smad1/5 and Smad4, elements of the canonical BMP pathway, is integral to osteoclast differentiation.¹⁰⁶ Specifically, loss of Smad1/5 or Smad4 expression was induced in osteoclasts precursors through infection with adenoviruses expressing Cre recombinase (Ad-CRE).¹⁰⁶ Compared with control, Ad-CRE cells exhibited decreased osteoclast differentiation, as indicated by the presence of fewer and smaller multinuclear cells.¹⁰⁶ Moreover, expression of osteoclast-enriched genes Dc-stamp and Cathepsin K was reduced in Ad-CRE cells.¹⁰⁶ To further assess the effect of BMP-2 on osteoclast activity, mature osteoclasts were treated with either BMP-2 or dorsomorphin, a BMP pathway inhibitor.¹⁰⁶ Whereas BMP-2 stimulated osteoclast resorption, dorsomorphin blocked this activity.¹⁰⁶ Thus, both BMP-2 and elements of the canonical BMP pathway, including Smad1/5 and Smad4, are crucial to osteoclast differentiation and osteoclast-mediated resorption.

Induction of adipogenesis

Many pro-osteogenic cytokines show converse antiadipogenic properties—however, BMP-2 is a notable exception. BMP-2 dose dependently induces PPARγ expression, the master transcriptional regulator of adipogenesis. Notably, Takada et al. recently described epigenetic crosstalk between BMP-2 and PPARγ.¹⁰⁷ After pretreatment with the PPARγ ligand troglitazone, BMP-2 upregulated mRNA levels of the adipocyte marker genes Fabp4 and Gpd1 in ST2 stromal cells by enhancing the levels of active histone markers on the promoter of the PPARγ target gene Fabp4.¹⁰⁷ Thus, BMP-2 not only affects pleiotropic MSCs by inducing both osteogenic and chondrogenic differentiation through the RUNX2 pathway¹⁰⁸ but can also promote adipogenic differentiation.

Of note, recent studies have shown that BMP receptor type dictates whether adipogenesis or osteogenesis is induced. Whereas signaling through BMPR-IA exerts adipogenic effects, BMPR-IB exerts osteogenic effects.^49,109,110 For example, expression of constitutively active BMPR-IA induces adipogenic differentiation, whereas overexpression of inactive BMPR-IA inhibits adipogenic differentiation.⁴⁹ The converse effects were obtained by manipulation of BMPR-IB expression. Namely, constitutive BMPR-IB activation induces osteogenic differentiation, whereas inactive BMPR-IB inhibited osteogenic differentiation.⁴⁹ In another study, McArdle et al. evaluated the osteogenic potential of BMPR-IB(+) versus BMPR-IB(−) human adipose-derived stromal cells (ASCs).¹⁰⁹ Compared with BMPR-IB(−) ASCs, BMPR-IB(+) ASCs significantly enhanced osteogenesis in vitro, as evidenced by increased alkaline phosphatase staining, extracellular matrix mineralization, and osteocalcin expression.¹⁰⁹ In vivo, BMPR-IB(+) cells resulted in a twofold increase in bone regeneration in murine critical-sized calvarial defects.¹⁰⁹ However, conflicting data do exist regarding the specificity of BMPRs for lineage differentiation. For example, osteoblast-selective interference of BMPR-IA demonstrated antiosteogenic effects, including irregular calcification and decreased bone mass.¹¹⁰

The presence of adipogenesis occurring during BMP-2-mediated osteogenesis results in formation of cyst-like bone voids filled with fatty marrow instead of the standard trabecular bone structure.¹⁰² Despite complete bone union, the increased concentrations of BMP-2 (150–600 μg/mL) that are required for human osteogenesis lead to inconsistent bone formation and decreased overall bone quality through activation of PPARγ and downregulation of the Wnt signaling pathway.¹⁰² Moreover, the formation of cystic bone voids from high-dosage BMP-2 can be repressed with the coapplication of Wnt signaling pathway activators.¹⁰²

Methods to Improve BMP-2 Use

With an increased knowledge secondary to clinical experience with BMP-2, numerous potential adverse events of BMP-2 have been elucidated, and a significant effort by investigators has been made to find a safer, cheaper, and more efficient pharmaceutical replacement for BMP-2. To date, no suitable alternative has been identified that would have similar or superior efficacy in inducing bone formation without the adverse effects that come with BMP-2.²²

Thus, the appropriate dosing, carriers, and location of BMP application pose as pharmacologic variables potentially resulting in such adverse events,¹¹¹ and as such, investigators have been subject to numerous studies. Several studies, highlighted hereunder, have sought to investigate alterations in dosage, scaffold, and the implementation of supplemental proteins or growth factors to mediate the nonspecific action of BMP-2.

Addition of vascular endothelial growth factor

Effective healing of bone fractures depends on not only osteogenesis but also angiogenesis to reestablish the inflow of oxygen and nutrients necessary to produce healthy bone. Vascular endothelial growth factor (VEGF) is a protein produced by endothelial cells that induces new vascular formation after injury.^112–114 VEGF has been shown to be essential for endochondral and intramembranous bone formation.¹¹⁵ Recent evidence suggests that not only does VEGF produce angiogenesis but there is also cross-talk between VEGF and the BMP signaling pathway that plays a role in osteoblastic differentiation of MSCs.¹¹⁶

Unlike BMP-2, the use of VEGF alone in bone fractures is not sufficient enough to lead to complete healing;¹¹⁷ however, when combined with BMP-2, VEGF increases the rate of complete union healing in defects in a synergistic manner in rat models with critical size defects.¹¹⁷ It must be noted, however, that although combination treatment with BMP-2 and VEGF possesses superior efficacy of bone formation, coimplantation and corresponding angiogenesis may result in aberrant migration of BMP-2 to unwanted areas and potentiate higher rates of ectopic bone formation.¹¹⁸

Addition of bisphosphonates

One of the many concerns with BMP-2 use is late osteoclast activation potential due to the effect BMP-2 has on both the number and functionality of osteoclasts. One budding solution to this increased activity of osteoclasts is to inhibit osteoclast function or number, thus effectively eliminating that particular side effect.¹¹⁹ Bisphosphonates are a class of drugs directed at preventing loss of bone mass and are clinically relevant by treating osteoporosis.^120–122

Use of bisphosphonates in conjunction with BMP-2 proved to significantly decrease the number of osteoclasts per bone surface area. In an experiment conducted on piglets with Legg–Calvé–Perthes disease, BMP-2 administered with bisphosphonate decreased bone resorption while maintaining increased new bone formation compared with BMP-2 alone.¹¹⁹ With this decrease, extreme bone loss associated with BMP-2 that could lead to osteolysis and subsidence can be avoided. Conversely, with a decreased quantity of osteoclasts, osteoblastic differentiation and functionality are much less regulated, and side effects such as heterotopic ossification are found much more frequently than with BMP-2 use alone.¹¹⁹

Addition of anti-inflammatory drugs

Supraphysiological dosing of BMP-2 causes a large inflammatory response as emphasized in the inflammation section. Major cytokines IL-6, IL-10, and TNF-α as well as the formation of inflammatory exudates, highlight the proinflammatory state that BMP-2 induces locally.¹²³ The administration of adjunctive corticosteroids along with BMP-2 is directed at decreasing the inflammatory reaction induced by BMP-2.¹²⁴

To curb such an inflammatory response, low-dose dexamethasone has been studied and shown to significantly reduce soft-tissue inflammation and cellular invasion.¹²⁴ As demonstrated by high-powered microscopy, the inflammatory region in a rat model after administration of rhBMP-2 or the combination of rhBMP-2 with different steroid doses had a significantly larger mean area solely in the rhBMP-2-only group than that in the control.¹²⁴ This suppression of the chemokines avoids the infiltration of monomorphic and polymorphic nucleated cells that are attracted to the local connective tissues as well as the edema that impedes the healing process.^125,126

Alternative carriers for BMP-2

Although an absorbable collagen sponge has been approved and used in humans, the optimal scaffold for BMP-2 delivery in humans has not been established. Of note, numerous organic and synthetic scaffolds have been tested for BMP-2 release, and a complete listing is not feasible here (see Haidar et al.^127,128 for a further discussion).

The four major categories of BMP-2 carriers include natural-origin polymers, inorganic materials, synthetic biodegradable polymers, and composites.¹²⁷ In addition to the previously discussed collagen sponge, natural-origin polymers include hyaluronic acid (HA) and chitosan (CH). Compared with collagen sponges, HA-based delivery vehicles possess greater BMP-2 retention capacity¹²⁹ and result in improved bone formation in mandibular defects.¹³⁰ Similarly, rhBMP-2-loaded CH has been reported to accelerate osteogenesis in a rat critical-sized mandibular defect.¹³¹

Inorganic BMP-2 carriers include hydroxyapatite (HAP) calcium phosphate-based cements, ceramics, and coatings (CPCs), and bioactive glasses (BGs). Thus far, only porous HAP has been evaluated as a scaffold for BMP-2; however, it is inefficient as a controlled release carrier because of high BMP affinity and lack of HAP resorption.¹³² Interestingly, studies have shown that CPCs require lower doses of BMPs than other carriers.¹³³ In addition, various studies have demonstrated that BGs enhance the effects of BMP and induce osteoblast differentiation.^134,135

Synthetic biodegradable polymers have also been deemed as favorable BMP-2 carriers because of lack of immunogenicity or disease transmission.¹²⁷ The most common polymers include polylactic acid (PLA), polyglycolide (PLG), and their copolymer, PLGA.¹²⁷ PLA, PGA, and PLGA have been effectively used as rhBMP-2 carriers to repair diverse bone defects, including mandibular defects in a rat model, and periodontal, maxillary alveolar cleft, and segmental ulnar long-bone defects in a canine model, among others.^136–139

Lastly, composites are derived from combinations of the carriers discussed previously. In comparison to carriers composed of solely HAP, composites consisting of HAP, tricalcium phosphates, and collagen have been found to result in improved local BMP delivery and orthotopic bone formation.^140,141

In summary, although there are many suitable delivery systems available, the ideal BMP-2 carrier remains elusive.

Summary and Conclusions

The proper use of BMP-2 in the treatment of some patients with impaired fusion capacity can be an immense medical benefit that can clinically alter the overall long-term outcome of a surgical procedure. Thus, circumstantial use of BMP-2 has the capability to decrease prevalence of repeated surgical re-entry, trauma, complications, and additional medical cost.¹⁴² However, with this benefit comes a set of risks due to the supraphysiological dosing requirements and off-label use for inappropriate indication or anatomical location.

Once BMP-2 was declared to be FDA approved for one particular surgical procedure coupled with reporting “near perfect” safety,¹⁴³ this caused an extensive escalation of use in a wide range of orthopaedic procedures. Larger clinical trials were able to unearth more clinically relevant adverse events previously unknown. Still unclear is the tentative link between BMP-2 and an increased risk of cancer development.

The ideal candidate and best practices for BMP-2 use are difficult to determine and are dependent on the source consulted. Based on the available evidence, BMP-2 is indicated as a second-line adjunct to spinal fusion in cases in which other alternatives are either not available or are not likely to lead to effective fusion. Indications for application in the pediatric population are more difficult to define, as guidelines for age, weight, and level-dependent dosage of rhBMP-2 have yet to be elucidated.¹⁴⁴ Application in specific anatomic locations is correlated with distinctive adverse events. Although use in the lumbar spine is efficacious, ALIF is associated with increased risk for implant displacement, subsidence, infection, and urogenital events. Likewise, PLIF is associated with radiculitis, ectopic bone formation, and osteolysis.²² Application in the cervical spine is associated with rare life-threatening complications and is contraindicated.^22,24

In conclusion, the application of BMP-2 from bench to bedside has revolutionized the study of bone biology. Although BMP-2 is associated with a concerning side effect profile, it is currently the only osteoinductive growth factor to receive FDA approval for use as a bone graft substitute. Clearly there is an appropriate clinical context for rhBMP-2 use, especially when alternative bone graft substitutes are lacking. Despite the multitudes of studies over the past decades that have already been performed, further study of BMP-2 will better assess long-term results, examine alternative biologics or scaffolds to be included with BMP-2, and examine the cost–benefit of BMP-2 to the healthcare system.

Acknowledgments

This work was supported by the NIH/NIAMS (grants R01 AR061399, R01 AR066782, and K08 AR068316), the UCLA Department of Pathology and Laboratory Medicine, the Translational Research Fund, the UCLA Daljit S. and Elaine Sarkaria Fellowship award, and the Orthopaedic Research and Education Foundation with funding provided by the Musculoskeletal Transplant Foundation.

Disclosure Statement

No competing financial interests exist.