Differential Vascularity in Genetic and Nonhereditary Heterotopic Ossification

Heterotopic ossification (HO) is the formation of bone outside of the normal skeleton. This process may occur in the skin, muscles, and soft tissues and can cause significant morbidity. Nonhereditary heterotopic ossification (NHO) is a common consequence of musculoskeletal trauma and surgery. NHO has also been reported following spinal cord injury, traumatic brain injury, and severe burns.^1-5 NHO has been reported to occur in up to 90% of patients following hip arthroplasty and acetabular fractures, and in up to 53% of patients with neurologic injury.^3,6 This condition causes significant morbidity as a result of joint contractures, loss of soft tissue, and chronic pain.^2-4 Histologically, NHO is a process that demonstrates temporospatial evolution. Early lesions demonstrate a nodular fasciitis (NF)-like fibroblastic proliferation followed by gradual bone production and osseous maturation over time.^1,2,4 Similar to bone morphogenesis⁷ or fracture repair,⁸ NHO may form through endochondral or intramembranous mechanisms.⁹ Reported frequencies of endochondral versus intramembranous ossification in NHO are highly variable.^1,2,4,5,10

Progressive osseous heteroplasia (POH) is a rare genetic form of HO characterized by predominantly intramembranous ossification (OMIM: 166350).^11-17 POH typically presents in infancy with dermal ossification that progresses to involve the subcutaneous and deep skeletal muscle and connective tissues.^11-22 Ossification may progress to ankylosis of the joints causing focal growth retardation of involved limbs.^17,21 POH is caused by inactivating mutations in the GNAS gene, which encodes the alpha subunit of the stimulatory G protein (G_sα) of adenylyl cyclase,^{11,13,14,18,23-32} and has been associated with autosomal dominant^17,18,21,32 inheritance with the mutation most frequently occurring on the paternally inherited allele,^{16,18,27,28,30,31} although many cases are sporadic.^11,12,17,18 The disorder exists on a spectrum of GNAS mutation-associated hereditary HO disorders, including Albright hereditary osteodystrophy.^{11,13,17,18,23,25,27} Disease progression typically slows during adulthood.^11,12 Patients have a normal level of intelligence and do not have significant congenital skeletal abnormalities or endocrine abnormalities.^12,17

Fibrodysplasia ossificans progressiva (FOP) is another rare genetic form of HO that presents in childhood caused by activating mutations in the ACVR1 gene (OMIM: 135100). In contrast with POH, FOP is characterized by predominantly endochondral ossification within muscle and deep connective tissues and spares the skin.^11,16,17,33 Patients with FOP usually have congenital malformations of the great toes and may have other congenital skeletal anomalies. In the absence of injury, HO generally arises bilaterally in a cranial to caudal, proximal to distal progression, and lesions are preceded by inflammatory tumor-like swellings.^{11-13,17,19,33} The disease begins in early childhood and continues to progress through adulthood, with most patients wheelchair-bound by the second decade of life. The median lifespan for FOP patients is approximately 40 years, and patients typically succumb to thoracic insufficiency syndromes.^33-37 Most cases of FOP are sporadic; however, some families show autosomal dominant inheritance. Both de novo and inherited cases are caused by activating mutations in the ACVR1 gene, which encodes activin A receptor type 1/activin-like kinase 2, a bone morphogenic protein type 1 receptor.^1-3,33

In a prior study, we characterized the vascular patterning of NHO.¹ We found that heterotopic bone formation and maturation is coupled with dynamic temporal vascular changes.¹ In this article, we report on the vascular patterning of NHO in comparison with inherited forms of HO (including both POH and FOP). We especially sought to characterize the differences in vascular modeling across the various disease processes and across the histologic spectrum of HO.

Heterotopic bone has diverse etiologies, and in this study, we sought to directly compare vascular patterns among HO with and without an underlying genetic driver. POH and FOP are rare genetic diseases that share a commonality, but have important differences in their site of bone formation, frequency of endochondral bone, studied signaling cascades, and even prognosis. Our qualitative and quantitative data in vascular patterning suggest that despite these differences, both genetic forms of HO demonstrate overall increased vascularity in comparison to NHO. Both genetic and NHO disorders show temporospatial variation in vascularity. To our knowledge, this is the first study comparing the vascular histomorphometric features of genetic and NHO.

The histology of heterotopic bone has been well described elsewhere, progressing from an early inflammatory phases through mature bone, which resembles normal skeletal elements. Overall and in this study, HO from all etiologies showed some a similar phasic disease process. NF-like areas were similar in appearance in both genetic forms (POH and FOP) and NHO. In contrast, the frequency and overall amount of cartilage varied greatly between genetic forms of HO as well as compared with NHO. POH has been characteristically described as having predominantly intramembranous ossification,^{2,11-14,17,23,26} and in agreement with this, we observed frank cartilage in only 1 of 6 cases of POH. In contrast, FOP is routinely described as predominantly an endochondral process,³⁸ and all 3 cases in our files showed prominent cartilage. Interestingly, in the present study and in agreement with our clinical experience, the frequency and amount of cartilage in cases of FOP greatly exceeded that of NHO.

Despite these differences, conserved features in vascularity were seen across HO types. As we observed in prior studies,¹ early NF-like lesions showed the highest number of small caliber blood vessels. As HO bone maturation occurs, vessels are less frequent but dilated in size. While the overall patterning of blood vessels was similar in POH, FOP, and NHO, notable differences in the degree of the vascularity were present. Among NF-like areas, vascular quantification showed a slight increase in vascularity within both forms of genetic HO as compared with NHO. These differences were most obvious in areas of woven bone, where genetic forms of HO both evidenced significantly increased vascularity in comparison to NHO. Most striking for the authors were foci of POH (as in Figure 2A), in which blood vessels within a given area actually eclipsed the bone itself in prominence. In our clinical experience, this prominence of vascularity is not seen in NHO at any stage in its evolution. Finally, these differences in vascularity between genetic and nonhereditary were not as prominent in areas of mature lamellar bone. In our experience, overtime human HO maturation progresses toward an appearance that resembles native skeletal elements. It seems that those distinguishing histologic features of genetic versus NHO (whether osseous, cartilaginous, or vascular) are lost with maturation of the lesion.

Studies have shown that osteogenesis is closely tied to vascularization.^3,39-45 Hypoxia-inducible factor 1α (Hif-1α) regulates responses to low oxygen availability and is necessary for normal osteogenesis.^44,45 Hif-1α causes an increase in vascular endothelial growth factor (VEGF) within osteoblasts, stimulating the angiogenesis that drives osteogenesis.⁴⁵ VEGF expression has been shown in areas of ectopic ossification,^39-41 suggesting that angiogenesis is also necessary for HO. Studies show that mice with increased Hif-1α activation in osteoblasts have markedly increased vascularity and increased bone production, and that those lacking Hif-1α have impaired angiogenesis and bone formation. This clinical study in subtypes of human HO provides yet more evidence of the proximal linkage between bone and vasculogenesis. Of note, experimental models of HO suggest that the lymphatic system may also be an important environmental contributor to the formation of HO.⁴⁶ In this context, surgical lymph node excision decreased HO formation in a trauma-induced mouse model.⁴⁶ An interesting future study would include the characterization of lymphatic ingrowth and distribution across human HO samples.

Current therapies for HO are largely ineffective, and HO remains a debilitating spectrum of diseases. Nonsteroidal anti-inflammatory drugs have been used to prevent NHO; however, these carry a risk of long-bone nonunion and gastrointestinal bleeding.³ Radiation has been used effectively as preventative therapy following hip arthroplasty,³ although this is a less feasible therapy in patients with genetic HO who may have progressive and widespread lesions. Bisphosphonates have been used by some, but their efficacy remains unclear. Lesions often recur following surgical excision, leading to soft tissue loss and increased morbidity.^2,3,11,12 In conjunction with previous studies showing the close relationship with angiogenesis, our findings suggest that antiangiogenic therapeutic agents that target vasculogenic growth factors, particularly Hif-1α or VEGF, may be useful in the treatment of HO. Neutralizing antibodies to VEGF (Bevacizumab, Avastin) have been studied in numerous examples of pathologic angiogenesis⁴⁷ and may be efficacious in HO; however, future experimental studies to assess targeting of VEGF signaling in HO are warranted. Such agents could lead to a critical reduction in vasculogenesis that is necessary for new heterotopic bone formation.

In summary, both genetic and nonhereditary forms of HO show temporospatial variation in vascularity. Both POH and FOP demonstrate a comparatively richer vascular environment than NHO. Further studies are necessary to explore the relationship between angiogenesis and osteogenesis in HO, and to determine whether targeting angiogenesis may be of therapeutic benefit.