Heterotopic ossification secondary to injury: insights from a novel zebrafish model

Abstract

Heterotopic ossification is the formation of bone, either solitary or multiple, in the extra-skeletal soft tissues of the body where it would not normally be present. While there are a few rare varieties of this disorder driven by underlying genetic mutations, others occurring purely in response to triggering events such as injury are relatively common and can potentially occur in anyone. There are two such conditions, collectively referred to as non-genetic or acquired disorders of heterotopic ossification: myositis ossificans traumatica (MOT) and neurogenic heterotopic ossification (NHO). MOT can occur following musculoskeletal trauma of any form, whereas NHO occurs following spinal cord injury or traumatic brain injury. Existing experimental models for studying heterotopic ossification have limitations, as they either lack mechanistic relevance to the human condition or are invasive and laborious. Thus, currently there are no experimentally amenable models for this disorder that can be leveraged for discovery of mechanisms and treatment. To address this research gap, the primary aim of this project is to establish a simple zebrafish model capable of accurately reproducing trauma-induced heterotopic ossification as observed in humans and use it to dissect and comprehend the intricate molecular and cellular mechanisms underlying the disorder. Additionally, the project seeks to employ mutant analyses and pharmacological testing to facilitate the identification of potential therapeutic targets and validate the efficacy of the model established.

To induce heterotopic ossification in zebrafish as a response to musculoskeletal trauma mimicking human injuries, targeted injuries were conducted in various sites, including the caudal peduncle, pectoral fin, and dorsal thoracic region. Both the pectoral fin and caudal peduncle regions exhibited heterotopic bone formation, with the former displaying a more consistent response. Particularly noteworthy was the microscopic bone injury in the pectoral fin rays, creating a raw area on the bone surface, contributed to the consistent heterotopic ossification response, resembling spurs and bridges of ectopic bone as observed in humans. This underscores the osteo-inductive nature of the injury microenvironment. Another intriguing finding was the significant proliferation and increased size of intermuscular bones at the caudal peduncle and dorsal thoracic region following contusions, suggesting that muscle injuries, even in the absence of bony injury, develop osteo-proliferative properties, resulting in enhanced bone size and density. Recognizing osteo-induction and proliferation as crucial aspects of bone development, the zebrafish injury microenvironment demonstrating both properties serves as a valuable tool for in-depth exploration into heterotopic bone formation.

To explore potential changes in the transcriptome associated with this process, particularly following repeated muscle contusions, preliminary targeted qRT-PCR assays were conducted, followed by comprehensive genome-wide bulk RNA sequencing (RNASeq) analysis. The results from Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment plots indicate that a robust immune response, characterized by apoptosis and upregulation of inflammatory mediators, is triggered after injury, and persists following recurring injuries. Specifically, subsequent to the initial upregulation of genes encoding Il-6 family cytokines, including il11a and il11b, an elevation in various osteoblastic differentiation markers was observed over time. These markers encompassed runx3, bglap, dlx2a, dlx2b, dlx3b, dlx4a, dlx5a, msx2a, spp1, fn1a, along with teleost-specific scpp1, scpp5, and scpp7. Furthermore, an increase in expression was noted for sall1a and sall1b, responsible for regulating region-specific morphogenesis; hoxa13a and hoxa13b, crucial for skeletal development; kcnk5b, which acts locally within the mesenchyme of fins and barbels to specify appendage size; and osteoclast-specific stat1b, ocstamp, csf1ra, and ctsk. These RNASeq findings were further validated through qRT-PCR analysis of selected genes.

Given that the caudal peduncle contusion site, from where samples for RNASeq were obtained, exhibited heterotopic ossification at a limited rate, yet showed a more pronounced hypertrophy of intermuscular bones following the injuries, the RNASeq findings closely correspond to the osteoproliferation phenotype. This sheds light on the differentially regulated genes at contusion sites with the potential to promote osteogenesis, despite the absence of bony injuries. Although the key regulators governing this process remain to be defined, the relevant genes and pathways identified can be designated as targets to be studied using genetic manipulations and pharmacological interventions to determine whether they exert any inhibitory effect on the heterotopic bone formation response. After assessing the transcriptome, experiments were conducted on csf1ra, kif7, and pycard mutants, as well as utilizing pharmacological agents like dexamethasone and alendronate. These experiments revealed no altered response in the frequency of heterotopic bone formation. However, kcnk5b and il11ra mutants exhibited striking variations, both in terms of frequency and quantitative assessments.

Specifically, gain-of-function mutants of Kcnk5b exhibited a significantly increased magnitude of heterotopic bone formation, as confirmed through micro-CT based volumetric assessment, suggesting a central role for potassium channel signaling in regulating the heterotopic bone formation response. In contrast, zebrafish carrying a loss-of-function mutation of the Interleukin 11 receptor alpha (Il11ra), associated with impaired regeneration, showed a substantially reduced heterotopic bone formation response. This contrast underscores the importance of interleukin signaling via Il11ra in the injury response mechanism leading to heterotopic bone formation. These findings advance our understanding of the molecular basis of heterotopic bone formation and offer potential insights for therapeutic strategies for human patients facing this debilitating condition. Further research is warranted to elucidate the intricate interplay between potassium channel signaling, IL-11, and other mechanisms influencing impaired tissue regeneration and skeletal repair leading to heterotopic ossification.