BMP Ligand Trap ALK3-Fc Attenuates Osteogenesis and Heterotopic Ossification in Blast-Related Lower Extremity Trauma

Abstract

Traumatic heterotopic ossification (tHO) commonly develops in wounded service members who sustain high-energy and blast-related traumatic amputations. Currently, no safe and effective preventive measures have been identified for this patient population. Bone morphogenetic protein (BMP) signaling blockade has previously been shown to reduce ectopic bone formation in genetic models of HO. In this study, we demonstrate the efficacy of small-molecule inhibition with LDN193189 (ALK2/ALK3 inhibition), LDN212854 (ALK2-biased inhibition), and BMP ligand trap ALK3-Fc at inhibiting early and late osteogenic differentiation of tissue-resident mesenchymal progenitor cells (MPCs) harvested from mice subjected to burn/tenotomy, a well-characterized trauma-induced model of HO. Using an established rat tHO model of blast-related extremity trauma and methicillin-resistant Staphylococcus aureus infection, a significant decrease in ectopic bone volume was observed by micro-computed tomography imaging following treatment with LDN193189, LDN212854, and ALK3-Fc. The efficacy of LDN193189 and LDN212854 in this model was associated with weight loss (17%-19%) within the first two postoperative weeks, and in the case of LDN193189, delayed wound healing and metastatic infection was observed, while ALK3-Fc was well tolerated. At day 14 following injury, RNA-Seq and quantitative reverse transcriptase-polymerase chain reaction analysis revealed that ALK3-Fc enhanced the expression of skeletal muscle structural genes and myogenic transcriptional factors while inhibiting the expression of inflammatory genes. Tissue-resident MPCs harvested from rats treated with ALK3-Fc exhibited reduced osteogenic differentiation, proliferation, and self-renewal capacity and diminished expression of genes associated with endochondral ossification and SMAD-dependent signaling pathways. Together, these results confirm the contribution of BMP signaling in osteogenic differentiation and ectopic bone formation and that a selective ligand-trap approach such as ALK3-Fc may be an effective and tolerable prophylactic strategy for tHO.

Keywords: ALK2/ALK3 kinase inhibitors; BMPRIA; bone morphogenic protein (BMP); heterotopic ossification; limb amputation; trauma-induced extremity injury.

FIG. 1.

BMP type I receptor kinase inhibitors limit early and late osteogenic differentiation of MPCs. Following burn/tenotomy injury, MPCs were harvested at the zone of injury on POD7 and cultured in ODM treated with LDN193189 (750 nM), LDN212854 (750 nM), or ALK3-Fc (750 nM). (A) Analysis of early osteogenic differentiation was performed by alkaline phosphatase staining following 6 days in ODM. (B) Pixel quantification of alkaline phosphatase staining. (C) Analysis of late osteogenic differentiation was performed by Alizarin red staining following 12 days in ODM. (D) Pixel quantification of alizarin red staining. Mean ± SEM. **P < 0.01, ***P < 0.001, compared with vehicle treatment. SEM, standard error of the mean; ODM, osteogenic differentiation medium; BMP, bone morphogenetic protein; MPC, mesenchymal progenitor cell; POD, postoperative day.

FIG. 2.

BMP ligand trap ALK3-Fc limits endochondral ossification and heterotopic ossification and is well tolerated without any side effects. Rats were subjected to trauma that simulates a blast-induced extremity injury by exposing rats to blast overpressure, femur fracture, and a soft tissue crush injury, followed by an amputation through the zone of injury and then wound inoculation with MRSA following wound closure. Rats were then treated with vehicle (PBS, n = 6), LDN193189 (oral lavage, 6 mg/kg daily for 21 days, n = 8), LDN212854 (oral lavage, 6 mg/kg daily for 21 days, n = 8), and ALK3-Fc (intraperitoneal injection, 3 mg/kg twice weekly for 28 days, n = 6). (A) Animals were weighed every other week. (B) Micro-computed tomography scans were performed on animals following blast injury at 12 weeks. Total new bone was determined by defining the difference between new bone and naive bone. (C) Representative micro-computed tomography scan of rats treated with vehicle and ALK3-Fc at 12 weeks. Arrows highlight ectopic bone formation. (D) Representative hematoxylin and eosin images of sections in the ectopic bone region demonstrating the presence of fibrous capsule (arrow) around the amputation site without distinct chondrocytes (arrowhead) observed in the vehicle-treated control. Mean ± SEM. *P < 0.05, compared with vehicle- and LDN193189-treated rats; ^#P < 0.05, ^##P < 0.01, compared with vehicle- and LDN212854-treated rats; ^ΦΦP < 0.01, compared with vehicle- and ALK3-Fc-treated rats. Scale bars, 20 μM. PBS, phosphate-buffered saline; MRSA, methicillin-resistant Staphylococcus aureus.

FIG. 3.

No systemic side effects following treatment with ALK3-Fc. Following blast-related polytraumatic extremity injury and MRSA infection, rats were treated with vehicle (PBS, n = 3) or ALK3-Fc (3 mg/kg, n = 3) twice a week with intraperitoneal injections and whole blood was analyzed on POD3, POD7, and POD14. Blood was collected from the tail vein and immediately subjected to analysis. (A) Complete blood count and (B) comprehensive metabolic profile were evaluated. Mean ± SEM. *P < 0.05, **P < 0.01, comparing vehicle and ALK3-Fc treatment. Gray box indicates normal references values. WBC, white blood count; AST, aspartate aminotransferase; ALT, alanine aminotransferase; LDH, lactic acid dehydrogenase; CK, creatine kinase, ALP, alkaline phosphatase.

FIG. 4.

ALK3-Fc reduces osteogenic differentiation, proliferation, and self-renewal capacity of MPCs. Following blast-related polytraumatic extremity injury and MRSA infection, rats were treated with either vehicle or ALK3-Fc (3 mg/kg) twice a weekly by intraperitoneal injections, and MPCs muscle biopsies at the zone of injury were harvested on POD14 and plated in stromal media or osteogenic media. (A) Analysis of early osteogenic differentiation by alkaline phosphatase activity following 6 days in stromal media or ODM. (B) Cell proliferation after 6 days in stromal media or ODM and evaluated by BrdU assay. (C) Quantification of MPC CFPs by crystal violet after 6 days in stromal media or ODM. Colonies containing >50 cells were counted. Mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, comparing vehicle and ALK3-Fc treatment. BrdU, bromodeoxyuridine.

FIG. 5.

ALK3-Fc treatment enhances muscle-like gene expression while reducing inflammatory genes. RNA-Seq analysis of local wound tissues from rats subjected to blast-related polytraumatic extremity injury and MRSA infection treated with vehicle or ALK3-Fc collected on POD14 and compared with uninjured, naive control rats. (A) Principal component analysis reveals clustering of samples by treatment groups based on PC1 and PC2. (B) A Venn diagram reveals the number of significantly DE genes from DESeq2 analysis comparing POD14 vehicle- and ALK3-Fc-treated with naive controls. Functional analysis (g:Profiler) by GO terms and adjusted P values (P_adj) reveals significantly upregulated genes from DE analysis comparing POD14 vehicle-treated and POD14 ALK3-Fc-treated rats, showing (C) the top 20 significant BP and (D) the significant CC. (E) Functional analysis of g:Profiler functional analysis results showing GO terms and P_adj using the list of significantly downregulated genes from the differential expression analysis comparing POD14 vehicle-treated rats with POD14 ALK3-Fc-treated animals, showing the top 20 significant BP. (F, G) Heat maps showing the fold change in expression of POD14 vehicle- and ALK3-Fc-treated rats compared with naive controls. (F) The set of genes from C were “intersected” with the biological process of “muscle structure development,” and the functional analysis and relative fold expression versus naive samples depicted by heat map according to a linear scale. (G) The set of genes from E were “intersected” with the biological process of “inflammatory response” and relative expression versus naive samples depicted by heat map according to a linear scale. PC, principal components; DE, differentially expressed; GO, Gene Ontology; BP, biological processes; CC, cellular components.

FIG. 5.

ALK3-Fc treatment enhances muscle-like gene expression while reducing inflammatory genes. RNA-Seq analysis of local wound tissues from rats subjected to blast-related polytraumatic extremity injury and MRSA infection treated with vehicle or ALK3-Fc collected on POD14 and compared with uninjured, naive control rats. (A) Principal component analysis reveals clustering of samples by treatment groups based on PC1 and PC2. (B) A Venn diagram reveals the number of significantly DE genes from DESeq2 analysis comparing POD14 vehicle- and ALK3-Fc-treated with naive controls. Functional analysis (g:Profiler) by GO terms and adjusted P values (P_adj) reveals significantly upregulated genes from DE analysis comparing POD14 vehicle-treated and POD14 ALK3-Fc-treated rats, showing (C) the top 20 significant BP and (D) the significant CC. (E) Functional analysis of g:Profiler functional analysis results showing GO terms and P_adj using the list of significantly downregulated genes from the differential expression analysis comparing POD14 vehicle-treated rats with POD14 ALK3-Fc-treated animals, showing the top 20 significant BP. (F, G) Heat maps showing the fold change in expression of POD14 vehicle- and ALK3-Fc-treated rats compared with naive controls. (F) The set of genes from C were “intersected” with the biological process of “muscle structure development,” and the functional analysis and relative fold expression versus naive samples depicted by heat map according to a linear scale. (G) The set of genes from E were “intersected” with the biological process of “inflammatory response” and relative expression versus naive samples depicted by heat map according to a linear scale. PC, principal components; DE, differentially expressed; GO, Gene Ontology; BP, biological processes; CC, cellular components.