Pathologic extraskeletal bone formation, or heterotopic ossification (HO), occurs following mechanical trauma, burns, orthopedic operations, and in patients with hyperactivating mutations of the type I bone morphogenetic protein receptor ACVR1 (Activin type 1 receptor). Extraskeletal bone forms through an endochondral process with a cartilage intermediary prompting the hypothesis that hypoxic signaling present during cartilage formation drives HO development and that HO precursor cells derive from a mesenchymal lineage as defined by Paired related homeobox 1 (Prx). Here we demonstrate that Hypoxia inducible factor-1α (Hif1α), a key mediator of cellular adaptation to hypoxia, is highly expressed and active in three separate mouse models: trauma-induced, genetic, and a hybrid model of genetic and trauma-induced HO. In each of these models, Hif1α expression coincides with the expression of master transcription factor of cartilage, Sox9 [(sex determining region Y)-box 9]. Pharmacologic inhibition of Hif1α using PX-478 or rapamycin significantly decreased or inhibited extraskeletal bone formation. Importantly, de novo soft-tissue HO was eliminated or significantly diminished in treated mice. Lineage-tracing mice demonstrate that cells forming HO belong to the Prx lineage. Burn/tenotomy performed in lineage-specific Hif1α knockout mice (Prx-Cre/Hif1α fl:fl ) resulted in substantially decreased HO, and again lack of de novo soft-tissue HO. Genetic loss of Hif1α in mesenchymal cells marked by Prx-cre prevents the formation of the mesenchymal condensations as shown by routine histology and immunostaining for Sox9 and PDGFRα. Pharmacologic inhibition of Hif1α had a similar effect on mesenchymal condensation development. Our findings indicate that Hif1α represents a promising target to prevent and treat pathologic extraskeletal bone.is the pathologic formation of extraskeletal bone in soft tissues. This process occurs in two separate patient populations: those with severe trauma, including large surface-area burns, musculoskeletal injury, orthopedic operations, and even spinal cord injury; and those with a genetic disease known as fibrodysplasia ossificans progressiva (FOP) (1-4). FOP is caused by a hyperactivating mutation in the type I bone morphogenetic protein (BMP) receptor ACVR1 (Activin type 1 receptor), and patients with FOP develop ectopic bone lesions in the absence of any substantial trauma. The clinical sequela of these pathologic ectopic bone formations, whether in the setting of trauma or genetic mutations, include nonhealing wounds, chronic pain, and joint immobility. In the case of FOP, progressive ossification may lead to death as a result of loss of thoracic cage compliance.Treatment options for HO are limited because bone often recurs following surgical resection, and some patients may have nonresectable HO because of its sensitive location. The risk of an operation may outweigh the benefits of excision, especially in the face of recurrence (5). Therefore, there is a need to identify therapeutic options ...

Witkop syndrome, also known as tooth and nail syndrome (TNS), is a rare autosomal dominant disorder. Affected individuals have nail dysplasia and several congenitally missing teeth. To identify the gene responsible for TNS, we used candidate-gene linkage analysis in a three-generation family affected by the disorder. We found linkage between TNS and polymorphic markers surrounding the MSX1 locus. Direct sequencing and restriction-enzyme analysis revealed that a heterozygous stop mutation in the homeodomain of MSX1 cosegregated with the phenotype. In addition, histological analysis of Msx1-knockout mice, combined with a finding of Msx1 expression in mesenchyme of developing nail beds, revealed that not only was tooth development disrupted in these mice, but nail development was affected as well. Nail plates in Msx1-null mice were defective and were thinner than those of their wild-type littermates. The resemblance between the tooth and nail phenotype in the human family and that of Msx1-knockout mice strongly supports the conclusions that a nonsense mutation in MSX1 causes TNS and that Msx1 is critical for both tooth and nail development.

PAX9, a paired domain transcription factor, has important functions in craniofacial and limb development. Heterozygous mutations of PAX9, including deletion, nonsense, or frameshift mutations that lead to a premature stop codon, and missense mutations, were previously shown to be associated with autosomal dominant oligodontia. Here, we report a novel missense mutation that lies in the highly conserved paired domain of PAX9 and that is associated with non-syndromic oligodontia in one family. The mutation, 83G-->C, is predicted to result in the substitution of arginine by proline (R28P) in the N-terminal subdomain of PAX9 paired domain. To rule out the possibility that this substitution is a rare polymorphism and to test whether the predicted amino acid substitution disrupts protein-DNA binding, we analyzed the binding of wild-type and mutant PAX9 paired domain to double-stranded DNA targets. The R28P mutation dramatically reduces DNA binding of the PAX9 paired domain and supports the hypothesis that loss of DNA binding is the pathogenic mechanism by which the mutation causes oligodontia.

To elucidate the transcriptional regulation of Bmp4 expression during organogenesis, we used phylogenetic footprinting and transgenic reporter analyses to identify Bmp4 cis-regulatory modules (CRMs). These analyses identified a regulatory region located ∼46 kb upstream of the mouse Bmp4 transcription start site that had previously been shown to direct expression in lateral plate mesoderm. We refined this regulatory region to a 396-bp minimal enhancer, and show that it recapitulates features of endogenous Bmp4 expression in developing mandibular arch ectoderm and incisor epithelium during the initiation-stage of tooth development. In addition, this enhancer directs expression in the apical ectodermal ridge (AER) of the developing limb and in anterior and posterior limb mesenchyme. Transcript profiling of E11.5 mouse incisor dental lamina, together with protein binding microarray (PBM) analyses, allowed identification of a conserved DNA binding motif in the Bmp4 enhancer for Pitx homeoproteins, which are also expressed in the developing mandibular and incisor epithelium. In vitro electrophoretic mobility shift assays (EMSA) and in vivo transgenic reporter mutational analyses revealed that this site supports Pitx binding and that the site is necessary to recapitulate aspects of endogenous Bmp4 expression in developing craniofacial and limb tissues. Finally, Pitx2 chromatin immunoprecipitation (ChIP) demonstrated direct binding of Pitx2 to this Bmp4 enhancer site in a dental epithelial cell line. These results establish a direct molecular regulatory link between Pitx family members and Bmp4 gene expression in developing incisor epithelium.

Abstract. Calciphylaxis is a painful, debilitating, and premorbid condition, which presents as calcified vasculature and soft tissues. Traditional diagnosis of calciphylaxis lesions requires an invasive biopsy, which is destructive, time consuming, and often leads to exacerbation of the condition and infection. Furthermore, it is difficult to find small calcifications within a large wound bed. To address this need, a noninvasive diagnostic tool may help clinicians identify ectopic calcified mineral and determine the disease margin. We propose Raman spectroscopy as a rapid, pointof-care, noninvasive, and label-free technology to detect calciphylaxis mineral. Debrided calciphylactic tissue was collected from six patients and assessed by microcomputed tomography (micro-CT). Micro-CT confirmed extensive deposits in three specimens, which were subsequently examined with Raman spectroscopy. Raman spectra confirmed that deposits were consistent with carbonated apatite, consistent with the literature. Raman spectroscopy shows potential as a noninvasive technique to detect calciphylaxis in a clinical environment. © The Authors. Published by SPIE under a Creative Commons Attribution 3.0 Unported License. Distribution or reproduction of this work in whole or in part requires full attribution of the original publication, including its DOI.

PURPOSE:Heterotopic ossification (HO) is extra-skeletal lamellar bone in soft tissues following trauma. Prior to HO deposition, patients develop inflammation and hypervascularization. Endothelial progenitor cells contribute to neo-angiogenesis by differentiating into mature endothelium. We hypothesized that angiogenesis establishes the niche necessary for HO, and that endothelial cells contribute to HO thereby providing a target for future inhibition. METHODS:Debrided tissue from 244 patients with >20% TBSA burn within 96 hours of burn was compared to 34 nonburn controls for vasculogenic gene expression. Separately, C57BL/6 mice underwent our proven HO model (Achilles' tenotomy + 30% total body surface area burn) or control tenotomy no burn injury (n=6 per group). The vasculogenic niche was assessed using flow cytometry, lineage tracing mice, in vivo confocal microscopy, histology, bioluminescensce, microCT, microcapture mRNA quantification, and protein quantification. Next, Cdh5+/+-Cre/RFP+/+ mice were used to visualize the presence of cells (expressing red fluorescent protein) of endothelial progenitor lineage at the HO site (3 and 6 weeks) after trauma. Laser capture microdissection (LCM) was performed to extract tissues of interest and RNA capture was performed with subsequent qRT-PCR to evaluate for gene expression. Protein was collected from the hind limb soft tissue and immunoblotted for VEGF. RESULTS:Tissue from burn patients demonstrated significant up-regulation of vasculogenic genes (VEGF/HIF-1alpha/ VE-cadherin/CD31) compared to unburned patients. In our mouse model, bioluminescent imaging demonstrated increased vascularity at the site of future HO when compared with the contralateral limb. Consistent with these findings, burned mice developed more HO at the site of trauma when compared to unburned mice. In vivo confocal microscopy using Cdh5+/0/ RFP+/+ mice confirmed the presence of robust vascularity at the site of HO when compared with the contralateral limb at 6 weeks. Flow cytometry of cells from the HO site showed 20% enrichment of mature endothelial cells (Tie2+/CD31+) and 100% enrichment of endothelial progenitor cells (CD34+/ CD133+) compared with the control uninjured leg at three weeks (p <0.05). 3wks after trauma, Cdh5+/0-Cre/RFP+/+ lineagetracing mice also demonstrated enrichment of endotheliallineage cells at the HO site compared to the contralateral control limb. However, HO osteoblasts did not express RFP, indicating they are not of endothelial-lineage. LCM sections showed more angiogenic RNA transcript (vegfa) and protein (VEGFA) in the burned mice compared with unburned mice. Western blot of protein from hind limb confirmed these findings. CONCLUSION:We demonstrate that the development of heterotopic ossification is dependent on the presence of a vasculogenic niche. Mice with robust HO demonstrate an enrichment of mature endothelial and endothelial precursor cells. Although cells of endothelial progenitor origin play a role in chondrogenesis, they do not contribute to HO osteoblasts ...

PURPOSE:Heterotopic ossification (HO) is the formation of bone in soft tissues following trauma. We hypothesized that early vascularity establishes the niche for HO, and that endothelial cells contribute to HO. METHODS:Tissue from 244 patients with >20% TBSA burn within 96 hours was compared to 34 nonburn controls for vasculogenic gene expression. Separately, C57BL/6 mice underwent Achilles' tenotomy with 30% TBSA burn or no burn. The vasculogenic niche of the tenotomy site was assessed using flow cytometry, lineage-tracing mice, bioluminescent and microCT imaging, and mRNA quantification using microcapture. RESULTS:Tissue from burn patients demonstrated significant upregulation of vasculogenic genes (VEGF/HIF-1alpha/ VE-cadherin/CD31) compared to controls. Flow cytometry of cells from our mouse model showed 20% enrichment of mature endothelial cells (Tie2+/CD31+) and 100% enrichment of endothelial progenitor cells (CD34+/CD133+) at the tenotomy site compared with uninjured leg(A;p < 0.05). At two weeks, bioluminescent imaging demonstrated increased vascularity at the future HO site compared to contralateral control limbs(B/C). Three weeks after trauma, Cdh5 +/0 -Cre/ RFP +/+ lineage-tracing mice demonstrated enrichment of endothelial progenitor cells at the tenotomy site compared with the contralateral control limb(D). At 6 weeks after injury, HO lacked cells of endothelial origin. CONCLUSIONS:An early, vasculogenic niche enhances HO. Mice with robust HO demonstrate enrichment of mature endothelial cells and endothelial precursor cells. Although endothelial progenitor cells contribute to vasculogenesis, they likely do not contribute to HO osteoblasts. PURPOSE:Abdominal wall vascularized composite tissue allotransplantation (AW-VCA) is a common form of VCA. Functional recovery is expected in VCA; however, this has never been demonstrated in AW-VCA. Our purpose was to create a translatable animal model for an innervated AW-VCA that retains form and function. METHODS:The rat donor's common iliac vessels were anastomosed to recipient's femoral vessels. Intercostal nerves T10/ L1 were coapted using nylon suture. Four groups (n=5/group) were included for study. Group 1=Intercostal nerves cut, not repaired. Group 2=Intercostal nerves cut, T10/L1 repaired. Group 3=Allogeneic AW-VCA, T10/L1 repaired. Group 4=Syngeneic AW-VCA, T10/L1 repaired. Animals were sacrificed on POD 60. Nerve regeneration was assessed using muscle weight, laminin cross sectional area (CSA), and neuromuscular junction percent reinnervation. RESULTS:Groups 2, 3, and 4 maintained a greater percentage of muscle weight when compared with Group 1 (.22, .21, .23 versus .16, p < 0.05). Group 1 had decreased CSA when compared with contralateral controls (3171 μm2 versus 4453 μm2, p < 0.05). Group 1 had decreased percent reinnervation of the alloflap when compared to internal controls (21% versus 91%, p < 0.05) There was no difference between Groups 3 or 4 with internal controls (80% versus 91%, p > 0.05; 82% versus 91%, p > 0.05). CONCLUSION:In a rodent m...

No abstract