Dynamics of skeletal muscle-resident stem cells during myogenesis in fibrodysplasia ossificans progressiva

Fibrodysplasia ossificans progressiva (FOP) is a rare genetic disease in which extraskeletal (heterotopic) bone forms within tissues such as skeletal muscles, often in response to injury. Mutations in the BMP type I receptor ACVR1/ALK2 cause FOP by increasing BMP pathway signaling. In contrast to the growing understanding of the inappropriate formation of bone tissue within the muscle in FOP, much is still unknown about the regenerative capacity of adult diseased muscles. Utilizing an inducible ACVR1R206H knock-in mouse, we found that injured Acvr1R206H/+ skeletal muscle tissue regenerates poorly. We demonstrated that while two resident stem cell populations, muscle stem cells (MuSCs) and fibro/adipogenic progenitors (FAPs), have similar proliferation rates after injury, the differentiation potential of mutant MuSCs is compromised. Although MuSC-specific deletion of the ACVR1R206H mutation does not alter the regenerative potential of skeletal muscles in vivo, Acvr1R206H/+ MuSCs form underdeveloped fibers that fail to fuse in vitro. We further determined that FAPs from Acvr1R206H/+ mice repress the MuSC-mediated formation of Acvr1R206H/+ myotubes in vitro. These results identify a previously unrecognized role for ACVR1R206H in myogenesis in FOP, via improper interaction of tissue-resident stem cells during skeletal muscle regeneration.

Role of the P2X7 Receptor in the Osteogenesis of Heterotopic Ossification of the Achilles Tendon

Background Heterotopic ossification (HO) refers to a painful and complex disease. HO occurs in the setting of persistent systemic inflammation and appears in flare-ups during inflammation, following injury. In the recent research, the P2X7 receptor (P2X7R) is tightly involved in the osteogenesis of periodontal ligament stem cells under the inflammatory conditions. The ionotropic P2X7 receptor (P2X7R) is an ATP-gated ion channel expressed in the majority of stem cells. However, the function of P2X7R in the pathological formation of HO is unclear. Here, this paper hypothesizes that in the model of Achilles tendon ectopic ossification, P2X7R is overexpressed in tendon-derived stem cells and promote osteogenesis of tendon-derived stem cells under inflammatory conditions. Methods The tenotomy puncture and burn injury-induced HO model was constructed. The qPCR and immunofluorescence were used to detect the expression of P2X7R at the site of injured Achilles tendon where HO occurs. Achilles tendon stem cells (SCs) from control group and experimental group sources were cultivated separately under inflammatory conditions. The cells from the two groups were cultured for osteogenic analysis. In addition, a specific antagonist of P2X7R, BBG was used to detect whether reversed the above process. At last, BBG was used to intervene in animal models of heterotopic ossification. Results Under inflammatory conditions, P2X7R expression of the Achilles tendon and osteogenic capability of SCs is higher in heterotopic ossification group (HOG) than in other two groups. The P2X7R expression was positive correlated with the capacity of osteogenesis of SCs. BBG can inhibit osteogenic differentiation and subsequent bone formation in the P2X7R overexpress of SCs. BBG impeded the heterotopic bone formation in animal model. Conclusions P2X7R is one of the crucial mediators in the formation of the HO, blocking which may represent a potential therapeutic target for HO.

Over-expression of wild-type ACVR1 in fibrodysplasia ossificans progressiva mice rescues perinatal lethality and inhibits heterotopic ossification

Fibrodysplasia ossificans progressiva (FOP) is a devastating disease of progressive heterotopic bone formation for which effective treatments are currently unavailable. FOP is caused by dominant gain-of-function mutations in the receptor ACVR1 (also known as ALK2), which render the receptor inappropriately responsive to activin ligands. In previous studies, we developed a genetic mouse model of FOP that recapitulates most clinical aspects of the disease. In this model, genetic loss of the wild-type Acvr1 allele profoundly exacerbated heterotopic ossification, suggesting the hypothesis that the stoichiometry of wild-type and mutant receptors dictates disease severity. Here, we tested this model by producing FOP mice that conditionally over-express human wild-type ACVR1. Injury-induced heterotopic ossification (HO) was completely blocked in FOP mice when expression of both the mutant and wild-type receptor were targeted to Tie2-positive cells, which includes fibro/adipogenic progenitors (FAPs). Perinatal lethality of Acvr1R206H/+ mice was rescued by constitutive ACVR1 over-expression and these mice survived to adulthood at predicted Mendelian frequencies. Constitutive over-expression of ACVR1 also provided protection from spontaneous HO, and the incidence and severity of injury-induced HO in these mice was dramatically reduced. Analysis of pSMAD1/5/8 signaling both in cultured cells and in vivo indicates that ACVR1 over-expression functions cell-autonomously by reducing osteogenic signaling in response to activin A. Manipulating the stoichiometry of FOP-causing and wild-type ACVR1 receptors may provide the foundation for novel therapeutic strategies to treat this devastating disease.

The miR-4739/DLX3 Axis Modulates Bone Marrow-Derived Mesenchymal Stem Cell (BMSC) Osteogenesis Affecting Osteoporosis Progression

Osteoporosis is a complex multifactorial disorder linked to various risk factors and medical conditions. Bone marrow-derived mesenchymal stem cell (BMSC) dysfunction potentially plays a critical role in osteoporosis pathogenesis. Herein, the study identified that miR-4739 was upregulated in BMSC cultures harvested from osteoporotic subjects. BMSCs were isolated from normal and osteoporotic bone marrow tissues and identified for their osteogenic differentiation potential. In osteoporotic BMSCs, miR-4739 overexpression significantly inhibited cell viability, osteoblast differentiation, mineralized nodule formation, and heterotopic bone formation, whereas miR-4739 inhibition exerted opposite effects. Through direct binding, miR-4739 inhibited distal-less homeobox 3 (DLX3) expression. In osteoporotic BMSCs, DLX3 knockdown also inhibited BMSC viability and osteogenic differentiation. Moreover, DLX3 knockdown partially attenuated the effects of miR-4739 inhibition upon BMSCs. Altogether, the miR-4739/DLX3 axis modulates the capacity of BMSCs to differentiate into osteoblasts, which potentially plays a role in osteoporosis pathogenesis. The in vivo and clinical functions of the miR-4739/DLX3 axis require further investigation.

Comment to: The utility of ultrasound examination in cubital tunnel syndrome caused by heterotopic ossification

Ultrasound (US) could visualize the pathological anatomy of HO and the enlargement site and compression location of the nerve in the cubital tunnel [1]. We read with great interest the article of Jačisko et al[2]. In addition, we report rare US images of HO in direct contact with the swollen ulnar nerve in the cubital tunnel that was not detected by plain radiography. A 60-year-old female presented with a six-month history of elbow pain. Her pain was located at the medial side of the right elbow joint and accompanied by numbness of the fifth finger. She had a history of excessive manual labor due to her occupation as a gardener over the past few decades. The numbness began with the fifth finger initially and gradually extended toward the medial side of the elbow joint. US images showed hyperechoic masses causing acoustic shadowing, in direct contact with the ulnar nerve in the cubital tunnel. The HO seems to be related to compression of the ulnar nerve. The ulnar nerve was swollen (Figure 1-a, b). The maximal cross-sectional-area was 0.10 cm2. Plain elbow radiographs demonstrated osteophyte formation in the coronoid process of the ulna, the coronoid fossa of the humerus, and in the radial head (Figure 1-c). Radiographic imaging showed no heterotopic bone formation in the soft tissues surrounding the medial side of the right elbow. We performed US-guided perineural injection with a mixture of 1 cc of 10 mg triamcinolone and 3 cc of 0.2 % ropivacaine. Her pain and numbness gradually diminished with no adverse effects. Her pain reduced by 70% after two weeks, with pain improvement sustained for 6 months after the injection. Jačisko et al[2]have presented some diagnostic US imaging on neuropathy caused by HO located close to the ulnar nerve in the cubital tunnel. Especially, this case showed definite heterotopic bone formation in the soft tissue surrounding the medial side of the elbow on plain radiography. The classic sonographic patterns of HO were defined by the presence of central hypoechoic area surrounded by foci of calcification [3, 4]. The distortion of normal soft tissue and the formation of hypoechoic areas, with or without foci of calcification can also be shown as early signs[3, 4]. The use of US for HO is highly sensitive and provides an earlier diagnosis compared with other radiologic modalities [3-5]. It can be an effective treatment strategy and may improve the prognosis of neuropathy. We highlight that US evaluation can provide early diagnostic information about ulnar nerve morphology and various HO formations even if plane radiographs did not show heterotopic bone formation in the soft tissues surrounding the medial side of the elbow.

The Treatment Advantage of Complex Acetabular Fractures by the Pararectus Approach

Background: The surgical treatment of complex acetabular fractures is one of the most challenging procedures for orthopedic surgeons. The Pararectus approach, as a reasonable alternative to the existing surgical procedures, was performed for the treatment of complex acetabular fractures involving the anterior column. This study aimed to evaluate outcome using the Pararectus approach for acetabular fractures involving anterior columns. Methods: Thirty-seven with displaced complex acetabular fractures involving anterior columns were treated between July 2016 and October 2019 using the Pararectus approach. The functional outcomes (using the Merle d Aubigné and Postel scoring system, WOMAC and modified Harris scoring), the quality of surgical reduction (using the Matta criteria), and postoperative complications were assessed with about 26 months follow-up.Results: Thirty-seven patients (mean age 53 years, range: 30-71; 28 male) underwent surgery. Mean intraoperative blood loss was 840 ml (rang: 400-2000 ml) and mean operating time was 210 min (rang: 140-500 min). The modified Merle d Aubigné score was excellent and good in 27 cases (73%), fair in 6 cases (16%), and poor in 3 cases (12%). The mean score was 88.5 (range:77-96) for the modified Harris Hip scores, and 22 (range:7-35) for the WOMAC scores after operation. Postoperative functional outcomes were significantly improved compared with preoperative outcomes (P<0.0001). The quality of reduction was anatomical in 21 cases (57%), satisfactory in 9 cases (24%), and unsatisfactory in 7 cases (20%). At follow-up, four patients developed a DVT, and heterotopic bone formation was observed in one patient. The hip osteoarthritis was not observed.Conclusion: The Pararectus approach achieved good functional outcomes and anatomical reduction in the treatment of complex acetabular fractures involving anterior column with minimal access morbidity.

P028 COMBINING SEVEN ITEMS OF THE TOOLBOX TO TREAT A LARGE INCISIONAL HERNIA WITH HETEROTOPIC OSSIFICATION: A CASE REPORT

Aim We present our approach of treating a W3 (EHS-Classification) incisional hernia with heterotopic ossification in the abdominal wall. Material and Methods a 62-years-old female patient presented with a hernia in her inverted-T incision (midline and transverse) after undergoing multiple laparotomies. The CT-scan showed calcified structures within the abdominal wall. We planned the extensive reconstruction after preoperative Botox injections. Results The 20x25 cm hernial sack contained parts of the stomach and colon. The dissection of the midline and transverse scars was challenging with the needed removal of scattered pieces of heterotopic bone tissues. After dissecting the retro-muscular space, the fascial edges were 25 cm apart. With bilateral transversus abdominis release (TAR), It was reduced to 20 cm. The posterior fascia was approximated, leaving a central 12 cm defect, and a smaller lateral defect, which we covered using open-IPOM and underlay techniques respectively. A 30x40 cm mesh in sublay position was placed and fascial traction was applied on the anterior fascia. With the resulting defect of 16 cm, a tension-free closure was still not possible, and we bridged the gap with a mesh in inlay position. Conclusions Despite combining pre-operative Botox injection and fascial traction with TAR, complete closure of the fascia was not possible. IPOM, sublay, underlay and inlay bridging were needed. Specialized hernia surgeons should be familiar with a wide range of different techniques to deal with such cases.

P068 Abstract tin soldiers Global FOP patient search

While looking for one, you may find another: Tin Soldiers and the search for undiagnosed individuals with Fibrodysplasia Ossificans Progressiva (FOP) Background FOP is an ultra-rare condition where heterozygous, gain-of-function missense mutations in the ACVR1 gene result in progressive heterotopic bone formation in ligaments, tendons and muscles and result in severe disability.1 FOP has an estimated incidence of 0.6–1.3 per million individuals 2,3 suggesting that currently there are ∼8000 patients living with FOP worldwide, however only about 900 patients are currently diagnosed worldwide The diagnosis is made clinically by identification of typical malformations of the great toes as well as inflammatory swellings (flare-ups) that result in progressive and episodic ossification of soft connective tissues, often triggered by trauma.4 Muscle biopsies, though contraindicated, are frequently performed mistakenly during the course of diagnosis, as FOP is not a well-known condition. There is an urgent need to identify individuals with FOP across the globe in order to avoid harmful biopsies and to provide a pathway to care for patients with FOP. Tin Soldiers is a global FOP patient search program utilizing multimedia campaigns aimed at educating and bringing attention to FOP, to find individuals across the globe and to connect them to pathways of care. The mission is to identify every person with FOP who is currently undiagnosed, as well as to deliver education and support to those living with a diagnosis, but not connected to support networks. Once found, all people living with FOP are connected to pathways to care. The aim is to describe the Tin Soldiers global FOP patient search program approach and report early results of the program. Methods Tin Soldiers creates multimedia campaigns to create awareness and to educate medical professionals, healthcare workers, general public and local communities on FOP. At the heart of the communication program is story-telling of people living with FOP, from a feature-length documentary to public service announcements, animated short films and an 8-part Global Master Series—all designed to bring attention to FOP in order to find patients and provide a pathway to diagnosis and care. Importantly diagnosis is not the end of the journey, it’s just the beginning. Results Since official operations commenced in March 2020, Tin Soldiers has trained 535 medical professionals; established an African Clinicians Council of 10 doctors with the intention of mentoring others across the continent; increased the number of African patients with a diagnosis from 25 patients in December 2020–32 in April 2021. Connected previously diagnosed (but not connected) patients to a robust support network and held the first African FOP Family Gathering with clinicians from both South Africa and Nigeria. On the journey, patients with other conditions have been discovered including Juvenile Idiopathic Arthritis (JIA), Progressive Osseus Heteroplasia (POH) and Multiple Osteochondromas (MO). These patients have been diagnosed and connected to both medical care and patient support. Another important outcome is the continued education of doctors globally with the uptake of the CME Master Series in Russia and planned rollouts in Algeria, Nigeria, Kenya, Namibia, Sweden (in partnership with the national patient organization) and Brazil (under the First Lady’s patronage). Conclusion Tin Soldiers offers an innovative model of patient identification, diagnosis, support and education at all levels of care, using the power of story-telling and multi-media marketing. Such a model could be considered for raising the profile of other musculoskeletal or rare conditions and connecting patients to a functioning pathway to care.