Heterotopic ossification (HO) is defined as aberrant bone formation in extraskeletal locations. In this process, local stromal cells of mesenchymal origin abnormally differentiate, resulting in pathologic cartilage and bone matrix deposition. However, the specific cell type and mechanisms beyond this process are not well understood, in part due to the heterogeneity of progenitor cells involved. Here, a combination of single cell RNA sequencing (scRNA-Seq) and lineage tracing, defined the extent to which synovial / tendon sheath progenitor cells contribute to HO. For this purpose, a Tppp3 (tubulin polymerization-promoting protein family member 3) inducible reporter model was used, in combination with either Scx (Scleraxis) or Pdgfra (Platelet derived growth factor receptor alpha) reporter animals. Both arthroplasty-induced and tendon injury-mouse experimental HO models were utilized. ScRNA-Seq of tendon-induced traumatic HO suggested that Tppp3 is a progenitor cell marker for either osteochondral or tendon or cells. After HO induction, Tppp3 reporter+ cell population expanded in number and contributed to cartilage and bone formation in tendon and joint-associated HO. Using double reporter animals, we found that both Pdgfra+Tppp3+ and Pdgfra+Tppp3- progenitor cells produced HO-associated cartilage. Finally, the examination of human samples showed a significant population of TPPP3+ cells overlapping with osteogenic markers in areas of HO. Overall, these results provide novel observations that peritenon and synovial progenitor cells undergo abnormal osteochondral differentiation and contribute to heterotopic bone formation after trauma

Abstract. Objectives. The term heterotopic ossification (HO) describes lamellar bone formation within soft tissues following injury. A genome-wide scan of patients after hip arthroplasty has identified that variation within the lncRNA CASC20 is associated with HO susceptibility. Previous findings in our lab have demonstrated upregulation of CASC20 during BMP2-induced osteodifferentiation of adipose-derived stem cells (hMAD) alongside osteodifferentiation markers, RUNX2 and OSX. We hypothesize that CASC20 is a novel regulator of bone formation and aim to investigate CASC20 function in bone formation. Methods. 1) We used miRanda prediction algorithm and the ENCORI database to respectively predict which miRNAs CASC20 interacts with and to select for experimentally validated miRNAs. 2) We characterized the expression and functional role of CASC20-interacting miRNAs by respectively analyzing publicly available datasets (GSE107279 and pubmed.ncbi.nlm.nih.gov/26175215/) and by using Gene Ontology (GO) analysis. 3) We overexpressed CASC20 in hMAD using a lentiviral system and tested the effect of CASC20 overexpression in osteodifferentiation and expression of putative CASC20-interacting miRNAs. Results. 1) We identified 64 experimentally validated miRNAs that are predicted to interact with CASC20. 2) GO analysis revealed that the most frequently targeted molecular functions included SMADs, MAPKK and other kinase activities known to play a central role in osteo and chondrogenesis. We found 10 miRNAs including hsa-miR-485-3p that demonstrated down-regulation in both osteo- and chondrogenesis. 3) We found that CASC20-overexpression augmented the osteodifferentiation of hMAD measured in mineralization using Alizarin Red S. CASC20 overexpression increased the expression of osteogenic marker ALP and decreased the expression of hsa-miR-485-3p. Conclusion. Here we show how CASC20 may regulate bone formation by acting as a competitive endogenous RNA (ceRNA). We are currently using CASC20 overexpression model in osteo- and chondrogenesis, and testing CASC20-miRNA interaction to establish the underlying mechanism for the observed associations

Introduction and Objective. Interest for direct anterior approach (DAA) in hip hemiarthroplasty (HHA) has greatly increased in recent years, however which is the best surgical approach in hip replacement treating femoral neck fractures (FNFs) is already unclear. The aim of this study is to perform a radiographic and perioperative complications analysis by comparing the direct anterior approach (DAA) with the direct lateral approach (DLA) in patients treated with hemiarthroplasty for FNFs. Materials and Methods. Patients with FNFs surgically treated between 2016–2020 with HHA were enrolled. The radiographical outcomes of DAA and DLA are compared. Several peri-operative and post-operative variables were evaluated: mean surgery time, complications as periprosthetic fractures or episodes of dislocation, the average of post-operative diaphyseal filling of the stem (Canal Fill Index, CFI), the extent of heterotopic ossification (HO) (simplified Broker classification) and metadiaphiseal bone loss (Paprosky classification) within one year from surgery. Results. 86 patients underwent HHA by DAA and 80 patients by DLA. The two groups are qualitatively comparable. No statistically significate differences were showed in all variables analyzed (p>0.05). The average of surgical time of DAA were 61 minutes compared to 67 of DLA. No differences were showed in the post-operative CFI (DAA 0.71 ± 6.1; DLA 0.76 ± 13.5), the extent of the HO (DAA 79.07% low; DLA 75% low) and metadiaphiseal bone loss (DAA Grade I 91.86%; DLA Grade I 93.75%). Regarding perioperative complications, we have discovered only one periprosthetic fracture each group. Although there was no statistically significant difference, we highlighted a higher number of dislocations in the group of DLA (2 episodes vs no one). Conclusions. In this study we have shown that the DAA is an adequate surgical choice comparing with the classical DLA for FNFs treated with HHA. The analysis of our radiographic parameters and perioperative complications have not shown a significant difference between the two surgical approach. This study is limited by a purely radiographic analysis without addition of clinical parameters

Heterotopic ossification (HO) is lamellar bone formation that occurs within tissues that do not normally have properties of ossification. The pathoaetiology of HO is poorly understood. We conducted a genome wide association study to better understand the genetic architecture of HO. 891 patients of European descent (410 HO cases) following THA for primary osteoarthritis were recruited from the UK. HO was assessed from plain AP radiographs of the pelvis. Genomic DNA was extracted, genotyped using the Illumina 610 beadchip and referenced using the 1000 Genome Project panel. HO susceptibility case-control analysis and an evaluation of disease severity in those with HO was undertaken using SNPTESTv2.3.0 on>10 million variants. We tested variants most strongly associated with HO in an independent UK THA replication cohort comprising 209 cases and 211 controls. The datasets were meta-analysed using PLINK. In the discovery cohort 70 signals with an index variant at p<9×10–5 were suggestively associated with HO susceptibility. The strongest signal lay just downstream of the gene ARHGAP18 (rs59084763, effect allele frequency (EAF) 0.19, OR1.87 [1.48–2.38], p=2.48×10–8), the second strongest signal lay within the long non-coding (LNC) RNA gene CASC20 (rs11699612, EAF 0.25, OR1.73 [1.1.40–2.16, p=9.3×10–8). In the discovery cohort 73 signals with an index variant at p<9×10–5 were associated with HO severity. At replication, 12 of the leading 14 susceptibility signals showed a concordant direction of allelic effect and 5 replicated at nominal significance. Following meta-analysis, the lead replicating susceptibility signal was the CASC20 variant rs11699612 (p=2.71×10–11). We identify consistent replicating association of variation within the LNC RNA CASC20 with HO susceptibility after THA. Although the function of CASC20 is currently unknown, possible mechanisms include transcriptional, post-transcriptional and epigenetic regulation of downstream target genes. The work presented here provides new avenues for the development of novel predictive and therapeutic approaches towards HO

Summary. Indomethacin has differential effects on chondrogencic outcome depending on differentiation stage. Introduction. Heterotopic ossification (HO) is the abnormal formation of bone in soft tissues and is a frequent complication of hip replacement surgery. The standard treatment to prevent HO is administration of the NSAID indomethacin. HOs are described to develop via endochondral ossification. As it is currently unknown how indomethacin prevents HO, we aimed to define whether indomethacin might influence HO via impairing the chondrogenic phase of endochondral ossification. Materials. ATDC5, human bone marrow stem cells (hBMSCs) and rabbit periosteal agarose cultures were employed as progenitor cell models; SW1353, human articular chondrocytes and differentiated ATDC5 cells were used as matured chondrocyte cell models. All cells were cultured in the presence of (increasing) concentrations of indomethacin. The action of indomethacin was confirmed by decreased PGE. 2. levels in all experiments, and was determined by specific PGE. 2. ELISA. Gene- and protein expression analyses were employed to determine chondrogenic outcome. Results. A dose-dependent decrease in expression of Col2a1, Col10a1 and GAG content was observed when progenitor ATDC5 cells differentiating in the chondrogenic lineage were treated with increasing concentrations of indomethacin. These results were confirmed on primary hBMSCs and ex vivo periosteal agarose cultures. Even when hypertrophic differentiation of ATDC5 cells was provoked by BMP-2 (30ng/ml) the addition of indomethacin resulted in decreased hypertrophic marker expression. Interestingly, when adult chondrocytes (SW1353 and primary human articular chondrocytes) were treated with indomethacin, a clear increase in Col2a1 expression was observed. Similarly, when ATDC5 cells were differentiated for 10 days to obtain a chondrocyte phenotype and indomethacin was added from this time point onwards, low concentrations of indomethacin also resulted in increased Col2a1 expression. Conclusions. Indomethacin (dose-dependently) prevents chondrogenic and hypertrophic differentiation from progenitor cells. In addition we found thatindomethacin (in low concentrations) is able to increase the chondrogenic phenotype of maturated chondrocytes. Together, these data indicate that indomethacin has differentiation stage-dependent effects on chondrogenic differentiation and part of the HO-preventing action of indomethacin might be contributed to inhibition of chondrogenic differentiation

Systemic factors are believed to be pivotal for the development of heterotopic ossification in severely-injured patients. In this study, cell cultures of putative target cells (human fibroblastic cells, osteoblastic cells (MG-63), and bone-marrow stromal cells (hBM)) were incubated with serum from ten consecutive polytraumatised patients taken from post-traumatic day 1 to day 21 and with serum from 12 healthy control subjects.

The serum from the polytraumatised patients significantly stimulated the proliferation of fibroblasts, MG-63 and of hBM cells. The activity of alkaline phosphatase in MG-63 and hBM cells was significantly decreased when exposed to the serum of the severely-injured patient. After three weeks in 3D cell cultures, matrix production and osteogenic gene expression of hBM cells were equal in the patient and control groups. However, the serum from the polytraumatised patients significantly decreased apoptosis of hBM cells compared with the control serum (4.3% vs 19.1%, p = 0.031).

Increased proliferation of osteoblastic cells and reduced apoptosis of osteoprogenitors may be responsible for increased osteogenesis in severely-injured patients.

We have developed an animal model to examine the formation of heterotopic ossification using standardised muscular damage and implantation of a beta-tricalcium phosphate block into a hip capsulotomy wound in Wistar rats. The aim was to investigate how cells originating from drilled femoral canals and damaged muscles influence the formation of heterotopic bone. The femoral canal was either drilled or left untouched and a tricalcium phosphate block, immersed either in saline or a rhBMP-2 solution, was implanted. These implants were removed at three and 21 days after the operation and examined histologically, histomorphometrically and immunohistochemically.

Bone formation was seen in all implants in rhBMP-2-immersed, whereas in those immersed in saline the process was minimal, irrespective of drilling of the femoral canals. Bone mineralisation was somewhat greater in the absence of drilling with a mean mineralised volume to mean total volume of 18.2% (sd 4.5) versus 12.7% (sd 2.9, p < 0.019), respectively.

Our findings suggest that osteoinductive signalling is an early event in the formation of ectopic bone. If applicable to man the results indicate that careful tissue handling is more important than the prevention of the dissemination of bone cells in order to avoid heterotopic ossification.