Pathological Ossification Research Articles

PERIPROSTHETIC JOINT INFECTION AND HETEROTOPIC OSSIFICATION AFTER TOTAL HIP ARTHROPLASTY: A NARRATIVE REVIEW

This meta analysis address the relationship between infection developing after total hip arthroplasty (THA) and heterotopic ossification (HO). To identify the gaps in available knowledge, we screened for full-length peer-reviewed research articles listed in PubMed, Embase, and Web of Science over the past 20 years. The following search terms and Boolean operators were used: heterotopic ossification AND infection AND (hip replacement OR hip arthroplasty). The search resulted in the identification of as few as 14 articles describing periprosthetic joint infection (PJI) and HO after THA. Data summarized from 6 studies suitable for further meta-analysis yielded a cumulative sample size of 753 observations, with 186 recorded events of HO. The pooled RR was estimated at 2.22 (95% CI: 1.00 to 4.91, p = 0.0497), suggesting a more than twofold risk of HO compared to the group without PJI. In conclusion, there is a clear association between a higher risk of HO and PJI. Basic research findings support the hypothesis that bacterial pathogen-associated molecular patterns (PAMPs) can lead to osteogenesis through a toll-like receptor (TLR) and nuclear factor kappa B (NF-κB) pathway in the course of HO development. Together, these results suggest that HO prophylaxis should always be prescribed in PJI after THA. Moreover, during revisions following THA for presumed non-septic reasons, the presence of HO warrants consideration for infection, as there is a potential heightened risk of pathologic ossification induced by PAMPs.Keywords: heterotopic ossification; total hip arthroplasty; total hip replacement; periprosthetic joint infection; bacteriaAuthors Ł. Pulik and P. Łęgosz contributed equally to this work.

Patients with ankylosing spondylitis present a distinct CD8 T cell subset with osteogenic and cytotoxic potential

ObjectivesAnkylosing spondylitis (AS) is a chronic inflammatory rheumatic disease affecting mainly the axial skeleton. Peripheral involvement (arthritis, enthesitis and dactylitis) and extra-musculoskeletal manifestations, including uveitis, psoriasis and bowel inflammation, occur...

Impact of immune regulation and differentiation dysfunction of mesenchymal stem cells on the disease process in ankylosing spondylitis and prospective analysis of stem cell transplantation therapy.

Abstract Ankylosing spondylitis (AS) is a rheumatic bone and joint disease caused by inflammation, erosion, and pathological bone formation. The pathological features of chronic inflammation, bone destruction, and pathological ossification occur due to the disruption of the body’s immune regulation and altered bone remodeling balance. Mesenchymal stem cells (MSCs) have multidirectional differentiation potential and immunomodulatory functions and play an important role in immune regulation and bone formation. The immune regulation and osteogenic capacity of MSCs in AS are altered by factors such as genetic background, internal environment, infection, and mechanical forces that drive disease development. This review further evaluates the role of MSCs dysfunction in inflammation and pathological bone formation by analyzing the effects of the above-mentioned factors on MSCs function and also looks forward to the prospects of MSCs in treating AS, providing some ideas for an in-depth study of inflammation and ectopic ossification. Key messages

Analysis of potential key ferroptosis genes in the pathogenesis of ankylosing spondylitis by bioinformatics

AbstractBackgroundAnkylosing spondylitis (AS) is a disabling chronic inflammatory disease. Mechanisms of ferroptosis in AS remain unclear. Using bioinformatics analysis, we aimed to identify key molecules involved in ferroptosis, provide potential therapeutic targets for AS, and further explore mechanisms of ferroptosis in AS.MethodsGSE25101 was downloaded from the Gene Expression Omnibus and intersected with a ferroptosis gene dataset. The ferroptosis‐relate differentially‐expressed genes were further subjected to functional enrichment analysis, protein interaction network analysis, and gene‐miRNA interaction network analysis, from which potential key ferroptosis genes in the pathogenesis of ankylosing spondylitis were screened.ResultsA total of 20 differentially expressed genes were screened, most of which are involved in phosphoinositide 3 kinase‐Akt or mitogen‐activated protein kinase (MAPK) signaling pathways or the endoplasmic reticulum stress response. The following target genes were identified through protein‐protein interaction network analysis and screening of key modules constructed from genes associated with PI3K‐Akt and MAPK signaling pathways: TP53, PTEN, TLR4, HSPB1, DDIT3, and XBP1. In addition, PI3K‐Akt and MAPK signaling were associated with oxidative stress, which may play a role in AS pathological ossification related to ferroptosis. Only hsa‐miR‐205‐5p was found to target at least two genes by gene‐miRNA interaction network analysis.ConclusionsFuture therapeutic drug development may intervene by modulating MAPK or PI3K‐Akt signaling pathways rather than directly affecting the interleukin 17 pathway. hsa‐miR‐205‐5p may be a potential novel biomarker for AS.

Verteporfin attenuates trauma-induced heterotopic ossification of Achilles tendon by inhibiting osteogenesis and angiogenesis involving YAP/β-catenin signaling.

Heterotopic ossification occurs as a pathological ossification condition characterized by ectopic bone formation within soft tissues following trauma. Vascularization has long been established to fuel skeletal ossification during tissue development and regeneration. However, the feasibility of vascularization as a target of heterotopic ossification prevention remained to be further clarified. Here, we aimed to identify whether verteporfin as a widely used FDA-approved anti-vascularization drug could effectively inhibit trauma-induced heterotopic ossification formation. In the current study, we found that verteporfin not only dose dependently inhibited the angiogenic activity of human umbilical vein endothelial cells (HUVECs) but also the osteogenic differentiation of tendon stem cells (TDSCs). Moreover, YAP/β-catenin signaling axis was downregulated by the verteporfin. Application of lithium chloride, an agonist of β-catenin, recovered TDSCs osteogenesis and HUVECs angiogenesis that was inhibited by verteporfin. In vivo, verteporfin attenuated heterotopic ossification formation by decelerating osteogenesis and the vessels densely associated with osteoprogenitors formation, which could also be readily reversed by lithium chloride, as revealed by histological analysis and Micro-CT scan in a murine burn/tenotomy model. Collectively, this study confirmed the therapeutic effect of verteporfin on angiogenesis and osteogenesis in trauma-induced heterotopic ossification. Our study sheds light on the anti-vascularization strategy with verteporfin as a candidate treatment for heterotopic ossification prevention.

Otosclerosis - analysis of factors influencing the improvement of hearing after surgical treatment.

<b>Introduction:</b> Otosclerosis is a disease that occurs only in humans, in the course of which there are foci of pathological ossification in the temporal bone. The etiology of the dise ase is not fully understood. Treatment of the conductive component of hearing loss is surgical. The results of the treatment are influenced by factors related to the surgery, the local condition of the middle ear and the function of the inner ear. </br></br> <b>Aim:</b> The aim of the study is to identify factors influencing the improvement of hearing in patients treated surgically due to otosclerosis. </br></br> <b>Material and methods:</b> The study included patients who underwent otosclerosis for the first middle ear surgery and under-went stapedotomy. Considering the factors that may affect the outcome of surgical treatment, the patients qualified for the analysis were divided into subgroups. All patients underwent a medical history and physical examination of otorhinolaryn-gology and a complete set of audiological examinations. </br></br> <b> Results:</b> A statistically significant reduction in cochlear reserve was observed in all patients after stapedotomy. The be-neficial effect of the performed treatment on the improvement of threshold values of bone conduction in patients with mild sensorineural hearing loss was also confirmed. Intraoperative removal of adhesions present in the tympanic cavity significantly improved hearing in terms of bone conduction values, especially at 500 Hz. </br></br> <b> Conclusions:</b> (1) The conducted study confirmed the influence of factors related to the local condition of the middle ear lining on the final result of otosclerosis surgery; (2) Audiometric markers of cochlear otosclerosis, observed before surgical treat-ment, are an unfavorable factor in the improvement of hearing after the performed treatment.

Effects of vitamin D with or without calcium on pathological ossification: A retrospective clinical study.

Vitamin D protects against the development and severity of several rheumatic diseases. However, the effect of vitamin D on the pathological ossification associated with rheumatic diseases remains unknown. The present retrospective study analyzed the clinical outcomes of vitamin D without calcium compared with vitamin D with calcium on pathological ossification in joints and ligaments. Data were collected from patients who were diagnosed with osteoarthritis, rheumatoid arthritis or spondylarthritis, and the presence of pathological ossification in joints or ligaments was confirmed by X-ray, computed tomography or magnetic resonance imaging examination. A total of 2,965 patients aged 18-75 years old were included, among who, 1,725 were included in the vitamin D alone group and 1,240 in the vitamin D with calcium group. Vitamin D was administered intramuscularly (300,000 IU) once every 7-10 days, 4-6 times in total. Patients who ingested an oral calcium supplement (1,000 mg/day; ≥5 days/week) were considered the vitamin D with calcium group. The clinical outcome was evaluated based on the imaging changes of pathological ossification, which were classified as alleviation, aggravation and unchanged. The bone mineral density (BMD) was determined, and the calcium concentration in the serum and urine was measured. The results revealed that vitamin D alone alleviated pathological ossification, while vitamin D combined with calcium aggravated pathological ossification in the majority of patients (P<0.0001) independent of disease type and patient age. BMD measurements demonstrated a decreasing trend in the vitamin D alone group, whereas they exhibited an increasing trend in the vitamin D combined with calcium group. The urine calcium concentration increased after vitamin D treatment alone. Therefore, it was concluded that vitamin D exerted both pro-resorptive and anti-resorptive actions on pathological ossification. The bidirectional action of vitamin D on bone metabolism may depend on exogenous calcium supplementation.

Participation of transcriptional factor ZBTB16 in the processes of physiological bone tissue formation and in pathological calcification of the aortic valve

Degenerative calcific aortic valve stenosis is the most common type of heart valve disease in the Western world. Patients with severe stenosis are associated with 50 percent chance of mortality within two years in the absence of intervention. Surgical interventions are the only treatment method for severe calcific aortic valve stenosis to date. Pharmacological approaches have so far failed to affect the course of the disease. Thus, there is an urgent need to develop novel treatment strategies that could slow down the progression of the stenosis. ZBTB16 is a zinc finger protein with N-term BTB/POZ domain (protein-protein interaction motif) and 9 zinc finger domains (DNA binding motif) in C-term. There is growing evidence proving the participation of ZBTB16 in skeletal development. ZBTB16 has been shown to play a role in the specification of limb and axial skeleton patterning. Moreover, the expression of ZBTB16 is increased in patients with ectopic bone formation. Nowadays, the evidence supports that the mechanisms that play key roles in the formation of bone tissue are similar to the processes occurring during the development of ectopic ossification of the aortic valve. Thus, it can be assumed that ZBTB16 is heavily involved in osteogenic transformation in the aortic valve. Understanding similarities and differences in the mechanisms that mediate osteogenic differentiation of stem cells during bone formation and pathological ossification of tissues can help to find the ways to control the osteogenic differentiation in the human body. The aim of this review is to summarize data on the role of ZBTB16 and its products in the regulation of differentiation and proliferation of cells involved in osteogenesis and in the development of ectopic calcification of the aortic valve. The study of the dynamic changes of ZBTB16 expression in aortic valve calcification is a new and relevant study field.

Trabecular Bone Score (TBS) in Patients with Early Ankylosing Spondylitis-Limited Utility.

Purpose: Ankylosing spondylitis (AS) not only results in pathological ossification of the spine, but can also be associated with osteoporosis. Due to the presence of syndesmophytes and possible involvement of the hip joints, classical dual X-ray absorptiometry (DXA) is of limited use in patients with advanced stages of AS. Trabecular bone score (TBS) is a method complementary to DXA, providing additional information about bone microarchitecture. There is a growing body of evidence for the usefulness of TBS in AS patients. The aim of this study was to assess the clinical utility of TBS in patients with AS. Methods: Patients with AS underwent DXA with additional TBS assessment. A cross-sectional analysis of the frequency of osteoporosis and bone microarchitecture deterioration and their association with patients’ characteristics was done. Results: A total of 51 male patients, mean age 40.7 years, were enrolled. Osteoporosis was diagnosed in seven patients (13.7%). Lumbar bone mineral density (BMD) was higher (p < 0.001) than femoral BMD, indicating abnormal BMD readings in the spine caused by syndesmophytes. Patients with DXA-diagnosed osteoporosis had lower TBS (p = 0.03) and TBS T-score (p = 0.043) values compared to patients without osteoporosis. However, disturbed bone microarchitecture (TBS < 1.23) was present in only three patients (5.9%). None of the patients had a history of an osteoporotic fracture. A lower TBS T-score (p = 0.032) was demonstrated in patients with sacroiliitis grade 4 than in patients with sacroiliitis grade 2, with no significant differences in BMD and T-score values. Conclusion: Among patients with early AS, the clinical utility of TBS is limited—it does not add value to DXA.

Tail suspension delays ectopic ossification in proteoglycan-induced ankylosing spondylitis in mice via miR-103/DKK1.

Ankylosing spondylitis (AS), characterized by inflammatory lesions and osteophyte formation, is a common immune rheumatic disease affecting the sacroiliac and axial joints. A high-intensity mechanical load is known to accelerate the heterotopic ossification associated with enthesitis in AS. Thus, the present study explored whether decreased mechanical load could delay the heterotopic ossification in AS. First, 24-week-old female BALB/c mice were induced with proteoglycan (PG) to establish an AS model. The AS-induced pathological and bone morphological changes of the sacroiliac joint were confirmed by hematoxylin and eosin staining and microCT analysis, respectively. Subsequently, the mice were treated with interventions of different mechanical loads. Using reverse transcription-quantitative PCR, it was revealed that expression levels of the osteogenesis-related genes bone morphogenetic protein-2, runt-related transcription factor 2 and osteocalcin were significantly reduced in sacroiliac bone tissue after intervention with a reduced mechanical load. The level of mechanosensory microRNA (miR)-103 increased in response to reduced mechanical loads. Consistently, in groups with reduced mechanical load, proteins with mechanical functions, including ρ-associated coiled-coil-containing protein kinase 1 (ROCK1), phosphorylated (p)-Erk1/2 and β-catenin, were reduced compared with the PG control. A dual-luciferase assay verified that miR-103 binds to the 3'-untranslated region end of Rock1 mRNA, thus negatively regulating the activity of Rock1 and affecting pathological ossification during AS. However, immunohistochemical staining indicated that the expression of dickkopf Wnt signaling pathway inhibitor 1, an inhibitor of the Wnt/β-catenin pathway, was increased in sacroiliac tissues. The results indicated that tail suspension decreased the mechanical load, thus reducing the bone formation in AS mice. Furthermore, tail suspension could inhibit the activation of mechanical kinase ROCK1 and p-Erk1/2 in the MAPK signaling pathway by upregulating miR-103, thereby inhibiting the classical osteogenesis-related Wnt/β-catenin pathway in AS. In summary, the present study uncovered the ameliorative effect of suspension on AS and its therapeutic potential for AS.

Local injection of a hexametaphosphate formulation reduces heterotopic ossification in vivo.

Heterotopic ossification (HO), the pathological formation of ectopic bone, is a debilitating condition which can cause chronic pain, limit joint movement, and prevent prosthetic limb fitting. The prevalence of this condition has risen in the military population, due to increased survivorship following blast injuries. Current prophylaxes, which aim to target the complex upstream biological pathways, are inconsistently effective ​and have a range of side-effects that make them unsuitable for combat-injured personnel. As such, many patients must undergo further surgery to remove the formed ectopic bone. In this study, a non-toxic, U.S. Food and Drug Administration (FDA) -approved calcium chelator, hexametaphosphate (HMP), is explored as a novel treatment paradigm for this condition, which targets the chemical, rather that biological, ​bone formation pathways. This approach allows not only prevention of pathological bone formation ​but also uniquely facilitates reversal, which current drugs cannot achieve. Targeted, minimally invasive delivery is achieved by loading HMP into an injectable colloidal alginate. These formulations significantly reduce ​the length of the ectopic bone formed in a rodent model of HO, with no effect on the adjacent skeletal bone. This study demonstrates the potential of localized dissolution as a new treatment ​and an alternative to surgery ​for pathological ossification and calcification conditions.

Labyrinthitis and Labyrinthitis Ossificans - A case report and review of the literature.

Labyrinthitis most commonly results from an infectious and less commonly from an inflammatory process of the inner ear, but it can be associated with temporal bone trauma, hemorrhage, or tumor. This inflammation (regardless of the etiology) disrupts the transmission of sensory information from the ear to the brain. Labyrinthitis ossificans is the pathological ossification of the membranous labyrinthine spaces in response to an insult to the inner ear involving membranous labyrinth or the endosteum of the otic capsule. Herein, we present a case of a 67-year-old female with progressive bilateral sensorineural hearing loss and positional vertigo.

Insulin Enhances the In Vitro Osteogenic Capacity of Flexor Tendon-Derived Progenitor Cells.

There is increased incidence of tendon disorders and decreased tendon healing capacity in people with diabetes mellitus (DM). Recent studies have also suggested pathological ossification in repair tendon of people with DM. Therefore, the objective of this study is to investigate the effects of insulin supplementation, an important pathophysiologic stimulus of DM, on tendon progenitor cell (TPC) proliferation and in vitro osteogenic capacity. Passage 3 TPCs were isolated from collagenase-digested, equine superficial digital flexor tendons. TPC proliferation was measured via MTT assay after 3 days of monolayer culture in medium supplemented with 0, 0.007, 0.07, and 0.7 nM insulin. In vitro osteogenic capacity of TPCs (Alizarin Red staining, osteogenic mRNA expression, and alkaline phosphatase bioactivity) was assessed with 0, 0.07, and 0.7 nM insulin-supplemented osteogenic induction medium. Insulin (0.7 nM) significantly increased TPC proliferation after 3 days of monolayer culture. Alizarin Red staining of insulin-treated TPC osteogenic cultures demonstrated robust cell aggregation and mineralized matrix secretion, in a dose-dependent manner. Runx2, alkaline phosphatase, and Osteonectin mRNA and alkaline phosphatase bioactivity of insulin-treated TPC cultures were significantly higher at day 14 of osteogenesis compared to untreated controls. Addition of picropodophyllin (PPP), a selective IGF-I receptor inhibitor, mitigated the increased osteogenic capacity of TPCs, indicating that IGF-I signaling plays an important role. Our findings indicate that hyperinsulinemia may alter TPC phenotype and subsequently impact the quality of repair tendon tissue.

Arthroscopic Resection of Symptomatic Bennett Lesions

Bennett lesions, also known as “thrower's exostosis” of the shoulder, involve ossification of the posteroinferior glenoid and are not uncommon in overhead throwing athletes. The literature surrounding the optimal operative management of the symptomatic Bennett lesion is limited. The purpose of this article is to describe the arthroscopic surgical technique for the visualization and excision of the symptomatic extra-articular pathologic ossification involving the posteroinferior glenoid. Because many surgeons may not be familiar with this problem or procedure, we present a straightforward method that allows for identification and excision of the exostosis through an arthroscopic posterior arthrotomy.

MiR-124 regulates osteoblast differentiation through GSK-3β in ankylosing spondylitis.

Ankylosing spondylitis (AS) is a spastic and spinal joint disease with the characteristic of pathological ossification. Bioinformatics analysis demonstrated that there is a complementary binding site between microRNA-124 (miR-124) and the 3'-UTR of glycogen synthase kinase-3β (GSK-3β) mRNA. We aimed to investigate the role of miR-124 in regulating GSK-3β expression, Wnt/β-catenin pathway activity, and osteoblast differentiation of spinal ligament fibroblasts. The ligament tissues of AS and the femoral neck fracture patients were collected. MiR-124 and GSK-3β mRNA expressions were detected by using quantitative Real-time PCR (qRT-PCR). GSK-3β and β-catenin protein expressions were detected by using Western blot. Ligament fibroblasts were isolated and induced to differentiate into osteoblasts. Alizarin red S staining (ARS) was used to identify osteoblast differentiation. Expressions of miR-124, GSK-3β, β-catenin, Osterix, and runt-related transcription factor 2 (RUNX2) were detected during differentiation. The cells were divided into two groups as agomiR-normal control (NC) transfection group and agomir miR-124 transfection group. Alkaline phosphatase (ALP) activity and Alizarin Red S staining were detected. MiR-124 and β-catenin expressions in the ligament of AS patients increased, while GSK-3β level reduced compared with control. MiR-124, β-catenin, Osterix, and RUNX2 expressions gradually elevated, whereas GSK-3β level gradually declined following increased osteoblasts differentiation. Antagomir miR-124 transfection significantly up-regulated the expression of GSK-3β in osteoblast differentiation, significantly decreased the expression of β-catenin, Osterix, and RUNX2, and significantly inhibited osteoblast differentiation. MiR-124 decreased and GSK-3β elevated in AS ligament tissue. Down-regulation of miR-124 expression enhanced GSK-3β expression, weakened Wnt/β-catenin pathway activity, and inhibited the differentiation of ligament fibroblasts into osteoblasts.

Zoning laminectomy for the treatment of ossification of thoracic ligamentum flavum

Objective To investigate the and effect of zoning laminectomy for the ossification of thoracic ligamentum flavum. Methods From November 2011 to December 2014, 34 patients (15 males, 19 females; 41-76 years old, average 55.0±8.1) with ossification of thoracic ligamentum flavum (OLF) were treated by zoning laminectomy. The course of disease ranged from 1 month to 123 months (average 16.5 months). According to the anatomical characteristics and the pathological ossification process of the thoracic ligamentum flavum, we proposed the concept of zoning, which divided each segmental thoracic OLF into three zones: safety zone, middle and risk zone. From the features of anatomy of LF and process of OLF development, we found there is no or less cerebrospinal fluid between spinal cord and the tip of each ossified nodular masses in severe OLF, any procedures using instruments in this area have the potential to cause irreversible spinal cord injury, we defined this area as risk zone, the null area of each lamina and lateral and dorsal side of nodular masses as safety zone, and the other area as middle zone. From safety to risk the spinal canal decreased gradually, different zone needs different surgical strategy: This surgical procedure first removed the null area of superior and inferior lamina and dorsal side of each segmental OLF. Next, partially or totally resected the middle zone, exploring the lateral side of nodular masses, and the risk was exposed and isolated. Finally, dissected the lateral side of nodular masses, and then the risk was floated and resected with a directly decompressing the spinal cord. Preoperative and postoperative modified Japanese Orthopedic Association (JOA) score and neurologic functional recovery ratio were used to evaluate the surgical outcomes. Results Of the total 83 decompressed OLF segments, 5 (6.0%) located in the upper thoracic spine (T1-T4), 8 (9.6%) in the midthoracic spine (T5-T8), and 70 (84.4%) in the lower thoracic spine (T9-L 1). The followed up ranged from 4 to 40 months, with an average of 21.7±9.9 months. The mean JOA score increased significant from 5.3±2.0 preoperatively to 8.8±1.8 at the final follow-up (t=11.566, P=0.001). Postoperative average JOA neurologic functional recovery rates were 63.2%±24.7%, including excellent in 15 cases, good in 11 cases and fair in 8 cases. The excellent and good rate was 76.5%. Twelve cases had transient CSF leakage because of dural defect. The dural defect was only treated by tightly suturing the paraspinal muscles, the subcutaneous tissue, and the skin layers. The CSF leakage lasted for 6 to 8 days after operation. Two cases with wound infection were treated with debridement and antibiotics and healed completely. One case with thoracic spinal cord transient incomplete paralysis due to a post-operative epidural hematoma was treated with an emergency operation and got recovered neurological function. Conclusion Zoning laminectomy has the advantages of safe manipulation and thorough decompression, which is an effective choice for the surgical treatment of thoracic OLF. Key words: Thoracic vertebrae; Ligamentum flavum; Ossification, heterotopic; Treatment outcome

In Vitro and In Vivo Osteogenesis of Human Orbicularis Oculi Muscle-Derived Stem Cells.

Cell-based therapies for treating bone defects require a source of stem cells with osteogenic potential. There is evidence from pathologic ossification within muscles that human skeletal muscles contain osteogenic progenitor cells. However, muscle samples are usually acquired through a traumatic biopsy procedure which causes pain and morbidity to the donor. Herein, we identified a new alternative source of skeletal muscle stem cells (SMSCs) without conferring morbidity to donors. Adherent cells isolated from human orbicularis oculi muscle (OOM) fragments, which are currently discarded during ophthalmic cosmetic surgeries, were obtained using a two-step plating method. The cell growth kinetics, immunophenotype and capabilities of in vitro multilineage differentiation were evaluated respectively. Moreover, the osteogenically-induced cells were transduced with GFP gene, loaded onto the porous β-tricalcium phosphate (β-TCP) bioceramics, and transplanted into the subcutaneous site of athymic mice. Ectopic bone formation was assessed and the cell fate in vivo was detected. OOM-derived cells were fibroblastic in shape, clonogenic in growth, and displayed phenotypic and behavioral characteristics similar to SMSCs. In particular, these cells could be induced into osteoblasts in vitro evidenced by the extracellular matrix calcification and enhanced alkaline phosphatase (ALP) activity and osteocalcin (OCN) production. New bone formation was found in the cell-loaded bioceramics 6weeks after implantation. By using the GFP-labeling technique, these muscle cells were detected to participate in the process of ectopic osteogenesis in vivo. Our data suggest that human OOM tissue is a valuable and noninvasive resource for osteoprogenitor cells to be used in bone repair and regeneration.

Defining the Balance between Regeneration and Pathological Ossification in Skeletal Muscle Following Traumatic Injury.

Heterotopic ossification (HO) is characterized by the formation of bone at atypical sites. This type of ectopic bone formation is most prominent in skeletal muscle, most frequently resulting as a consequence of physical trauma and associated with aberrant tissue regeneration. The condition is debilitating, reducing a patient's range of motion and potentially causing severe pathologies resulting from nerve and vascular compression. Despite efforts to understand the pathological processes governing HO, there remains a lack of consensus regarding the micro-environmental conditions conducive to its formation, and attempting to define the balance between muscle regeneration and pathological ossification remains complex. The development of HO is thought to be related to a complex interplay between factors released both locally and systemically in response to trauma. It develops as skeletal muscle undergoes significant repair and regeneration, and is likely to result from the misdirected differentiation of endogenous or systemically derived progenitors in response to biochemical and/or environmental cues. The process can be sequentially delineated by the presence of inflammation, tissue breakdown, adipogenesis, hypoxia, neo-vasculogenesis, chondrogenesis and ossification. However, exactly how each of these stages contributes to the formation of HO is at present not well understood. Our previous review examined the cellular contribution to HO. Therefore, the principal aim of this review will be to comprehensively outline changes in the local tissue micro-environment following trauma, and identify how these changes can alter the balance between skeletal muscle regeneration and ectopic ossification. An understanding of the mechanisms governing this condition is required for the development and advancement of HO prophylaxis and treatment, and may even hold the key to unlocking novel methods for engineering hard tissues.

PDGF is a potent initiator of bone formation in a tissue engineered model of pathological ossification.

Heterotopic ossification (HO) is a debilitating condition defined by the rapid formation of bone in soft tissues. What makes HO fascinating is first the rate at which bone is deposited, and second the fact that this bone is structurally and compositionally similar to that of a healthy adult. If the mechanisms governing HO are understood, they have the potential to be exploited for the development of potent osteoinductive therapies. With this aim, a tissue‐engineered skeletal muscle was used model to better understand the role of inflammation on this debilitating phenomenon. It was shown that myoblasts could be divided into two distinct populations: myogenic cells and undifferentiated ‘reserve’ cells. Gene expression analysis of myogenic and osteoregulatory markers confirmed that ‘reserve’ cells were primed for osteogenic differentiation but had a reduced capacity for myogenesis. Osteogenic differentiation was significantly enhanced in the presence of platelet‐derived growth factor (PDGF)‐BB and bone morphogenetic protein 2 (BMP2), and correlated with conversion to a Sca‐1+/CD73+ phenotype. Alizarin red staining showed that PDGF‐BB promoted significantly more mineral deposition than BMP2. Finally, it was shown that PDGF‐induced mineralization was blocked in the presence of the pro‐inflammatory cytokines tumour necrosis factor‐α and interleukin 1. In conclusion, the present study identified that PDGF‐BB is a potent osteoinductive factor in a model of tissue‐engineered skeletal muscle, and that the osteogenic capacity of this protein was modulated in the presence of pro‐inflammatory cytokines. These findings reveal a possible mechanism by which HO develops following trauma. Importantly, these findings have implications for the induction and control of bone formation for regenerative medicine. © 2016 The Authors Journal of Tissue Engineering and Regenerative Medicine Published by John Wiley & Sons Ltd.

Endochondral Growth Defect and Deployment of Transient Chondrocyte Behaviors Underlie Osteoarthritis Onset in a Natural Murine Model.

ObjectiveTo explore whether aberrant transient chondrocyte behaviors occur in the joints of STR/Ort mice (which spontaneously develop osteoarthritis [OA]) and whether they are attributable to an endochondral growth defect.MethodsKnee joints from STR/Ort mice with advanced OA and age‐matched CBA (control) mice were examined by Affymetrix microarray profiling, multiplex polymerase chain reaction (PCR) analysis, and immunohistochemical labeling of endochondral markers, including sclerostin and MEPE. The endochondral phenotype of STR/Ort mice was analyzed by histologic examination, micro–computed tomography, and ex vivo organ culture. A novel protocol for quantifying bony bridges across the murine epiphysis (growth plate fusion) using synchrotron x‐ray computed microtomography was developed and applied.ResultsMeta‐analysis of transcription profiles showed significant elevation in functions linked with endochondral ossification in STR/Ort mice (compared to CBA mice; P < 0.05). Consistent with this, immunolabeling revealed increased matrix metalloproteinase 13 (MMP‐13) and type X collagen expression in STR/Ort mouse joints, and multiplex quantitative reverse transcriptase–PCR showed differential expression of known mineralization regulators, suggesting an inherent chondrocyte defect. Support for the notion of an endochondral defect included accelerated growth, increased zone of growth plate proliferative chondrocytes (P < 0.05), and widespread type X collagen/MMP‐13 labeling beyond the expected hypertrophic zone distribution. OA development involved concomitant focal suppression of sclerostin/MEPE in STR/Ort mice. Our novel synchrotron radiation microtomography method showed increased numbers (P < 0.001) and mean areal growth plate bridge densities (P < 0.01) in young and aged STR/Ort mice compared to age‐matched CBA mice.ConclusionTaken together, our data support the notion of an inherent endochondral defect that is linked to growth dynamics and subject to regulation by the MEPE/sclerostin axis and may represent an underlying mechanism of pathologic ossification in OA.