Fetuin A is down-regulated in rats during heterotopic ossification after Achilles tenotomy

We investigated the role of fetuin A during heterotopic ossification (HO) in rats following Achilles tenotomy. We performed a right midpoint Achilles tenotomy on 24 rats. At 5 and 10 h after surgery, we investigated the formation of ectopic bone using X-ray and histological examination. We evaluated the mRNA level of fetuin A using real-time PCR. Presence of fetuin A in the Achilles tendon was assessed by immunohistochemical staining. We also measured the serum concentration of fetuin A using enzyme linked immunosorbent assay (ELISA). The expression of fetuin A was significantly decreased in both the liver and Achilles tendon during HO. ELISA showed a small amount of fetuin A in blood throughout the development of HO. Immunohistochemical staining showed that fetuin A was abundant in the ectopic bone. Fetuin A appears to be involved in the formation of ectopic bone induced by Achilles tenotomy, and a deficiency of fetuin A plays a role in the development of HO.     

Inhibition of IL-17 prevents the progression of traumatic heterotopic ossification

Traumatic heterotopic ossification (HO) is the abnormal formation of bone in soft tissues as a consequence of injury. However, the pathological mechanisms leading to traumatic HO remain unknown. Here, we report that aberrant expression of IL-17 promotes traumatic HO formation by activating β-catenin signalling in mouse model. We found that elevated IL-17 and β-catenin levels are correlated with a high degree of HO formation in specimens from patients and HO animals. We also show that IL-17 initiates and promotes HO progression in mice. Local injection of an IL-17 neutralizing antibody attenuates ectopic bone formation in a traumatic mouse model. IL-17 enhances the osteoblastic differentiation of mesenchymal stem cells (MSCs) by activating β-catenin signalling. Moreover, inhibition of IL-17R or β-catenin signalling by neutralizing antibodies or drugs prevents the osteogenic differentiation of isolated MSCs and decreases HO formation in mouse models. Together, our study identifies a novel role for active IL-17 as the inducer and promoter of ectopic bone formation and suggests that IL-17 inhibition might be a potential therapeutic target in traumatic HO.     

Berberine and aspirin prevent traumatic heterotopic ossification by inhibition of BMP signalling pathway and osteogenic differentiation

Heterotopic ossification (HO) is a pathological process that often occurs in soft tissues following severe trauma. There is no effective therapy for HO. The BMP signalling pathway plays an essential role in the pathogenesis of HO. Our previous study showed that AMPK negatively regulates the BMP signalling pathway and osteogenic differentiation. The present study aims to study the effect of two AMPK activators berberine and aspirin on osteogenic differentiation and HO induced by traumatic injury. The effects of two AMPK activators, berberine and aspirin, on BMP signalling and osteogenic differentiation were measured by western blot, ALP and Alizarin red S staining in C3H10T1/2 cells. A mouse model with Achilles tenotomy was employed to assess the effects of berberine and aspirin on HO using μCT and histological analysis. First, our study showed that berberine and aspirin inhibited phosphorylation of Smad1/5 induced by BMP6 and the inhibition was attributed to the down-regulation of ALK2 expression. Second, the combination of berberine and aspirin yielded more potent effects on BMP signalling. Third, we further found that there was an additive effect of berberine and aspirin combination on osteogenic differentiation. Finally, we found that berberine and aspirin blocked trauma-induced ectopic bone formation in mice, which may be through suppression of phosphorylation of Smad1/5 in injured tissues. Collectively, these findings indicate that berberine and aspirin inhibit osteogenic differentiation in C3H10T1/2 cells and traumatic HO in mice, possibly through the down-regulation of the BMP signalling pathway. Our study sheds a light on prevention and treatment of traumatic HO using AMPK pharmacological activators berberine and aspirin.     

Chondrocyte phenotype and ectopic ossification in collagenase-induced tendon degeneration

We report chondrocyte phenotype and ectopic ossification in a collagenase-induced patellar tendon injury model. Collagenase or saline was injected intratendinously in one limb. The patella tendon was harvested for assessment at different times. There was an increase in cellularity, vascularity, and loss of matrix organization with time after collagenase injection. The tendon did not heal histologically until week 32. Ectopic mineralization as indicated by von Kossa staining started from week 8. Tendon calcification was mediated by endochondral ossification, as shown by expression of type X collagen. viva CT imaging and polarization microscopy showed characteristic bony porous structures and collagen fiber arrangement, respectively, in the calcific regions. Marrow-like cells and blood vessels were observed inside calcific deposits. Chondrocyte-like cells as indicated by morphology, expression of type II collagen, and sox 9 were seen around and embedded inside the calcific deposits. Fibroblast-like cells expressed type II collagen and sox 9 at earlier times, suggesting that erroneous differentiation of healing tendon fibroblasts may account for failed healing and ossification in collagenase-induced tendon degeneration. Because this animal model replicates key histopathological changes in calcific tendinopathy, it can be used as a model for the study of its pathogenesis at the patellar tendon.     

Patellar tendon and hamstring moment-arms and cross-sectional area in patients with anterior cruciate ligament reconstruction and controls

The purpose of this study was to examine the moment-arm and cross-sectional area (CSA) of the patellar tendon (PT) and the hamstrings after anterior cruciate ligament (ACL) reconstruction. The right knee of five males who underwent ACL reconstruction with a PT graft and five age-matched controls was scanned using magnetic resonance image scans. Based on three-dimensional (3D) solids of the PT, CSAs and moment-arms of semitendinous (ST), biceps femoris (BF) long head and semimembranosus (SM) were estimated. Analysis of variance indicated no significant group differences in muscle moment-arms (p>0.05). 3D moment-arms of PT, ST and BF were significantly lower than the corresponding 2D values (p < 0.05). The ACL group displayed a significantly higher maximum BF CSA, a lower ST CSA (p < 0.05) but similar PT and SM CSAs compared with controls. It is concluded that any alterations in PT properties 1 year after harvesting do not affect knee muscle moment-arms compared with age-matched controls. Moment-arm estimation differed between 3D and 2D data, although it did not affect comparisons between ACL reconstruction group and controls. Design of rehabilitation programmes should take into consideration a potential alteration in hamstring morphology following surgery with a PT graft.     

Neurotrophin‐3 acts on the endothelial‐mesenchymal transition of heterotopic ossification in rats

Despite the fact that extensive studies have focused on heterotopic ossification (HO), its molecular mechanism remains unclear. The endothelial‐mesenchymal transition (EndMT), which may be partially modulated by neuroendocrine cytokines is thought to play a major role in HO. Neurotrophin‐3 (NT‐3), which has neuroendocrine characteristics is believed to promote skeletal remodeling. Herein, we suggest that that NT‐3 may promote HO formation through regulation of EndMT. Here, we used an in vivo model of HO and an in vitro model of EndMT induction to elucidate the effect and underlying mechanism of NT‐3 on EndMT in HO. Our results showed that heterotopic bone and cartilage arose from EndMT and NT‐3 promoted HO formation in vivo. Our in vitro results showed that NT‐3 up‐regulated mesenchymal markers (FSP‐1, α‐SMA and N‐cadherin) and mesenchymal stem cell (MSC) markers (STRO‐1, CD44 and CD90) and down‐regulated endothelial markers (Tie‐1, VE‐cadherin and CD31). Moreover, NT‐3 enhanced a chondrogenesis marker (Sox9) and osteogenesis markers (OCN and Runx2) via activation of EndMT. However, both EndMT specific inhibitor and tropomyosin‐related kinase C (TrkC) specific inhibitor rescued NT‐3‐induced HO formation and EndMT induction in vivo and in vitro. In conclusion, our findings demonstrate that NT‐3 promotes HO formation via modulation of EndMT both in vivo and in vitro, which offers a new potential target for the prevention and therapy of HO.     

Proteomic differences between native and tissue‐engineered tendon and ligament

Tendons and ligaments (T/Ls) play key roles in the musculoskeletal system, but they are susceptible to traumatic or age‐related rupture, leading to severe morbidity as well as increased susceptibility to degenerative joint diseases such as osteoarthritis. Tissue engineering represents an attractive therapeutic approach to treating T/L injury but it is hampered by our poor understanding of the defining characteristics of the two tissues. The present study aimed to determine differences in the proteomic profile between native T/Ls and tissue engineered (TE) T/L constructs. The canine long digital extensor tendon and anterior cruciate ligament were analyzed along with 3D TE fibrin‐based constructs created from their cells. Native tendon and ligament differed in their content of key structural proteins, with the ligament being more abundant in fibrocartilaginous proteins. 3D T/L TE constructs contained less extracellular matrix (ECM) proteins and had a greater proportion of cellular‐associated proteins than native tissue, corresponding to their low collagen and high DNA content. Constructs were able to recapitulate native T/L tissue characteristics particularly with regard to ECM proteins. However, 3D T/L TE constructs had similar ECM and cellular protein compositions indicating that cell source may not be an important factor for T/L tissue engineering.     

In vivo assessment of the interaction of patellar tendon tibial shaft angle and anterior cruciate ligament elongation during flexion

A potential cause of non-contact anterior cruciate ligament (ACL) injury is landing on an extended knee. In line with this hypothesis, studies have shown that the ACL is elongated with decreasing knee flexion angle. Furthermore, at low flexion angles the patellar tendon is oriented to increase the anterior shear component of force acting on the tibia. This indicates that knee extension represents a position in which the ACL is taut, and thus may have an increased propensity for injury, particularly in the presence of excessive force acting via the patellar tendon. However, there is very little in vivo data to describe how patellar tendon orientation and ACL elongation interact during flexion. Therefore, this study measured the patellar tendon tibial shaft angle (indicative of the relative magnitude of the shear component of force acting via the patellar tendon) and ACL length in vivo as subjects performed a quasi-static lunge at varying knee flexion angles. Spearman rho rank correlations within each individual revealed that flexion angles were inversely correlated to both ACL length (rho = −0.94 ± 0.07, mean ± standard deviation, p < 0.05) and patellar tendon tibial shaft angle (rho = −0.99 ± 0.01, p < 0.05). These findings indicate that when the knee is extended, the ACL is both elongated and the patellar tendon tibial shaft angle is increased, resulting in a relative increase in anterior shear force on the tibia acting via the patellar tendon. Therefore, these data support the hypothesis that landing with the knee in extension is a high risk scenario for ACL injury.     

A biochemical strategy for simulation of endochondral and intramembranous ossification

Following the assumption that parathyroid hormone related protein and Indian hedgehog form a biochemical regulatory loop for the endochondral process and bone morphogenetic protein 2 and Noggin in the intramembranous process, this paper implements these regulatory mechanisms. For this purpose, we use a set of reaction–diffusion equations that are widely used in morphogenesis, in which biochemical factors are assumed to be secreted by precursor cells, mesenchymal cells and chondrocytes, in endochondral and intramembranous ossification, respectively. The solution leads to the so-called Turing patterns, which represent these processes of ossification in a very approximate way.     

Tendon and ligament: development, repair and disease

Tendons and ligaments (T/L) are very similar fibrous tissues that respectively connect muscle to bone and bone to bone. They are comprised of fibroblasts that produce large amounts of extra-cellular matrix, resulting in a dense and hypocellular structure. The complex molecular organization of T/L, together with high water content, are responsible for their viscoelastic properties, hence insuring their mechanical function. We will first review recent work on tendon embryology and discuss ligament formation, which has been less documented. We will next summarize our current knowledge of T/L molecular architecture, alterations of which are a major cause for disease. We will finally focus on T/L repair after injury and on genetic diseases responsible for T/L defects.     

Metformin attenuates trauma‐induced heterotopic ossification via inhibition of Bone Morphogenetic Protein signalling

AMP‐activated protein kinase (AMPK) is an intracellular sensor of energy homoeostasis that is activated under energy stress and suppressed in energy surplus. AMPK activation leads to inhibition of anabolic processes that consume ATP. Osteogenic differentiation is a process that highly demands ATP during which AMPK is inhibited. The bone morphogenetic proteins (BMPs) signalling pathway plays an essential role in osteogenic differentiation. The present study examines the inhibitory effect of metformin on BMP signalling, osteogenic differentiation and trauma‐induced heterotopic ossification. Our results showed that metformin inhibited Smad1/5 phosphorylation induced by BMP6 in osteoblast MC3T3‐E1 cells, concurrent with up‐regulation of Smad6, and this effect was attenuated by knockdown of Smad6. Furthermore, we found that metformin suppressed ALP activity and mineralization of the cells, an event that was attenuated by the dominant negative mutant of AMPK and mimicked by its constitutively active mutant. Finally, administration of metformin prevented the trauma‐induced heterotopic ossification in mice. In conjuncture, AMPK activity and Smad6 and Smurf1 expression were enhanced by metformin treatment in the muscle of injured area, concurrently with the reduction of ALK2. Collectively, our study suggests that metformin prevents heterotopic ossification via activation of AMPK and subsequent up‐regulation of Smad6. Therefore, metformin could be a potential therapeutic drug for heterotopic ossification induced by traumatic injury.     

Simulating localised cellular inflammation and substrate properties in a strain energy density based bone remodelling algorithm for use in modelling trauma

Bone responds to mechanical stimulus and a range of pre-existing finite element models have been suggested to reproduce the internal physiological structure of bone. Inflammation effects are not included in these models, yet inflammation is a key component of bone repair in trauma. Therefore, a model is proposed and tested here that extends these methods to include parameters that could be considered to represent the behaviour of bone remodelling when influenced by inflammation. The proposed model regulates remodelling based on findings from recent studies into the nature of heterotopic ossification, the formation of heterotopic bone, which have revealed information about the nature of bone after high levels of trauma. These parameters include consideration of the distance from the zone of trauma, the density of mesenchymal stem cells, and substrate stiffness as a trigger for cells becoming osteogenic. The method is tested on a two-dimensional plate model and shows that the new extended algorithm can produce a range of structures depending on inputs that could be used in the future to replicate physiological scenarios.     

Heterotopic autotransplantation of ovarian cortex in cynomolgus monkeys

In recent years, removal of ova or ovaries before chemotherapy or radiation therapy has been investigated in young female cancer patients to avoid the adverse effects of treatment. Orthotopic autotransplantation of ovarian cortex has advantages such as easy collection of ova and the possibility of spontaneous pregnancy. Although children have been born after successful orthotopic autotransplantation into the residual ovaries, some patients cannot undergo this procedure such as those who need bilateral ovariectomy or pelvic radiation therapy, therefore it is still necessary to investigate suitable heterotopic autotransplantation sites. The present study was performed in primates (cynomolgus monkeys) with the objective of determining the optimum site for heterotopic autotransplantation of ovarian cortex to enhance the clinical application of this method. The retroperitoneal iliac fossa and omentum were selected as sites for heterotopic autotransplantation. Two cynomolgus monkeys were subjected to laparotomy under anesthesia. After resection of the bilateral adnexae, the ovaries were cut into 0.5 cm cubes that were transplanted. Blood levels of follicle-stimulating hormone, luteinizing hormone, estradiol, and progesterone were monitored, and monkeys with a regular estrus cycle underwent superovulation and egg collection. In both monkeys studied, recovery of a regular estrus cycle was confirmed after heterotopic autotransplantation of ovarian tissue. MII phase ova were successfully collected from tissues transplanted into the retroperitoneal iliac fossa or omentum. Development to the early blastocyst stage was confirmed after microfertilization. We established an appropriate method of heterotopic autotransplantation using ovarian cortex into the retroperitoneal iliac fossa or omentum in primates.     

T-cell cytokine induction of BMP-2 regulates human mesenchymal stromal cell differentiation and mineralization

How T-cells, attracted to local sites of inflammation in arthritides, affect heterotopic ossification is presently unknown. Here, we tested the hypothesis that T-cell cytokines play a role in the differentiation of human mesenchymal stromal cells (HMSC) into the osteoblast phenotype by inducing autologous BMP-2, providing a possible mechanism for heterotopic ossification. HMSC from multiple donor bones were treated with either activated T-cell conditioned medium (ACTTCM) or physiological concentrations of the major inflammatory cytokines, TNF-alpha, TGF-beta, IFN-gamma, and IL-17 (TTII), individually or in combinations. ACTTCM induced BMP-2 protein in a time-dependent manner over a 48 h period and alkaline phosphatase (AlkP) within 7 days. In combination, TTII, like ACTTCM, induced AlkP and synergistically induced BMP-2 protein. Either individually, or in combinations of up to three, the T-cell cytokines failed to induce BMP-2 above control levels while a combination of all four cytokines synergistically induced BMP-2 10-fold as assessed by ELISA. TTII induced mineralized matrix as effectively as dexamethasone. Inhibition of p38 MAPK completely inhibited TTII-induced BMP-2 production and matrix mineralization. Real time RT-PCR analysis demonstrated a striking early (within 4 h) increase in BMP-2 gene expression by TTII, which was suppressed by p38 MAP kinase inhibition. In localized chronic inflammatory diseases, T-cell cytokines released at localized sites of inflammation may be the driving force for differentiation of local mesenchymal stromal cells into the osteoblast phenotype thereby playing a significant role in the heterotopic ossification observed in these diseases.     

Cell-based gene therapy for repair of critical size defects in the rat fibula

More than a decade has passed since the first experiments using adenovirus-transduced cells expressing bone morphogenetic protein 2 were performed for the synthesis of bone. Since this time, the field of bone gene therapy has tackled many issues surrounding safety and efficacy of this type of strategy. We present studies examining the parameters of the timing of bone healing, and remodeling when heterotopic ossification (HO) is used for bone fracture repair using an adenovirus gene therapy approach. We use a rat fibula defect, which surprisingly does not heal even when a simple fracture is introduced. In this model, the bone quickly resorbs most likely due to the non-weight bearing nature of this bone in rodents. Using our gene therapy system robust HO can be introduced at the targeted location of the defect resulting in bone repair. The HO and resultant bone healing appeared to be dose dependent, based on the number of AdBMP2-transduced cells delivered. Interestingly, the HO undergoes substantial remodeling, and assumes the size and shape of the missing segment of bone. However, in some instances we observed some additional bone associated with the repair, signifying that perhaps the forces on the newly forming bone are inadequate to dictate shape. In all cases, the HO appeared to fuse into the adjacent long bone. The data collectively indicates that the use of BMP2 gene therapy strategies may vary depending on the location and nature of the defect. Therefore, additional parameters should be considered when implementing such strategies.     

Cellular and morphological aspects of fibrodysplasia ossificans progressiva

Fibrodysplasia ossificans progressiva (FOP) is a rare congenital disease that causes bone formation within the muscles, tendons, ligaments and connective tissues. There is no cure for this disorder and only treatment of the symptoms is available. The purpose of this study was to review the literature and describe the clinical, cellular and molecular aspects of FOP. The material used for the study was obtained by reviewing scientific articles published in various literature-indexed databases. In view of its rarity and of the lack of insightful information and the unpredictability of its course, FOP is a challenging disorder for professionals who are confronted by it. However, this rare disease raises a great deal of interest because understanding the mechanism of mature bone formation can encourage research lines related to bone regeneration and the prevention of heterotopic ossification.