Ossification Front Research Articles

Osteochondritis Dissecans of the Medial Femoral Condyle: MRI Findings of Instability.

BACKGROUND: Osteochondritis dissecans (OCD) of the medial femoral condyle (MFC), a common cause of pediatric chronic knee pain, is often found to be stable at arthroscopy. OBJECTIVE: To investigate the performance of MRI for identifying instability of MFC OCD in children. METHODS: This retrospective study included 59 children (37 boys, 22 girls; mean age, 12.5±2.2 years) with 69 MFC OCD lesions, who underwent knee MRI from January 2016 to October 2023. Two radiologists independently reviewed examinations to determine regional findings (effusion, skeletal maturity, secondary physis visibility, progeny composition), as well as direct (osteochondral defect, intraarticular body, cartilage alteration, bone plate disruption, receded ossification front) and indirect (progeny-parent bone interface, radius of curvature, parent-bone low-signal intensity (SI) marginal rim, perilesional marrow edema) findings of instability; disagreements were resolved through consensus. Two investigators assessed an additional indirect finding, cyst(s), in consensus. Demographic characteristics, and lesion volume were collected. The reference standard for lesion stability was arthroscopy (n=52) or follow-up evaluation (based on symptoms and imaging findings) in patients managed conservatively (n=17). Stable and unstable lesions were compared. RESULTS: Forty-nine lesions were stable, and 20 were unstable. Sensitivity and specificity for instability, among features showing significant associations with instability, were 70.0% and 100.0% for skeletal maturity, 75.0% and 75.5% for osteochondral progeny composition, 100.0% and 85.7% for cartilage alteration, 85.0% and 81.6% for presence of progeny-parent bone interface, 30.0% and 100.0% for extensive interface, 90.0% and 34.7% for cyst(s), 45.0% and 91.8% for altered radius of curvature, 80.0% and 89.8% for parent-bone low-SI rim, and 50.0% and 81.6% for extensive perilesional marrow edema. In multivariable analysis, factors independently associated with instability included larger lesion volume and indirect signs (presence of interface, cyst(s), altered radius of curvature, parent-bone low-SI rim, and extensive perilesional marrow edema). Frequency of instability was 0.0%, 7.4%, 16.7%, 75.0%, 83.3%, and 100.0% for lesions with 0, 1, 2, 3, 4, and 5 indirect signs. CONCLUSION: Lesion instability was associated with an increasing number of indirect MRI signs. CLINICAL IMPACT: Assessment of lesion stability by MRI can help guide decisions between arthroscopy and conservative management for MFC OCD in children.

The Immature Pediatric Appendicular Skeleton.

Growth and maturation occur in a predictable pattern throughout the body and within each individual bone. In the appendicular skeleton, endochondral ossification predominates in long bones and growth plates. The ends of these long bones are sites of relative weakness in the immature skeleton and prone to injury from acute insult and overuse. We present the normal histoanatomy and physiology of the growth plate complex, highlighting the unique contribution of each component and shared similarities between primary and secondary complexes. Components of the growth plate complex include the physis proper, subjacent vascularity within the growth cartilage, and the ossification front. The second section describes imaging considerations and features of normal and abnormal growth. Finally, we review the Salter-Harris classification for acute fractures and offer examples of characteristic overuse injury patterns involving the epiphyseal (proximal humerus and distal radius), apophyseal (medial epicondyle and tibial tubercle), and secondary growth plate complexes (medial femoral condyle and capitellar osteochondritis dissecans). This article provides a foundation and basic framework to better understand and anticipate potential complications and growth disturbances and to ensure optimal follow-up and early intervention when treatment can be less invasive.

Increased Cellular Expression of Interleukin-6 in Patients With Ossification of the Posterior Longitudinal Ligament.

We performed histologic, immunohistochemical, immunoblot examination and suspension array analyses of cytokine expression in cultured cells derived from human cervical ossification of the posterior longitudinal ligament (OPLL). To determine the roles of interleukin-6 (IL-6) during the maturation of osteoblasts and chondrocytes associated with the development of OPLL. Ectopic OPLL affects ~3% of the general population, with a higher incidence in Asian ethnic groups. Alterations in cytokine profiles may influence osteoblast differentiation, but the mechanisms and signaling pathways associated with the ossification process remain unclear. Samples were collected from 14 patients with OPLL who had undergone spinal surgery and seven with cervical spondylotic myelopathy without OPLL. Tissue sections were used for histologic and immunohistochemical studies, and primary cells from ligamentum samples were used for cytokine array and immunoblotting. A suspension array was used to measure the concentrations of 27 inflammatory cytokines or growth factors. Suspension array and immunoblot analyses revealed significantly elevated levels of IL-6 in OPLL patients. Alterations in IL-6 concentrations were found to alter the expression of the genes Sox9 , Runx2 , and SIRT1 . In addition, immunohistochemical analysis revealed that these factors are present in mesenchymal cells within the degenerative portion of the ligament matrix that is adjacent to the ossification front. IL-6 plays a profound role in the osteoblast differentiation process along with the induction of chondrocyte hypertrophy and cell apoptosis in the early stages of ossification in OPLL. These changes in cytokine profiles are essential factors for regulation of the ectopic ossified plaque in OPLL.

What's New in Musculoskeletal Basic Science.

What's New in Musculoskeletal Basic Science.

Poster 200: High Prevalence of Abnormal Imaging Findings in Clinically Asymptomatic Siblings Of JOCD Patients On 3T Knee MRI

Objectives:Endochondral ossification is a skeletal developmental process in children by which the embryonic cartilaginous model of bones undergoes gradual ossification over time until skeletal maturity is reached. Juvenile Osteochondritis Dissecans (JOCD) is a developmental disease characterized by focal areas of chondronecrosis within the epiphyseal cartilage and subsequent delay of the ossification front. JOCD lesions can become loose bodies and lead to premature Osteoarthritis. The etiology of JOCD is not yet well understood, however, there is mounting evidence of a genetic predisposition. Recently, positive familial history, including familial OCD and a genome-wide association study in humans identified genetic loci related to juvenile OCD. In this longitudinal pilot study, we examined siblings of JOCD patients utilizing clinical 3T MRI data to evaluate morphological integrity of the epiphyseal cartilage and the advancing ossification front in the distal femoral condyles in bilateral knees. We hypothesized that asymptomatic, clinically “silent” JOCD lesions could be detected in young siblings of patients affected by JOCD.Methods:In this longitudinal study, 44 MRI datasets from seven skeletally immature participants with no clinical symptoms (7 siblings of JOCD patients; 3 females; 12 bilateral knees; average age at first visit=8.7 years; range=6-10 years) were evaluated. This study was approved by the institutional review board and informed consent was obtained. All images were acquired at 3T MRI system with a 1Tx/15Rx knee coil. Sagittal PD-weighted TSE images without fat suppression (TR/TE = 3900/40 ms, resolution = 0.36x0.36 mm2, scanning time = 1min:49s) and with fat suppression (TR/TE = 3530/40 ms, resolution = 0.36x0.36 mm2, scanning time = 3min:46s) were acquired bilaterally at first visit and at multiple MRI follow-up studies ranging from 6-21 months. The images were reviewed by an experienced musculoskeletal radiologist using the following MRI criteria: (i) integrity of the ossification front; (ii) presence/absence of bone marrow edema; (iii) intact/disrupted secondary physis; and (iv) T2 hyperintensity in epiphyseal cartilage.Results:Five of seven participants (71%) met at least one of the MRI criteria defining a JOCD lesion. Six of the seven participants (86%) had an irregular ossification front at the distal aspect of the medial and/or lateral femoral epicondyles, focal areas of T2 hyperintensities in the epiphyseal cartilage, and a disrupted secondary physis. Of the 13 imaged knees, two knees (both lateral and medial epicondyle) from the same participant and two knees (only lateral epicondyle) had no abnormal MRI findings (= 6 normal epicondyles). Abnormal findings in the medial and lateral epicondyles of the remaining bilateral knees resolved over time in 11 epicondyles (Fig. 1), whereas in the remaining 9 epicondyles the abnormal findings persisted or progressed (Fig. 2).Conclusions:The most important finding of this pilot study is the high prevalence (6 out of 7, 86%) of morphological changes during skeletal development in this group of first-degree relatives of JOCD patients. Furthermore, 71% (5 out of 6) of participants met the imaging criteria for early JOCD lesions at least at one time point during the study without developing any clinical symptoms. There is mounting evidence of a genetic contribution to the etiology of JOCD. In the literature, so called ossification variants are considered morphological variants of bone shape with an overall prevalence of 12% and are believed to resolve naturally over time. While 55% of the epicondyles with abnormalities in the ossification front in our cohort resolved over time on MRI, during the course of this study – typically 8-21 months, the remaining 45% did not resolve or occurred at the last imaging time point with associated marrow edema and needed clinical surveillance. A high incidence of MRI abnormalities in the ossification front, epiphyseal cartilage and secondary physis in a clinically asymptomatic study group of first degree relatives of patients with JOCD supports the hypothesis a) that there is a strong genetic predisposition for this disease, b) clinically “silent” initial stages of the disease could be overlooked, challenging the common current understanding of normal physiologic “ossification variants”.Figure 1.Sagittal PD-weighted TSE w/o fat-suppression (FS) of the lateral femoral condyle in a 10-year-old boy. The follow-up studies demonstrate complete resolution of the irregular ossification front (arrowhead), absence of bone marrow edema.Figure 2.Sagittal PD-weighted TSE w/o fat-suppression (FS) of the lateral femoral condyle in a 10-year-old boy. The baseline study demonstrates a focal T2-hyperintense lesion (arrow) and a delayed ossification front with associated bone marrow edema (*) meeting the MR imaging criteria for an early JOCD lesion. The asymptomatic sibling was clinically surveilled and subsequent follow-up studies demonstrated resolution of the MR imaging findings.

Influence of Hoxa11 and Hoxd11 on Calcaneus Growth and Ossification

Hox genes are key developmental patterning genes that impact segmental identity and skeletal patterning. While Hox11 genes are known to be expressed around the developing calcaneus bone of the ankle, previous studies on mice with Hox11 mutations have indicated that calcaneus morphology is not affected until both Hoxa11 and Hoxd11are knocked out, at which point the calcaneus and talus fail to form.The pisiform bone, a wrist bone that is paralogous to the calcaneus, exhibits substantial morphological and growth plate alterations with Hox11 mutations. We have previously shown that some length differences are present in the adult calcanei of mice with Hoxa11 and Hoxd11 loss-of-function mutations. The present study investigates whether or not the calcaneus growth plate is altered by Hoxa11 and Hoxd11 loss-of-function mutation. We conducted histological analysis of the calcaneus growth plate in juvenile mice with Hoxa11 and Hoxd11 loss-of-function mutations and compared them to ossification patterns observed in whole-mount specimens that were cleared and stained with alizarin red and alcian blue to visualize bone and cartilage, respectively. Histological analysis reveals that early calcaneus growth plates preserve the hypertrophic and proliferative growth plate zones. This is in contrast to the pisiform and likely a result of Hoxc gene expression in the hind limb but not the forelimb. The shape of the epiphyseal cartilage, however, differs greatly in mice with a combined three loss-of-function alleles between Hoxa11 and Hoxd11. In these mice, the calcaneus epiphyseal cartilage is conical shaped with an elongated region of reserve zone chondrocytes. The ossification front and calcaneal tendon insertion are also altered compared to wild type specimens. The first evidence of calcaneal epiphysis ossification appears at P9 in some Hox11 mutant mice, while it typically appears at P11 in wild type specimens. By P17, the epiphysis appears to be larger in specimens with both Hoxa11 and Hoxd11 mutations compared to wild type. These results indicate that the calcaneus growth plate is more resilient to Hox11 mutations than the pisiform, but that the calcaneus exhibits morphological changes and evidence of altered ossification timing with fewer loss-of-function alleles than identified by previous studies.

Differential Impact of Hoxa11 and Hoxd11 on Pisiform Ossification and Epiphysis Formation

Hox genes are key developmental patterning genes that impact segmental identity and skeletal patterning. Hox11 genes are known to impact wrist and ankle development and are expressed around the developing pisiform and calcaneus. These paralogous bones in the wrist and ankle are the only carpal and tarsal to form a growth plate in mammals, although humans have lost this growth plate and the associated primary ossification center in the pisiform. Loss-of-function mutations to Hoxa11 and Hoxd11 result in pisiform truncation and appear to also cause at least some disorganization of the growth plate cartilage; however, little is known about the nature of this disorganization or if ossification timing is impacted by Hox11 genes. The present study investigates the role of Hoxa11 and Hoxd11 in pisiform growth plate organization and ossification timing. We conducted histological analysis of the pisiform growth plate in juvenile mice with Hoxa11 and Hoxd11 loss-of-function mutations and compared them to ossification patterns observed in age- and genotype-matched whole-mount specimens that were cleared and stained with Alizarin red and Alcian blue to visualize bone and cartilage, respectively. Histological analysis reveals a dosage-dependent impact of Hox11 mutations on pisiform ossification to both the primary and secondary ossification center. As the number of Hox11 mutation alleles increase, less bone is present in the early primary ossification center compared to age-matched specimens. In specimens with three loss-of-function alleles, no trabeculae or growth plate organization are visible at P9, when both are well established in wild type specimens. Cleared and stained specimens indicate a possible pseudo epiphysis forming with Hoxd11 mutation, while Hoxa11 knockout specimens have not formed any visible epiphysis or calcification by P9. These results indicate that ossification timing and patterns, along with growth plate organization, are affected by Hox11 mutations during early pisiform ossification. Furthermore, Hoxa11 and Hoxd11 alter the pisiform epiphysis differently, suggesting that each plays a specific role in formation of the ossification front and epiphysis ossification either by influencing timing, ossification progression, or both. Further work is needed to understand the mechanisms by which Hox genes impact ossification patterns and timing, as well as the differential roles of Hoxa11 and Hoxd11 in growth plate organization and epiphysis formation.

Radiological, vascular osteochondrosis occurs in the distal tarsus, and may cause osteoarthritis.

Osteochondrosis occurs due to failure of the blood supply to growth cartilage. Osteochondrosis lesions have been identified in small tarsal bones and suggested to cause distal tarsal osteoarthritis; however, it has not been determined whether distal tarsal osteochondrosis lesions were the result of vascular failure. To perform post-mortem arterial perfusion and micro-computed tomography (CT) of the central (CTB) and third tarsal bones (TIII) of fetuses and foals up to 5months old, to describe tarsal development and any lesions detected. Descriptive, nonconsecutive case series. Twenty-three animals that died or were euthanased from 228days of gestation to 5months old were collected, comprising two fetuses and nine foals of miscellaneous breeds and 12 Icelandic Horse foals, a breed with high prevalence of distal tarsal osteoarthritis. One hindlimb from each foal was perfused arterially with barium, and the CTB and TIII were examined with micro-CT. Perfusion yielded partial information from 41% of the animals. The CTB and TIII were supplied by nutrient arteries and perichondrial vessels with vertical, transverse and circumferential configurations. Fourteen of the 23 (61%) animals had focal defects in the ossification front, that is, radiological osteochondrosis. The majority of lesions matched the configuration and development of vertical vessels. Additionally, full-thickness, cylindrical defects matched transverse vessels, and the long axes of some dorsal lesions matched circumferential vessels. Lack of histological validation. Post-mortem perfusion was poor for examination of the blood supply to the growth cartilage of the CTB and TIII. Radiological osteochondrosis lesions were compatible with vascular failure because they were focal, and because lesion geometry matched vessel configuration. The relationship between osteochondrosis and distal tarsal osteoarthritis warrants further investigation.

Quantifying the hydroxyapatite orientation near the ossification front in a piglet femoral condyle using X-ray diffraction tensor tomography

While a detailed knowledge of the hierarchical structure and morphology of the extracellular matrix is considered crucial for understanding the physiological and mechanical properties of bone and cartilage, the orientation of collagen fibres and carbonated hydroxyapatite (HA) crystallites remains a debated topic. Conventional microscopy techniques for orientational imaging require destructive sample sectioning, which both precludes further studies of the intact sample and potentially changes the microstructure. In this work, we use X-ray diffraction tensor tomography to image non-destructively in 3D the HA orientation in a medial femoral condyle of a piglet. By exploiting the anisotropic HA diffraction signal, 3D maps showing systematic local variations of the HA crystallite orientation in the growing subchondral bone and in the adjacent mineralized growth cartilage are obtained. Orientation maps of HA crystallites over a large field of view (~ 3 × 3 × 3 mm3) close to the ossification (bone-growth) front are compared with high-resolution X-ray propagation phase-contrast computed tomography images. The HA crystallites are found to predominantly orient with their crystallite c-axis directed towards the ossification front. Distinct patterns of HA preferred orientation are found in the vicinity of cartilage canals protruding from the subchondral bone. The demonstrated ability of retrieving 3D orientation maps of bone-cartilage structures is expected to give a better understanding of the physiological properties of bones, including their propensity for bone-cartilage diseases.

Cyclic tensile strain facilitates ossification of the cervical posterior longitudinal ligament via increased Indian hedgehog signaling

The pathomechanisms of initiation and progression of ossification of the posterior longitudinal ligament (OPLL) are unclear. Indian hedgehog (Ihh) and related signaling molecules are key factors in normal enchondral ossification. The purpose of this study is to investigate the contribution of mechanical strain to OPLL and the relationship of Ihh with OPLL. Sections of the posterior longitudinal ligament (PLL) were obtained from 49 patients with OPLL and from 7 patients without OPLL. Cultured PLL cells were subjected to 24 hours of cyclic tensile strain. To identify differentially expressed genes associated with cyclic tensile strain, microarray analysis was performed. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis identified upregulation of various genes, particularly of the Hedgehog signaling pathway; Ihh and related genes had increased expression compared with controls after 24-hour cyclic tensile strain. In immunoblotting analysis, Ihh, Runx2, Sox9, Gli2, Gli3, and smoothened (SMO) had significantly increased expression after 6- or 12-hour cyclic tensile strain. OPLL samples were strongly immunopositive for Ihh, Sox9, Runx2, Gli2, Gli3, and SMO in the ossification front of OPLL. These results suggest that cyclic tensile strain induces abnormal activation of Ihh and related signaling molecules, and this might be important in the ossification process in OPLL.

The Pathogenesis of Osteochondrosis in Humans and Animals

Osteochondrosis is a disease involving failure of endochondral ossification that occurs in growing animals and children in highly specific predilection sites, resulting in the formation of a cartilaginous or osseocartilaginous flap that causes pain and locking of the joint. In veterinary medicine, the term “osteochondrosis” (OC) is used for all stages of the disease and all affected sites. In human medicine, “osteochondritis dissecans” (OCD), is the preferred term for clinical disease involving the distal femur/knee joint. In other affected sites in humans, the disease is named after the physician(s) who first recognized it at that site; the disease in humans is further subdivided into adult and juvenile forms. Until very recently, subclinical disease was not recognized in humans as the only available tissue for study was the flap removed surgically from the affected joint, which often had been present for months to years. Subclinical disease, however, is well recognized in animals due to study of predilection sites at ages prior to the age of onset of clinical disease, work that are not possible in humans. Because the appearances of the subclinical and clinical disease are completely different, and because only end‐state tissue has been available for study of the disease in humans, the pathogenesis of the disease has been poorly understood in human medicine. In pigs and horses, it has been well demonstrated that subclinical lesions of osteochondrosis involving the articular‐epiphyseal cartilage complex are composed of areas of necrotic epiphyseal cartilage that often surround necrotic cartilage canal blood vessels. The overlying articular cartilage and subjacent subchondral bone are not initially affected; however, as endochondral ossification proceeds these areas of necrotic cartilage cause a delay in the ossification front that is visible in imaging studies. Study of both naturally‐occurring and surgically‐induced disease in animals have unequivocally demonstrated that failure of cartilage canal blood supply is the cause of these lesions. Traumatic forces to the surface of the articular cartilage, even when minimal in severity, may result in collapse of the necrotic epiphyseal cartilage and cleft formation through the overlying articular cartilage, resulting in the well‐recognized clinical disease. Recent development of magnetic resonance imaging methods to identify cartilage canal vessels and areas of cartilage necrosis, as well as access to human cadaveric knee specimens, have provided strong evidence that the pathogenesis of OC/OCD in animals and humans is identical and that subclinical disease in humans occurs years before clinical disease is evident. This work provides the potential for the development of new methods of early detection of disease in humans and methods for monitoring its response to treatment.Support or Funding InformationNIH/NIAMS R01AR070020; NIH/OD K01OD021293; WM Keck FoundationThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Maturation of the medial femoral condyle epiphyseal growth plate and osteochondral junction

Purpose: The osteochondral junction region has an important role in osteoarthritis and many early pathophysiological features (e.g. chondrocyte hypertrophy) of the disease recapitulate development events. Femorotibial osteoarthritis arises most commonly in the medial compartment in humans and horses. Furthermore, juvenile osteochondrosis (JOCD) also develops in this region, and if not treated successfully, the persisting incongruency can lead to secondary osteoarthritis. The maturation of the epiphyseal growth cartilage and the osteochondral junction of the medial femoral condyle (MFC) of the distal femur is poorly described in the literature. Horses are also increasingly recognized to be appropriate large animal models for cartilage repair investigations and information on maturation of the osteochondral junction is lacking. Our goal was to describe the normal development of the equine MFC osteochondral junction from birth to 2 years old. Methods: Distal femurs were collected from Thoroughbred horses (n=61, 0-2 years old), presented for post-mortem examination for reasons unrelated to this study. Radiographs and tomodensitometry were performed to screen for healthy specimens without focal osteochondral junction lesions. Micro-tomodensitometry (μ-CT), histology and immunohistochemistry were performed on the selected MFCs (n=29). μ-CT: A region of interest (ROI) was selected for study in a weight bearing area of the MFC that also incorporated developmental disease (JOCD and equine cysts/osteochondrosis) prone sites. Cartilage thickness (Cth) and trabecular bone volume were measured. 3D images were also constructed and the osteochondral junction morphology was assessed. Histology: MFC epiphyseal growth plate structure was studied on hematoxylin phloxine and saffron (HPS) stained sections. Collagen structure (picrosirius red) and proteoglycan content (Safranin O fast green) were also evaluated. Chondrocyte lacunae diameters were measured in predefined regions below the articular surface and near the osteochondral junction as an indicator of chondrocyte hypertrophy. Collagen X (Col X) immunohistochemical staining was also employed as a marker of endochondral ossification. Results: μ-CT: The Cthranged from 3.8mm in the youngest specimens to 1.2mm at 2 years of age. The Cthdecreased from birth to 7 months and remained constant thereafter (regression into 2 segments, p<0.0001). From birth to 6 months of age, the ossification front was porous on 3D images (fig 1.a) and later had a smooth appearance (fig 1. d). The trabecular bone volume did not change with age and was significantly higher at the periphery of the condyle, near the cartilage, compared to the center (p<0.0001). Histology: The structure of the epiphyseal growth cartilage differed from that reported for the physis as there was no columnar organization of the chondrocytes. The safranin O fast green and picrosirius stains showed a stratification of the extracellular matrix content that varied with age. The osteochondral unit was immature up to 6 months of age: Cartilage canals containing blood vessels crossed the ossification front (fig 1.b) and the chondrocytes were undergoing hypertrophy near the cartilage-bone junction. The chondrocyte lacunar volume increased 14-fold beside the osteochondral junction in the youngest specimens (fig 1.c). Expression of Col X was evident in the extracellular matrix surrounding hypertrophic chondrocytes in site matched specimens in the juvenile specimens alone. At six months of age, the absence of chondrocyte hypertrophy and Col X immunostaining indicated the conclusion of endochondral ossification. This coincided with the smallest Cth, similar to mature articular cartilage and the appearance of a narrow band of compact bone indicating formation of a subchondral cortical bone plate (fig 1.e). Conclusions: The equine MFC osteochondral junction is structurally immature and undergoes endochondral ossification up to 6 months of age. At this time, the histological structure of a subchondral cortical bone plate emerges. Cartilage canals and hypertrophic chondrocytes are no longer present and the cartilage thickness is stable and consistent with a mature articular cartilage. Although we have explored the structural and matrix maturation events, additional studies are now required to assess the biomechanical maturation of the region. New information is provided on MFC osteochondral junction development that may help towards the understanding of the remodeling events occurring in osteoarthritic joints.

Facilitation of Chemotaxis Activity of Mesenchymal Stem Cells via Stromal Cell-Derived Factor-1 and Its Receptor May Promote Ectopic Ossification of Human Spinal Ligaments.

Mesenchymal stem cells (MSCs) have been used to elucidate the pathogenesis of numerous diseases. Our recent study showed that MSCs may conduce to the ossification of spinal ligaments. Stromal cell-derived factor-1 (SDF-1) and CXC chemokine receptor 4 (CXCR4) regulate MSC migration. Moreover, their expression is elevated in sites of damage and remodeling in pathologic states. We explored the possible role of the SDF-1/CXCR4 axis in the chemotactic behavior of MSCs in the ossification of spinal ligaments. Specimens of thoracic vertebra ossified ligamentum flavum (OLF) and non-OLF plaques were received from patients in whom we had performed spine surgery. Paraffin-embedded tissue sections were prepared for immunohistochemical staining. Cultured MSCs from the ligamentum flavum were prepared for in vitro analyses. We observed SDF-1 and CXCR4 localization immunohistochemically in the perivascular area and collagenous matrix of ligaments and in chondrocytes near the ossification front of OLF. And then, immunohistochemical staining showed a close relationship between MSCs and the SDF-1/CXCR4 axis. In the in vitro analyses, expression of the SDF-1/CXCR4 and the migratory capacity of MSCs in OLF were remarkably higher compared with non-OLF MSCs. Furthermore, the migration of MSCs was upregulated by SDF-1 and downregulated by treatment with AMD3100 (C28H54N88HCl), a specific antagonist for CXCR4. All in vitro test data showed a significant difference in MSCs from OLF compared with non-OLF MSCs. Our results reveal that the SDF-1/CXCR4 axis may contribute to an MSC-mediated increase in the ossification process, indicating that the SDF-1/CXCR4 axis may become a potential target for a novel therapeutic strategy for ossification of spinal ligaments.

Juvenile osteochondritis dissecans of the knee is a result of failure of the blood supply to growth cartilage and osteochondrosis

Juvenile osteochondritis dissecans (JOCD) is similar to osteochondrosis dissecans (OCD) in animals, which is the result of failure of the cartilage canal blood supply, ischemic chondronecrosis and delayed ossification, or osteochondrosis. The aim of the current study was to determine if osteochondrosis lesions occur at predilection sites for JOCD in children. Computed tomographic (CT) scans of 23 knees (13 right, 10 left) from 13 children (9 male, 4 female; 1 month to 11 years old) were evaluated for lesions consisting of focal, sharply demarcated, uniformly hypodense defects in the ossification front. Histological validation was performed in 11 lesions from eight femurs. Thirty-two lesions consisting of focal, uniformly hypodense defects in the ossification front were identified in the CT scans of 14 human femurs (7 left, 7 right; male, 7-11 years old). Defects corresponded to areas of ischemic chondronecrosis in sections from all 11 histologically validated lesions. Intra-cartilaginous secondary responses comprising proliferation of adjacent chondrocytes and vessels were detected in six and two lesions, whereas intra-osseous responses including accumulation of chondroclasts and formation of granulation tissue occurred in 10 and six lesions, respectively. One CT cyst-like lesion contained both a pseudocyst and a true cyst in histological sections. Changes identical to osteochondrosis in animals were detected at predilection sites for JOCD in children, and confirmed to represent failure of the cartilage canal blood supply and ischemic chondronecrosis in histological sections.

Ultrasonographic screening for subclinical osteochondrosis of the femoral trochlea in foals (28-166 days old): a prospective farm study.

Extensive osteochondritis dissecans (OCD) lesions of the lateral ridge of the trochlea of the femur (LRTF), the most common OCD-affected site in the stifle, have a poor outcome with surgical debridement and can be career ending. The early detection of osteochondrosis lesions and their conservative management holds the promise to enhance outcome. We hypothesise that ultrasonography is a valuable field screening tool to detect and monitor early subclinical LRTF osteochondrosis. The goals were to 1) describe the normal ultrasonographic features of the LRTF in foals of different ages and 2) screen a foal cohort at the farm for early subclinical osteochondrosis lesions. Prospective cohort study. The LRTF of both hindlimbs of Thoroughbred foals (n = 46, 27-166 days old) were imaged once with ultrasonography and radiography (lateromedial and caudolateral-craniomedial oblique views). Cartilage thickness, ossification front indentation of the chondro-osseous junction and epiphyseal vascularisation were assessed on ultrasonography. Follow-up radiographs were taken as yearlings. The cartilage thickness, ossification front indentation and epiphyseal vascularisation significantly decreased with advancing maturity. Subclinical osteochondrosis lesions, characterised by semicircular indentations in the ossification front (indirect evidence of focal failure of ossification and retained cartilage) were detected in six foals (28-145 days old), both with radiography and ultrasonography. Ultrasonography provided a better overall subjective assessment of the osteochondrosis lesion topography (length, depth and the width) compared with radiography. Post-mortem validation of lesions was not possible. Ultrasonography of the LRTF is a practical, inexpensive and reliable technique to discriminate physiological from pathological events at the LRTF in young foals. It revealed the complex topography of the chondro-osseous junction permitting a rapid, comprehensive assessment of the subclinical osteochondrosis lesions in very young foals.

Wnt signaling pathway correlates with ossification of the spinal ligament: A microRNA array and immunohistochemical study.

Ossification of the posterior longitudinal ligament or the ligamentum flavum parallels endochondral ossification. Cell differentiation at the ossification front is known to be important during this process, although the factors regulating its initiation and progression are still unclear. The purpose of this study was to identify factors important for the regulation of chondrocyte/osteoblast differentiation during spinal ossification. Ligamentum flavum tissue was isolated from 25 patients who underwent decompressive surgery for cervical ossification of the posterior longitudinal ligament. Tissue sections were used for invitro culture to obtain primary cells through migration methods. To identify microRNAs associated with ossification of the posterior longitudinal ligament, cultured cells were prepared from the ligamentous tissue (n=4; continuous type) or from control ligamentous samples harvested from patients with cervical spondylosis without spinal ossification, and analyzed using a microRNA array. The ligamentous sections were also examined by immunohistochemistry for the expression of candidate microRNA target genes. The microRNA array identified 177 factors; 12 of which were expressed at significantly different levels in patients with ossification of the posterior longitudinal ligament compared to those in control patients. The hsa-miR-487b-3p was down-regulated in patients with ossification of the posterior longitudinal ligament, which met the false discovery rate of <0.05. This microRNA was predicted to regulate the expression of genes involved in Wnt signaling. Furthermore, immunohistochemistry of Wnt signaling proteins, including Wnt 3a, LRP5/6, and beta-catenin, revealed positive expression in mesenchymal cells and/or premature chondrocytes at the ossification front. Our results suggested that down-regulation of miR-487b-3p plays an important role in the initiation of Wnt signaling during the ossification process. Wnt signaling may regulate both chondrocyte and osteoblast differentiation and the specification of endochondral ossification in the pathogenesis of ossification of the posterior longitudinal ligament or the ligamentum flavum.

Ossification Variants in the Femoral Condyles and Trochleae are Caused by Lesions of Osteochondrosis Manifesta in Children

Objectives:Over the last 50 years, the genesis of OCD lesions has been thought secondary to a primary bone necrosis origin. This theory has been challenged recently, as histological studies performed on specimens obtained from asymptomatic mammals, have revealed that ischemic necrosis of epiphyseal cartilage (rather than bone) is the precursor lesion of osteochondrosis dissecans (OCD) in several animal species. The earliest lesion identified histologically is osteochondrosis (OC) latens, in which the area of necrosis is confined to epiphyseal cartilage. As the ossification front advances, the area of necrosis causes a delay/failure in endochondral ossification that is visible radiographically and by CT and is termed OC manifesta. Juvenile osteochondritis dissecans (JOCD) in children has many similarities to OCD in animals; however, subclinical disease in children has not been studied due to the invasive nature of this work and the difficulty in obtaining appropriate cadaveric specimens. Lesions of OC latens are not recognized in children, and lesions resembling OC manifesta identified by CT are often considered to be normal ossification variants. Previous work by our group identified 32 suspected OC manifesta lesions in the medial and lateral femoral condyles (MFC and LFC) and the lateral trochlear ridge (LTR) in 14 cadaveric specimens from children ranging in age from 7 to 11 years. Our aim was to determine if skeletally immature human knees contained histological evidence of delayed endochondral ossification occurring secondary to ischemic necrosis of epiphyseal cartilage.Methods:Eleven sites (4 MFC, 4LFC, 3LTR) containing suspect OC manifesta lesions identified on CT from five male children (age range 7-11 years) were decalcified in 10% EDTA. Areas corresponding to the CT lesions were trimmed into 3-mm thick slabs (n=2-4 slabs/site), processed into paraffin, sectioned at 5 µm, and stained with hematoxylin and eosin. All sections (n= 30 total) were examined by four veterinary pathologists/radiologists with extensive experience in the study of OC in pigs and horses.Results:All sites examined contained at least one section containing histological evidence of one or more areas of OC manifesta Lesions were evidenced by focal failure of endochondral ossification accompanied by remnants of necrotic blood vessels, chondrocyte necrosis with matrix degeneration. Some lesions were accompanied by evidence of repair/healing response, including chondrocyte clusters, proliferating blood vessels, and fibrous connective tissue.Conclusion:Similar to other mammals, human OCD lesions appear to develop as a primary defect secondary to avascular necrosis of epiphyseal cartilage, rather than primary avascular necrosis in bone. Our findings strongly support a common pathogenesis of OCD in humans and mammals. Historically, ossification variants have been considered a developmental anomaly with a benign clinical course. Our histologic findings suggest that ‘ossification variants’ may in face be delayed presentations of a continuum of OCD lesion development that starts years earlier due to primary epiphyseal cartilage necrosis. Some of these ‘variants’ may progress to healing, but many may progress to frank ‘osteochondritis dissecans’, suggesting that these lesions that are idenfitifed on xray or with MRI sequences require close follow-up.

Validation of the ultrasonographic assessment of the femoral trochlea epiphyseal cartilage in foals at osteochondrosis predilected sites with magnetic resonance imaging and histology.

Noninvasive imaging tools are needed to screen foal femoropatellar joints to detect subclinical osteochondrosis lesions due to focal failure of endochondral ossification to enhance early management to optimise intrinsic healing events. Recently investigations employing 3T susceptibility-weighted magnetic resonance imaging (3T SWI MRI) and CT have demonstrated their capacity for early osteochondrosis diagnosis, but these technologies are not practical for field screening. We postulate that ultrasonography is a valuable field tool for the detection of subclinical osteochondrosis lesions. The goals were to 1) describe the ultrasonographic features of the femoral trochlea of healthy and osteochondrosis-predisposed neonatal foals, 2) validate the capacity of ultrasound to assess cartilage canal vascular archictecture and the ossification front and 3) evaluate field feasibility in a pilot study. Experimental study. Ultrasonographic evaluation of osteochondrosis predisposed (n = 10) and control (n = 6) femoral trochleas was performed ex vivo and compared with site-matched histological sections and 3T SWI MRI. The articular and epiphyseal cartilage thickness, ossification front indentation and cartilage canal vascular archictecture were assessed at each ROI. Femoral trochleae of foals (n = 3) aged ≈ 1, 3 and 6 months were also evaluated with ultrasonography in field. Ultrasonographic measurements strongly correlated with the histological measurements. There was no difference in the cartilage thickness or ossification front indentation between control and osteochondrosis-predisposed specimens. The cartilage canal vascular archictecture on ultrasonograms corresponded with the vessel pattern observed on site matched histology and 3T SWI MRI. The number of specimens for study was limited and no early osteochondrosis lesions were present within the predilected group, but a field study is now underway. Ultrasonographic examination of the femoral trochlea permitted accurate evaluation of cartilage thickness, cartilage canal vascular archictecture and ossification front indentation in young foals and is a promising, practical tool for screening subclinical osteochondrosis and monitoring and managing lesions at important clinical sites.

MiR-132-3p Regulates the Osteogenic Differentiation of Thoracic Ligamentum Flavum Cells by Inhibiting Multiple Osteogenesis-Related Genes.

Ossification of the ligamentum flavum (OLF) is a disorder of heterotopic ossification of spinal ligaments and is the main cause of thoracic spinal canal stenosis. Previous studies suggested that miR-132-3p negatively regulates osteoblast differentiation. However, whether miR-132-3p is involved in the process of OLF has not been investigated. In this study, we investigated the effect of miR-132-3p and its target genes forkhead box O1 (FOXO1), growth differentiation factor 5 (GDF5) and SRY-box 6 (SOX6) on the osteogenic differentiation of ligamentum flavum (LF) cells. We demonstrated that miR-132-3p was down-regulated during the osteogenic differentiation of LF cells and negatively regulated the osteoblast differentiation. Further, miR-132-3p targeted FOXO1, GDF5 and SOX6 and down-regulated the protein expression of these genes. Meanwhile, FOXO1, GDF5 and SOX6 were up-regulated after osteogenic differentiation and the down-regulation of endogenous FOXO1, GDF5 or SOX6 suppressed the osteogenic differentiation of LF cells. In addition, we also found FOXO1, GDF5 and SOX6 expression in the ossification front of OLF samples. Overall, these results suggest that miR-132-3p inhibits the osteogenic differentiation of LF cells by targeting FOXO1, GDF5 and SOX6.

Notch signaling pathways in human thoracic ossification of the ligamentum flavum

This study investigated the pathological process of Notch signaling in the osteogenesis of ligamentum flavum tissues and cells, and the associated regulatory mechanisms. Notch receptors, ligands, and target genes were identified by quantitative polymerase chain reaction (qPCR) in ligamentum flavum cells and immunohistochemistry in ligamentum flavum sections from ossification of the ligamentum flavum (OLF) patients and controls. The temporospatial expression patterns of JAG1/Notch2/HES1 in human ligamentum flavum cells during osteogenic differentiation were determined by qPCR. Lentiviral vectors for Notch2 overexpression and knockdown were constructed and transfected into ligamentum flavum cells before osteogenic differentiation to examine the function of Notch signaling pathways in the osteogenic differentiation of ligamentum flavum cells. Alkaline phosphatase, Runx2, Osterix, osteocalcin, and osteopontin mRNA levels, alkaline phosphatase activity, and Alizarin Red staining were used as indicators of osteogenic differentiation. JAG1/Notch2/HES1 mRNA levels were up-regulated in ligamentum flavum cells from OLF patients, which increased during osteogenic differentiation. Immunohistochemical analysis suggested positive Notch2 expression at the ossification front. Down-regulation of Notch2 expression decelerated osteogenic differentiation of ligamentum flavum cells, and Notch2 overexpression promoted osteogenic differentiation of ligamentum flavum cells. Expression of Runx2 and Osterix increased in a manner similar to that of Notch2 during osteogenic differentiation of ligamentum flavum cells, and Notch2 knockdown and overexpression influenced their expression levels. Notch signaling plays an important role in OLF, and Notch may affect the osteogenic differentiation of ligamentum flavum cells via interactions with Runx2 and Osterix.© 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:1481-1491, 2016.