Aged Callus Skeletal Stem/Progenitor Cells Contain an Inflammatory Osteogenic Population With Increased IRF and NF-κB Pathways and Reduced Osteogenic Potential.

Skeletal stem/progenitor cells (SSPCs) are critical for fracture repair by providing osteo-chondro precursors in the callus, which is impaired in aging. However, the molecular signatures of callus SSPCs during aging are not known. Herein, we performed single-cell RNA sequencing on 11,957 CD45-CD31-Ter119- SSPCs isolated from young and aged mouse calluses. Combining unsupervised clustering, putative makers, and DEGs/pathway analyses, major SSPC clusters were annotated as osteogenic, proliferating, and adipogenic populations. The proliferating cluster had a differentiating potential into osteogenic and adipogenic lineages by trajectory analysis. The osteoblastic/adipogenic/proliferating potential of individual clusters was further evidenced by elevated expression of genes related to osteoblasts, adipocytes, or proliferation. The osteogenic cluster was sub-clustered into house-keeping and inflammatory osteogenic populations that were decreased and increased in aged callus, respectively. The majority of master regulators for the inflammatory osteogenic population belong to IRF and NF-κB families, which was confirmed by immunostaining, RT-qPCR, and Western blot analysis. Furthermore, cells in the inflammatory osteogenic sub-cluster had reduced osteoblast differentiation capacity. In conclusion, we identified 3 major clusters in callus SSPCs, confirming their heterogeneity and, importantly, increased IRF/NF-κB-mediated inflammatory osteogenic population with decreased osteogenic potential in aged cells.

The E3 ubiquitin ligase Itch limits the progression of post-traumatic osteoarthritis in mice by inhibiting macrophage polarization.

OBJECTIVE: Osteoarthritis (OA) is characterized by articular cartilage loss, associated with synovial inflammation. We recently reported increased pro-inflammatory macrophages in murine post-traumatic OA (PTOA) joints, and blockade of the ubiquitin-proteasome system alleviates PTOA progression. However, the mechanisms whereby protein ubiquitination influences PTOA pathology are not well studied. We hypothesized that loss of the negative regulator of inflammation, E3 ligase Itch, in macrophages contributes to joint OA tissue damage by promoting pro-inflammatory polarization of macrophages. METHODS: Mice deficient Itch in macrophages (MΔItch) were generated by crossing Itchfl/fl mice with LysM-Cre mice. PTOA surgery was performed on global Itch knockout, Itch-/-, mice and MΔItch mice. Joint tissue damage and synovial macrophages were examined. Itch-/- cells were treated with IL-1 and pro-inflammatory polarization was determined. Expression of Itch protein and mRNA in PTOA synovium were assessed at different time points post PTOA. RESULTS: Similar to Itch-/- mice, MΔItch mice developed more severe joint damage than control mice following PTOA surgery (mean difference of OARSI score: 1.17 (95% CI 0.31-2.03) between MΔItch and Itchfl/fl mice), accompanied by increased the inflammatory macrophage infiltration in the synovium (mean difference of % F4/80 + CD86 + CD36-inflammatory macrophages: 14.81 (95% CI 8.90-20.73) between MΔItch and Itchfl/fl mice). Itch-/- macrophages exerted pro-inflammatory phenotype in response to IL-1ß treatment. Itch protein, but not mRNA levels decreased during PTOA progression. CONCLUSION: The negative regulator of inflammation, Itch, limits PTOA progression by inhibiting macrophage pro-inflammatory polarization. Itch protein degradation may contribute to PTOA pathology.

Posttreatment Imaging in Patients with Head and Neck Cancer without Clinical Evidence of Recurrence: Should Surveillance Imaging Extend Beyond 6 Months?

BACKGROUND AND PURPOSE: Early detection of residual or recurrent disease is important for effective salvage treatment in patients with head and neck cancer. Current National Comprehensive Cancer Network guidelines do not recommend standard surveillance imaging beyond 6 months unless there are worrisome signs or symptoms on clinical examination and offer vague guidelines for imaging of high-risk patients beyond that timeframe. Our goal was to evaluate the frequency of clinically occult recurrence in patients with head and neck squamous cell carcinoma with positive imaging findings (Neck Imaging Reporting and Data Systems scores of 2-4), especially after 6 months. MATERIALS AND METHODS: This institutional review board-approved, retrospective data base search queried neck CT reports with Neck Imaging Reporting and Data Systems scores of 2-4 from June 2014 to March 2018. The electronic medical records were reviewed to determine outcomes of clinical and radiologic follow-up, including symptoms, physical examination findings, pathologic correlation, and clinical notes within 3 months of imaging. RESULTS: A total of 255 cases, all with Neck Imaging Reporting and Data Systems scores of 2 or 3, met the inclusion criteria. Fifty-nine patients (23%) demonstrated recurrence (45 biopsy-proven, 14 based on clinical and imaging progression), and 21 patients (36%) had clinically occult recurrence (ie, no clinical evidence of disease at the time of the imaging examination). The median overall time to radiologically detected, clinically occult recurrence was 11.4 months from treatment completion. CONCLUSIONS: Imaging surveillance beyond the first posttreatment baseline study was critical for detecting clinically occult recurrent disease in patients with head and neck squamous cell carcinoma. More than one-third of all recurrences were seen in patients without clinical evidence of disease; and 81% of clinically occult recurrences occurred beyond 6 months.

An update on the validation of whole slide imaging systems following FDA approval of a system for a routine pathology diagnostic service in the United States.

Pathologists have used light microscopes and glass slides to interpret the histologic appearance of normal and diseased tissues for more than 150 years. The quality of both microtomes used to cut tissue sections and microscopes has improved significantly during the past few decades, but the process of rendering diagnoses has changed little. By contrast, major advances in digital technology have occurred since the introduction of hand held electronic devices, including the development of whole slide imaging (WSI) systems with software packages that can convert microscope images into virtual (digital) slides that can be viewed on computer monitors and via the internet. To date, however, these technological developments have had minimal impact on the way pathologists perform their daily work, with the exception of using computers to access electronic medical records and scholarly web sites for pertinent information to assist interpretation of cases. Traditional practice is likely to change significantly during the next decade, especially since the Federal Drug Administration in the USA has approved the first WSI system for routine diagnostic practice. I review here the development and slow acceptance of WSI by pathology departments. I focus on recent advances in validation of WSI systems that is required for routine diagnostic reporting of pathology cases using this technology.

PETE Doctoral Institutions: Programs, Faculty, and Doctoral Students.

PURPOSE: The present study of doctoral physical education teacher education (D-PETE) programs was part of a longitudinal study that provided an extensive description of demographics including: (a) doctoral program characteristics, (b) faculty, and (c) doctoral students/graduates. METHOD: This trend study incorporated 3 data sets (2005-2006, 2008-2009, and 2011-2012) that described the characteristics of D-PETE programs. Academic heads of D-PETE programs provided demographic information on their doctoral students, faculty, and institutional characteristics for the 2005-2006, 2008-2009, and 2011-2012 academic years and selected summary data from 1996-1997 through 2011-2012. RESULTS/CONCLUSION: As a result of this longitudinal data collection, the following trends were revealed. First, there was a decrease in the number of D-PETE programs and an increase of nontenured and part-time pedagogy faculty. Second, initial teacher licensure programs remained in existence at the vast majority of D-PETE programs. Third, funding for doctoral students at D-PETE programs was decreasing. Fourth, racial composition of doctoral graduates and current doctoral students remained largely skewed toward Caucasians. Fifth, there was a slight decline in the percentage of doctoral graduates entering higher education, but employment rates were exceptionally high. Sixth, non-U.S. doctoral students and ABDs were marketable in the United States.

Whole slide imaging: uses and limitations for surgical pathology and teaching.

Advances in computer and software technology and in the quality of images produced by digital cameras together with development of robotic devices that can take glass histology slides from a cassette holding many slides and place them in a conventional microscope for electronic scanning have facilitated the development of whole slide imaging (WSI) systems during the past decade. Anatomic pathologists now have opportunities to test the utility of WSI systems for diagnostic, teaching and research purposes and to determine their limitations. Uses include rendering primary diagnoses from scanned hematoxylin and eosin stained tissues on slides, reviewing frozen section or routine slides from remote locations for interpretation or consultation. Also, WSI can replace physical storage of glass slides with digital images, storing images of slides from outside institutions, presenting slides at clinical or research conferences, teaching residents and medical students, and storing fluorescence images without fading or quenching of the fluorescence signal. Limitations include the high costs of the scanners, maintenance contracts and IT support, storage of digital files and pathologists' lack of familiarity with the technology. Costs are falling as more devices and systems are sold and cloud storage costs drop. Pathologist familiarity with the technology will grow as more institutions purchase WSI systems. The technology holds great promise for the future of anatomic pathology.

Advances in the regulation of osteoclasts and osteoclast functions.

Osteoclasts are derived from mononuclear hematopoietic myeloid lineage cells, which are formed in the bone marrow and are attracted to the bloodstream by factors, including sphingsine-1 phosphate. These circulating precursors are attracted to bone surfaces undergoing resorption by chemokines and other factors expressed at these sites, where they fuse to form multinucleated bone-resorbing cells. All aspects of osteoclast formation and functions are regulated by macrophage-colony-stimulating factor (M-CSF) and receptor activator of NF-κB ligand (RANKL), cytokines essential for osteoclast formation and expressed by a variety of cell types, including osteoblast lineage cells. Since the discovery of RANKL in the mid-1990s, mouse genetic and molecular studies have revealed numerous signaling pathways activated by RANKL and M-CSF. More recent studies indicate that osteoclasts and their precursors regulate immune responses and osteoblast formation and functions by means of direct cell-cell contact through ligands and receptors, such as ephrins and Ephs, and semaphorins and plexins, and through expression of clastokines. There is also growing recognition that osteoclasts are immune cells with roles in immune responses beyond mediating the bone destruction that can accompany them. This article reviews recent advances in the understanding of the molecular mechanisms regulating osteoclast formation and functions and their interactions with other cells in normal and pathologic states.

Use of a whole-slide imaging system to assess the presence and alteration of lymphatic vessels in joint sections of arthritic mice.

We investigated the presence and alteration of lymphatic vessels in joints of arthritic mice using a whole-slide imaging system. Joints and long bone sections were cut from paraffin blocks of two mouse models of arthritis: meniscal-ligamentous injury (MLI)-induced osteoarthritis (OA) and TNF transgene (TNF-Tg)-induced rheumatoid arthritis (RA). MLI-OA mice were fed a high fat diet to accelerate OA development. TNF-Tg mice were treated with lymphatic growth factor VEGF-C virus to stimulate lymphangiogenesis. Sections were double immunofluorescence stained with anti-podoplanin and alpha-smooth muscle actin antibodies. The area and number of lymphatic capillaries and mature lymphatic vessels were determined using a whole-slide imaging system and its associated software. Lymphatic vessels in joints were distributed in soft tissues mainly around the joint capsule, ligaments, fat pads and muscles. In long bones, enriched lymphatic vessels were present in the periosteal areas adjacent to the blood vessels. Occasionally, lymphatic vessels were observed in the cortical bone. Increased lymphatic capillaries, but decreased mature lymphatic vessels, were detected in both OA and RA joints. VEGF-C treatment increased lymphatic capillary and mature vessel formation in RA joints. Our findings suggest that the lymphatic system may play an important role in arthritis pathogenesis and treatment.

The challenge and importance of standardizing pre-analytical variables in surgical pathology specimens for clinical care and translational research.

The introduction of targeted cancer therapies into clinical practice, in which patients are selected for novel treatments based on results of companion molecular testing of their tumor specimens, has created significant new challenges for the surgical pathology laboratory. These include standardization of tissue handling and sample preparation with accurate documentation to ensure optimal quality of clinical samples to reduce the risk of errors in molecular biology tests. The assay of tumor tissues for biomarkers that can provide predictive data for prognosis or treatment should enable selection of the most appropriate therapies (Yaziji et al. 2008, Hicks and Kulkarni 2008). Major advances have been made in the ability to profile clinical samples for research at the DNA, RNA and protein levels. To translate this new information into the clinical setting, however, the quality of the starting material, in this case the tumor tissue, determines the accuracy and reliability of companion diagnostic assay results and therefore optimal therapeutic strategies. Inaccurate results owing to compromised tissue quality can lead to false positive or false negative results with therapeutic consequences that can harm patients and affect their eventual outcome.

Polymicrobial periodontal pathogen transcriptomes in calvarial bone and soft tissue.

Porphyromonas gingivalis, Treponema denticola, and Tannerella forsythia are consistently associated with adult periodontitis. This study sought to document the host transcriptome to a P. gingivalis, T. denticola, and T.forsythia challenge as a polymicrobial infection using a murine calvarial model of acute inflammation and bone resorption. Mice were infected with P. gingivalis, T. denticola, and T. forsythia over the calvaria, after which the soft tissues and calvarial bones were excised. A Murine GeneChip(®) array analysis of transcript profiles showed that 6997 genes were differentially expressed in calvarial bones (P < 0.05) and 1544 genes were differentially transcribed in the inflamed tissues after the polymicrobial infection. Of these genes, 4476 and 1035 genes in the infected bone and tissues were differentially expressed by upregulation. Biological pathways significantly impacted by the polymicrobial infection in calvarial bone included leukocyte transendothelial migration (LTM), cell adhesion molecules, adherens junction, major histocompatibility complex antigen, extracellular matrix-receptor interaction, and antigen processing and presentation resulting in inflammatory/cytokine/chemokine transcripts stimulation in bone and soft tissue. Intense inflammation and increased activated osteoclasts were observed in calvarias compared with sham-infected controls. Quantitative real-time RT-PCR analysis confirmed that the mRNA level of selected genes corresponded with the microarray expression. The polymicrobial infection regulated several LTM and extracellular membrane pathway genes in a manner distinct from mono-infection with P. gingivalis, T. denticola, or T. forsythia. To our knowledge, this is the first definition of the polymicrobially induced transcriptome in calvarial bone and soft tissue in response to periodontal pathogens.

An inverse finite element method for determining the anisotropic properties of the cornea.

An inverse finite element method was developed to determine the anisotropic properties of bovine cornea from an in vitro inflation experiment. The experiment used digital image correlation (DIC) to measure the three-dimensional surface geometry and displacement field of the cornea at multiple pressures. A finite element model of a bovine cornea was developed using the DIC measured surface geometry of the undeformed specimen. The model was applied to determine five parameters of an anisotropic hyperelastic model that minimized the error between the measured and computed surface displacement field and to investigate the sensitivity of the measured bovine inflation response to variations in the anisotropic properties of the cornea. The results of the parameter optimization revealed that the collagen structure of bovine cornea exhibited a high degree of anisotropy in the limbus region, which agreed with recent histological findings, and a transversely isotropic central region. The parameter study showed that the bovine corneal response to the inflation experiment was sensitive to the shear modulus of the matrix at pressures below the intraocular pressure, the properties of the collagen lamella at higher pressures, and the degree of anisotropy in the limbus region. It was not sensitive to a weak collagen anisotropy in the central region.

Tannerella forsythia infection-induced calvarial bone and soft tissue transcriptional profiles.

Tannerella forsythia is associated with subgingival biofilms in adult periodontitis, although the molecular mechanisms contributing to chronic inflammation and loss of periodontal bone remain unclear. We examined changes in the host transcriptional profiles during a T. forsythia infection using a murine calvarial model of inflammation and bone resorption. Tannerella forsythia was injected into the subcutaneous soft tissue over calvariae of BALB/c mice for 3 days, after which the soft tissues and calvarial bones were excised. RNA was isolated and Murine GeneChip (Affymetrix, Santa Clara, CA) array analysis of transcript profiles showed that 3226 genes were differentially expressed in the infected soft tissues (P < 0.05) and 2586 genes were differentially transcribed in calvarial bones after infection. Quantitative real-time reverse transcription-polymerase chain reaction analysis of transcription levels of selected genes corresponded well with the microarray results. Biological pathways significantly impacted by T. forsythia infection in calvarial bone and soft tissue included leukocyte transendothelial migration, cell adhesion molecules (immune system), extracellular matrix-receptor interaction, adherens junction, and antigen processing and presentation. Histologic examination revealed intense inflammation and increased osteoclasts in calvariae compared with controls. In conclusion, localized T. forsythia infection differentially induces transcription of a broad array of host genes, and the profiles differ between inflamed soft tissues and calvarial bone.

Molecular characterization of Treponema denticola infection-induced bone and soft tissue transcriptional profiles.

Treponema denticola is associated with subgingival biofilms in adult periodontitis and with acute necrotizing ulcerative gingivitis. However, the molecular mechanisms by which T. denticola impacts periodontal inflammation and alveolar bone resorption remain unclear. Here, we examined changes in the host transcriptional profiles during a T. denticola infection using a murine calvarial model of inflammation and bone resorption. T. denticola was injected into the subcutaneous soft tissue over the calvaria of BALB/c mice for 3 days, after which the soft tissues and the calvarial bones were excised. RNA was isolated and analysed for transcript profiling using Murine GeneChip arrays. Following T. denticola infection, 2905 and 1234 genes in the infected calvarial bones and soft tissues, respectively, were differentially expressed (P

Porphyromonas gingivalis infection-induced tissue and bone transcriptional profiles.

Porphyromonas gingivalis has been associated with subgingival biofilms in adult periodontitis. However, the molecular mechanisms of its contribution to chronic gingival inflammation and loss of periodontal structural integrity remain unclear. This investigation aimed to examine changes in the host transcriptional profiles during a P. gingivalis infection using a murine calvarial model of inflammation and bone resorption. P. gingivalis FDC 381 was injected into the subcutaneous soft tissue over the calvaria of BALB/c mice for 3 days, after which the soft tissues and calvarial bones were excised. RNA was isolated from infected soft tissues and calvarial bones and was analysed for transcript profiles using Murine GeneChip((R)) arrays to provide a molecular profile of the events that occur following infection of these tissues. After P. gingivalis infection, 6452 and 2341 probe sets in the infected soft tissues and calvarial bone, respectively, were differentially expressed (P

Repair of full-thickness femoral condyle cartilage defects using allogeneic synovial cell-engineered tissue constructs.

OBJECTIVE: Synovium-derived stem cells (SDSCs) have proven to be superior in cartilage regeneration compared with other sources of mesenchymal stem cells. We hypothesized that conventionally passaged SDSCs can be engineered in vitro into cartilage tissue constructs and the engineered premature tissue can be implanted to repair allogeneic full-thickness femoral condyle cartilage defects without immune rejection. METHODS: Synovial tissue was harvested from rabbit knee joints. Passage 3 SDSCs were mixed with fibrin glue and seeded into non-woven polyglycolic acid (PGA) mesh. After 1-month incubation with growth factor cocktails, the premature tissue was implanted into rabbit knees to repair osteochondral defects with Collagraft as a bone substitute in the Construct group. Fibrin glue-saturated PGA/Collagraft composites were used as a Scaffold group. The defect was left untreated as an Empty group. RESULTS: SDSCs were engineered in rotating bioreactor systems into premature cartilage, which displayed the expression of sulfated glycosaminoglycan (GAG), collagen II, collagen I, and macrophages. Six months after implantation with premature tissue, cartilage defects were full of smooth hyaline-like cartilage with no detectable collagen I and macrophages but a high expression of collagen II and GAG, which were also integrated with the surrounding native cartilage. The Scaffold and Empty groups were resurfaced with fibrous-like and fibrocartilage tissue, respectively. CONCLUSION: Allogeneic SDSC-based premature tissue constructs are a promising stem cell-based approach for cartilage defects. Although in vitro data suggest that contaminated macrophages affected the quality of SDSC-based premature cartilage, effects of macrophages on in vivo tissue regeneration and integration necessitate further investigation.

A nonlinear anisotropic viscoelastic model for the tensile behavior of the corneal stroma.

Tensile strip experiments of bovine corneas have shown that the tissue exhibits a nonlinear rate-dependent stress-strain response and a highly nonlinear creep response that depends on the applied hold stress. In this paper, we present a constitutive model for the finite deformation, anisotropic, nonlinear viscoelastic behavior of the corneal stroma. The model formulates the elastic and viscous response of the stroma as the average of the elastic and viscous response of the individual lamellae weighted by a probability density function of the preferred in-plane lamellar orientations. The result is a microstructure-based model that incorporates the viscoelastic properties of the matrix and lamellae and the lamellar architecture in the response of the stroma. In addition, the model includes a fully nonlinear description of the viscoelastic response of the lamellar(fiber) level. This is in contrast to previous microstructure-based models of fibrous soft tissues, which relied on quasilinear viscoelastic formulations of the fiber viscoelasticity. Simulations of recent tensile strip experiments show that the model is able to predict, well within the bounds of experimental error and natural variations, the cyclic stress-strain behavior and nonlinear creep behavior observed in uniaxial tensile experiments of excised strips of bovine cornea.

Stress-controlled viscoelastic tensile response of bovine cornea.

The viscoelastic response of bovine corneas was characterized using in vitro load-controlled uniaxial tension experiments. Specifically, two types of tests were employed: cycled ramp tests over a range of loading rates and creep tests over a range of hold stresses. Multiple replicates of each were used to quantify natural variability as well as mean trends. A preconditioning protocol was used to obtain a unique reference state before testing and to overcome the effects of non-physiological loading. A quasi-linear viscoelastic model incorporating a representation of the microstructure of the cornea was compared to the experimental results. For low stresses and moderate durations this model compares favorably, but overall the material displays non-linearities that cannot be represented within the quasi-linear framework.

Tumor necrosis factor prevents alendronate-induced osteoclast apoptosis in vivo by stimulating Bcl-xL expression through Ets-2.

OBJECTIVE: To investigate why bisphosphonates are less effective at preventing focal bone loss in rheumatoid arthritis (RA) patients than in those with generalized osteoporosis, and the mechanisms involved. METHODS: The response of osteoclasts to alendronate (ALN) in tumor necrosis factor-transgenic (TNF-Tg) mice that develop erosive arthritis and in wild-type littermates was studied. TNF-Tg and wild-type mice were given ALN, and the osteoclast numbers in the inflamed joints and in the long bones were compared. The expression levels of Bcl-xL in the osteoclasts of TNF-Tg and wild-type mice were examined by immunostaining. The effect of overexpression of Bcl-xL and Ets-2 proteins on ALN-induced osteoclast apoptosis was determined using an in vitro osteoclast survival assay and retrovirus transfer approach. RESULTS: ALN reduced osteoclast numbers in the metaphyses by 97%, but by only 46% in the adjacent inflamed joints. Bcl-xL expression was markedly higher in osteoclasts in the joints than in those in the metaphyses of TNF-Tg mice. Bcl-xL or Ets-2 overexpression protected osteoclasts from ALN-induced apoptosis, and TNF stimulated Bcl-xL and Ets-2 expression in osteoclasts. Overexpression of Ets-2 increased Bcl-xL messenger RNA in osteoclasts, while a dominant-negative form of the Ets-2 blocked the protective effect of Bcl-xL or TNF on ALN-induced apoptosis. CONCLUSION: The reduced efficacy of bisphosphonates to stop bone erosion in the inflamed joints of RA patients may result from local high levels of TNF up-regulating Ets-2 expression in osteoclasts, which in turn stimulates Bcl-xL expression in them and reduces their susceptibility to bisphosphonate-induced apoptosis.