The subacromial bursa modulates tendon healing after rotator cuff injury in rats.

Rotator cuff injuries result in more than 500,000 surgeries annually in the United States, many of which fail. These surgeries typically involve repair of the injured tendon and removal of the subacromial bursa, a synovial-like tissue that sits between the rotator cuff and the acromion. The subacromial bursa has been implicated in rotator cuff pathogenesis and healing. Using proteomic profiling of bursa samples from nine patients with rotator cuff injury, we show that the bursa responds to injury in the underlying tendon. In a rat model of supraspinatus tenotomy, we evaluated the bursa's effect on the injured supraspinatus tendon, the uninjured infraspinatus tendon, and the underlying humeral head. The bursa protected the intact infraspinatus tendon adjacent to the injured supraspinatus tendon by maintaining its mechanical properties and protected the underlying humeral head by maintaining bone morphometry. The bursa promoted an inflammatory response in injured rat tendon, initiating expression of genes associated with wound healing, including Cox2 and Il6. These results were confirmed in rat bursa organ cultures. To evaluate the potential of the bursa as a therapeutic target, polymer microspheres loaded with dexamethasone were delivered to the intact bursae of rats after tenotomy. Dexamethasone released from the bursa reduced Il1b expression in injured rat supraspinatus tendon, suggesting that the bursa could be used for drug delivery to reduce inflammation in the healing tendon. Our findings indicate that the subacromial bursa contributes to healing in underlying tissues of the shoulder joint, suggesting that its removal during rotator cuff surgery should be reconsidered.

A role for TGFß signaling in Gli1+ tendon and enthesis cells.

The development of musculoskeletal tissues such as tendon, enthesis, and bone relies on proliferation and differentiation of mesenchymal progenitor cells. Gli1+ cells have been described as putative stem cells in several tissues and are presumed to play critical roles in tissue formation and maintenance. For example, the enthesis, a fibrocartilage tissue that connects tendon to bone, is mineralized postnatally by a pool of Gli1+ progenitor cells. These cells are regulated by hedgehog signaling, but it is unclear if TGFß signaling, necessary for tenogenesis, also plays a role in their behavior. To examine the role of TGFß signaling in Gli1+ cell function, the receptor for TGFß, TbR2, was deleted in Gli1-lineage cells in mice at P5. Decreased TGFß signaling in these cells led to defects in tendon enthesis formation by P56, including defective bone morphometry underlying the enthesis and decreased mechanical properties. Immunohistochemical staining of these Gli1+ cells showed that loss of TGFß signaling reduced proliferation and increased apoptosis. In vitro experiments using Gli1+ cells isolated from mouse tail tendons demonstrated that TGFß controls cell proliferation and differentiation through canonical and non-canonical pathways and that TGFß directly controls the tendon transcription factor scleraxis by binding to its distant enhancer. These results have implications in the development of treatments for tendon and enthesis pathologies.

Hedgehog Activation for Enhanced Rotator Cuff Tendon-to-Bone Healing.

BACKGROUND: Rotator cuff repair is a common orthopaedic procedure, yet the rate of failure to heal after surgery is high. Repair site rupture is due to poor tendon-to-bone healing and lack of regeneration of the native fibrocartilaginous enthesis. During development, the enthesis is formed and mineralized by a pool of progenitors activated by hedgehog signaling. Furthermore, hedgehog signaling drives regenerative enthesis healing in young animals, in contrast to older animals, in which enthesis injuries heal via fibrovascular scar and without participation of hedgehog signaling. HYPOTHESIS: Hedgehog activation improves tendon-to-bone healing in an animal model of rotator cuff repair. STUDY DESIGN: Controlled laboratory study. METHODS: A total of 78 adult Sprague-Dawley rats were used. Supraspinatus tendon injury and repair were completed bilaterally, with microsphere-encapsulated hedgehog agonist administered to right shoulders and control microspheres administered to left shoulders. Animals were sacrificed after 3, 14, 28, or 56 days. Gene expression and histological, biomechanical, and bone morphometric analyses were conducted. RESULTS: At 3 days, hedgehog signaling pathway genes Gli1 (1.70; P = .029) and Smo (2.06; P = .0173), as well as Runx2 (1.69; P = .0386), a transcription factor of osteogenesis, were upregulated in treated relative to control repairs. At 14 days, transcription factors of tenogenesis, Scx (4.00; P = .041), and chondrogenesis, Sox9 (2.95; P = .010), and mineralized fibrocartilage genes Col2 (3.18; P = .031) and Colx (1.85; P = .006), were upregulated in treated relative to control repairs. Treatment promoted fibrocartilage formation at the healing interface by 28 days, with improvements in tendon-bone maturity, organization, and continuity. Treatment led to improved biomechanical properties. The material property strength (2.43 vs 1.89 N/m2; P = .046) and the structural property work to failure (29.01 vs 18.09 mJ; P = .030) were increased in treated relative to control repairs at 28 days and 56 days, respectively. Treatment had a marginal effect on bone morphometry underlying the repair. Trabecular thickness (0.08 vs 0.07 mm; P = .035) was increased at 28 days. CONCLUSION: Hedgehog agonist treatment activated hedgehog signaling at the tendon-to-bone repair site and prompted increased mineralized fibrocartilage production. This extracellular matrix production and mineralization resulted in improved biomechanical properties, demonstrating the therapeutic potential of hedgehog agonism for improving tendon-to-bone healing after rotator cuff repair. CLINICAL RELEVANCE: This study demonstrates the therapeutic potential of hedgehog agonist treatment for improving tendon-to-bone healing after rotator cuff injury and repair.

Solubilized curcuminoid complex prevents extensive immunosuppression through immune restoration and antioxidant activity: Therapeutic potential against SARS-CoV-2 (COVID-19).

The therapeutic benefits of curcuminoids in various diseases have been extensively reported. However, little is known regarding their preventive effects on extensive immunosuppression. We investigated the immunoregulatory effects of a curcuminoid complex (CS/M), solubilized with stevioside, using a microwave-assisted method in a cyclophosphamide (CTX)-induced immunosuppressive mouse model and identified its new pharmacological benefits. CTX-treated mice showed a decreased number of innate cells, such as dendritic cells (DCs), neutrophils, and natural killer (NK) cells, and adaptive immune cells (CD4 and CD8 T cells) in the spleen. In addition, CTX administration decreased T cell activation, especially that of Th1 and CD8 T cells, whereas it increased Th2 and regulatory T (Treg) cell activations. Pre-exposure of CS/M to CTX-induced immunosuppressed mice restored the number of innate cells (DCs, neutrophils, and NK cells) and increased their activity (including the activity of macrophages). Exposure to CS/M also led to the superior restoration of T cell numbers, including Th1, activated CD8 T cells, and multifunctional T cells, suppressed by CTX, along with a decrease in Th2 and Treg cells. Furthermore,CTX-injected mice pre-exposed to CS/M were accompanied by an increase in the levels of antioxidant enzymes (superoxide dismutase, catalase, and glutathione peroxidase), which play an essential role against oxidative stress. Importantly, CS/M treatment significantly reduced viral loads in severe acute respiratory syndrome coronavirus2-infected hamsters and attenuated the gross pathology in the lungs. These results provide new insights into the immunological properties of CS/M in preventing extensive immunosuppression and offer new therapeutic opportunities against various cancers and infectious diseases caused by viruses and intracellular bacteria.

Enhanced Tendon-to-Bone Healing via IKKß Inhibition in a Rat Rotator Cuff Model.

BACKGROUND: More than 450,000 rotator cuff repairs are performed annually, yet healing of tendon to bone often fails. This failure is rooted in the fibrovascular healing response, which does not regenerate the native attachment site. Better healing outcomes may be achieved by targeting inflammation during the early period after repair. Rather than broad inhibition of inflammation, which may impair healing, the current study utilized a molecularly targeted approach to suppress IKKß, shutting down only the inflammatory arm of the nuclear factor κB (NF-κB) signaling pathway. PURPOSE: To evaluate the therapeutic potential of IKKß inhibition in a clinically relevant model of rat rotator cuff repair. STUDY DESIGN: Controlled laboratory study. METHODS: After validating the efficacy of the IKKß inhibitor in vitro, it was administered orally once a day for 7 days after surgery in a rat rotator cuff repair model. The effect of treatment on reducing inflammation and improving repair quality was evaluated after 3 days and 2, 4, and 8 weeks of healing, using gene expression, biomechanics, bone morphometry, and histology. RESULTS: Inhibition of IKKß attenuated cytokine and chemokine production in vitro, demonstrating the potential for this inhibitor to reduce inflammation in vivo. Oral treatment with IKKß inhibitor reduced NF-κB target gene expression by up to 80% compared with a nontreated group at day 3, with a subset of these genes suppressed through 14 days. Furthermore, the IKKß inhibitor led to enhanced tenogenesis and extracellular matrix production, as demonstrated by gene expression and histological analyses. At 4 weeks, inhibitor treatment led to increased toughness, no effects on failure load and strength, and decreases in stiffness and modulus when compared with vehicle control. At 8 weeks, IKKß inhibitor treatment led to increased toughness, failure load, and strength compared with control animals. IKKß inhibitor treatment prevented the bone loss near the tendon attachment that occurred in repairs in control. CONCLUSION: Pharmacological inhibition of IKKß successfully suppressed excessive inflammation and enhanced tendon-to-bone healing after rotator cuff repair in a rat model. CLINICAL RELEVANCE: The NF-κB pathway is a promising target for enhancing outcomes after rotator cuff repair.

Development of a new thiol-reactive prosthetic group for site-specific labeling of biomolecules with radioactive iodine.

In this report, we describe the radiosynthesis of a new thiol-targeting prosthetic group for efficient radioactive iodine labeling of biomolecules. Radioiodination using the precursor 3 was performed to obtain 125I-labeled tetrazole 4b with high radiochemical yield (73%) and radiochemical purity. Using the radiolabeled 4b, a single free cysteine containing peptide and human serum albumin were labeled with 125I in modest-to-good radiochemical yields (65-99%) under mildly reactive conditions. A biodistribution study of [125I]7 in normal ICR mice exhibited lower thyroid uptake values than those of 125I-labeled human serum albumin prepared via a traditional radiolabeling method. Thus, [125I]7 could be employed as an effective radiotracer for molecular imaging and biodistribution studies. The results clearly demonstrate that 4b has the potential to be effectively implemented as a prosthetic group in the preparation of radiolabeled biomolecules.

Tart Cherry Prevents Bone Loss through Inhibition of RANKL in TNF-Overexpressing Mice.

Current drugs for the treatment of rheumatoid arthritis-associated bone loss come with concerns about their continued use. Thus, it is necessary to identify natural products with similar effects, but with fewer or no side effects. We determined whether tart cherry (TC) could be used as a supplement to prevent inflammation-mediated bone loss in tumor necrosis factor (TNF)-overexpressing transgenic (TG) mice. TG mice were assigned to a 0%, 5%, or 10% TC diet, with a group receiving infliximab as a positive control. Age-matched wild-type (WT) littermates fed a 0% TC diet were used as a normal control. Mice were monitored by measurement of body weight. Bone health was evaluated via serum biomarkers, microcomputed tomography (µCT), molecular assessments, and mechanical testing. TC prevented TNF-mediated weight loss, while it did not suppress elevated levels of interleukin (IL)-1ß and IL-6. TC also protected bone structure from inflammation-induced bone loss with a reduced ratio of receptor activator of nuclear factor kappa-B ligand (RANKL)/osteoprotegerin (OPG) to a degree comparable to infliximab. Furthermore, unlike with infliximab, TC exhibited a moderate improvement in TNF-mediated decline in bone stiffness. Thus, TC could be used as a prophylactic regimen against future fragility fractures in the context of highly chronic inflammation.

Calreticulin inhibits inflammation-induced osteoclastogenesis and bone resorption.

Osteoclasts play key roles in bone remodeling and pathologic osteolytic disorders such as inflammation, infection, bone implant loosening, rheumatoid arthritis, metastatic bone cancers, and pathological fractures. Osteoclasts are formed by the fusion of monocytes in response to receptor activators of NF-κB-ligand (RANKL) and macrophage colony stimulating factor 1 (M-CSF). Calreticulin (CRT), a commonly known intracellular protein as a calcium-binding chaperone, has an unexpectedly robust anti-osteoclastogenic effect when its recombinant form is applied to osteoclast precursors in vitro or at the site of bone inflammation externally in vivo. Externally applied Calreticulin was internalized inside the cells. It inhibited key pro-osteoclastogenic transcription factors such as c-Fos and nuclear factor of activated T cells, cytoplasmic 1 (NFATc1)-in osteoclast precursor cells that were treated with RANKL in vitro. Recombinant human Calreticulin (rhCRT) inhibited lipopolysaccharide (LPS)-induced inflammatory osteoclastogenesis in the mouse calvarial bone in vivo. Cathepsin K molecular imaging verified decreased Cathepsin K activity when rhCalreticulin was applied at the site of LPS application in vivo. Recombinant forms of intracellular proteins or their derivatives may act as novel extracellular therapeutic agents. We anticipate our findings to be a starting point in unraveling hidden extracellular functions of other intracellular proteins in different cell types of many organs for new therapeutic opportunities. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:2658-2666, 2017.

Subclinical Hypothyroidism in Childhood Cancer Survivors.

PURPOSE: In childhood cancer survivors, the most common late effect is thyroid dysfunction, most notably subclinical hypothyroidism (SCH). Our study evaluated the risk factors for persistent SCH in survivors. MATERIALS AND METHODS: Survivors (n=423) were defined as patients who survived at least 2 years after cancer treatment completion. Thyroid function was assessed at this time and several years thereafter. Two groups of survivors with SCH were compared: those who regained normal thyroid function during the follow-up period (normalized group) and those who did not (persistent group). RESULTS: Overall, 104 of the 423 survivors had SCH. SCH was observed in 26% of brain or nasopharyngeal cancer survivors (11 of 43) and 21.6% of leukemia survivors (35 of 162). Sixty-two survivors regained normal thyroid function, 30 remained as persistent SCH, and 12 were lost to follow-up. The follow-up duration was 4.03 (2.15-5.78) years. Brain or nasopharyngeal cancer and Hodgkin disease were more common in the persistent group than in the normalized group (p=0.002). More patients in the persistent group received radiation (p=0.008). Radiation to the head region was higher in this group (2394±2469 cGy) than in the normalized group (894±1591 cGy; p=0.003). On multivariable analysis, lymphoma (p=0.011), brain or nasopharyngeal cancer (p=0.039), and head radiation dose ≥1800 cGy (p=0.039) were significant risk factors for persistent SCH. CONCLUSION: SCH was common in childhood cancer survivors. Brain or nasopharyngeal cancer, lymphoma, and head radiation ≥1800 cGy were significant risk factors for persistent SCH.

Symptom Interval and Patient Delay Affect Survival Outcomes in Adolescent Cancer Patients.

PURPOSE: Unique features of adolescent cancer patients include cancer types, developmental stages, and psychosocial issues. In this study, we evaluated the relationship between diagnostic delay and survival to improve adolescent cancer care. MATERIALS AND METHODS: A total of 592 patients aged 0-18 years with eight common cancers were grouped according to age (adolescents, ≥10 years; children, <10 years). We retrospectively reviewed their symptom intervals (SIs, between first symptom/sign of disease and diagnosis), patient delay (PD, between first symptom/sign of disease and first contact with a physician), patient delay proportion (PDP), and overall survival (OS). RESULTS: Mean SI was significantly longer in adolescents than in children (66.4 days vs. 28.4 days; p<0.001), and OS rates were higher in patients with longer SIs (p=0.001). In children with long SIs, OS did not differ according to PDP (p=0.753). In adolescents with long SIs, OS was worse when PDP was ≥0.6 (67.2%) than <0.6 (95.5%, p=0.007). In a multivariate analysis, adolescents in the long SI/PDP ≥0.6 group tended to have a higher hazard ratio (HR, 6.483; p=0.069) than those in the long SI/PDP <0.6 group (HR=1, reference). CONCLUSION: Adolescents with a long SI/PDP ≥0.6 had lower survival rates than those with a short SI/all PDP or a long SI/PDP <0.6. They should be encouraged to seek prompt medical assistance by a physician or oncologist to lessen PDs.

Poor bone health at the end of puberty in childhood cancer survivors.

BACKGROUND: Although the survival rate following childhood cancer is >80%, late effects are a major concern. We aimed to determine the clinical factors affecting bone health after puberty in childhood cancer survivors at risk for low bone mineral density (BMD). PROCEDURES: We performed dual-energy X-ray absorptiometry at the lumbar spine, femoral neck, and total hip regions for survivors with the following bone densitometry indications (BDIXs): brain or nasopharyngeal cancer, head or neck area radiotherapy, or corticosteroid treatment (N = 92). Additionally, we evaluated 16 survivors without these BDIXs but with other clinical factors that could affect bone health. We assessed the effects of these factors on BMD using univariate and logistic regression analyses. Moderate BMD deficit was defined as a Z-score of <-1.0 and ≥-2.0, and severe BMD deficit was defined as <-2.0. RESULTS: Severe BMD deficits were found in 18 survivors (16.7%) and moderate BMD deficits were in 39 (36.1%) in at least one bone region. BMD deficits tended to increase as the number of BDIXs increased (P < 0.010). There were no severe BMD deficits in survivors without BDIXs. The duration since cancer treatment completion was correlated with higher BMD (P < 0.05). Endocrine dysfunction was a significant risk factor for decreased BMD in univariate and multivariate analyses (P < 0.05 for both). CONCLUSIONS: Decreased BMD was prevalent in our study cohort. Endocrine dysfunction was found to be a significant risk factor, and it should be managed in survivors to ensure future bone health.

CA-074Me compound inhibits osteoclastogenesis via suppression of the NFATc1 and c-FOS signaling pathways.

The osteoclast is an integral cell of bone resorption. Since osteolytic disorders hinge on the function and dysfunction of the osteoclast, understanding osteoclast biology is fundamental to designing new therapies that curb osteolytic disorders. The identification and study of lysosomal proteases, such as cathepsins, have shed light on mechanisms of bone resorption. For example, Cathepsin K has already been identified as a collagen degradation protease produced by mature osteoclasts with high activity in the acidic osteoclast resorption pits. Delving into the mechanisms of cathepsins and other osteoclast related compounds provides new targets to explore in osteoclast biology. Through our anti-osteoclastogenic compound screening experiments we encountered a modified version of the Cathepsin B inhibitor CA-074: the cell membrane-permeable CA-074Me (L-3-trans-(Propylcarbamoyl) oxirane-2-carbonyl]-L-isoleucyl-L-proline Methyl Ester). Here we confirm that CA-074Me inhibits osteoclastogenesis in vivo and in vitro in a dose-dependent manner. However, Cathepsin B knockout mice exhibited unaltered osteoclastogenesis, suggesting a more complicated mechanism of action than Cathepsin B inhibition. We found that CA-074Me exerts its osteoclastogenic effect within 24 h of osteoclastogenesis stimulation by suppression of c-FOS and NFATc1 pathways.

Lipoma of the cerebral convexity.

A computerised tomographic scan revealed a space-occupying lesion in the right parieto-occipital region of a 34-year-old male patient. He had suffered frequent generalized convulsions and a left-sided hemiparesis since early childhood, but with normal physical and intellectual development. Total resection of the tumour was performed and the pathological finding confirmed the diagnosis of lipoma.