Angiogenic conditioning of peripheral blood mononuclear cells promotes fracture healing.

OBJECTIVES: The objective of this study was to investigate the therapeutic effect of peripheral blood mononuclear cells (PBMNCs) treated with quality and quantity control culture (QQ-culture) to expand and fortify angiogenic cells on the acceleration of fracture healing. METHODS: Human PBMNCs were cultured for seven days with the QQ-culture method using a serum-free medium containing five specific cytokines and growth factors. The QQ-cultured PBMNCs (QQMNCs) obtained were counted and characterised by flow cytometry and real-time polymerase chain reaction (RT-PCR). Angiogenic and osteo-inductive potentials were evaluated using tube formation assays and co-culture with mesenchymal stem cells with osteo-inductive medium in vitro. In order to evaluate the therapeutic potential of QQMNCs, cells were transplanted into an immunodeficient rat femur nonunion model. The rats were randomised into three groups: control; PBMNCs; and QQMNCs. The fracture healing was evaluated radiographically and histologically. RESULTS: The total number of PBMNCs was decreased after QQ-culture, however, the number of CD34+ and CD206+ cells were found to have increased as assessed by flow cytometry analysis. In addition, gene expression of angiogenic factors was upregulated in QQMNCs. In the animal model, the rate of bone union was higher in the QQMNC group than in the other groups. Radiographic scores and bone volume were significantly associated with the enhancement of angiogenesis in the QQMNC group. CONCLUSION: We have demonstrated that QQMNCs have superior potential to accelerate fracture healing compared with PBMNCs. The QQMNCs could be a promising option for fracture nonunion.Cite this article: K. Mifuji, M. Ishikawa, N. Kamei, R. Tanaka, K. Arita, H. Mizuno, T. Asahara, N. Adachi, M. Ochi. Angiogenic conditioning of peripheral blood mononuclear cells promotes fracture healing. Bone Joint Res 2017;6: 489-498. DOI: 10.1302/2046-3758.68.BJR-2016-0338.R1.

The relationship between central motor conduction time and spinal cord compression in patients with cervical spondylotic myelopathy.

STUDY DESIGN: Retrospective study. OBJECTIVES: Few studies have reported a relationship between central motor conduction time (CMCT), which evaluates corticospinal function, and degree of spinal cord compression in patients with myelopathy. Thus, there is no consensus on predicting the degree of prolonged CMCT on the basis of the degree of spinal cord compression. If a correlation exists between CMCT and spinal cord compression, then spinal cord compression may be a useful noninvasive clinical indicator of corticospinal function. Therefore, this study evaluated the relationship between CMCT and cervical spinal cord compression measured by magnetic resonance imaging (MRI) in patients with cervical spondylotic myelopathy (CSM). SETTING: Hiroshima University Hospital in Japan. METHODS: We studied 33 patients undergoing laminoplasty. Patients exhibited significant cervical spinal cord compression on both MRI and intraoperative electrophysiological examination. We assessed transcranial magnetic stimulation measurement of CMCT; spinal cord compression parameters such as area, lateral diameter, anteroposterior diameter and flattening of the spinal cord at the lesion site and C2/3 levels on MRI; and pre- versus postoperative Japanese Orthopaedic Association (JOA) scores. RESULTS: Correlations between CMCT and flattening as well as anteroposterior diameter of the spinal cord at the lesion level were observed. Strong correlations between CMCT and the ratio of the flattening and anteroposterior diameter parameters at the lesion level to that at the C2/3 level were also observed. CONCLUSIONS: Measurement of spinal cord compression may be useful for the evaluation of corticospinal function as a proxy for CMCT in patients with CSM.

Resection of spinous processes can cause spinal cord injury in patient with ossification of the posterior longitudinal ligament in the thoracic spine.

STUDY DESIGN: Case report. OBJECTIVE: To report intraoperative spinal cord injury by resection of spinous processes in a 73-year-old man with ossification of the posterior longitudinal ligament (OPLL) in the thoracic spine. METHODS: A 73-year-old man presented with cervicothoracic OPLL with bilateral numbness and clumsiness of his hand, weakness of his lower extremities and severe gait disturbance. His Japanese Orthopaedic Association (JOA) score was 7.5 out of 17. Cervical laminoplasty (C2-6), cervicothoracic laminectomy (C7-T10) and posterior fusion (C7-T10) were performed in the prone position with electrophysiologic monitoring of the spinal cord-evoked potentials (SCEPs). RESULTS: The spinal processes with supra- and interspinous ligaments between C7 and T10 were resected. After resection, the amplitude of SCEP waveforms decreased rapidly to <10% of control levels. Laminectomy was performed, and, after 80 min of SCEP deterioration, an instrumented fusion with correction for kyphosis was completed. The SCEP amplitude recovered gradually. Immediately after surgery, the patient suffered severe motor loss in both lower limbs. His neurological recovery progressed gradually from 2 days after surgery, and he was able to walk at 3 months after surgery. At 6 years after surgery, the JOA score was 11. CONCLUSION: Our results suggest that intraoperative spinal cord injury can occur before posterior decompression by resection of spinal processes with supra- and interspinous ligaments. The timing of the instrumented stabilization using a temporary rod is important and should be considered immediately after posterior exposure of the spine.

Circulating microRNAs as biomarkers for evaluating the severity of acute spinal cord injury.

STUDY DESIGN: An in vivo study in mouse models of spinal cord contusion. OBJECTIVES: To develop a novel indicator to anticipate the severity of spinal cord injury (SCI) during the acute phase and for the assessment of the efficacy of novel therapies. MicroRNAs (miRNAs) circulating in the peripheral blood are reported to modulate signaling between cells, and to be diagnostic markers for cancers. The purpose of this study was to identify circulating miRNAs for predicting the severity of SCI in the acute phase. SETTING: Department of Orthopaedic Surgery, Graduate School of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan. METHODS: Mouse SCI models were made using Infinite Horizon impactor with 50 or 70 kdyn compressing power following thoracic laminectomy. The mice were then divided into four groups: normal (without surgery), sham (laminectomy only), mild (50 kdyn), and severe (70 kdyn). TaqMan low-density array analysis and real-time PCR were performed to identify candidate miRNAs that were increased in the serum relative to the severity of SCI. RESULTS: The expression levels of miR-9*, miR-219 and miR-384-5p in the serum were significantly increased relative to the severity of SCI 12 h after injury. The expression of miR-9* was also significantly increased relative to injury severity at 3 and 24 h after injury. CONCLUSION: Serum miR-9*, miR-219 and miR-384-5p might be promising biomarkers for predicting the severity of SCI.

In vivo bioluminescence imaging of transplanted bone marrow mesenchymal stromal cells using a magnetic delivery system in a rat fracture model.

For the treatment of ununited fractures, we developed a system of delivering magnetic labelled mesenchymal stromal cells (MSCs) using an extracorporeal magnetic device. In this study, we transplanted ferucarbotran-labelled and luciferase-positive bone marrow-derived MSCs into a non-healing femoral fracture rat model in the presence of a magnetic field. The biological fate of the transplanted MSCs was observed using luciferase-based bioluminescence imaging and we found that the number of MSC derived photons increased from day one to day three and thereafter decreased over time. The magnetic cell delivery system induced the accumulation of photons at the fracture site, while also retaining higher photon intensity from day three to week four. Furthermore, radiological and histological findings suggested improved callus formation and endochondral ossification. We therefore believe that this delivery system may be a promising option for bone regeneration.

Low serum levels of total and high-molecular-weight adiponectin predict the development of metabolic syndrome in Japanese-Americans.

BACKGROUND: Adiponectin is thought to play a significant role in the development of both insulin resistance and metabolic syndrome. Yet, there is very few evidence about the association plasma adiponectin and metabolic syndrome in the prospective study. Adiponectin exists as multimers in serum, and high-molecular-weight (HMW) adiponectin is particularly considered to be the active form of the protein. AIM: We investigated whether serum HMW adiponectin as well as total adiponectin is associated with the development of metabolic syndrome in a longitudinal study. SUBJECTS AND METHODS: We enrolled 224 men and 312 women of Japanese- Americans without metabolic syndrome at baseline who were followed for an average of 3.2 yr. The association of plasma total and HMW adiponectin with a progression to metabolic syndrome was examined. RESULTS: Subjects who developed metabolic syndrome had significantly lower plasma total and HMW adiponectin levels at baseline than those who did not develop metabolic syndrome. In a Cox proportional hazards model, lower total and HMW adiponectin levels were independent risk factors for the development of metabolic syndrome after adjusting for age, body mass index, classification of 75-g glucose tolerance test, and homeostasis model assessment (hazards ratio: total, 0.684, p=0.017, in men; 0.606, p=0.003, in women; HMW, 0.687, p=0.014, in men; 0.704, p=0.029, in women, respectively). CONCLUSIONS: Low circulating levels of total and HMW adiponectin may be a possible predictor for the development of metabolic syndrome.

Responses of microRNAs 124a and 223 following spinal cord injury in mice.

STUDY DESIGN: We investigated microRNA (miRNA) expression after spinal cord injury (SCI) in mice. OBJECTIVES: The recent discovery of miRNAs suggests a novel regulatory control over gene expression during plant and animal development. MiRNAs are short noncoding RNAs that suppress the translation of target genes by binding to their mRNAs, and play a central role in gene regulation in health and disease. The purpose of this study was to examine miRNA expression after SCI. SETTING: Department of Orthopaedic Surgery, Graduate School of Biomedical Sciences, Hiroshima University. METHODS: We examined the expression of miRNA (miR)-223 and miR-124a in a mouse model at 6 h, 12 h, 1 day, 3 days and 7 days after SCI using quantitative PCR. The miRNA expression was confirmed by in situ hybridization. RESULTS: Quantitative PCR revealed two peaks of miR-223 expression at 6 and 12 h and 3 days after SCI. MiR-124a expression decreased significantly from 1 day to 7 days after SCI. In situ hybridization demonstrated the presence of miR-223 around the injured site. However, miR-124a, which was present in the normal spinal cord, was not observed at the injured site. CONCLUSION: Our results indicate a time-dependent expression pattern of miR-223 and miR-124a in a mouse model of SCI. In this study, the time course of miRNA-223 expression may be related to inflammatory responses after SCI, and the time course of decreased miR-124a expression may reflect cell death.

Chondroitinase ABC promotes corticospinal axon growth in organotypic cocultures.

STUDY DESIGN: Organotypic coculture model using brain cortex and spinal cord of neonatal rats was used to test the effect of chondroitinase ABC (ChABC) on corticospinal axon growth. OBJECTIVE: Chondroitin sulfate proteoglycan (CSPG) is neurite outgrowth inhibitory factor that combines with reactive astrocyte at the lesion site to form a dense scar that acts as a barrier to regenerating axons. ChABC is a bacteria enzyme that digests the glycosaminoglycan side chain of CSPG. We investigated the effect of ChABC on corticospinal axon growth quantitatively using the organotypic cocultures of brain cortex and spinal cord. SETTING: Department of Orthopaedic Surgery, Graduate School of Biomedical Sciences, Hiroshima University. METHOD: We used organotypic cocultures with neonatal brain cortex and spinal cord as an in vitro assay system for assessing axon growth. After administering ChABC, we counted the number of axons passing through a reference line running parallel to the junction between the brain cortex and spinal cord 500 and 1000 microm from the junction. The immunoreactivity of CSPG was assessed. RESULT: The average number of axons after ChABC administration was significantly greater than in the control group. Administration of ChABC decreased CSPG expression in this coculture system. CONCLUSION: ChABC induces axonal regeneration by degrading CSPG after central nerve system injury. ChABC has great potential for future therapeutic use in spinal cord-injured patients.

The effect of polymorphism in the intestinal fatty acid-binding protein 2 gene on fat metabolism is associated with gender and obesity amongst non-diabetic Japanese-Americans.

AIM: The role of the codon 54 polymorphism of the fatty acid-binding protein 2 (FABP2) gene on fat metabolism has been controversial. Assuming that the effects of the polymorphism were modulated by gender and obesity which were related to lipid and glucose metabolism, we investigated this polymorphism and its effect on fat metabolism according to such factors. METHODS: Subjects were Japanese-Americans (123 men and 126 women) who were diagnosed as non-diabetic by a 75 g oral glucose tolerance test at the baseline. RESULTS: During approximately 7.8 years, 49 (24 men and 25 women) were diagnosed with type 2 diabetes. In a Cox proportional hazards model, this polymorphism was not a significant variable in the incidence of diabetes in either gender. Amongst non-obese men with the Thr54 allele, there was a significant elevation of triglycerides (TGs) (p=0.033) compared with alanine (Ala) homozygotes. Women with the Thr54 allele had significantly elevated total cholesterol (p=0.033) and low-density lipoprotein-cholesterol (LDL-C) (p=0.023) compared with Ala54 homozygotes. CONCLUSIONS: These results therefore suggested that the effects of the FABP2 polymorphism on TG, LDL-C and body mass index were associated with gender difference and obesity amongst non-diabetic Japanese-American subjects.

Erectile dysfunction is strongly linked with decreased libido in diabetic men.

Erectile dysfunction frequently occurs with diabetes mellitus. A survey of diabetic men was conducted by anonymous questionnaire to investigate the associations of erectile dysfunction with various predictive factors. A total of 112 diabetic males without an obvious history of erectile dysfunction were available for analyses. The mean age and duration of diabetes were 53.7 +/- 12.2 years and 10.2 +/- 8.6 years (mean +/- standard deviation), respectively. The questionnaire included questions on the presence or absence of smoking, hypertension, libido and subjective symptoms of diabetic neuropathy that may be associated with erectile dysfunction. Analysis of the answers to the questionnaire revealed that 40% of the patients complained of erectile dysfunction (erection 'always insufficient'). Erectile dysfunction was significantly correlated with age (p = 0.005), but not with duration of diabetes (p = 0.25), adjusted for age. Erectile dysfunction was also associated with sensory neuropathy and reduced libido, independently of age. The logistic regression analysis revealed that erectile dysfunction was positively associated with reduced libido and age. The odds ratio of erectile dysfunction for reduced compared to unreduced libido was 18.21, suggesting that psychogenic factors have a marked influence on erectile dysfunction. It is concluded that the presence of erectile dysfunction should be considered when symptoms related to diabetic neuropathy are observed; psychological approaches, such as sexual counseling, could be applied for the treatment of erectile dysfunction.

A rapidly diverging EGF protein regulates species-specific signal transduction in early sea urchin development.

The macromolecules mediating species-specific events during fertilization and early development and their molecular evolution are only beginning to be understood. We screened sea urchin ovary mRNA for species-specific gene products using representational differential analysis to identify unique transcripts in Strongylocentrotus franciscanus that are absent or divergent from a closely related species, S. purpuratus. One of the transcripts identified by this screening process is SfEGF-II, which contains four EGF repeats. SfEGF-II is orthologous to the previously reported genes S. purpuratus SpEGF-II and Anthocidaris crassispina AcEGF-II, encoding exogastrulation-inducing peptides (EGIP). EGF peptides derived from EGIP induce exogastrulation, a classical developmental defect, when added to embryos prior to gastrulation. The first three EGF repeats (EGF1-3) share 50 to 60% identity among the three species, but the fourth repeat (EGF4) is more divergent, displaying only 30% identity. Analysis of the sequence divergence indicates that the EGF-II genes display a relatively high nonsynonymous-to-synonymous ratio, a significant excess of radical compared to conservative amino acid substitutions, and a lack of polymorphism within SfEGF-II, indicating that these genes have been subjected to positive Darwinian selection. Recombinant EGF3 from S. franciscanus induces exogastrulation in both S. franciscanus and S. purpuratus. In contrast, recombinant EGF4 from both S. franciscanus and S. purpuratus induces exogastrula in a species-specific manner. In hybrid embryos, both species of EGF4 induce exogastrulation, suggesting that the receptor for this EGF molecule is expressed from both parental genomes during development. Both EGF3 and EGF4 induce the phosphorylation of membrane proteins of the blastula stage embryos, but EGF4 stimulates phosphorylation of proteins only in membranes prepared from homologous embryos, suggesting that it utilizes a unique pathway involving a species-specific receptor for EGF4. Thus, species-specific events of gastrulation and early development may be controlled by these rapidly diverging EGF molecules, through a novel species-specific signal transduction pathway.

Identification of a 97-kDa heat shock protein from S. franciscanus ovaries with 94% amino acid identity to the S. purpuratus egg surface receptor for sperm.

To identify species-specific regions of the sea urchin egg surface receptor for sperm, we cloned and sequenced the cDNA from S. franciscanus (Sf) ovary mRNA that is homologous to the S. purpuratus (Sp) sperm receptor sequence. The Sf cDNA contains an 886-amino-acid open reading frame (ORF) that is 96% identical at the nucleotide level to the Sp sperm receptor sequence over 2.9 kb. In contrast to the published Sp sequence, the Sf sequence does not encode a signal peptide or transmembrane domain. However, like the Sp sperm receptor sequence, the Sf protein has substantial similarity to the 70-kDa heat shock family of proteins and appears to encode a member of a newly identified subfamily of hsp70-related proteins designated hsp110/SSE. A BLAST search using the 5' end of the published Sp cDNA sequence as a query indicates that a segment of approximately 400 bp, which encodes the putative signal sequence, is 95% identical to Sp mitochondrial DNA. Resequencing of the Sp cDNA clone failed to confirm the presence of the published transmembrane and cytoplasmic domains. These results suggest that the published sequence of the Sp egg receptor for sperm may contain errors in the critical regions that were believed to encode an amino-terminal signal sequence, transmembrane domain, and cytoplasmic tail and that the protein products encoded by these cDNAs are highly related to mammalian cytoplasmic hsp110s.

Multiple sclerosis with bilateral continuous cystic lesions along lateral ventricles and caudate-callosal angles (Wetterwinkel).

We report a 53-year-old female autopsy case of multiple sclerosis with bilateral continuous cystic lesions along the lateral ventricles and caudate-callosal angles (Wetterwinkel). The pathophysiological mechanisms underlying these peculiar huge cystic lesions can be explained by the appearance of necrotic tissue during the recurrent relapsing stages of the disease, and then, by the absorption and scavenging of activated microglias. Poor astrocytic gliosis, which might be an effect of frequent use of corticosteroids during the clinical course makes the cavities bigger.

Levels of serum IgE, serum soluble-Fc epsilon RII, and Fc epsilon RII(+) peripheral blood lymphocytes in atopic dermatitis.

Levels of serum IgE, serum soluble-Fc epsilon RII (S-Fc epsilon RII), and Fc epsilon RII(+) peripheral blood lymphocytes (PBL) were examined in 73 patients with atopic dermatitis (AD) and 17 control subjects with no atopic disease, in order to investigate the correlation of these parameters with AD. AD patients showed increases in IgE, S-Fc epsilon RII and Fc epsilon RII(+)PBL as compared with control subjects. In AD patients, levels of serum IgE and Fc epsilon RII(+)PBL increased as the extent of dermatitis became more severe, while levels of serum S-Fc epsilon RII showed no correlation with the extent of dermatitis. In 8 of the 73 AD patients who showed an improvement in their symptoms with treatment with topical corticosteroids or antihistamine, IgE, Fc epsilon RII(+)PBL, and S-Fc epsilon RII were measured before and after treatment. Fc epsilon RII(+)PBL correlated with disease activity; IgE and S-Fc epsilon RII did not show any such correlation. Patients with elevated IgE levels (IgE greater than 5,000 U/ml) showed low levels of S-Fc epsilon RII. Severely affected cases with a history of respiratory atopy also showed decreased S-Fc epsilon RII levels. It is believed that S-Fc epsilon RII binds to IgE in serum and may neutralize or down-regulate IgE mediated allergic reactions. A low level of S-Fc epsilon RII may cause an elevation of IgE and an exacerbation of the disease.

[An autopsy case of multiple sclerosis with bilateral continuous cystic lesions along lateral ventricles and caudate-callosal angles (Wetterwinkel)].

A 47-year old female had a fever about 39 degrees C of unknown origin for 2 days. Soon she developed pain in the bilateral lower extremities followed by gait disturbance and vesicorectal disorder. Prednisolone was administered with an improvement. However, she developed paresthesia in the upper extremities 1 month later, and then gradually paraplegia another 5 month later. Nystagmus, painful tonic spasm, facial spasm, and visual disorder also appeared. These symptoms repeatedly exacerbated and remitted with administration of prednisolone. We examined this patient at age 53, CBC, blood chemistry, urinalysis, ECG and chest X-ray were normal. Serum IgG and IgA level were decreased. CSF protein content and IgG level were remarkably increased. EEG showed diffuse theta activities. MRI studies revealed high intensity signals in the putamen, deep frontal and periventricular white matter region. Pulse therapy of methylprednisolone was performed effectively for several times. She died of respiratory and heart failure 6 years after the onset. Autopsy revealed bilateral continuous cystic lesions along the lateral ventricles extending from the frontal tips of anterior horns to the occipital tips of posterior, and further, to the temporal tips of lateral horns; the caudate-callosal angeles (Wetterwinkel) were more severely and widely affected bilaterally. There were also old and fresh demyelinated lesions scattered in the cerebral white matter, brainstem, cerebellum, and spinal cord. Although this case is considered to have typical MS from clinical and pathological findings, there have been only a few reports of MS with such continuous cystic lesions in the cerebral hemispheres as seen in this case.

[Enzyme immunoassay for secretory immunoglobulin A on skin surface and in sweat].

Recently secretory IgA (S-IgA) was found to be secreted from the eccrine gland. By using a sandwich enzyme immunoassay, we measured the concentration of S-IgA 1) in sweat (sweat S-IgA) and 2) in the extract buffer obtained by pipetting++ on the skin (skin surface S-IgA). Skin surface hydration and skin surface lipid were measured at the sites where the samples of skin surface S-IgA were collected. (These measurements were taken immediately after the buffer was pipetted). The quantity of both sweat S-IgA and skin surface S-IgA differed according to the sites where they were collected. Sites in order of decreasing sweat S-IgA level: face, chest, forearm. Sites in order of decreasing skin surface S-IgA level: face, chest, palm, forearm, sole. (Skin surface S-IgA levels on palm and forearm were approximately equal). Although the amount of skin surface S-IgA was not related to the skin surface hydration, there was a significant correlation between skin surface S-IgA and skin surface lipid.

Supranuclear paralysis preventing lid closure in amyotrophic lateral sclerosis.

We report a case of amyotrophic lateral sclerosis (ALS) in which the ability to close the eyes on command or voluntarily, was lost in spite of retention of reflex activity. A electrophysiological study of the blink reflex revealed a prominent R1 component with normal latency, which confirmed that the blink reflex was exactly preserved and also suggested a hemispherical lesion. Postmortem examination disclosed prominent cortical and subcortical lesions of the precentral areas on both sides. These lesions seem to be very closely related to the inability to initiate lid closing.

Distal myopathy with rimmed vacuole formation. A follow-up study.

A long-term follow-up study of patients with familial distal myopathy with rimmed vacuole formation and a review of the literature indicates that the prognosis of the disorder was extremely poor as to daily life. Although the initial symptom appearing in early adulthood was muscular wasting and weakness in the legs, especially the distal muscles, severe generalized skeletal muscle involvement with sparing of the facial, extraocular, bulbar, intercostal and diaphragm muscles was recognized in the advanced stage. The disease is probably inherited as an autosomal recessive trait, while there is a considerable female preponderance, the female-to-male ratio being 2:1. The disorder is distinguishable from various types of distal myopathy on the basis of clinical and pathological findings, and other myopathies with rimmed vacuole formation, including inclusion body myositis, from a prognostic viewpoint.