The Role of Cytokines in the Metastasis of Solid Tumors to the Spine: Systematic Review.

Although many studies have investigated the role of cytokines in bone metastases, our knowledge of their function in spine metastasis is limited. Therefore, we performed a systematic review to map the available evidence on the involvement of cytokines in spine metastasis in solid tumors. A PubMed search identified 211 articles demonstrating a functional link between cytokines/cytokine receptors and bone metastases, including six articles confirming the role of cytokines/cytokine receptors in spine metastases. A total of 68 cytokines/cytokine receptors were identified to mediate bone metastases; 9 (mostly chemokines) played a role in spine metastases: CXC motif chemokine ligand (CXCL) 5, CXCL12, CXC motif chemokine receptor (CXCR) 4, CXCR6, interleukin (IL) 10 in prostate cancer, CX3C motif chemokine ligand (CX3CL) 1 and CX3C motif chemokine receptor (CX3CR) 1 in liver cancer, CC motif chemokine ligand (CCL) 2 in breast cancer, and transforming growth factor (TGF) ß in skin cancer. Except for CXCR6, all cytokines/cytokine receptors were shown to operate in the spine, with CX3CL1, CX3CR1, IL10, CCL2, CXCL12, and CXCR4 mediating bone marrow colonization, CXCL5 and TGFß promoting tumor cell proliferation, and TGFß additionally driving bone remodeling. The number of cytokines/cytokine receptors confirmed to mediate spinal metastasis is low compared with the vast spectrum of cytokines/cytokine receptors participating in other parts of the skeleton. Therefore, further research is needed, including validation of the role of cytokines mediating metastases to other bones, to precisely address the unmet clinical need associated with spine metastases.

Addition of Popular Exogenous Antioxidant Agent, PBN, to Culture Media May Be an Important Step to Optimization of Myogenic Stem/Progenitor Cell Preparation Protocol.

The aim of the study was to modify human skeletal muscle-derived stem/progenitor cells (SkMDS/PCs) and demonstrate the optimal cell preparation protocol for application in post-infarction hearts. We used conditioned SkMDS/PC culture medium with α-phenyl-N-tert-butyl nitrone (PBN). SkMDS/PCs were cultured under hypoxic conditions and the results were compared to the standard ones. We observed a significant increase of CD-56 positive phenotypic marker the ability to form functional myotubes, increase in the proportion of young cells in cell primary suspensions, and a decrease in the percentage of apoptotic cells among PBN-conditioned cells in normoxia an hypoxia. We also observed significantly higher levels of SOD3 expression; maintained expression of SOD1, SOD2, and CAT; a higher level of BCL2 gene expression; and a rather significant decrease in Hsp70 gene expression in PBN-conditioned SkMDS/PCs compared to the WT population under hypoxic conditions. In addition, significant increase of myogenic genes expression was observed after PBN addition to culture medium, compared to WT population under hypoxia. Interestingly, PBN addition significantly increased the lengths of telomeres under hypoxia. Based on the data obtained, we can postulate that PBN conditioning of human SkMDS/PCs could be a promising step in improving myogenic cell preparation protocol for pro-regenerative treatment of post-infarction hearts.

Transient and Stable Overexpression of Extracellular Superoxide Dismutase Is Positively Associated with the Myogenic Function of Human Skeletal Muscle-Derived Stem/Progenitor Cells.

In the present study, the genetic modification of human skeletal muscle-derived stem/progenitor cells (SkMDS/PCs) was investigated to identify the optimal protocol for myogenic cell preparation for use in post-infarction heart therapy. We used two types of modifications: GFP-transfection (using electroporation) and SOD3 transduction (using a lentiviral vector). SkMDS/PCs were cultured under different in vitro conditions, including standard (21% oxygen) and hypoxic (3% oxygen), the latter of which corresponded to the prevailing conditions in the post-infarction heart. Transfection/transduction efficacy, skeletal myogenic cell marker expression (CD56), cellular senescence, and apoptosis, as well as the expression of antioxidant (SOD1, SOD2, and SOD3), anti-aging (SIRT1 and FOXO), anti-apoptotic (BCL2), and myogenic (MyoD and MyoG) genes, were evaluated. The percentage of GFP-positive SkMDS/PCs was determined as an indicator of the efficacy of transfection, which reached 55%, while transduction showed better efficiency, reaching approximately 85% as estimated by fluorescence microscopy. The CD56-positive SkMDS/PCs were present in approximately 77% of the tested cells after transient transfection and approximately 96% after transduction. Under standard in vitro culture conditions, the ability of the differentiated, transfected SkMDS/PCs to form myotubes was greater than that of the wild type (WT) cell population (p < 0.001), while the cells transduced with the SOD3 gene exhibited an increase in cell fusion under both standard (p < 0.05) and hypoxic conditions (p < 0.001). In transduced SkMDS/PCs, we observed a positive influence of SOD3 overexpression on cell ageing and apoptosis. We observed an increase in the percentage of young cells under standard (p < 0.05) and hypoxic (p < 0.001) in vitro culture conditions, with a notable decrease in the percentage of senescent and advanced senescent cells in the SOD3-overexpressing cell population detected compared to that observed for the untransduced muscle-derived cells. A lower percentage of apoptotic cells was observed for transduced SkMDS/PCs than that for WT cells under hypoxic in vitro culture conditions. In transiently transfected SkMDS/PCs, we observed significantly higher gene expression levels of SOD2 (almost 40-fold) (p < 0.001) and FOXO (p < 0.05) (approximately 3-fold) under both normoxic and hypoxic culture conditions and of BCL2 under hypoxia compared to those observed in untreated cells (WT). In addition, myogenic genes showed a significant increase in MyoD (almost 18-fold) expression under standard culture conditions (p < 0.0001) and decreased MyoG expression (approximately 2-fold) after transfection (p < 0.05) compared with that detected in the WT skeletal muscle-derived cell control. Taken together, these results demonstrate that SOD3-tranduced skeletal muscle-derived cells may have potential for use in the regenerative treatment of the post-infarction heart.

Chromatin and transcriptome changes in human myoblasts show spatio-temporal correlations and demonstrate DPP4 inhibition in differentiated myotubes.

Although less attention was paid to understanding physical localization changes in cell nuclei recently, depicting chromatin interaction maps is a topic of high interest. Here, we focused on defining extensive physical changes in chromatin organization in the process of skeletal myoblast differentiation. Based on RNA profiling data and 3D imaging of myogenic (NCAM1, DES, MYOG, ACTN3, MYF5, MYF6, ACTN2, and MYH2) and other selected genes (HPRT1, CDH15, DPP4 and VCAM1), we observed correlations between the following: (1) expression change and localization, (2) a gene and its genomic neighbourhood expression and (3) intra-chromosome and microscopical locus-centromere distances. In particular, we demonstrated the negative regulation of DPP4 mRNA (p < 0.001) and protein (p < 0.05) in differentiated myotubes, which coincided with a localization change of the DPP4 locus towards the nuclear lamina (p < 0.001) and chromosome 2 centromere (p < 0.001). Furthermore, we discuss the possible role of DPP4 in myoblasts (supported by an inhibition assay). We also provide positive regulation examples (VCAM1 and MYH2). Overall, we describe for the first time existing mechanisms of spatial gene expression regulation in myoblasts that might explain the issue of heterogenic responses observed during muscle regenerative therapies.

Tissue-specific promoter-based reporter system for monitoring cell differentiation from iPSCs to cardiomyocytes.

The possibility of using stem cell-derived cardiomyocytes opens a new platform for modeling cardiac cell differentiation and disease or the development of new drugs. Progress in this field can be accelerated by high-throughput screening (HTS) technology combined with promoter reporter system. The goal of the study was to create and evaluate a responsive promoter reporter system that allows monitoring of iPSC differentiation towards cardiomyocytes. The lentiviral promoter reporter system was based on troponin 2 (TNNT2) and alpha cardiac actin (ACTC) with firefly luciferase and mCherry, respectively. The system was evaluated in two in vitro models. First, system followed the differentiation of TNNT2-luc-T2A-Puro-mCMV-GFP and hACTC-mcherry-WPRE-EF1-Neo from transduced iPSC line towards cardiomyocytes and revealed the significant decrease in both inserts copy number during the prolonged in vitro cell culture (confirmed by I-FISH, ddPCR, qPCR). Second, differentiated and contracting control cardiomyocytes (obtained from control non-reporter transduced iPSCs) were subsequently transduced with TNNT2-luc-T2A-Puro-CMV-GFP and hACTC-mcherry-WPRE-EF1-Neo lentiviruses to observe the functionality of obtained cardiomyocytes. Our results indicated that the reporter modified cell lines can be used for HTS applications, but it is essential to monitor the stability of the reporter sequence during extended cell in vitro culture.

Short-Term Effects of Kinesio Taping® on Electromyographic Characteristics of Paraspinal Muscles, Pain, and Disability in Patients With Lumbar Disk Herniation.

Context: Kinesio taping® (KT) is a therapeutic modality frequently used in the clinical practice for the treatment of various musculoskeletal disorders. It is often applied in patients with chronic low back pain to decrease pain and improve functional capacity. However, it is not known, whether thoracolumbar fascia KT technique can decrease back pain, restore normal activity of paraspinal muscles, and improve functional capacity in patients with lumbar disk herniation (LDH). Objective: To evaluate the impact of 7-day new KT stabilizing application on lumbar paraspinal muscles function, pain perception, and disability in patients with LDH. Design: A randomized controlled trial. Setting: Human Performance Laboratory. Patients: A number of 38 patients with LDH were randomized into KT (n = 19) and placebo taping (n = 19) groups. Interventions: Both groups received the same "x" type application running over the back along fibers of superficial lamina of the posterior layer of thoracolumbar fascia. Main Outcome Measures: The primary outcome measures were flexion-relaxation and extension-relaxation ratios calculated from electromyographic activity of lumbar multifidus and longissimus thoracic muscles. Pain intensity rating (Quadruple Visual Analogue Scale), pressure pain thresholds of the lower back, Roland-Morris Disability Questionnaire score, back extension force, and flexion range of motion (ROM) were among secondary outcomes. Results: KT application did not affect the lumbar multifidus and longissimus thoracic muscles flexion-relaxation and extension-relaxation ratios, lower back pressure pain thresholds, back flexion ROM, and back extension force (no group × time interaction [GTI]). KT and placebo taping comparably decreased disability level (time effect: F1,36 = 22.817, P < .001; GTI: F1,36 = 0.189, P = .67), average pain (time effect: F1,36 =39.648, P < .001; GTI: F1,36 = 2.553, P = .12), and the worst pain (time effect: F1,36 = 36.039, P < .001; GTI: F1,36 = 0.003, P = .96) intensity. Conclusion: Seven-day KT does not normalize lumbar paraspinal muscle function and is not superior to placebo in reducing disability and pain intensity in patients with LDH.

The impact of in vitro cell culture duration on the maturation of human cardiomyocytes derived from induced pluripotent stem cells of myogenic origin.

Ischemic heart disease, also known as coronary artery disease (CAD), poses a challenge for regenerative medicine. iPSC technology might lead to a breakthrough due to the possibility of directed cell differentiation delivering a new powerful source of human autologous cardiomyocytes. One of the factors supporting proper cell maturation is in vitro culture duration. In this study, primary human skeletal muscle myoblasts were selected as a myogenic cell type reservoir for genetic iPSC reprogramming. Skeletal muscle myoblasts have similar ontogeny embryogenetic pathways (myoblasts vs. cardiomyocytes), and thus, a greater chance of myocardial development might be expected, with maintenance of acquired myogenic cardiac cell characteristics, from the differentiation process when iPSCs of myoblastoid origin are obtained. Analyses of cell morphological and structural changes, gene expression (cardiac markers), and functional tests (intracellular calcium transients) performed at two in vitro culture time points spanning the early stages of cardiac development (day 20 versus 40 of cell in vitro culture) confirmed the ability of the obtained myogenic cells to acquire adult features of differentiated cardiomyocytes. Prolonged 40-day iPSC-derived cardiomyocytes (iPSC-CMs) revealed progressive cellular hypertrophy; a better-developed contractile apparatus; expression of marker genes similar to human myocardial ventricular cells, including a statistically significant CX43 increase, an MHC isoform switch, and a troponin I isoform transition; more efficient intercellular calcium handling; and a stronger response to ß-adrenergic stimulation.

Biological and Pro-Angiogenic Properties of Genetically Modified Human Primary Myoblasts Overexpressing Placental Growth Factor in In Vitro and In Vivo Studies.

Cardiovascular diseases are a growing problem in developing countries; therefore, there is an ongoing intensive search for new approaches to treat these disorders. Currently, cellular therapies are focused on healing the damaged heart by implanting stem cells modified with pro-angiogenic factors. This approach ensures that the introduced cells are capable of fulfilling the complex requirements of the environment, including the replacement of the post-infarction scar with cells that are able to contract and promote the formation of new blood vessels that can supply the ischaemic region with nutrients and oxygen. This study focused on the genetic modification of human skeletal muscle cells (SkMCs). We chose myoblast cells due to their close biological resemblance to cardiomyocytes and the placental growth factor (PlGF) gene due to its pro-angiogenic potential. In our in vitro studies, we transfected SkMCs with the PlGF gene using electroporation, which has previously been proven to be efficient and generate robust overexpression of the PlGF gene and elevate PlGF protein secretion. Moreover, the functionality of the secreted pro-angiogenic proteins was confirmed using an in vitro capillary development assay. We have also examined the influence of PlGF overexpression on VEGF-A and VEGF-B, which are well-known factors described in the literature as the most potent activators of blood vessel formation. We were able to confirm the overexpression of VEGF-A in myoblasts transfected with the PlGF gene. The results obtained in this study were further verified in an animal model. These data were able to confirm the potential therapeutic effects of the applied treatments.

The Content of the 14 Metals in Cancellous and Cortical Bone of the Hip Joint Affected by Osteoarthritis.

The aim of the study was to determine the content of particular elements Ca, Mg, P, Na, K, Zn, Cu, Fe, Mo, Cr, Ni, Ba, Sr, and Pb in the proximal femur bone tissue (cancellous and cortical bone) of 96 patients undergoing total hip replacement for osteoarthritis using ICP-AES and FAAS analytical techniques. The interdependencies among these elements and their correlations depended on factors including age, gender, place of residence, tobacco consumption, alcohol consumption, exposure to environmental pollution, physical activity, and type of degenerative change which were examined by statistical and chemometric methods. The factors that exerted the greatest influence on the elements in the femoral head and neck were tobacco smoking (higher Cr and Ni content in smokers), alcohol consumption (higher concentrations of Ni, Cu in people who consume alcohol), and gender (higher Cu, Zn, and Ni concentrations in men). The factors influencing Pb accumulation in bone tissue were tobacco, alcohol, gender, and age. In primary and secondary osteoarthritis of the hip, the content and interactions of elements are different (mainly those of Fe and Pb). There were no significant differences in the concentrations of elements in the femoral head and neck that could be attributed to residence or physical activity.

Comparison of trace element concentration in bone and intervertebral disc tissue by atomic absorption spectrometry techniques.

BACKGROUND: Trace element (TE) analysis in human tissue has the dual purpose of assessing environmental pollution and metabolism. In literature, bone TE analysis is common, but studies in intervertebral disc (IVD) tissue are lacking. The aim of the study was evaluation of the difference of TE concentration in intervertebral disc and bone in patients with degenerative changes. The comparison of the tissues differing in metabolism, blood perfusion, or separateness from adjoining tissues but playing similar biomechanical role and presenting some common morphological traits may shed new light on metabolism nuances, degenerative process, as well as accumulation potential of IVD in respect to bone. METHODS: In the study, we analyzed two types of samples: intervertebral disc (n =30, from 22 patients operated due to degenerative disc disease) and femoral bone (n =26, separately femoral head and neck, from 26 patients, acquired in total hip arthroplasty procedure in course of idiopathic osteoarthritis of the hip joint). In the samples we analyzed, with atomic absorption spectrometry, the concentrations of Pb, Ni, Mo, Cu, Mg, and Zn. RESULTS: The element concentrations identified in bone are comparable to those presented in the literature. In the case of Pb, Ni, Mo, Mg, and Zn, the concentration in the bone was 2 to 25.8 times higher than that observed in the disc. Only the Cu concentration was higher in disc tissue than in bone. In disc tissue, fewer samples had TE concentrations below the detection threshold. We found significant differences in TE profiles in the compared tissues. CONCLUSIONS: The results show that the disc could serve as a more stable compartment for evaluating TE concentration, especially for TEs that are environmentally related.

Intraobserver and interobserver reproducibility of the novel transcription method for selection of potential nerve root compression in MRI study in degenerative disease of the lumbar spine.

BACKGROUND: Degenerative disease of the lumbar spine is characterized by symptoms related to the affected nerve root. A recently described method allows the classification of the roots in relation to the occurrence of compression on its course. This method can serve as a clinical selection tool and decision support for semi-invasive pain therapy in back pain patients. MATERIAL AND METHODS: We examined 40 lumbar spine MRIs in 3 sessions of transcription each, according to the method being evaluated. Every MRI evaluation was performed by each of 3 different observers. Intra- and interobserver reproducibility was calculated using chance-corrected agreement using a weighted kappa value with quadratic weights to assess reliability for each nerve root separately. RESULTS: We found high intraobserver agreement in indication of the root with most pronounced interference due to potential compression by degenerative changes, at the level mean kappa=0.81 (with 95% CI, range 0.04). Less agreement was observed in the interobserver evaluation test with the mean kappa=0.75 (95% CI within the range not exceeding 0.03), although it still reached the substantial agreement. CONCLUSIONS: This zstudy provides evidence for substantial inter- and intraobserver agreement for the decision support method allowing selection of the most serious nerve structure compression in degenerative disease of the lumbar spine based on of the MRI description.

Operative treatment of Schwannoma, the primary sacral tumor--case presentation.

Sacral bone tumors constitute a separate group in spinal surgery among all other neoplastic dis-orders due to the specificity of clinical symptoms and methods of surgical treatment. In the majority of cases, in the initial phase, symptoms are nonspecific for neoplastic disease, which is a sig-nificant factor delaying the final diagnosis. At the moment of diagnosis, most tumors are so large in size that removing the whole mass carries a risk related to the extent of surgery and maintaining the stability of pelvic girdle and sacro-lumbar junction. In this publication we present a case of a 38-year-old female patient with primary sacral tumor (S2-S4) undergoing surgical treatment at the Spinal Surgery, Oncological Orthopedics and Traumatology Clinic at Poznan University of Medical Sciences. The patient was referred to an orthopedic outpatient clinic following gynecological surgery for removal of a suspected tumor of the uterus confirmed in an ultrasound examination. In a control magnetic resonance examination (MRI) there was a pathological mass visualized within the sacral bone infiltrating the presacral region and compressing the distal part of the colon. The patient underwent subtotal sacrectomy from a posterior approach. In order to reconstruct the posterior pelvic wall, biological material Permacol™ was used. There was a transient anal sphincter atony and urinary bladder paresis as well as sensory disturbances in S3-S5 dermatomes after the surgery. Several days after surgery, the patient was diagnosed with a deep cutaneous fistula and a significant volume of fluid in the postoperative cavity (in control CT). VAC treatment was implemented following a few days of passive drainage and significant improvement as well as secondary closing of the fistula were observed within a short period of time.