Jakub Głowacki

Co‑culture of human nucleus pulposus cells with multipotent mesenchymal stromal cells from human bone marrow reveals formation of tunnelling nanotubes

Degeneration of the intervertebral disc (IVD) is the main cause of age-related damage of spinal tissues. Using multipotent mesenchymal stromal cells (MSCs) regenerative medicine intends to restore the IVD components of annulus fibrosus (AF) and nucleus pulposus (NP). In the present study NP cells (NPCs) and MSCs obtained from adolescent patients suffering from scoliosis were used. IVDs and vertebrae were obtained during surgery and subsequently processed in order to establish cultures of NPCs and MSCs. The two cell types were co-cultured in 1-µm pore size insert system (indirect co-culture) or on one surface (direct co-culture). Prior to co-culture in these systems one of the cell types was stained by lipophilic fluorescent dye DiD (red). The results demonstrated that regardless of the cell type, the flow of DiD from stained to non-stained cells was more efficient in the direct co-culture in comparison with the insert system. Moreover, in the direct system the DiD flow was more efficient from MSCs towards NPCs compared with that in the opposite direction. These data indicated that the membrane interchange between the two cell types was asymmetric. To discriminate the subpopulation of cells that underwent membrane interchange, cells were double stained with DiD and DiO (green). In the first part of the experiment NPCs were stained by DiO and MSCs by DiD. In the second, NPCs were stained by DiD and MSCs by DiO. The cells were co-cultured in the direct system for 8 days and subsequently analyzed by flow cytometry and confocal microscopy. This analysis revealed that >50% of cells were stained by the DiO and DiD dyes. NPCs and MSCs formed structures similar to tunnelling nanotubes (TnT). In conclusion, the formation of TnT-like structures is able to promote, phenotypic changes during the direct co-culture of NPCs with MSCs.

Kinesiophobia in Pre-Operative Patients with Cervical Discopathy and Coexisting Degenerative Changes in Relation to Pain-Related Variables, Psychological State and Sports Activity

Background No research group has ever investigated the level of kinesiophobia in a well defined group of preoperative patients treated due to cervical discopathy and degenerative spine disease, confirmed by X-ray and magnetic resonance imaging (MRI) examinations. We aimed to investigate the degree of kinesiophobia and the differences in pain-related and psychosocial characteristics between patients with high and low levels of kinesiophobia, in relation to factors commonly associated with neck pain. Material/Methods Sixty-five consecutive patients with cervical discopathy and coexisting degenerative changes were assessed pre-surgically. The mean pain duration was 31.7 SD 34.0 months. Patients completed the Polish versions of the Tampa Scale for Kinesiophobia (TSK-PL) on 2 occasions, and the following once: Neck Disability Index (NDI-PL), State-Trait Anxiety Inventory (STAI-PL), Coping Strategies Questionnaire (CSQ-PL), and the Visual Analogue Scale (VAS-PL). Results A high level of kinesiophobia was indicated in 81.5% and 87.7% of patients in first and second completion, respectively. Patients with high and low kinesiophobia differ in regards to the recreation section of NDI-PL (p=0.012), gender (p=0.043), and sports activity (p=0.024). Correlations were identified between TSK-PL and marital status (p=0.023) and sports activity (p=0.024). Conclusions Kinesiophobia levels are higher in patients with chronic cervical pain before surgical treatment. Fear of movement tends to be higher in women and among patients avoiding sports recreation before surgical treatment. Although sports activity and socio-demographic data are predictors of kinesiophobia, psychological, pain-related, and clinical data are not. These findings should be considered when planning rehabilitation after surgical treatment of cervical discopathy and coexisting degenerative changes.

A longitudinal study of alexithymia in relation to physical activity in adolescent females with scoliosis subjected to cheneau brace treatment: preliminary report.

Study Design. A longitudinal pilot study of changes in levels of alexithymia among females with adolescent idiopathic scoliosis (AIS) under brace treatment. Objective. To investigate the prevalence of alexithymia and to evaluate changes in alexithymia levels among female patients with AIS treated with a Cheneau brace, in comparison with healthy female adolescents. Summary of Background Data. Alexithymia is a personality trait incorporating the following core characteristics: difficulty in identifying and describing feelings, difficulty in distinguishing between feelings and the physical sensation of emotional arousal, limited imaginal processes, and an externally oriented cognitive style. Alexithymia can be common among adolescents and young adults with severe idiopathic scoliosis. Methods. Thirty-six female patients with AIS, aged 13.4 years (standard deviation [SD], 1.7) at the beginning of the study, completed the Polish version of the Toronto Alexithymia Scale-26 (TAS-26). The second and third evaluations took place at 6 and 12 months, respectively, after the beginning of the study. Thirty-six healthy female controls were also included. Results. The mean TAS-26 total score was 63.4 (SD, 9.3), 59.5 (SD, 12.1), and 59.6 (SD, 12.5) during the first, second, and last patient evaluation, whereas in healthy females 60.0 (SD, 10.9). With regard to the TAS-26 total score, results differed significantly between the first and the second (P = 0.007) and between the first and the third patient evaluation (P = 0.007). Regression analysis revealed that during the second and third patient evaluation, duration of recreational sports activity had a statistically significant (P = 0.029 and P = 0.005, respectively) influence on the probability of females reporting no alexithymia. Conclusion. The prevalence of alexithymia in healthy female controls is the same as in patients with scoliosis subjected to underarm brace treatment. Statistically significant changes with regard to TAS-26 total score and externally oriented thinking domain were found in patient sample. Physical activity in females with AIS treated nonoperatively coexists with lower scores in the TAS-26. Level of Evidence: 2

Effects of living environment on the postoperative Scoliosis Research Society-24 results in females with adolescent idiopathic scoliosis

Summary Background There are many factors influencing postoperative health-related quality of life of adolescent idiopathic scoliosis patients, including the degree of the deformity, culture, differences in geography, rural versus urban living environments, and social factors. The objective of this study was to analyze the significance of geographic factors and their differences influencing the postoperative quality of life in females with adolescent idiopathic scoliosis residing in urban and rural environments, by use of the Polish version of the SRS-24 questionnaire. Material/Methods Forty urban and 20 rural postoperative patients with adolescent scoliosis with a minimum 2-year follow-up period after surgery were included in the study. The process of cross-cultural adaptation was performed according to the IQOLA Project. Results General results of the Polish SRS-24 equalled 4.1 (SD 0.5) and 4.0 (SD.0.5) in the rural and urban groups of patients, respectively. The 2 groups do not differ in incidence of floor and ceiling effects. The Cronbach’s alpha values are excellent for the general result of SRS-24 in urban and rural groups (0.85 and 0.85, respectively). The sub-groups differed significantly in the self-image after surgery domain (p=0.048). Conclusions Patients from the rural group scored higher in the self-image after surgery domain but reported higher pain levels when compared to urban patients. The associations between SRS-24 results and radiographic parameters in the rural group of patients were strong, compared with moderate relations reported in the urban group.

The Pediatric Outcomes Data Collection Instrument for a Polish sample with juvenile idiopathic arthritis: psychometric properties of proxy version.

The aims of the study were to investigate the psychometric properties of the Pediatric Outcomes Data Collection Instrument (PODCI) for parents of children aged 2–10 years, in a Polish sample of juvenile idiopathic arthritis and to compare the results from children with chronic arthritis to the results from a normative sample.

Long-term effects of conservative treatment of Milwaukee brace on body image and mental health of patients with idiopathic scoliosis

We aimed to provide a complex assessment of adult females with adolescent idiopathic scoliosis (AIS) after a minimum of 23 years after completed Milwaukee brace treatment. In the present study, a comparison between healthy female and AIS patients’ perception of trunk disfigurement, self-image, mental health, pain level and everyday activity was made. Thirty AIS patients with a mean of 27.77 yrs (SD 3.30) after the treatment were included in the study. The control group consisted of 42 females, matching the age profile of the patient group. Study participants from both groups were examined using the same protocol, except for the radiological evaluation. Patients and healthy controls completed the Polish versions of the Scoliosis Research Society (SRS-22) and Spinal Appearance Questionnaire (SAQ). Patients additionally filled the Bad Sobberheim Stress Questionnaire-Deformity (BSSQ-Deformity) and Bad Sobberheim Stress Questionnaire-Brace (BSSQ-Brace). The study group’s SAQ results differ significantly in regard to the total score and all individual domains, indicating better functioning among healthy controls. Except for the General domain (p = 0.002), among the remaining subscales the study group’s results differed significantly at p<0.001. Considering SRS-22 results, it was revealed that the patient group scored higher, signaling better functioning with reference to pain level (p = 0.016), function/activity (p<0.001) and the total score (p<0.001). The findings add to the complexity of long-term effect evaluations of AIS, particularly amongst females treated with a Milwaukee brace. Long-term results were not conclusive in terms of nonverbal assessment of body image and emotional tension regarding the experiences of brace-wearing. Future patients can be reassured that scoliosis treated conservatively does not negatively affect everyday activity, pain level, childbearing and mental health. Subjects who declared to have psychological problems due to scoliosis had a bigger curve size after treatment and in this study than the other AIS patients.

Back and neck pain and function in females with adolescent idiopathic scoliosis: A follow-up at least 23 years after conservative treatment with a Milwaukee brace

We aimed to explore the long-term outcomes of back and neck pain and functionality in adult females with adolescent idiopathic scoliosis (AIS), who had been treated with a Milwaukee brace, in a follow-up study a minimum of 23 years after the completion of the treatment, using radiological, clinical and socio-demographical data. Thirty AIS patients (scoliosis group–SG), were included in the study based on an extensive search of Pediatric Orthopedics and Traumatology Clinic charts. All treatments were successfully completed between 1974 and 1990. In all cases, scoliosis had not been detected before the age of 10 and was not combined with any major spinal deformities at the time when the brace treatment was implemented. In those patients, the Risser sign 4 and minimum two years post-menarche was defined as a maturity, after that time the brace treatment was completed. Patients were excluded from the study if they, at the time of the follow-up examinations, suffered from any other disease leading to trunk deformity. Forty patients met the criteria for inclusion, but due to change some personal details, not all of them were contacted. Finally, 30 women returned for a follow-up evaluation. Patients’ follow-up period was mean 27.77 yrs. ± SD 3.30 (range 23–35). Curvature change from the end of the treatment until the present day was mean 9.1 degrees ± SD 7.64 (range 0–27). A control group of 42 healthy females (healthy controls group—HG) matching the age profile of the patient group was randomly selected for comparative purposes.Both SG and HG completed the Polish versions of the Revised Oswestry Lower Back Pain Disability Index (RODI), the Rolland-Morris Questionnaire (RMQ), the Quebec Back Pain Disability Scale (QDS), the Neck Disability Index (NDI) and the Copenhagen Neck Functional Disability Scale (CNFDS). Descriptive statistics were calculated for demographics and baseline questionnaire scores. To determine if the investigated sample sizes are equivalent, the chi-square test was used. The chi-square test was used to compare qualitative features between persons with scoliosis and healthy controls. In addition, a Mann-Whitney test was utilized to compare differences between both groups in regard to quantitative characteristics. To establish relations between quantitative data such as e.g. age, duration of brace application, apical translation, Cobb angle, and questionnaire results, we used Spearmans rank correlation (marked as rS). To determine dependency between quantitative and qualitative characteristics, e.g. between questionnaire numerical data and marital status, place of residence or curve type, ANOVA Kruskal-Wallis test was used. A p<0.05 indicates statistical significance. Statistical calculations were performed by Statistica software. In regards to RODI, RMQ, QDS, NDI and CNFDS (both for total scores and particular sub-sections), statistically significant differences (p <0.001) between both samples were found, indicating higher levels of pain and neck and lower back pain-related disability among persons with scoliosis. Associations exist between RODI and RMQ (rS = 0.76) QDS (rS = 0.70), NDI (rS = 0.69) and CNFDS (rS = 0.60). RMQ was associated with QDS (rS = 0.71) and NDI (rS = 0.69), whereas QDS correlated with NDI (rS = 0.80) and CNFDS (rS = 0.60). NDI was also associated with CNFDS (rS = 0.81). Persons with scoliosis treated in adolescence with a Milwaukee brace display significant restrictions in everyday activities, due to lower back pain (LBP) and neck-related disabilities, compared to healthy controls. In addition, back pain is associated with curve progression in long-term follow-up after conservative treatment. Moreover, LBP-related disability coexists with restrictions experienced due to neck pain.

Quantification of the asymmetric migration of the lipophilic dyes, DiO and DiD, in homotypic co-cultures of chondrosarcoma SW-1353 cells

DiO and DiD are lipophilic cell labelling dyes used in the staining of cells in vivo and in vitro. The aim of the present study was to quantify the asymmetrical distribution of dyes in co-cultured cells and to measure the intercellular transfer of DiO and DiD. DiO and DiD were applied separately to stain two identical populations of SW-1353 human chondrosarcoma cells that were subsequently co-cultured (homotypic co-culture). The intercellular migration of dyes in the co-cultured cells was measured by flow cytometry and recorded under a fluorescent microscope. DiD and DiO caused no effect on the proliferation of cells, the degradation rate of the two dyes was comparable and crossover effects between dyes were negligible. The results of the present study suggested that asymmetrical intercellular migration of DiD and DiO was responsible for the asymmetrical distribution of these dyes in co-cultured cells. To take advantage of the lipophilic dyes migration in the double-stained co-cultured cells we suggest to apply mixed-dyes controls prior to the flow cytometric analysis. These controls are performed by staining cells with a 1:1 mix of the two dyes and would enable the estimation of the intensity of intercellular contact in co-culture systems. A 1:1 premix of DiO and DiD was applied to estimate cellular effect on intercellular exchange of lipid dyes in co-cultures incubated with cycloheximide and cytochalasin B. The cellular effect contributed 6–7% of intercellular migration of the lipophilic dyes, DiO and DiD. The majority of the observed intercellular transfer of these dyes was due to non-cellular, passive transfer.