Birender Balain

Isolation and Characterisation of Mesenchymal Stem Cells from Different Regions of the Human Umbilical Cord

Umbilical cords as a source of stem cells are of increasing interest for cell therapies as they present little ethical consideration and are reported to contain immune privileged cells which may be suitable for allogeneic based therapies. Mesenchymal stem cells (MSCs) sourced from several different cord regions, including artery, vein, cord lining, and Whartons jelly, are described in the literature. However, no one study has yet isolated and characterised MSCs from all regions of the same cord to determine the most suitable cells for cell based therapeutics.

Viability, growth kinetics and stem cell markers of single and clustered cells in human intervertebral discs: implications for regenerative therapies

PurposeThere is much interest in the development of a cellular therapy for the repair or regeneration of degenerate intervertebral discs (IVDs) utilising autologous cells, with some trials already underway. Clusters of cells are commonly found in degenerate IVDs and are formed via cell proliferation, possibly as a repair response. We investigated whether these clusters may be more suitable as a source of cells for biological repair than the single cells in the IVD.MethodsDiscs were obtained at surgery from 95 patients and used to assess the cell viability, growth kinetics and stem or progenitor cell markers in both the single and clustered cell populations.ResultsSixty-nine percent (±15) of cells in disc tissue were viable. The clustered cell population consistently proliferated more slowly in monolayer than single cells, although this difference was only significant at P0–1 and P3–4. Both populations exhibited progenitor or notochordal cell markers [chondroitin sulphate epitopes (3B3(−), 7D4, 4C3 and 6C3), Notch-1, cytokeratin 8 and 19] via immunohistochemical examination; stem cell markers assessed with flow cytometry (CD73, 90 and 105 positivity) were similar to those seen on bone marrow-derived mesenchymal stem cells.ConclusionsThese results confirm those of previous studies indicating that progenitor or stem cells reside in adult human intervertebral discs. However, although the cell clusters have arisen via proliferation, there appear to be no greater incidence of these progenitor cells within clusters compared to single cells. Rather, since they proliferate more slowly in vitro than the single cell population, it may be beneficial to avoid the use of clustered cells when sourcing autologous cells for regenerative therapies.

Does walking change the Romberg sign

The Romberg sign helps demonstrate loss of postural control as a result of severely compromised proprioception. There is still no standard approach to applying the Romberg test in clinical neurology and the criteria for and interpretation of an abnormal result continue to be debated. The value of this sign and its adaptation when walking was evaluated. Detailed clinical examination of 50 consecutive patients of cervical myelopathy was performed prospectively. For the walking Romberg sign, patients were asked to walk 5xa0m with their eyes open. This was repeated with their eyes closed. Swaying, feeling of instability or inability to complete the walk with eyes closed was interpreted as a positive walking Romberg sign. This test was compared to common clinical signs to evaluate its relevance. Whilst the Hoffman’s reflex (79%) was the most prevalent sign seen, the walking Romberg sign was actually present in 74.5% of the cases. The traditional Romberg test was positive in 17 cases and 16 of these had the walking Romberg positive as well. Another 21 patients had a positive walking Romberg test. Though not statistically significant, the mean 30xa0m walking times were slower in patients with traditional Romberg test than in those with positive walking Romberg test and fastest in those with neither of these tests positive. The combination of either Hoffman’s reflex and/or walking Romberg was positive in 96% of patients. The walking Romberg sign is more useful than the traditional Romberg test as it shows evidence of a proprioceptive gait deficit in significantly more patients with cervical myelopathy than is found on conventional neurological examination. The combination of Hoffman’s reflex and walking Romberg sign has a potential as useful screening tests to detect clinically significant cervical myelopathy.

Incidence of numerical variants and transitional lumbosacral vertebrae on whole-spine MRI

AbstractObjectivesThis study sets out to prospectively investigate the incidence of transitional vertebrae and numerical variants of the spine.Materials and methodsOver a period of 28xa0months, MRIs of the whole spine were prospectively evaluated for the presence of transitional lumbosacral vertebrae and numerical variants of the spine.ResultsMRI of the whole spine was evaluated in 420 patients, comprising 211 female and 209 male subjects. Two patients had more complex anomalies. Lumbosacral transitional vertebrae were seen in 12 patients: eight sacralised L5 (3 male, 5 female) and four lumbarised S1 (3 male, 1 female). The incidence of transitional vertebrae was approximately 3.3. % (14/418). Thirty-two (7.7xa0%) of 418 patients had numerical variants of mobile vertebrae of the spine without transitional vertebrae. The number of mobile vertebrae was increased by one in 18 patients (12 male, 6 female), and the number was decreased by one in 14 patients (4 male, 10 female).ConclusionsNumerical variants of the spine are common, and were found to be almost 2.5 times as frequent as transitional lumbosacral vertebrae in the study population. Only whole-spine imaging can identify numerical variants and the anatomical nature of transitional vertebrae. The tendency is toward an increased number of mobile vertebrae in men and a decreased number in women.n Main messages• Numerical variants of the spine are more common than transitional vertebrae.• Spinal numerical variants can be reliably identified only with whole-spine imaging.• Increased numbers of vertebrae are more common in men than women.• Transitional lumbosacral vertebrae occurred in about 3.3xa0% of the study population.• The incidence of numerical variants of the spine was about 7.7xa0%.

Mesenchymal stromal cells derived from whole human umbilical cord exhibit similar properties to those derived from Wharton's jelly and bone marrow

Mesenchymal stromal cells (MSC) can be isolated from several regions of human umbilical cords, including Whartons jelly (WJ), artery, vein or cord lining. These MSC appear to be immune privileged and are promising candidates for cell therapy. However, isolating MSC from WJ, artery, vein or cord lining requires time‐consuming tissue dissection. MSC can be obtained easily via briefly digesting complete segments of the umbilical cord, likely containing heterogenous or mixed populations of MSC (MC‐MSC). MC‐MSC are generally less well characterized than WJ‐MSC, but nevertheless represent a potentially valuable population of MSC. This study aimed to further characterize MC‐MSC in comparison to WJ‐MSC and also the better‐characterized bone marrow‐derived MSC (BM‐MSC). MC‐MSC proliferated faster, with significantly faster doubling times reaching passage one 8.8 days sooner and surviving longer in culture than WJ‐MSC. All MSC retained the safety aspect of reducing telomere length with increasing passage number. MSC were also assessed for their ability to suppress T‐cell proliferation and for the production of key markers of pluripotency, embryonic stem cells, tolerogenicity (CD40, CD80, CD86 and HLA‐DR) and immunomodulation (indoleamine 2,3‐dioxygenase [IDO] and HLA‐G). The MC‐MSC population displayed all of the positive attributes of WJ‐MSC and BM‐MSC, but they were more efficient to obtain and underwent more population doublings than from WJ, suggesting that MC‐MSC are promising candidates for allogeneic cell therapy in regenerative medicine.

The outcome of decompression alone for lumbar spinal stenosis with degenerative spondylolisthesis

PurposeLumbar spinal stenosis in the presence of degenerative spondylolisthesis is generally treated by means of surgery. The role of lumbar decompression without fusion is not clear. Therefore, the aim of this study was to assess whether patients who undergo decompression alone have a favourable outcome without the need for a subsequent fusion.MethodsThis is a prospective cohort study with single blinding of 83 consecutive patients with lumbar stenosis and degenerative spondylolisthesis treated by decompression, without fusion, using a spinous process osteotomy. Blinded observers collected pre- and post-operative Oswestry Disability Index (ODI), EuroQol Five Dimensions (EQ-5D), and visual analogue scale (VAS) for back and leg pain scores prospectively. Failures for this study were those patients who required a subsequent lumbar fusion procedure at the decompressed levels. Statistical analysis was performed using paired t test and Mann–Whitney test.ResultsThere were 36 males and 47 females with a mean age of 66xa0years (range 35–82). The mean follow-up was 36xa0months (range 19–48xa0months). The mean pre-operative ODI, EQ-5D, and VAS scores were 52 [standard deviation (SD) 18], 0.25 (SD 0.30), and 61 (SD 22), respectively. All mean scores improved post-operatively to 38 (SD 23), 0.54 (SD 0.34) and 36 (SD 27), respectively. There was a statistically significant improvement in all scores (pxa0≤xa00.0001). Nine patients (11xa0%) required a subsequent fusion procedure and five patients (6xa0%) required revision decompression surgery alone.ConclusionOur study’s results show that a lumbar decompression procedure without arthrodesis in a consecutive cohort of patients with lumbar spinal stenosis with degenerative spondylolisthesis had a significant post-operative improvement in ODI, EQ-5D, and VAS. The rate of post-operative instability and subsequent fusion is not high. Only one in 10 patients in this group ended up needing a subsequent fusion at a mean follow-up of 36xa0months, indicating that fusion is not always necessary in these patients.

Temporal Analyses of the Response of Intervertebral Disc Cells and Mesenchymal Stem Cells to Nutrient Deprivation

Much emphasis has been placed recently on the repair of degenerate discs using implanted cells, such as disc cells or bone marrow derived mesenchymal stem cells (MSCs). This study examines the temporal response of bovine and human nucleus pulposus (NP) cells and MSCs cultured in monolayer following exposure to altered levels of glucose (0, 3.15, and 4.5u2009g/L) and foetal bovine serum (0, 10, and 20%) using an automated time-lapse imaging system. NP cells were also exposed to the cell death inducers, hydrogen peroxide and staurosporine, in comparison to serum starvation. We have demonstrated that human NP cells show an initial “shock” response to reduced nutrition (glucose). However, as time progresses, NP cells supplemented with serum recover with minimal evidence of cell death. Human NP cells show no evidence of proliferation in response to nutrient supplementation, whereas MSCs showed greater response to increased nutrition. When specifically inducing NP cell death with hydrogen peroxide and staurosporine, as expected, the cell number declined. These results support the concept that implanted NP cells or MSCs may be capable of survival in the nutrient-poor environment of the degenerate human disc, which has important clinical implications for the development of IVD cell therapies.

Only walking matters-assessment following lumbar stenosis decompression.

PurposeMultiple outcome measures exist to evaluate the outcomes of spinal decompression surgery; however, these tend to be complex and are difficult to express to the patient pre-operatively to accurately guide their expectations. We present outcomes, in terms of walking distance measurement, of a prospective single surgeon series of 76 consecutive patients with spinal stenosis.Methods76 patients (mean age 68.8xa0years; 48–91xa0years) had decompression surgery using spinous process osteotomy. Accurate measurement of walking distance was used as an outcome measure, and factors that affect it were evaluated. Walking distance was measured pre-operatively, post-operatively and at 3xa0months follow-up using a measuring wheel. The minimum follow-up was 5xa0years.ResultsThe mean distances walked were 78.1, 419.9 and 1285xa0m, respectively. Pre-operative disc height (pxa0=xa00.023) and male gender (pxa0=xa00.039) predicted a significant improvement in walking distance, while age (pxa0=xa00.23), ASA grade (pxa0=xa00.39) and the number of levels operated on (pxa0=xa00.89) did not significantly affect the increase in walking distance. 12 patients experienced post-operative complications (15.8%), and at last clinical follow-up (6.3xa0years, 5.1–6.9xa0years) 27 patients (35.5%) had residual leg symptoms and 8 had undergone further revision procedures (10.5%).ConclusionThis study demonstrates that walking distance is an accurate and accessible method of determining surgical outcomes.

Is Osteogenic Differentiation of Human Nucleus Pulposus Cells a Possibility for Biological Spinal Fusion

Objective The purpose of this study was to investigate whether a simple, biologically robust method for inducing calcification of degenerate intervertebral discs (IVD) could be developed to provide an alternative treatment for patients requiring spinal fusion. Design Nucleus pulposus (NP) cells isolated from 14 human IVDs were cultured in monolayer and exposed to osteogenic medium, 1,25-dihydroxyvitamin D3 (VitD3), parathyroid hormone (PTH), and bone morphogenic proteins (BMPs) 2/7 to determine if they could become osteogenic. Similarly explant cultures of IVDs from 11 patients were cultured in osteogenic media with and without prior exposure to VitD3 and BMP-2. Osteogenic differentiation was assessed by alkaline phosphatase activity and areas of calcification identified by alizarin red or von Kossa staining. Expression of osteogenic genes during monolayer culture was determined using polymerase chain reaction and explant tissues assessed for BMP inhibitors. Human bone marrow–derived mesenchymal stromal cells (MSCs) were used for comparison. Results Standard osteogenic media was optimum for promoting mineralization by human NP cells in monolayer. Some osteogenic differentiation was observed with 10 nM VitD3, but none following application of PTH or BMPs. Regions of calcification were detected in 2 of the eleven IVD tissue explants, one cultured in osteogenic media and one with the addition of VitD3 and BMP-2. Conclusions Human NP cells can become osteogenic in monolayer and calcification of the extracellular matrix can also occur, although not consistently. Inhibitory factors within either the cells or the extracellular matrix may hinder osteogenesis, indicating that a robust biological fusion at this time requires further optimization.