Christopher J. Hunter

Cytomorphology of notochordal and chondrocytic cells from the nucleus pulposus: a species comparison

The nuclei pulposi of the intervertebral discs (IVDs) contain a mixed population of cell types at various stages of maturation. This tissue is formed either by or with the help of cells from the embryonic notochord, which appear to be replaced during development by a population of chondrocyte‐like cells of uncertain origin. However, this transition occurs at widely varying times, depending upon the species – or even breed – of the animal being examined. There is considerable debate among spine researchers as to whether the presence of these residual notochordal cells has a significant impact upon IVD degeneration models, and thus which models may best represent the human condition. The present study examines several different species commonly used in lumbar spine investigations to explore the variability of notochordal cells in the IVD.

The three-dimensional architecture of the notochordal nucleus pulposus: novel observations on cell structures in the canine intervertebral disc

Cells from the nucleus pulposus of young (< 2 years) and old (> 5 years) non‐chondrodystrophoid dogs were studied using routine histology, confocal laser scanning microscopy and transmission electron microscopy. The architecture of cell structures – from the tissue scale down to subcellular scale – was reported. Clusters of notochordal cells were observed in young nuclei pulposi, ranging from 10 to 426 cells each. These clusters resisted mechanical disruption and showed evidence of cell–cell signalling via gap junctions. Cells (30–40 µm in diameter) within the clusters had a physaliferous appearance, containing numerous large inclusions which ranged from 1 to 20 µm in diameter. The inclusions were surrounded by a dense actin cortex but were not contained by a lipid bilayer. The contents of the inclusions were determined not to be predominantly carbohydrate or neutral lipid as assessed by histochemical staining, but the exact composition of the contents remained uncertain. There were striking differences in the cell architecture of young vs. old nuclei pulposi, with a loss of both cell clusters and physaliferous cells during ageing. These observations demonstrate unique cell structures, which may influence our understanding of the differences between notochordal and chondrocytic cells in the nucleus pulposus. Such differences could have substantial impact upon how we think about development, degeneration and repair of the intervertebral disc.

Chordoma and chondrosarcoma gene profile: implications for immunotherapy

Chordoma and chondrosarcoma are malignant bone tumors characterized by the abundant production of extracellular matrix. The resistance of these tumors to conventional therapeutic modalities has prompted us to delineate the gene expression profile of these two tumor types, with the expectation to identify potential molecular therapeutic targets. Furthermore the transcriptional profile of chordomas and chrondrosarcomas was compared to a wide variety of sarcomas as well as to that of normal tissues of similar lineage, to determine whether they express unique gene signatures among other tumors of mesenchymal origin, and to identify changes associated with malignant transformation. A HG-U133A Affymetrix Chip platform was used to determine the gene expression signature in 6 chordoma and 14 chondrosarcoma lesions. Validation of selected genes was performed by qPCR and immunohistochemistry (IHC) on an extended subset of tumors. By unsupervised clustering, chordoma and chondrosarcoma tumors grouped together in a genomic cluster distinct from that of other sarcoma types. They shared overexpression of many extracellular matrix genes including aggrecan, type II & X collagen, fibronectin, matrillin 3, high molecular weight-melanoma associated antigen (HMW-MAA), matrix metalloproteinaseMMP-9, and MMP-19. In contrast, T Brachyury and CD24 were selectively expressed in chordomas, as were Keratin 8,13,15,18 and 19. Chondrosarcomas are distinguished by high expression of type IX and XI collagen. Because of its potential usefulness as a target for immunotherapy, the expression of HMW-MAA was analyzed by IHC and was detected in 62% of chordomas and 48% of chondrosarcomas, respectively. Furthermore, western blotting analysis showed that HMW-MAA synthesized by chordoma cell lines has a structure similar to that of the antigen synthesized by melanoma cells. In conclusion, chordomas and chondrosarcomas share a similar gene expression profile of up-regulated extracellular matrix genes. HMW-MAA represents a potential useful target to apply immunotherapy to these tumors.

The Functional Significance of Cell Clusters in the Notochordal Nucleus Pulposus : Survival and Signaling in the Canine Intervertebral Disc

Study Design. Cell viability was assessed in relation to cell clustering, and mechanisms of cell–cell signaling in the clusters were investigated. Objectives. To explore the functional role of cell clustering in the notochordal nucleus pulposus. Summary of Background Data. The intervertebral disc of some species contains residual cells from the embryonic notochord. These cells form large three-dimensional clusters in the young, healthy disc but are replaced by chondrocyte-like cells during aging and degeneration. Methods. Forty nucleus pulposi of adult dog lumbar intervertebral discs were isolated, and were left untreated, mechanically disrupted through a syringe, or enzymatically digested. The presence of functional gap junctions was determined by the fluorescence recovery after photobleaching method. Cell viability was also assessed over 20 days in vitro. Results. The cell clusters were interconnected via functional gap junctions. Mechanical disruption of the tissue had little effect on long-term cell viability, but enzymatic disruption of the tissue had a substantial negative impact on cell survival. Conclusions. These results demonstrate that the notochordal cells in adult dog nucleus pulposi are able to communicate via cytoplasmic signals and that such communications may influence the functionality of these cells in the young disc.

Improved expansion of human bone marrow-derived mesenchymal stem cells in microcarrier-based suspension culture.

Human bone marrow‐derived mesenchymal stem cells (hBM‐MSCs) have potential clinical utility in the treatment of a multitude of ailments and diseases, due to their relative ease of isolation from patients and their capacity to form many cell types. However, hBM‐MSCs are sparse, and can only be isolated in very small quantities, thereby hindering the development of clinical therapies. The use of microcarrier‐based stirred suspension bioreactors to expand stem cell populations offers an approach to overcome this problem. Starting with standard culture protocols commonly reported in the literature, we have successfully developed new protocols that allow for improved expansion of hBM‐MSCs in stirred suspension bioreactors using CultiSpher‐S microcarriers. Cell attachment was facilitated by using intermittent bioreactor agitation, removing fetal bovine serum, modifying the stirring speed and manipulating the medium pH. By manipulating these parameters, we enhanced the cell attachment efficiency in the first 8 h post‐inoculation from 18% (standard protocol) to 72% (improved protocol). Following microcarrier attachment, agitation rate was found to impact cell growth kinetics, whereas feeding had no significant effect. By serially subculturing hBM‐MSCs using the new suspension bioreactor protocols, we managed to obtain cell fold increases of 103 within 30 days, which was superior to the 200‐fold increase obtained using the standard protocol. The cells were found to retain their defining characteristics after several passages in suspension. This new bioprocess represents a more efficient approach for generating large numbers of hBM‐MSCs in culture, which in turn should facilitate the development of new stem cell‐based therapies. Copyright

Is vibration truly an injurious stimulus in the human spine

Epidemiological data at one time was taken to suggest that chronic vibrations--for example operating vehicles with low-quality seats--contributed to intervertebral disc degeneration and lower back pain. More recent discussions, based in part upon extended twin studies, have cast doubt upon this interpretation, and question how much of the vibration is actually transmitted to the spine during loading. This review summarizes our recent survey of the current state of knowledge. In particular, we note that current studies are lacking a detailed factorial exploration of frequency, amplitude, and duration; this may be the primary cause for inconclusive and/or contradictory studies. It is our conclusion that vibrations are still an important consideration in discogenic back pain, and further controlled studies are warranted to definitively examine the underlying hypothesis: that chronic vibration can influence IVD cell biology and tissue mechanics.

Optimizing gelling parameters of gellan gum for fibrocartilage tissue engineering.

Gellan gum is an attractive biomaterial for fibrocartilage tissue engineering applications because it is cell compatible, can be injected into a defect, and gels at body temperature. However, the gelling parameters of gellan gum have not yet been fully optimized. The aim of this study was to investigate the mechanics, degradation, gelling temperature, and viscosity of low acyl and low/high acyl gellan gum blends. Dynamic mechanical analysis showed that increased concentrations of low acyl gellan gum resulted in increased stiffness and the addition of high acyl gellan gum resulted in greatly decreased stiffness. Degradation studies showed that low acyl gellan gum was more stable than low/high acyl gellan gum blends. Gelling temperature studies showed that increased concentrations of low acyl gellan gum and CaCl₂ increased gelling temperature and low acyl gellan gum concentrations below 2% (w/v) would be most suitable for cell encapsulation. Gellan gum blends were generally found to have a higher gelling temperature than low acyl gellan gum. Viscosity studies showed that increased concentrations of low acyl gellan gum increased viscosity. Our results suggest that 2% (w/v) low acyl gellan gum would have the most appropriate mechanics, degradation, and gelling temperature for use in fibrocartilage tissue engineering applications.

Identifying Mechanisms for Therapeutic Intervention in Chordoma : c-Met Oncoprotein

Study Design. A human sacral chordoma cell line, CCL3, was established and in vitro characterization of c-Met oncoprotein in chordoma cells was performed. Objective. Determination of whether c-Met plays an important role in chordoma’s malignancy. Summary of Background Data. Chordomas are malignant life-threatening tumors that arise from the remnants of the notochord. c-Met is an oncoprotein that is expressed by a variety of solid tumors, including chordomas, and HGF is its high affinity ligand. In the present study, we investigated c-Met and HGF expression, localization, and function in human chordoma cells. Methods. SDS-PAGE, Western blotting, immunofluorescence techniques, and cell migration functional assays were used to asses c-Met and HGF expression, localization, and functional activity. Results. Intracellular protein tyrosine phosphorylation was enhanced on HGF binding, and an increase in the amount of 50 kDa &agr;-chain of c-Met was detected in HGF-stimulated cells. Immunostaining of c-Met and HGF revealed membrane/cytoplasmic localization in nonstimulated cells, and perinuclear colocalization in HGF-stimulated cells. Positive chemotactic and migration activity in response to HGF was also demonstrated. Conclusion. Our data supports our hypothesis that the c-Met oncoprotein plays a leading role in the metastatic process in chordomas, and that a c-Met-HGF pair is involved in chordoma malignancy. Taking into consideration the very limited treatment options and an extremely poor prognosis for the chordoma patients, our results are a valuable and promising addition to the current situation in managing chordomas.

The role of extracellular factors in human metastatic chordoma cell growth in vitro.

Study Design. Human metastatic chordoma cells were isolated, and initial in vitro characterization was performed. Biochemical and physiologic changes were observed in response to pH, oxygen, and glucose. Objective. The extracellular microenvironment directly affects metastatic chordoma cell phenotype in vitro. Summary of Background Data. Chordomas are primary bone tumors that usually occur in the spine or skull. Chordomas arise from embryonic notochordal remnants along the axial skeleton, most commonly the sacrum, followed by the base of the skull and the mobile spine. Due to a high degree of resistance to radiation and chemotherapy, chordomas eventually cause death by direct growth or by metastasizing to other organs. Methods. Extracellular pH, oxygen, and glucose levels in the culture medium were controlled, and cell response was assessed using MTT staining, SDS-PAGE, Western blotting, tandem mass spectrometry, TUNEL, immunofluorescence, and flow cytometry. Results. In this study, we present a new chordoma cell line established from metastatic tissue and novel data characterizing some aspects of chordoma cell phenotype in different conditions in vitro. Chordoma biologic markers were expressed in the new cell line. Alkaline pH dramatically increased intracellular protein tyrosine phosphorylation, metabolic activity, and albumin accumulation in the cells, while acidic pH caused apoptosis. Conclusion. The level of proliferation, apoptosis, and tyrosine phosphorylation, as well as the overall protein expression profile, strongly depended on extracellular media pH and oxygen/glucose levels. Chordomas preferred extracellular microenvironment in vitro was rather alkaline, with an optimum at pH 8.5, and apoptotic changes were induced at acidic pH. We found that bovine serum albumin was accumulated by chordoma cells from the incubation media, and this accumulation depended on extracellular pH, with the highest accumulation at alkaline pH.

In situ cell-matrix mechanics in tendon fascicles and seeded collagen gels: implications for the multiscale design of biomaterials.

Designing biomaterials to mimic and function within the complex mechanobiological conditions of connective tissues requires a detailed understanding of the micromechanical environment of the cell. The objective of our study was to measure the in situ cell–matrix strains from applied tension in both tendon fascicles and cell-seeded type I collagen scaffolds using laser scanning confocal microscopy techniques. Tendon fascicles and collagen gels were fluorescently labelled to simultaneously visualise the extracellular matrix and cell nuclei under applied tensile strains of 5%. There were significant differences observed in the micromechanics at the cell–matrix scale suggesting that the type I collagen scaffold did not replicate the pattern of native tendon strains. In particular, although the overall in situ tensile strains in the matrix were quite similar (∼2.5%) between the tendon fascicles and the collagen scaffolds, there were significant differences at the cell–matrix boundary with visible shear across cell nuclei of >1 μm measured in native tendon which was not observed at all in the collagen scaffolds. Similarly, there was significant non-uniformity of intercellular strains with relative sliding observed between cell rows in tendon which again was not observed in the collagen scaffolds where the strain environment was much more uniform. If the native micromechanical environment is not replicated in biomaterial scaffolds, then the cells may receive incorrect or mixed mechanical signals which could affect their biosynthetic response to mechanical load in tissue engineering applications. This study highlights the importance of considering the microscale mechanics in the design of biomaterial scaffolds and the need to incorporate such features in computational models of connective tissues.