Pauline Baird

Nerve growth factor expression and innervation of the painful intervertebral disc

Following a previous description of nociceptive nerve fibre growth into usually aneural inner parts of painful intervertebral disc (IVD), this study has investigated whether nociceptive nerve ingrowth into painful IVD is stimulated by local production of neurotrophins. Immunohistochemistry and in situ hybridization have been used to investigate expression of the candidate neurotrophin, nerve growth factor (NGF), and its high‐ and low‐affinity receptors trk‐A and p75, respectively, in painful IVD excised for the management of low back pain. IVD from patients with back pain were of two types: those that when examined by discography reproduced the patient symptoms (pain level IVD) and those that did not (non‐pain level IVD). Microvascular blood vessels accompanied nerve fibres growing into pain level IVD and these expressed NGF. The adjacent nerves expressed the high‐affinity NGF receptor trk‐A. These vessels entered the normally avascular IVD through the discal end plates. NGF expression was not identified in non‐pain level or control IVD. Some non‐pain level IVD had vessels within them, which entered through the annulus fibrosus. These did not express NGF nor did nerves accompany them. These findings show that nociceptive nerve ingrowth into painful IVD is causally linked with NGF production by blood vessels growing into the IVD, from adjacent vertebral bodies. Copyright

Expression of chondrocyte markers by cells of normal and degenerate intervertebral discs

Aims: To investigate the phenotype of cells in normal and degenerate intervertebral discs by studying the expression of molecules characteristic of chondrocytes in situ. Methods: Human intervertebral discs taken at surgery were graded histologically, and classified on this basis as normal or degenerate. Eighteen of each type were selected, and in situ hybridisation was performed for the chondrocytic markers Sox9 and collagen II using 35S labelled cDNA probes. Aggrecan was located by immunohistochemistry, using the monoclonal antibody HAG7E1, and visualised with an avidin–biotin peroxidase system. Results: In the normal discs, strong signals for Sox9 and collagen II mRNA, and strong staining for the aggrecan protein were seen for the cells of the nucleus pulposus (NP), but reactions were weak or absent over the cells of the annulus fibrosus (AF). In degenerate discs, the Sox9 and collagen II mRNA signals remained visible over the cells of the NP and were again absent in the AF. Aggrecan staining was not visible in the NP cells, and was again absent in the AF. Conclusions: Cells of the normal NP showed expression of all three markers, clearly indicating a chondrocytic phenotype. In degeneration, there was evidence of a loss of aggrecan synthesis, which may contribute to the pathogenesis of disc degeneration. AF cells showed no evidence of a chondrocytic phenotype in either normal or degenerate discs.

Demonstration of estrogen receptor mRNA in bone using in situ reverse-transcriptase polymerase chain reaction.

Falling estrogen levels affect the female skeleton profoundly. Following menopause, estrogen lack is a major cause of osteoporosis. The site of estrogen action in human bone, however, is unclear, but responsive cells must express the estrogen receptor (ER). One obstacle to localizing these cells is that mRNA for ER is expressed in low copy number. Hence, conventional molecular techniques are either too insensitive to detect receptor transcripts (in situ hybridization) or necessitate amplification of RNA extracted from tissue [Northern analysis and polymerase chain reaction (PCR)], thus failing to identify the specific target cells within the mixed-cell population of bone. In situ PCR (IS-PCR) is a technique that combines the sensitivity of PCR with the localization of conventional in situ hybridization. The technique has previously been used primarily to detect single-copy genes and viral DNA within cells. More recently, incorporation of a reverse-transcriptase reaction (IS-RT-PCR) has allowed the technique to be used to identify rare mRNAs within tissues. We have therefore applied the technique of IS-RT-PCR to localize ER mRNA first in human breast tumors, a known positive tissue, and then in bone. Using conventional riboprobe in situ hybridization, ER transcripts were not detectable in any bone cells within sections taken from normal bone and several actively remodeling bone tissues, namely, Pagets disease, renal hyperparathyroidism, and healing fracture callus. The technique of IS-RT-PCR, however, allowed amplification of transcripts to a detectable level. Following two cycles of amplification, hybridization signal was observed in osteoblasts and to a lower level in osteoclasts and occasional osteocytes. This positive signal was more obvious after five cycles, particularly in osteoclasts and osteocytes. After ten cycles, although signal was increased in osteoclasts and osteocytes, it appeared to be decreased in osteoblasts, suggesting that overamplification leads to loss of target complex from these cells. We conclude that several cell types in human bone express ER mRNA in vivo.

An in vitro study investigating the survival and phenotype of mesenchymal stem cells following injection into nucleus pulposus tissue.

IntroductionThe decreased disc height characteristic of intervertebral disc (IVD) degeneration has often been linked to low back pain, and thus regeneration strategies aimed at restoring the disc extracellular matrix and ultimately disc height have been proposed as potential treatments for IVD degeneration. One such therapy under investigation by a number of groups worldwide is the use of autologous mesenchymal stem cells (MSCs) to aid in the regeneration of the IVD extracellular matrix. To date, however, the optimum method of application of these cells for regeneration strategies for the IVD is unclear, and few studies have investigated the direct injection of MSCs alone into IVD tissues. In the present article, we investigated the survival and phenotype of human MSCs, sourced from aged individuals, following injection into nucleus pulposus (NP) tissue explant cultures.MethodsHuman MSCs extracted from bone marrow were expanded in monolayer culture and, after labelling with adenoviral vectors carrying the green fluorescent protein transcript, were injected into NP tissue explants (sourced from bovine caudal discs) and maintained in culture for 2, 7, 14 and 28 days post injection. Following fixation and paraffin embedding, cell viability was assessed using in situ hybridisation for polyA-mRNA and using immunohistochemistry for caspase 3. Immunohistochemistry/fluorescence for aggrecan, Sox-9 and types I, II and X collagen together with Alizarin red staining was employed to investigate the MSC phenotype and matrix formation.ResultsMSCs were identified in all injected tissue samples and cell viability was maintained for the 4 weeks investigated. MSCs displayed cellular staining for Sox-9, and displayed cellular and matrix staining for aggrecan and type II collagen that increased during culture. No type I collagen, type X collagen or Alizarin red staining was observed at any time point.ConclusionsMSCs from older individuals differentiate spontaneously into chondrocyte-like NP cells upon insertion into NP tissue in vitro, and thus may not require additional stimulation or carrier to induce differentiation. This is a key finding, as such a strategy would minimise the level of external manipulation required prior to insertion into the patient, thus simplifying the treatment strategy and reducing costs.

Expression of parathyroid hormone-related protein and its receptor in bone metastases from prostate cancer

Studies of breast cancer suggest that parathyroid hormone‐related protein (PTHrP) is important in the development of bone metastases. To determine whether PTHrP expression is important in prostate cancer metastasis, immunohistochemistry and in situ hybridization were used to assess the expression of PTHrP and its receptor in primary prostate cancer and bone metastases from both prostate and non‐prostate cancers. PTHrP was expressed in more prostate primary tumours than bone metastases (p=0.003, Fishers exact test). All bone metastases from non‐prostate cancers expressed PTHrP. In contrast, PTHrP receptor was expressed in all bone metastases, but in only 19% of primary prostate tumours (p=0.001). The receptor to PTHrP was found to be highly expressed in bone metastases from prostate and other primaries, whereas PTHrP protein was found to have lower expression in the bone metastases than in the primary tumours. In conclusion, the expression of the receptor to PTHrP is increased in bone metastases from prostate cancer and may play an important role in their formation. Copyright

Expression of receptors for putative anabolic growth factors in human intervertebral disc: implications for repair and regeneration of the disc.

Low back pain (LBP) is a common, debilitating and economically important disorder. Current evidence implicates loss of intervertebral disc (IVD) matrix consequent upon ‘degeneration’ as a major cause of LBP. Degeneration of the IVD involves increases in degradative enzymes and decreases in the extracellular matrix (ECM) component in a process that is controlled by a range of cytokines and growth factors. Studies have suggested using anabolic growth factors to regenerate the normal matrix of the IVD, hence restoring disc height and reversing degenerative disc disease. However, for such therapies to be successful it is vital that the target cells (i.e. the disc cells) express the appropriate receptors. This immunohistochemical study has for the first time investigated the expression and localization of four potentially beneficial growth factor receptors (i.e. TGFβRII, BMPRII, FGFR3 and IGFRI) in non‐degenerate and degenerate human IVDs. Receptor expression was quantified across regions of the normal and degenerate disc and showed that cells of the nucleus pulposus (NP) and inner annulus fibrosus (IAF) expressed significantly higher levels of the four growth factor receptors investigated. There were no significant differences between the four growth factor expression in non‐degenerate and degenerate biopsies. However, expression of TGFβRII, FGFR3 and IGFRI, but not BMP RII, were observed in the ingrowing blood vessels that characterize part of the disease aetiology. In conclusion, this study has demonstrated the expression of the four growth factor receptors at similar levels in the chondrocyte‐like cells of the NP and IAF in both non‐degenerate and degenerate discs, implicating a role in normal disc homeostasis and suggesting that the application of these growth factors to the degenerate human IVD would stimulate matrix production. However, the expression of some of the growth factor receptors on ingrowing blood vessels might be problematic in a therapeutic approach. Copyright

Effect of ovarian steroid deficiency on oestrogen receptor alpha expression in bone.

The mechanism by which oestrogen and hormone replacement therapy (HRT) maintain bone mass in women is still unclear. It has previously been shown that cells of osteoblast lineage in vivo, particularly osteocytes, express oestrogen receptor α (ERα). Nevertheless, it is still debatable whether oestrogen and the ovarian steroids have a direct affect on osteocytes. If they could regulate osteocyte ERα expression, this would be strong evidence for the involvement of these cells in the hormonal regulation of bone mass. This study therefore aimed to compare bone biopsies from women who were replete with ovarian steroids (pre‐ovariectomy or post‐HRT) with those from the same women when hormone‐deficient (post‐ovariectomy or pre‐HRT) for cellular localization of ERα protein or mRNA expression by indirect immunofluorescence, or by in situ hybridization combined with reverse transcriptase‐polymerase chain reaction (IS‐RT‐PCR) respectively. Image analysis showed that proportions of osteocytes positive for immunodetectable ERα were higher in hormone‐replete than in hormone‐deficient women (25 ± SEM 3 per cent, 12 ± SEM 4 per cent, respectively; n = 5), with similar but non‐statistically significant changes in osteoblasts. This was observed even when HRT was commenced 18 years after menopause. In contrast, grain volume/unit cell area of osteoblast mRNA signal was markedly higher when hormone‐deficient (0·055 ± 0·01) than when hormone‐replete (0·016 ± 0·004), with similar but non‐significant differences in osteocytes. This preliminary study indicates up‐regulation of osteocyte ERα protein by ovarian steroids in these patients, which is accompanied by decreased osteoblast ERα mRNA expression, providing further evidence for the involvement of osteocytes in the regulation of skeletal structure by ovarian steroids. Copyright

Degenerate human nucleus pulposus cells promote neurite outgrowth in neural cells.

Innervation of nociceptive nerve fibres into the normally aneural nucleus pulposus (NP) of the intervertebral disc (IVD) occurs during degeneration resulting in discogenic back pain. The neurotrophins nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF), which are associated with stimulation of axonal outgrowth and nociception by neuronal cells, are both expressed by NP cells, with BDNF levels increasing with disease severity. However the mechanism of interaction between human NP cells and neural cells has yet to be fully elucidated. Therefore the aim of this study was to determine whether non-degenerate or degenerate human NP cells inhibit or stimulate neural outgrowth and whether any outgrowth is mediated by NGF or BDNF. Human NP cells from non-degenerate and degenerate IVD were cultured in alginate beads then co-cultured for 48 hours with human SH-SY5Y neuroblastoma cells. Co-culture of non-degenerate NP cells with neural cells resulted in both an inhibition of neurite outgrowth and reduction in percentage of neurite expressing cells. Conversely co-culture with degenerate NP cells resulted in an increase in both neurite length and percentage of neurite expressing cells. Addition of anti-NGF to the co-culture with degenerate cells resulted in a decrease in percentage of neurite expressing cells, while addition of anti-BDNF resulted in a decrease in both neurite length and percentage of neurite expressing cells. Our findings show that while non-degenerate NP cells are capable of inhibiting neurite outgrowth from human neural cells, degenerate NP cells stimulate outgrowth. Neurotrophin blocking studies demonstrated that both NGF and BDNF, secreted by degenerate NP cells, may play a role in this stimulation with BDNF potentially playing the predominant role. These findings suggest that NP cells are capable of regulating nerve ingrowth and that neoinnervation occurring during IVD degeneration may be stimulated by the NP cells themselves.

Canine bone marrow cell cultures infected with canine distemper virus: an in vitro model of Paget's disease.

We have previously shown that the canine paramyxovirus, canine distemper virus (CDV), is a possible aetiologic agent in Pagets disease of bone and in the canine bone disorder, metaphyseal osteopathy. More recently, we have examined the effects of CDV on the formation of multinucleated, tartrate resistant acid phosphatase positive, calcitonin receptor positive, osteoclast-like cells in cultures of canine bone marrow mononuclear cells, and shown that both in vitro and in vivo infection with CDV produced a dose dependent increase in the number and size of osteoclast-like cells. We have now extended these results to show that CDV infection induces interleukin-6 and c-Fos mRNA in these cells, similar to our recent findings in pagetic bone cells. These results further support the hypothesis that CDV might be involved in the aetiopathogenesis of Pagets disease and metaphyseal osteopathy and suggest that canine marrow culture systems will prove useful as an in vitro model to examine the disease processes in more detail.

Thermally-triggered gelation of PLGA dispersions: towards an injectable colloidal cell delivery system.

In this study the properties of poly(D,L-lactide-co-glycolide) (PLGA) dispersions containing a thermoresponsive cationic copolymer were investigated. The PLGA dispersions were prepared by interfacial deposition in aqueous solution and were rendered thermoresponsive by addition of a cationic poly(N-isopropyl acrylamide) (PNIPAm) graft copolymer. The copolymers used had the general composition PDMA(x)(+)-g-(PNIPAm(n))(y). DMA(+) is quarternarized N,N-dimethylaminoethyl methacrylate. The PDMA(x)(+)-g-(PNIPAm(n))(y) copolymers have x and y values that originate from the macroinitiator used for their preparation; values for n correspond to the PNIPAm arm length. The thermoresponsive dispersions were characterised using photon correlation spectroscopy, turbidity measurements and electrophoretic mobility measurements. A strong electrostatic attraction between the anionic PLGA particles and cationic copolymer was present and the dispersions showed thermally-triggered gelation at total polymer volume fractions as low as 0.015. These new PLGA gels, which formed at about 32 degrees C, had elastic modulus values that could be controlled using dispersion composition. Scanning electron micrographs of the gels showed high porosity and interconnectivity of elongated pores. Remarkably, the gels were flexible and had critical yield strains as high as 160%. The ability of the gels to support growth of bovine nucleus pulposus cells was investigated using two-dimensional cell culture. The cells proliferated and remained viable on the gels after 3days. The results suggest that this general family of biodegradable thermogelling PLGA dispersions, introduced here for the first time, may have longer-term application as an injectable colloidal cell delivery system.