Jun Li

ISSLS Prize Winner : LMP-1 Upregulates Intervertebral Disc Cell Production of Proteoglycans and BMPs In Vitro and In Vivo

Study Design. Experiments using both in vitro tissue culture and in vivo rabbit methods were used to study the effect of Lim Mineralization Protein-1 (LMP-1) on intervertebral disc (IVD) cell production of proteoglycans and bone morphogenetic proteins (BMPs). Objectives. To determine the effect of LMP-1 overexpression in IVD cells on the production of proteoglycans and BMPs both in vitro and in vivo and to show that LMP-1 mediates the control of proteoglycan production through its action on BMPs. Summary of Background Data. Because BMPs are known to increase proteoglycan synthesis and LMP-1 is known to upregulate BMPs in certain cells, it was hypothesized that LMP-1 may increase proteoglycan production in IVD cells. Methods. DMMB, real-time polymerase chain reaction, and ELISA methods were used to quantitate proteoglycan, mRNA, and protein levels, respectively. Noggin was used to block the effect of the adenovirus carrying LMP-1 (AdLMP-1) on proteoglycan synthesis. In vivo experiments using intradiscal AdLMP-1 injection were performed with New Zealand White rabbits. Three weeks later, the mRNA levels of LMP-1, aggrecan, BMP-2, and BMP-7 were measured. Results. In vitro experiments revealed that the sulfated glycosaminoglycan (sGAG) and aggrecan mRNA levels were significantly increased with AdLMP-1 treatment. Similarly, BMP-2 and BMP-7 mRNA and protein levels increased significantly, but BMP-4 and BMP-6 levels were unchanged. Noggin blocked the upregulation of proteoglycan by AdLMP-1. In vivo discs injected with AdLMP-1 had significantly elevated levels of LMP-1, BMP-2, and BMP-7 mRNA. Conclusions. LMP-1 overexpression increases disc cellproduction of proteoglycan, BMP-2, and BMP-7. LMP-1mediates the control of proteoglycan production through its action on BMP.

The Effect of Bone Morphogenetic Protein-2 on Rat Intervertebral Disc Cells in Vitro

Study Design. An in vitro experiment to determine the molecular and cellular effect of recombinant human bone morphogenetic protein-2 on cultured rat intervertebral disc cells was performed. Objectives. To determine the effect of recombinant human bone morphogenetic protein-2 on cell proliferation, production of sulfated-glycosaminoglycan, and the expression of genes specific for chondrocytes (Type II collagen, aggrecan, and Sox9) in cultured rat intervertebral disc cells. Summary of Background Data. Intervertebral disc degeneration is associated with cellular and biochemical changes, which include decreased synthesis of cartilage specific gene products such as Type II collagen and aggrecan. Although bone morphogenetic protein-2 is known to induce chondrogenesis during new bone formation, the effects on intervertebral disc cells have not been characterized. Method. Cells were isolated from the anulus fibrosus and transition zones of lumbar discs from Sprague-Dawley rats. The cells were grown in monolayer and treated with recombinant human bone morphogenetic protein-2 (0, 10, 100, 1000 ng/mL) in Dulbecco’s Modified Eagle Medium/F-12 with 1% fetal bovine serum (day 0). On days 2, 4, and 7 after recombinant human bone morphogenetic protein-2 treatment, sulfated-glycosaminoglycan content in the media was quantified using 1,9-dimethylmethylene blue staining. The results were normalized according to culture duration and cell number. On day 7, mRNA was extracted for reverse transcriptase-polymerase chain reaction and real-time polymerase chain reaction to quantitate mRNAs of Type I collagen, Type II collagen, aggrecan, Sox9, osteocalcin, and glyceraldehyde phosphate dehydrogenase. Cell number was determined with a hemocytometer. Results. Recombinant human bone morphogenetic protein-2 at 100 and 1000 ng/mL yielded a 17% and 42% increase in cell number on day 4, and a 59% and 79% on day 7, respectively. Recombinant human bone morphogenetic protein-2 at 10 ng/mL had no effect on cell number. Sulfated-glycosaminoglycan increase was greatest at day 7, increasing by 1.3-, 2.1-, and 3.6-fold with recombinant human bone morphogenetic protein-2 treatments of 10, 100, and 1000 ng/mL, respectively. Increases in mRNA levels of Type II collagen, aggrecan, Sox9, and osteocalcin were observed with recombinant human bone morphogenetic protein-2 concentrations of 100 and 1000 ng/mL on day 7 as determined by reverse transcriptase-polymerase chain reaction. No detectable increase in mRNA level of Type I collagen was observed with any levels of recombinant human bone morphogenetic protein-2. Real-time polymerase chain reaction showed the greatest effect at 1000 ng/mL recombinant human bone morphogenetic protein-2, leading to an 11.5-fold increase in aggrecan, a 4.6-fold increase in Type II collagen, a 5.3-fold increase in Sox9, and a 1.9-fold increase in osteocalcin mRNA above untreated controls at day 7. Conclusion. The results of this study show that recombinant human bone morphogenetic protein-2 enhances disc matrix production and chondrocytic phenotype of intervertebral disc cells. Recombinant human bone morphogenetic protein-2 increases cell proliferation and sulfated-glycosaminoglycan (proteoglycan) synthesis. It increases mRNA of Type II collagen, aggrecan, and Sox9 genes (chondrocyte specific genes), and osteocalcin, but not Type I collagen or glyceraldehyde phosphate dehydrogenase.

Disc degeneration in the rabbit: a biochemical and radiological comparison between four disc injury models.

Study Design. A biochemical and radiologic comparison of 4 disc injury models to produce disc degeneration in the rabbit was carried out in 2 experiments. Objectives. To develop a reliable animal model of intervertebral disc degeneration. Summary of Background Data. In order to study various interventions for retarding or preventing disc degeneration, a reliable animal model of disc degeneration is needed. Methods. First experiment: 7 New Zealand white rabbits (1 year old, 3.5–4.5 kg body weight) were used to test 4 different disc injury models; intradiscal injection of Camptothecin (an apoptotic agent) using a 23-gauge needle at L2–L3, nucleus aspiration using a 21-gauge needle at L3–L4, 3 anulus punctures using a 21-gauge needle at L4–L5, and 1 anulus puncture using a 18-gauge needle at L5–L6. The L1–L2 level was used as a control. Rabbits were killed 12 weeks later. Lumbar spinal magnetic resonance images were assessed using 4 grades of disc degeneration. The water content of the nucleus was measured. Dimethylmethylene blue (DMMB) assay was used to measure the sulfated-glycosaminoglycan content. Second experiment: the 21-gauge 3-puncture and the 18-gauge 1-puncture models, thought most effective in producing disc degeneration in the first experiment, were again used in a second study. Six rabbits were killed 8 weeks later, the water and sulfated-glycosaminoglycan contents being measured as in the first experiment. Results. In the first experiment, the water content in the aspiration and puncture models was significantly decreased. Only the sulfated-glycosaminoglycan content in the aspiration model showed a significant decrease as compared to the control. Disc heights and magnetic resonance grades documented significant degeneration occurring in the aspiration and puncture models. In the second experiment, the water content showed a significant decrease in the 21-gauge 3-puncture model, whereas neither of the results for the sulfated-glycosaminoglycancontent showed a significant difference as compared to the control data. Conclusion. In the first experiment, the 21-gauge 3-puncture and the 18-gauge 1-puncture models produced the most consistent disc degeneration in the rabbit lumbar spine. When these 2 models were again studied in the second experiment, the 21-gauge 3-puncture technique was superior in producing disc degeneration over a shorter period of time.

Effect of bone morphogenetic protein-2 (BMP-2) on matrix production, other BMPs, and BMP receptors in rat intervertebral disc cells.

Objective An in vitro experiment study using rat disc cells was carried out to determine the effect of bone morphogenetic protein-2 (BMP-2) on extracellular matrix production, other BMPs, and BMP receptors (BMPRs). Methods Cells from the anulus fibrosus and transition zone were harvested and cultured. When the cells reached 80% confluence, BMP-2 was added to reach a final concentration of 200 ng/mL. Three days later, the culture media were collected for the assay of sulfated glycosaminoglycans (sGAG) and collagen types I and II. The cells were harvested for RNA extraction to determine the genes expressed. All experiments were performed at least three times to ensure repeatability. Results BMP-2 significantly increased aggrecan and collagen type II mRNA expression 8.30 and 4.61 times, respectively, and decreased versican mRNA expression 0.54 times as compared with control. Collagen type I production and mRNA level were not changed. BMP-2 significantly increased transforming growth factor-β1 (TGFβ1) and BMP-7 mRNA expression 2.32 and 2.45 times, respectively, compared with control. There was no significant change in BMP-6 mRNA expression. BMPR type IB and II mRNA expressions were increased and BMPR type 1A mRNA expression was decreased, but none of these differences was significant. Conclusions The results of this study show that in rat intervertebral disc cells, BMP-2 increases aggrecan and collagen type II mRNA expression and decreases versican gene expression. BMP-2 also up-regulates mRNA expression for BMP-7 and TGFβ but has no significant effect on the BMPRs.

Effect of tail suspension (or simulated weightlessness) on the lumbar intervertebral disc: study of proteoglycans and collagen.

Study Design. An experiment to measure the proteoglycan and collagen content of the lumbar intervertebral discs of rats that had been tail-suspended for up to 4 weeks. Objectives. To determine the effect of tensile force (or simulated weightlessness) on the intervertebral disc. Summary of Background Data. During space flight the intervertebral disc experiences low compressive force (because of so-called “weightlessness”), which, in turn, produces, among other things, low hydrostatic pressure acting on the disc cells. Although disc cells respond (in vitro) to changes in hydrostatic pressure, it is unclear what effect low levels of hydrostatic pressure have in vivo and whether they lead to a degenerative catabolic process. The rat tail-suspension model is appropriate for studying the effects of tensile force on the disc. The disc (especially the anulus) is subjected to tension during various body movements (e.g., bending stretches the posterior anulus, and twisting tensions the whole anulus). Methods. Thirty-two Sprague–Dawley rats were tail-suspended for either 2 weeks (16 rats) or 4 weeks (16 rats). Sixteen other rats were left unsuspended for 4 weeks; these were used as controls. At the end of 2 or 4 weeks, as appropriate, the rats were killed and their lumbar spines were removed. In each rat the six lumbar discs were bisected and the discs (anulus and nucleus together) were carefully removed. The six lumbar discs from one rat were pooled with the six lumbar discs of a second matching rat (i.e., from the same group) to give one sample. The disc samples were then assessed using enzyme-linked immunosorbent assays. Results. There was a 35% statistically significant decrease in proteoglycan content going from the control group down to the 4-week group, but no significant differences between the control group and the 2-week group or between the 2-week group and the 4-week group. There were no statistically significant differences between the three groups for collagen I or collagen II. Conclusions. These findings clearly establish a link between decreased proteoglycan content and tension on the disc, as modeled by the tail-suspended rat.

The effect of blocking a nutritional pathway to the intervertebral disc in the dog model.

Background The hypothesis that injecting bone cement adjacent to one or both endplates would bring about degeneration in the intervening disc was tested. Methods In 11 dogs, bone cement was injected just below the superior endplates of L1, L2, and L3 to block the nutritional supply through these endplates to the three intervertebral discs T13–L1, L1–L2, and L2–L3. In one other dog, both the superior and the inferior endplates of the same discs (T13–L1, L1–L2, and L2–L3) were blocked with bone cement. All 12 dogs were euthanized between 31 and 70 weeks after the surgery. The three experimental discs (T13–L1, L1–L2, and L2–L3) and two control discs (T12–T13 and L4–L5) were excised and assessed using enzyme-linked immunosorbent assay (ELISA) and histology. Results Radiographs of the lumbar spine at the time of death did not show any signs of disc bulging, disc space narrowing, or peripheral osteophyte formation in any of the 12 dogs. The experimental discs as well as the control discs appeared normal in every dog. After the discs were bisected, they were carefully inspected for any visible signs of degeneration. The experimental discs showed no clear signs of disc degeneration and were not distinguishable from the control discs on a gross level. The numerical results from the ELISA showed that in the experimental discs as opposed to the control discs, there were significant increases in proteoglycan content in both the nucleus (P = 0.033) and annulus (P = 0.01) and clear histologic changes in some of the discs. Conclusion The results show that injecting bone cement adjacent to one or both endplates for up to 70 weeks does not produce degeneration in any visible form in the intervening disc. There were no disc bulging, no apparent annular fissures, and no disc spacing narrowing. There were, however, increases in protoglycan content in both the nucleus and the annulus and clear histologic changes in some of the discs.

A comparison of three cell types as potential candidates for intervertebral disc therapy: annulus fibrosus cells, chondrocytes, and bone marrow derived cells.

OBJECTIVES Candidate cell types for disc cell transplantation therapy include anulus fibrosus (AF) cells, chondrocytes, and bone marrow derived cells (BMDCs). We compared the disc matrix production in these three types of cells, before and after stimulation with rhBMP-2. There is no study extant that compares these three cell types to determine the best candidate for the disc cell therapy. METHODS AF cells, chondrocytes, and BMDCs (iliac crest and femur) were isolated and grown in monolayer. They were treated for 3 days with rhBMP-2. After 3 days, proteoglycan (sGAG) content in the media was quantified. The results were normalized by cell numbers. The mRNA expression of aggrecan, type I collagen, and type II collagen was measured using real-time PCR. Each cell type was also cultured in chamber slides and immunostained for aggrecan, type I collagen, and type II collagen after 3 days of treatment with rhBMP-2. RESULTS (1) Without rhBMP-2 the chondrocytes produced more proteoglycan (sGAG) as compared to the other two cell types (AF cells and BMDCs). After stimulation with rhBMP-2 the chondrocytes produce even more proteoglycan than the other two cell types. (2) As compared to the other two cell types, in terms of mRNA expression, the chondrocytes expressed more aggrecan, type I collagen, and type II collagen before stimulation with rhBMP-2. After rhBMP-2 stimulation, the chondrocytes expressed even more aggrecan, type I collagen, and type II collagen in proportion to the concentration of rhBMP-2. For the BMDCs there were no changes in type I and II collagen. (3) rhBMP-2 stimulation produced increases in the protein levels of aggrecan, type I and II collagen in all three types of cells. CONCLUSIONS On balance, according to these results, it would seem that chondrocytes are the best candidate for the disc cell therapy.

The AdLMP-1 transfection in two different cells; AF cells, chondrocytes as potential cell therapy candidates for disc degeneration

SummaryBackground. LMP-1 is known to increase proteoglycan production through the upregulating the BMPs and it is also known that BMP-2 acts on anulus fibrosus cells and chondrocytes to increase proteoglycan production. Method. We carried out an experiment, the effect of AdLMP-1 transfection on AF cells and chondrocytes in the production of sulfated-glycosaminoglycans, mRNA expression (aggrecan, type I, II collagen, LMP-1, BMP-2, and BMP-7), and immunofluorescence staining.AF cells and chondrocytes were grown in monolayer and treated for 6 days with AdLMP1-green fluorescence protein (GFP) (10, 20, and 30 multiplicity of infection [MOI]). After 6 days, the sGAG content in the media was quantified using 1,9-dimethylmethylene blue staining. The mRNA expression was measured with real-time PCR after 20 MOI infection of AdLMP1-GFP. The each cells treated with 20 MOI infection of AdGFP was used as a control group for the mRNA expression.The each cell group was immunofluorescence stained with each antibodies in the chamber slide at 3 × 104 cells/chamber. Findings. 1) The sGAG production was maximum in 20 MOI AdLMP1-GFP infection on the AdLMP-1 treatment for both of AF cells and chondrocytes. 2) The mRNA expression of aggrecan, type I collagen, type II collagen, LMP-1, BMP-2, and BMP-7 is increased in both AF cells and chondrocytes in 20 MOI AdLMP1-GFP infection. 3) On the immunofluorescence staining results, the positive immunofluorescence stained cell numbers are increased after 20 MOI AdLMP1-GFP infection concordant with upregulation of mRNA expression. Conclusions. The AdLMP-1 treatments in AF cells and chondrocytes may be useful for cell transplantation therapy in disc degeneration.