3D-Printing of Meso-structurally Ordered Carbon Fiber/Polymer Composites with Unprecedented Orthotropic Physical Properties.

Here we report the first example of a class of additively manufactured carbon fiber reinforced composite (AMCFRC) materials which have been achieved through the use of a latent thermal cured aromatic thermoset resin system, through an adaptation of direct ink writing (DIW) 3D-printing technology. We have developed a means of printing high performance thermoset carbon fiber composites, which allow the fiber component of a resin and carbon fiber fluid to be aligned in three dimensions via controlled micro-extrusion and subsequently cured into complex geometries. Characterization of our composite systems clearly show that we achieved a high order of fiber alignment within the composite microstructure, which in turn allows these materials to outperform equivalently filled randomly oriented carbon fiber and polymer composites. Furthermore, our AM carbon fiber composite systems exhibit highly orthotropic mechanical and electrical responses as a direct result of the alignment of carbon fiber bundles in the microscale which we predict will ultimately lead to the design of truly tailorable carbon fiber/polymer hybrid materials having locally programmable complex electrical, thermal and mechanical response.

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.

BMP-2 and CDMP-2: stimulation of chondrocyte production of proteoglycan.

A hallmark of intervertebral disc degeneration is loss of proteoglycans. Cytokines may be used to stimulate proteoglycan production in the disc to reverse or prevent disc degeneration. The effects of bone morphogenetic protein 2 (BMP-2) and cartilage-derived morphogenetic protein 2 (CDMP-2) (singly and jointly) on proteoglycan synthesis by a chondrocytic cell line (MC615) were studied. MC615 cells were dosed with BMP-2, CDMP-2, or both, cultured for 6 days, and then assayed as follows: (1). The proteoglycan content of the medium and extracellular matrix were determined by dimethyl-methylene blue staining; (2). cell numbers were determined after 6 days of culture using the Hoechst dye DNA assay; (3). aggrecan mRNA was measured with the reverse transcription-polymerase chain reaction and Northern blotting assays. Both BMP-2 and CDMP-2 significantly enhanced proteoglycan production and aggrecan mRNA expression in a dose-dependent manner, although BMP-2 is more effective than CDMP-2 in increasing sulfated proteoglycan production. But BMP-2 and CDMP-2 have only a slight, nonsignificant effect on cell proliferation. There is no evidence of antagonism or synergy between the two growth factors in the aggrecan gene expression.

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.

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.