Degenerative grade affects the responses of human nucleus pulposus cells to link-N, CTGF, and TGFß3.

STUDY DESIGN: Cells isolated from moderately and severely degenerated human intervertebral disks (IVDs) cultured in an alginate scaffold. OBJECTIVE: To compare the regenerative potential of moderately versus severely degenerated cells using 3 proanabolic stimulants. SUMMARY OF BACKGROUND DATA: Injection of soluble cell signaling factors has potential to slow the progression of IVD degeneration. Although degenerative grade is thought to be an important factor in targeting therapeutic interventions it remains unknown whether cells in severely degenerated IVDs have impaired metabolic functions compared to lesser degenerative levels or if they are primarily influenced by the altered microenvironment. METHODS: Nucleus pulposus (NP) cells were cultured in alginate for 21 days and treated with 3 different proanabolic stimulants: a growth factor/anti-inflammatory combination of transforming growth factor ß3 (TGFß3)+dexamethasone (Dex), or matricellular proteins connective tissue growth factor (CTGF) or Link-N. They were assayed for metabolic activity, DNA content, glycosaminoglycan, and qRT-PCR gene profiling. RESULTS: Moderately degenerated cells responded to stimulation with increased proliferation, decreased IL-1ß, MMP9, and COL1A1 expression, and upregulated HAS1 as compared with severely degenerated cells. TGFßR1 (ALK5) receptors were expressed at greater levels in moderately than severely degenerated cells. TGFß3+Dex had a notable stimulatory effect on moderately degenerated NP cells with increased anabolic gene expression and decreased COL1A1 and ADAMTS5 gene expression. Link-N and CTGF had similar responses in all assays, and both treatments upregulated IL-1ß expression and had a more catabolic response than TGFß3+Dex, particularly in the more severely degenerated group. All groups, including different degenerative grades, produced similar amounts of glycosaminoglycan. CONCLUSIONS: Proanabolic stimulants alone had limited capacity to overcome the catabolic and proinflammatory cytokine expression of severely degenerated NP cells and likely require additional anti-inflammatory treatments. Moderately degenerated NP cells had greater TGFß receptor 1 expression and better responded to anabolic stimulation.

Perioperative Family Updates Reduce Anxiety and Improve Satisfaction: A Randomized Controlled Trial.

PURPOSE: This study aimed to determine if standardized updates at specific perioperative events affect anxiety and satisfaction of the family members and if the length of surgical procedure affects the satisfaction with updates. METHODS: This study was a randomized control trial. In the control group, surgeons communicated with the family only once near the completion of the procedure. In the intervention group, families received electronic updates at 3 significant perioperative events during the procedure. A postoperative survey rating family member satisfaction and anxiety levels, using a Likert scale of 0-5, was administered. RESULTS: Mean level of overall satisfaction did not differ between groups (intervention: 4.68 ± 0.69 [95% CI: 4.50, 4.87]; control: 4.61 ± 0.78 [95% CI: 4.40, 4.82]; P=0.69). Mean anxiety levels were lower in the intervention group (2.48 ± 1.43 [2.10, 2.86]) than in the control group (3.12 ± 1.32 [2.77, 3.47]; P=0.01). Mean satisfaction with perioperative updates was higher in the intervention (4.48 ± 0.83 [4.26, 4.70]) versus control group (3.16 ± 1.89 [2.67, 3.65]; P=0.0001). For all subjects, there was positive correlation between procedure time and anxiety (Spearman's rho: 0.34; P=0.0002) and negative correlation between procedure time and overall satisfaction (Spearman's rho: -0.23; P=0.01). CONCLUSIONS: Anxiety and satisfaction with perioperative updates were significantly improved by additional perioperative updates. These findings indicate that updating families during significant standardized strategic perioperative events can reduce the anxiety of loved ones and are preferred by most families.

Cervical transforaminal injection and the radicular artery: variation in anatomical location within the cervical intervertebral foramina.

BACKGROUND: Recent articles have detailed the adverse events associated with transforaminal steroid injections into the radicular arteries. Guidelines on strict transforaminal epidural techniques have been described to limit complications. There remains limited information regarding the cervical level of entry, location within the intervertebral foramina, and prevalence of the radicular arteries within the population. METHODS: With the aid of premortem angiography and postmortem latex-injected vasculature, a single detailed cadaveric dissection was performed to elucidate the specific anatomic location of the radicular arteries within the intervertebral foramina and the anastomoses of the arteries to the anterior spinal artery. RESULTS: Five radicular arteries (C5, C6, two at C7, C8) were traced entering the foramina either anteriorly or posteriorly to supply the anterior and posterior spinal arteries. Radicular arteries received blood supply from either the deep cervical (C8) or vertebral arteries. The C8 radicular artery entered the lateral aspect of the foramen and penetrated the dural sleeve within the inferior portion of the foramen, directly inferior to the exiting spinal nerve, to supply the anterior spinal artery. This artery was of a large enough caliber to be entered by a 22-gauge needle. CONCLUSIONS: A larger population is necessary to characterize the range of anatomic variations in arterial supply within the foramina. Available studies support the current technique of fluoroscopic needle insertion. Yet, there is wide anatomic variation in the origin and location of these vessels, and even with strict adherence to technique, it is feasible that a properly placed needle could penetrate a radicular artery.

Regenerative potential of TGFß3 + Dex and notochordal cell conditioned media on degenerated human intervertebral disc cells.

Injection of soluble cell signaling factors into degenerated intervertebral discs (IVDs) offers a minimally invasive treatment that could limit the processes of degeneration by stimulating native matrix repair. This study evaluated the regenerative capacity of degenerated nucleus pulposus (NP) cells obtained from patients undergoing anterior interbody fusions by measuring metabolic activity, DNA content, glycosaminoglycan (GAG) content, and cellular phenotype using qRT-PCR profiling with a custom array of 42 genes. NP cells were cultured in alginate for 7 days with 4 treatment groups: transforming growth factor beta 3 (TGFß3) + dexamethasone (Dex), soluble factors released from notochordal cells (NCs) cultured in alginate (NCA), soluble factors released from NCs in their native tissue environment (NCT), and basal media. TGFß3 + Dex stimulated degenerated human NP cells to proliferate and exhibit an anti-catabolic gene expression profile (with a decrease in ADAMTS5 and MMP1 compared to basal, and an increase in SOX9, decrease in ADAMTS5, MMP1, collagen I and collagen III compared to day 0), while NCA stimulated the greatest GAG per cell. We conclude that degenerated human NP cells exhibit regenerative potential, and that an optimal treatment will likely require treatments, such as TGFß3 + Dex, which were able to increase cell metabolism and reduce catabolism, as well as treatments with factors found in NC conditioned medium, that were able to produce high amounts of GAG per cell. Additional studies to optimize NC culture conditions are required to determine if NC conditioned medium can be made with the capacity to enhance NP cell proliferation and metabolism.

Effects of torsion on intervertebral disc gene expression and biomechanics, using a rat tail model.

STUDY DESIGN: In vitro and in vivo rat tail model to assess effects of torsion on intervertebral disc biomechanics and gene expression. OBJECTIVE: Investigate effects of torsion on promoting biosynthesis and producing injury in rat caudal intervertebral discs. SUMMARY OF BACKGROUND DATA: Torsion is an important loading mode in the disc and increased torsional range of motion is associated with clinical symptoms from disc disruption. Altered elastin content is implicated in disc degeneration, but its effects on torsional loading are unknown. Although effects of compression have been studied, the effect of torsion on intervertebral disc gene expression is unknown. METHODS: In vitro biomechanical tests were performed in torsion on rat tail motion segments subjected to 4 treatments: elastase, collagenase, genipin, control. In vivo tests were performed on rats with Ilizarov-type fixators implanted to caudal motion segments with five 90 minute loading groups: 1 Hz cyclic torsion to ± 5 ± 15° and ± 30°, static torsion to + 30°, and sham. Anulus and nucleus tissues were separately analyzed using qRT-PCR for gene expression of anabolic, catabolic, and proinflammatory cytokine markers. RESULTS: In vitro tests showed decreased torsional stiffness following elastase treatment and no changes in stiffness with frequency. In vivo tests showed no significant changes in dynamic stiffness with time. Cyclic torsion upregulated elastin expression in the anulus fibrosus. Up regulation of TNF-α and IL-1ß was measured at ±30°. CONCLUSION: We conclude that strong differences in the disc response to cyclic torsion and compression are apparent with torsion increasing elastin expression and compression resulting in a more substantial increase in disc metabolism in the nucleus pulposus. Results highlight the importance of elastin in torsional loading and suggest that elastin remodels in response to shearing. Torsional loading can cause injury to the disc at excessive amplitudes that are detectable biologically before they are biomechanically.

In vivo intervertebral disc remodeling: kinetics of mRNA expression in response to a single loading event.

Kinetics of mRNA expression following a single loading event was measured using an in vivo rat tail model. Animals were instrumented and loaded in compression for 1.5 h at 1 MPa and 1 Hz. Real-time RT-PCR was used to measure mRNA levels 0, 8, 24 and 72 h after mechanical stimulation for genes associated with matrix proteins (aggrecan, collagen-I, collagen-II), proteases (MMP-2, MMP-3, MMP-13, ADAMTS-4), and their inhibitors (TIMP-1, TIMP-3) in anulus fibrosus and nucleus pulposus regions. Baseline mRNA levels were of greatest abundance for matrix proteins and lowest for proteases. The mRNA levels reached maximum levels 24 h following mechanical stimulation for the majority of genes evaluated, but some had maximum levels 8 and 72 h following loading. The mRNA levels returned to baseline levels for all genes in the nucleus 72 h following loading, but the majority of genes in the anulus remained upregulated. Results support a coordinated strategy of relative mRNA expression that varied over time beginning with inhibition of tissue breakdown, followed by synthesis of aggrecan and matrix degrading enzymes, and eventually collagen metabolism days following loading. Consequently, optimal time for tissue harvest for mRNA measurements depends on genes of interest. Results suggest attempts at anabolic remodeling must be given adequate time for metabolic processes and protein synthesis to occur, and that changes in TIMP and MMP levels may have greater potency in affecting structural protein abundance than direct changes in the structural protein messages. Results have important implications for disc remodeling and tissue engineering.