J. Antoniou

Blood transfusion in primary total hip and knee arthroplasty. Incidence, risk factors, and thirty-day complication rates.

BACKGROUNDnThe aim of this study was to analyze NSQIP (National Surgical Quality Improvement Program) data to better understand the incidence, risk factors, and thirty-day complication rates associated with transfusions in primary total hip and knee arthroplasty.nnnMETHODSnWe identified 9362 total hip and 13,662 total knee arthroplasty procedures from the database and separated those in which any red blood-cell transfusion was performed within seventy-two hours after surgery from those with no transfusion. Patient demographics, comorbidities, preoperative laboratory values, intraoperative variables, and postoperative complications were compared between patients who received a transfusion and those who did not. Multivariate logistic regression was used to identify independent risk factors for receiving a transfusion as well as for associated postoperative complications (thirty-day incidences of infection, venous thromboembolism, and mortality).nnnRESULTSnThe transfusion rate after total hip arthroplasty was 22.2%. Significant risk factors for receiving a transfusion were age (OR [odds ratio] per ten years = 10.1), preoperative anemia (OR = 3.6), female sex (OR = 2.0), BMI (body mass index) of <30 kg/m(2) (OR = 1.4), and ASA (American Society of Anesthesiologists) class of >2 (OR = 1.3). Multivariate logistic regression analysis indicated that adjusted odds of infection, venous thromboembolism, and mortality did not differ significantly between patients who received a transfusion and those who did not. The transfusion rate after total knee arthroplasty was 18.3%. Risk factors for receiving a transfusion were age (OR per ten years = 10.2), preoperative anemia (OR = 3.8), BMI of <30 kg/m(2) (OR = 1.4), female sex (OR = 1.3), and ASA class of >2 (OR = 1.3). Multivariate logistic regression indicated that a transfusion was significantly associated with mortality (OR = 2.7) but not with infection or venous thromboembolism.nnnCONCLUSIONSnWe did not find a strong association between perioperative red blood-cell transfusion and thirty-day incidences of infection, venous thromboembolism, or mortality; however, the odds of mortality were higher in patients who received a transfusion during total knee arthroplasty.nnnLEVEL OF EVIDENCEnTherapeutic Level III. See Instructions for Authors for a complete description of levels of evidence.

Femoral Head Size Does Not Affect Ion Values in Metal-on-Metal Total Hips

BackgroundMetal-on-metal articulations can release substantial amounts of particles containing cobalt and chromium into the surrounding milieu, causing concern for cellular toxicity and adverse local soft tissue reactions. The diameter of the femoral head has been one of the variables that inversely affects wear of metal-on-metal total hip arthroplasty (THA). The oxidative stress of increased metal ions can be measured with serum markers. It is still controversial if larger femoral head diameters decrease wear rates in patients with metal-on-metal THA and if the increased metal ions alter the body’s antioxidant status.Questions/purposesWe therefore (1) determined whole blood metal ions in patients with small (28xa0mm and 36xa0mm) and large (40xa0mm and 44xa0mm) diameter femoral heads; (2) measured oxidative stress markers (total antioxidants, nitrotyrosine, and peroxides); and (3) determined whether acetabular version or inclination influenced ion levels.MethodsOne hundred four patients were retrospectively studied. We recorded Harris hip scores and UCLA activity scores. All patients were followed at 1xa0year.ResultsThe activity scores were similar in the two groups. There was no difference in metal ion levels or oxidative stress markers between patients with small- or large-diameter femoral heads. Acetabular inclination and anteversion had no effect on the metal ion levels.ConclusionsThe data suggest there is no difference in ion values in patients with large or small metal-on-metal THA and the increased metal ions do not alter the oxidant status of the patient.Level of EvidenceLevel III, retrospective comparative study. See Guidelines for Authors for a complete description of levels of evidence.

Shorter Hospital Stay and Lower 30-Day Readmission After Unicondylar Knee Arthroplasty Compared to Total Knee Arthroplasty

BACKGROUNDnReducing hospital stay and unplanned hospital readmission of arthroplasty patients has been a topic of recent interest. The aim of the present study was to query the National Surgical Quality Improvement Program database to compare the length of hospital stay (LOS) and the subsequent 30-day hospital readmission rates in patients undergoing primary unicondylar knee arthroplasty (UKA) and total knee arthroplasty (TKA).nnnMETHODSnWe identified 1340 UKAs and 36,274 TKAs over a 2-year period (2011-2012). Patient demographics, comorbidities, LOS, 30-day postoperative complications, and readmission rates were compared between the groups. Multivariate regression analysis was used to determine the effect of procedure type on LOS and readmission rates.nnnRESULTSnUnicondylar knee arthroplasty patients had a median LOS of 2 days compared to 3 days for TKAs (P < .001). The readmission rate in the TKA group was nearly double that of the UKA group (4.1% vs 2.2%) (P < .0001). Multivariate regression analysis identified that undergoing a UKA was predictive for a shorter LOS (coefficient -1 day) and was protective for 30-day readmission (odds ratio, 0.60; 95% confidence interval, 0.41-0.88).nnnCONCLUSIONnPatients undergoing UKA had a shorter LOS and a lower 30-day readmission rate compared to TKA patients. After adjusting for selected cofounders, we demonstrated that undergoing a UKA is a protective factor for 30-day readmission.

Effect of nitrogen-rich cell culture surfaces on type X collagen expression by bovine growth plate chondrocytes

BackgroundRecent evidence indicates that osteoarthritis (OA) may be a systemic disease since mesenchymal stem cells (MSCs) from OA patients express type X collagen, a marker of late stage chondrocyte hypertrophy (associated with endochondral ossification). We recently showed that the expression of type X collagen was suppressed when MSCs from OA patients were cultured on nitrogen (N)-rich plasma polymer layers, which we call PPE:N (N-doped plasma-polymerized ethylene, containing up to 36 atomic percentage (at.% ) of N.MethodsIn the present study, we examined the expression of type X collagen in fetal bovine growth plate chondrocytes (containing hypertrophic chondrocytes) cultured on PPE:N. We also studied the effect of PPE:N on the expression of matrix molecules such as type II collagen and aggrecan, as well as on proteases (matrix metalloproteinase-13 (MMP-13) and molecules implicated in cell division (cyclin B2). Two other culture surfaces, hydrophilic polystyrene (PS, regular culture dishes) and nitrogen-containing cation polystyrene (Primaria®), were also investigated for comparison.ResultsResults showed that type X collagen mRNA levels were suppressed when cultured for 4 days on PPE:N, suggesting that type X collagen is regulated similarly in hypertrophic chondrocytes and in human MSCs from OA patients. However, the levels of type X collagen mRNA almost returned to control value after 20 days in culture on these surfaces. Culture on the various surfaces had no significant effects on type II collagen, aggrecan, MMP-13, and cyclin B2 mRNA levels.ConclusionHypertrophy is diminished by culturing growth plate chondrocytes on nitrogen-rich surfaces, a mechanism that is beneficial for MSC chondrogenesis. Furthermore, one major advantage of such intelligent surfaces over recombinant growth factors for tissue engineering and cartilage repair is potentially large cost-saving.

Link-N Peptide: A Stepping Stone towards Biological Repair of Intervertebral Disk Degeneration

Introduction Back pain is a fairly common problem which affects a large portion of the population across all ages and has an impact on quality of life. Intervertebral disk (IVD) degeneration is the single most common implicated cause of back pain. Presently, there is no medical treatment or therapeutic agent to address this problem and surgery is the only offered option. Link-N peptide represents the 16 amino acid sequence from the N-terminus of the link protein that stabilizes the proteoglycan aggregates present in cartilage and disk. Link-N peptide is released from the link protein as a result of proteolysis, and has been suggested to play a role in matrix homeostasis by promoting new matrix synthesis. We evaluated its regenerative potential in intact human IVD. Materials and Methods Lumbar IVDs were obtained through organ donations via Transplant Quebec. Disks from seven individuals, five disks per spine, were harvested. Cells were isolated from nucleus pulposus (NP) and inner annulus fibrosus regions of the disks. Single cells were beaded in 1.2% alginate and cultured in DMEM containing 1 g/L glucose and 10% FBS. Alginate beads were exposed to 10–10000 ng/mL Link-N peptide for 48 hours. Intact disks were prepared for organ culture by parallel cuts through the adjacent vertebral bodies close to the end plates, and the remaining bone and the calcified part of the cartilage endplates were removed using a high-speed bone burr. Disks were maintained and cultured with no external load applied in DMEM containing 1 g/L glucose and supplemented with 1% FBS. Link-N was conjugated with 5-TAMRA dye then injected into the center of the disk. The distribution of Link-N in the medium and within the disk was studied to determine whether Link-N is retained in the disk. Disks from adjacent levels were matched for the degree of degeneration and were injected in their NP region with 50µCi 35SO4 along with 0.1 mg or 1 mg of Link-N in 100 µL of medium per disk and harvested after 48 hours. Sustained effect of Link-N was evaluated by injecting the disk with Link-N and injecting 35SO4 1 week later. Proteoglycan synthesis was evaluated by measuring 35SO4 incorporation. Results When human lumbar disk cells from NP and iAF regions beaded in alginate were exposed to Link-N peptide for 48 hours, proteoglycan synthesis was observed to increase in a dose-dependent manner with the maximal response at 1000 ng/mL Link-N. Fluorescently labelled Link-N peptide was injected into the disks to determine if Link-N is retained in the disks matrix or freely diffuses throughout the tissue and equilibrates with surrounding medium. Samples were taken continually from the surrounding medium and from the disk tissue at the termination of the experiment. Fluorescent-Link-N was detectable in the medium at 24 hours and reached equilibrium after 48 hours. The fluorescent peptide was found in the NP and NP/iAF junction but not in the remaining AF. Thus loss of Link-N appears to occur by diffusion through the endplates (Fig. 1). Cell viability was maintained in the NP, at >96%, after injection of 1 mg of Link-N/disk. Disks injected with Link-N showed increased proteoglycan synthesis in the NP and iAF compared to adjacent level control disks matched for grade of degeneration. To evaluate the duration of the effect, disks were injected with 35SO4 1 week after the injection of Link-N. Proteoglycan synthesis remained elevated in Link-N injected disks compared to adjacent level control disks suggesting a sustained effect. Conclusion Link-N peptide has previously been shown to promote matrix protein synthesis by bovine disk cells in monolayer and pellet cultures. In this work, we show that Link-N can promote proteoglycan synthesis not only in human disk cells cultured in 3D constructs, but also in intact adult human disks where the cells are in their native environment. Recently, an increase in disk height measured by MRI was shown in an in vivo rabbit model, where degenerated disks were injected with Link-N. If a similar restoration of disk function could be achieved in the human disks, then Link-N could be a promising candidate for biologically induced disk repair, and could provide an alternative to surgical intervention for early stage disk degeneration. Link-N has a significant cost advantage over growth factors, such as BMP7, TGFβ and, GDF5. Based on prior in vivo studies in the rabbit, Link-N is over 100 times less expensive than recombinant growth factors that have a similar repair response. Thus, Link-N peptide injection could be both effective and cost-efficient therapy for retarding the ongoing degenerative process in early stage disk disease and help relieve back pain. I confirm having declared any potential conflict of interest for all authors listed on this abstract Yes Disclosure of Interest None declared

Link-N Exerts Its Growth Factor-Like Effects through Activation of Smad 1/5 Signaling in Human IVD Cells

Introduction Back pain is a fairly common problem, which affects a large portion of the population and has an impact on quality of life. Intervertebral disc (IVD) degeneration is one of the most common causes of back pain. Link-N peptide represents the 16 amino acid sequence (DHLSDNYTLDHDRAIH) from the N-terminus of the link protein that stabilizes the proteoglycan aggregates present in cartilage and IVDs. We have previously shown that Link-N can stimulate collagen and proteoglycan synthesis in IVD cells in vitro1 and in intact human IVDs ex vivo,2 as well as increase disc height in a rabbit model of disc degeneration.3 Recently, BMPII receptor has been shown to interact with Link-N in rabbit IVD cells.4 The purpose of this study was to determine the mechanism by which Link-N exerts its beneficial effects by promoting matrix production in human IVD cells. Materials and Methods Human lumbar spines were retrieved through the Transplant Quebec organ donation program. IVD cells were isolated from nucleus pulposus (NP) and annulus fibrosus (AF) regions. The cells were cultured to 90% confluence in NP Cell Medium (NPCM, Cat. No.4801, ScienCell). Then the cells were incubated overnight in serum-free DMEM followed by treatment with Link-N (1 μg/mL), TGF-β (10 ng/mL) or BMP-2 (25 ng/mL) for 10 minutes to 4 hours. TGF-β (10 ng/mL) or BMP-2 (25 ng/mL) was used as positive controls. Cell extracts were prepared using NP40 lysis buffer. Total protein in the cell extracts was determined by the bicinchoninic acid assay (BCA). Protein expression was analyzed by immunoblotting using specific antibodies. Western blot images were quantified using ImageJ (NIH) software. For gene expression studies, cells were incubated for 24 hours with Link-N (1 μg/mL) in the presence or absence of human recombinant noggin (100 ng/mL) and total RNA was isolated using Trizol reagent and relative gene expression was determined by RT PCR. For receptor-binding studies, AF and NP cells were treated with biotinylated Link-N (1 μg/mL) with or without untagged Link-N (10 μg/mL) for competitive displacement. Scrambled biotinylated Link-N (1 μg/mL) was used as a specificity control. After treatments, cells were fixed in 2% paraformaldehyde. Fixed cells were treated with fluorescent-dye conjugated streptavidin (Alexa Fluor 700 conjugate, streptavidin). We assessed whether noggin directly binds with biotinylated Link-N by dot-blot technique. Results Link-N addition to human IVD cells did not promote the phosphorylation of Smads 2 and 3. This suggested that Link-N signaling does not function in a fashion similar to TGF-β, which stimulates the phosphorylation/activation of Smads 2 and 3. Further, similar to BMP-2, Link-N promoted the phosphorylation of Smad ? (Fig. 1). These results suggest that Link-N exerts its growth factor-like effects via a BMP-signaling mechanism rather than TGF-β. Noggin is a known physiological negative modulator of BMP signaling and noggin levels are elevated in disease conditions. As we observed that Link-N signaling is similar to BMP, it is essential to assess if noggin can interfere with the function of Link-N. Dot-blot results showed that noggin does not directly bind with Link-N. Therefore, noggin inhibition of Link-N signaling is unlikely because of noggin binding with Link-N. This suggests that Link-N may have an added advantage as a therapeutic agent over BMPs. In addition, Link-N increased BMP 4 expression significantly in both AF and NP cells, with the increase in AF cells being significantly higher than that in NP cells. Specific binding (also internalization) of biotinylated Link-N was found both in AF and NP cells, as this binding was competitively displaced by nonbiotinylated Link-N. No binding was seen with scrambled biotinylated Link-N. Conclusion Link-N represents a potential economic growth factor with beneficial effects on disc repair and the identification of the intracellular signaling pathways that it transduces is a step further toward clinical trials on NP repair in the degenerate human IVD. Disclosure of Interest None declared References Mwale F, Demers CN, Petit A, et al. A synthetic peptide of link protein stimulates the biosynthesis of collagens II, IX and proteoglycan by cells of the intervertebral disc. J Cell Biochem 2003;88(6):1202–1213 Gawri R, Antoniou J, Ouellet J, et al. Best paper NASS 2013: link-N can stimulate proteoglycan synthesis in the degenerated human intervertebral discs. Eur Cell Mater 2013;26:107-119, discussion 119 Mwale F, Masuda K, Pichika R, et al. The efficacy of Link N as a mediator of repair in a rabbit model of intervertebral disc degeneration. Arthritis Res Ther 2011;13(4):R120 Wang Z, Weitzmann MN, Sangadala S, Hutton WC, Yoon ST. Link protein N-terminal peptide binds to bone morphogenetic protein (BMP) type II receptor and drives matrix protein expression in rabbit intervertebral disc cells. J Biol Chem 2013;288(39):28243–28253

Link-N Is Cleaved by Human Annulus Fibrosus Cells Generating a Fragment with Retained Biological Activity

Introduction Back pain affects a large portion of the population across all ages, and intervertebral disc (IVD) degeneration is its most common cause. Presently, there are no established treatments to prevent, stop, or even retard disc degeneration, and surgery is often the offered option. Previous studies have shown that Link-N can act as a growth factor and stimulate the synthesis of proteoglycans and collagens in articular cartilage, in bovine IVD in vitro, and in intact human IVDs ex vivo, as well as increase disc height in a rabbit model of disc degeneration. However, the sequences in Link-N involved in modulating cellular activity and the stability of Link-N in the presence of IVD cells are not well understood. Materials and Methods Human lumbar spines were obtained through the Transplant Quebecs organ donation program. Six spinal segments were retrieved (mean age 30 years) and IVD cells were isolated from the nucleus pulposus (NP) and inner annulus fibrosus (iAF) regions by sequential enzymatic digestion. IVD cells were cultured in monolayer in the presence of Link-N to assess its stability in culture. Media was collected at designated time points from the cultures and was analyzed by MALDI-TOF and MS-MS mass spectrophotometry to evaluate the stability of Link-N peptide. IVD cells embedded in 1.2% alginate were exposed to the identified Link-N fragments and to the native scrambled or reversed Link-N peptides in the absence and presence of IL-1β to assess the biological activity of the peptides. SO4 incorporation was used to assess proteoglycan synthesis in response to the peptides. All peptides were used at equimolar concentrations. Results Mass spectrometric analysis revealed that a peptide spanning residues 1 to 8 was generated in the presence of human AF, while the original 16 amino acids peptide remained intact in the presence of NP cells. Link-N 1-8 significantly induced proteoglycan production in the absence and presence of IL-1β in both human NP and AF cells, confirming that the biological effect is maintained within the first 8 amino acids of the peptide. A reversed or scrambled Link-N peptide, as well as residues 9-16 of Link-N, had no biological effect. Conclusion In conclusion, Link-N 1-8 peptide is a promising therapeutic alternative for treating early disc degeneration before major collagen degradation has occurred. One advantage in using this shorter 8 amino acid peptide rather than the original 16 amino acid Link-N in therapy is the production cost. More important, this small size should be amenable to medicinal chemists allowing them to optimize the structure for maximal biological activity and stability. Thus Link-N 1-8 could be a promising candidate for biologically induced disc repair, and the identification of such a stable specific peptide may facilitate the design of compounds to promote disc repair and provide alternatives to surgical intervention for early stage disc degeneration. Disclosure of Interest None declared

Effect of Link N on the Expression of Neurotrophins and Substance P Release by Human Intervertebral Disc Cells Stimulated with Proinflammatory Cytokines

Introduction Although there are different causes of low back pain, intervertebral disc (IVD) degeneration is one of them. In healthy discs, nociceptive nerve fibers and mechanoreceptors penetrate up to the outer annulus fibrosus (AF). In contrast, the endplates are heavily innervated. However, neither the inner AF nor the nucleus pulposus (NP) are innervated. Discogenic pain can occur due to degenerated discs acquiring cracks and fissures (annulogenic pain) or endplate damage (vertebrogenic pain) resulting in increased nerve fibers that penetrate the inner AF and NP. The increased expression of neurotrophins nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) has been identified in human and animal models of degenerating IVDs.1 Proinflammatory cytokines interleukin (IL)-1β and tumor necrosis factor-α (TNF-α) have been shown to trigger the expression of NGF, BDNF, and substance P in human NP and AF cells. In related studies, bone morphogenetic protein (BMP) was shown to suppress innervation while overexpression of the BMP inhibitor noggin resulted in a significant increase in nerve fibers.2 However, the use of growth factors in clinical practice is limited by their high cost. Using synthetic peptides, such as link N, which stimulate BMP signaling, can circumvent this cost. The purpose of the present study was to evaluate the effect of link N on neurotrophins and substance P by human IVD cells stimulated with proinflammatory cytokines as well as in injured bovine IVDs. Materials and Methods Lumbar IVDs were obtained through organ donations within 6 hours after death. The procedure was approved by the local Research Ethics Board. Cells were isolated from NP and AF regions of the discs by sequential digestion with pronase followed by collagenase IA digestion for NP and collagenase II digestion for AF, respectively. They were cultured in monolayers and stimulated with TNF-α (100 ng/mL) and IL-1β (10 ng/mL) in the presence or absence of link N (1 μg/mL) for 48 hours. Total RNA was isolated and gene expression was measured using reverse-transcriptase polymerase chain reaction. The release of substance P into the culture media was measured after cells were stimulated by TNF-α (100-ng/mL) and IL-1β (10 ng/mL) in the presence or absence of link N (1 μg/mL) at different time points (1, 2, 4,and 24 hours). Coccygeal IVDs from the tails of adult bovine steers (20-25 months) were used for disc isolation. Four discs with cartilage end plates were isolated and were treated (control, capsaicin [1.5 μg/mL], punctured with a 16G needle, link N [10 μg/mL] treated) after preconditioning for 24 hours in complete DMEM. Disc culture media was collected at different time points for analysis. Substance P in the media was concentrated by solid phase extraction and was assayed by ELISA. Results Link N is known to induce proteoglycan synthesis by isolated disc cells and in degenerate rabbit and human discs, and to enhance chondrogenesis of MSCs in vitro. It is, however, not known if link N can suppress inflammatory mediators and neurotransmitters in disc degeneration. Without intervention or with link N supplementation alone only trace amounts of NGF gene expression in human lumbar disc cells from AF regions was observed. When human lumbar disc cells from AF regions were exposed to TNF-α for 48 hours, NGF gene expression was observed to increase significantly (p < 0.05). However, supplementing 1.0 µg/mL link N to this media led to a significant downregulation of NGF gene expression. Further, link N significantly suppressed substance P release from punctured bovine discs after 24 hours of treatment as compared with the untreated punctured disc (p < 0.05) (Fig. 1). We also showed that link N can suppress TNF-α and IL-1β-induced messenger RNA levels of brain-derived neurotrophic factor (BDNF) and TAC 1 in AF cells. Thus, link N appears to reduce inflammatory mediators and neurotransmitters in human AF cells from normal and degenerated discs, as well as in injured bovine IVDs. Fig. 1 Link N treatment significantly reduced substance P release into culture medium from punctured IVDs isolated from adult bovine tails. Conclusion Link N is a promising agent for biological repair of degenerated human discs but an affective treatment for discogenic pain will also depend on stopping inflammation. Previous studies have shown that BMPs can suppress peripheral innervation in the skin.2 Because BMPs and link N both share the Smad 1/5 signaling pathway, it was not unreasonable to assume that link N could also suppress inflammatory mediators associated with discogenic pain. The present study indicates that link N can also suppress NGF, BDNF, and TAC 1 in human disc cells as well as substance P release in injured bovine discs. This suggests that link N has the potential to inhibit pain induced by neuronal innervation caused by disc degeneration. Disc degeneration is often associated with low back pain; link N represents a potential economical growth factor with beneficial effects on disc repair. It would be of clinical significance to see if link N has any potential in reducing the pain caused by neuronal invasion during disc degeneration. Disclosure of Interest None declared References García-Cosamalón J, del Valle ME, Calavia MG, et al. Intervertebral disc, sensory nerves and neurotrophins: who is who in discogenic pain? J Anat 2010;217(1):1–15 Guha U, Gomes WA, Samanta J, Gupta M, Rice FL, Kessler JA. Target-derived BMP signaling limits sensory neuron number and the extent of peripheral innervation in vivo. Development 2004;131(5):1175–1186

Can Quantitative Magnetic Resonance Imaging Predict Mechanical Behavior of Human Intervertebral Disks with Different Grades of Degeneration

Introduction The dramatic changes in morphology, composition, and structure that occur in the intervertebral disk (IVD) with aging and degeneration are accompanied by specific changes in mechanical properties of the disk material.1,2 Evaluation of these changes in the IVD hinges on the ability to objectively and noninvasively assess the IVD matrix composition and integrity. Different studies on human IVDs have correlated IVD matrix composition and integrity to the longitudinal magnetization recovery T1, the transverse magnetization decay T2, the magnetization transfer ratio (MTR), and apparent diffusion coefficient (ADC).3,4 Correlations and multiple linear regressions have been also identified between quantitative magnetic resonance imaging (qMRI) parameters, biochemical, and mechanical parameters of targeted enzyme matrix denaturation and buffer-treated bovine IVDs. To this end, qMRI analysis can be used to correlate MRI signal to the mechanical properties of NP and AF tissue in order to predict structural changes in IVDs with degeneration. The aim of the present study was to determine how quantitative MRI parameters can predict biomechanical properties in human IVDs with different grades of degeneration. Materials and Methods Experimental Groups Ten whole lumbar spine specimens, 5 disks per spine, were obtained through organ donations via Héma-Québec within 24 hours after death. Age of donors was from 32 to 77 years. The samples were vacuum sealed in plastic bags for MRI to maintain hydration. MRI Procedure The MRI examinations were carried out in a 1.5T whole-body Siemens’ Avanto system using the standard circularly polarized head coil. The samples were placed in a sagittal orientation and T1, T2, MTR, and ADC were measured as previously described.1 All disks (nu2009=u200950) were then graded from T2-weighted images according to the classification system described by Pfirrmann. Numerical analysis of quantitative MRI was performed using a custom code written in MATLAB (MathWorks, Natick, MA, USA) allowing the selection of the regions of interest (ROI) and the calculation of average signal intensities from all images. ROI were traced manually as polygonal shapes with no contact with the endplate tissues and were reproduced identically on all T1, T2, Ms/Mo ratio, and diffusion images. Mechanical Testing Procedure Confined compression tests were performed on 5-mm-diameter cylindrical plugs of tissue using a custom built axial testing machine. Material parameters (aggregate modulus HA and permeability k) were obtained from a linear biphasic fit. Dynamic shear testing was carried out using a rheometer (TA Instruments). Steady-state dynamic shear modulus and phase angle were calculated at each point of the frequency and strain sweeps and fitted with exponential functions. Statistical Methods Correlations between qMRI and mechanical parameters were investigated using Pearson test performed on GraphPad Prism software (GraphPad Software, La Jolla, CA, USA). Correlation between a mechanical parameter and an MR parameter in the same region of the disk was considered significant with p. Results Significant correlations for the NP tissue were found between T2 and shear modulus |G*| (ru2009=u2009−0.465, pu2009=u20090.022), and between diffusion ADC and αδ (ru2009=u20090.4, pu2009=u20090.047) (Fig. 1). Significant correlations for the AF tissue were found between T1 and αδ (ru2009=u20090.372, pu2009=u20090.047) and between T1 and permeability k (ru2009=u2009−0.468, pu2009=u20090.043) (Fig. 2). No correlations were found between MTR and any mechanical parameters for both AF and NP tissues. Conclusion The results of the present study are consistent with our previous studies in bovine model and indicate sensitivity to distinct changes at varying levels of degeneration. In the AF, permeability and phase angle were predicted by T1 while in the NP tissue, T2 was a stronger determinant of the tissue integrity (reflected by shear modulus). This may relate to the fact that T1 has been predominantly correlated to water content, while T2 is influenced by tissue anisotropy (orientation of collagen fibers), collagen concentration, and water content in tissues. These results prove that it is possible to develop correlations and multiple linear regressions in human IVDs which are essential for developing quantitative MRI as a diagnostic tool in determining the functional state of the disk. I confirm having declared any potential conflict of interest for all authors listed on this abstract Yes Disclosure of Interest None declared Mwale F, et al. Journal of Magnetic Resonance Imaging 2008; 27:563–573 Iatridis J, et al. Journal of Biomechanics 1998; 31:535–544 Antoniou, J. et al. Magnetic Resonance in Medicine1998; 40(6):900–907 Antoniou, J. et al. Journal of Magnetic Resonance Imaging 2004; 22:963–972

The Potential of Nitrogen Rich Surfaces to Distinguish between Cells of the Nucleus Pulposus and Annulus Fibrosus

Introduction Mesenchymal stem cells (MSCs) have been proposed for the purposes of tissue engineering and repair of intervertebral disk (IVD) tissue. These cells would be induced to differentiate into cells with a disc-cell like phenotype, as an alternative treatment for against disk degeneration. One major drawback is that there currently exists no simple way to distinguish a nucleus pulposus (NP) cell from its annulus fibrosus (AF) or hyaline cartilage counterparts. In addition, immature NP cells produce similar macromolecules to those of hyaline cartilage1; therefore, it is difficult to confirm MSCs differentiation into a disk cell phenotype. Our previous studies have shown that surfaces rich in primary amines, -NH2, can influence the adhesion properties of cells.2 In this study, we created reproducible NH2-rich surfaces by low-pressure plasma polymerization of ethylene-ammonia mixture (so-called “L-PPE: N”) to test the hypothesis that selective adhesion of disk cells can be used to distinguish between an NP and an AF phenotype. This would serve as a marker to distinguish an appropriate NP, generated by tissue engineering techniques, from other cartilaginous phenotypes that may be inappropriate for function in the disc. Materials and Methods Surface preparation L-PPE: N coatings approximately 100 nm thick were prepared as previously described.3 These deposits, which contain about 7.5% NH2, were deposited on poly(ethylene terephthalate) (PET) film in a low-pressure (L) capacitively coupled radio-frequency glow-discharge plasma reactor. However, half of the films surfaces were masked, so as to remain uncoated, bare PET. Cell isolation and culture Adult bovine tails (2 to 3 years old) were obtained 2 hours after slaughter at a local abattoir. The IVDs were dissected from their adjacent vertebral bodies and separated into nucleus pulposus (NP) and AF. Both tissues of the AF and the NP were first weighed (wet weight) and then subjected to a pronase digestion (0.2% W/V), followed by a digestion of collagenase 1A (0.04% W/V) for NP tissue and collagenase II (0.04% W/V) for AF tissue. After isolation, the AF cells and the NP cells were separately cultured on the half-coated surfaces in DMEM high glucose, supplemented with 10% FBS and 1% penicillin/streptomycin in a concentration of 1u2009×u2009106 cells/mL. Regular polystyrene culture dishes (TCPS) were used as a control. After 4 days of culture, cells were washed with ddH20, fixed and stained with Safranin-O. Images were captured of both cell types before and after wash, using an optical microscope. Results AF cells adhered preferentially to the L-PPE: N-coated surface, but did not adhere to the bare part of the PET surface (Fig. 1). NP cells, however, indiscriminately adhered to both PET and L-PPE: N surfaces. Both NP and AF cells adhered to regular TCPS dishes after 4 days. After washing with ddH20, which was meant to remove any nonadherent cells, and to provide a clearer image to be captured, AF cells remained bound to L-PPE:N surfaces; however, NP cells were completely removed from both surfaces. Conclusion In this study, we show that AF cells adhered preferentially to the substrates portion that was L-PPE: N-coated, whereas NP cells did not manifest any preference to coated or uncoated portions. This preferential attachment to an amine-containing surface can be used to distinguish NP from AF cells; together with the production of a high GAG-to-hydroxyproline (proteoglycan-to-collagen) ratio by NP cells, it could help in identifying an NP-like phenotype. I confirm having declared any potential conflict of interest for all authors listed on this abstract Yes Disclosure of Interest None declared Mwale F, et al. European Cells and Materials 2004;8:58–64 Girard-Lauriault PL, et al. Macromolecular Bioscience 2009;9:911–921 Girard-Lauriault PL, et al. Plasma Processes and Polymers 2005;2:263–270