- Email: [email protected]
Degenerated intervertebral discs contain increased proportion of α-smooth muscle actin positive cells
Abstracts / Osteoarthritis and Cartilage 24 (2016) S63eS534
fraction has been previously described for lumbar 4/5 facet joints of healthy individuals. Here, we sought to characterize trabecular bone structural parameters and subchondral bone-resident mesenchymal stromal cells (MSC) of osteoarthritic facet joints and compare these with regard to age, gender and healthy controls. Methods: Twenty-one patients scheduled for decompression and stabilization surgery for degenerative spinal stenosis were recruited for this study. Facet joint specimens were harvested during surgery and either fixed in formalin for microcomputed tomography (n ¼ 15) or isolation of MSC by collagenase digestion (n ¼ 6). MicroCT was performed spatial resolution of 20.5 mm. Bone volume fraction (BV/TV), trabecular thickness (Tb.Th), trabecular separation (Tb.Sp) and trabecular number (Tb.N) were evaluated using CT Analyser. Trabecular bone structural parameters of healthy subjects were taken from Wilke and co-workers (J. Anat. 2012). Statistical analysis of differences between gender and age groups (40e60 and 60e80 years) were performed using two-way analysis of variance (ANOVA). Early passage MSC were subjected to adipogenic and osteogenic differentiation for three weeks and evaluated using alkaline phosphatase assay, Alizarin red and Oil red O staining and RT-PCR. Results: Trabecular BV/TV was 0.36 ± 0.14 and 0.35 ± 0.17 for osteoarthritic lumbar facet joints of male and female individuals, respectively. Contrastingly, mean BV/TV in age-matched healthy controls was 0.32 (male) and 0.25 (female). Tb.Th was in the same range for both genders and age-groups (overall mean 0.20 ± 0.07 mm) and did not differ from healthy controls. The overall Tb.Sp averaged 0.52 ± 0.16 mm, which was lower than averages observed for age-matched healthy females (0.60) and males (0.71). Accordingly, Tb.N in osteoarthritic facet joints of male (1.9 ± 1.3/mm) and female (1.7 ± 0.8/mm) patients with lumbar spinal stenosis was higher than reported for healthy controls (1.5e1.0/mm). Statistical analysis did not reveal any age- or genderspecific differences in trabecular bone structural parameters of osteoarthritic lumbar facet joints. Facet joint MSC showed a strongly upregulated alkaline phosphatase activity upon adipogenic (8.1-fold) and osteogenic (4.6-fold) differentiation. Only a low number of lipid vacuoles were detected after adipogenesis. Alizarin red staining revealed varying degrees of in vitro matrix mineralization in MSC, which ranged from blunted to strong mineralization. Conclusions: Subchondral trabecular bone remodeling in lumbar facet joint osteoarthritis is characterized by an increase in bone volume fraction due to a higher trabecular number, but not an increase of trabecular thickness. MSC from osteoarthritic facet joints display impaired adipogenesis and abnormal mineralization during osteogenesis. These data provide a strong rationale for targeting increased bone remodeling in lumbar facet joint osteoarthritis. 828 OBJECTIVE MEASUREMENT OF FREE-LIVING PHYSICAL ACTIVITY (PERFORMANCE) IN LUMBAR SPINAL STENOSIS: ARE PHYSICAL ACTIVITY GUIDELINES BEING MET? J. Norden y, M. Smuck y, A. Sinha y, R. Hu z, C. Tomkins-Lane x. y Stanford Univ., Stanford, CA, USA; z Univ. of Calgary, Calgary, AB, Canada; x Mount Royal Univ., Calgary, AB, Canada Purpose: Lumbar spinal stenosis (LSS) is a painful form of spine arthritis, characterized by neurogenic claudication and mobility limitations. Research suggests that people with LSS would benefit from increased physical activity. Yet, to date we do not have disease specific activity guidelines for LSS, and the nature of free-living physical activity (performance) in LSS remains unknown. LSS care providers could endorse the 2008 United States Physical Activity Guidelines, however, we do not know if this is realistic. The aim of this study was to determine the proportion of individuals with LSS meeting the 2008 Physical Activity Guidelines. A secondary goal was to better understand the nature of physical performance in LSS. Methods: This was a retrospective study of the Lumbar Spinal Stenosis Accelerometry Database. All participants had both radiographic and clinical LSS and were seeking various treatments for their symptoms. Outcome measures included 7-day accelerometry and demographics. For this study, we analyzed only baseline data that was obtained prior to any new treatments for patients. We included only those participants who had at least 4 valid days of accelerometry data. We determined the proportion of individuals with LSS meeting the 2008 US Physical Activity Guidelines of at least 150 minutes of moderate-vigorous (MV) physical activity per week in bouts of 10 minutes or more. We also used
S481
the novel Physical Performance analysis designed by our group to determine time spent in varying intensities of activity. Results: We analyzed data from 75 individuals with a mean age of 68 (SD 9), and 37% male. Three people (4%) were considered Meeting Guidelines (at least 150 MV minutes/week), and 56 (75%) were considered Inactive with not even one MV minute/week. With the 10minute bout requirement removed, 10/75 (13%) achieved the 150minute threshold. The average time spent in sedentary activity was 82%, and of time spent in non-sedentary activity, 99.6% was in the light activity range. Of time spent in light activity, 53% was in the Performance Light 1 range, the lowest of light intensities. The percent of time spent in moderate-vigorous activity was 0.26% for males, 0.13% for females and 0.14% overall. Conclusions: People with LSS are extremely inactive with only 4% meeting current physical activity guidelines. It appears that people with LSS are largely sedentary, with 99.6% of non-sedentary time spent in light activity. Less than 1% of total activity time is spent in moderate or vigorous activity. There is an urgent need for interventions aimed at reducing sedentary behaviour and increasing activity in LSS both to improve function and prevent diseases of inactivity. However, the existing 2008 US Physical Activity Guidelines are difficult for this population to achieve. These results suggest that a focus on light intensity activity and shorter bouts of activity are more appropriate and realistic in this population. As this study reveals the need for disease specific activity guidelines and the value of physical performance measurement in LSS, it is an important step in a personalized medicine approach to people with LSS that is focused on increasing physical function through targeted and realistic activity goals. 829 DEGENERATED INTERVERTEBRAL DISCS CONTAIN INCREASED PROPORTION OF a-SMOOTH MUSCLE ACTIN POSITIVE CELLS F. Lv, F. Lim, V.Y. Leung, K.M. Cheung. The Univ. of Hong Kong, Hong Kong, China Purpose: Low back pain (LBP) is a common disease which affects 80% of people around the world for at least once in their life time. Our previous study based on Hong Kong cohort demonstrated that intervertebral disc (IVD) degenerates with aging and is a major contribution to LBP. To date, the molecular changes in the IVD during degeneration progress is not fully illustrated. Previously, Melrose et al reported that a proportion of NP (the inner compartment of IVD) and AF (the surrounding lamella tissue surrounding NP) cells expressed a-smooth muscle actin (a-SMA). Here we aim to evaluate if a-smooth muscle actin (a-SMA) expression is associated with disc degeneration and its source of expression. Methods: Human degenerated IVD were collected from patients with disc degeneration (graded IV-V at the Schneiderman scale) undergoing discectomy (n ¼ 5). Human non-degenerated IVD were collected from adolescent scoliosis patients during corrective scoliosis surgery (n ¼ 5). The corresponding informed patient consent and institutional review board (IRB) approval was obtained from the relevant committee. IVD tissues were carefully dissected into NP and AF, embedded into paraffin and cut into 6 um sections. The expression of a-SMA and MMP12 (matrix metalloproteinase 12) was investigated by immunochemical staining. The positivity was determined as the number of positive cells divided by the total number of cells per whole section. Results: a-SMA was mainly located in cell cytoplasm. Its expression was detected in both NP and AF of degenerated and non-degenerated human IVD. Degenerated human NP contained significantly higher proportion of a-SMA positive cells when compared with non-degenerated human NP (D-NP vs. ND-NP: 21.89 ± 3.79% vs. 4.42 ± 1.47%). Similarly, degenerated human AF also contained more a-SMA positive cells compared to non-degenerated human NP (D-AF vs. ND-AF: 29.05 ± 6.55% vs. 7.83 ± 2.46%). Interestingly, human degenerated NP and AF also had significantly higher percentage of MMP12(þ) cells than their non-degenerated counterparts. Analysis of the positivity for aSMA and MMP12 in all samples showed that the positivity for a-SMA is significantly correlated with the positivity for MMP12 (NP: p < 0.01; AF: p < 0.01). Conclusions: In this study, we found that the positivity of a-SMA increased in degenerated human NP and AF. The role of a-SMA in IVD cells is yet not clear. a-SMA is reported to aid wound contraction, it also helps to resist high mechanical stress and maintain cell shape. Besides, a-SMA is a marker characteristically expressed by myofibroblasts, which is a major type of cells contributing to tissue fibrosis. In our study,
S482
Abstracts / Osteoarthritis and Cartilage 24 (2016) S63eS534
the correlation of a-SMA expression with MMP12, a pre-fibrotic mediator, may indicate the on-going fibrosis in IVD degeneration. 830 SILICA NANOCARRIER AS A SUSTAINED DELIVERY SYSTEM OF GDF5 FOR INTERVERTEBRAL DISC REGENERATIVE MEDICINE H. Nina y, z, J. Clouet y, x, P. Colombier y, E. Gautron z, B. Humbert z, J. Le Bideau z, C. Le Visage y, J. Guicheux y, k. y INSERM, UMRS 791, Ctr. for Osteoarticular and Dental Tissue Engineering (LIOAD), Nantes, France; z CNRS, UMR 6502, Inst. of Materials Jean Rouxel (IMN), Nantes, France; x CHU Nantes, PHU 11 Pharmacie, Pharmacie Centrale, Nantes, France; k Universit e de Nantes, UFR Sci. Biologiques et Pharmaceutiques, Nantes, France Purpose: Intervertebral disc (IVD) degeneration, and notably its central part the nucleus pulposus (NP), is one of the leading causes of low back pain (LBP). Currently, LBP is mainly managed by pharmacological treatments and for advanced cases by invasive surgical procedures. To clinically address LBP early in the degenerative cascade, regenerative medicine strategies based on the use of smart biomaterials, cells or bioactive molecules are considered a less invasive and alternative to spinal reconstructive surgery. Among these strategies, the biomaterial-assisted intradiscal delivery of biofactors able to promote regenerative processes is notably contemplated with interest. Interestingly, we recently demonstrated that growth differentiation factor 5 (GDF5) is able to support the differentiation of human adipose stromal cells (hASC) into cells with a NPlike phenotype. In addition, mesoporous silica have for long been investigated as scaffolds for the development of drug delivery systems. In this context, we recently demonstrated that nanofibers of mesoporous silica (MSNFs) are biocompatible and exhibit a high specific surface making them an ideal candidate for intradiscal delivery. We thus aim at using MSNFs as a carrier for the intradiscal delivery of GDF5. The loading and release capability of MSNFs was first characterized. Then we deciphered the protein-silica interactions using a model protein mimicking GDF5 physicochemical properties. Finally, we studied the delivery and biological activity of GDF5. Methods: A model protein (lysozyme) was incubated in presence of MSNFs (50 500 nm) under constant stirring in different conditions of pH (4, 7, 10), time (from 1 to 48 h) and concentrations (from 1 to 200 mg/mL) to determine the optimal adsorption conditions. Analysis of protein-silica interactions were performed with transmission electron microscopy (TEM), attenuated total reflectance infrared (ATR/IR), thermogravimetric analysis coupled with mass spectrometry (TGA/MS) and zeta potential (ZP). Release assays were performed in PBS, pH 7.2 at 37 C for 20 days. Lysozyme concentration and biological activity were measured with BCA method and enzymatic assay respectively. GDF5 was incubated with MSNFs at pH7 for 48 h at concentrations from 1e4 mg/mL. Release was performed in PBS, pH7.2 at 37 C for 48 h. GDF5 concentration and bioactivity were measured by ELISA and Smad 1/5/8 phosphorylation in hASC by western blotting, respectively. Nucleopulpogenic hASC differentiation was evaluated by RT-qPCR and immunohistology. Results: Lysozyme was successfully adsorbed on MSNFs. The highest amount of adsorbed lysozyme was obtained at pH 10, initial concentration of 200 mg/mL and 48h of incubation, corresponding to a 20% adsorption efficiency. We obtained a 100% adsorption efficiency in 1h for concentrations 10 mg/mL. We hypothesized that when in contact with silica fibers, lysozyme could first adsorb quickly, creating a protein monolayer. Then, additional layers would be stacked on the monolayer. Different lysozyme morphologies were observed by TEM and precise information was obtained from the ATR/FTIR study by comparison of amides, Si-O-Si, Si-OH and water bands between lysozyme, MSNFs alone and MSNFs/lysozyme. Combined with ZP results, our data suggest that protein-silica interactions should be mostly driven by hydrogen bound involving the amide II of the protein. Release experiments showed a burst within the 1st hour, representing from 31% to 81% of the total released amount for concentrations ranging from 5 to 200 mg/mL, respectively. The release rates slowdown in the following hours leading to a release plateau after 3 days for high concentrations and up to 20 days for an initial concentration of 5 mg/mL. These kinetics results support the hypothesis of a two-step adsorption of the protein onto the MSNFs. Overall, the lysozyme release efficiency was related to the initial concentration of lysozyme. Our data also
indicated the maintenance of the biological activity of the released protein at any time points. In a first set of preliminary experiments, we also demonstrated that GDF5 could be adsorbed and released from MSNFs. The GDF5 bioactivity was evidenced by the phosphorylation of Smad 1/5/8. Conclusions: We have demonstrated that MSNFs are suitable nanocarriers for the loading and release of proteins. Protein silica interactions were extensively described providing us with a better understanding of the adsorption phenomenon involved and revealing a hydrogen bound-mediated interactions. Further experiments using GDF5 as a nucleopulpogenic factor are now under intense investigation.
Therapy - Non-Pharmacologic 831 THE IMPACT OF OBESITY ON KNEE OSTEOARTHRITIS SYMPTOMS AND RELATED BIOMARKER PROFILES IN A BARIATRIC SURGERY COHORT J. Samuels y, T. Mukherjee y, E. Wilder y, F. Bonfim y, K. Toth y, S. Aharon y, V. Chen y, L. Browne y, R. La Rocca Vieira y, J. Patel y, C. Ren-Fielding z, M. Parikh z, S.B. Abramson y, M. Attur y. y NYU Langone Hosp. for Joint Diseases, New York, NY, USA; z NYU Langone Med. Ctr., New York, NY, USA Purpose: Obesity is a modifiable risk factor of knee osteoarthritis (KOA). Surgical weight loss may be more effective than diet and exercise in delaying or avoiding joint replacement, as retrospective studies have in fact shown sustained improvement in KOA pain after bariatric surgery. We prospectively evaluated painful KOA in the obese population, tracked whether weight loss after bariatric surgery affects KOA-related pain and function, and studied serum and plasma levels of adipokines and (published) OA biomarkers in this subset of KOA patients. Methods: We screened consecutive patients prior to laparoscopic adjustable gastric banding (LAGB), sleeve gastrectomy, or gastric bypass at NYU and Bellevue. Patients age 18 with knee pain for 1 month and a visual analog scale pain score 30 mm were enrolled, excluding those with lupus, rheumatoid arthritis, psoriatic arthritis, or psoriasis. Baseline pre-op assessments included X-Rays for OA severity by KellgreneLawrence (KL) grade and completion of the Knee Injury and Osteoarthritis Outcome Score (KOOS). They repeated the questionnaire and were re-weighed to calculate percent excess weight loss (%EWL) at 1, 3, 6 and 12 month post-op intervals. Patients were consented for optional tissue collection (blood, urine and intra-operative adipose samples) for biomarker analysis at all visits, with analyses using existing data from controls and a non-obese OA cohort for comparison. Results: Of 537 patients planning to have bariatric surgery, 309 (58%) reported knee pain. 176 enrolled having met criteria and consented for the study: 91.5% female, mean BMI 43.6 kg/m2 ± 7 (31.6e60.6), and mean age of 42.4 ± 11 (18e73). For radiographic severity, KL0 ¼ 43 (25%), KL1 ¼ 34 (19%), KL2 ¼ 38 (22%), KL3 ¼ 34 (19%), KL4 ¼ 27 (15%).
fraction has been previously described for lumbar 4/5 facet joints of healthy individuals. Here, we sought to characterize trabecular bone structural parameters and subchondral bone-resident mesenchymal stromal cells (MSC) of osteoarthritic facet joints and compare these with regard to age, gender and healthy controls. Methods: Twenty-one patients scheduled for decompression and stabilization surgery for degenerative spinal stenosis were recruited for this study. Facet joint specimens were harvested during surgery and either fixed in formalin for microcomputed tomography (n ¼ 15) or isolation of MSC by collagenase digestion (n ¼ 6). MicroCT was performed spatial resolution of 20.5 mm. Bone volume fraction (BV/TV), trabecular thickness (Tb.Th), trabecular separation (Tb.Sp) and trabecular number (Tb.N) were evaluated using CT Analyser. Trabecular bone structural parameters of healthy subjects were taken from Wilke and co-workers (J. Anat. 2012). Statistical analysis of differences between gender and age groups (40e60 and 60e80 years) were performed using two-way analysis of variance (ANOVA). Early passage MSC were subjected to adipogenic and osteogenic differentiation for three weeks and evaluated using alkaline phosphatase assay, Alizarin red and Oil red O staining and RT-PCR. Results: Trabecular BV/TV was 0.36 ± 0.14 and 0.35 ± 0.17 for osteoarthritic lumbar facet joints of male and female individuals, respectively. Contrastingly, mean BV/TV in age-matched healthy controls was 0.32 (male) and 0.25 (female). Tb.Th was in the same range for both genders and age-groups (overall mean 0.20 ± 0.07 mm) and did not differ from healthy controls. The overall Tb.Sp averaged 0.52 ± 0.16 mm, which was lower than averages observed for age-matched healthy females (0.60) and males (0.71). Accordingly, Tb.N in osteoarthritic facet joints of male (1.9 ± 1.3/mm) and female (1.7 ± 0.8/mm) patients with lumbar spinal stenosis was higher than reported for healthy controls (1.5e1.0/mm). Statistical analysis did not reveal any age- or genderspecific differences in trabecular bone structural parameters of osteoarthritic lumbar facet joints. Facet joint MSC showed a strongly upregulated alkaline phosphatase activity upon adipogenic (8.1-fold) and osteogenic (4.6-fold) differentiation. Only a low number of lipid vacuoles were detected after adipogenesis. Alizarin red staining revealed varying degrees of in vitro matrix mineralization in MSC, which ranged from blunted to strong mineralization. Conclusions: Subchondral trabecular bone remodeling in lumbar facet joint osteoarthritis is characterized by an increase in bone volume fraction due to a higher trabecular number, but not an increase of trabecular thickness. MSC from osteoarthritic facet joints display impaired adipogenesis and abnormal mineralization during osteogenesis. These data provide a strong rationale for targeting increased bone remodeling in lumbar facet joint osteoarthritis. 828 OBJECTIVE MEASUREMENT OF FREE-LIVING PHYSICAL ACTIVITY (PERFORMANCE) IN LUMBAR SPINAL STENOSIS: ARE PHYSICAL ACTIVITY GUIDELINES BEING MET? J. Norden y, M. Smuck y, A. Sinha y, R. Hu z, C. Tomkins-Lane x. y Stanford Univ., Stanford, CA, USA; z Univ. of Calgary, Calgary, AB, Canada; x Mount Royal Univ., Calgary, AB, Canada Purpose: Lumbar spinal stenosis (LSS) is a painful form of spine arthritis, characterized by neurogenic claudication and mobility limitations. Research suggests that people with LSS would benefit from increased physical activity. Yet, to date we do not have disease specific activity guidelines for LSS, and the nature of free-living physical activity (performance) in LSS remains unknown. LSS care providers could endorse the 2008 United States Physical Activity Guidelines, however, we do not know if this is realistic. The aim of this study was to determine the proportion of individuals with LSS meeting the 2008 Physical Activity Guidelines. A secondary goal was to better understand the nature of physical performance in LSS. Methods: This was a retrospective study of the Lumbar Spinal Stenosis Accelerometry Database. All participants had both radiographic and clinical LSS and were seeking various treatments for their symptoms. Outcome measures included 7-day accelerometry and demographics. For this study, we analyzed only baseline data that was obtained prior to any new treatments for patients. We included only those participants who had at least 4 valid days of accelerometry data. We determined the proportion of individuals with LSS meeting the 2008 US Physical Activity Guidelines of at least 150 minutes of moderate-vigorous (MV) physical activity per week in bouts of 10 minutes or more. We also used
S481
the novel Physical Performance analysis designed by our group to determine time spent in varying intensities of activity. Results: We analyzed data from 75 individuals with a mean age of 68 (SD 9), and 37% male. Three people (4%) were considered Meeting Guidelines (at least 150 MV minutes/week), and 56 (75%) were considered Inactive with not even one MV minute/week. With the 10minute bout requirement removed, 10/75 (13%) achieved the 150minute threshold. The average time spent in sedentary activity was 82%, and of time spent in non-sedentary activity, 99.6% was in the light activity range. Of time spent in light activity, 53% was in the Performance Light 1 range, the lowest of light intensities. The percent of time spent in moderate-vigorous activity was 0.26% for males, 0.13% for females and 0.14% overall. Conclusions: People with LSS are extremely inactive with only 4% meeting current physical activity guidelines. It appears that people with LSS are largely sedentary, with 99.6% of non-sedentary time spent in light activity. Less than 1% of total activity time is spent in moderate or vigorous activity. There is an urgent need for interventions aimed at reducing sedentary behaviour and increasing activity in LSS both to improve function and prevent diseases of inactivity. However, the existing 2008 US Physical Activity Guidelines are difficult for this population to achieve. These results suggest that a focus on light intensity activity and shorter bouts of activity are more appropriate and realistic in this population. As this study reveals the need for disease specific activity guidelines and the value of physical performance measurement in LSS, it is an important step in a personalized medicine approach to people with LSS that is focused on increasing physical function through targeted and realistic activity goals. 829 DEGENERATED INTERVERTEBRAL DISCS CONTAIN INCREASED PROPORTION OF a-SMOOTH MUSCLE ACTIN POSITIVE CELLS F. Lv, F. Lim, V.Y. Leung, K.M. Cheung. The Univ. of Hong Kong, Hong Kong, China Purpose: Low back pain (LBP) is a common disease which affects 80% of people around the world for at least once in their life time. Our previous study based on Hong Kong cohort demonstrated that intervertebral disc (IVD) degenerates with aging and is a major contribution to LBP. To date, the molecular changes in the IVD during degeneration progress is not fully illustrated. Previously, Melrose et al reported that a proportion of NP (the inner compartment of IVD) and AF (the surrounding lamella tissue surrounding NP) cells expressed a-smooth muscle actin (a-SMA). Here we aim to evaluate if a-smooth muscle actin (a-SMA) expression is associated with disc degeneration and its source of expression. Methods: Human degenerated IVD were collected from patients with disc degeneration (graded IV-V at the Schneiderman scale) undergoing discectomy (n ¼ 5). Human non-degenerated IVD were collected from adolescent scoliosis patients during corrective scoliosis surgery (n ¼ 5). The corresponding informed patient consent and institutional review board (IRB) approval was obtained from the relevant committee. IVD tissues were carefully dissected into NP and AF, embedded into paraffin and cut into 6 um sections. The expression of a-SMA and MMP12 (matrix metalloproteinase 12) was investigated by immunochemical staining. The positivity was determined as the number of positive cells divided by the total number of cells per whole section. Results: a-SMA was mainly located in cell cytoplasm. Its expression was detected in both NP and AF of degenerated and non-degenerated human IVD. Degenerated human NP contained significantly higher proportion of a-SMA positive cells when compared with non-degenerated human NP (D-NP vs. ND-NP: 21.89 ± 3.79% vs. 4.42 ± 1.47%). Similarly, degenerated human AF also contained more a-SMA positive cells compared to non-degenerated human NP (D-AF vs. ND-AF: 29.05 ± 6.55% vs. 7.83 ± 2.46%). Interestingly, human degenerated NP and AF also had significantly higher percentage of MMP12(þ) cells than their non-degenerated counterparts. Analysis of the positivity for aSMA and MMP12 in all samples showed that the positivity for a-SMA is significantly correlated with the positivity for MMP12 (NP: p < 0.01; AF: p < 0.01). Conclusions: In this study, we found that the positivity of a-SMA increased in degenerated human NP and AF. The role of a-SMA in IVD cells is yet not clear. a-SMA is reported to aid wound contraction, it also helps to resist high mechanical stress and maintain cell shape. Besides, a-SMA is a marker characteristically expressed by myofibroblasts, which is a major type of cells contributing to tissue fibrosis. In our study,
S482
Abstracts / Osteoarthritis and Cartilage 24 (2016) S63eS534
the correlation of a-SMA expression with MMP12, a pre-fibrotic mediator, may indicate the on-going fibrosis in IVD degeneration. 830 SILICA NANOCARRIER AS A SUSTAINED DELIVERY SYSTEM OF GDF5 FOR INTERVERTEBRAL DISC REGENERATIVE MEDICINE H. Nina y, z, J. Clouet y, x, P. Colombier y, E. Gautron z, B. Humbert z, J. Le Bideau z, C. Le Visage y, J. Guicheux y, k. y INSERM, UMRS 791, Ctr. for Osteoarticular and Dental Tissue Engineering (LIOAD), Nantes, France; z CNRS, UMR 6502, Inst. of Materials Jean Rouxel (IMN), Nantes, France; x CHU Nantes, PHU 11 Pharmacie, Pharmacie Centrale, Nantes, France; k Universit e de Nantes, UFR Sci. Biologiques et Pharmaceutiques, Nantes, France Purpose: Intervertebral disc (IVD) degeneration, and notably its central part the nucleus pulposus (NP), is one of the leading causes of low back pain (LBP). Currently, LBP is mainly managed by pharmacological treatments and for advanced cases by invasive surgical procedures. To clinically address LBP early in the degenerative cascade, regenerative medicine strategies based on the use of smart biomaterials, cells or bioactive molecules are considered a less invasive and alternative to spinal reconstructive surgery. Among these strategies, the biomaterial-assisted intradiscal delivery of biofactors able to promote regenerative processes is notably contemplated with interest. Interestingly, we recently demonstrated that growth differentiation factor 5 (GDF5) is able to support the differentiation of human adipose stromal cells (hASC) into cells with a NPlike phenotype. In addition, mesoporous silica have for long been investigated as scaffolds for the development of drug delivery systems. In this context, we recently demonstrated that nanofibers of mesoporous silica (MSNFs) are biocompatible and exhibit a high specific surface making them an ideal candidate for intradiscal delivery. We thus aim at using MSNFs as a carrier for the intradiscal delivery of GDF5. The loading and release capability of MSNFs was first characterized. Then we deciphered the protein-silica interactions using a model protein mimicking GDF5 physicochemical properties. Finally, we studied the delivery and biological activity of GDF5. Methods: A model protein (lysozyme) was incubated in presence of MSNFs (50 500 nm) under constant stirring in different conditions of pH (4, 7, 10), time (from 1 to 48 h) and concentrations (from 1 to 200 mg/mL) to determine the optimal adsorption conditions. Analysis of protein-silica interactions were performed with transmission electron microscopy (TEM), attenuated total reflectance infrared (ATR/IR), thermogravimetric analysis coupled with mass spectrometry (TGA/MS) and zeta potential (ZP). Release assays were performed in PBS, pH 7.2 at 37 C for 20 days. Lysozyme concentration and biological activity were measured with BCA method and enzymatic assay respectively. GDF5 was incubated with MSNFs at pH7 for 48 h at concentrations from 1e4 mg/mL. Release was performed in PBS, pH7.2 at 37 C for 48 h. GDF5 concentration and bioactivity were measured by ELISA and Smad 1/5/8 phosphorylation in hASC by western blotting, respectively. Nucleopulpogenic hASC differentiation was evaluated by RT-qPCR and immunohistology. Results: Lysozyme was successfully adsorbed on MSNFs. The highest amount of adsorbed lysozyme was obtained at pH 10, initial concentration of 200 mg/mL and 48h of incubation, corresponding to a 20% adsorption efficiency. We obtained a 100% adsorption efficiency in 1h for concentrations 10 mg/mL. We hypothesized that when in contact with silica fibers, lysozyme could first adsorb quickly, creating a protein monolayer. Then, additional layers would be stacked on the monolayer. Different lysozyme morphologies were observed by TEM and precise information was obtained from the ATR/FTIR study by comparison of amides, Si-O-Si, Si-OH and water bands between lysozyme, MSNFs alone and MSNFs/lysozyme. Combined with ZP results, our data suggest that protein-silica interactions should be mostly driven by hydrogen bound involving the amide II of the protein. Release experiments showed a burst within the 1st hour, representing from 31% to 81% of the total released amount for concentrations ranging from 5 to 200 mg/mL, respectively. The release rates slowdown in the following hours leading to a release plateau after 3 days for high concentrations and up to 20 days for an initial concentration of 5 mg/mL. These kinetics results support the hypothesis of a two-step adsorption of the protein onto the MSNFs. Overall, the lysozyme release efficiency was related to the initial concentration of lysozyme. Our data also
indicated the maintenance of the biological activity of the released protein at any time points. In a first set of preliminary experiments, we also demonstrated that GDF5 could be adsorbed and released from MSNFs. The GDF5 bioactivity was evidenced by the phosphorylation of Smad 1/5/8. Conclusions: We have demonstrated that MSNFs are suitable nanocarriers for the loading and release of proteins. Protein silica interactions were extensively described providing us with a better understanding of the adsorption phenomenon involved and revealing a hydrogen bound-mediated interactions. Further experiments using GDF5 as a nucleopulpogenic factor are now under intense investigation.
Therapy - Non-Pharmacologic 831 THE IMPACT OF OBESITY ON KNEE OSTEOARTHRITIS SYMPTOMS AND RELATED BIOMARKER PROFILES IN A BARIATRIC SURGERY COHORT J. Samuels y, T. Mukherjee y, E. Wilder y, F. Bonfim y, K. Toth y, S. Aharon y, V. Chen y, L. Browne y, R. La Rocca Vieira y, J. Patel y, C. Ren-Fielding z, M. Parikh z, S.B. Abramson y, M. Attur y. y NYU Langone Hosp. for Joint Diseases, New York, NY, USA; z NYU Langone Med. Ctr., New York, NY, USA Purpose: Obesity is a modifiable risk factor of knee osteoarthritis (KOA). Surgical weight loss may be more effective than diet and exercise in delaying or avoiding joint replacement, as retrospective studies have in fact shown sustained improvement in KOA pain after bariatric surgery. We prospectively evaluated painful KOA in the obese population, tracked whether weight loss after bariatric surgery affects KOA-related pain and function, and studied serum and plasma levels of adipokines and (published) OA biomarkers in this subset of KOA patients. Methods: We screened consecutive patients prior to laparoscopic adjustable gastric banding (LAGB), sleeve gastrectomy, or gastric bypass at NYU and Bellevue. Patients age 18 with knee pain for 1 month and a visual analog scale pain score 30 mm were enrolled, excluding those with lupus, rheumatoid arthritis, psoriatic arthritis, or psoriasis. Baseline pre-op assessments included X-Rays for OA severity by KellgreneLawrence (KL) grade and completion of the Knee Injury and Osteoarthritis Outcome Score (KOOS). They repeated the questionnaire and were re-weighed to calculate percent excess weight loss (%EWL) at 1, 3, 6 and 12 month post-op intervals. Patients were consented for optional tissue collection (blood, urine and intra-operative adipose samples) for biomarker analysis at all visits, with analyses using existing data from controls and a non-obese OA cohort for comparison. Results: Of 537 patients planning to have bariatric surgery, 309 (58%) reported knee pain. 176 enrolled having met criteria and consented for the study: 91.5% female, mean BMI 43.6 kg/m2 ± 7 (31.6e60.6), and mean age of 42.4 ± 11 (18e73). For radiographic severity, KL0 ¼ 43 (25%), KL1 ¼ 34 (19%), KL2 ¼ 38 (22%), KL3 ¼ 34 (19%), KL4 ¼ 27 (15%).