- Email: [email protected]
The effect of bone morphogenetic protein-2 on chondrocyte-like cells derived from degenerated human and canine intervertebral discs
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Abstracts / Osteoarthritis and Cartilage 24 (2016) S63eS534
Purpose: Exercise-based therapy is fundamental for the management of knee osteoarthritis (OA). Exercise is necessary for enhancing muscle strength and lower extremity multijoint strength. The objective of this study was to compare the effects of supervised resistive exercise training with home-based strength training on lower limb muscle strength in patients with knee OA. Methods: Twenty-six patients with knee OA (Kellgren & Lawrence grade 2 or 3), aged 53,86 ± 5,33 years participated in this study. All of the patients were informed about OA risk factors, pathogenesis, prognosis, modification of daily living activities during a 2-hour long patient education lecture. Participants were then assigned at random to one of two groups as home-based strength training group (HEG) (n ¼ 13) and resistive exercise training group (REG) (n ¼ 13). Both exercise programs include the same or similar exercises and aimed at increasing lower limb strength (hip, knee and ankle muscles). All subjects performed three months exercise training. The subjects in the HEG were given strength exercise training and instructed to perform the exercise program three times a week for three months at home. And also, they received one session monthly supervised exercise training for progression. Progression in exercise training obtained with elastic bant. The REG received a supervised program three times a week for three months at indoor resistive exercise station. Patients in both groups performed 10 minutes warm-up period and cool-down period. The strength of quadriceps femoris, hamstrings, hip abductors, hip adductors, and gastrocnemius muscles assessed by using handheld dynamometry by the same investigator. Muscle strength of participants was assessed at baseline, 12 weeks and 24 weeks (follow-up). Results: Four patients in the HEG dropped out during the program (one patient had humerus fracture, and three patients did not attend to scheduled follow-up). One patient in the REG was excluded because of insufficient participation in the exercise program. After exercise training period, strength of hip abductors, quadriceps femoris, gastrocnemius muscles were increased in HEG (p < 0.05) and strength of quadriceps femoris, hamstrings, hip abductors, hip adductors, and gastrocnemius muscles were increased in the REG (p < 0.05). Conclusions: Supervised resistive exercises were more effective in strengthening lower extremity muscles when compared to home-based exercise training in patients with knee OA. However, the increase in muscle strength was not observed at long-term follow-up in both groups.
Spine & Intervertebral Disc 820 FACET CARTILAGE FROM PATIENTS WITH LUMBAR SPINE OSTEOARTHRITIS EXHIBIT REDUCED AUTOPHAGY AND ENHANCED EXPRESSION OF CELL DEATH, INFLAMMATORY AND CATABOLIC MEDIATORS A. Nakamura y, R. Rampersaud z, B. Wu y, E. Rossomacha y, S.J. Lewis z, M. Kapoor z. y Toronto Western Res. Inst., Toronto, ON, Canada; z Toronto Western Hosp./Univ. of Toronto, Toronto, ON, Canada Purpose: Specific mechanisms associated with facet cartilage degeneration during facet joint osteoarthritis (OA) are largely unknown. In this study, we obtained facet joint cartilage from patients undergoing surgery for lumbar spinal canal stenosis (LSCS) and from patients with lumbar intervertebral disc herniation (LDH) (control group) to determine the degree of facet cartilage degeneration and expression of autophagy, apoptosis, inflammatory, catabolic and anabolic markers. Methods: The severity of degeneration in the facet joint and intervertebral disc was assessed by the MRI grading scores as described by Weishaupt et al and Pfirrmann et al, respectively. The degree of facet cartilage degeneration was further assessed histologically using the Osteoarthritis Research Society International (OARSI) grading system. Facet joint cartilage (L3 to S1; medial aspect) was collected from patients with LSCS and patients with LDH. Protein and RNA extracted from facet cartilage was subjected to quantitative real time PCR and Western blot analysis. Results: Our results showed that all patients with LDH exhibited a degenerative score of grade 0 (normal) or grade 1, whereas all patients with LSCS exhibited scores of grade 2 or 3 for the facet joint. In the lumbar disc, all patients in both groups exhibited moderate to severe disc degeneration. Histological analysis using OARSI grading further revealed a significant degree of facet cartilage degeneration in LSCS
patients (OARSI score ¼ 5.5 þ 0.2) compared to LDH patients (OARSI score ¼ 0.9 ± 0.3). Our results further showed that LSCS facet cartilage (in comparison to the LDH control group) exhibited a significant decrease in the expression of chondroprotective autophagy markers (LC3 and ULK1), increase in mammalian target of rapamycin (mTOR, negative regulator of autophagy), increase in cell death (apoptosis) markers (PARP-1 and caspase-3), increase in inflammatory factors (IL1b, TNF-a, IL-6, COX-2, MCP-1), increase in catabolic factors (MMP-3, MMP-13 and ADAMTS-5) and decrease in the expression anabolic matrix molecules (aggrecan and type II collagen). Conclusions: These results clearly show that the LSCS facet cartilage exhibits an enhanced degree of facet cartilage degeneration associated with reduced expression of autophagy (chondroprotective) and anabolic markers; and enhanced expression of apoptosis, inflammatory and catabolic markers. Our uniqueness of using severely degenerated facet cartilage from patients undergoing LSCS surgery and facet cartilage from patients with LDH (as controls) allows us to understand the endogenous mechanisms associated with facet cartilage degeneration. 821 THE EFFECT OF BONE MORPHOGENETIC PROTEIN-2 ON CHONDROCYTE-LIKE CELLS DERIVED FROM DEGENERATED HUMAN AND CANINE INTERVERTEBRAL DISCS F. Bach y, A. Miranda Bedate y, F. van Heel y, M. Müller y, L. Creemers z, K. Ito x, K. Benz k, B. Meij y, M. Tryfonidou y. y Utrecht Univ., Utrecht, Netherlands; z Univ. Med. Ctr. Utrecht, Utrecht, Netherlands; x Eindhoven Univ. of Technology, Eindhoven, Netherlands; k Univ. of Tuebingen, Reutlingen, Germany Purpose: Both humans and dogs experience low back pain, which is related to intervertebral disc (IVD) degeneration. Biologic repair of the degenerated IVD is mainly based on growth factors that exert anabolic matrix effects, and mesenchymal stromal cells (MSCs) to replenish the cell population of the degenerated IVD. Thus far, the anabolic effects of different growth factors have not been compared. The aim of this study was to study the effect of the frequently used growth factors transforming growth factor beta 1 (TGF-b1) and bone morphogenetic protein-2 (BMP-2) on canine and human chondrocyte-like cells (CLCs) from degenerated IVDs alone and in combination with MSCs. Methods: CLCs from degenerated human, canine chondrodystrophic (CD) and non-chondrodystrophic (NCD) IVDs (Thompson score III) were cultured in micro-aggregates in base culture medium (negative control), or supplemented with TGF-ß1 (10 ng/mL) or BMP-2 (100 or 250 ng/mL) for 28 days. The additive effect of MSCs was studied in CD CLCs. Canine male CD CLCs were cultured in an albumin-based hydrogel (3*106 cells/mL) with or without the addition of female bone marrowderived MSCs (BMSCs) (CLC:BMSC 1:1) in control or 250 ng/mL BMP-2supplemented culture medium for 28 days. Read out parameters were extracellular matrix (ECM) production (RT-qPCR, glycosaminoglycan (GAG) production, Safranin O/Fast Green staining, immunohistochemistry), cell proliferation (DNA content, RT-qPCR) and apoptosis (RTqPCR). Results: TGF-ß1 treatment increased GAG deposition in human and canine CLC micro-aggregates, but also induced collagen type I deposition and a fibrotic rim. The latter was not observed in BMP-2-treated micro-aggregates. 250 ng/mL BMP-2 was more potent than 100 ng/mL BMP-2 in increasing GAG deposition and DNA content in canine and human micro-aggregates. Similarly, in the hydrogel culture system, BMP-2 induced GAG and collagen type II deposition and a higher DNA content compared with untreated controls. DNA and GAG content of BMSCþCLC hydrogels was higher than hydrogels with CLCs alone in the absence of BMP-2. In the BMP-2-treated hydrogels, DNA content of BMSCþCLC was higher than hydrogels with CLCs alone; GAG deposition or release was comparable between BMSCþCLC and CLC alone in the presence of BMP-2. Conclusions: In two different 3D culture systems, BMP-2 exerted comparable regenerative effects as TGF-b1 on human and canine CLCs in terms of GAG deposition and cell proliferation, but BMP-2 did not induce fibrotic (re)differentiation as observed with TGF-ß1 treatment. Moreover, in the BMP-2-treated BMSC:CLC-containing hydrogels, where only half of the amount of CLCs were seeded compared with CLC alone, the DNA content was higher and an equal amount of GAGs was deposited, indicating that the BMSCs exerted an additional, regenerative effect in addition to BMP-2 treatment. PCR for SRY:GAPDH genes on DNA will indicate the ratio male(CLCs):female(BMSCs) present at the
Abstracts / Osteoarthritis and Cartilage 24 (2016) S63eS534
end of the study. This will indicate whether the BMSCs exerted trophic effects or chondrogenically differentiated. 822 CARTILAGE b-CATENIN SIGNALING PLAYS A KEY ROLE IN THE DEVELOPMENT OF SPONDYLOARTHRITIS D. Chen, T. Hui, W. Xie, C. Oh, L. Liao, S. Li, S. Zhang, J.S. Kroin, H.-J. Im. Rush Univ., Chicago, IL, USA Purpose: Spondyloarthritis (SpA) is a group of autoimmune disease affecting spine and joint. Cartilage is the major tissue involved in this disease. Up-regulation of Wnt/b-catenin signaling has been reported in patients with SpA. In this study, we will determine if activation of bcatenin signaling in chondrocytes is the key event leading to bony overgrowth phenotype of the spine during the development of SpA. Methods: 1) Chondrocyte-specific b-catenin conditional activation mice, b-catenin(ex3)Col2ER, were generated by breeding b-catenin(ex3)flox /flox mice with Col2-CreERT2 transgenic mice. Deletion of exon3 of the b-catenin gene leads to the production of truncated b-catenin protein which is resistant to the GSK-3b-induced phosphorylation and subsequent ubiquitination and proteaome degradation. 2) Histology and histomorphometric analyses were performed in 3- and 6-month-old mice and changes in disc tissue were analyzed. Histological changes in facet joints were also analyzed in 4- and 9-month-old mice. 3) mCT analysis was performed to determine osteophyte formation in the spine. 4) Immortalized annular fibrosus (AF) and nucleus pulposus (NP) cells were generated. The cells maintain proliferation and differentiation properties and respond to growth factors even after 50 passages. These cells were used for b-catenin signaling studies. 5) Painrelated behavior tests were performed in 4e9 month old b-catenin(ex3)Col2ER and Cre-negative control mice. Results: 1) We found that severe destruction of disc tissues in 3- and 6month-old b-catenin(ex3)Col2ER mice, including severe loss of cartilage tissues in endplate, disorganized AF and NP tissues and large amounts of osteophyte formation in the entire spine. 2) Using immortalized rat AF and NP cell lines, we found that treatment with BIO, a GSK-3b inhibitor (mimic b-catenin signaling activation), stimulated Top-flash reporter activity (6-15 folds) in both AF and NP cells after 24 h treatment. BIO induced Mmp13 (8 and 11 folds) and Ccl2 (6 and 11 folds) expression in AF and NP cells. 3) To determine if Mmp13 is a key downstream target of b-catenin signaling, we generated b-catenin(ex3)/ Mmp13Col2ER double mutant mice and found that deletion of Mmp13 significantly reversed defects observed in disc tissue of b-catenin(ex3)Col2ER mice. 4) In addition to the disc tissues, we also observed severe damage and loss of proteoglycan and defects in cartilage structure in the facet joint in 4- and 9-month-old b-catenin(ex3)Col2ERmice. 5) CCL2 has been demonstrated as a key mediator of osteoarthritis (OA) pain. In the present studies we found that Ccl2 was significantly upregulated by the activation of b-catenin signaling in both AF and NP cells. We then determined changes in pain-related behavior in 4e9 month old b-catenin(ex3)Col2ER mice. The b-catenin(ex3)Col2ER mice had significantly increased pain sensitivity as evidenced by lower von Frey thresholds, and this pain behavior sustained throughout the entire experimental period (9 months). The rearing as well as ambulation counts were also reduced in 4e9 month old b-catenin(ex3)Col2ER mice compared to Cre-negative controls. Conclusions: Taking consideration of our previous observations from bcatenin(ex3)Col2ER mice with OA-like phenotype in knee joint and temporomandibular joint (TMJ), the findings observed in disc and facet joint tissues of b-catenin(ex3)Col2ER mice largely resemble key features found in patients with SpA. Our findings suggest that activation of bcatenin signaling in cartilage tissue may be the key event leading to the bony overgrowth of the spine in patients with SpA. 823 THE SPECIES-SPECIFIC REGENERATIVE EFFECT OF NOTOCHORDAL CELL-CONDITIONED MEDIUM ON DEGENERATED HUMAN NUCLEUS PULPOSUS CELLS F. Bach y, S. de Vries z, F. van Heel y, A. Krouwels x, L. Creemers x, K. Ito z, B. Meij y, M. Tryfonidou y. y Utrecht Univ., Utrecht, Netherlands; z Eindhoven Univ. of Technology, Eindhoven, Netherlands; x Univ. Med. Ctr. Utrecht, Utrecht, Netherlands Purpose: During human intervertebral disc (IVD) degeneration, the main cell type shifts from notochordal cells (NCs) to nucleus pulposus
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cells (NPCs). NCs secrete factors with regenerative potential, making them an interesting focus for regenerative strategies. Research into these strategies employs non-human donors due to easy availability of their NC-rich nucleus pulposus (NP) tissue. To determine the feasibility of translating these strategies towards clinical application, this study aimed to delineate whether NC-secreted factors of different species have a cross-species regenerative effect on degenerated human NPCs. Methods: Healthy human, porcine and canine NC-rich NP tissues (derived from Thompson grade I IVDs) were compared by histology, collagen, DNA and glycosaminoglycan (GAG) content per mg wet tissue weight. NP tissue of each species was cultured for 4 days and NC-conditioned medium (NCCM) was collected. Human NPC micro-aggregates from degenerated IVDs (Thompson grade III) were cultured for 28 days in human, canine or porcine NCCM. GAG content (corrected for DNA) was determined, and Safranin O/Fast Green staining, collagen type I, II and X IHC and gene expression profiling were performed. Results: Canine and porcine NPs were richer in NCs than human NPs. Human NPs contained the highest collagen content, whereas the DNA and GAG content of canine NPs was significantly higher than human or porcine NPs. NCCM from all species significantly increased the DNA and GAG content of the human NPC micro-aggregates. Porcine and canine NCCM were significantly more potent than human NCCM in inducing GAG deposition, whereas only human NCCM induced collagen type II production. Conclusions: Secreted factors from human, canine and porcine NC-rich NPs exerted regenerative effects on human NPCs, indicating a crossspecies effect. The species-specific NP properties appear to influence the regenerative capacity of NCCM on human NPCs. Bioactive compound(s) with treatment potential for human IVD degeneration are present in NCCM of different species, implying that strategies based on NC-technology employing canine or porcine models have potential for successful translation into humans. 824 HIF-1 ALPHA IS ESSENTIAL FOR DEVELOPMENT OF THE NUCLEUS PULPOSUS C. Merceron y, z, L. Mangiavini z, T. Wilson z, A. Robling x, J. Guicheux y, k, I. Shapiro ¶, M. Risbud ¶, E. Schipani z, #. y LIOAD- Inserm UMRS 791, Nantes, France; z Univ. of Michigan, Med. Sch., Dept. of Orthopaedic Surgery, Ann Arbor, MI, USA; x Indiana Univ., Indianapolis, IN, USA; k Nantes Univ. Hosp., PHU4 OTONN, Nantes, France; ¶ Thomas Jefferson Univ., Philadelphia, PA, USA; # Univ. of Michigan, Med. Sch., Dept. of Med., Div. of Endocrinology, Ann Arbor, MI, USA Purpose: The intervertebral disc is one of the largest avascular organs in vertebrates. It consists of an external ring formed by multi-lamellar collagenous layers called the annulus fibrosus (AF). The AF surrounds the nucleus pulposus (NP), a highly hydrated gelatinous structure. This assembly is covered on both the top and bottom sides by cartilaginous endplates (CEP). The NP is a unique tissue within the IVD as it results from the differentiation of notochordal cells, whereas, AF and CEP derive from the sclerotome. The NP cells or nucleopulpocytes are hypoxic and express the transcription factor HIF-1alpha (Hypoxia Inducible Factor), which is a major mediator of cellular adaptation to low oxygen tension. To address the function of HIF-1alpha in NP development and homeostasis and to decipher the molecular mechanisms of notochordal cell differentiation into nucleopulpocytes, we analyzed the consequences of the conditional deletion of HIF-1alpha in the notochord. Methods: Foxa2-Cre transgenic mice were bred with mice homozygous for a floxed HIF-1alpha allele (HIF-1alphaf/f) in order to generate FOXA2-Cre; HIF-1alphaf/f mutant mice and control littermates. Development of the nucleus pulposus upon the loss of HIF-1alpha was studied from E13.5 up to 4 months. Immunohistochemistry for HIF1alpha, histological analysis, in situ hybridizations, cell lineage studies and TUNEL assay were performed. Finally, to assess the functional consequences of HIF-alpha deletion in the NP, biomechanical testing of mutant IVD was performed. Results: Mutant mice in which HIF-1alpha had been conditionally inactivated in the NP were born with the expected mendelian frequency, reached adulthood and, macroscopically, did not exhibit any obvious phenotypic difference in comparison to control mice. As evidenced by H&E staining at E13.5, both control and mutant notochords exhibited similar morphologic features. They both displayed undistinguishable brachyury mRNA patterns of expression, which is a
Abstracts / Osteoarthritis and Cartilage 24 (2016) S63eS534
Purpose: Exercise-based therapy is fundamental for the management of knee osteoarthritis (OA). Exercise is necessary for enhancing muscle strength and lower extremity multijoint strength. The objective of this study was to compare the effects of supervised resistive exercise training with home-based strength training on lower limb muscle strength in patients with knee OA. Methods: Twenty-six patients with knee OA (Kellgren & Lawrence grade 2 or 3), aged 53,86 ± 5,33 years participated in this study. All of the patients were informed about OA risk factors, pathogenesis, prognosis, modification of daily living activities during a 2-hour long patient education lecture. Participants were then assigned at random to one of two groups as home-based strength training group (HEG) (n ¼ 13) and resistive exercise training group (REG) (n ¼ 13). Both exercise programs include the same or similar exercises and aimed at increasing lower limb strength (hip, knee and ankle muscles). All subjects performed three months exercise training. The subjects in the HEG were given strength exercise training and instructed to perform the exercise program three times a week for three months at home. And also, they received one session monthly supervised exercise training for progression. Progression in exercise training obtained with elastic bant. The REG received a supervised program three times a week for three months at indoor resistive exercise station. Patients in both groups performed 10 minutes warm-up period and cool-down period. The strength of quadriceps femoris, hamstrings, hip abductors, hip adductors, and gastrocnemius muscles assessed by using handheld dynamometry by the same investigator. Muscle strength of participants was assessed at baseline, 12 weeks and 24 weeks (follow-up). Results: Four patients in the HEG dropped out during the program (one patient had humerus fracture, and three patients did not attend to scheduled follow-up). One patient in the REG was excluded because of insufficient participation in the exercise program. After exercise training period, strength of hip abductors, quadriceps femoris, gastrocnemius muscles were increased in HEG (p < 0.05) and strength of quadriceps femoris, hamstrings, hip abductors, hip adductors, and gastrocnemius muscles were increased in the REG (p < 0.05). Conclusions: Supervised resistive exercises were more effective in strengthening lower extremity muscles when compared to home-based exercise training in patients with knee OA. However, the increase in muscle strength was not observed at long-term follow-up in both groups.
Spine & Intervertebral Disc 820 FACET CARTILAGE FROM PATIENTS WITH LUMBAR SPINE OSTEOARTHRITIS EXHIBIT REDUCED AUTOPHAGY AND ENHANCED EXPRESSION OF CELL DEATH, INFLAMMATORY AND CATABOLIC MEDIATORS A. Nakamura y, R. Rampersaud z, B. Wu y, E. Rossomacha y, S.J. Lewis z, M. Kapoor z. y Toronto Western Res. Inst., Toronto, ON, Canada; z Toronto Western Hosp./Univ. of Toronto, Toronto, ON, Canada Purpose: Specific mechanisms associated with facet cartilage degeneration during facet joint osteoarthritis (OA) are largely unknown. In this study, we obtained facet joint cartilage from patients undergoing surgery for lumbar spinal canal stenosis (LSCS) and from patients with lumbar intervertebral disc herniation (LDH) (control group) to determine the degree of facet cartilage degeneration and expression of autophagy, apoptosis, inflammatory, catabolic and anabolic markers. Methods: The severity of degeneration in the facet joint and intervertebral disc was assessed by the MRI grading scores as described by Weishaupt et al and Pfirrmann et al, respectively. The degree of facet cartilage degeneration was further assessed histologically using the Osteoarthritis Research Society International (OARSI) grading system. Facet joint cartilage (L3 to S1; medial aspect) was collected from patients with LSCS and patients with LDH. Protein and RNA extracted from facet cartilage was subjected to quantitative real time PCR and Western blot analysis. Results: Our results showed that all patients with LDH exhibited a degenerative score of grade 0 (normal) or grade 1, whereas all patients with LSCS exhibited scores of grade 2 or 3 for the facet joint. In the lumbar disc, all patients in both groups exhibited moderate to severe disc degeneration. Histological analysis using OARSI grading further revealed a significant degree of facet cartilage degeneration in LSCS
patients (OARSI score ¼ 5.5 þ 0.2) compared to LDH patients (OARSI score ¼ 0.9 ± 0.3). Our results further showed that LSCS facet cartilage (in comparison to the LDH control group) exhibited a significant decrease in the expression of chondroprotective autophagy markers (LC3 and ULK1), increase in mammalian target of rapamycin (mTOR, negative regulator of autophagy), increase in cell death (apoptosis) markers (PARP-1 and caspase-3), increase in inflammatory factors (IL1b, TNF-a, IL-6, COX-2, MCP-1), increase in catabolic factors (MMP-3, MMP-13 and ADAMTS-5) and decrease in the expression anabolic matrix molecules (aggrecan and type II collagen). Conclusions: These results clearly show that the LSCS facet cartilage exhibits an enhanced degree of facet cartilage degeneration associated with reduced expression of autophagy (chondroprotective) and anabolic markers; and enhanced expression of apoptosis, inflammatory and catabolic markers. Our uniqueness of using severely degenerated facet cartilage from patients undergoing LSCS surgery and facet cartilage from patients with LDH (as controls) allows us to understand the endogenous mechanisms associated with facet cartilage degeneration. 821 THE EFFECT OF BONE MORPHOGENETIC PROTEIN-2 ON CHONDROCYTE-LIKE CELLS DERIVED FROM DEGENERATED HUMAN AND CANINE INTERVERTEBRAL DISCS F. Bach y, A. Miranda Bedate y, F. van Heel y, M. Müller y, L. Creemers z, K. Ito x, K. Benz k, B. Meij y, M. Tryfonidou y. y Utrecht Univ., Utrecht, Netherlands; z Univ. Med. Ctr. Utrecht, Utrecht, Netherlands; x Eindhoven Univ. of Technology, Eindhoven, Netherlands; k Univ. of Tuebingen, Reutlingen, Germany Purpose: Both humans and dogs experience low back pain, which is related to intervertebral disc (IVD) degeneration. Biologic repair of the degenerated IVD is mainly based on growth factors that exert anabolic matrix effects, and mesenchymal stromal cells (MSCs) to replenish the cell population of the degenerated IVD. Thus far, the anabolic effects of different growth factors have not been compared. The aim of this study was to study the effect of the frequently used growth factors transforming growth factor beta 1 (TGF-b1) and bone morphogenetic protein-2 (BMP-2) on canine and human chondrocyte-like cells (CLCs) from degenerated IVDs alone and in combination with MSCs. Methods: CLCs from degenerated human, canine chondrodystrophic (CD) and non-chondrodystrophic (NCD) IVDs (Thompson score III) were cultured in micro-aggregates in base culture medium (negative control), or supplemented with TGF-ß1 (10 ng/mL) or BMP-2 (100 or 250 ng/mL) for 28 days. The additive effect of MSCs was studied in CD CLCs. Canine male CD CLCs were cultured in an albumin-based hydrogel (3*106 cells/mL) with or without the addition of female bone marrowderived MSCs (BMSCs) (CLC:BMSC 1:1) in control or 250 ng/mL BMP-2supplemented culture medium for 28 days. Read out parameters were extracellular matrix (ECM) production (RT-qPCR, glycosaminoglycan (GAG) production, Safranin O/Fast Green staining, immunohistochemistry), cell proliferation (DNA content, RT-qPCR) and apoptosis (RTqPCR). Results: TGF-ß1 treatment increased GAG deposition in human and canine CLC micro-aggregates, but also induced collagen type I deposition and a fibrotic rim. The latter was not observed in BMP-2-treated micro-aggregates. 250 ng/mL BMP-2 was more potent than 100 ng/mL BMP-2 in increasing GAG deposition and DNA content in canine and human micro-aggregates. Similarly, in the hydrogel culture system, BMP-2 induced GAG and collagen type II deposition and a higher DNA content compared with untreated controls. DNA and GAG content of BMSCþCLC hydrogels was higher than hydrogels with CLCs alone in the absence of BMP-2. In the BMP-2-treated hydrogels, DNA content of BMSCþCLC was higher than hydrogels with CLCs alone; GAG deposition or release was comparable between BMSCþCLC and CLC alone in the presence of BMP-2. Conclusions: In two different 3D culture systems, BMP-2 exerted comparable regenerative effects as TGF-b1 on human and canine CLCs in terms of GAG deposition and cell proliferation, but BMP-2 did not induce fibrotic (re)differentiation as observed with TGF-ß1 treatment. Moreover, in the BMP-2-treated BMSC:CLC-containing hydrogels, where only half of the amount of CLCs were seeded compared with CLC alone, the DNA content was higher and an equal amount of GAGs was deposited, indicating that the BMSCs exerted an additional, regenerative effect in addition to BMP-2 treatment. PCR for SRY:GAPDH genes on DNA will indicate the ratio male(CLCs):female(BMSCs) present at the
Abstracts / Osteoarthritis and Cartilage 24 (2016) S63eS534
end of the study. This will indicate whether the BMSCs exerted trophic effects or chondrogenically differentiated. 822 CARTILAGE b-CATENIN SIGNALING PLAYS A KEY ROLE IN THE DEVELOPMENT OF SPONDYLOARTHRITIS D. Chen, T. Hui, W. Xie, C. Oh, L. Liao, S. Li, S. Zhang, J.S. Kroin, H.-J. Im. Rush Univ., Chicago, IL, USA Purpose: Spondyloarthritis (SpA) is a group of autoimmune disease affecting spine and joint. Cartilage is the major tissue involved in this disease. Up-regulation of Wnt/b-catenin signaling has been reported in patients with SpA. In this study, we will determine if activation of bcatenin signaling in chondrocytes is the key event leading to bony overgrowth phenotype of the spine during the development of SpA. Methods: 1) Chondrocyte-specific b-catenin conditional activation mice, b-catenin(ex3)Col2ER, were generated by breeding b-catenin(ex3)flox /flox mice with Col2-CreERT2 transgenic mice. Deletion of exon3 of the b-catenin gene leads to the production of truncated b-catenin protein which is resistant to the GSK-3b-induced phosphorylation and subsequent ubiquitination and proteaome degradation. 2) Histology and histomorphometric analyses were performed in 3- and 6-month-old mice and changes in disc tissue were analyzed. Histological changes in facet joints were also analyzed in 4- and 9-month-old mice. 3) mCT analysis was performed to determine osteophyte formation in the spine. 4) Immortalized annular fibrosus (AF) and nucleus pulposus (NP) cells were generated. The cells maintain proliferation and differentiation properties and respond to growth factors even after 50 passages. These cells were used for b-catenin signaling studies. 5) Painrelated behavior tests were performed in 4e9 month old b-catenin(ex3)Col2ER and Cre-negative control mice. Results: 1) We found that severe destruction of disc tissues in 3- and 6month-old b-catenin(ex3)Col2ER mice, including severe loss of cartilage tissues in endplate, disorganized AF and NP tissues and large amounts of osteophyte formation in the entire spine. 2) Using immortalized rat AF and NP cell lines, we found that treatment with BIO, a GSK-3b inhibitor (mimic b-catenin signaling activation), stimulated Top-flash reporter activity (6-15 folds) in both AF and NP cells after 24 h treatment. BIO induced Mmp13 (8 and 11 folds) and Ccl2 (6 and 11 folds) expression in AF and NP cells. 3) To determine if Mmp13 is a key downstream target of b-catenin signaling, we generated b-catenin(ex3)/ Mmp13Col2ER double mutant mice and found that deletion of Mmp13 significantly reversed defects observed in disc tissue of b-catenin(ex3)Col2ER mice. 4) In addition to the disc tissues, we also observed severe damage and loss of proteoglycan and defects in cartilage structure in the facet joint in 4- and 9-month-old b-catenin(ex3)Col2ERmice. 5) CCL2 has been demonstrated as a key mediator of osteoarthritis (OA) pain. In the present studies we found that Ccl2 was significantly upregulated by the activation of b-catenin signaling in both AF and NP cells. We then determined changes in pain-related behavior in 4e9 month old b-catenin(ex3)Col2ER mice. The b-catenin(ex3)Col2ER mice had significantly increased pain sensitivity as evidenced by lower von Frey thresholds, and this pain behavior sustained throughout the entire experimental period (9 months). The rearing as well as ambulation counts were also reduced in 4e9 month old b-catenin(ex3)Col2ER mice compared to Cre-negative controls. Conclusions: Taking consideration of our previous observations from bcatenin(ex3)Col2ER mice with OA-like phenotype in knee joint and temporomandibular joint (TMJ), the findings observed in disc and facet joint tissues of b-catenin(ex3)Col2ER mice largely resemble key features found in patients with SpA. Our findings suggest that activation of bcatenin signaling in cartilage tissue may be the key event leading to the bony overgrowth of the spine in patients with SpA. 823 THE SPECIES-SPECIFIC REGENERATIVE EFFECT OF NOTOCHORDAL CELL-CONDITIONED MEDIUM ON DEGENERATED HUMAN NUCLEUS PULPOSUS CELLS F. Bach y, S. de Vries z, F. van Heel y, A. Krouwels x, L. Creemers x, K. Ito z, B. Meij y, M. Tryfonidou y. y Utrecht Univ., Utrecht, Netherlands; z Eindhoven Univ. of Technology, Eindhoven, Netherlands; x Univ. Med. Ctr. Utrecht, Utrecht, Netherlands Purpose: During human intervertebral disc (IVD) degeneration, the main cell type shifts from notochordal cells (NCs) to nucleus pulposus
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cells (NPCs). NCs secrete factors with regenerative potential, making them an interesting focus for regenerative strategies. Research into these strategies employs non-human donors due to easy availability of their NC-rich nucleus pulposus (NP) tissue. To determine the feasibility of translating these strategies towards clinical application, this study aimed to delineate whether NC-secreted factors of different species have a cross-species regenerative effect on degenerated human NPCs. Methods: Healthy human, porcine and canine NC-rich NP tissues (derived from Thompson grade I IVDs) were compared by histology, collagen, DNA and glycosaminoglycan (GAG) content per mg wet tissue weight. NP tissue of each species was cultured for 4 days and NC-conditioned medium (NCCM) was collected. Human NPC micro-aggregates from degenerated IVDs (Thompson grade III) were cultured for 28 days in human, canine or porcine NCCM. GAG content (corrected for DNA) was determined, and Safranin O/Fast Green staining, collagen type I, II and X IHC and gene expression profiling were performed. Results: Canine and porcine NPs were richer in NCs than human NPs. Human NPs contained the highest collagen content, whereas the DNA and GAG content of canine NPs was significantly higher than human or porcine NPs. NCCM from all species significantly increased the DNA and GAG content of the human NPC micro-aggregates. Porcine and canine NCCM were significantly more potent than human NCCM in inducing GAG deposition, whereas only human NCCM induced collagen type II production. Conclusions: Secreted factors from human, canine and porcine NC-rich NPs exerted regenerative effects on human NPCs, indicating a crossspecies effect. The species-specific NP properties appear to influence the regenerative capacity of NCCM on human NPCs. Bioactive compound(s) with treatment potential for human IVD degeneration are present in NCCM of different species, implying that strategies based on NC-technology employing canine or porcine models have potential for successful translation into humans. 824 HIF-1 ALPHA IS ESSENTIAL FOR DEVELOPMENT OF THE NUCLEUS PULPOSUS C. Merceron y, z, L. Mangiavini z, T. Wilson z, A. Robling x, J. Guicheux y, k, I. Shapiro ¶, M. Risbud ¶, E. Schipani z, #. y LIOAD- Inserm UMRS 791, Nantes, France; z Univ. of Michigan, Med. Sch., Dept. of Orthopaedic Surgery, Ann Arbor, MI, USA; x Indiana Univ., Indianapolis, IN, USA; k Nantes Univ. Hosp., PHU4 OTONN, Nantes, France; ¶ Thomas Jefferson Univ., Philadelphia, PA, USA; # Univ. of Michigan, Med. Sch., Dept. of Med., Div. of Endocrinology, Ann Arbor, MI, USA Purpose: The intervertebral disc is one of the largest avascular organs in vertebrates. It consists of an external ring formed by multi-lamellar collagenous layers called the annulus fibrosus (AF). The AF surrounds the nucleus pulposus (NP), a highly hydrated gelatinous structure. This assembly is covered on both the top and bottom sides by cartilaginous endplates (CEP). The NP is a unique tissue within the IVD as it results from the differentiation of notochordal cells, whereas, AF and CEP derive from the sclerotome. The NP cells or nucleopulpocytes are hypoxic and express the transcription factor HIF-1alpha (Hypoxia Inducible Factor), which is a major mediator of cellular adaptation to low oxygen tension. To address the function of HIF-1alpha in NP development and homeostasis and to decipher the molecular mechanisms of notochordal cell differentiation into nucleopulpocytes, we analyzed the consequences of the conditional deletion of HIF-1alpha in the notochord. Methods: Foxa2-Cre transgenic mice were bred with mice homozygous for a floxed HIF-1alpha allele (HIF-1alphaf/f) in order to generate FOXA2-Cre; HIF-1alphaf/f mutant mice and control littermates. Development of the nucleus pulposus upon the loss of HIF-1alpha was studied from E13.5 up to 4 months. Immunohistochemistry for HIF1alpha, histological analysis, in situ hybridizations, cell lineage studies and TUNEL assay were performed. Finally, to assess the functional consequences of HIF-alpha deletion in the NP, biomechanical testing of mutant IVD was performed. Results: Mutant mice in which HIF-1alpha had been conditionally inactivated in the NP were born with the expected mendelian frequency, reached adulthood and, macroscopically, did not exhibit any obvious phenotypic difference in comparison to control mice. As evidenced by H&E staining at E13.5, both control and mutant notochords exhibited similar morphologic features. They both displayed undistinguishable brachyury mRNA patterns of expression, which is a