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Mesenchymal stem cells-containing alginate particles for intra-articular injection in osteoarthritis
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Abstracts / Osteoarthritis and Cartilage 25 (2017) S76eS444
more frequent media change regimen must be implemented to obtain optimal results. In this regard, bioreactor culture can lead to improved chondrogenesis. Glucose consumption during chondrogenesis can be used to predict the biochemical properties of the resultant tissue. 629 EFFECT OF GRAVITATIONAL FORCE AND HYDROSTATIC PRESSURE ON THE DEVELOPMENT OF ARTICULAR NEOCARTILAGE €rinne, J. Piltti, C. Qu, M.J. Lammi. Umeå Univ., Umeå, J.O. Prittinen, J.H. Yla Sweden Purpose: Articular cartilage has an extremely limited ability for selfrepair and damaged tissue has a high risk of deteriorating further if not intervened. A promising treatment method could beosteochondral implants but construction of such implants requires further knowledge of articular cartilage development. Here we investigate a novel culture system for manufacturing osteochondral implants that also allows mechanical loading of the construct during in vitro culturing. Methods: Bovine articular chondrocytes from five individual cell isolations were cultured at high density (6106 cells) on top of defatted bone disks in an agarose well created into a centrifugation tube (A and B). A calcium phosphate layer was precipitated on the bone disk to prevent the chondrocytes from moving through the pores. The cultures were centrifuged 0, 1 or 3 times a day for 15 min. At 770 g, this program asserted a hydrostatic pressure of 2.9 MPa on the tissue constructs. Total mRNA and samples for glycosaminoglycan (GAG) and hydroxyproline content analysis were collected at 1, 2 and 4 weeks. Results: The centrifuged tissues appeared flatter and more uniform than the non-centrifuged controls. Although the total GAG content was slightly lower in the centrifuged tissue, there were no significant differences. Hydroxyproline measurement showed highest collagen content in the once and three times centrifuged tissue cultures. Gene expression analysis suggests higher type II procollagen expression in the non-centrifuged and once centrifuged tissue cultures in comparison with the three times centrifuged. On the other hand, the three times centrifuged samples had higher aggrecan expression in comparison to the non-centrifuged tissues. The type I procollagen/type II procollagen -ratio increased with time in all samples and was the highest in the three times centrifuged samples.
Conclusions: The culture system supports tissue growth and produces articular cartilage-like tissue. Both centrifugation regimes produced tissue that had a flatter and a more uniform surface (C) and the tissue also felt noticeably stiffer when handling, although the biomechanical properties still need to be analysed. There seems to be little to no benefit from centrifuging the samples three times a day, since tissues centrifuged once per day tissues maintained the expression of genes typical for hyaline cartilage formation. 630 MESENCHYMAL STEM CELLS-CONTAINING ALGINATE PARTICLES FOR INTRA-ARTICULAR INJECTION IN OSTEOARTHRITIS A. Smith y, M. Marquis z, C. Vinatier y, A. des Rieux x, k, D. Renard z, J. Guicheux y, ¶, C. Le Visage y. y INSERM UMR 1229-RMeS, Regenerative Med. and Skeleton, STEP Team, Univ. of Nantes, UFR odontology, Nantes eres Interactions Assemblages), F-44042, France; z UR1268 BIA (Biopolym INRA, 44300 Nantes, France; x Louvain Drug Res. Inst., Advanced Drug
Delivery and Biomaterials, Universit e Catholique de Louvain, 1200 Bruxelles, Belgium; k Inst. of Condensed Matter and NanoSci.s, Universit e Catholique de Louvain, 1048 Louvain-la-Neuve, Belgium; ¶ CHU Nantes, PHU 4 OTONN, Nantes F-44042, France Purpose: Osteoarthritis (OA) is a degenerative and inflammatory joint disease that affects cartilage, subchondral bone and joint tissues. Mesenchymal Stem Cells (MSCs) ability to secrete anti-inflammatory and immuno-modulatory factors represents an attractive tool in the treatment of osteoarthitis. Considering the risk of cell leakage and the massive cell death upon intra-articular injection, MSCs encapsulation therefore could prevent cell death, avoid cell effusion outside the articular space, and supply a suitable micro-environment supporting the biological activity of MSCs. Previously, we have demonstrated that alginate particles support MSCs viability and bioactivity. Nevertheless these particles obtained through a dropwise method had an average size of 1.5 ± 0.2 mm and were thus too large to be injected into joints. Here, we propose to develop a method of cell encapsulation in alginate compatible with intra-articular injection through a 26G needle. Methods: Alginate particles were generated through micromolding method. We first manufactured wafers patterned by soft lithography with a photocrosslinkable resin. The wafers had protruding 100 mm high patterns with square and circle shapes of 100, 150 and 200 mm of side or diameter. Polydimethylsiloxane (PDMS) molds were then generated by pouring 10% PDMS onto the wafers and curing it for 20 min at 70 C. Finally, the PDMS molds were peeled from the wafers and stored 1 night at 60 C. Before their use, the molds were hydrophilized by plasma treatment. A solution of 2% alginate was then deposited on the molds and alginate particles were crosslinked with an agarose gel charged with CaCl2. After manufacture, the size and shape of the microparticles were assessed using phase-contrast microscopy and digital imaging. To encapsulate cells, human adipose-derived mesenchymal stem cells (ASC) were suspended at 1 and 3 millions of cells per mL of the 2% alginate solution and deposited on PDMS molds containing 3200 circle wells with a diameter of 200 mm. Number of encapsulated cells were then determined by a PicoGreen assay. Results: We successfully obtained alginate microparticles reproducing the micromold shapes. Both cylindrical and cubic particles exhibited however a reduced size as compared to the original molds. For instance, cylindrical particles molded into 100 mm cycle molds had an average size of 73 ± 0.8 mm (n ¼ 3) and those molded from 150 and 200 mm diameter molds presented a size of 103 ± 0.7 and 136 ± 0.7 mm, respectively (n ¼ 3). Similarly, for cubic particles, we measured a size of 72 ± 0.7 mm, 107 ± 0.7 mm (n ¼ 3) and 137 ± 0.8 mm (n ¼ 3) for the particles molded into 100, 150 and 200 mm molds, respectively. We then selected the 200 mm cylindrical molds and performed cell encapsulation. The presence of cells into the alginate solution had no impact on the size or shape of the particles. Using optical microscopy, we easily identified that cells were homogeneously distributed within the transparent alginate particles. Preliminary results indicate that the number of encapsulated cells by particle depends on the initial cell concentration in the alginate solution. Conclusions: We first succeeded in generating alginate particles that could be injectable through a 26 G needle. In addition, micromoldingderived particles of alginate allowed the successful encapsulation of viable MSC. Our work will now focus on demonstrating the ability of encapsulated MSC to secrete immunomodulatory and anti-inflammatory factors. In a future step, we will consider intra-articular injection in an animal model of osteoarthritis. 631 IMMUNOMODULATORY EFFECT OF MESENCHYMAL STEM CELLS FOLLOWING INTRA-ARTICULAR INJECTION IN A MODEL OF OSTEOARTHRITIS: A POTENTIAL ROLE FOR APOPTOSIS P. Mancuso, M.J. Murphy, F. Barry. REMEDI (NUI Galway), Galway, Ireland Purpose: Mesenchymal stem cells (MSCs) promote regeneration when intra-articularly (IA) administered in pre-clinical models of osteoarthritis (OA). Their mechanism of action, however, is still poorly understood. Preliminary data show low engraftment of GFP-expressing syngeneic MSCs in mouse knee joints, correlating with published data using molecular techniques. As cells were not detected in other organs, we hypothesise that implanted cells may undergo apoptosis or cell death. There is growing evidence that apoptotic MSCs exert an antiinflammatory effect, by direct action on phagocytes or by release of immunosuppressive cytokines. This study aims to investigate the fate
Abstracts / Osteoarthritis and Cartilage 25 (2017) S76eS444
more frequent media change regimen must be implemented to obtain optimal results. In this regard, bioreactor culture can lead to improved chondrogenesis. Glucose consumption during chondrogenesis can be used to predict the biochemical properties of the resultant tissue. 629 EFFECT OF GRAVITATIONAL FORCE AND HYDROSTATIC PRESSURE ON THE DEVELOPMENT OF ARTICULAR NEOCARTILAGE €rinne, J. Piltti, C. Qu, M.J. Lammi. Umeå Univ., Umeå, J.O. Prittinen, J.H. Yla Sweden Purpose: Articular cartilage has an extremely limited ability for selfrepair and damaged tissue has a high risk of deteriorating further if not intervened. A promising treatment method could beosteochondral implants but construction of such implants requires further knowledge of articular cartilage development. Here we investigate a novel culture system for manufacturing osteochondral implants that also allows mechanical loading of the construct during in vitro culturing. Methods: Bovine articular chondrocytes from five individual cell isolations were cultured at high density (6106 cells) on top of defatted bone disks in an agarose well created into a centrifugation tube (A and B). A calcium phosphate layer was precipitated on the bone disk to prevent the chondrocytes from moving through the pores. The cultures were centrifuged 0, 1 or 3 times a day for 15 min. At 770 g, this program asserted a hydrostatic pressure of 2.9 MPa on the tissue constructs. Total mRNA and samples for glycosaminoglycan (GAG) and hydroxyproline content analysis were collected at 1, 2 and 4 weeks. Results: The centrifuged tissues appeared flatter and more uniform than the non-centrifuged controls. Although the total GAG content was slightly lower in the centrifuged tissue, there were no significant differences. Hydroxyproline measurement showed highest collagen content in the once and three times centrifuged tissue cultures. Gene expression analysis suggests higher type II procollagen expression in the non-centrifuged and once centrifuged tissue cultures in comparison with the three times centrifuged. On the other hand, the three times centrifuged samples had higher aggrecan expression in comparison to the non-centrifuged tissues. The type I procollagen/type II procollagen -ratio increased with time in all samples and was the highest in the three times centrifuged samples.
Conclusions: The culture system supports tissue growth and produces articular cartilage-like tissue. Both centrifugation regimes produced tissue that had a flatter and a more uniform surface (C) and the tissue also felt noticeably stiffer when handling, although the biomechanical properties still need to be analysed. There seems to be little to no benefit from centrifuging the samples three times a day, since tissues centrifuged once per day tissues maintained the expression of genes typical for hyaline cartilage formation. 630 MESENCHYMAL STEM CELLS-CONTAINING ALGINATE PARTICLES FOR INTRA-ARTICULAR INJECTION IN OSTEOARTHRITIS A. Smith y, M. Marquis z, C. Vinatier y, A. des Rieux x, k, D. Renard z, J. Guicheux y, ¶, C. Le Visage y. y INSERM UMR 1229-RMeS, Regenerative Med. and Skeleton, STEP Team, Univ. of Nantes, UFR odontology, Nantes eres Interactions Assemblages), F-44042, France; z UR1268 BIA (Biopolym INRA, 44300 Nantes, France; x Louvain Drug Res. Inst., Advanced Drug
Delivery and Biomaterials, Universit e Catholique de Louvain, 1200 Bruxelles, Belgium; k Inst. of Condensed Matter and NanoSci.s, Universit e Catholique de Louvain, 1048 Louvain-la-Neuve, Belgium; ¶ CHU Nantes, PHU 4 OTONN, Nantes F-44042, France Purpose: Osteoarthritis (OA) is a degenerative and inflammatory joint disease that affects cartilage, subchondral bone and joint tissues. Mesenchymal Stem Cells (MSCs) ability to secrete anti-inflammatory and immuno-modulatory factors represents an attractive tool in the treatment of osteoarthitis. Considering the risk of cell leakage and the massive cell death upon intra-articular injection, MSCs encapsulation therefore could prevent cell death, avoid cell effusion outside the articular space, and supply a suitable micro-environment supporting the biological activity of MSCs. Previously, we have demonstrated that alginate particles support MSCs viability and bioactivity. Nevertheless these particles obtained through a dropwise method had an average size of 1.5 ± 0.2 mm and were thus too large to be injected into joints. Here, we propose to develop a method of cell encapsulation in alginate compatible with intra-articular injection through a 26G needle. Methods: Alginate particles were generated through micromolding method. We first manufactured wafers patterned by soft lithography with a photocrosslinkable resin. The wafers had protruding 100 mm high patterns with square and circle shapes of 100, 150 and 200 mm of side or diameter. Polydimethylsiloxane (PDMS) molds were then generated by pouring 10% PDMS onto the wafers and curing it for 20 min at 70 C. Finally, the PDMS molds were peeled from the wafers and stored 1 night at 60 C. Before their use, the molds were hydrophilized by plasma treatment. A solution of 2% alginate was then deposited on the molds and alginate particles were crosslinked with an agarose gel charged with CaCl2. After manufacture, the size and shape of the microparticles were assessed using phase-contrast microscopy and digital imaging. To encapsulate cells, human adipose-derived mesenchymal stem cells (ASC) were suspended at 1 and 3 millions of cells per mL of the 2% alginate solution and deposited on PDMS molds containing 3200 circle wells with a diameter of 200 mm. Number of encapsulated cells were then determined by a PicoGreen assay. Results: We successfully obtained alginate microparticles reproducing the micromold shapes. Both cylindrical and cubic particles exhibited however a reduced size as compared to the original molds. For instance, cylindrical particles molded into 100 mm cycle molds had an average size of 73 ± 0.8 mm (n ¼ 3) and those molded from 150 and 200 mm diameter molds presented a size of 103 ± 0.7 and 136 ± 0.7 mm, respectively (n ¼ 3). Similarly, for cubic particles, we measured a size of 72 ± 0.7 mm, 107 ± 0.7 mm (n ¼ 3) and 137 ± 0.8 mm (n ¼ 3) for the particles molded into 100, 150 and 200 mm molds, respectively. We then selected the 200 mm cylindrical molds and performed cell encapsulation. The presence of cells into the alginate solution had no impact on the size or shape of the particles. Using optical microscopy, we easily identified that cells were homogeneously distributed within the transparent alginate particles. Preliminary results indicate that the number of encapsulated cells by particle depends on the initial cell concentration in the alginate solution. Conclusions: We first succeeded in generating alginate particles that could be injectable through a 26 G needle. In addition, micromoldingderived particles of alginate allowed the successful encapsulation of viable MSC. Our work will now focus on demonstrating the ability of encapsulated MSC to secrete immunomodulatory and anti-inflammatory factors. In a future step, we will consider intra-articular injection in an animal model of osteoarthritis. 631 IMMUNOMODULATORY EFFECT OF MESENCHYMAL STEM CELLS FOLLOWING INTRA-ARTICULAR INJECTION IN A MODEL OF OSTEOARTHRITIS: A POTENTIAL ROLE FOR APOPTOSIS P. Mancuso, M.J. Murphy, F. Barry. REMEDI (NUI Galway), Galway, Ireland Purpose: Mesenchymal stem cells (MSCs) promote regeneration when intra-articularly (IA) administered in pre-clinical models of osteoarthritis (OA). Their mechanism of action, however, is still poorly understood. Preliminary data show low engraftment of GFP-expressing syngeneic MSCs in mouse knee joints, correlating with published data using molecular techniques. As cells were not detected in other organs, we hypothesise that implanted cells may undergo apoptosis or cell death. There is growing evidence that apoptotic MSCs exert an antiinflammatory effect, by direct action on phagocytes or by release of immunosuppressive cytokines. This study aims to investigate the fate