Culture and differentiation of human bone marrow-derived mesenchymal stem cells in poly (L-lactic acid) scaffolds

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1 Culture and differentiation of human bone marrow-derived mesenchymal stem cells in poly (L-lactic acid) scaffolds e-poster: P155 Congress: ICRS 2009 Type: Electronic Poster Topic: Basic Science / Scaffolds Authors: I. Izal Azcárate 1, P. Ripalda Cemboráin 1, P. Aranda 1, G. Mora 1, R. Escribano 1, H. Deplaine 2, J.L. Gómez-Ribelles 2, G. Gallego Ferrer 2, V. Acosta 3, I. Ochoa 3, J.M. García-Aznar 3, M. Doblaré 3, F. Prosper 1 ; 1 Pamplona/ES, 2 Valencia/ES, 3 Zaragoza/ES Keywords: Tissue engineering, Scaffolds, Stem cells Any information contained in this pdf file is automatically generated from digital material submitted to e-poster by third parties in the form of scientific presentations. References to any names, marks, products, or services of third parties or hypertext links to third-party sites or information are provided solely as a convenience to you and do not in any way constitute or imply ICRS s endorsement, sponsorship or recommendation of the third party, information, product, or service. ICRS is not responsible for the content of these pages and does not make any representations regarding the content or accuracy of material in this file. As per copyright regulations, any unauthorised use of the material or parts thereof as well as commercial reproduction or multiple distribution by any traditional or electronically based reproduction/publication method is strictly prohibited. You agree to defend, indemnify, and hold ICRS harmless from and against any and all claims, damages, costs, and expenses, including attorneys fees, arising from or related to your use of these pages. Please note: Links to movies, ppt slideshows and any other multimedia files are not available in the pdf version of presentations.

2 1. Abstract Introduction: The lack of inflammatory response and the feasibility for controlling mechanical, morphological and degradation properties are attractive features of poly (L-lactic acid) (PLLA) for cartilage repair. Our aim was to determine the biocompatibility between Mesenchymal Stromal Cells (MSC) and PLLA and their effect on the mechanical properties of the scaffolds. Methods and Materials: MSC were cultured in PLLA films to study adherence and proliferation. We loaded 10x106 and 2x10 6 MSCs into PLLA scaffolds (1 and 6 mm in thickness), either by injection or aspiration. Scaffolds were maintained in expansion or differentiation media for 21 days. Seeding efficiency and cell distribution and viability were analyzed after the seeding while chondrocyte differentiation was assessed at 21 days. Results: Results showed an adhesion of MSC to PLLA films of 27,0±0,15 and 37,7±8,7 % of seeded cells at 1 and 4 hours. Proliferation was detected, but ceases at day 10. An efficiency of 80,8±4,1 and 71,5±9,4 % for 1 and 6 mm scaffolds was detected after the seeding. Retaining of cells were more efficient in the 6 mm scaffolds, where cells were poorly spread. Nevertheless, cells homogeneously saturated 1 mm scaffolds. Final results show the production of matrix composed by collagens I, and X, that produced a 2,6-fold increase in the Young modulus. Conclusions: We have developed biocompatible PLLA scaffolds that can be efficiently loaded with MSCs. We also show that differentiation of MSCs can be successful in PLLA and that it modify the mechanical properties of the PLLA itself. 2. Purpose The aim of this work is to study the chondrogenic differentiation potential of human bone marrow-derived mesenchymal stem cells cultured in PLLA scaffolds and the influence of the seeding technique and scaffold architecture. 3. Methods and Materials Human bone marrow-derived MSC were first assayed for their capacity to differentiate to adipose, bone and cartilaginous tissue. Two sizes of PLLA derived scaffolds were used, both 6 mm in diameter and 1 and 6 mm in thickness. 6 mm thick scaffolds were seeded by inyection with 2 millions MSCs, while 1 mm scaffolds were seeded aspirating 10 millions MSC through the scaffold. Seeding efficiency were measured by quantification of DNA, and distribution and density by fluorescence immunostaining of beta-actin. Retention of cells inside the scaffold at 21 days was also studied. Differentiation of MSC inside the scaffolds was performed by culturing them suspended in chondrogenic medium (CM) containing TGF-beta3 during 21 days. Matrix production was assesed by Massons tricrome staining and fluorescence immunostaining of aggrecan and collagens type I, II and X. Finally the mechanical properties of the differentiated scaffolds (Young modulus) was determined performing unconfined compression tests. 4. Results 1. Differentiation potential of human MSC (figure 1). MSCs showed a potentiality to differentiate into adipose and bone tissue in monolayer, and to cartilage in micromass structure as it is shown in the figure. 2. Monolayer culture of human MSC on PLLA (figure 2) Results show that 27,0±0,15 % of cells seeded anchored to the surface of the material after 1 hour.

3 When allowed for 4 hours, adherence reached a 37,71±8,7 %. Cells bound to PLLA did proliferate as it is shown in the graph. Cell proliferation beggins on day 3, peaks at day 10 and finally decreases againg from day 10 to day 14, without reaching a confluent state in the well (data not shown). 3. Three dimensional culture of MSC in PLLA scaffolds (figure 3) Both seeding methods used resulted to be highly effective (80,6±4,1 and 71,5±9,4 % of cells for 6 and 1 mm thickness scaffolds respectively), but produced a considerably different distribution and density of cells. In 6 mm thick scaffolds, distribution of cells were highly restricted to the center of the scaffold (in the site of injection). On the other hand cells tend to spread over all the volume of 1 mm scaffolds. According to cell density, the poor distribution in 6 mm scaffolds combines with a high density, while a better distribution in 1 mm scaffolds, with a lower density. During a 21 days experiment, the quantity of DNA remains stable in 6 mm thick scaffolds but showed a progresive decrease in the 1 mm scaffolds. 4. Differentiation of MSC in PLLA scaffolds (figure 4) When scaffolds were cultivated in the presence of CM, cells appeared surrounded by extracellular fibers that were positively stained using massons trichrome. The chemical nature of these fibers was studied and resulted in a predominant immunostaining for collagen type X in both 1 and 6 mm thick scaffolds. Aggrecan staining was also positive for both sizes, but showed a more intense pattern in 6 mm scaffolds. To a lesser extent, matrix fibers were also composed by collagen type I. Finally, the mechanical properties (Young modulus) were calculated. During the culture in expansion medium, the modulus significantly decreased only for 6 mm scaffolds, but increased with chondrogenic medium for both sizes. Figure 1 Figure 1. Assesment of the differentiation potential of bone marrow-derived human MSCs. Cells were pelleted and cultured in micromass spheres using CM to asses chondrogenic potential. Photomicrography shows staining using toluidine blue of a representative sphere after 21 days in culture (A). Alternatively MSCs were cultured in monolayer in osteogenic and adipogenic medium during 21 days. Osteogenic differentiation was assesed using Alizarin Red and Alkaline Phosphatase staining (B), while adipogenic differentiation was shown using oil red staining (C).

4 Figure 2 Figure 2. Biocompatibility of PLLA to human MSCs. A total of cells were allowed to adhere to PLLA films in 96 well plates during 1 and 4 hours. Average of the percentage of cells adhered (n=3) was calculated using MTS staining and represented in (A). The same number of cells were seeded and allowed to proliferate in the films cultivated in expansion medium. The average number (n=3) of viable cells, estimated by MTS, at different time points shows the pattern of proliferation of MSCs in PLLA (B). Error bars: standard deviation

5 Figure 3 Figure 3. Three-dimensional culture of human MSCs in PLLA scaffolds. Two different protocols were used, injecting 2 millions of MSCs in 6 mm thick scaffolds and aspirating 10 millions in 1 mm thick scaffolds as it is indicated in the text. Images show the distribution of cells inside the scaffold for 6 (A) and 1 mm (B) scaffolds. (C) Seeding efficiency was calculated as the average of the quantity of DNA in homogeneized scaffolds (n=3) referred to the quantity in 2 or 10 millions of MSCs (for 6 and 1 mm scaffolds respectively). (D) The three dimensional distribution of MSCs inside scaffolds was studied calculating the average (n=3) of the the area occupied by MSCs in different sections of the scaffold. (E) Survival of MSCs in PLLA scaffolds was determined measuring the DNA content in a 21 days experiment. Average for every time point (n=3) is represented and referred to day 0. Error bars: standard deviation.

6 Figure 4 Figure 4. Chondrogecnic differentiation of MSCs in PLLA scaffolds. (A) Masson s trichrome staining of sections from 6 and 1 mm thick scaffolds showing the presence of matrix fibers. (B) Unconfined compression tests were used to determine the mechanical resistance of control and differentiated scaffolds compared. The average (n=5) is represented, compared with the same measurement in unseeded scaffolds (labeled as PLLA). (C) Immunofluorescence analysis of sections from 6 and 1 mm thick scaffolds used for the detection of Aggrecan, and Collagens type I, II and X. 5. Conclusions 1. Human bone marrow derived mesenchymal stem cells (MSC) have the potential to differentiat to chondrocyte phenotype when cultured in PLLA derived scaffolds as it is shown by the production of extracellular matrix (ECM). This matrix significantly improves the mechanical properties of the construct. 2. ECM produced by MSC in PLLA scaffolds differentiated suspended in chondrogenic medium is composed mainly by aggrecan and collagens type I and X. We suggest the lack of mechanical stimuli as the cause of the poor content in type II collagen. 3. The architecture of the scaffold and the seeding technique are issues that strongly influence the final construct, as it is shown by a higher production of aggrecan in 6 mm scaffolds (with a higher density of cells in the site of injection) and by the different mechanical behaviour observed in scaffolds used in the study. 6. References

7 Brittberg M, Lindahl A, Nilsson A, Ohlsson C, Isaksson O, Peterson L. Treatment of deep cartilage defects in the knee with autologous chondrocyte transplantation. New Engl J Med 1994;331: Timothy SH, Scott DG, Lars P. Treatment of patellofemoral articular cartilage injuries with autologous chondrocyte implantation. Oper Tech Sports Med 2002;10: Breinan HA, Minas T, Hsu HP, Nehrer S, Shortkroff S, Spector M. Autologous chondrocyte implantation in a canine model: Change in composition of reparative tissue with time. J Orthop Res 2001;19: Holtzer, H., Abbott, J., Lash, J., and Holtzer, A. (1960) The loss of phenotypic traits by differentiated cells in vitro, I. Dedifferentiation of cartilage cells. Proc Natl Acad Sci U S A 46, von der Mark, K., Gauss, V., von der Mark, H., and Muller, P. (1977) Relationship between cell shape and type of collagen synthesised as chondrocytes lose their cartilage phenotype in culture. Nature 267, Thorogood, P., Bee, J., and von der Mark, K. (1986) Transient expression of collagen type II at epitheliomesenchymal interfaces during morphogenesis of the cartilaginous neurocranium. Dev Biol 116, Mayne, R., Vail, M. S., Mayne, P. M., and Miller, E. J. (1976) Changes in type of collagen synthesized as clones of chick chondrocytes grow and eventually lose division capacity. Proc Natl Acad Sci U S A 73, Benya, P. D., and Shaffer, J. D. (1982) Dedifferentiated chondrocytes reexpress the differentiated collagen phenotype when cultured in agarose gels. Cell 30, Pittenger MF, Mackay AM, Beck SC, Jaiswal RK, Douglas R, Mosca JD, et al. Multilineage potential of adult human mesenchymal stem cells. Science 1999;284: Haynesworth SE, Goshima J, Goldberg VM, Caplan AI. Characterization of cells with osteogenic potential from human marrow. Bone 1992;13:81 8. Jaiswal N, Haynesworth SE, Caplan AI, Bruder SP. Osteogenic differentiation of purified, culture-expanded human mesenchymal stem cells in vitro. J Cell Biochem 1997;64: Mackay AM, Beck SC, Murphy JM, Barry FP, Chichester CO, Pittenger MF. Chondrogenic differentiation of cultured human mesenchymal stem cells from marrow. Tissue Engineering 1998;4: Young RG, Butler DL, Weberm W, Caplan AI, Gordon SL, FinkDJ. Use of mesenchymal stem cells in a collagen matrix for Achilles tendon repair. J Orthop Res 1998;16: Barry F, Boynton RE, Liu B, Murphy JM. Chondrogenic differentiation of mesenchymal stem cells from bone marrow: differentiation- dependent gene expression of matrix components. Exp Cell Res 2001;268: Toma C, Pittenger MF, Cahill KS, Byrne BJ, Kessler PD. Human mesenchymal stem cells differentiate to a cardiomyocyte phenotype in the adult murine heart. Circulation 2002;105:93 8. Langer, R., and Vacanti, J. P. (1993) Tissue engineering. Science 260, Bryant, S. J., and Anseth, K. S. (2002) Hydrogel properties influence ECM production by chondrocytes photoencapsulated in poly(ethylene glycol) hydrogels. J Biomed Mater Res 59, Lee, C. R., Breinan, H. A., Nehrer, S., and Spector, M. (2000) Articular cartilage chondrocytes in type I and type II collagen-gag matrices exhibit contractile behavior in vitro. Tissue Eng 6, Shastri, V. P., Martin, I., and Langer, R. (2000) Macroporous polymer foams by hydrocarbon templating. Proc Natl Acad Sci U S A 97, Freed, L. E., Marquis, J. C., Nohria, A., Emmanual, J., Mikos, A. G., and Langer, R. (1993) Neocartilage formation in vitro and in vivo using cells cultured on synthetic biodegradable polymers. J Biomed Mater Res 27, 11-23

8 7. Mediafiles Figure 1 Figure 1. Assesment of the differentiation potential of bone marrow-derived human MSCs. Cells were pelleted and cultured in micromass spheres using CM to asses chondrogenic potential. Photomicrography shows staining using toluidine blue of a representative sphere after 21 days in culture (A). Alternatively MSCs were cultured in monolayer in osteogenic and adipogenic medium during 21 days. Osteogenic differentiation was assesed using Alizarin Red and Alkaline Phosphatase staining (B), while adipogenic differentiation was shown using oil red staining (C). Figure 2 Figure 2. Biocompatibility of PLLA to human MSCs. A total of cells were allowed to adhere to PLLA films in 96 well plates during 1 and 4 hours. Average of the percentage of cells adhered (n=3) was calculated using MTS staining and represented in (A). The same number of cells were seeded and allowed to proliferate in the films cultivated in expansion medium. The average number (n=3) of viable cells, estimated by MTS, at different time points shows the pattern of proliferation of MSCs in PLLA (B). Error bars: standard deviation

9 Figure 3 Figure 3. Three-dimensional culture of human MSCs in PLLA scaffolds. Two different protocols were used, injecting 2 millions of MSCs in 6 mm thick scaffolds and aspirating 10 millions in 1 mm thick scaffolds as it is indicated in the text. Images show the distribution of cells inside the scaffold for 6 (A) and 1 mm (B) scaffolds. (C) Seeding efficiency was calculated as the average of the quantity of DNA in homogeneized scaffolds (n=3) referred to the quantity in 2 or 10 millions of MSCs (for 6 and 1 mm scaffolds respectively). (D) The three dimensional distribution of MSCs inside scaffolds was studied calculating the average (n=3) of the the area occupied by MSCs in different sections of the scaffold. (E) Survival of MSCs in PLLA scaffolds was determined measuring the DNA content in a 21 days experiment. Average for every time point (n=3) is represented and referred to day 0. Error bars: standard deviation.

10 Figure 4 Figure 4. Chondrogecnic differentiation of MSCs in PLLA scaffolds. (A) Masson s trichrome staining of sections from 6 and 1 mm thick scaffolds showing the presence of matrix fibers. (B) Unconfined compression tests were used to determine the mechanical resistance of control and differentiated scaffolds compared. The average (n=5) is represented, compared with the same measurement in unseeded scaffolds (labeled as PLLA). (C) Immunofluorescence analysis of sections from 6 and 1 mm thick scaffolds used for the detection of Aggrecan, and Collagens type I, II and X.