DISCUSSION

Until this date, scaffolds based on collagen or caprolactone urethane (PU) have been developed to regenerate partial defects in human menisci13,20_{. These scaffolds}

are implanted without cells. Results are promising for partial defect repair, but not applicable for all patients. Furthermore, it is not possible to treat defects involving the rim of the meniscus with these scaffolds. To repair such a defect with a scaffold, it is worthwhile to invest the possibility to enhance the biological and mechanical properties by seeding cells prior implantation. These pre-cultured scaffolds are able to integrate with the host tissue stronger and faster23_{. To supply suffi cient volume for}

cells to grow and synthesize matrix a 95%, highly interconnective porous scaffold was used.

The fi rst aim of this study was to investigate the biological properties of cell seeded scaffolds. Scaffolds made from 50/50 P(D/L)LA / PCL with BDI were seeded with MFCs and cultured up to 6 weeks. This study shows that MFCs can be cultured on this scaffold. However, they did not proliferate and ECM synthesis did not increase in time. Also the number of cells in the center of the scaffold was limited. This could be due to the hydrophobic nature of the scaffold, or by a lack of nutrition in the center43_{. Another explanation could be that passage 4 MFCs lose the ability to}

proliferate or synthesize matrix.

To enhance biological response, 10 ng/ml TGF-β was added to standard medium. TGF-β is known to induce proliferation and matrix syntheses. Addition of TGF-β increased proliferation of MFCs cultured in these scaffolds. This effect of TGF-β is in line with results described by others30,44,45_{. Moreover, collagen and GAG, which are}

two main components of the meniscus ECM, also increased after TGF-β stimulation. Increase in matrix was not only due to an increase in cell numbers. As indicated with the picogreen assay total cell number was 3.75 times higher compared to non- stimulated cultures. Collagen synthesis was increased more than 15 times compared

Figure 5: The compressive Young’s modulus was determined by compressing the scaffolds till 20% at 1 mm/min in PBS at 37O_{C. No signifi cant decrease or}

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to non-stimulated cultures. This means that collagen production per cell was also strongly increased by TGF-β. In addition, this proves that passage 4 MFCs are still able to proliferate, if correct stimuli are presented to the cells. Which indicates that the aforementioned loss of proliferation is not the case. The result that MFCs will not proliferate in the scaffold, under normal culture conditions, points out that the surface characteristics of this scaffold is not bio-inductive for these cells. A study from Gunja et al. reported that environmental characteristic have a great effect on MFCs. They showed that with increasing passage number, up to passage 4, MFCs alter gene expressions for collagen and GAG’s. Reversal effects could be initiated if re-cultured on a collagen or GAG coated surface46_{. For future applications of this}

scaffold it could be advantageous to coat the scaffolds surface with bioactive markers such as collagen or GAG. This would improve correct gene expression, and in the end, lead to native like meniscal tissue.

The second aim of this study was not realized. The initial Young’s Modulus of the scaffold was 26 kPa and did not reduce significantly after 6 weeks of culture due to degradation. This short term degradation stability is in line with previous studies. In here it was shown that scaffolds remained physically intact up till 20 weeks47_.

The initial compressive stiffness measured is below that of human meniscal tissue. Others showed that native menisci vary in mechanical stiffness between 30 – 100 kPa depending on sample site48,49_{. Since cell proliferation and matrix deposition can}

increase stiffness, the cell seeded scaffolds were expected to have a higher stiffness50_.

The TGF-β stimulated group, which synthesized the most ECM, did not increase significantly in mechanical stiffness. However, the mean Youngs modulus of these cultures after 6 weeks was 43 kPa. Possibly, if culture times prolonges matrix synthesis will increase, thereby the Youngs modulus will increase further and could become similar as native meniscus tissue. On the other hand, perhaps initial cell density was too low. Many methods have been described to increase cell density. The most obvious would be to increase total number of seeding cells. Alternatively, optimizing seeding efficiency can increase initial cell density and distribution. Cell seeding techniques can be divided into static or dynamic systems. Examples of static systems are droplet and cell suspension51_{. Recently more dynamic seeding systems have been described}

which uses specially developed devices such as perfusion systems, spinner flasks of bioreactors51_{. These dynamic seeding techniques yield higher densities and better}

homogeneous cell distribution51_{. Also bioreactors can be promising to increase the}

biological properties. When dynamic mechanical loading is applied on cell seeded scaffolds, proliferation and matrix synthesize increase compared to static cultures52_.

Furthermore the produced matrix was unorganized while in the meniscus collagen fibers are highly oriented. Methods to increase biological properties further, could lead to more in vivo like mechanical properties. New methods to increase mechanical properties are in vivo cultures of cell seeded constructs. If pre-seeded scaffolds are cultured subcutaneously, the mechanical modulus becomes higher when compared to in vitro pre-seeded constructs53_{. On the other hand, scaffolds could be adapted to}

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In summary, meniscal fibrochondrocytes can be cultured in PU scaffolds made from poly D/L lactide and caprolactone and 1,4-butanediisocyanate. However, the scaffold did not permit proliferation of these cells. Addition of TGF-β will increase the biological properties significantly. The mechanical properties, however, remained similar. Further research needs to focus on changing the scaffolds properties to increase biological and mechanical behavior during culture. Methods to change these properties could be coating the scaffolds surface with biologic cues such as proteins. This can increase the cellular response during cell cultures.

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