Introduction: Tissue engineering (TE) strategies using acellular scaffolds for meniscus replacement have limitations given that final tissue is not similar to native meniscus. Advanced scaffolds for meniscus TE should be able to mimic biomechanics and preserve the asymmetric vascular network. This study aims to enhance knowledge of meniscus biology leading to development of a novel silk-fibroin scaffold and combine it with the methacrylated gellan gum hydrogel (iGG-MA) hydrogel, which is able to prevent the ingrowth of endothelial cells and blood vessels into the hydrogels.
Material and Methods: Morphologically intact menisci were collected from 44 human donors. All menisci (30 lateral and 14 medial) were divided into anterior, middle and posterior segments. Human meniscus cells (HMC´s) were isolated using an enzymatic standard protocol. Dynamic mechanical analysis (DMA) was performed using fresh tissue samples. HMC’s were seeded onto the different silk scaffolds. HMC´s adhesion, viability and proliferation were investigated by scanning electron microscopy (SEM), calcein-AM assay and DNA quantification tests. The mechanical properties of cell-loaded scaffolds were also evaluated by DMA.
Results: DMA analysis has shown that fresh medial meniscus has significantly higher stiffness (E' and Tan d) than lateral meniscus. There is also significant regional variation from anterior to posterior menisci segments regarding biomechanical features. Age, gender and bone mass index (BMI) also influences meniscus stiffness. HMC’s maintained their phenotype for 21 days when cultured in tissue culture polystyrene plate (2D). The micro-CT analysis revealed that the human freeze-dried meniscus possessed a mean porosity of 58.0±20.3%. HMC´s were viable and proliferated well when cultured onto both silk-10 and silk-12 scaffolds. DMA analysis has shown that the moduli of the acellular scaffolds were 27.6 ± 7.9 kPa and 61.1 ± 0.4 at 10 Hz, for silk-10 e silk-12, respectively. By its turn, the moduli determined at 10 Hz of the cell-laden scaffolds after 14 days of culturing were 48.2± 19.8 and 140.1 ± 15.6 kPa, for silk-10 and silk-12, respectively. The in vivo study have shown that iGG-MA hydrogel prevented the blood vessels infiltration into the HMC’s hydrogel/silk scaffolds, even in the presence of VEGF.
Conclusions: This study showed that silk scaffolds can support proliferation of HMC’s. Cells increased the biomechanical features of acellular scaffolds. Medial and lateral menisci present different biomechanical properties which are also influenced by age, gender and BMI. Silk scaffolds combined with the iGG-MA hydrogel enabled controlling the segmental vascularization. This study has contributed for developing a novel strategy based on combining silk scaffolds with hydrogels and cells, and possibly mimic the native vasculature architecture during meniscus regeneration.
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