Comparative evaluation of in vivo biocompatibility and biodegradability of regenerated silk scaffolds reinforced with/without natural silk fibers

Nowadays, exceptional advantages of silk fibroin over synthetic and natural polymers have impelled the scientists to application of this biomaterial for tissue engineering purposes. Recently, we showed that embedding natural degummed silk fibers in regenerated Bombyx mori silk-based scaffold significantly increases the mechanical stiffness, while the porosity of the scaffolds remains the same. In the present study, we evaluated degradation rate, biocompatibility and regenerative properties of the regenerated 2% and 4% wt silk-based composite scaffolds with or without embedded natural degummed silk fibers within 90 days in both athymic nude and wild-type C57BL/6 mice through subcutaneous implantation. In all scaffolds, a suitable interconnected porous structure for cell penetration was seen under scanning electron microscopy. Compressive tests revealed a functional relationship between fiber reinforcement and compressive modulus. In addition, the fiber/fibroin composite scaffolds support cell attachment and proliferation. On days 30 to 90 after subcutaneous implantation, the retrieved tissues were examined via gross morphology, histopathology, immunofluorescence staining and reverse transcription-polymerase chain reaction as shown in Figure 1. Results showed that embedding the silk fibers within the matrix enhances the biodegradability of the matrix resulting in replacement of the composite scaffolds with the fresh connective tissue. Fortification of the composites with degummed fibers not only regulates the degradation profile but also increases the mechanical performance of the scaffolds. This report also confirmed that pore size and structure play an important role in the degradation rate. In conclusion, the findings of the present study narrate key role of additional surface area in improving in vitro and in vivo biological properties of the scaffolds and suggest the potential ability of these fabricated composite scaffolds for connective tissue regeneration

Evaluation of multipotency and chromosomal stability of isolated MenSCs <i>versus</i> BMSCs.

<p>(A) MenSC and BMSCs differentiation into osteoblasts (ii), chondrocytes (iii) and adipocytes (iiii) judged by Alizarin red staining, immuostaining of Collagen type II and Oil red O staining, respectively; Scale bar: 100 µm. (B) Chromatograms illustrating no chromosomal aberrations in MenSCs at passage 12 compared to cells at passage 2. GeneMarker plots showing results of MLPA analysis. Green lines illustrated the upper and lower limits of acceptable ranges of variations in MLPA analysis. Green dots show the chromosomal locations which are balanced and the red dots in the upper side of the plots show chromosomal gain and red dots in lower side of the plots show chromosomal loss.</p

Schematic diagram of three-stage differentiation protocols.

<p>Both MenSCs and BMSCs were sequentially treated by three combinations of cytokines, growth factors, and hormones under commitment, differentiation and maturation steps.</p

Phenotypic characterization of the MenSCs and BMSCs.

<p>(A) Morphological appearance of cultured MenSCs and BMSCs at passage 1; Scale bar: 100 µm. (B) Representative histograms of MenSC and BMSCs immunophenotyping by flow cytometry. CD markers are demonstrated in gray and the respective isotype control is shown as colorless. The results are presented as median (range) of 3–5 independent experiments. (C) Immunofluorescence staining of OCT-4, vimentin and GFAP in cultured MenSCs and BMSCs. Scale bar: 100 µm.</p

Immunofluorescent staining of differentiated MenSCs and BMSCs.

<p>Expression of ALB in differentiated MenSCs and BMSCs (A) and expression of CK-18 (B) and AFP (C) in differentiated MenSCs was examined by immunofluorescence staining with DAPI nuclear staining; scale bar: 100 µm. As negative control, cells were treated in parallel with the mouse irrelevant IgG2a for ALB and AFP and IgG1 for CK-18. Human HepG2 hepatoma cells were considered as positive control.</p

Sequences of the primers used for analysis of cells differentiation into hepatocyte-like cells.

<p>ALB: Albumin, CK-18: Cytokeratin-18, CK-19: Cytokeratin-19, CYP7A1: Cytochrome P450 7A1, TAT: Tyrosine aminotransferase, GAPDH: Glyceraldehyde 3-phosphate dehydrogenase.</p

Quantitative RT-PCR results of differentiated cells using three protocols.

<p>(A) Data of differentiated MenSCs and BMSCs were normalized to corresponding GAPDH and calculated in reference to undifferentiated cells. Each bar in each differentiation protocol represents the gene expression in ratio to undifferentiated cells (the first three pairs of bars). The second three pairs of bars represent comparisons of a given marker expression between two protocols in each stem cell. † indicates significant difference between differentiated and undifferentiated status of the same stem cell (<i>P</i><0.05), ‡ indicates significant difference (<i>P</i><0.05) between MenSCs and BMSCs. (B) Relative gene expression of differentiated cells compared to isolated adult hepatocytes. Results are shown as % of hepatocyte expression level.</p

Functionality characteristics of differentiated MenSCs compared to BMSCs.

<p>(A) ALB levels (ng/ml/48 h) in cell supernatants at days 0, 10, 15, and 25 of differentiation. † indicates significant difference between specified day and the previous time period of differentiation in the same stem cell (<i>P<0.05</i>), ‡ indicates significant difference (<i>P<0.05</i>) between differentiated MenSCs and BMSCs at last day of differentiation. (B) PAS staining of glycogen storage in fetus liver and HepG2 as positive control, undifferentiated and differentiated MenSCs and BMSCs by various protocols (P1–P3). (C) Expression pattern of Cytochrome P450 7A1 (<i>CYP7A1</i>) in reference to GAPDH in differentiated MenSCs and BMSCs by various protocols. Undif: undifferentiated cells, W: water.</p

<i>In vivo</i> assay of tumorigenicity and immunological reaction of MenSCs.

<p>(A) 2×10⁶ cells were subcutaneously injected in the dorsolateral part of the flank of nude mice (i), No tumor formation was observed in treated mice (ii), The excised tissues were fixed in buffered formalin, embedded in paraffin, and sectioned in 5-µm sections (iii). (B) The sectioned tissues were evaluated using H & E staining. (C) Immunoreactivity of mice sera to cultured MenSCs was evaluated using immunofluorescence staining. The cells (2×10⁴ cells per slide) were fixed in acetone at −20°C for 5 min and were incubated for 1 hour at 4°C with mice sera. Subsequently, the cells were washed three times with PBS and incubated with FITC-labeled sheep anti-mouse IgG at RT for 45 min in the dark. DAPI was used for nuclear staining.</p

Morphology of MenSCs compared to BMSCs during differentiation by three protocols.

<p>The gradual change of MenSC morphology compared with BMSCs under each differentiation protocol (P1–P3) has been shown by phase contrast photographs. Scale bar: 100 µm.</p