Quantifying Osteogenic Cell Degradation of Silk Biomaterials

Abstract

The degradation of silk protein films by human mesenchymal stem cells (hMSCs), osteoblasts and osteoclasts, cells involved in osteogenic functions in normal and diseased bone, was assessed in vitro. The involvement of specific matrix metalloproteinases (MMPs) and integrin signaling in the degradation process was determined. Scanning electron microscopy (SEM) and atomic force microscopy (AFM) were used to quantitatively compare degradation by the different cell types using surface patterned silk films. Osteoblasts and osteoclasts demonstrated significant degradation of the silk films in vitro in comparison to the hMSCs and the film controls without cells. The osteoclasts degraded the silk films the most and also generated the highest level of MMPs 1 and 2. The osteoblasts upregulated integrins α5 and β1 while the osteoclasts upregulated integrins α2 and β1. There was significant contrast in responses on the silk matrices between osteogenic cells vs undifferentiated hMSCs to illustrate in vitro the role of cell type on matrix remodeling. These are important issues in matching biomaterial matrix features and studies in vitro to remodeling in vivo, in both normal and disease tissue systems. Cell populations and niche factors impact tissue regeneration, wound healing and physiological state and the ability to better understand the role of different cell types is critical to overall regenerative outcomes.

Silk structure and degradation

“…Shang et al [25] found the membranous materials prepared with regenerated silk fibroin containing low amounts (17-18%) of ß-sheets were easily degraded in water after pretreatment with proteinase XIV and co-culturing with fibroblasts (human corneal fibroblasts). These conclusions are in agreement with our findings, because regenerated silk fibroin [4][5][6][7][8][9][10][11][12]14,15,19,20,25] has a different inner structure and degradation properties to that of natural silk fibroin. Furthermore, serum contains a variety of enzymes that play synergic and antagonist actions.…”

“…Pritcharda et al [10] prepared a sustained release system using regenerated silk fibroin, and investigated the effect of EDTA on inhibiting its degradation in the presence of proteinase XIV, thereby regulating its drug-release rate. Sengupta et al [20] explored the effect of steogenic cells on matrix metalloproteinases and the integrin signaling pathway in an in vitro degradation process of regenerated silk fibroin. Shang et al [25] found the membranous materials prepared with regenerated silk fibroin containing low amounts (17-18%) of ß-sheets were easily degraded in water after pretreatment with proteinase XIV and co-culturing with fibroblasts (human corneal fibroblasts).…”

“…As a natural biological material, the immunogenicity of silk is a topical issue for investigation [4][5][6][7][8][9][10][11][12][13][14][15]20]. The majority of studies have used regenerated fibroin, in which the main component is silk fibroin.…”

“…In the degradation process, cells play a significant role in both the in vitro and in vivo environments [13,20,25]. At present, only a few studies have focused directly on investigating the degradation mechanism of the materials or the role tissue solution and cells play during the in vivo degradation process.…”

“…Degradation of degummed silk [17,18], degummed silkprepared surgical sutures [19], or regenerated silk fibroin [1,2,[4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20]25,26] is attributed to the degradation of silk fibroin. While the degradation properties of silk fibroin have been the subject of intensive investigation, the degradation properties of raw silk remain unclear.…”