RESUMO
Silk fibroin biomaterials are highly versatile in terms of materials formation and functionalization, with applications in tissue engineering and drug delivery, but necessitate modifications for optimized biological activity. Herein, a facile, avidin-based technique is developed to noncovalently functionalize silk materials with bioactive molecules. The ability to adsorb avidin to silk surfaces and subsequently couple biotinylated macromolecules via avidin-biotin interaction is described. This method better preserved functionality than standard covalent coupling techniques using carbodiimide cross-linking chemistry. The controlled release of avidin from the silk surface was demonstrated by altering the adsorption parameters. Application of this technique to culturing human foreskin fibroblasts (hFFs) and human mesenchymal stem cells (hMSCs) on arginine-glycine-aspartic-acid-modified (RGD-modified) silk showed increased cell growth over a seven-day period. This technique provides a facile method for the versatile functionalization of silk materials for biomedical applications including tissue engineering, drug delivery, and biological sensing.
Assuntos
Avidina/química , Fibroínas/química , Alicerces Teciduais/química , Adsorção , Células Cultivadas , Fibroblastos/efeitos dos fármacos , Fibroblastos/fisiologia , Humanos , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/fisiologia , Oligopeptídeos/química , Alicerces Teciduais/efeitos adversosRESUMO
In vitro liver models are necessary tools for the development of new therapeutics. HepaRG cells are a commonly used cell line to produce hepatic progenitor cells and hepatocytes. This study demonstrates for the first time the suitability of 3% silk scaffolds to support HepaRG growth and differentiation. The modulus and pore size of 3% silk scaffolds were shown to be within the desired range for liver cell growth. The optimal seeding density for HepaRG cells on silk scaffolds was determined. The growth and maturation of scaffolded HepaRG cells was evaluated for 28 days, where the first 14 days of culture were a proliferation period and the last 14 days of culture were a differentiation period using dimethyl sulfoxide (DMSO) treatment. After the first 14 days of culture, the scaffolded HepaRG cells exhibited increased metabolic activity and albumin secretion compared to monolayer cultured controls and preserved these attributes through the duration of culture. Additionally, after the first 14 days of culture, the scaffolded HepaRG cells displayed a significantly reduced expression of genes associated with hepatocyte maturation. This difference in expression was no longer apparent after 28 days of culture, suggesting that the cells underwent rapid differentiation within the scaffold. The functionalization of silk scaffolds with extracellular matrix (ECM) components (type I collagen and/or an arginylglycylaspartic acid (RGD)-containing peptide) was investigated to determine the impact on HepaRG cell attachment and maturation. The inclusion of ECM components had no noticeable impact on cell attachment but did significantly influence CYP3A4 expression and albumin secretion. Finally, the matrix support provided by the 3% silk scaffolds could prime the HepaRG cells for steatosis liver model applications, as evidenced by lipid droplet accumulation and expression of steatosis-related genes after 24 h of exposure to oleic acid. Overall, our work demonstrates the utility of silk scaffolds in providing a modifiable platform for liver cell growth.
Assuntos
Fibroínas , Fibroínas/farmacologia , Alicerces Teciduais , Fígado/metabolismo , Seda , Albuminas/metabolismoRESUMO
Lyophilization of protein solutions, such as silk fibroin (silk), produces porous scaffolds useful for tissue engineering (TE). The impact of modifying lyophilization primary drying parameters on scaffold properties has not yet been explored previously. In this work, changes to primary drying duration and temperature were investigated using 3%, 6%, 9%, and 12% (w/v) silk solutions, via protocols labeled as Long Hold, Slow Ramp, and Standard. The 9% and 12% scaffolds were not successfully fabricated using the Standard protocol, while the Long Hold and Slow Ramp protocols resulted in scaffolds from all silk solution concentrations. Scaffolds fabricated using the Long Hold protocol had higher Young's moduli, smaller pore Feret diameters, and faster degradation. To investigate the utility of the different lyophilized scaffolds for in vitro cell culturing, the HepaRG liver cell line was cultured in the 3% to 12% scaffolds fabricated using the Long Hold protocol. The HepaRG cells grown in 3% scaffolds initially had greater lipid accumulation and metabolic activity than the other groups, although these differences were no longer apparent by Day 28. The deoxyribonucleic acid content of the HepaRG cells grown in 3% scaffold group was also initially significantly higher than the other groups. Significant differences in gene expression by 9% scaffolded HepaRG cells (CK19, HNFα) were seen on Day 14 while significant differences by 12% scaffolded HepaRG cells (ALB, APOA4) were seen on Day 28. Overall, modifying the primary drying parameters and silk concentration resulted in lyophilized scaffolds with tunable properties useful for TE applications.
Assuntos
Fibroínas , Seda , Porosidade , Alicerces Teciduais , Temperatura , Engenharia Tecidual , LiofilizaçãoRESUMO
Development of a 3D, biomaterials-based model for clear cell renal cell carcinoma (ccRCC) would be advantageous for understanding disease progression in vitro. This study demonstrated the development of lyophilized silk scaffolds that mechanically match the experimentally determined Young's modulus for ex vivo ccRCC samples and normal kidney tissue. Scaffolds fabricated from silk solutions ranging from 3 to 12% (w/v) were evaluated through mechanical testing. Following mechanical characterization of ccRCC samples, it was demonstrated that 6% silk scaffolds mechanically matched ccRCC samples. No impact of pathological grade and stage on the calculated ccRCC modulus was observed and all tumors evaluated mechanically matched the 6% silk scaffold formulation. Stratifying tissue specimens based upon histological observations (e.g. evidence of high levels of collagen deposition) resulted in no significant differences between groups. To investigate the impact of a mechanically matched culturing environment on in vitro ccRCC disease characteristics a model ccRCC cell line, 786-O, was utilized. Scaffolded 786-O cells demonstrated increased lipid droplet accumulation, a hallmark of ccRCC, compared to standard two-dimensional (2D) culture conditions. Additionally, scaffolded 786-O cells demonstrated increased expression of genes associated with ccRCC aggressiveness (ex. VEGFA, TNF, and IL-6) or immune markers under investigation as therapeutic targets (ex. PDL1, CTLA4). Comparison with 786-O cells grown on non-mechanically matched scaffolds demonstrated that these improved ccRCC characteristics were driven by scaffold modulus. Overall, our findings support the use of silk scaffolds in replicating physiologic tumor behavior for clear cell renal cell carcinoma and provide a platform for investigating disease progression.
Assuntos
Carcinoma de Células Renais , Neoplasias Renais , Materiais Biocompatíveis , Proliferação de Células , Colágeno , Humanos , Seda , Alicerces TeciduaisRESUMO
Recent identification and isolation of suture stem cells capable of long-term self-renewal, clonal expanding, and differentiating demonstrate their essential role in calvarial bone development, homeostasis, and injury repair. These bona fide stem cells express a high level of Axin2 and are able to mediate bone regeneration and repair in a cell autonomous fashion. The importance of Axin2 is further demonstrated by its genetic inactivation in mice causing skeletal deformities resembling craniosynostosis in humans. The fate determination and subsequent differentiation of Axin2+ stem cells are highly orchestrated by a variety of evolutionary conserved signaling pathways including Wnt, FGF, and BMP. These signals are often antagonistic of each other and possess differential effects on osteogenic and chondrogenic cell types. However, the mechanisms underlying the interplay of these signaling transductions remain largely elusive. Here we identify Rap1b acting downstream of Axin2 as a signaling interrogator for FGF and BMP. Genetic analysis reveals that Rap1b is essential for development of craniofacial and body skeletons. Axin2 regulates Rap1b through modulation of canonical BMP signaling. The BMP-mediated activation of Rap1b promotes chondrogenic fate and chondrogenesis. Furthermore, by inhibiting MAPK signaling, Rap1b mediates the antagonizing effect of BMP on FGF to repress osteoblast differentiation. Disruption of Rap1b in mice not only enhances osteoblast differentiation but also impairs chondrocyte differentiation during intramembranous and endochondral ossifications, respectively, leading to severe defects in craniofacial and body skeletons. Our findings reveal a dual role of Rap1b in development of the skeletogenic cell types. Rap1b is critical for balancing the signaling effects of BMP and FGF during skeletal development and disease. © 2017 American Society for Bone and Mineral Research.