Understanding mesenchymal stromal cells (MSCs) growth mechanisms in response to surface chemistries is essential to optimize culture methods for high‐quality and robust cell yields in cell manufacturing applications. Heparin (HEP) and collagen 1 (COL) substrates have been reported to enhance cell adhesion, growth, viability, and secretory potential in MSCs. However, the biomolecular mechanisms underlying the benefits of combined HEP/COL substrates are unknown. This work used HEP/COL bilayered surfaces to investigate the role of integrin‐HEP interactions in the advantages of MSC culture. The layer‐by‐layer approach (LbL) was used to create HEP/COL bilayers, which were made up of stacks of 8 and 9 layers that combined HEP and COL in an alternate arrangement. Surface spectroscopic investigations and laser scanning microscopy evaluations verified the biochemical fingerprint of each component and a total stacked bilayer thickness of roughly 150 nm. Cell growth and apoptosis in response to IC50and IC75levels of BTT‐3033 and Cilengitide, α2β1 and αvβ3 integrin inhibitors respectively, were evaluated on HEP/COL coated surfaces using two bone marrow‐derived MSC donors. While integrin activity did not affect cell growth rates, it significantly affected cell adhesion and apoptosis on HEP/COL surfaces. HEP‐ending HEP/COL surfaces significantly increased FAK‐ERK½ phosphorylation and endogenous cell COL deposition compared to COL, COL‐ending HEP/COL and uncoated surfaces. BTT‐3033 but not Cilengitide treatment markedly affected FAK‐ERK½ activity levels on HEP‐ending HEP/COL surfaces supporting a major role for α2β1 activity. BTT‐3033 treatment on HEP‐ending bilayers reduced MSC‐mediated macrophage inhibitory activity and altered the cytokine profile of co‐cultures. Overall, this study supports a novel role for HEP in regulating the survival and potency of MSCs via enhancing the α2β1‐FAK‐ERK½ signaling mechanism.
In this study, layer‐by‐layer coatings composed of heparin and collagen are proposed as an extracellular mimetic environment on nerve guide conduits (NGC) to modulate the behavior of Schwann cells (hSCs). The authors evaluated the stability, degradation over time, and bioactivity of six bilayers of heparin/collagen layer‐by‐layer coatings, denoted as (HEP/COL)6. The stability study reveals that (HEP/COL)6is stable after incubating the coatings in cell media for up to 21 days. The impact of (HEP/COL)6on hSCs viability, protein expression, and migration is evaluated. These assays show that hSCs cultured in (HEP/COL)6have enhanced protein expression and migration. This condition increases the expression of neurotrophic and immunomodulatory factors up to 1.5‐fold compared to controls, and hSCs migrated 1.34 times faster than in the uncoated surfaces. Finally, (HEP/COL)6is also applied to a commercial collagen‐based NGC, NeuraGen, and hSC viability and adhesion are studied after 6 days of culture. The morphology of NeuraGen is not altered by the presence of (HEP/COL)6and a nearly 170% increase of the cell viability is observed in the condition where NeuraGen is used with (HEP/COL)6. Additionally, cell adhesion on the coated samples is successfully demonstrated. This work demonstrates the reparative enhancing potential of extracellular mimetic coatings.
more » « less- PAR ID:
- 10493883
- Publisher / Repository:
- Wiley Blackwell (John Wiley & Sons)
- Date Published:
- Journal Name:
- Advanced Healthcare Materials
- Volume:
- 13
- Issue:
- 14
- ISSN:
- 2192-2640
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
More Like this
-
more » « less
Abstract -
Human mesenchymal stromal cells (hMSCs) are multipotent cells that have been proposed for the treatment of immune-mediated diseases. Culturing hMSCs on tissue culture plastic reduces their therapeutic potential in part due to the lack of extracellular matrix components. The aim of this study is to evaluate multilayers of heparin and poly(L-lysine) (HEP/PLL) as a bioactive surface for hMSCs stimulated with soluble interferon gamma (IFN‐γ). Multilayers were formed, via layer-by-layer assembly, with HEP as the final layer and supplemented with IFN-γ in the culture medium. Multilayer construction and chemistry were confirmed using Azure A staining, quartz crystal microbalance (QCM), and X-ray photoelectron spectroscopy. hMSCs adhesion, viability, and differentiation, were assessed. Results showed that (HEP/PLL) multilayer coatings were poorly adhesive for hMSCs. However, performing chemical crosslinking using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide (EDC/NHS) significantly enhanced hMSCs adhesion and viability. The immunosuppressive properties of hMSCs cultured on crosslinked (HEP/PLL) multilayers were confirmed by measuring the level of indoleamine 2,3-dioxygenase (IDO) secretion. Lastly, hMSCs cultured on crosslinked (HEP/PLL) multilayers in the presence of soluble IFN- γ successfully differentiated towards the osteogenic and adipogenic lineages as confirmed by Alizarin red, and oil-red O staining, as well as alkaline phosphatase activity. This study suggests that crosslinked (HEP/PLL) films can modulate hMSCs response to soluble factors, which may improve hMSCs-based therapies aimed at treating several immune diseases.more » « less
-
more » « less
Abstract The instructive capabilities of extracellular matrix components in progenitor cell differentiation have recently generated significant interest in the development of bioinspired materials for regenerative applications. Previously, a correlation was described between the osteogenic capabilities of nanoparticulate mineralized collagen glycosaminoglycan scaffolds (MC‐GAG) and an autogenous activation of small mothers against decapentaplegic ( Smad1/5) in the canonical bone morphogenetic protein receptor (BMPR) pathway with a diminished extracellular signal regulated kinase 1/2 (ERK1/2) activation when compared to nonmineralized collagen glycosaminoglycan scaffolds (Col‐GAG). This work utilizes a canonical BMPR inhibitor (dorsomorphin homologue 1, DMH1) and an inhibitor of the mitogen activated protein kinase/ERK kinase (MEK)/(ERK) cascade (PD98059) to characterize the necessity of each pathway for osteogenesis. While DMH1 inhibits runt‐related transcription factor 2 (Runx2) and bone sialoprotein II (BSPII) gene expression of primary human mesenchymal stem cells (hMSCs) on MC‐GAG, PD98059 inhibits BSPII expression on Col‐GAG independent of Runx2 expression. DMH1 inhibits mineralization on both Col‐GAG and MC‐GAG, however, PD98059 only inhibits mineralization on Col‐GAG. DMH1 inhibits both Smad1/5 phosphorylation and Runx2 protein expression, whereas PD98059 inhibits ERK1/2 and c‐Jun amino‐terminal kinase 1/2 (JNK1/2) phosphorylation without affecting Runx2. Thus, activation of the canonical BMPR signaling is necessary for osteogenic differentiation and mineralization of hMSCs on Col‐GAG or MC‐GAG. The MEK/ERK cascade, intimately tied to JNK activation, is necessary for Runx2‐independent osteogenesis on Col‐GAG, while completely dispensable in osteogenesis on MC‐GAG.
-
more » « less
Abstract Liver fibrosis is a wound healing process marked by excessive accumulation of extracellular matrix in the liver. A poly(rC)‐binding protein 2 (PCBP2) siRNA that reverses fibrogenesis in activated hepatic stellate cells (HSCs) has been recently discovered. However, targeted delivery of siRNAs to HSCs still remains a daunting challenge. Herein, a new strategy is developed to fabricate a multicomponent nanocomplex using siRNA/peptide nucleic acid (PNA) hybrid instead of chemically conjugated siRNA, thus increasing the scalability and feasibility of the siRNA nanocomplex for animal studies. The nanocomplex is modified with an insulin growth factor 2 receptor ‐specific peptide, which specifically binds to activated HSCs. The siRNA nanocomplex demonstrates a controllable size, high serum stability, and high cellular uptake in activated HSCs in vitro and in vivo. Anti‐fibrotic activity of the siRNA nanocomplex is evaluated in rats with carbon tetrachloride‐induced liver fibrosis. Treatment with the PCBP2 siRNA nanocomplex significantly inhibits the mRNA expressions of PCBP2 and type I collagen in fibrotic liver. The histology study reveals that the siRNA nanocomplex efficiently reduces the protein level of type I collagen and reverses liver fibrosis. The data suggests that the nanocomplex efficiently delivers the siRNA to fibrotic liver and produces a potent anti‐fibrotic effect.
-
more » « less
Abstract Collagen I interactions with integrins α1and α2are known to support human mesenchymal stem cell (hMSC) osteogenesis. Nonetheless, elucidating the relative impact of specific integrin interactions has proven challenging, in part due to the complexity of native collagen. In the present work, we employed two collagen‐mimetic proteins—Scl2‐2 and Scl2‐3— to compare the osteogenic effects of integrin α1versus α2signaling. Scl2‐2 and Scl2‐3 were both derived from Scl2‐1, a triple helical protein lacking known cell adhesion, cytokine binding, and matrix metalloproteinase sites. However, Scl2‐2 and Scl2‐3 were each engineered to display distinct collagen‐based cell adhesion motifs: GFPGER (binding integrins α1and α2) or GFPGEN (binding only integrin α1), respectively. hMSCs were cultured within poly(ethylene glycol) (PEG) hydrogels containing either Scl2‐2 or Scl2‐3 for 2 weeks. PEG‐Scl2‐2 gels were associated with increased hMSC osterix expression, osteopontin production, and calcium deposition relative to PEG‐Scl2‐3 gels. These data indicate that integrin α2signaling may have an increased osteogenic effect relative to integrin α1. Since p38 is activated by integrin α2but not by integrin α1, hMSCs were further cultured in PEG‐Scl2‐2 hydrogels in the presence of a p38 inhibitor. Results suggest that p38 activity may play a key role in collagen‐supported hMSC osteogenesis. This knowledge can be used toward the rational design of scaffolds which intrinsically promote hMSC osteogenesis. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 2594–2604, 2018.
