Mesenchymal stem cell adhesion but not plasticity is affected by high substrate stiffness

The acknowledged ability of synthetic materials to induce cell-specific responses regardless of biological supplies provides tissue engineers with the opportunity to find the appropriate materials and conditions to prepare tissue-targeted scaffolds. Stem and mature cells have been shown to acquire distinct morphologies in vitro and to modify their phenotype when grown on synthetic materials with tunable mechanical properties. The stiffness of the substrate used for cell culture is likely to provide cells with mechanical cues mimicking given physiological or pathological conditions, thus affecting the biological properties of cells. The sensitivity of cells to substrate composition and mechanical properties resides in multiprotein complexes called focal adhesions, whose dynamic modification leads to cytoskeleton remodeling and changes in gene expression. In this study, the remodeling of focal adhesions in human mesenchymal stem cells in response to substrate stiffness was followed in the first phases of cell–matrix interaction, using poly-ε-caprolactone planar films with similar chemical composition and different elasticity. As compared to mature dermal fibroblasts, mesenchymal stem cells showed a specific response to substrate stiffness, in terms of adhesion, as a result of differential focal adhesion assembly, while their multipotency as a bulk was not significantly affected by matrix compliance. Given the sensitivity of stem cells to matrix mechanics, the mechanobiology of such cells requires further investigations before preparing tissue-specific scaffolds.     

三维成球培养优化间充质干细胞的研究进展

间充质干细胞(mesenchymal stem cells,MSCs)源于发育早期的中胚层,因其来源广泛、具有多向分化潜能、低免疫原性和自我更新能力,在组织工程和再生医学应用中显示出巨大的潜力,也是当前基础研究和临床研究中应用最多的一类干细胞。然而,间充质干细胞的临床应用面临许多挑战,比如治疗所需细胞数量巨大,细胞质量存在异质性,细胞体内移植后存活率低,以及二维(two-dimensional,2D)贴壁培养导致间充质干细胞特征衰减等。三维(three-dimensional,3D)成球培养可以更好地模拟体内微环境,且大量的研究证明,3D成球培养增强了间充质干细胞的细胞存活和因子分泌能力,促进了干细胞特征维持、细胞迁移和血管生成,在临床医学领域具有广阔的应用前景。基于此,综述了体外3D成球培养的方法、3D成球培养优化的间充质干细胞的生物学特性及应用,并对3D成球培养未来的研究方向进行展望。     

Factors Ruling the Uptake of Silica Nanoparticles by Mesenchymal Stem Cells: Agglomeration Versus Dispersions,Absence Versus Presence of Serum Proteins

The results of a systematic investigation of the role of serum proteins on the interaction of silica nanoparticles (NP) doped in their bulk with fluorescent molecules (IRIS Dots, 50 nm in size), with human mesenchymal stem cells (hMSCs) are reported. The suspension of IRIS Dots in bare Dulbecco‐modified Eagle's medium results in the formation of large agglomerates (≈1.5 μm, by dynamic light scattering), which become progressively smaller, down to ≈300 nm in size, by progressively increasing the fetal bovine serum (FBS) content of the solutions along the series 1.0%, 2.5%, 6.0%, and 10.0% v/v. Such difference in NP dispersion is maintained in the external cellular microenvironment, as observed by confocal microscopy and transmission electron microscopy. As a consequence of the limited diffusion of proteins in the inter‐NP spaces, the surface of NP agglomerates is coated by a protein corona independently of the agglomerate size/FBS concentration conditions (ζ‐potential and UV circular dichroism measurements). The protein corona appears not to be particularly relevant for the uptake of IRIS Dots by hMSCs, whereas the main role in determining the internalization rate is played by the absence/presence of serum proteins in the extracellular media.     

Tailoring RGD local surface density at the nanoscale toward adult stem cell chondrogenic commitment

Arginine-glycine-aspartic acid (RGD) dendrimer-based nanopatterns on poly(L-lactic acid) were used as bioactive substrates to evaluate the impact of the RGD local surface density on the chondrogenic induction of adult human mesenchymal stem cells.During chondrogenic commitment,active extracellular matrix (ECM) remodeling takes place,playing an instructive role in the differentiation process.Although three-dimensional environments such as pellet or micromass cultures are commonly used for in vitro chondrogenic differentiation,these cultures are rather limited with respect to their ability to interrogate cells in cell-ECM interactions.In the present study,the nanopatterns of the tunable RGD surface density were obtained as a function of the initial dendrimer concentration.The local RGD surface density was quantified through probability contour plots for the minimum interparticle distance,constructed from the corresponding atomic force microscopy images,and correlated with the cell adhesion and differentiation response.The results revealed that the local RGD surface density at the nanoscale acts as a regulator of chondrogenic commitment,and that intermediate adhesiveness of cells to the substrates favors mesenchymal cell condensation and early chondrogenic differentiation.     

Enhanced mesenchymal stromal cell recruitment via natural killer cells by incorporation of inflammatory signals in biomaterials

An exacerbated inflammatory response questions biomaterial biocompatibility, but on the other hand, inflammation has a central role in the regulation of tissue regeneration. Therefore, it may be argued that an ‘ideal’ inflammatory response is crucial to achieve efficient tissue repair/regeneration. Natural killer (NK) cells, being one of the first populations arriving at an injury site, can have an important role in regulating bone repair/regeneration, particularly through interactions with mesenchymal stem/stromal cells (MSCs). Here, we studied how biomaterials designed to incorporate inflammatory signals affected NK cell behaviour and NK cell–MSC interactions. Adsorption of the pro-inflammatory molecule fibrinogen (Fg) to chitosan films led to a 1.5-fold increase in adhesion of peripheral blood human NK cells, without an increase in cytokine secretion. Most importantly, it was found that NK cells are capable of stimulating a threefold increase in human bone marrow MSC invasion, a key event taking place in tissue repair, but did not affect the expression of the differentiation marker alkaline phosphatase (ALP). Of significant importance, this NK cell-mediated MSC recruitment was modulated by Fg adsorption. Designing novel biomaterials leading to rational modulation of the inflammatory response is proposed as an alternative to current bone regeneration strategies.     

Poly(lactic acid) porous scaffold with calcium phosphate mineralized surface and bone marrow mesenchymal stem cell growth and differentiation

This work aims to modify the surface of a poly(lactic acid) (PLA) porous scaffold with calcium phosphate (CaP) via a simple solution-based technique, and to evaluate the effects of this modification on the responses of rat bone marrow mesenchymal stem cells (rBMMSCs). Under appropriate modification conditions involving stepwise-treatments in the Ca-and-P supersaturated solution under gentle agitation, a thin, poorly crystallized CaP layer was deposited. The BMMSCs derived from adult rats were shown to adhere quite well to the CaP-coated scaffold, and to proliferate actively with culturing time, although some down-regulation was noted with regard to the unmodified PLA scaffold. The osteogenic differentiation of rBMMSCs was significantly higher on the CaP-modified scaffold than on the unmodified scaffold, as confirmed by alkaline phosphatase (ALP) activity. Moreover, the expression of genes associated with bone, including collagen type I, osteopontin and bone sialoprotein, was stimulated better on the CaP-modified PLA scaffold. Based on these results, the currently used CaP-treatment was deemed effective in stimulating the osteogenic development of rBMMSCs on the PLA-based scaffold, and the CaP-treated PLA scaffold may be useful for future bone tissue engineering.