Mesenchymal stromal cells (MSCs) have been the object of extensive research for decades, due to their intrinsic clinical value. Mesangiogenic progenitor cells (MPCs), instead, were only recently discovered and characterized. They were firstly co-isolated, in different percentages, with MSCs in human adult bone marrow cultures applying autologous serum (Petrini et al., 2009); later, the establishment of specific culture conditions selective for MPCs allowed the isolation of these cells with a high grade of purity (>95%). Specifically, when human bone marrow mononuclear cells (hBM-MNCs) are cultured in basal medium supplemented with human serum (HS) on hydrophobic surfaces, it is possible to select slow-cycling MPCs after 6 days of culture (Trombi et al., 2009) because this was the only cell population present in the initial preparation capable of attaching on hydrophobic plastic dishes.

Phenotypically, MPCs are easily distinguishable from MSCs for their fried egg-shape morphology and peculiar immuophenotype characterized by the positivity to CD18, CD31 and nestin as well as negativity to the MSC markers as CD73 and CD90. Interestingly, as MPCs initially showed to retain mesengenic, cardiomyogenic, and angiogenic potential, these cells were firstly named “mesodermal” progenitor cells. Later on, the mesengenic potential was definitively demonstrated and finely described in two steps of differentiation, once cultured in specific pre-formulated media for MSC expansion. Namely, MPCs firstly differentiate into slow cycling CD90⁺/nestin⁺ cells (earlyMSCs), activating non-canonical Wnt signaling, that generate exponentially growing MSC-like cells (lateMSCs) after prolonged culture time (Fazzi et al., 2011). Similarly, angiogenic potential of MPCs was demonstrated. Also in this case the process involves two steps: first, MPCs are induced to VEGF-stimulated angiogenic sprouting; then they complete differentiation by forming capillary tube-like structures in 3D-cultures. Conversely, the cardiomyogenic potential was not definitively demonstrated, suggesting changing the MPC nomenclature into “mesangiogenic progenitor cells” (Montali et al., 2016) in spite of “mesodermal” progenitor cells. MPCs have been demonstrated deriving from a unique bone marrow cell population named Pop#8 (Pacini et al., 2016). This cell population has been sorted from adult human bone marrow as CD45^lowCD31^brightCD64^brightCD14^neg and showed similarities to monocytoid progenitors. Moreover, MPC morphology and phenotype partially resemble macrophages suggesting that these mesangiogenic progenitors and their in vivo counterparts (Pop#8) could belong to the hemopoietic compartment. Similarly, plasticity of peripheral monocytes and macrophages has been demonstrated. Some groups reported evidences showing that these cells could differentiate into fibroblast-like and collagen-producing cells called “fibrocytes” which participate in tissue repair, mainly sustaining fibrosis (reviewed in Bucala, 2015). Nonetheless, demonstration of genuine mesengenic potential (osteogenic, adipogenic, and chondrogenic) of fibrocytes is still lacking, restricting their tissue repairing potential to scar formation, while sustaining to angiogenesis has been reported for these cells but only as paracrine secretion of pro-angiogenic factors (Grieb et al., 2011). Moreover, fibrocytes derived from peripheral CD45^brightCD14⁺ monocytes also characterized for the expression of CD11b, C11c, CD13, and CD16 (Pilling et al., 2009), which have not been detected on bone marrow-derived CD45^lowCD14^neg Pop#8.

As MPCs can be considered progenitors for MSCs, they potentially represent a promising cell population to apply for skeletal tissue regeneration applications. Cell based medicinal products obtained by hBM-MNCs cultured in autologous serum were applied in the treatment of upper limb nonunions (Giannotti et al., 2013). In this paper, authors reported consistent percentages of MPCs (1–10%) within the expected MSC population, although not all of the conditions for MPC selection were complained. Nonetheless, it was suggested that the MPC fraction possibly contributed to the long-term healing reported in the treated patients. This hypothesis arose from the reported MPC plasticity suggesting that, once implanted, these cells could support osteogenesis differentiating into earlyMSCs, as well as contributing to the neo-vascularization of the engineered construct thank to their angiogenic potential.

In order to better investigate and exploit this possible therapeutic scenario in the field of orthopedics, further experiments should be performed in vitro in order to predict the MPC differentiation fate resulting from different physico-chemical stimulations that selectively mimic specific aspects of the in vivo micro-environments.

Bone tissue homeostasis represents a complex biological process finely regulated by humoral stimuli as hormones, growth factors and cytokines as well as by cell-cell and cell-matrix contact interactions (Florencio-Silva et al., 2015). Thus, optimal bone regenerative therapy should enhance mineralized tissue healing through enrichment of the bone defect with a micro/nanostructured matrix scaffold to support the wound, with cells that will give raise to osteoprogenitors and proper biochemical stimuli. Recently, a factor controlling the fate of many osteocompentent cells has been introduced, taking in consideration that micro- e nano-topography of the bone architectures could have a role in the regulation of the activity of the bone cells trough the activation of cellular mechanotransduction mechanisms mediated by adhesion molecules (Green et al., 1995; Zohar, 2012). In particular, this complex bone architecture resulted mainly sustained by collagen and hydroxyapatite (HA), which together form a highly aligned composite matrix that contribute to the toughness and strength of bone itself (Weiner et al., 1999; Kerschnitzki et al., 2011). Collagen triple helices are typically around 300 nm long and 1.5 nm in diameter (Weiner et al., 1999) conferring a linear topography to the bone structure at the nanoscale. Many studies reported the influence of nanotopography to the biology of osteoprogenitors (Dalby et al., 2007; McMurray et al., 2011; Janson et al., 2014) suggesting to control their differentiation applying nanostructured surfaces of orthopedic implants.

MPCs showed particular adhesion properties sustained by podosome-like structures, that were applied for the definition of a MPC selective culture method (Trombi et al., 2009). Further studies demonstrated that gelsolin-served F-actin podosomial structures were re-organizedn in paxillin-served F-actin stress fibers, during the mesengenic differentiation of MPCs (Pacini et al., 2013), suggesting that topographical stimuli could play a crucial role in the MPC fate.

The aim of this study is evaluating the effects of surface nanostructuring on MPCs, considering the possibility that the nanotopography alone can trigger the MPC differentiation toward the mesenchymal lineage. In particular, we exploited nanogratings to deliver a mechanical, directional stimulus by contact interaction to promote cell morphological polarization and stretching. Following this interaction, we studied the MPC-MSC transition by (i) analyzing the change in cell morphotype by immunostaining of the key cell-adhesion structures and confocal fluorescence microscopy, and (ii) quantifying the expression of cell-phenotype characterizing markers by flow cytometry.

Implant osteointegration represents the key of success in most of the orthopedic procedures. It has been defined as the formation of structural and functional interconnection between the implant and the host bone (Albrektsson et al., 1981). In order to promote osteointegration, many materials and new fabrication processes have been proposed to obtain a high porous implant surface on which the bone cells could adhere and growth (Agarwal and García, 2015). Nowadays, the gold standard is represented by devices made by trabecular metal, which can mimic the bone microstructure and make available an ideal space for cellular colonization and revascularization of the neoformed bone tissue. However, an important limit of the implants and materials currently used in orthopedic surgery is that they present micron-sized textures while the bone is a nanostructured tissue. Collagen and hydroxyapatite, for example, provide a unique nanostructured scaffold for proteins and bone cell interactions. For these reasons, nanomaterials have been proposed with the aim of improving surface properties and creating an environment more conducive for osteoblast function and bone ingrowth (Tran and Webster, 2009; Parchi et al., 2013). Sub-micrometer sized features on traditional implants can control protein absorption and decrease scar tissue growth, bacterial infection and promote appropriate tissue growth. Moreover, they can also guide cell differentiation through physical stimulation, by a process called mechanotrasduction in which physical forces are converted into biochemical signals that are the finally integrated to give specific cellular response (Duncan and Turner, 1995).

Substrate nanotopography has been demonstrated to affect the biology of MSCs in vitro and the possibility of controlling the phenotype of these cells by just physically modifying the substrate topography represents one of the most promising innovations in the field. However, the majority of the studies was conducted with bone marrow derived mesenchymal stromal cells (BM-MSCs). Very recently our group demonstrated that nanogratings, alternate lines of sub-micrometer sized ridges and grooves, can effectively polarize BM-MSCs by pure contact interaction (Antonini et al., 2014), and promote enhanced osteo-differentiation with respect to flat surfaces (Antonini et al., 2016). There is indeed a large consensus considering BM-MSCs as the progenitors of the skeletal tissue related cell lineages for their ability to differentiate into adipocytes, osteoblasts and chondroblasts (Keating, 2012). This study also shows that after interaction with nanograting, BM-MSCs retain their osteodifferentiation potential.

Nonetheless, BM-MSCs represent a heterogeneous cell population isolated in vitro, in which the composition and cell biology are strongly affected by the isolating and expanding procedures (Phinney, 2012). Alongside the variability related to donors (Deasy et al., 2007; Siegel et al., 2013) and culture methods (Sharma et al., 2014), multiple origins of BM-MSCs were hypothesized and supported by several experiments demonstrating that genuine MSC cultures can be obtained by different BM subpopulations in vivo and in vitro, (reviewed in Pacini, 2014).

Remarkably, the MSC frequency in human normal bone marrow has been estimated only about the 0.001 and 0.01% of the mononuclear cells. Conversely, the MPCs frequency, and its in vivo counterpart denominated Pop#8, results from two to three logs higher (Trombi et al., 2009; Pacini et al., 2016).

Therefore, it is reasonable to hypothesize that the effects exerted by the nanostructured surfaces on MPCs can more strongly affect implant osteointegration than those on very small cell populations such us “skeletal stem cells” (SSCs) (Bianco and Robey, 2015) or CD271⁺CD140^low/− (Li et al., 2016), today considered the bona fide ancestors of BM-MSCs in vivo. Our results indicate that nanogratings can promote MPC to MSC transition, suggesting an in vivo scenario in which osteointegration is possibly promoted following MPC to MSC transition and nanograting-driven MSC osteogenic differentiation.

It is known that the substrate directionality stimulus is optimally delivered to many kind of undifferenziated cells, including MSCs, which in turn elongate and align to the nanograting lines. This polarization occurs also at level of cytoskeleton fibers and, though to a lesser extent, of nuclei. In this context, an elegant mechanistic model was proposed suggesting that an increased binding of integrins to ECM proteins would lead to increased FAK recruitment to the adhesion plaque inducing downstream ERK-dependent differentiation (Yee et al., 2008) This model provides an insight into the mechanisms of focal adhesion-dependent differentiation that might apply also to the MPC-MSC transition, and shows that nanotopographical surface modifications may directly regulate stem cell differentiation. Substrate topography can, indeed, interfere with focal-adhesion maturation and shaping, which in turn reflects on cellular mechanical stress distribution and shaping.

Interestingly, here we have demonstrated that the MPC mesengenic differentiation could be induced by the solely interaction with the T2 NGs, in absence of any other external stimuli. This aspect opens an interesting issue regarding the heterogeneity of culture expanded MSCs. In fact, it has been hypothesized that expanding MSCs from unfractionated “crude” bone marrow cell suspensions, in uncontrolled open culture systems, could lead to significantly different cell products with unpredictable biological properties because of mild modifications in the culture determinants or even to environmental fluctuations during cell expansion (Pacini, 2014). Data presented here suggest that differences in the intrinsic nanotopography of the culture surfaces, correlated to the materials or to the production processes, should be consider an additional culture determinant that might increase the variability of the final product. Thus, it might be possible that some pre-clinical data collected on MSCs expanded in flasks from bone marrow are not predictive for the therapeutic value of the same cell population cultured in bioreactors (i.e., equipped with hollow fibers), as their proliferation and differentiation potential could be altered by the specific micro- and/or nanotopography of the contact surfaces.

Concluding, the seminal findings reported here sustain the fascinating hypothesis of a topographic control of progenitor cells fate by specific designs of nanostructured surfaces. This aspect is of particular interest in the case of multipotent progenitors as MPCs that, retaining both mesengenic and angiogenic potential, possess a high clinical appeal.

SA and MM: Conception and design, Data collection, assembly, analysis, and interpretation; EJ: Data collection, assembly, analysis, and interpretation; SM: Microfabrication of nanostrucutres; PP, SB, FP: Data collection, assembly, analysis, and interpretation; SP: Conception and design, Data collection, assembly, analysis, and interpretation. Manuscript writing; IP: Manuscript writing. MC: Conception and design, Data collection, assembly, analysis, and interpretation. Manuscript writing.

This work was partially funded by “Centro per l'Uso Clinico delle Cellule Staminali” (CUCCS) as part of the project “Impiego di cellule stromali mesenchimali di origine midollare nelle pseudoatrosi, cisti ossee di astragalo e osteotomie in plus delle ossa lunghe” (project number 539999_2014_Petrini_CUCCS).