Nanotechnology to Drive Stem Cell Commitment

Review

Nanotechnology to drive stem cell commitment

Stem cells (SCs) are undifferentiated cells responsible for the growth, homeostasis and repair of many tissues. The maintenance and survival of SCs is strongly influenced by several stimuli from the local microenvironment. The majority of signaling molecules interact with SCs at the nanoscale level. Therefore, scaffolds with surface nanostructures have potential applications for SCs and in the field of regenerative medicine. Although some strategies have already reached the field of cell biology, strategies based on modification at nanoscale level are new players in the fields of SCs and tissue regeneration. The introduction of the possibility to perform such modifications to these fields is probably due to increasing improvements in nanomaterials for biomedical applications, as well as new insights into SC biology. The aim of the present review is to exhibit the most recent applications of nanostructured materials that drive the commitment of adult SCs for potential clinical applications.

1,2 Keywords: adipogenesis n adipose-derived stem cells n adult stem cells Eriberto Bressan , n differentiation n mesenchymal stem cell n nanoparticles n nanotechnology Amedeo Carraro3, Letizia n nanotubes n neurogenesis n osteogenesis Ferroni4, Chiara Gardin4, Luca Sbricoli1, Riccardo Nanotechnology represents a fascinating new SCs and are responsible for the generation of Guazzo1, Edoardo outlook on regenerative medicine that could cells with different properties. These cells can Stellini1, Marco Roman5, promote extensive research and lead to the either multiply (progenitors or transit amplify- Paolo Pinton2, Stefano realization of interesting and innovative tools ing cells) or be committed to terminal differ- Sivolella1,2 & Barbara [1,2] to improve and restore tissue function . entiation. Progenitors and transit amplifying 4 Worldwide interest in both adult stem cells cells have a limited lifespan and, therefore, can Zavan* (SCs) and embryonic SCs in the fields of tis- only reconstitute a tissue for a short period of 1Department of Neurosciences, University of Padova, Via Venezia 90, sue engineering and regenerative medicine has time when transplanted. By contrast, SCs are 35100 Padova, Italy 2 grown tremendously in recent years [3,4]. SCs self-renewing and, therefore, can generate any Department of Experimental & Diagnostic Medicine, Section of have been identified as having the potential tissue for a lifetime. This is a key property for a General Pathology, Interdisciplinary capacity to replace cells that are damaged or successful therapy. The capacity to expand SCs Center for the Study of Inflammation (ICSI) and LTTA Center, University of diseased and to restore vital functions, mak- in culture is an indispensable step for regen- Ferrara, Ferrara 44100, Italy ing them key players in tissue regeneration. erative medicine, and a considerable effort has 31st General Surgery, Department of Furthermore, the decreased immunogenicity been made to evaluate the consequences of General Surgery & Odontoiatrics, University Hospital of Verona, P.le A. and potential ‘immunomodulatory’ properties the cultivation on SC behavior [8]. Classically, Stefani 1, 37126, Verona, Italy that have been observed in various populations the control of SC fate either in vivo or in vitro 4Department of Biomedical Sciences, University of Padova, Via G. Colombo 3, of adult SCs may also facilitate allogenic trans- has been attributed to genetic and molecular 35100 Padova, Italy plantation, providing advantageous sources for mediators (e.g., growth factors or transcrip- 5IDPA-CNR, Institute for the Dynamics of Environmental Processes, Calle cell-based therapies [3–5]. Recent insights into tion factors). However, increasing evidence Author ProofLarga S. Marta 2137, 30123, Venice, the multilineage potential and inherited plastic- has revealed that a different array of additional Italy ity of progenitor cells have also created oppor- environmental factors, which belong to the *Author for correspondence: Tel.: +39 827 6096 tunities, dictated by an increased need for new ‘nanodimension’, may contribute to the overall Fax: +39 827 6079 cell-based therapies, to enable the regulation control of the activity of SCs; this may herald [email protected] of cell growth, differentiation and phenotypic the advent of new perspectives in biomedical expression through the modulation of SCs [6,7]. research [9–11] . The integration of nanotechno- A SC is defined as a cell that can continuously logical biomimetic materials and translational produce unaltered daughters and, furthermore, medicine could provide the chance to produce has the ability to generate cells with different surfaces (e.g., bone, vasculature, heart tissue, and more restricted properties. SCs can divide cartilage, bladder tissue and brain tissue), struc- either symmetrically (allowing an increase in tures and systems with nanoscale features that SC number) or asymmetrically. Asymmetric can mimic the natural cellular environment divisions maintain the number of unaltered and quickly promote cellular events, such as part of

adhesion, mobility and differentiation [12–14]. recruit numerous proteins such as FAK, vincu- Further improvements, stemming from the lin, paxillin, talin and p130Cas, among others optimization of nanomaterials by the continu- [18]. The process of this concentration and the ous introduction of nanotechnology platforms, topography of cell-adhesion sites in the ECM will boost the development of innovative cell- are critical to integrin clustering and activation based therapeutics. The aim of this review is to (Figures 2 & 3). For this reason, parameters includ- evaluate the current strategies and the emerging ing size, lateral spacing, surface chemistry and applications of nanotechnology and its goals in the geometry of nanofeatures are important SC and regenerative medicine research. variables that guide SC behavior [19–21] . Moreover, Seo et al. demonstrated, after cul- Commitment by nanopatterned turing bone marrow murine mesenchymal SCs substrates (MSCs) on both flat and microscale or nanoscale Extracellular environment & cell patterned topographies, that the formation and adhesion maturation of FAs is highly dependent on the The selection of a good-quality scaffold is an topography of the substrate, while the shape, essential strategy for tissue engineering. Ideally, morphology and spreading of cells on different the scaffold should be a functional and structural substrates were not significantly different[22] . platform able to mimic the native extracellular The preconditioning of cellular function at matrix (ECM) and support the morphogenesis nanostructured interfaces may result from direct of multiple tissues; on this basis, 3D nanofi- influence on cellular responses or an altered brous scaffolds appear to be the most capable of ECM layer deposited on the surface and a con- influencing cellular behavior. Nanotechnology, sequent change in the availability of binding as defined by the US National Nanotechnology sites [23,24]. Initiative (US NNI 2010), involves “structures With the inherent plasticity and multilineage with dimensions between approximately 1 and potential provided by SCs comes an increased 100 nanometers.” We can assume that all stud- need for regulating cell differentiation, growth ies focused on the interactions between cells and phenotypic expression. Classically, the con- and nanoscale materials are nanotechnology, trol of SC fate, either in vivo or in vitro, has been and their further in vivo applications can be attributed principally to genetic and molecular consequently called nanomedicine [15] . mediators (e.g., growth factors and transcrip- The interactions between cells and ECM tion factors). However, increasing evidence components strongly influence cell growth, has revealed that a diverse array of additional guide cell mobility and differentiation, and environmental factors contribute to the overall affect general cellular behavior; as a conse- control of SC activity. In particular, fascinating quence, cell–substratum interaction maintains data continue to mount on the important influ- a central role in many biological phenomena ence of the ‘solid-state’ environment, in other (Figure 1). Knowledge of these interactions is cru- words, the influence ECM has on SC fate, with cial to the understanding of many fundamental particular emphasis on the interactions of ECM biological questions and to the design of medical ligands with cell surface receptors [25]. However, devices. The complex structures of soluble and it is now clear that ECM‑based control of the immobilized biomacromolecules in the ECM, cell may also occur through multiple physical including collagens, glycoproteins and glyco mechanisms, such as ECM geometry at the Authorsaminoglycans, range from several to hundreds Proof microscale and nanoscale, and ECM elastic- of nanometers in size. For example, collagens ity or mechanical signals transmitted from the have a hierarchical structure composed of fibrils ECM to the cells. In addition to the influence ranging from 10 to 300 nm in size to collagen that an artificial ECM may have on cell shape, fibers that can be up to several microns in size. there is significant evidence that other physical At the other end of the spectrum, the base- properties of the ECM may also contribute to ment membrane includes a complex mixture SC fate or lineage commitment [26–28]. Cells that of pores, ridges and fibers that are nanometers attach to a substrate have been shown to exert in size [16] . Cell adhesion to the ECM is medi- contractile forces, resulting in tensile stresses in ated by important transmembrane proteins the cytoskeleton [29]. Interestingly, the relation- called integrins. Cell spreading determines the ship between these forces and the mechanical clustering of integrins into focal adhesion (FA) stiffness, or elasticity, of the ECM can have a complexes and the activation of intracellular major influence on cell behaviors, such as migra- signaling cascades [17] . In turn, FA complexes tion [30,31], apoptosis [32] and proliferation [33].

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Soluble factors Extracellular matrix Mechanical forces Migration Cell–cell interaction

Self renewal Differentiation Apoptosis

Figure 1. Interactions between stem cells and the extracellular environment. Soluble factors (e.g., growth factors and hormones) and extracellular matrix components (e.g., collagen, fibronectin and laminin) mechanically stimulate and influence cell growth, apoptosis and differentiation, and affect the general behavior of cells.

The mechanisms by which nanotopographic manipulation of SCs [39–43].. Typically, SCs cul- cues influence SC proliferation and differentia- tured on nanofiber scaffolds differ in morphol- tion are not well studied, but appear to involve ogy, viability and migration behavior compared changes in cytoskeletal organization and struc- with cultures grown on conventional substrates. ture, potentially in response to the geometry For example, human MSCs (hMSCs) grown on and size of the underlying features of the ECM 500–1000-nm nanofibers are flatter and dem- [34]. That is, changes in the feature size of the onstrate significantly higher cell viability and substrate may influence the clustering of inte- lower cell mobility than control cells grown on grins and other cell adhesion molecules, thus tissue culture polystyrene [44]. Nanofiber scaf- altering the number and distribution of FAs. For folds offer great potential for SC applications, example, previous studies have demonstrated a fact that is also supported by recent studies that the precise spacing between nanoscale demonstrating the responses of mammalian adhesive islands on a substrate can modulate cells to nanoscale surface stimuli [45,46,47]. An the clustering of the associated integrins, and application of such approaches is important in the formation of FA and actin stress fibers, and studies of hair cells. The biological hair cell is can, therefore, control the adhesion and spread- a modular building block of a rich variety of ing of cells [31] . These studies and others clearly biological sensors. Liu et al., using micro- and demonstrate that physicalAuthor interactions with nano-fabrication technology,Proof developed an the ECM significantly influence SC behavior, equivalent artificial hair cell sensor, imitating the thanks to the interaction with chemical (i.e., structure and transfer function of the biological composition), molecular (i.e., soluble mediators) hair cell. The artificial hair cells can be made or genetic (cell-type) factors to regulate cell fate. of hybrid semiconductor, metal and polymers Importantly, the ability to engineer artificial [48]. The paper discusses a number of strategies, ECMs that, through physical as well as molecu- using representative material systems, for build- lar interactions, enable directed control of SC ing artificial hair cell sensors and briefly outlines behavior, may further extend our capabilities in the fabrication method and performance. The engineering functional tissue substitutes [34–38]. motivation for imitating the biological hair cell is also discussed to provide a background for this Nanofibers work. In this context, Schroeder et al. imple- Nanotechnology is also capable of enhancing mented artificial cilia on giant magnetoresis- the reparative potential of tissue without direct tive multilayer sensors for a biomimetic sensing

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Extracellular matrix

α β Integrins

Fak Tln Src Pxn Tns Vcl Actn Focal contact

Cytoskeleton

Nucleus

Figure 2. Integrin signaling. Following the integration of integrins and extracellular matrix components, the intracellular signaling pathways triggered by integrins are directed to several functions: for example, organization of the actin cytoskeleton; regulation of the fate of the cell mechanosensing; adhesion; migration; and tissue invasion. Integrins are catalytically inactive and translate positional cues into biochemical signals by direct and/or functional association with intracellular adaptors, cytosolic tyrosine kinases or growth factors and cytokine receptors. The attachment of the cell takes place through the formation of cell-adhesion complexes, which consist of integrins and many cytoplasmic proteins, such as TLN, VCL, PXN and ACTN. These act by directing kinases, such as FAK and Src kinase family members to phosphorylate substrates such as p130Cas. These adhesion complexes attach to the actin cytoskeleton. The integrins therefore serve to link two networks across the plasma membrane: the extracellular matrix and the intracellular actin filamentous system.

approach using polypyrrole-based nanowires [49]. via the application of a high electric field and the The arrays were tagged with a magnetic material, presentation of a grounded region some distance the stray field of which changes relative to the away [54–57]. underlying sensor as a consequence of mechanical stimuli that are delivered by a piezoactua- Nanopatterned surfaces tor. The principle resembles balance sensing in A crucial element of tissue engineering is to cre- mammals. Measurements of the sensor output ate a favorable extracellular microenvironment, voltage suggest a proof of concept at frequencies mainly the ECM, to guide cell differentiation of approximately 190 kHz and a tag thickness and tissue regeneration [58,59]. of approximately 300 nm. Characterization was In addition to topography, the extracellular performed by scanning electron microscopy and microenvironment may also provide signal- Authormagnetic force microscopy, and micromagnetic Proof ing cues to the anchorage-dependent cells via and finite-element simulations were conducted a feedback of local matrix stiffness [60]. Matrix to assess basic sensing aspects. elasticity can direct hMSCs to differentiate With regard to nanofibers, there are currently into specific lineages: a soft matrix induces three manufacturing approaches to fabricating a neurogenic phenotype, while increasingly nanofibrous scaffolds: electrospinning [50], phase stiffer matrices induce myogenic and osteogenic separation [51] and self-assembly [52]. Structures phenotypes accordingly [61]. Taken together, the created by each of these approaches are quite observations of nanotopography-induced and different and, therefore, have their own unique stiffness-directed differentiation suggest that advantages. For example, the phase separation physical interactions between the cells and the technique allows for control of pore architectures extracellular environment, either in the form [53]. The most common method for fabricating of topography or stiffness, or a combination nanofibers is electrospinning. In this process, of these can, therefore, modulate cell function nanofibers are produced from polymer solutions and SC differentiation [61] . The application

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of nanotechnology to cell surfaces involves a application will require 3D structures. In this number of different arrangements. Most of all, review, the authors describe three such struc- a great variety of techniques are used to pro- tures: nanotubes, nanoparticles and nanofibers, duce nanotopographies on biomaterial surfaces. and their effect on biological regulation. Methods leading to ordered topographies with regular, controlled patterns and methods lead- Commitment based on SC surface ing to unordered topographies with random interaction orientations and organization have both been Osteogenic commitment developed. These in turn can be divided into There is currently an unmet need for the supply of chemical and physical processes. autologous, patient-specific SCs for regenerative Chemical modifications involve chemical reactions where parameters such as tempera- Nanopattern Unpatterned control ture, duration and composition of solutes can be adjusted to improve upon the number and depth of nanopits produced. Nanosurfaces are obtained through anodic oxidation or a combination of acids (and bases) and oxidants. Physical methods generate porous layers through colli- sions with microscopic particles (blasting) or by coating with small particles (plasma spray). In some cases, a combination of chemical and physical methods has also been used [62–64]. For example, the combination of particle blasting and hydrofluoric acid treatment has been used to create a commercial endosseous Ti implant with microrough surfaces and superimposed uncharacterized features ranging in size from 50 to 200 nm. Nanoscale features are able to orient cells, control cell spreading by limiting the surface area available for cell attachment, and modulate FA patterns and resultant stress fiber organization. For example, Teixeira et al. demonstrated that epithelial cell morphology was dictated by PDMS precisely controlled nanogroove and nanoridge patterns [51] . The nanotopographical surface was created with 400–4000-nm wide pitches and 150–600-nm deep grooves, and coated with silicon oxide to eliminate any effect from surface chemistry. The authors found that epithelial cells aligned and elongated along the

nanoridges (Figure 1A), while cells on smooth TCPS surface substrates remainedAuthor predominantly Proof round. Furthermore, a greater percentage of aligned cells were observed in deeper grooves. In addition, cells extended lamellipodia and filopodia primarily along ridges and down to Figure 3. Focal adhesion. (A) F‑actin cytoskeleton visualized by Oregon®-green- groove floors (Figure 1B). Finally, the size of the labeled phallodin in human mesenchymal stem cells on PDMS with 350-nm FAs was dependent on the ridge width, with gratings or unpatterned substrates. (B) Distribution of focal adhesions visualized by wider ridges allowing for larger FAs to form. immunofluorescence staining of tyrosine‑397 phosphorylated FAK (red) and F‑actin Together, these data suggest that nanoscale sur- (green). (C) Distribution of focal adhesions visualized by immunofluorescence staining of vinculin (red). Images of (A) are taken with fluorescence microscopy; face features can have profound effects on cell images of (B & C) are taken with confocal microscopy. Boxes indicate the area of morphology. the magnified views shown in the insert figures; scale bar is 10 µm in the insert While 2D surfaces are valuable tools for study- figures. ing basic cellular response to nanotopography, PDMS: Polydimethylsiloxane; TCPS: Tissue culture polystyrene. translation of these findings towards clinical Reproduced with permission from [144] © Elsevier (2010).

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therapies in the clinic. MSC differentiation can composed of grafted chitosan polymers and car- be driven by the material–cell interface, sug- bon nanotubes was incorporated into composites gesting a unique strategy to manipulate SCs in for bone regeneration to increase the mechanical the absence of complex soluble chemistries or strength of these composites [70]. cellular reprogramming. However, so far the A key tenet of BTE is the development of derivation and identification of surfaces that scaffold materials that can stimulate SC differ- allow retention of multipotency of this key entiation in the absence of chemical treatment regenerative cell type have remained elusive. to become osteoblasts without compromising Adult SCs spontaneously differentiate in cul- material properties. At present, conventional ture, resulting in a rapid diminution of the mul- implant materials fail owing to encapsulation tipotent cell population and their regenerative by soft tissue, rather than direct bone bonding. capacity. Bone fractures, healing critically sized Indeed, in 2007, Dalby et al. demonstrated the segmental defects and regeneration of articular use of nanoscale disorder to stimulate hMSCs cartilage in degenerative joint diseases owing to produce bone mineral in vitro, in the absence to various traumas or natural aging, represent of osteogenic supplements [71]. This approach typical aspects of tissue malfunction. Surgical has similar efficiency to that of cells cultured treatment frequently requires the implantation with osteogenic media. In addition, the current of a temporary or permanent prosthesis, which studies demonstrate that topographically treated represents a challenge for orthopedic surgeons, MSCs have a distinct differentiation profile com- especially in the case of large bone defects [66]. pared with those treated with osteogenic media, An understanding of how mechanics influences which has implications for cell therapies. A few tissue differentiation during repair and regen- years later, the same group performed a more eration crucially requires spatial and temporal detailed analysis that identified a nanostructured knowledge of the local mechanical environment; surface that retains SC phenotype and main- it is now evident that mechanical boundary tains SC growth over 8 weeks. Furthermore, conditions influence the regeneration of bone. the study implicates a role for small RNAs in Mimicking the structures of natural ECM may repressing key cell signaling and metabolomic lead to the successful regeneration of damaged pathways, demonstrating the potential of sur- tissue [67]. As mentioned above, cell adhesion to faces as noninvasive tools with which to address the ECM strongly influences cellular events; a the SC niche [72]. recapitulation of bone topography enriched with The use of HA nanocrystals has been exten- nanoadhesion sites might guide SC fates and sively studied by Bigi et al. [73]. This group coated prove useful for regenerative treatments for bone a biocompatible, nanostructured titanium alloy,

healing [68]. The literature offers some examples. Ti13Nb13Zr, with a thin layer of HA nanocrystals Chitosan, a natural polymer (a linear polysac- and investigated the response of cultured human charide composed of randomly distributed bone marrow-derived mesenchymal cells to this b‑(1–4)-linked d‑glucosamine (deacetylated material. Supersaturated solutions with ionic unit) and N‑acetyl-d‑glucosamine (acetylated compositions similar to that of human plasma unit) obtained from chitin, has played a major have been widely employed with the aim of role in bone tissue engineering (BTE) over the mimicking the mineralization process of bone. last two decades. Chitosan’s interesting charac- With this view, a slightly supersaturated cal- teristics that make it suitable for tissue recon- cium phosphate solution was used for coating, Authorstruction are its minimal foreign body reaction, Proof resulting in a fast deposition of nanocrystalline its intrinsic antibacterial nature and its ability to HA that reached thicknesses of 1–2 µm after be molded into various geometries and forms, 3 h of soaking. The same coating, deposited on

including porous structures suitable for in cell Ti6Al4V, was examined for comparison after a growth and osteoconduction [69]. Owing to its culture period of up to 35 days. Although the favorable gelling properties, chitosan can even presence of a HA coating slightly reduces cell deliver morphogenic factors and pharmaceuti- proliferation, it also strongly induces the differ- cal agents in a controlled fashion, presenting an entiation of MSCs toward a phenotypic osteo- ideal method for gene delivery strategies (owing blastic lineage, in agreement with the increased to its additional capacity to complex with DNA expression of osteopontin, osteonectin and col- molecules) [70]. Composites of chitosan, includ- lagen type I, as evaluated by reverse transcrip- ing hydroxyapatite (HA), are also very popular tion‑PCR. Type I collagen expression has been

because of their biodegradability and biocom- demonstrated to be higher in Ti13Nb13Zr MSC

patibility with nature. Recently, a material culture as compared with Ti6Al4V, representing

6 Nanomedicine (2013) 8(3) future science group Review Bressan, Carraro, Ferroni et al. Nanotechnology to drive stem cell commitment Review

Nanopatterned surface Smooth surface

Fibroblast

Osteoblast

Focal contact Focal contact

Figure 4. The topographies of nanoadhesion sites in tissue engineering scaffolds are able

to guide stem cell fate. The original study compared different diameter TiO2 nanotubes, ranging from 15 to 100 nm. On the 100 nm nanotube, diameter adhesion is significantly reduced and cell death by anoikis (lack of adhesion) was observed. Reproduced from [74].

a more efficient ECM deposition. In terms of The development of nanofibers with nano‑HA topography, Park et al. demonstrated that the (n‑HA) has enhanced the scope of scaffold fab- differentiation of rat MSCs (RMSCs) into an rication that mimics the architecture of natural osteogenic lineage occurs most frequently on bone tissue. Nanofibrous substrates supporting

vertically aligned TiO2 nanotubes with diam- the adhesion, proliferation and differentiation eters of 15 nm; this size approximates the pre- of cells and the incorporation of HA induce dicted lateral spacing of integrin receptors in cells to form organic mineralized and non- contact with the ECM (Figure 4). The phosphory- mineralized matrices [78]. Biocomposite poly- lation of FAK and ERK, which is a target of the meric nanofibers containing ‑n HA fabricated FAK signaling pathway, confirmed these data, by electrospinning could be promising scaf- while significantly poorer results were obtained folds for bone reconstruction: nanofibrous scaf- with increased nanotube diameters (100 nm) folds of poly-l‑lactide (PLLA; 860 ± 110 nm), [74]. Compared with flat substrates, structures PLLA/HA (845 ± 140 nm) and PLLA/col- with nanoscale features and different chem- lagen/HA (310 ± 125 nm) were proposed istries (e.g., silica, alumina and poly[methyl by Prabhakaran et al. [79]. For this purpose, methacrylate]) have been reported to enhance Prabhakaran et al. evaluated the morphological, adhesion, growth and osteogenic differentia- chemical and mechanical characteristics of the tion of hMSCs and marrow stromal cells, and nanofibers using scanning electron microscopy, could have potential applications as osteogenic Fourier transform infrared spectroscopy and coatings for orthopedic implants [74–77]. tensile testing, respectively [79]. The synergistic Electrospun nanofiber-basedAuthor synthetic and effect of the presence Proof of an ECM protein, col- natural polymer scaffolds are being explored lagen and HA in PLLA/collagen/HA nanofi- as scaffolds similar to natural ECM for tissue bers allows these nanofibers to act as temporary engineering applications. Nanostructured mate- templates for bone regeneration that actively rials for bone scaffolds are smaller in size, in stimulate the attachment of osteoprogenitor the 1–100 nm range, and have specific proper- cells. This approach, providing cell recognition ties and functions related to the sizes of natural sites together with apatite, significantly improves materials (e.g., HA). Bone contains consider- cell proliferation and osteoconduction, as well as able amounts of minerals and proteins: HA optimizing the mineralization process and bone

Ca10(PO4)6(OH)2 is one of the most stable forms formation [79]. of calcium phosphate, and it occurs in bones A combination of several biomaterials has also as a major component (60–65%), along with been used by Ravichandran et al. who fabricated other materials, including collagen, chondroitin nanofibers based on PLLA/poly-benzyl-l‑glu- sulfate, keratin sulfate and lipids. tamate (PBLG)/collagen by electrospinning

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and deposited n-HA by the calcium phosphate features have been investigated for bioactivity dipping method for BTE [80]. Adipose tissue and cellular responses; microstructures and could be used as a source of SCs, in this case micronanoscale surface morphology have been they are called adipose-derived SCs (ADSCs). controlled by adding a hydroxycarboxylic acid ADSCs were cultured on these scaffolds and (citric acid) in the sol-gel process [82]. Results were induced to undergo osteogenic differ- have demonstrated that the addition of citric entiation in the presence of PBLG/n‑HA for acid induces the formation of nanoscale surface BTE. The cell–biomaterial interactions were structures and increases the specific surface analyzed using cell proliferation, scanning elec- areas, pore volumes and pore sizes of the par- tron microscopy and 5‑chloromethylfluorescein ticles. In particular, the use of citric acid with diacetate dye-extraction techniques. Osteogenic low-concentration-derived sol-gel bioactive differentiation of ADSC was confirmed using glass resulted in enhanced apatite formation in alkaline phosphatase activity, mineralization simulated body fluids, as compared with normal and dual immunofluorescent staining using bioactive glass [83,84]. both an ADSC marker protein and osteo- Combinations of nanostructures with growth calcin, which is a bone-specific protein. The factors are alternative strategies adopted by utmost significance of this study is the bioac- Schofer et al., who evaluated the influence of tive PBLG/n‑HA biomolecule introduced on the 3D PLLA nanofiber scaffolds on bone forma- polymeric nanofibers to regulate and improve tion in vivo and analyzed whether incorporated specific biological functions, such as adhesion, BMP‑2 could enhance their efficacy[85] . PLLA proliferation and differentiation of ADSC into nanofiber scaffolds were demonstrated to facili- osteogenic lineage. The observed results proved tate cell immigration and, therefore, to achieve that the PLLA/PBLG/collagen/n‑HA scaffolds high cell densities. However, they lacked ade- promoted greater osteogenic differentiation of quate osteogenic stimuli to allow further dif- ADSC, as evident from the enzyme activity and ferentiation of those cells. The incorporation of mineralization profiles for BTE. The authors recombinant human BMP‑2 into PLLA nano- conclude that the importance of this study is the fibers could overcome this problem. Therefore, application of bioactive macromolecules PBLG/ PLLA/BMP‑2 implants were able to close n‑HA that have been introduced on the surface critical-size calvarial defects within 8 weeks. of polymeric nanofibers, to regulate and improve Increased expression of osteocalcin, BMP‑2 and specific biological functions, such as adhesion, Smad5 suggests a subsequent activation of the proliferation and differentiation. Smart materi- osteoblast lineage. Therefore, PLLA/BMP‑2 als such as PLLA/PBLG/collagen/n‑HA scaf- nanofiber scaffolds combine a suitable matrix for folds that can also elicit therapeutic effects by cell migration with an osteoinductive stimulus. incorporating biosignaling molecules within In the field of osteogenic commitment, dental the nanofibers, such as proteins and genes, hold pulp represents another highly specialized mes- great promise as scaffolds for BTE with drug enchymal tissue that has a limited regeneration delivery application. Owing to their abundance capacity due to anatomical arrangement and the and accessibility, ADSC cells may prove to be post-mitotic nature of odontoblastic cells. Dental desirable cell therapeutics for bone repair and caries remain one of the most prevalent infec- regeneration. tious diseases in the world, with a demonstrat- An innovative tool using magnetic nano able pharmaceutical impact. Available treatment Authorparticles was recently developed: Kanczler Proofet al. methods rely on the replacement of decayed soft have investigated remote magnetic field activa- and mineralized tissue with inert biomaterials. tion of magnetic nanoparticle-tagged mecha- Regenerative endodontics promises innovative nosensitive receptors on the cell membrane of results using SCs associated with scaffolds and human bone marrow stromal cells for use in responsive molecules. Wang et al. investigated osteoprogenitor cell-delivery systems and for the odontogenic differentiation of human dental the activation of differentiation, in vitro and pulp SCs on nanofibrous PLLA scaffolds in vitro in vivo, toward an osteochondral lineage [81] . and in vivo [86]. The combination of a phase- Other strategies take advantage of the ability separation technique and a porogen-leaching of bioactive glass to bond directly with bone. method recapitulated the architecture of colla- Clinical applications of these materials are likely gen type I fibers in the design of a highly porous to use their particulate form. Microstructured nanofibrous PLLA scaffold [86,87]. Positive apatite-forming bioactive glass particle scaf- immunohistochemical staining for dentin sia- folds with nanoscale or non-nanoscale surface loprotein, together with other assays confirmed

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the more effective differentiation of dental pulp marrow-derived SCs (BMSCs) were the primary SCs into odontoblast-like cells with the capacity source of SCs for tissue engineering applications to regenerate dental pulp and dentin. [95]. Recent studies have demonstrated that sub- cutaneous adipose tissue provides a clear advan- Chondrogenic commitment tage over other SC sources owing to the ease with One of the most important application fields which adipose tissue can be accessed (under local of cartilage tissue engineering is for osteoar- anesthesia and with minimal patient discom- thritis treatments. Osteoarthritis is the most fort), as well as the ease of isolating SCs from the prevalent musculoskeletal disease in humans, harvested tissue [96]. Moreover, the SC frequency causing pain, loss of joint motility and function, is significantly higher in adipose tissue than in and severely reducing the standard of living of bone marrow and the maintenance of prolif- patients. Cartilage tissue engineering attempts erative ability in culture seems to be superior in to repair the damaged tissue of individuals suf- ADSCs as compared with BMSCs [97]. fering from osteoarthritis by providing mechan- Adipogenic differentiation of SCs can also ical support to the joint as new tissue regener- be efficiently induced by physical factors and ates. The combination of SC biology with nano- modulation of ECM nanostructures. The litera- technology has been used by Fong et al. who ture offers several different methods to induce used human umbilical cord Wharton’s jelly on such differentiation through the use of high- poly(e‑caprolactone) (PCL)/collagen nanoscaf- quality nanoparticles of various chemical com- folds in the presence of a two-stage sequential positions. Miyagawa et al. reported the highly complex/chondrogenic medium for 21 days [88]. efficient in vitro differentiation of human bone In separate experiments the authors demon- marrow-derived mesenchymal stem/progenitor strated that the 16 ng/ml of bFGF present in the cells using a novel nanotechnology-based culture complex medium may have contributed to driv- plate, called the NanoCulture® Plate (Infinite ing chondrogenesis. Chondrogenic commitment Bio, Inc., CA, USA) [98]. The NanoCulture Plate of SCs could be driven by nanotubes as reported is composed of uneven microfabricated elements by Erisken et al., who obtained chondrogenic (with diameters of ~2–3 µm) arranged in a hon- differentiation of human adipose-derived stro- eycomb pattern on the surface. At first, human mal cells using a combination of twin-screw mesenchymal stem/progenitor cells cultured in extrusion and an electrospinning generated 3D culture using the NanoCulture Plate system nanofibrous scaffold suitable for inducing SC rapidly form adhesive spheroids. These spheroi- commitment [89,90]. The results of the imple- dal clusters demonstrate enhanced adipogenic mentation of this scaffold were selective dif- differentiation characterized by a more rapid ferentiation of human adipose-derived stromal accumulation of triglycerides than in 2D culture. cells toward a chondrogenic lineage. An alternative strategy to achieve commitment to an adipogenic lineage is laser-assisted Adipogenic commitment bioprinting, which permits the production of Adipose tissue pathologies and defects have computer-generated 3D tissue grafts. In a recent always represented a reconstructive challenge study, laser-assisted bioprinted human ADSCs for plastic surgeons. Contour defects resulting embedded in a hydrogel environment in a free- from resections of tumors, trauma and congeni- scalable 3D grid pattern [99]. The authors dem- tal abnormalities not only affect patients cos- onstrated that the biological behavior of the SCs metically but may also impairAuthor function, making was affected by theProof nanotechnological proce- adipose tissue restoration a clinical need [91,92]. dure; in fact, after 10 days of enhancing the The emerging field of adipose tissue engi- adipogenic lineage, quantitative assessments of neering aims to develop biological substitutes adipogenic markers demonstrated that the 3D that promote regeneration and restore function, grafts resembled cell lineages of natural adipose through the application of the principles and tissue. methods of engineering and the life sciences [93]. Recent advances have resulted in an increas- The development of adipose tissue-engineering ing interest in the development of bioconju- strategies requires investigation of all key aspects gated carriers for the delivery of bioactive of the tissue engineering process, including the molecules to SCs. The novel properties of selection of a cell source, scaffold biomaterial these nanoparticles are intended to favor and and microenvironment to provide the appropri- modulate SC differentiation. Liu et al. used ate cues and signals for cell growth and adipose this method to promote the adipogenic dif- tissue formation [94]. For many years, bone ferentiation of RMSCs in vitro, reporting

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biocompatible silica nanoparticle (SiNP)–insu- In this light, modification of 3D electrospun lin conjugates [100] . After the biocompatibility PCL nanofiber scaffolds by fiber alignment and of SiNPs with RMSCs was tested, a cell viabil- aminolyzation is superior to classical 2D culture- ity assay was performed to screen the SiNP con- ware in promoting the in vitro proliferation and centration for its cytotoxicity toward RMSCs. differentiation of cortical cells. Horne et al. After obtaining the optimum SiNP concentra- demonstrated that tethering the BDNF onto tion that induced minor cytotoxicity on the modified nanofibers is superior to culturing in RMSC phenotype, SiNP–insulin conjugates the presence of soluble BDNF [103] . Functional were used for RMSC adipogenic differentia- immobilization of BDNF onto polymer nano- tion, resulting in the prompt differentiation of fibers enhances neural SC proliferation and RMSCs into adipocytes when cultured in the directs cell fate toward neuronal and oligoden- presence of insulin-conjugated SiNPs. drocyte specifications, essential for neural tissue repair. These findings indicate that modi- Neuronal commitment fied PCL nanofibrous 3D scaffolds are capable Biotechnology is being increasingly used to reca- of supporting neural SCs and their derivatives pitulate specific aspects of brain niches able to and may present a new avenue for encouraging promote regeneration and repair damaged neu- neural repair in the future. Carbon nanotubes ronal pathways with SC therapies. Many of these have electrical, mechanical and chemical prop- approaches are gaining momentum because erties that make them one of the most promis- nanotechnology allows for greater control ing materials for applications in neuroscience. over material–cell interactions. This, in turn, Single- and multi-walled carbon nanotubes have allows for the induction of specific developmen- been increasingly used as scaffolds for neuronal tal processes and cellular responses including growth and, more recently, for neural SC growth differentiation, migration and outgrowth. and differentiation. They are also used in inter- Many studies have examined the importance faces with neurons, where they can detect neu- of exogenous soluble factors in promoting cell ronal electrical activity and also deliver electrical fate specification. Soleimaniet al. experimented stimulation to these cells. Therefore, in the near with a 3D nanofibrous scaffold fabricated from future, they could be used in brain–machine aligned PLLA, studying its ability to support interfaces [104] . neurogenic, and hinder dopaminergic, differentiation of conjunctiva MSCs in vitro [101] . Hepatocyte-like SCs Neurogenic lineages were induced by cultur- It is well known that tissue engineering proves ing cells in specific differentiation media. The to be a temporary treatment for patients suffer- tested nanofibrous PLLA scaffold has been dem- ing from hepatic failure. For successful tissue onstrated to be a potential cell carrier in neural regeneration, the cells constituting the tissues tissue engineering applications with the partial to be regenerated are necessary. Considering inhibition of the dopaminergic differentiation of the proliferation activity and differentiation conjunctiva MSCs [101] . potential of cells, SCs are promising. Human Alternative strategies have been proposed by BMSCs (hBMSCs) have great potential for liver Cho et al. who developed a NGF‑conjugated tissue engineering because autologous BMSCs aligned nanofibrous mesh-based method for can be harvested, expanded extensively ex vivo neuronal differentiation of MSCs [102] . Amine- and differentiated into a hepatic phenotype Authorterminated poly(ethylene glycol) was conjugated Proof for transplantation back into the patient [104] . to PCL to produce amine-functionalized copo- Differentiation of hBMSCs into hepatocyte- lymers, which were then electrospun in a rotat- like cells (HLCs) in standard monolayer or 2D ing drum to obtain aligned nanofibrous meshes. cultures is now well established. However, the NGF was chemically linked to the amine challenge remains to develop robust protocols to groups of the nanofibrous meshes in the aque- generate functional hepatocytes from hBMSCs ous phase. In vitro release profiles of the NGF suitable for transplantation [105] . In recent years, were then investigated; the growth factor physi- with respect to nanofibers for tissue engineering cally adsorbed on the nanofibrous meshes and purposes, a wide variety of nanofibrous scaffolds demonstrated an initial burst release in MSCs have been produced [106,107]. The experimental cultured for 5 days [102] . results have demonstrated that, although syn- Regarding CNS tissue repair strategies, thetic biodegradable PCL supports cell growth, achieving the desired results depends on the res- to increase proliferation and encourage cell toration of appropriate neuronal connectivity. ingrowth for better integration between cells and

10 Nanomedicine (2013) 8(3) future science group Review Bressan, Carraro, Ferroni et al. Nanotechnology to drive stem cell commitment Review

the scaffold, the biologically inert PCL nanofi- properties of nanofibers enhance differentiation bers need effective hybridization with bioactive of HLCs from MSCs and maintain their func- molecules. It has been reported that electrospin- tion in long-term culture, which has implications ning of PCL with collagen gives encouraging for cell therapies. The authors concluded that results in improving the cell–scaffold interac- nanofibers have the capability not only to drive tions [107] . SC commitment into hepatocyte-like SCs but Moreover, polyethersulfone has many fasci- also to maintain stable differentiation, thereby nating properties including favorable mechani- achieving transplantable hepatocytes. cal strength, thermal and chemical resistance, and excellent biocompatibility. Therefore, the Other lineage polymer blend of PCL/collagen/polyethersul- Shi et al. investigated the effects of substrate fone can overcome the shortcomings of natu- nanotopography on the endothelial differen- ral and synthetic polymers, resulting in a new tiation of ADSCs [112] . The authors compared biomaterial with good biocompatibility and two nanograting substrates with periods (ridge improved mechanical, physical and chemical and groove length) of approximately 250 properties [108] . and 500 nm, respectively, with a flat surface. In a study of Kazemnejad et al., the potential Endothelial differentiation of ADSCs on both of hBMSCs to differentiate into functional hepa- flat and nanograting substrates can be induced tocytes within designed PCL/collagen/poly- with VEGF. PCR analysis demonstrated sig- ethersulfone nanofibers has been investigated nificantly enhanced upregulation of vWF, [109] . Cytochemical, biochemical and molecular PECAM‑1 and VE‑cadherin at the gene level features of HLCs differentiated from hBMSCs in ADSCs grown on the nanograting substrates. on the scaffold were used to show the role of In vitro angiogenesis assays on Matrigel™ (BD nanofibrous structure to support differentiation. Biosciences, NJ, USA) showed that nanograting Using similar PCL nanofiber scaffolds, Hashemi substrates enhanced capillary tube formation. et al. tested the in vitro differentiation of human Another cellular lineage studied is myogenic cord blood-derived unrestricted somatic SCs into differentiation. Tian et al. used growth factors HLCs [110] . In their study, the authors focused on to stimulate SCs into smooth muscle-like cells the ability of PCL nanofiber scaffolds to support [113] . Growth factors alone or combined with and maintain hepatic differentiation in vitro. either bladder ECM coatings or a dynamic cul- Unrestricted somatic SCs and self-renewing plu- ture system induced BMSCs to express smooth ripotent cells, were isolated from human cord muscle-specific genes and proteins in vitro. A blood. The electrospun PCL nanofiber porous nanofibrous 3D PLLA polymer porous scaffold scaffold was constructed of uniform, randomly provides an optimal microenvironment for facili- oriented nanofibers. Unrestricted somatic SCs tating cell-matrix penetration and retention of were seeded onto PCL nanofiber scaffolds, and myogenic-differentiated BMSCs, thereby pro- were induced to differentiate into hepatogenic moting tissue remolding with rich capillary for- lineages by culturing with differentiation factors mation in vivo. Yu et al. stressed the important for 6 weeks. role of FA in driving SC commitment [114] . The Ultra-web® (Donaldson, Leuven, Belgium) authors postulated that differentiation outcomes nanofibers (nano+ and nano-) have indeed been can be controlled by modulating FA morphology used by Piryaei et al. to better differentiate and and distribution. Gekas et al. investigated the maintain the function andAuthor engraftment of dif- behavior of human SCsProof obtained from amniotic ferentiating MSCs both in vitro and in vivo [111]. fluid [115] . When cultured in vitro in myogenic- MSCs, early and late HLCs in both nano- and specific induction media, these SCs were able nano+ culture conditions that were transplanted to differentiate as expected. However, when by an intravenous route caused a decrease in liver transplanted into the skeletal muscle of mice, fibrosis when engrafted in the recipient liver and differentiation into tubular glandular-like tissue were able to differentiate into functional hepa- occurred. tocytes (albumin +), with the exception of late HLCs in the nano- group. Late HLCs trans- Commitment through intracellular planted in the nano+ group were more effective delivery of small particles in rescuing liver failure, enhancing serum albu- Commitment of SCs as reported above could be min, homing transplanted cells and undergoing induced by either the ECM or soluble factors. The functional engraftment than the other groups. final results are the ‘reprogrammations’ of the These results demonstrated that topographic genome of the cells and its reprogramming. The

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technique appointed to do this is gene therapy. number of publications on the in vitro behavior Gene therapy is a technique for correct- of gene vectors such as cationic polymer/nucleic ing missing, defective or overexpressed genes acid complexes (lipoplexes), cationic liposomes that are responsible for disease development. or polymeric nanoparticles, and recently several Although viral vectors can efficiently transfect studies on cationic solid lipid nanoparticles have cells, their clinical application is limited by been reported [123] . the related risks for patients [116,117]. Nonviral The induction of SC differentiation by delivery systems are a safer approach, easier drugs and growth factors has been the objec- to manufacture, more versatile and more cost tive of many studies aiming to optimize meth- effective. Nevertheless, their transfection effi- ods for the regeneration of new tissues or the ciency is low compared with that of viral vec- repair of degenerated tissues via transplanta- tors. Many groups have dedicated considerable tion. Park et al., for example, used drugs and effort to improving the efficiency of nonviral growth factors with a high potential for tissue gene delivery systems, and particular attention repair embedded in hMSCs; cell differentiation has been devoted to investigating complexes into chondrogenic, osteogenic and adipogenic composed of DNA and soft materials, such lineages was subsequently enhanced [124]. This as lipids, polymers, peptides, dendrimers and culture condition, enriched with microspheres gemini surfactants. The theoretical approach coated and loaded with drugs and growth factors, in designing these nanoparticles considers dif- demonstrated proliferation and, as expected, ferent components essential for assuring high induced differentiation of transplanted hMSCs levels of transfection, biocompatibility and into the desired specific cell types. tissue-targeting ability [118]. In this context, polymeric nanoparticles Several advanced in vitro studies have proved are promising gene delivery systems because the broad potential of cationic solid lipid they offer stability and controlled release, have nanoparticles as synthetic nucleic acid vectors the capacity to encapsulate large amounts of that have been proposed as an alternative to genetic material, allow for codelivery and can liposomes. Certainly, results are closely related readily be surface modified to enhance stabil- to their transfection performances [119,120] . ity, transport properties, targeting or uptake Nanovectors already play a very important role [125–129]. Polymers that are biodegradable, in pharmaceutical applications for the delivery biocompatible and nontoxic make attractive of drugs or other biologically active materials. candidates for constructing in vivo delivery Solid lipid nanoparticles are basically com- vehicles. Chitosan, cyclodextrin, polyethyl- posed of a solid lipid core in nanometer ranges eneimine (PEI), poly(lactic-co-glycolic) acid, stabilized by a layer of emulsifier; they can be dendrimers and metallic-core nanoparticles prepared by using lipids with a relatively high have become popular for use in delivery sys- melting point (i.e., triglycerides, hard fat types, tems, although none of these materials possess partial glycerides, steroids and waxes). Among all of the desirable properties [130] . Chitosan is these lipids, glycerides, which are composed a natural, cationic polysaccharide harvestable of fatty acids, can be employed in injection from crustacean exoskeletons. It is an exten- form since they are already used in parenteral sively studied biomaterial due to its biocompat- nutrition. The development of innovative ibility, mucoadhesive properties and nuclease DNA‑based medicines for incurable disorders resistance [30]. Optimal cationic charge for Author(gene therapy) is an important component Proof of maximal siRNA encapsulation in chitosan can pharmaceutical advancement. Gene therapy be attained by tuning the ratio of amines to vectors can be categorized into two groups, phosphates. In two separate studies, optimized biological (viral) and nonbiological (nonviral) chitosan–siRNA nanoparticles have been suc- systems, and each group has its own advantages cessfully administered intranasally to silence and limitations [121] . Synthetic nonviral vectors, GAPDH and EGFP in the lungs of mice [131] . although being less efficient in bringing about Extensive branching and dense cationic cellular transfection, enjoy several advantages charge gives synthetic polymer PEI the capacity over biological vehicles, such as immunological to condense siRNAs, protect them from deg- inertia and a large degree of flexibility in the radation by RNases, and facilitate their cellu- design of their properties [122] . Nonviral vectors lar uptake via endocytosis. An added feature such as nanoparticle-mediated gene delivery sys- is the ability of PEI to act as a proton sponge, tems have become a hot area of research both in because its extensive amine groups buffer the academia and industry. Indeed, there is a large acidic inner compartment of an endosome

12 Nanomedicine (2013) 8(3) future science group Review Bressan, Carraro, Ferroni et al. Nanotechnology to drive stem cell commitment Review

causing water to swell the endosome to the an implant. As a consequence of this, the authors point of rupture, thereby facilitating endo- predict that complex tissues and organs can be somal escape of its encapsulated siRNA. Some engineered by managing the in situ development wariness surrounds PEI use in vivo, however, of multiple cell types reprogrammed by spatially due to in vitro evidence of high cytotoxicity restricted nanoparticles. [132]. In an effort to reduce the toxic effects In the end, it is important to find the right of PEI, the polymer has been modified with combination of good nanostructures with high- polyethylene glycol (previously demonstrated transfection efficiency. A well-written review by to slow clearance and reduce toxicity) and the Adler et al. explains that the use of surface nan- PEI–polyethylene glycol/siRNA complex was otechnology to modify particulate parameters demonstrated to exhibit decreased toxicity, but has gained well-deserved attention in nonviral drastically increased particle size [133–135]. gene delivery, as these parameters are becom- Dendrimers are heavily branched polymeric ing increasingly well-understood modulators of molecules that can be engineered to form modu- uptake and transfection efficiency[143] . Another lar, nanosized, spherical structures for siRNA approach worth considering is the engineering of delivery. Packaging siRNAs in dendrimer struc- desirable endocytic cellular phenotypes through tures can be accomplished by positively charg- substrate surface nanotechnology. Optimiza ing the core while abolishing surface charge tion of nonviral gene delivery has so far mostly [136] . Alternatively, siRNAs can be caged within focused on design of particulate carriers that are dendrimer polyplexes via disulfide linkages, endowed with desirable membrane targeting, which incidentally also provide for controlled internalization and endosomal escape properties. release in the reducing intracellular milieu. These structures can be additionally stabilized Conclusion & future perspective through the incorporation of polyethylene glycol The creation of complex tissues and organs is [137–139]. The modularity of dendrimers allows the ultimate goal of tissue engineering, and engi- for dendrimer–siRNA polyplexes to be further neered morphogenesis necessitates the spatially improved for siRNA delivery by combining controlled development of multiple cell types them with targeting ligands and technologies within a biomaterial-based scaffold. The cur- that provide for endosomal release. rent scenario of directly transplanting adult SCs Another siRNA delivery strategy involves in vivo for the treatment of different diseases or metallic core nanoparticles [140] . Metal cores of for direct delivery to injured sites is increasingly iron oxide, iron cobalt, iron gold or iron nickel changing. Recent progress in nanotechnology are coated with a layer of sugars or other poly- and a better understanding of the molecular mers generating a core shell structure to which pathways that control differentiation have led siRNA can be externally conjugated through us to combine biocompatible scaffolds with linking molecules, such as thiols [141], dextran adult SCs. As SC technologies transition from [140], cationic polymers [137] or biotin–strepta- the research laboratory to clinical applications, vadin [30]. Contingent upon the metal used, the there will be an increasing need for robust cul- cores of these particles can impart properties that ture systems that consistently control SC growth allow for the study of biodistribution upon injec- and differentiation. tion using MRI or targeting to specific tissues by Concerns regarding the use of nanoparticles applying external magnets. in the commitment of SCs and the protection Andersen et al. presentedAuthor a novel method of the bioactive molecules Proof against environmen- involving adhering nanoparticles containing dif- tal degradation are still valid; moreover, it is ferent siRNAs onto nanostructured scaffolds [142] . essential to better control the delivery factors in This allows for spatial retention of the siRNAs a dose- and time-correct manner. As discussed, within nanopores until their cellular delivery. particle delivery systems have been conceived to The released siRNAs were capable of silencing provide improvements in SC commitment such BCL2L2 and TRIB2 genes in MSCs and enhanc- as the ability to enhance the differentiation and ing osteogenic and adipogenic differentiation, stability of SCs. However, further investigation respectively. This approach for enhancing a single is necessary to better determine therapeutic type of differentiation is immediately applica- concentrations, combinations of molecules and ble to all areas of tissue engineering. Different methods for controlled release of factors. Recent nanoparticles localized to spatially distinct loca- advances in biotechnology, SC biology, polymer tions within a single implant allow for two differ- chemistry and nanotechnology are now open- ent tissue types to develop in controllable areas of ing up exciting possibilities for the improvement

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and restoration of tissue function, while mini- financial interest in or financial conflict with the subject mizing adverse effects and improving patient matter or materials discussed in the manuscript. This compliance. includes employment, consultancies, honoraria, stock own‑ ership or options, expert testimony, grants or patents received Financial & competing interests disclosure or pending, or royalties. The authors have no relevant affiliations or financial No writing assistance was utilized in the production of involvement with any organization or entity with a this manuscript.

Executive summary Osteogenic commitment Commitment of stem cells into the osteogenic lineage is highest on:

– TiO2 nanotubes vertically aligned with a diameter of 15 nm; – Nanofibers with nanohydroxyapatite fabricated by electrospinning; – Hydrogel scaffolds combined with peptide–amphiphile tailored by incorporation of the cell-adhesion sequence RGD and an enzyme-cleavable site; – Bioactive glass particles with nanoscale surface features; – Chitosan natural polymer with carbon nanotubes incorporated to increase the mechanical strength; – Scaffolds embedded with nanoparticles containing different siRNAs or growth factors. Adipose commitment Commitment of mesenchymal stem cells into adipose features is improved by the in vitro cultivations in: – Nanotechnology-based culture plate, composed of uneven microfabrications (diameters of ~2–3 µm) arranged in a honeycomb pattern on the cell surface; – Biocompatible silica nanoparticle–insulin conjugates; – Scaffolds embedded with nanoparticles containing different siRNAs or growth factors. Neuronal commitment Neuronal commitment of adult stem cells is enhanced by the applications of: – Nanofibrous scaffold fabricated from aligned poly-l‑lactic acid; – NGF‑conjugated aligned nanofibrous meshes; – Electrospun poly-e‑caprolactone nanofiber scaffolds.

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