Stimulation of 3D osteogenesis by mesenchymal stem cells using a nanovibrational bioreactor.

Bone grafts are one of the most commonly transplanted tissues. However, autologous grafts are in short supply, and can be associated with pain and donor-site morbidity. The creation of tissue-engineered bone grafts could help to fulfil clinical demand and provide a crucial resource for drug screening. Here, we show that vibrations of nanoscale amplitude provided by a newly developed bioreactor can differentiate a potential autologous cell source, mesenchymal stem cells (MSCs), into mineralized tissue in 3D. We demonstrate that nanoscale mechanotransduction can stimulate osteogenesis independently of other environmental factors, such as matrix rigidity. We show this by generating mineralized matrix from MSCs seeded in collagen gels with stiffness an order of magnitude below the stiffness of gels needed to induce bone formation in vitro. Our approach is scalable and can be compatible with 3D scaffolds.

Publisher Correction: Stimulation of 3D osteogenesis by mesenchymal stem cells using a nanovibrational bioreactor.

In the version of this Article originally published, in Fig. 4f, the asterisk was missing; in Fig. 6a-c, the labels 'Wnt/ß-catenin signalling', 'Wnt/Ca+ pathway' and 'ERK' and their associated lines/arrows were missing; and in Fig. 6d and in the sentence beginning "In MSCs that were...", 'myosin' and 'nanostimulated', respectively, were spelt incorrectly. These errors have now been corrected in all versions of the Article.

Use of nanoscale mechanical stimulation for control and manipulation of cell behaviour.

The ability to control cell behaviour, cell fate and simulate reliable tissue models in vitro remains a significant challenge yet is crucial for various applications of high throughput screening e.g. drug discovery. Mechanotransduction (the ability of cells to convert mechanical forces in their environment to biochemical signalling) represents an alternative mechanism to attain this control with such studies developing techniques to reproducibly control the mechanical environment in techniques which have potential to be scaled. In this review, the use of techniques such as finite element modelling and precision interferometric measurement are examined to provide context for a novel technique based on nanoscale vibration, also known as "nanokicking". Studies have shown this stimulus to alter cellular responses in both endothelial and mesenchymal stem cells (MSCs), particularly in increased proliferation rate and induced osteogenesis respectively. Endothelial cell lines were exposed to nanoscale vibration amplitudes across a frequency range of 1-100 Hz, and MSCs primarily at 1 kHz. This technique provides significant potential benefits over existing technologies, as cellular responses can be initiated without the use of expensive engineering techniques and/or chemical induction factors. Due to the reproducible and scalable nature of the apparatus it is conceivable that nanokicking could be used for controlling cell behaviour within a wide array of high throughput procedures in the research environment, within drug discovery, and for clinical/therapeutic applications. STATEMENT OF SIGNIFICANCE: The results discussed within this article summarise the potential benefits of using nanoscale vibration protocols for controlling cell behaviour. There is a significant need for reliable tissue models within the clinical and pharma industries, and the control of cell behaviour and stem cell differentiation would be highly beneficial. The full potential of this method of controlling cell behaviour has not yet been realised.

Production of Nanoscale Vibration for Stimulation of Human Mesenchymal Stem Cells.

Mechanical stimulation is becoming a common technique for manipulating cell behaviour in bioengineering with applications in tissue engineering and possibly regenerative therapy. Living organisms show biological responses in vivo and in vitro to various types of mechanical stimulation including vibration. The development of apparatus to produce vertical motions of nanoscale amplitude is detailed and their effect on mouse endothelial (Le2) and human mesenchymal stem cells (hMSCs) is investigated. Piezo ceramic actuators and aluminium reinforcement were utilised along with laser interferometry to ensure amplitude consistency at the nanometre level across a cell culture substrate. Peak force applied to the cells was estimated to be of nN magnitude at frequencies of 500 and 1000 Hz. Morphological changes in the cytoskeleton were found for both cell types along with increased MSC proliferation after 1 week of stimulation at 500 Hz. Changes in the nuclear size of MSCs after stimulation were also found.

Nanoscale stimulation of osteoblastogenesis from mesenchymal stem cells: nanotopography and nanokicking.

AIM: Mesenchymal stem cells (MSCs) have large regenerative potential to replace damaged cells from several tissues along the mesodermal lineage. The potency of these cells promises to change the longer term prognosis for many degenerative conditions currently suffered by our aging population. We have endeavored to demonstrate our ability to induce osteoblatogenesis in MSCs using high-frequency (1000-5000 Hz) piezo-driven nanodisplacements (16-30 nm displacements) in a vertical direction. MATERIALS & METHODS: Osteoblastogenesis has been determined by the upregulation of osteoblasic genes such as osteonectin (ONN), RUNX2 and Osterix, assessed via quantitative real-time PCR; the increase of osteocalcin (OCN) and osteopontin (OPN) at the protein level and the deposition of calcium phosphate determined by histological staining. RESULTS: Intriguingly, we have observed a relationship between nanotopography and piezo-stimulated mechanotransduction and possibly see evidence of two differing osteogenic mechanisms at work. These data provide confidence in nanomechanotransduction for stem cell differentiation without dependence on soluble factors and complex chemistries. CONCLUSION: In the future it is envisaged that this technology may have beneficial therapeutic applications in the healthcare industry, for conditions whose overall phenotype maybe characterized by weak or damaged bones (e.g., osteoporosis and bone fractures), and which can benefit from having an increased number of osteoblastic cells in vivo.

Cell interactions at the nanoscale: piezoelectric stimulation.

Nanometric movements of the substrate on which endothelial cells are growing, driven by periodic sinusoidal vibration from 1 Hz to 50 Hz applied by piezo actuators, upregulate endothelin-1 and Kruppel-like factor 2 expression, and increase cell adhesion. These movements are in the z (vertical) axis and ranges from 5 to 50 nm and are similar in vertical extent to protrusions from the cells themselves already reported in the literature. White noise vibrations do not to produce these effects. Vibrational sweeps, if suitably confined within a narrow frequency range, produce similar stimulatory effects but not at wider sweeps. These effects suggest that coherent vibration is crucial for driving these cellular responses. In addition to this, the applied stimulations are observed to be close to or below the random seismic noise of the surroundings, which may suggest stochastic resonance is being employed. The stimulations also interact with the effects of nanometric patterning of the substrates on cell adhesion and Kruppel-like factor 2 and endothelin-1 expression thus linking cell reactions to nanotopographically patterned surfaces with those to mechanical stimulation.

Osteogenesis of mesenchymal stem cells by nanoscale mechanotransduction.

It is likely that mesenchymal stem cells will find use in many autologous regenerative therapies. However, our ability to control cell stem growth and differentiation is presently limited, and this is a major hurdle to the clinical use of these multipotent cells especially when considering the desire not to use soluble factors or complex media formulations in culture. Also, the large number of cells required to be clinically useful is currently a hurdle to using materials-based (stiffness, chemistry, nanotopography, etc.) culture substrates. Here we give a first demonstration of using nanoscale sinusoidal mechanotransductive protocols (10-14 nm displacements at 1 kHz frequency), "nanokicking", to promote osteoblastogenesis in human mesenchymal stem cell cultures. On the basis of application of the reverse piezo effect, we use interferometry to develop the optimal stem cell stimulation conditions, allowing delivery of nanoscale cues across the entire surface of the Petri dishes used. A combination of immunofluorescence, PCR, and microarray has then been used to demonstrate osteoblastogenesis, and the arrays implicate RhoA as central to osteoblastic differentiation in agreement with materials-based strategies. We validate this with pharmacological inhibition of RhoA kinase. It is easy to envisage such stimulation protocols being up-scaled to form large-scale osteoblast bioreactors as standard cell culture plates and incubators are used in the protocol.

The role of microtopography in cellular mechanotransduction.

Mechanotransduction is crucial for cellular processes including cell survival, growth and differentiation. Topographically patterned surfaces offer an invaluable non-invasive means of investigating the cell response to such cues, and greater understanding of mechanotransduction at the cell-material interface has the potential to advance development of tailored topographical substrates and new generation implantable devices. This study focuses on the effects of topographical modulation of cell morphology on chromosomal positioning and gene regulation, using a microgrooved substrate as a non-invasive mechanostimulus. Intra-nuclear reorganisation of the nuclear lamina was noted, and the lamina was required for chromosomal repositioning. It appears that larger chromosomes could be predisposed to such repositioning. Microarrays and a high sensitivity proteomic approach (saturation DiGE) were utilised to identify transcripts and proteins that were subject to mechanoregulated changes in abundance, including mediators of chromatin remodelling and DNA synthesis linked to the changes in nucleolar morphology and the nucleoskeleton.

A hierarchical response of cells to perpendicular micro- and nanometric textural cues.

In this paper, we report on the influence of shallow micro- and nanopatterned substrata on the attachment and behavior of a human fibroblast [human telomerase transfected immortalized (hTERT)] cells. We identify a hierarchy of textural guidance cues with respect to cell alignment on these substrates. Cells were seeded and cultured for 48 h on silicon substrates patterned with two linear textures overlaid at 90 degrees, both with 24 microm pitch: a micrograting and a nanopattern of rows of 140- nm-diameter pits arranged in a rectangular array with 300 nm centre-to-centre spacing. The cell response to these textures was shown to be highly dependent on textural feature dimensions. We show that cells align to the stripes of nanopits. Stripes of 30-nm deep nanopits were also shown to elicit a stronger response from cells than 160-nm deep nanopits.

Microtopography of metal surfaces influence fibroblast growth by modifying cell shape, cytoskeleton, and adhesion.

Stainless Steel (SS), titanium (cpTi), and Ti-6Al-7Nb (TAN) are frequently used metals in fracture fixation, which contact not only bone, but also soft tissue. In previous soft tissue cytocompatibility studies, TAN was demonstrated to inhibit cell growth in its "standard" micro-roughened state. To elucidate a possible mechanism for this inhibition, cell area, shape, adhesion, and cytoskeletal integrity was studied. Only minor changes in spreading were observed for cells on electropolished SS, cpTi, and TAN. Cells on "standard" cpTi were similarly spread in comparison with electropolished cpTi and TAN, although the topography influenced the cell periphery and also resulted in lower numbers and shorter length of focal adhesions. On "standard" microrough TAN, cell spreading was significantly lower than all other surfaces, and cell morphology differed by being more elongated. In addition, focal adhesion numbers and mean length were significantly lower on standard TAN than on all other surfaces, with 80% of the measured adhesions below a 2-microm threshold. Focal adhesion site location and maturation and microtubule integrity were compromised by the presence of protruding beta-phase microspikes found solely on the surface of standard TAN. This led us to propose that the impairment of focal adhesion numbers, maturation (length), and cell spreading to a possibly sufficient threshold observed on standard TAN blocks cell cycle progress and eventually cell growth on the surface. We believe, as demonstrated with standard cpTi and TAN, that a difference in surface morphology is influential for controlling cell behavior on implant surfaces.

Nanomechanotransduction and interphase nuclear organization influence on genomic control.

The ability of cells to alter their genomic regulation in response to mechanical conditioning or through changes in morphology and the organization of the interphase nuclei are key questions in cell biology. Here, two nanotopographies have been used as a model surfaces to change cell morphology in order to investigate spatial genomic changes within the nuclei of fibroblasts. Initially, centromeres for chromosome pairs were labeled and the average distance on different substrates calculated. Further to this, Affymetrix whole genome GeneChips were used to rank genomic changes in response to topography and plot the whereabouts on the chromosomes these changes were occurring. It was seen that as cell spreading was changed, so were the positions along the chromosomes that gene regulations were being observed. We hypothesize that as changes in cell and thus nuclear morphology occur, that this may alter the probability of transcription through opening or closing areas of the chromosomes to transcription factors.

Nanotopographical stimulation of mechanotransduction and changes in interphase centromere positioning.

We apply a recently developed method for controlling the spreading of cultured cells using electron beam lithography (EBL) to create polymethylmethacrylate (PMMA) substrata with repeating nanostructures. There are indications that the reduced cell spreading on these substrata, compared with planar PMMA, results from a reduced adhesivity since there are fewer adhesive structures and fewer of their associated stress fibres. The reduced cell spreading also results in a reduced nuclear area and a closer spacing of centrosomes within the nucleus, suggesting that the tension applied to the nucleus is reduced as would be expected from the reduction in stress fibres. In order to obtain further evidence for this, we have used specific inhibitors of components of the cytoskeleton and have found effects comparable with those induced by the new substrata. We have also obtained evidence that these subtrata result in downregulation of gene expression which suggests that this may be due to the changed tension on the nucleus: an intriguing possibility that merits further investigation.

Nanotopographical control of human osteoprogenitor differentiation.

Current load-bearing orthopaedic implants are produced in 'bio-inert' materials such as titanium alloys. When inserted into the reamed bone during hip or knee replacement surgery the implants interact with mesenchymal populations including the bone marrow. Bio-inert materials are shielded from the body by differentiation of the cells along the fibroblastic lineage producing scar tissue and inferior healing. This is exacerbated by implant micromotion, which can lead to capsule formation. Thus, next-generation implant materials will have to elicit influence over osteoprogenitor differentiation and mesenchymal populations in order to recruit osteoblastic cells and produce direct bone apposition onto the implant. A powerful method of delivering cues to cells is via topography. Micro-scale topography has been shown to affect cell adhesion, migration, cytoskeleton, proliferation and differentiation of a large range of cell types (thus far all cell types tested have been shown to be responsive to topographical cues). More recent research with nanotopography has also shown a broad range of cell response, with fibroblastic cells sensing down to 10 nm in height. Initial studies with human mesenchymal populations and osteoprogenitor populations have again shown strong cell responses to nanofeatures with increased levels of osteocalcin and osteopontin production from the cells on certain topographies. This is indicative of increased osteoblastic activity on the nanotextured materials. Looking at preliminary data, it is tempting to speculate that progenitor cells are, in fact, more responsive to topography than more mature cell types and that they are actively seeking cues from their environment. This review will investigate the range of nanotopographies available to researchers and our present understanding of mechanisms of progenitor cell response. Finally, it will make some speculations of the future of nanomaterials and progenitor cells in tissue engineering.

Nanoprinting onto cells.

Growing cells on surfaces bearing nanotopography signals makes many changes in cell gene expression and downstream changes in phenotype but the mechanisms for this have, so far, been obscure. We consider the question of whether the topography directly nanoimprints onto the cell as a component of the signal transduction system. Evidence we present from SEM, TEM and fluorescence detection of the arrangements of cytoskeletal components is consistent with the possibility that cells are nanoimprinted by the substrate. The nanoprinting does not interfere with integrin-mediated adhesion processes and may perhaps work through them. Time-lapse video studies of cells moving from areas bearing nanotopography to flat areas and vice versa suggests that the nanoimprinting takes 1-6h to appear on the cell and a similar time to disappear when the cell moves from a flat surface to a nanotopographic one and back. This nanoprinting of cells would appear to be a novel type of cell signalling.

The effects of colloidal nanotopography on initial fibroblast adhesion and morphology.

Colloidal lithography offers a simple, inexpensive method of producing irregular nanotopographies, a pattern not easily attainable utilizing conventional serial writing processes. Colloids with 20- or 50-nm diameter were utilized to produce such an irregular topography and were characterized by calculating the percentage area coverage of particles. Interparticle and nearest neighbor spacing were also assessed for the individual colloids in the pattern. Two-way analysis of variance (ANOVA) indicated significant differences between the number of fibroblasts adhering to planar, 20-, and 50-nm-diameter colloidal topographies, the number of fibroblasts adhering to the substrates at the time intervals studied, namely 20 min, 1 h, and 3 h and significant interaction between time and topography on fibroblast adhesion (P < 0.01). Tukey tests were utilized for sensitive identification of the differences between the sample means and compounded ANOVA results. Cytoskeletal and general cell morphology were investigated on planar and colloidal substrates, and indicated cells in contact with irregular nanotopographies exhibit many peripheral protrusions while such protrusions are absent in cells on planar control surfaces. These protrusions are rich in microtubules on 20-nm-diameter colloidal surfaces while microfilaments are prevalent on 50-nm-diameter surfaces. Moreover, by 3 h, cells on the colloidal substrates initiate cell-cell adhesions, also absent in controls.

Nanoparticle targeting at cells.

Gold nanoparticles have been used for analytical and biomedical purposes for many years. In fact, the labeling of targeting molecules with nanoparticles has revolutionized the visualization of cellular or tissue components by electron microscopy. We report in this study the derivatization of tiopronin-protected nanoparticles with ethylenediamine and poly(ethylene glycol) bis(3-aminopropyl) terminated and their functionalization with the GRGDSP peptide sequence by a straightforward and economical methodology. The particles were subsequently tested in vitro with a human fibroblast cell line to determine the biocompatibility, and the cell-particle interactions, using fluorescence and scanning electron microscopies. The results indicate that tiopronin gold nanoparticles aggregate due to culture medium proteins, whereas the tiopronin gold nanoparticles derivatized with ethylenediamine induce endocytosis, and the same nanoparticles derivatized with poly(ethylene glycol) derivative promote particle-cell adhesion.

Cell signaling arising from nanotopography: implications for nanomedical devices.

This review discusses the roles in signaling to cells by nanochemical, nanostructural (nanotopography) and mechanical means, as well as recent work and trends in nanobioscience that are relevant to therapeutic applications. It is suggested that the mechanical results may often integrate the other two types of signal. Although the field is still in an almost embryonic but rapidly developing state, it is possible to envisage potential medical devices. Nanoparticle-based therapies are recognized as having some appreciable hazards, while those based on extended nanofeatured surfaces probably have fewer risks.

Human fibroblast reactions to standard and electropolished titanium and Ti-6Al-7Nb, and electropolished stainless steel.

Stainless steel (SS), titanium (cpTi), and Ti-6Al-7Nb (TAN) are frequently used metals in orthopedic internal fracture fixation. Although reactivity to SS and cpTi are noted in reference, the soft tissue compatibility of TAN has not been comprehensively studied. This study focuses on the in vitro soft tissue compatibility of TAN in comparison to SS and cpTi using a human fibroblast model. The industrial standard surface finishes of these three materials vary considerably in view of their use in similar applications. To distinguish between material parameters of topography and chemistry, we have included electropolished (e.p) counterparts of the standard preparations of cpTi and TAN in the study (standard SS is e.p). All materials were characterized using atomic force microscopy, profilometry, and scanning electron microscopy. Our findings demonstrate that cell morphology and growth rate was similar for SS, and e.p. cpTi and TAN, with cells well spread and forming a confluent monolayer by 10 days. Cell growth on standard cpTi was similar to the electropolished samples; however, they showed a less spread morphology with more filopodia and surface ruffling present. Cell morphology on standard TAN was rounded or elongated and proliferation was inhibited at all time points, with possible cell necrosis by day 10. We found evidence of endocytosis of beta-phase particles originating from the standard TAN surface. We believe that the particle uptake coupled with the characteristic surface topography contribute to the noncytocompatibility of fibroblasts on standard TAN.

Neutralisation of TGF beta or binding of VLA-4 to fibronectin prevents rat tendon adhesion following transection.

Following tendon injury, severe loss of function often occurs either as a result of obliteration of the synovial canal with fibrous scar tissue or from rupture of the repaired tendon. The role of cell engineering in tendon repair is to promote strong and rapid healing of tendon whilst at the same time facilitating rapid reconstitution of the synovial canal. Modification of the immediate inflammatory response around healing tendon has been found to be of value. Experimentally this has been achieved by neutralisation of transforming growth factor-beta over the first 3 days following injury, or by blockade of inflammatory cell binding to the CS-1 locus on fibronectin with an anti-VLA-4 antibody, or with the synthetic VLA-4 inhibitor, CS-1 peptide, in a rat model of tendon transection. It is concluded from this pilot study that the treatments described hold promise in improving outcomes of the common clinical problem of tendon injury in man.