What if a bone-regenerating scaffold could do more than just hold cells in place? This study combines a piezoelectric polymer, a fibronectin-forming coating, and vibrations only nanometers wide to influence stem-cell behavior.

There is an unmet clinical need to provide viable bone grafts for clinical use. Autologous bone is commonly transplanted, but it is associated with donor site morbidity. Tissue engineering strategies could differentiate an autologous cell source, such as mesenchymal stromal cells, into a potential bone-graft material.

But osteogenesis of these cells can require long culture periods that are impractical in a clinical setting and can lead to significant cost. The proposed strategy uses the piezoelectric copolymer poly(vinylidene fluoride–trifluoroethylene), functionalized with a poly(ethyl acrylate) coating that drives fibronectin network formation, to enhance MSC adhesion and present growth factors in the solid phase.

Dynamic electrical cues are incorporated through a nanovibrational bioreactor, and the MSC response to electromechanical stimulation is investigated. Bone regeneration continues to be challenging in the clinic, even though autologous bone grafts are the gold standard graft material and possess properties that promote bone repair.

Autologous bone has limited supply, and harvesting it requires a second surgical procedure associated with donor site morbidity. Decellularized grafts also have limitations, including donor quality and the risk of host immunogenic responses. In stressed bone, electrical potentials depend on mechanical deformation.

Nanoscale mechanical force has also been shown as a viable approach to stimulating osteogenesis of mesenchymal stromal cells. Using a nanovibrational bioreactor, MSCs undergo robust osteogenic commitment in two-dimensional and three-dimensional collagen gels. However, nanokicking typically requires more than twenty-eight days of stimulation to drive osteogenesis.

That turnaround time is impractical in a clinical setting and would require extensive equipment hours at significant cost, while longer culture times are associated with infection, cell phenotypic drift, and viability concerns. The study therefore applies mechanical forces from the nanovibrational bioreactor to PVDF-TrFE, aiming to incorporate dynamic electrical cues and investigate its potential to promote osteogenesis of MSCs.

The system introduces piezoelectric stimulation through PVDF-TrFE into the NK bioreactor system. PEA and FN coatings are incorporated to enhance cell adhesion and potentially present osteogenic growth factors to cells in the solid phase.

By incorporating multiple stimuli native to bone, the system is investigated for its potential to promote osteogenesis of MSCs. PVDF-TrFE is capable of producing physiologically relevant piezoelectric cues. The PVDF-TrFE copolymer was dissolved in a N,N-dimethylformamide and acetone solution and cast onto glass to form films twenty to thirty micrometers thick.

The films were then annealed at one hundred twenty degrees Celsius for twelve hours. Electrical poling was carried out using direct-current voltage: thin films were clamped between steel electrodes and subjected to an electric field of one hundred volts per micrometer for five minutes.

Custom cell culture plates were magnetically attached to a vibration plate secured to an array of low-profile, multilayer piezo actuators. A custom power supply provided a one-thousand-hertz sine-wave modulation. This setup made the vibration plate oscillate at an amplitude of thirty nanometers and a frequency of one thousand hertz.

Figure one shows how the PVDF-TrFE films are integrated into a six-well culture plate and driven by the nanovibrational bioreactor at one kilohertz. Panel B maps the measured z-displacement across the bioreactor plate and culture wells, while panel C records voltage generated by poled and unpoled films during vibration.

Together, these panels establish the setup’s mechanical stimulation and electrical output, which matters because the study links nanoscale deformation and electromechanical signaling to cell culture responses. The volcano plate was optimized for further experiments to minimize variation.

The voltage generated by poled and unpoled PVDF-TrFE films mounted on volcano culture plates was then measured under one-kilohertz vibration. The measured peak-to-peak voltage was zero point six plus or minus zero point one millivolts for unpoled films and one point six plus or minus zero point zero three millivolts for poled films.

Figure two shows XPS core-level spectra for unpoled and poled PVDF-TrFE, followed by PVDF-TrFE coated with plasma-polymerized PEA. The panels track bromine, carbon, nitrogen, and oxygen binding environments within roughly the top ten nanometers, with fitted peaks marking different chemical conformations.

After polymerization, the spectra for the two films are described as identical, and the carbon and oxygen signals represent pPEA, confirming the intended surface modification. The total surface density of fibronectin showed no significant variation on any surface.

The analysis also measured the availability of specific cryptic fibronectin binding domains. The synergy domain, PHSRN, and the growth-factor-binding domain showed a similar trend: poled surfaces with or without pPEA presented higher availability of these binding domains.

Poled PVDF-TrFE without pPEA had the highest degree of hydrophobicity, retained even after fibronectin treatment, and this condition showed a relative increase in the presentation of cryptic fibronectin binding domains. Figure three links surface structure to fibronectin function.

AFM scans show that plasma PEA changes the film topography, with fibronectin nanonetworks especially visible on poled, treated surfaces; the water-contact-angle measurements also show how these treatments alter surface hydrophobicity. Importantly, total fibronectin density remains comparable across conditions, while the synergy and growth-factor-binding domains show increased availability in poled conditions, suggesting that polarization and surface treatment can influence how cells access adhesive signals.

MSCs were cultured on the films for up to seven days. On unpoled PVDF-TrFE without pPEA or fibronectin, initial viability was high at twenty-four hours, decreased at seventy-two hours, and no attached cells were detected by day seven. Cell-number quantification indicates that the initially high viability resulted from a low number of attaching cells.

Cell viability and number were high on the other unpoled films, including unpoled films with pPEA but without fibronectin. pPEA without fibronectin led to low cell viability and adhesion on poled films at all time points, and poling led to significant reductions in cell viability and number.

When poled PVDF-TrFE was coated with pPEA and fibronectin, viability significantly increased. This supports the potential use of pPEA and fibronectin on poled films for bioengineering applications. Figure four tracks MSC cell viability and average cell number per frame on unpoled and poled PVDF-TrFE films, with or without pPEA coating and fibronectin, at twenty-four hours, seventy-two hours, and seven days.

Panel A uses live/dead analysis, while panel B reports cell counts; bars show means with standard deviations, and significance is evaluated by two-way ANOVA. This matters because it tests whether the surface modifications support MSC survival and retention over one week, motivating the later analysis of focal adhesions and osteogenic commitment.

Nanovibration, or nanokicking, has been shown to promote commitment to osteogenesis. After pPEA and fibronectin increased cell viability and focal-adhesion formation, nanokicking was incorporated with the aim of driving osteogenic commitment.

Poled and unpoled PVDF-TrFE with pPEA and fibronectin were mounted onto custom-made volcano tissue-culture plates. Under one-kilohertz nanokicking, the resulting amplitude was eighty-seven point one nanometers. Figure five examines early adhesion of mesenchymal stem cells after four hours on poled PVDF-TrFE films, comparing pPEA and fibronectin coatings.

Actin images and quantification show that the combined pPEA-plus-fibronectin coating is associated with greater cell spreading and lower circularity in both poling conditions, while vinculin staining shows significantly longer focal adhesions. The lack of a poling effect on focal-adhesion length suggests that surface chemistry, rather than electrical orientation alone, strongly shapes these initial cell–material interactions.

Figure six examines how nanokicking changes mesenchymal stem-cell attachment on pPEA- and fibronectin-modified PVDF-TrFE, comparing poled and unpoled films. The images show nuclei, actin, and vinculin, while the charts quantify cell density, area, circularity, focal-adhesion length, and focal-adhesion size distributions after three days.

Together, these measurements connect the mechanical stimulus and film poling state to cell spreading and adhesion organization, supporting the authors’ strategy for improving early cell interactions in the bioreactor. To evaluate osteogenic potential, BMP-2 was adsorbed to PVDF-TrFE with pPEA and fibronectin to support a low-dose, one-hundred-nanograms-per-milliliter, solid-phase presentation to MSCs.

Cells were cultured for fourteen days, and relative fluorescent intensity was measured for the early osteogenic markers ON and OSX. Expression of both ON and OSX increased only on poled, pPEA-and-fibronectin-coated films subjected to nanomechanical stimulation.

Expression was increased relative to static poled, pPEA-and-fibronectin-coated films, suggesting that nanovibration could enhance osteogenic commitment on these surface-modified films. Figure seven shows how pPEA-coated PVDF-TrFE films organize fibronectin, exposing growth-factor and integrin-binding domains that can present low-dose BMP-2 in the solid phase.

The heatmap then reports osteogenic marker fluorescence for MSCs after fourteen days, comparing pole, pPEA, and nanokicking conditions. The authors observed increased osteocalcin and osterix expression in the positively poled, pPEA- and fibronectin-coated films subjected to nanokicking, highlighting the system’s osteogenic potential.

Taken together, the data suggest that poled PVDF responds to mechanical stimulation, yet pristine poled PVDF-TrFE does not support cell–material interactions. Enhancing adhesive interactions through surface modification with pPEA and fibronectin allowed low-amplitude, high-frequency nanokicking to be incorporated into the system.

The piezoelectric films require a degree of deformation to fully optimize the charge induced from poling, meaning that deformation is necessary to maximize the charge generated through the poling process. PVDF-TrFE films have been shown to synergize with one-hertz, thirty-nanometer-amplitude nanokicking, producing approximately thirty-six picocoulombs per newton of charge and a modified amplitude of eighty-seven point one nanometers.

Because nanovibrational stimulation appeared to promote early osteogenic-marker expression, nanoscale coatings such as pPEA and fibronectin have the potential to increase the bioactivity of smart materials such as PVDF-TrFE. The central takeaway is that PEA and fibronectin coatings made poled PVDF-TrFE more supportive of MSC adhesion, while nanomechanical stimulation increased early osteogenic-marker expression under the tested conditions.

That combination points toward a more bioactive smart material.