PZLA
Piezoelectric Lactic Acid
In collaboration with Jerry Scheinbeim PhD & Carlos Palant MD

Introduction & Background

Demonstration of PZLA

Mechanical deformation of piezoelectrically active PLLA (PZLA) produces a redistribution of surface charge.

    Polymeric materials have been used for biomedical and biomaterials applications for centuries, dating back to the first uses of wood for the support of fractured human exoskeleton and flora for wound healing. Synthetic polymers have been employed in surgical and tissue engineering modalities for several decades. One such polyester, Poly(L-lactic acid) (PLLA) has been used in its fibrous conformation as a biodegradable suture since the 1950s, and more recently as a substrate for cell regenerative therapies.

    PLLA is not only biocompatible, but completely bioresorbable (hydrolyzing to water and lactic acid, a natural by-product of skeletal muscle activity), and also intrinsically piezoelectric (PZ). This inherent property of PLLA enables the polymer to become PZ-active simply by stretching the material. The PZ activity of PLLA produces a change in the internal polar nature of the material, yielding a redistribution of surface charge which can be felt by cells on the surface, which are sensitive to electrochemical gradients.

Fibroblasts on PLLA and PZLA

Rat dermal fibroblasts seeded onto PLLA (top) and PZLA (bottom). Cells seeded onto PZLA exhibited greater density of cell growth at 24 hours.

Preliminary Cell Culture Results

Enhanced fibroblast outgrowth
    Rat dermal fibroblasts were seeded onto sterilized PLLA (unstretched/PZ-inactive) films and stretched, PZ-active PZLA films cemented to the bottom of the well-plate with a methacrylate-based adhesive. Plate tissue culture polystyrene (TCPS) served as the control condition. Cells were made to express Green Fluorescent Protein (GFP) and seeded at a density of 100 000/mL in 0.5mL of serum solution (88% DMEM, 10% FBS, 1% L-Glutamine, 1% Pen/Strep). Cultures were incubated in physiological conditions (37deg C, 100% humidity, 5% pCO2). The well plate was also subject to an external stimulation protocol wherein the culutures were vibrated at 30Hz at a moderate amplitude (~20 mV) by a 1.5% audio speaker situated in an insulated polycarbonate platform, starting at 6H post-seeding time to allow for proper attachment. Cell proliferation was determined as the percentage of frame (fluorescent microscopy images) pixellation after a minimum-value thresholding.

    Fibroblasts showed considerably greater growth on the PLLA (3.82X amplification in outgrowth @ T=72 hours vs T=20 hours) and PZLA (2.99X) as compared to controls (2.56X). Though cells on PZLA proliferated at a slower rate in the first 48 hours, cell growth on the PZ-active films in the 3rd day was observed to be greater than 2X that of PLLA and controls.

Mesangial cell migration & alignment
    SV40-transformed mesangial cells were seeded onto PLLA, PZLA, and glass, and bathed in serum-free media. Cell cultures were incubated in physiological conditions and evaluated using time lapse bright field microscopy (24 hours @ 15-minute intervals). Cells were analyzed using image processing of bright-field frames. Cell bodies were acquired via a Gaussian sliding operator with neighborhood standard deviation output, thresholding operation for a minimum luminescence value (selected for artifact minimization), and binary dilation (disk strelation). Bounding box, centroid, and cell area acquisition were performed via inline feature extraction operations. Cell adhesion time was defined as the step-wise increaes in either bounding box area or in area fraction (the ratio of cell area to bounding box area) commensurate with the spread of fibrous processes from the soma. Cell velocities were calculated as the point-wise square root of the sum of the squares of the discrete derivative in the 2-dimensional motion of the cell's centroid.

    A greater fraction of the cells seeded onto TCPS and PZLA surfaces attached and spread processes, developing a cell network. Proto-tissue (synctitia) was observed in the PZLA condition after ~18 hours. Cell migration velocities wer maximal on PLLA, moving 3X as fast as cells seeded onto PZLA and control. Cell adhesion time, however, was shortest (13.5 Hours) in the PLLA condition, slightly slower on PZLA (14.35 Hours), and longest on controls (15.8 Hours).

Cells on glass slide control: 18 Hours Mesangial cells on unstretched PLLA: 18 Hours Mesangial cells on stretched PZLA: 18 Hours

Mesangial cells seeded on glass slide controls (left), unstretched PLLA (middle), and stretched PZLA (right). Click on images to see time-lapse microscopy of the cells over 18 hours.

Preliminary Material Characterization

Dynamic mechanical and piezoelectric properties
Piezoelectric strain: PZLA vs PLLA

Piezoelectric strain coefficients greatly increased for uniaxially stretched PLLA films (2.23pC/N) over the undrawn condition (0.06pC/N).

    PLLA films were melt-pressed from powder and drawn uniaxially to a ratio of 3.5:1 (final length:initial length) at a rate of 0.25mm/min in a thermal bath of 80deg C, well above the polymer's glass transition temperature (~60deg C). Films were permitted to relax at room temperature overnight, and draw ratio was confirmed by fiducial markers along the films' edges, thus ensuring uniform stretching of the material. Gold electrodes were sputtered at room temperature and samples were placed into a rheolograph solid mechanical tester @ 100Hz at room temperature.

    Piezoelectric strain (picoColoumbs of charge per Newton of strain) were 2 orders of magnitude greater in the stretched (3.5:1) condition than in the unstretched condition. Whereas no polarizing of the films was performed, this 100-fold increase in PZ strain confirms that mere uniaxial stretching is sufficient to induce PLLA's PZ activity. PZ strains of 2.23pC/N (3.5:1) may pose considerably more stimulation to cells on PZLA than those on traditional PLLA (PZ strain = 0.06pC/N).