1998 MRS Fall Meeting & Exhibit

Chairs

Elizabeth Chen, Johnson & Johnson Professional
Cato Laurencin, Allegheny Univ of Health Sciences
Michele Marcolongo, Drexel University
Grace Picciolo, Food & Drug Administration

Symposium Support 

• Johnson & Johnson Professional, Inc.
• Reprogenesis
• Telios Pharmaceuticals, Inc. 

* Invited paper

SESSION HH1: LOCALIZED PATTERNING FOR CELLULAR RESPONSE 
Chair: Elizabeth Chen 
Monday Morning, November 30, 1998 
Essex West (W)

9:00 AM *HH1.1 FROM CELL STRUCTURE TO BIOMIMETIC MATERIALS. Donald Ingber , Harvard Medical School and Childrens Hospital, and Molecular Geodesits, Inc. Abstract Not Available 

9:30 AM HH1.2 
MICROLITHOGRAPHIC PATTERNING OF POLYMER SUBSTRATES FOR DIRECTED NEURONAL GROWTH STUDIES. Kristine Schmalenberg , Rakhee Parikh, Youssef Travaly, Kathryn Uhrich, Rutgers University, Department of Chemistry, Piscataway, NJ; Tamanna Patel, Helen Buettner, Rutgers University, Department of Chemical and Biochemical Engineering, Piscataway, NJ. 

Microlithography is a technique that has long been used in the computer industry for patterning microchips. Recently, this method has been applied towards patterning inorganic substrates (e.g., glass) for neuron growth. Because most cells need a directional scaffold to promote phenotypical and genotypical expression, microlithography can provide a precise pattern along which cells can align, function and grow. Although there are many groups using inorganic substrates, there is little work on organic or even polymeric substrates. A polymeric material used for micropatterning must possess several qualities in order to be useful in vivo. It must be nontoxic, biodegradable, processable, transparent for microscopic analysis, and mechanically stable to provide a scaffold for cell growth. Poly(methyl methacrylate), poly(styrene) and poly(lactide-co-glycolide) were evaluated as substrates. The polymers were compressed into thin films using a heated Carver Laboratory press. Conventional microlithographic techniques were adapted for micropatterning these polymeric substrates. The patterned areas were filled with laminin to encourage neuronal growth. Using fluorescence microscopy neuronal alignment on the patterned surfaces was observed. To ensure that the lithographic process did not significantly change the polymer surface, we evaluated the polymeric substrates using x-ray photoelectron spectroscopy. 

9:45 AM HH1.3 
PROTEIN ADSORPTION AND CONFORMATION AND CELLULAR ATTACHMENT ON BIOMATERIALS SURFACES AS A FUNCTION OF SURFACE CHARGE. Michele S. Marcolongo , Drexel Univ, Dept of Materials Engineering, Philadelphia, PA; N. John DiNardo, Drexel Univ, Dept of Physics, Philadelphia, PA; Noreen J. Hickok, Thomas Jefferson Univ, Philadelphia, PA; Kambiz Pourezzaei, Richard B. Beard, Drexel Univ, Dept of Electrical and Computer Engineering, Philadelphia, PA; Daniel J. Brennan, Drexel Univ, Dept of Physics, Philadelphia, PA; Paul S. Heipp, Drexel Univ, School of Biomedical Engineering, Science and Health Systems, Philadelphia, PA; Tuan Phan, Drexel Univ, Dept of Chemical Engineering, Philadelphia, PA. 

Growth of tissues in biomaterials scaffolds is initially dependent on protein and cell attachment. Different biomaterials elicit cellular attachment to various degrees. When considering bone tissue, hydroxyapatite has a high degree of osteoblast attachment and bonding to bone tissue in vivo, while alumina will only elicit a fibrous encapsulation in vivo. The different biological responses to these materials can be attributed to their specific surface characteristics, including surface charge, chemistry, topography, and energy. Surface charge is thought to play an important role in influencing the electrostatic interaction between protein and surface, resulting in protein adsorption. In this work, we have evaluated the electrostatic influence of differently charged surfaces on protein and cell attachment. To examine the effect of surface charge on protein adsorption and conformation, we incubated fluorescently labeled fibronectin onto thin films of titanium of various surface charges of different polarities and magnitudes. Fibronectin adsorption was quantified fluorescently and conformation identified using atomic force microscopy in tapping mode. Similarly, primary human osteoblastic cells were cultured on the titanium thin films of various surface charges for six hours. Osteoblast attachment was quantified by adhesion assay and cell spreading was identified by confocal microscopy. Through a systematic understanding of surface interactions we can then engineer surfaces that enhance cellular attachment to biomaterials scaffolds for tissue engineering. 

SESSION HH2: BIOACTIVE SURFACES 
Chair: David H. Kohn 
Monday Morning, November 30, 1998 
Essex West (W)

10:30 AM *HH2.1 
BIOMIMETIC HYDROGELS FOR TISSUE ENGINEERING. Kevin E. Healy , Ali Rezania and Ranne A. Stile, Northwestern Univ., Div. of Biological Materials and Dept. of Biomedical Engineering, Chicago, IL. 

A major limitation in the performance of materials used in tissue engineering is that they lack the ability to integrate with biological systems through either molecular or cellular pathways. The inability to interact with biological systems has relegated biomaterials to a passive role dictated by the constituents of a particular environment, leading to unfavorable outcomes and suboptimal results. We have designed and synthesized model biomimetic materials that can be used to test hypotheses regarding the effect of cell-materials interactions on bone regeneration. We can control cell behavior chemically through specific ligand-receptor interactions. Materials grafted with mixed peptides from the cell and putative heparin-binding domains of bone sialoprotein (RGD/FHRRIKA) demonstrated enhanced strength of osteoblast attachment, spreading, and mineralization of the deposited matrix compared to surfaces grafted with either homogenous peptides or adsorbed vitronectin. Knowledge gained from these two dimensional experiments have been utilized to synthesize injectable thermo-reversible hydrogels that exploit these signals in three dimensions. The implications of this work are that the chemistry of a material can be modified to alter the kinetics of differentiation of mammalian cells, and may ultimately be used to control tissue regeneration within hydrogel scaffolds. 

11:00 AM HH2.2 
SPATIALLY CONTROLLED CELL ADHESION ON PATTERNED BIODEGRADABLE POLYMER SURFACES. Nikin Patel, Giles H.W. Saunders, Martyn C. Davies, Clive J. Roberts, Saul J.B. Tendler, Kevin M. Shakesheff , School of Pharmaceutical Sciences, The University of Nottingham, UK; Robert Padera, Scott M. Cannizzaro, Robert Langer, Department of Chemical Engineering, MIT, Cambridge MA. 

Controlling receptor-mediated interactions between cells and template surfaces is fundamental in many tissue engineering procedures. Biomaterial surfaces engineered to present cell adhesion ligands undergo integrin-mediated molecular interactions with cells, stimulating cell spreading and differentiation. In addition, the regeneration of human tissue structures such as nerves and blood vessels will require templates that control the spatial distribution of these ligands. Further, these new applications require the formation of ligand patterns on biocompatible and biodegradable templates, that control tissue regeneration processes, before removal by metabolism. Patterning technology promises to facilitate such spatially controlled tissue engineering. Here we present the development of a method for generating micron-scale patterns of any biotinylated ligand on the surface of a biodegradable block copolymer, polylactide-poly(ethylene glycol) (PLA-PEG). The technique achieves control of biomolecule deposition with nanometer precision by utilizing the technique of micro-fluidic networks. Spatial control over cell development has been observed when using these templates to culture bovine aortic endothelial cells and PC12 nerve cells. Furthermore, neurite extension on the biodegradable polymer surface is directed by pattern features composed of peptides containing the IKVAV sequence, suggesting that directional control over nerve regeneration on biodegradable biomaterials can be achieved. In addition, this technology can be adapted to promote ordering in heterogenous cell populations, with implications in the engineering of ordered structures in liver regeneration and vasculogenesis. 

11:15 AM HH2.3 
TOWARDS A TISSUE ENGINEERED BLADDER WALL PATCH. Jons Hilborn , Andre Laurent, EPFL, Polymers Laboratory, Lausanne, SWITZERLAND; Isabelle Bisson, Peter Frey, CHUV, Laboratory of Experimental Pediatric Surgery, Lausanne, SWITZERLAND. 

The design of biomaterials containing specific ligands on the surface offers the possibility of creating materials that can interact with and potentially control mammalian cell behavior. Biodegradable materials further provide the significant advantage that the polymer will disappear in vivo, obviating long-term negative tissue responses as well as the need for retrieval. A promotion of selective interactions that will target specific cellular response is the key. The base for this are molecular biological models of receptor - ligand binding. It is therefore a desire to combine the bulk properties of certain polymers with the cell interactive properties of proteins or peptides only on the surface. While UV, e-beam or g-irradiation and plasma are reliable methods for surface modification they suffer from molecular unspecificity and are accompanied by alterations of the base polymer. Here we report on a method that allows peptides or proteins that regulates cell adhesion to be covalently immobilized onto readily available and mechanically robust polyesters such as polyethyleneterephtalate (PET) or degradable poly(lactic acid). The technique being simple, roboust and straight forward allows for control of surface composition and cellular response. This scaffold preparation makes a part of a project aimed to tissue engineer a bladder wall patch for bladder augmentation or partial replacement treating congenital malformations of the urinary tract in children. Surface functionalization, characterization and results of human cell culture will be discussed. 

11:30 AM HH2.4 
DESIGN OF CELL-SIGNALING SCAFFOLDS FOR HEPATOCYTE TRANSPLANTATION. Darrell J. Irvine , A.M. Mayes, Massachusetts Institute of Technology, Dept. of Mater. Sci. and Eng., Cambridge, MA; Mark J. Powers, Linda G. Griffith, Massachusetts Institute of Technology, Dept. of Chemical Eng., Cambridge, MA. 

Scaffolds for cell transplantation which provide cell-signaling combined with protein resistance may provide the means for successful tissue engineering and implant therapies. To this end, branched copolymers having a comb architecture have been synthesized. The teeth of the comb are short hydrophilic poly(ethylene glycol) chains to which small peptide ligands may be selectively tethered, while the backbone consists of poly(lactide) segments. These copolymers provide protein-resistant surfaces which present cell-signaling ligands with tunable lateral distribution. Further, the amphiphilic nature of the copolymer has been exploited to use this copolymer as a surface segregating additive in poly(lactide), providing a self-assembling coating on poly(lactide) devices. We have studied this system for the design of cell scaffolds for tissue engineering, focusing in particular on the case of hepatocyte transplantation. The comb copolymer/PLA mixture allows the creation of porous scaffolds which resist nonspecific cell adhesion and concomitantly present tethered ligands for interaction with seeded cells. The incorporation of both adhesive peptides containing the RGD amino acid sequence and the growth factor EGF have been studied for the promotion of growth and maintenance of differentiated function of hepatocytes in vitro. 

SESSION HH3: ORTHOPAEDIC AND DENTAL APPLICATIONS 
Chair: Cato T. Laurencin 
Monday Afternoon, November 30, 1998 
Essex West (W)

1:30 PM *HH3.1 
TISSUE ENGINEERING WITH SMALL INTESTINE SUBMUCOSA: A XENOGRAPHIC SCAFFOLD FOR TISSUE REGENERATION.  Dick Tarr , DePuy, Inc. 

Abstract Not Available 

2:00 PM HH3.2 
EVOLVING STANDARDS FOR TISSUE ENGINEERED MEDICAL PRODUCTS. Grace Lee Picciolo , Kiki B. Hellman, Food and Drug Administration, Rockville, MD; Peter C. Johnson, Pittsburgh Tissue Engineering Initiative, Pittsburgh, PA. 

After a year of effort, progress has been made in the development of a number of draft standards for Tissue Engineered Medical Products (TEMPs). Operating within the consensus mechanism of the American Society for Testing and Materials (ASTM), a broad effort has resulted in draft standards in areas such as terminology, cell processing, tissue engineered biomaterials, animal models and other important aspects of tissue engineering technology development. The task groups represent a balance among industry, academia and government. The most accepted and useful specifications, characterization and test methods for product components are being selected on a consensus basis by members of all affected groups, as is the method used for standards acceptance worldwide. 
The TEMP standards effort was begun when it became clear that tissue engineering technology development would be rapid and would require multidisciplinary focus for both product development and regulatory review. It was felt that the engagement of a real time consensus building process would aid product development in this emerging field and hasten regulatory review of TEMPs. 
Research scientists, developers, industry and government representatives are invited to participate in this process, which is supported by electronic standards development forums through ASTM. The Task Groups meet twice yearly at ASTM meetings but will also be meeting more frequently at other venues. It is envisioned that the TEMP standards effort will provide a common ground for all disciplines whether from academia, industry and government to ensure that the field of tissue engineering progresses in a participatory and effective fashion. 

2:15 PM HH3.3 
TISSUE ENGINEERED SCAFFOLDS FOR ORTHOPAEDIC APPLICATIONS: IN VITRO CELLUAR REPSONSE. Frank K. Ko ^1,3, Cato T. Laurencin ^1,2,4, Mark D. Borden³, Mohamed A. Attawia⁴ and Darrell Reneker⁵, ¹Department of Materials Engineering, ²Department of Chemical Engineering, and ³School of Biomedical Engineering, Science, and Health Systems, Drexel University, Philadelphia, PA; ⁴Department of Orthopaedic Surgery, Allegheny University of the Health Sciences, Philadelphia, PA; ⁵Department of Polymer Science, University of Akron, Akron, OH. 

As a result of the progress made in biotechnology and biomaterials in the recent years, tissue engineering is quickly becoming a method of choice for the development of implants in orthopaedic surgery. In order to create porous 3-D scaffolds conducive for cell deposition and cell proliferation, there is a need for an understanding of the dynamic interaction between cell and matrix substrates. In our lab, we are examining several scaffolds for use in bone, ligament and cartilage reconstruction Of particular interest is the response of these types of cells to various tissue engineered structures. The basis of these scaffolds is the biodegradable co-polymer poly(lactide-co-glycolide) [PLAGA]. Five distinct matrices were created using either PLAGA microspheres or PLAGA fibers: 1) a 3-D fibrous braid 2) a nano-fiber nonwoven mesh, 3) a sintered microsphere matrix 4) a gel microsphere matrix, and 5) a solvent cast microsphere matrix. The cellular response of osteoblasts (bone repair), ligament fibroblasts (ligament repair), and chondrocytes (cartilage repair) was evaluated on each of the five matrices. Cells were cultured in vitro for 1, 3, 7, 14, and 21 days. The cellular response was assessed by cell proliferation, morphology, and phenotypic expression. Cell proliferation was measured by DNA assay, while morphology was examined by scanning electron microscopy. Since phenotypic expression is based on cell type, cellular mineralization was measured for osteoblasts, and collagen synthesis was measured for ligament fibroblasts and chondrocytes. 

2:30 PM HH3.4 
ATOMIC FORCE MICROSCOPY TO QUANTIFY LOCAL MECHANICAL PROPERTIES OF ENGINEERED TISSUE. Stan Tsai ^1,2, R. Bruce Rutherford ³, Brian H. Clarkson ³, David H. Kohn ^1,2, University of Michigan, Departments of ¹ Biologic and Materials Sciences, ² Biomedical Engineering, ³ Cariology, Restorative Sciences and Endodontics, Ann Arbor, MI. 

For regenerated tissue to be functional, it must exhibit the mechanical integrity of normal tissue. Because of the composite nature of tissues, regional variation in properties, and fact that macroscopic mechanical testing is not always feasible with regenerated tissue, microscopic approaches are needed to better delineate structure-function relations. We have been utilizing atomic force microscopy (AFM) to determine local, sub-micron level mechanical properties of normal and regenerated tissues. In this paper, we discuss the technique and demonstrate that property variations within the 1um spatial resolution of AFM are less than variations based on anatomic location. We modified an AFM by replacing the Si3N4 probe and silicon cantilever with a diamond pico-indenter tip. Mechanical measurements were made by placing the tip in a desired location within an image and applying a force sufficient to plastically indent the sample. Hardness was determined by relating the applied force to the projected contact area. Stiffness is the slope of the initial portion of the unloading curve and is related to an effective modulus, which is a function of the elastic constants of the tissue and probe tip. AFM indentations of normal and regenerated bone and dentin were made at multiple locations across perpendicular diameters of 3 mm thick annular sections. As representative data, regional stiffnesses (in GPa) of dentin are presented: Facial Quadrant: Pulp 18.5 (0.9), Middle 19.0 (0.4), DEJ 24.5 (1.5); Lingual Quadrant: Pulp 19.1 (3.1), Middle 24.4 (4.5), DEJ 24.9 (1.3); Mesial Quadrant: Pulp 16.6 (1.3), Middle 23.9 (0.8), DEJ 21.5 (1.8); Distal Quadrant: Pulp 15.5 (1.4), Middle 19.6 (3.8), DEJ 23.0 (2.4). Repeated measures ANOVA indicated that moduli were site-specific. However, local variations at a given point (e.g. properties at < 1um spatial displacement) were less than site to site variations, validating the efficacy of the technique. Hardness and elasticity may be considered quantitative measures of degree of mineralization, enabling linkage between mechanical, structural and biological measures of tissue regeneration. 

2:45 PM HH3.5 
MECHANICAL AND STRUCTURAL COUPLING AT DENTIN-ENAMEL JUNCTION FOR ENAMEL BIOMIMETICS. H. Fong and M. Sarikaya, Materials Science and Engineering, University of Washington, Seattle, WA; S. White and M. Snead, Center for Craniofacial Molecular Biology, Univ. of Southern California, Los Angeles. 

The human tooth is an inorganic/organic composite material containing collagen and hydroxyapatite (HA). Two sections of the hard tissue, dentin and enamel, are connected by dentin-enamel junction (DEJ), whose structure and mechanical properties are critical parameters for rational biomimetic design of teeth. The DEJ is where structural interlocking takes place and which provides mechanical coupling between the hard (enamel) and the soft (dentin) regions forming a functionally-gradient biogenic tool. AFM and TEM investigations reveal structures of the surfaces and interfaces, respectively, over several orders of dimension (from nanometer to macroscale) including the crystallography and morphology of the inorganic component. Furthermore, nanomechanical testing using a nanoindentor attached to an AFM, reveal a detailed hardness and elastic modulus profiles across the DEJ. For example, the hardness profile reveals a hard exterior region (4.2 GPa) that decreases (3.2 GPa) just before the DEJ, and then, with a step function, down to a soft region (0.86 GPa) in dentin that continuously decreases (down to 0.64 GPa) in the interior of the tissue. These results and those from controlled biomineralization using transgenic mice (affects of enamel proteins) are directed toward rational enamel biomimetics using polymeric scaffolds. 

SESSION HH4: SCAFFOLD AND CELL CHARACTERIZATION TECHNIQUES 
Chair: Kevin E. Healy 
Monday Afternoon, November 30, 1998 
Essex West (W)

3:30 PM HH4.1 
SYNCHROTRON INFRARED MICROSPECTROSCOPY FOR ASSESSMENT OF MUTAGENICITY OF METAL IMPLANTS. Miqin Zhang , Univ of California, Dept of Materials Science, Berkeley, CA; Hoi-Ying N. Holman, Lawrence Berkeley National Laboratory, Berkeley, CA; Mauro Ferrari, Univ of California, Dept of Materials Science, Berkeley, CA; Jennie C. Huntercevera, Lawrence Berkeley National Laboratory, Berkeley, CA. 

A major concern in the development and implementation of metal implants for the clinical use is the assessment of material-induced mutagenesis. In this study we used synchrotron microspectroscopy in the mid-infrared region (4000-400 cm-1) to non-invasively assess the in situ human cell responses to metal surfaces. Specifically, we examined the subtle genetic aberrations of cells as they responsed to a range of metals commonly used in the metal prosthetic devices. Relative band intensities and band intensity ratios for functional groups of biomolecules that are inherent to the experimental system were examined. The molecular components of the biomolecules as they were perturbed by interactions with metals were investigated. Mapping of the spectral markers for the interactions at the biological metal interfaces will be presented and discussed. The changes of infrared spectra were related to the changes of cell morphology observed using conventional light microscopy. These results demonstrate the potential use of synchrotron FTIR microspectroscopy to screen the mutagenicity of metal implants. 

3:45 PM HH4.2 
NONDESTRUCTIVE EVALUATION OF SYNTHETIC TISSUE SCAFFOLDS WITH NMR. Leoncio Garrido , Biomaterials Laboratory, NMR Center, Department of Radiology, Massachusetts General Hospital and Harvard Medical School, Charlestown, MA. 

The design of functional biodegradable tissue scaffolds requires detailed knowledge of the morphology of the porous matrix and the interaction between implant and tissue. Under physiological conditions, this interaction is a dynamic heterogeneous process. The degradation rate of most polymers is not constant and usually is not confined to the interface between implant and tissue. Water and other active agents (e.g., enzymes) penetrate into the matrix, initiating the hydrolytic reaction that leads to the breakdown of the polymer chains and disintegration of the synthetic scaffold. Destructive methods, such as histology, may introduce artifacts in the evaluation of implant architecture, as well as in the study of implant-tissue interaction. As a solution to these problems, we are developing non-invasive and non-destructive characterization nuclear magnetic resonance (NMR) methods that may provide the required information. The application of NMR-based methodology to measure in situ the changes in porosity and tissue infiltration with aging in synthetic biodegradable composites of polyalkanoates will be presented. 

4:00 PM HH4.3 
NORMAL AND CANCER LYMPHOCYTES INVESTIGATED BY TIME DOMAIN DIELECTRIC SPECTROSCOPY. Yulia Polevaya , Irina Ermolina, Yuri Feldman, The Department of Applied Physics, The Hebrew University of Jerusalem, Jerusalem, ISRAEL; Michael Schlesinger, Hadassa Medical School, Hebrew University of Jerusalem, Jerusalem, ISRAEL; Ben-Zion Ginzburg, Institute of Life Science, Hebrew University of Jerusalem, Jerusalem, ISRAEL. 

An interesting and important subject in biophysics is the investigation of the dielectric properties of cells and it's structural components (membranes, cytoplasm etc.). These can bring valuable knowledge about different cell structures, their functions and metabolic mechanism. The aim of this contribution is a comprehensive theoretical and experimental study by means of time domain dielectric spectroscopy (TDDS) of static and dynamic dielectric properties of normal and malignant blood cells of different types. A suspensions of normal, transformed (Magala) and leukemic (Farage, Raji, Daudi, Bjab, Peer and HDMar) lymphocytes of T and B types were investigated at the low volume fractions (3-10%) where the cellular interactions are negligible. The measurements were performed at a wide frequency range from 200 KHz to 1 GHz at room temperature 25C. The effect of electrode polarization in TDDS experiment was corrected The T-normal Iymphocytes were isolated from the human blood, whereas B-normal ones were segregated from tonsils. The transformed and malignant cells were grown in cell cultures. Cell radii and volume fractions were measured by independent methods. An analysis of the dielectric spectra was performed by the use of Maxwell-Wagner mixture model and the double-shell model of single cell to obtain the dielectric parameters of cell components The capacitance of outer cell membranes were calculated by the Hanai-Asami-Koizumi equation. This value ranged from 0.7 to 1.6 F/cm² for investigated cell lines. 

4:15 PM HH4.4 
INITIAL CELLULAR ADHESION AND FGF-SIMULATED GROWTH OF ENDOTHELIAL CELLS MONITORED IN REAL TIME USING THE QUARTZ CRYSTAL MICROBALANCE (QCM). Tiean Zhou ^*, Susan J. Braunhut, Diane Medeiros and Kenneth A. Marx^*, Center for Intelligent Biomaterials^*, Departs of Chemistry^* and Biological Sciences, University of Massachusetts, Lowell, MA. 

Edothelial cells (ECs), lining the blood vessels, function to regulate blood flow, and are normally stringently growth inhibited. However, during inflammation and injury, new vessels rapidly form under the influence of growth factors such as FGF. Alterations in the degree of adhesion, cellular spreading and cellular mass distribution of ECs accompanies this change in phenotype from one of growth inhibition to growth stimulation. We have applied the QCM technique to continuously record the processes of EC adhesion, spreading and cellular mass distribution changes during initial cell to gold-coated quartz cyrstal surface contact, homeostatic attachment to the crystal and challenge with a mitogen. ECs (50,000) were layered onto a set volume of media in the QCM device and sedimented through a specific distance to the QCM surface. Simultaneously, ECs were sedimented in the same manner through an identical distance in a mock device used for subsequent photomicroscopy. As cells were observed in the mock device to contact (20 min) and extend processes on the surface, in the QCM device we measured a continuous decrease in crystal frequency and increase in crystal resistance. By 1 hr., all cells were observed to contact the surface in the mock device. In parallel, we measured a continuous decrease in QCM frequency and continuous increase in resistance, acheiving maximum values at one hr (as high as 1400 Hz frequency change; 1400  resistance change). These frequency and resistance values stabilized over the next 12 hrs. by QCM measurement (to a 700 Hz decrease, to a 700  increase), as the cells were observed to uniformly spread, covering a larger surface area in the mock device.