Biomedical subjects

Kevin E Healy

Publications and source records attributed to Kevin E Healy.

At least 19 recordsLinked to original sources

PubMed2007

The effect of enzymatically degradable IPN coatings on peri-implant bone formation and implant fixation.

Short-term osseointegration of orthopedic implants is critical for the long-term stability of the implant-bone interface. To improve initial implant stability, one strategy under consideration involves the presentation of adhesion ligands on the implant surface to stimulate bone regeneration in the peri-implant region. To assess the relative effects of implant surface chemistry and topography on osseointegration within the rat femoral ablation implant model, a nonfouling, enzymatically degradable interpenetrating polymer network (edIPN) of poly(AAm-co-EG/AAc) amenable to presenting the cell signaling domain Arg-Gly-Asp (RGD), was developed. Moderate enhancement of peri-implant bone formation was found after 28 days using the edIPN without peptide modification (p = 0.032). However, no data supported a benefit of peptide modification, as bone-implant contact, normalized bone volume and normalized fixation strength was equivalent or poorer than dual acid-etched (DAE) treated implants after 28 days. Surface topography was determined to be the dominant factor in modulating osseointegration, as DAE implants produced equivalent roughness-normalized fixation strength versus previously reported data on plasma-sprayed hydroxyapatite/tricalcium phosphate-coated implants (Barber et al., J Biomed Mater Res A, forthcoming). An ideal osseointegrated implant will require optimization of all three aforementioned parameters, and may take the form of biomolecule delivery from thin degradable polymer networks.

Analysis of Variance↗

PubMed2007

Peri-implant bone formation and implant integration strength of peptide-modified p(AAM-co-EG/AAC) interpenetrating polymer network-coated titanium implants.

Interpenetrating polymer networks (IPNs) of poly (acrylamide-co-ethylene glycol/acrylic acid) functionalized with an -Arg-Gly-Asp- (RGD) containing 15 amino acid peptides, derived from rat bone sialoprotein (bsp-RGD(15), were grafted to titanium implants in an effort to modulate bone formation in the peri-implant region in the rat femoral ablation model. Bone-implant contact (BIC) and bone formation within the medullary canal were determined using microcomputed tomography at 2 and 4 weeks postimplantation. BIC for bsp-RGD(15)-IPN implants was enhanced relative to hydroxyapatite tricalcium phosphate (HA-TCP) coated implants, but was similar to all other groups. Aggregate bone formation neither indicated a dose-dependent effect of bsp-RGD(15) nor a meaningful trend. Mechanical testing of implant fixation revealed that only the HA-TCP coated implants supported significant (>1 MPa) interfacial shear strength, despite exhibiting lower overall BIC, an indication that bone ingrowth into the rougher coating was the primary mode of implant fixation. While no evidence was found to support the hypothesis that bsp-RGD(15)-modified IPN coated implants significantly impacted bone-implant bonding, these results point to the lack of correlation between in vitro studies employing primary osteoblasts and in vivo wound healing in the peri-implant region.

Acrylates↗

PubMed2006

Biomimetic artificial ECMs stimulate bone regeneration.

We demonstrate that a biomimetic polymer network is capable of affecting bone regeneration in vivo. Starting with a foundation consisting of an environmentally responsive poly(N-isopropylacrylamide-co-acrylic acid) hydrogel, we incorporated matrix metalloproteinase-13 (MMP-13) degradable crosslinkers and peptides containing integrin-binding domains (i.e., Arg-Gly-Asp) to create a biomimetic matrix designed to encourage osteoblast migration and proliferation. We independently tuned matrix stiffness and peptide concentration to generate a response surface model of osteoblast proliferation on different types of matrices. Osteoblast proliferation was significantly influenced by matrix stiffness (i.e., its complex modulus) and peptide concentration. When implanted in a rat femoral ablation model, these matrices induced bone regeneration only when protease degradable crosslinks were used to create the network. For the matrices with MMP-13 degradable crosslinkers, the bone formed had a trabecular-like structure and was distributed throughout the marrow space. Based on the correlated effects of matrix stiffness and ligand concentration, the response surface model will facilitate improvements in the regenerative capacity of these artificial extracellular matrices.

Animals↗

PubMed2006

Theoretical impact of the injection of material into the myocardium: a finite element model simulation.

BACKGROUND: To treat cardiac injuries created by myocardial infarcts, current approaches seek to add cells and/or synthetic extracellular matrices to the damaged ventricle to restore function. Because definitive myocardial regeneration remains undemonstrated, we propose that cardiac changes observed from implanted materials may result from altered mechanisms of the ventricle. METHODS AND RESULTS: We exploited a validated finite element model of an ovine left ventricle with an anteroapical infarct to examine the short-term effect of injecting material to the left ventricular wall. The model's mesh and regional material properties were modified to simulate expected changes. Three sets of simulations were run: (1) single injection to the anterior border zone; (2) therapeutic multiple border zone injections; and (3) injection of material to the infarct region. Results indicate that additions to the border zone decrease end-systolic fiber stress proportionally to the fractional volume added, with stiffer materials improving this attenuation. As a potential therapy, small changes in wall volume (approximately 4.5%) reduce elevated border zone fiber stresses from mean end-systole levels of 28.2 kPa (control) to 23.3 kPa (treatment), similar to levels of 22.5 kPA computed in remote regions. In the infarct, injection improves ejection fraction and the stroke volume/end-diastolic volume relationship but has no effect on the stroke volume/end-diastolic pressure relationship. CONCLUSIONS: Simulations indicate that the addition of noncontractile material to a damaged left ventricular wall has important effects on cardiac mechanics, with potentially beneficial reduction of elevated myofiber stresses, as well as confounding changes to clinical left ventricular metrics.

Animals↗

PubMed2006

Hydrogels as artificial matrices for human embryonic stem cell self-renewal.

Human embryonic stem cells (hESCs) have the potential to differentiate into all cell types in the body and hold great promise for regenerative medicine; however, large-scale expansion of undifferentiated hESCs remains a major challenge. Self-renewal of hESCs requires culturing these cells on either mouse or human fibroblast cells (i.e., a feeder layer of cells), or on artificial extracellular matrices (ECMs) while supplementing the media with soluble growth factors. Here we report a completely synthetic ECM system composed of a semi-interpenetrating polymer network (sIPN), a polymer hydrogel, which was designed to allow the independent manipulation of cell adhesion ligand presentation and matrix stiffness. In the short term, hESCs that were cultured on the sIPN adhered to the surface, remained viable, maintained the morphology, and expressed the markers of undifferentiated hESCs. This was the first demonstration that a completely synthetic ECM can support short-term self-renewal of hESCs.

Biocompatible Materials↗

PubMed2006

In vitro characterization of peptide-modified p(AAm-co-EG/AAc) IPN-coated titanium implants.

Interpenetrating polymer networks (IPNs) of poly(acrylamide-co-ethylene glycol/acrylic acid) [p(AAm-co-EG/AAc)] functionalized with an -Arg-Gly-Asp- containing peptide derived from rat bone sialoprotein [bsp-RGD(15)] were grafted to titanium implants in an effort to modulate osteoblast behavior in vitro. Surface characterization data were consistent with the presence of an IPN, and ligand density measurements established that the range of peptide density on the modified implants spanned three orders of magnitude (0.01-20 pmol/cm2). In vitro biological characterization of the modified implants employing the primary rat calvarial osteoblast (RCO) model resulted in the identification of a critical ligand density (0.01<Gammacrit<0.1 pmol/cm2) for maximal support of the osteoblast phenotype. After 14 and 21 days, mineralization was greater on the 0.1 and 10 pmol/cm2 bsp-RGD(15) modified implants compared to the base titanium and other control surfaces. The observed effects were attributed to specific interactions with bsp-RGD(15) and support the concept that peptide-modified implants can enhance the kinetics of differentiation of the cells they contact. These results suggest that in vivo biological performance evaluation of these biomimetic implant surfaces is merited.

Acrylic Resins↗

PubMed2005

The effect of ligand type and density on osteoblast adhesion, proliferation, and matrix mineralization.

Polystyrene surfaces grafted with a nonfouling interfacial interpenetrating polymer network (IPN) of poly(acrylamide-co-ethylene glycol/acrylic acid) [p(AAm-co-EG/AAc)] were modified with several peptide ligands adapted from bone sialoprotein (BSP). IPNs were modified with both single ligands and ligand blends to study the correlation between a simple metric, ligand-receptor adhesion strength, and the extent of matrix mineralization for osteoblast like cells (rat calvarial osteoblasts). The ligands studied included RGD cell-binding [CGGNGEPRGDTYRAY (l-RGD), CGGEPRGDTYRA (s2-RGD), CGPRGDTYG (lc-RGD), cyclic(CGPRGDTYG) (c-RGD), and CGGPRGDT (s-RGD)], heparin binding (CGGFHRRIKA), and collagen binding (CGGDGEAG) peptides, with the appropriate controls. Adhesion strength scaled with ligand density (1-20 pmol/cm(2)) and was dependent on ligand type with the following trend: l-RGD > s2-RGD approximately c-RGD >> s-RGD approximately lc-RGD >>> FHRRIKA approximately DGEA. Independent of ligand density, % matrix mineralization varied with ligand type resulting in the following trend: lc-RGD > s2-RGD > l-RGD approximately c-RGD >> s-RGD >>> FHRRIKA. The Tyr (Y) residue immediately following the RGD cell-binding domain proved to be critical for stable cell proliferation and mineralization, since removal of this residue resulted in erratic cell attachment and mineralization behavior. The minimum BSP sequence necessary for strong adhesion and extensive mineralization was CGGEPRGDTYRA; the minimal sequence suitable for extensive mineralization but lacking strong adhesion was CGPRGDTYG. The cyclic peptide (c-RGD) had much greater adhesion strength compared to its linear counterpart (lc-RGD). The calculated characteristic adhesion strength (F(70)) obtained using a centrifuge adhesion assay proved to be a poor metric for predicting % mineralized area; however, in general, surfaces possessing a F(70) > 100g promoted extensive matrix mineralization. Percent mineralization and number of mineralized nodules scaled with number of cells seeded suggesting a critical dependence on the initial number of osteoprogenitors in culture. This study demonstrates matrix mineralization dependence on ligand type, ligand density, and adhesion strength. The high-throughput character of these surfaces allowed efficient investigation of multiple ligands at multiple densities providing an excellent tool for studying ligand-receptor interactions under normal cell culture conditions with serum present.

Animals↗

PubMed2005

Synthetic MMP-13 degradable ECMs based on poly(N-isopropylacrylamide-co-acrylic acid) semi-interpenetrating polymer networks. I. Degradation and cell migration.

Thermoresponsive and injectable semi-interpenetrating polymer networks (sIPNs) containing a biospecific cell-adhesive signal and proteolytically degradable domains were developed as a synthetic equivalent of the extracellular matrix (ECM). The sIPNs synthesized define a modular hydrogel ECM where different properties of the matrix can be manipulated independently, thus creating a system where parametric analysis of the effect of hydrogel properties on cell proliferation and differentiation is possible. sIPNs composed of poly(N-isopropylacrylamide-co-acrylic acid) [p(NIPAAm-co-AAc)] and RGD-grafted poly(acrylic acid) linear chains [p(AAc)-g-RGD] were synthesized with peptide crosslinkers containing a matrix metalloproteinase-13 (MMP-13, collagenase-3) degradable domain. The lower critical solution temperature (LCST) of peptide-crosslinked p(NIPAAm-co-AAc) sIPNs was not influenced by the addition of either linear p(AAc) or peptide-modified p(AAc) chains ( approximately 34 degrees C) in PBS. Degradation of peptide-crosslinked hydrogels and sIPNs was enzyme specific and concentration dependent. Exposure of rat calvarial osteoblast (RCO) culture to the degradation products from the peptide-crosslinked hydrogels did not significantly affect cell viability. Migration of RCOs into the sIPNs was dependent upon the presence of both a cell-adhesive RGD peptide (Ac-CGGNGEPRGDTYRAY-NH2) and proteolytically-degradable crosslinks; however, there was greater dependence on the latter. The sIPNs synthesized are versatile materials for assessing cell fate in synthetic ECM constructs in vitro and tissue regeneration in vivo.

Acrylamides↗

PubMed2005

Analysis of sterilization protocols for peptide-modified hydrogels.

Concerns about the efficacy of ethanol disinfection for implanted biomaterials prompted investigation of an alternative sterilization process, ultraviolet irradiation, for terminal sterilization of N-isopropylacrylamide-based hydrogels containing biomimetic peptides. Ultraviolet irradiation is more easily applied on a laboratory scale than gamma irradiation or electron beam, two commercially utilized methods; thus, UVC irradiation was investigated as a low-cost sterilization procedure that might be performed in laboratories prior to in vivo studies. UVC irradiation at 400 muW/cm(2) for up to 15 h did not prevent growth of Escherichia coli within the hydrogels, while ethanol disinfection did prevent growth for the duration of the experiment (120 h). Furthermore, UVC irradiation caused progressive degradation of peptides containing the Arg-Gly-Arg (RGD) domain. UVC irradiation cannot be used as a terminal sterilization process for peptide-modified materials. The system used in this study is not intended to be adequate for evaluating the sterility of medical devices in accordance with current Good Manufacturing Practice (cGMP); however, it remains a useful, low-cost system for the preliminary evaluation of sterilization procedures in terms of their ability to eliminate pathogenic organisms while preserving the structure of biologically active molecules within in a laboratory setting. Ethanol treatment is still the preferred method for disinfection of bioactive materials containing peptides or UV-degradable groups.

Acrylamides↗

PubMed2005

Ligand density characterization of peptide-modified biomaterials.

A simple fluorescence based characterization method was developed to assess ligand density on peptide-modified biomaterials. The method exploits the exquisite sensitivity of proteolysis for the purpose of liberating a fluorescently labeled probe fragment from an immobilized peptide. The released fragment can then be detected in solution using high-throughput fluorometry. In silico screening tools identified the enzyme chymotrypsin as a promising candidate for releasing a detectable probe fragment from the fluorescently labeled peptide, Ac-CGGNGEPRGDTYRAYK(FITC)GG-NH(2). After chymotrypsin digestion of the peptide in solution was first characterized using mass spectrometry and HPLC, a basic enzyme mediated release protocol was developed and implemented to generate peptide-binding isotherms on various peptide-modified biomaterials. The new method is sensitive, has good signal-to-noise ratio (S/N), and is easily standardized. Furthermore, the technique can be applied independent of material chemistry and geometry, making it a suitable alternative to radiolabeling for a wide range of biomaterial applications.

Binding Sites↗

PubMed2004

Inhibition of macrophage development and foreign body giant cell formation by hydrophilic interpenetrating polymer network.

The ability of monocytes to adhere, differentiate into macrophages, and fuse to form foreign body giant cells (FBGCs) on an implanted material surface is a critical step toward biomaterial degradation. Novel homogeneous surfaces were utilized to mediate adhesion. These surfaces consisted of N-(2 aminoethyl)-3-aminopropyltrimethoxysilane (EDS) and an interpenetrating polymer network (IPN) of polyacrylamide and poly(ethylene glycol). These surfaces were designed to control cell adhesion and morphology and mediate cell differentiation, activation, metabolic ability, and apoptosis, resulting in a reduced or controlled inflammatory response. The EDS surface promotes cell adhesion and the IPN minimizes protein adsorption and subsequent cell adhesion. Both surfaces had similar cellular adhesion rates at each respective time point. However, the adherent macrophage morphology was similar at 2 h and day 3, and at days 7 and 10 adherent macrophages on the EDS surface formed FBGCs (46% at day 7 and 40% at day 10). Adherent cells on the IPN surface did not form FBGCs but instead formed monocyte aggregates (73% of adherent cells formed aggregates at day 7 and 63% at day 10). It is indicated that the two surface chemistries differentially controlled monocyte differentiation into macrophages and subsequent macrophage fusion to form FBGCs.

Acrylic Resins↗

PubMed2004

Compositional regulation of poly(lysine-g-(lactide-b-ethylene glycol))-DNA complexation and stability.

The search for an effective nonviral gene therapy vector has revealed several significant hurdles, such as transient expression and cytotoxicity, that impede the success of these systems. A terpolymer of poly(lysine-g-(lactide-b-ethylene glycol)) [pK-g-p(LL-b-EG)] has been developed, which is capable of stably packaging DNA with significantly less polycation than unmodified polylysine (pK) systems. A comparison of unmodified pK (DP=14, M(w)=2930) to pK grafted with p(LL-b-EG) (M(w tot)=7500) showed that the minimum amine/phosphate ratio (N:P(min)) needed for complete DNA condensation was reduced by 50%. However, when the molecular weight of pK was reduced (DP=4, M(w)=838), there was evidence of terpolymer interference with DNA condensation. Increasing the number of grafted p(LL-b-EG) chains produced a similar result of incomplete DNA condensation. All terpolymer formulations produced complexes with DNA that had greater resistance to salt-induced dissociation and short-term exchange with excess DNA. Terpolymer-DNA complexes exhibited approximately zero-order plasmid release profiles over a period of 6 weeks. The rate of release was dependent on the complex N:P ratio as well as the molecular weights of pLL and pK. The ability to use terpolymer composition to control complex stability and controlled release can provide a means for system optimization for sustained expression profiles of exogenous DNA.

DNA↗

PubMed2004

Patterning, prestress, and peeling dynamics of myocytes.

As typical anchorage-dependent cells myocytes must balance contractility against adequate adhesion. Skeletal myotubes grown as isolated strips from myoblasts on micropatterned glass exhibited spontaneous peeling after one end of the myotube was mechanically detached. Such results indicate the development of a prestress in the cells. To assess this prestress and study the dynamic adhesion strength of single myocytes, the shear stress of fluid aspirated into a large-bore micropipette was then used to forcibly peel myotubes. The velocity at which cells peeled from the surface, V(peel), was measured as a continuously increasing function of the imposed tension, T(peel), which ranges from approximately 0 to 50 nN/ micro m. For each cell, peeling proved highly heterogeneous, with V(peel) fluctuating between 0 micro m/s ( approximately 80% of time) and approximately 10 micro m/s. Parallel studies of smooth muscle cells expressing GFP-paxillin also exhibited a discontinuous peeling in which focal adhesions fractured above sites of strong attachment (when pressure peeled using a small-bore pipette). The peeling approaches described here lend insight into the contractile-adhesion balance and can be used to study the real-time dynamics of stressed adhesions through both physical detection and the use of GFP markers; the methods should prove useful in comparing normal versus dystrophic muscle cells.

Animals↗

PubMed2004

Poly(N-isopropylacrylamide)-based semi-interpenetrating polymer networks for tissue engineering applications. Effects of linear poly(acrylic acid) chains on rheology.

Semi-interpenetrating polymer networks (semi-IPNs), comprised of poly(N-isopropylacrylamide-co-acrylic acid) (p(NIPAAm-co-AAc)) hydrogels and linear p(AAc) chains, were synthesized, and the effects of the p(AAc) chains on semi-IPN rheology were examined. Oscillatory shear rheometry studies were performed and the rheological data were analyzed as a function of temperature, frequency, and p(AAc) chain amount (weight average molecular weight (Mw) 4.5 x 10(5) g/mol). At 22 degrees C, the semi-IPNs, as well as control p(NIPAAm-co-AAc) hydrogels, demonstrated rheological data that were representative of soft, loosely cross-linked solids. Furthermore, only the highest p(AAc) chain amount tested affected the rigidity of the p(NIPAAm-co-AAc)-based semi-IPNs, as compared to the p(NIPAAm-co-AAc) hydrogels. At 37 degrees C, the complex shear moduli (G*) demonstrated by the p(NIPAAm-co-AAc)-based semi-IPNs were significantly greater than G* exhibited by the p(NIPAAm-co-AAc) hydrogels, and the semi-IPN G* values significantly increased with increasing p(AAc) chain amount. These results can be used to develop p(NIPAAm)-based semi-IPNs with tailored mechanical properties that may function as scaffolds in tissue engineering initiatives.

Acrylates↗

PubMed2003

The role of alpha3beta1 integrin in determining the supramolecular organization of laminin-5 in the extracellular matrix of keratinocytes.

Analyses of mice with targeted deletions in the genes for alpha3 and beta1 integrin suggest that the alpha3beta1 integrin heterodimer likely determines the organization of the extracellular matrix within the basement membrane of skin. Here we tested this hypothesis using keratinocytes derived from alpha3 integrin-null mice. We have compared the organizational state of laminin-5, a ligand of alpha3beta1 integrin, in the matrix of wild-type keratinocytes with that of laminin-5 in the matrix of alpha3 integrin-null cells. Laminin-5 distributes diffusely in arc structures in the matrix of wild-type mouse keratinocytes, whereas laminin-5 is organized into linear, spike-like arrays by the alpha3 integrin-null cells. The fact that alpha3 integrin-null cells are deficient in their ability to assemble a proper laminin-5 matrix is also shown by their failure to remodel laminin-5 when plated onto surfaces coated with purified laminin-5 protein. In sharp contrast, wild-type keratinocytes organize exogenously added laminin-5 into discrete ring-like organizations. These findings led us next to assess whether differences in laminin-5 organization in the matrix of the wild-type and alpha3 integrin-null cells impact cell behavior. Our results indicate that alpha3 integrin-null cells are more motile than their wild-type counterparts and leave extensive trails of laminin-5 over the surface on which they move. Moreover, HEK 293 cells migrate significantly more on the laminin-5-rich matrix derived from the alpha3 integrin-null cells than on the wild-type keratinocyte laminin-5 matrix. In addition, alpha3 integrin-null cells show low strength of adhesion to surfaces coated with purified laminin-5 compared to wild-type cells although both the wild type and the alpha3 integrin-null keratinocytes adhere equally strongly to laminin-5 that has been organized into arrays by other epithelial cells. These data suggest: (1) that alpha3beta1 integrin plays an important role in determining the incorporation of laminin-5 into its proper higher-order structure within the extracellular matrix of keratinocytes and (2) that the organizational state of laminin-5 has an influence on laminin-5 matrix function.

Animals↗

PubMed2003

Peptide-modified p(AAm-co-EG/AAc) IPNs grafted to bulk titanium modulate osteoblast behavior in vitro.

Interpenetrating polymer networks (IPNs) of poly(acrylamide-co-ethylene glycol/acrylic acid) (p(AAm-co-EG/AAc) applied to model surfaces prevent protein adsorption and cell adhesion. Subsequently, IPN surfaces functionalized with the RGD cell-binding domain from rat bone sialoprotein (BSP) modulated bone cell adhesion, proliferation, and matrix mineralization. The objective of this study was to utilize the same biomimetic modification strategy to produce functionally similar p(AAm-co-EG/AAc) IPNs on clinically relevant titanium surfaces. Contact angle goniometry and X-ray photoelectron spectroscopy (XPS) data were consistent with the presence of the intended surface modifications. Cellular response was gauged by challenging the surfaces with primary rat calvarial osteoblast (RCO) surfaces in serum-containing media. IPN modified titanium and negative control (RGE-IPN) surfaces inhibit cell adhesion and proliferation, while RGD-modified IPNs on titanium supported osteoblast attachment and spreading. Furthermore, the latter surfaces supported significant mineralization despite exhibiting lower levels of proliferation than positive control surfaces. These results suggest that with the appropriate optimization, this approach may be practical for surface engineering of osseous implants.

Amino Acid Sequence↗

PubMed2002

Sequential robust design methodology and X-ray photoelectron spectroscopy to analyze the grafting of hyaluronic acid to glass substrates.

Sequential Robust Design experiments and X-ray photoelectron spectroscopic (XPS) studies were performed to examine the immobilization of hyaluronic acid (HA) on glass substrates chemisorbed with N-(2-aminoethyl)-3-aminopropyl-trimethoxysilane (EDS). Numerous reaction conditions were investigated, including the concentrations of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), N-hydroxysulfosuccinimide (Sulfo-NHS), and HA, and the reaction buffer type, concentration, and pH. The elemental surface compositions of carbon and silicon (C/Si ratio) were used to assess the extent of HA immobilization, leading to the identification of critical HA-binding reaction conditions and the determination of an optimum surface chemistry. The optimum chemistry consisted of 200 mM EDC, 50 mM Sulfo-NHS, 10 mM N-(2-hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid) (HEPES) buffer at a pH of 7.0, and 3 mg/mL HA. This work emphasizes the advantages of using Robust Design methods over traditional statistical experimental design, particularly when large numbers of variables are examined and costly analytical techniques are employed.

Adhesiveness↗

PubMed2002

Engineering gene expression and protein synthesis by modulation of nuclear shape.

The current understanding of the relationships between cell shape, intracellular forces and signaling, nuclear shape and organization, and gene expression is in its infancy. Here we introduce a method for investigating gene-specific responses in individual cells with controlled nuclear shape and projected area. The shape of the nuclei of primary osteogenic cells were controlled on microfabricated substrata with regiospecific chemistry by confining attachment and spreading of isolated cells on adhesive islands. Gene expression and protein synthesis were altered by changing nuclear shape. Collagen I synthesis correlated directly with cell shape and nuclear shape index (NSI), where intermediate values of nuclear distension (6 < NSI < 8) promoted maximum synthesis. Osteocalcin mRNA, a bone-specific differentiation marker, was observed intracellularly by using reverse transcription in situ PCR at 4 days in cells constrained by the pattern and not detected in unconstrained cells of similar projected area, but different NSI. Our data supports the concept of gene expression and protein synthesis based on optimal distortion of the nucleus, possibly altering transcription factor affinity for DNA, transport to the nucleus, or nuclear matrix organization. The combination of microfabricated surfaces, reverse transcription in situ PCR, and NSI measurement is an excellent system to study how transcription factors, the nuclear matrix, and the cytoskeleton interact to control gene expression and may be useful for studying a wide variety of other cell shape/gene expression relationships.

Active Transport, Cell Nucleus↗