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Morshed Khandaker,1,4 Shahram Riahinezhad,1 Fariha Sultana,1 Melville B Vaughan,2,4 Joshua Knight,2 Tracy L Morris3,4 1Department of Engineering & Physics, 2Department of Biology, 3Department of Mathematics and Statistics, 4Center for Interdisciplinary Biomedical Education and Research, University of Central Oklahoma, Edmond, OK, USA Abstract: Implant failure due to poor integration of the implant with the surrounding biomaterial is a common problem in various orthopedic and orthodontic surgeries. Implant fixation mostly depends upon the implant surface topography. Micron to nanosize circular-shaped groove architecture with adequate surface roughness can enhance the mechanical interlock and osseointegration of an implant with the host tissue and solve its poor fixation problem. Such groove architecture can be created on a titanium (Ti) alloy implant by laser peening treatment. Laser peening produces deep, residual compressive stresses in the surfaces of metal parts, delivering increased fatigue life and damage tolerance. The scientific novelty of this study is the controlled deposition of circular-shaped rough spot groove using laser peening technique and understanding the effect of the treatment techniques for improving the implant surface properties. The hypothesis of this study was that implant surface grooves created by controlled laser peen treatment can improve the mechanical and biological responses of the implant with the adjoining biomaterial. The objective of this study was to measure how the controlled laser-peened groove architecture on Ti influences its osteoblast cell functions and bonding strength with bone cement. This study determined the surface roughness and morphology of the peen-treated Ti. In addition, this study compared the osteoblast cell functions (adhesion, proliferation, and differentiation) between control and peen-treated Ti samples. Finally, this study measured the fracture strength between each kind of Ti samples and bone cement under static loading. This study found that laser peen treatment on Ti significantly changed the surface architecture of the Ti, which led to enhanced osteoblast cell adhesion and differentiation on Ti implants and fracture strength of Ti–bone cement interfaces compared with values of untreated Ti samples. Therefore, the laser peen treatment method has the potential to improve the biomechanical functions of Ti implants. Keywords: titanium, cement, interface, PMMA, fracture strength, orthopedics, laser peen, orthodontics

Morshed Khandaker,1 Melville B Vaughan,2 Tracy L Morris,3 Jeremiah J White,1 Zhaotong Meng1 1Department of Engineering and Physics, 2Department of Biology, 3Department of Mathematics and Statistics, University of Central Oklahoma, Edmond, OK, USA Abstract: The most common bone cement material used clinically today for orthopedic surgery is poly(methyl methacrylate) (PMMA). Conventional PMMA bone cement has several mechanical, thermal, and biological disadvantages. To overcome these problems, researchers have investigated combinations of PMMA bone cement and several bioactive particles (micrometers to nanometers in size), such as magnesium oxide, hydroxyapatite, chitosan, barium sulfate, and silica. A study comparing the effect of these individual additives on the mechanical, thermal, and cell functional properties of PMMA would be important to enable selection of suitable additives and design improved PMMA cement for orthopedic applications. Therefore, the goal of this study was to determine the effect of inclusion of magnesium oxide, hydroxyapatite, chitosan, barium sulfate, and silica additives in PMMA on the mechanical, thermal, and cell functional performance of PMMA. American Society for Testing and Materials standard three-point bend flexural and fracture tests were conducted to determine the flexural strength, flexural modulus, and fracture toughness of the different PMMA samples. A custom-made temperature measurement system was used to determine maximum curing temperature and the time needed for each PMMA sample to reach its maximum curing temperature. Osteoblast adhesion and proliferation experiments were performed to determine cell viability using the different PMMA cements. We found that flexural strength and fracture toughness were significantly greater for PMMA specimens that incorporated silica than for the other specimens. All additives prolonged the time taken to reach maximum curing temperature and significantly improved cell adhesion of the PMMA samples. The results of this study could be useful for improving the union of implant-PMMA or bone-PMMA interfaces by incorporating nanoparticles into PMMA cement for orthopedic and orthodontic applications. Keywords: poly(methyl methacrylate), magnesium oxide, hydroxyapatite, chitosan, barium sulfate, silica, flexural strength, fracture toughness, curing temperature, cell viability

Missions into Deep Space are planned this decade. Yet the health consequences of exposure to microgravity and galactic cosmic radiation (GCR) over years-long missions on indispensable visceral organs such as the kidney are largely unexplored. We performed biomolecular (epigenomic, transcriptomic, proteomic, epiproteomic, metabolomic, metagenomic), clinical chemistry (electrolytes, endocrinology, biochemistry) and morphometry (histology, 3D imaging, miRNA-ISH, tissue weights) analyses using samples and datasets available from 11 spaceflight-exposed mouse and 5 human, 1 simulated microgravity rat and 4 simulated GCR-exposed mouse missions. We found that spaceflight induces: 1) renal transporter dephosphorylation which may indicate astronauts' increased risk of nephrolithiasis is in part a primary renal phenomenon rather than solely a secondary consequence of bone loss; 2) remodelling of the nephron that results in expansion of distal convoluted tubule size but loss of overall tubule density; 3) renal damage and dysfunction when exposed to a Mars roundtrip dose-equivalent of simulated GCR., (© 2024. The Author(s).)

The objective of this study was to create a nanofiber-based skin graft with an antimicrobial bandage that could accelerate the healing of an open wound while minimizing infection. To this end, we prepared a bi-layer construct where the top layer acts as bandage, and the bottom layer acts as a dermal equivalent graft. A collagen (CG) gel was combined without and with an electrospun polycaprolactone (PCL) membrane to prepare CG and CG-PCL dermal equivalent constructs. The antibacterial properties of PCL with and without an antibacterial agent (MgO nanoparticles) against Staphylococcus aureus (ATCC 6538) was also examined. Human dermal fibroblasts were cultured in each construct to make the dermal equivalent grafts. After culturing, keratinocytes were plated on top of the tissues to allow growth of an epidermis. Rheological and durability tests were conducted on in vitro dermal and skin equivalent cultures, and we found that PCL significantly affects CG-PCL graft biological and mechanical strength (rheology and durability). PCL presence in the dermal equivalent allowed sufficient tension generation to activate fibroblasts and myofibroblasts in the presence of transforming growth factor-beta. During culture of the skin equivalents, optical coherence tomography (OCT) showed layers corresponding to dermal and epidermal compartments in the presence or absence of PCL; this was confirmed after fixed specimens were histologically sectioned and stained. MgO added to PCL showed antibacterial activity against S. aureus. In vivo animal studies using a rat skin model showed that a polycaprolactone nanofiber bandage containing a type I collagen skin graft has potential for wound healing applications.

In the current study, we designed and manufactured a scaffold and fixation system for the reconstruction of long-bone segmental defects in a rabbit tibia model. We used biocompatible and biodegradable materials, polycaprolactone (PCL) and PCL soaked with sodium alginate (PCL-Alg) to manufacture the scaffold, interlocking nail and screws using a phase separation casing method. Degradation and mechanical tests on the PCL and PCL-Alg scaffolds indicated that both were suitable for faster degradation and early weight-bearing capacity. PCL scaffold surface porosity facilitated the infiltration of alginate hydrogel through the scaffold. Cell viability results showed that the number of cells increased on Day 7 and decreased marginally by Day 14. For accurate placement of the scaffold and fixation system, a surgical jig was designed and 3D-printed using biocompatible resin in a stereolithography (SLA) 3D printer, then cured with UV light for increased strength. Our cadaver tests using New Zealand White rabbit confirmed our novel jigs' potential for accurate placement of the bone scaffold, intramedullary nail and the alignment of the fixation screws in future reconstructive surgeries on rabbit long-bone segmental defects. Additionally, the cadaver tests confirmed that our designed nails and screws were strong enough to carry the surgical insertion force. Therefore, our designed prototype has the potential for further clinical translational study using the rabbit tibia model.

The study's aim was to develop a dermal equivalent scaffold that can mimic the architecture and biological performance of the human dermis. Poly ε-caprolactone (PCL) electrospun nanofiber material (ENF) was assembled with polyethylene glycol diacrylate (PEGDA), sodium alginate (SA) and type I collagen (CG1) to develop three groups of dermal equivalent scaffolds. These scaffolds were named PEGDA-PCL, SA-PCL and CG1-PCL. Scanning electron microscopy (SEM) images of cell-free scaffolds' top and cross-sectional surface were collected and analyzed to examine internal morphology, specifically the adhesiveness of PCL fibers with the different scaffolds. Human dermal fibroblasts were cultured on each of the scaffolds. Cell viability studies including cell adhesion, cell differentiation and stress fiber production were conducted on each scaffold. Furthermore, the architectural integrity of each scaffold was verified by degradation analysis for 2 weeks by soaking each scaffold in phosphate-buffered saline (PBS) solution. Finally, we conducted rheological characteristics of each scaffold. Based on our results from the above analysis, the study concluded that CG1-PCL is best suitable for the dermal equivalent model and has potential to be used as a graft for skin repair.

The flow diverting stent (FDS) has become a promising endovascular device for the treatment of aneurysms. This research presents a novel biodegradable and non-braided Polycaprolactone (PCL) FDS. The PCL FDS was designed and developed using an in-house fabrication unit and coated on two ends with BaSO 4 for angiographic visibility. The mechanical flexibility and quality of FDS surfaces were examined with the UniVert testing machine, scanning electron microscope (SEM), and 3D profilometer. Human umbilical vein endothelial cell (HUVEC) adhesion, proliferation, and cell morphology studies on PCL FDS were performed. The cytotoxicity and NO production by HUVECs with PCL FDS were also conducted. The longitudinal tensile, radial, and bending flexibility were found to be 1.20 ± 0.19 N/mm, 0.56 ± 0.11 N/mm, and 0.34 ± 0.03 N/mm, respectively. The FDS was returned to the original shape and diameter after repeated compression and bending without compromising mechanical integrity. Results also showed that the proliferation and adhesion of HUVECs on the FDS surface increased over time compared to control without FDS. Lactate dehydrogenase (LDH) release and NO production showed that PCL FDS were non-toxic and satisfactory. Cell morphology studies showed that HUVECs were elongated to cover the FD surface and developed an endothelial monolayer. This study is a step forward toward the development and clinical use of biodegradable flow diverting stents for endovascular treatment of the aneurysm.

Laser micromachining technique offers a promising alternative method for rapid production of microfluidic devices. However, the effect of process parameters on the channel geometry and quality of channels on common microfluidic substrates has not been fully understood yet. In this research, we studied the effect of laser system parameters on the microchannel characteristics of Polydimethylsiloxane (PDMS), polymethyl methacrylate (PMMA), and microscope glass substrate-three most widely used materials for microchannels. We also conducted a cell adhesion experiment using normal human dermal fibroblasts on laser-machined microchannels on these substrates. A commercial CO 2 laser system consisting of a 45W laser tube, circulating water loop within the laser tube and air cooling of the substrate was used for machining microchannels in PDMS, PMMA and glass. Four laser system parameters - speed, power, focal distance, and number of passes were varied to fabricate straight microchannels. The channel characteristics such as depth, width, and shape were measured using a scanning electron microscope (SEM) and a 3D profilometer. The results show that higher speed produces lower depth while higher laser power produces deeper channels regardless of the substrate materials. Unfocused laser machining produces wider but shallower channels. For the same speed and power, PDMS channels were the widest while PMMA channels were the deepest. Results also showed that the profiles of microchannels can be controlled by increasing the number of passes. With an increased number of passes, both glass and PDMS produced uniform, wider, and more circular channels; in contrast, PMMA channels were sharper at the bottom and skewed. In rapid cell adhesion experiments, PDMS and glass microchannels performed better than PMMA microchannels. This study can serve as a quick reference in material-specific laser-based microchannel fabrications.

The anchored fibroblast-populated collagen matrix (aFPCM) is an appropriate model to study fibrocontractive disease mechanisms. Our goal was to determine if aFPCM height reduction (compaction) during development is sufficient to predict tension generation. Compaction was quantified daily by both traditional light microscopy and an optical coherence tomography (OCT) system. Contraction in aFPCM was revealed by releasing them from anchorage. We found that aFPCM contraction increase was correlated to the compaction increase. Cytochalasin D treatment reversibly inhibited compaction. Therefore, we demonstrated that aFPCM height reduction efficiently measures compaction, contraction, and relative maturity of the collagen matrix during development or treatment. In addition, we showed that OCT is suitable for effectively imaging the cross-sectional morphology of the aFPCM in culture. This study will pave the way for more efficient studies on the mechanisms of (and treatments that target) migration and contraction in wound healing and Dupuytren's contracture in a tissue environment.

Fibrotic diseases, such as Dupuytren's contracture (DC), involve excess scar tissue formation. The differentiation of fibroblasts into myofibroblasts is a significant mechanism in DC, as it generates tissue contraction in areas without wound openings, leading to the deposition of scar tissue, and eventually flexing one or more fingers in a restrictive fashion. Additionally, DC has a high recurrence rate. Previously, we showed that N -dihydrogalactochitosan (GC), an immunostimulant, inhibited myofibroblast differentiation in a DC fibroblast culture. Our goal of this study was to expand our previous study to include other DC and normal cell lines and other chitosan derivatives (GC and single-walled carbon nanotube-conjugated GC) to determine the specific mechanism of inhibition. Derivative-incorporated and vehicle control (water) anchored fibroblast-populated collagen matrices (aFPCM) were used to monitor compaction (anchored matrix height reduction) using microscopy and optical coherence tomography (OCT) for six days. Fibroblasts were unable to compact chitosan derivative aFPCM to the same extent as vehicle control aFPCM in repeated experiments. Similarly, chitosan derivative aFPCM contracted less than control aFPCM when released from anchorage. Proliferative myofibroblasts were identified by the presence of alpha smooth muscle actin via myofibroblast proliferative assay. In all tested conditions, a small percentage of myofibroblasts and proliferative cells were present. However, when aFPCM were treated with transforming growth factor-beta 1 (TGF-β1), all tested samples demonstrated increased myofibroblasts, proliferation, compaction, and contraction. Although compaction and contraction were reduced, there was sufficient tension present in the chitosan derivative aFPCM to allow exogenous stimulation of the myofibroblast phenotype.

Poly(methyl methacrylate) (PMMA) bone cement has limited biocompatibility. Polycaprolactone (PCL) electrospun nanofiber (ENF) has many applications in the biomedical field due to its excellent biocompatibility and degradability. The effect of coating PCL ENF on the surface topography, biocompatibility, and mechanical strength of PMMA bone cement is not currently known. This study is based on the hypothesis that the PCL ENF coating on PMMA will increase PMMA roughness leading to increased biocompatibility without influencing its mechanical properties. This study prepared PMMA samples without and with the PCL ENF coating, which were named the control and ENF coated samples. This study determined the effects on the surface topography and cytocompatibility (osteoblast cell adhesion, proliferation, mineralization, and protein adsorption) properties of each group of PMMA samples. This study also determined the bending properties (strength, modulus, and maximum deflection at fracture) of each group of PMMA samples from an American Society of Testing Metal (ASTM) standard three-point bend test. This study found that the ENF coating on PMMA significantly improved the surface roughness and cytocompatibility properties of PMMA ( p < 0.05). This study also found that the bending properties of ENF-coated PMMA samples were not significantly different when compared to those values of the control PMMA samples ( p > 0.05). Therefore, the PCL ENF coating technique should be further investigated for its potential in clinical applications., Competing Interests: The authors have no conflicts of interest.

Titanium (Ti) alloys have been widely used in orthopedics and orthodontic surgeries as implants because of their beneficial chemical, mechanical, and biological properties. Improvement of these properties of a Ti alloy, Ti-6Al-4V Eli, is possible by the use of plasma nitriding treatment on the Ti alloy. The novelty of this study is the evaluation of a DC glow discharge nitrogen plasma treatment method on the surface, mechanical and biological properties of Ti alloy. Specifically, this study measured the chemical states, roughness, hardness, and biocompatibility of plasma nitride treated Ti-6Al-4V Eli as well as determined the effect of plasma treatment on the fracture strength between the Ti alloy and bone clement. This study hypothesized that DC glow discharge nitrogen plasma treatment may alter the surface chemical and mechanical states of the Ti alloy that may influence the fracture strength of implant/cement interfaces under static load. This study found that plasma nitride treatment on Ti alloy does not have effect on the roughness and biocompatibility (P value > 0.5), but significantly effect on the hardness and fracture strength of Ti-bone cement interfaces compared to those values of untreated Ti samples (P value < 0.5). Therefore, the DC glow discharge nitrogen plasma treated Ti alloy can potentially be used for orthopedic applications.

Myofibroblasts are resident cells of wound healing, contractures and fibroses; these tissues are often referred to as fibroproliferative. Whether myofibroblasts themselves proliferate is of interest. Since many in vitro cultures are heterogeneous, staining in situ is required to identify the myofibroblast. We have tested a newly available fluorescent staining kit using ethynyl deoxyuridine (EdU) and click chemistry to identify EdU incorporation into the replicated DNA of proliferative cells. The proliferation stain was combined with the definitive myofibroblast immunostain for alpha smooth muscle actin (α-sma). Fibroblasts were grown on coverslips and within attached collagen lattices. Cultures were pulsed with EdU 4 h prior to fixation. Different standard methods of fixation and permeabilization were used to test the effects of these variables on EdU and α-sma labeling. Images of the stained samples were quantified as the total percentage of proliferative cells, as well as the proportion of fibroblasts and myofibroblasts that were proliferating. Proliferative myofibroblasts were identified in both culture conditions and with all preparation methods tested. Proliferation within the fibroblast population was greater than within the myofibroblast population in both culture conditions. Fixation and permeabilization had little effect on EdU or α-sma labeling. This method of identifying proliferative myofibroblasts will be useful in future studies of myofibroblast proliferation within heterogeneous populations., (© 2014 International Federation for Cell Biology.)

Background: Normal, healthy human breast tissue from a variety of volunteer donors has become available for research thanks to the establishment of the Susan G. Komen for the Cure® Tissue Bank at the IU Simon Cancer Center (KTB). Multiple epithelial (K-HME) and stromal cells (K-HMS) were established from the donated tissue. Explant culture was utilized to isolate the cells from pieces of breast tissue. Selective media and trypsinization were employed to select either epithelial cells or stromal cells. The primary, non-transformed epithelial cells, the focus of this study, were characterized by immunohistochemistry, flow cytometry, and in vitro cell culture., Results: All of the primary, non-transformed epithelial cells tested have the ability to differentiate in vitro into a variety of cell types when plated in or on biologic matrices. Cells identified include stratified squamous epithelial, osteoclasts, chondrocytes, adipocytes, neural progenitors/neurons, immature muscle and melanocytes. The cells also express markers of embryonic stem cells., Conclusions: The cell culture conditions employed select an epithelial cell that is pluri/multipotent. The plasticity of the epithelial cells developed mimics that seen in metaplastic carcinoma of the breast (MCB), a subtype of triple negative breast cancer; and may provide clues to the origin of this particularly aggressive type of breast cancer. The KTB is a unique biorepository, and the normal breast epithelial cells isolated from donated tissue have significant potential as new research tools.

Background: Ras proteins affect both proliferation and expression of collagen-degrading enzymes, two important processes in cancer progression. Normal skin architecture is dependent both on the coordinated proliferation and stratification of keratinocytes, as well as the maintenance of a collagen-rich basement membrane. In the present studies we sought to determine whether expression of H-ras in skin keratinocytes would affect these parameters during the establishment and maintenance of an in vitro skin equivalent., Methodology/principal Findings: Previously described cdk4 and hTERT immortalized foreskin keratinocytes were engineered to express ectopically introduced H-ras. Skin equivalents, composed of normal fibroblast-contracted collagen gels overlaid with keratinocytes (immortal or immortal expressing H-ras), were prepared and incubated for 3 weeks. Harvested tissues were processed and sectioned for histology and antibody staining. Antigens specific to differentiation (involucrin, keratin-14, p63), basement-membrane formation (collagen IV, laminin-5), and epithelial to mesenchymal transition (EMT; e-cadherin, vimentin) were studied. Results showed that H-ras keratinocytes produced an invasive, disorganized epithelium most apparent in the lower strata while immortalized keratinocytes fully stratified without invasive properties. The superficial strata retained morphologically normal characteristics. Vimentin and p63 co-localization increased with H-ras overexpression, similar to basal wound-healing keratinocytes. In contrast, the cdk4 and hTERT immortalized keratinocytes differentiated similarly to normal unimmortalized keratinocytes., Conclusions/significance: The use of isogenic derivatives of stable immortalized keratinocytes with specified genetic alterations may be helpful in developing more robust in vitro models of cancer progression.

During wound healing and fibrocontractive diseases fibroblasts acquire a smooth muscle cell-like phenotype by differentiating into contractile force generating myofibroblasts. We examined whether regulation of myofibroblast contraction in granulation tissue is dominated by Ca2+-induced phosphorylation of myosin light chain kinase or by Rho/Rho kinase (ROCK)-mediated inhibition of myosin light chain phosphatase, similar to that of cultured myofibroblasts. Strips of granulation tissue obtained from rat granuloma pouches were stimulated with endothelin-1 (ET-1), serotonin, and angiotensin-II and isometric force generation was measured. We here investigated ET-1 in depth, because it was the only agonist that produced a long-lasting and strong response. The ROCK inhibitor Y27632 completely inhibited ET-1-promoted contraction and the phosphatase inhibitor calyculin elicited contraction in the absence of any other agonists, suggesting that activation of the Rho/ROCK/myosn light chain phosphatase pathway is critical in regulating in vivo myofibroblast contraction. Membrane depolarization with K+ also stimulated a long-lasting contraction of granulation tissue; however, the amount of force generated was significantly less compared to ET-1. Moreover, K+-induced contraction was inhibited by Y27632. These results are consistent with inhibition of myosin light chain phosphatase by the Rho/ROCK signaling pathway, which would account for the long-duration contraction of myofibroblasts necessary for wound closure.

A therapeutic approach being investigated for a variety of pathologies is tissue regeneration using a patient's own cells. Such studies have been hampered due to the difficulty in growing epithelial cells for prolonged periods in culture. Replicative senescence due to short telomeres and p16 induced by culture stress work together to inhibit cell growth. Forced expression of telomerase (hTERT) can prevent replicative senescence, and expression of the cell cycle protein cdk4 can sequester p16, thereby immortalizing epithelial cells in culture. In the present study, we used this method to immortalize human bronchial epithelial cells (HBECs) to determine whether immortalized HBECs retain the ability to differentiate normally. HBECs were plated atop contracted collagen gels containing lung fibroblasts. This three-dimensional (3D) tissue model was cultured initially submerged, then raised to the air/liquid interface for up to 28 days. Normal differentiation was assessed by the presence of ciliated cells, goblet (mucin-producing) cells, and basal epithelial cells. Scanning electron microscopic observations revealed both ciliated and non-ciliated cells in these 3D tissues. Histological examination revealed the presence of mucin-producing cells, and immunohistochemistry using antibodies against p63 and keratin 14 showed the presence of basal cells. These results demonstrate that immortalized HBECs retain the capacity to differentiate into each of three cell types: basal, mucin-producing, and columnar ciliated epithelial cells. Such cells will be useful cellular reagents for research in aging, cancer progression, as well as normal bronchial epithelial differentiation and will help progress the use of engineered cells to enhance tissue regeneration.

We evaluated the contribution of three genetic alterations (p53 knockdown, K-RAS(V12), and mutant EGFR) to lung tumorigenesis using human bronchial epithelial cells (HBEC) immortalized with telomerase and Cdk4-mediated p16 bypass. RNA interference p53 knockdown or oncogenic K-RAS(V12) resulted in enhanced anchorage-independent growth and increased saturation density of HBECs. The combination of p53 knockdown and K-RAS(V12) further enhanced the tumorigenic phenotype with increased growth in soft agar and an invasive phenotype in three-dimensional organotypic cultures but failed to cause HBECs to form tumors in nude mice. Growth of HBECs was highly dependent on epidermal growth factor (EGF) and completely inhibited by EGF receptor (EGFR) tyrosine kinase inhibitors, which induced G1 arrest. Introduction of EGFR mutations E746-A750 del and L858R progressed HBECs toward malignancy as measured by soft agar growth, including EGF-independent growth, but failed to induce tumor formation. Mutant EGFRs were associated with higher levels of phospho-Akt, phospho-signal transducers and activators of transcription 3 [but not phospho-extracellular signal-regulated kinase (ERK) 1/2], and increased expression of DUSP6/MKP-3 phosphatase (an inhibitor of phospho-ERK1/2). These results indicate that (a) the HBEC model system is a powerful new approach to assess the contribution of individual and combinations of genetic alterations to lung cancer pathogenesis; (b) a combination of four genetic alterations, including human telomerase reverse transcriptase overexpression, bypass of p16/RB and p53 pathways, and mutant K-RAS(V12) or mutant EGFR, is still not sufficient for HBECs to completely transform to cancer; and (c) EGFR tyrosine kinase inhibitors inhibit the growth of preneoplastic HBEC cells, suggesting their potential for chemoprevention.

By expressing two genes (hTERT and Cdk4), we have developed a method to reproducibly generate continuously replicating human bronchial epithelial cell (HBEC) lines that provide a novel resource to study the molecular pathogenesis of lung cancer and the differentiation of bronchial epithelial cells. Twelve human bronchial epithelial biopsy specimens obtained from persons with and without lung cancer were placed into short-term culture and serially transfected with retroviral constructs containing cyclin-dependent kinase (Cdk) 4 and human telomerase reverse transcriptase (hTERT), resulting in continuously growing cultures. The order of introduction of Cdk4 and hTERT did not appear to be important; however, transfection of either gene alone did not result in immortalization. Although they could be cloned, the immortalized bronchial cells did not form colonies in soft agar or tumors in nude mice. The immortalized HBECs have epithelial morphology; express epithelial markers cytokeratins 7, 14, 17, and 19, the stem cell marker p63, and high levels of p16(INK4a); and have an intact p53 checkpoint pathway. Cytogenetic analysis and array comparative genomic hybridization profiling show immortalized HBECs to have duplication of parts of chromosomes 5 and 20. Microarray gene expression profiling demonstrates that the Cdk4/hTERT-immortalized bronchial cell lines clustered together and with nonimmortalized bronchial cells, distinct from lung cancer cell lines. We also immortalized several parental cultures with viral oncoproteins human papilloma virus type 16 E6/E7 with and without hTERT, and these cells exhibited loss of the p53 checkpoint and significantly different gene expression profiles compared with Cdk4/hTERT-immortalized HBECs. These HBEC lines are a valuable new tool for studying of the pathogenesis of lung cancer.

Many stimuli causing 'stress' or DNA damage in cells can produce phenotypes that overlap with telomere-based replicative senescence. In epithelial systems, the p16/RB pathway may function as a stress senescence-signaling pathway independent of telomere shortening. Overexpressing cyclin-dependent kinase 4 (Cdk4) in human epidermal keratinocytes and human mammary epithelial cells not only prevents the p16(INK4a)-associated premature growth arrest due to telomere-independent stress (e.g., inadequate culture conditions), but also bypasses the ensuing telomere-dependent senescence (M1). Overexpressed Cdk4 in epithelial cells induces a dramatic upregulation of p16(INK4a) and milder upregulation of p53 and p21(WAF1), which become unresponsive to UV irradiation. Despite the high levels of these checkpoint factors, Cdk4-overexpressing cells divide in an apparently normal regulated fashion, are able to respond to changes in calcium levels, retain the stem cell phenotype, and fully differentiate and stratify. These results suggest that the differentiation pathways in Cdk4-overexpressing cells remain intact.

The myofibroblast is responsible for the generation of contractile force associated with wound contraction and pathological contractures and is characterized by the presence of alpha-smooth muscle (alpha-sm) actin-containing stress fibers, vinculin-containing fibronexus adhesion complexes, and fibronectin fibrils containing the ED-A splice variant. Transforming growth factor-beta1 (TGF-beta1) can promote the expression of alpha-sm actin in myofibroblasts, but the functional significance of this increased expression is unclear. In this study, we demonstrate, using the stress-relaxed collagen lattice contraction assay, that TGF-beta1 promoted a dose-dependent increase in the generation of contractile force in myofibroblasts and a concomitant increase in the expression of alpha-sm actin. We also demonstrate that TGF-beta1 enhanced the formation of the structural elements important in myofibroblast contractile force generation and transmission, including stress fibers, vinculin-containing fibronexus adhesion complexes, and fibronectin fibrils, and that this enhancement occurred prior to, and independent of, alpha-sm actin expression. This differentiated myofibroblast phenotype was not stable. Removal of TGF-beta1 resulted in reduced expression of alpha-sm actin as well as a decreased assembly of stress fibers and vinculin-containing adhesion complexes; however, there was no reduction in fibronectin fibrils. We conclude that TGF-beta1 promotes the morphological and functional differentiation of the myofibroblast by first enhancing the formation of the structural elements characteristic of the myofibroblast followed by increased expression of alpha-sm actin and contractile force generation.

Purpose: The contractile properties of in vitro cultured bladder smooth muscle cells (SMC) are unknown. This study characterized the in vitro contractile response of human and rat bladder SMC to several pharmacological agonists known to induce in vivo contraction of intact bladder muscle., Materials and Methods: Human and rat bladder SMC were seeded separately within attached collagen lattices. Contractility of SMC was analyzed by measuring alterations in lattice diameter after exposure and release to the following contractile agonists: carbachol (10(-7)-10(-3) microM), calcium-ionophore (10 microM), lysophosphatidic acid (LPA) (1 microM), endothelin (0.1 microM), KCl (3.33 mmicroM) angiotensin II (10 microM), and serotonin (100 microM). Results were recorded as a mean reduction of the lattice diameter. In addition, immunohistochemical analysis for phenotypic markers of smooth muscle cell differentiation was performed on bladder SMC cultured within collagen lattices. Human palmar fascia fibroblasts, which have been previously well characterized by in vitro contractility and immunohistochemistry, were tested in parallel and used as controls for all the above experiments., Results: Human SMC had significant contractile responses to calcium-ionophore (31% +/- 4 relative percent contraction, p <0.05), LPA (34% +/- 4, p <0.05), and endothelin (37 +/- 5%, p <05). There was no significant contraction in response to carbachol, angiotensin II, KCl, or serotonin. Rat bladder SMC had a similar contractile response but did not contract in response to endothelin. In contrast to human and rat bladder SMC, fibroblasts did not contract to calcium-ionophore., Conclusions: In vitro cultured bladder SMC demonstrate loss of contractile response to normal in vivo pharmacologic agonists. Both human and rat bladder SMC can be distinguished in vitro from fibroblasts based upon their lack of contractile response to calcium- ionophore. These results demonstrate the ability to further characterize cultured bladder SMC with in vitro contractility. Further characterization is essential if we are to advance our understanding of the clinical applicability of in vitro studies utilizing cultured bladder SMC.

Numerous studies support the idea that the myofibroblast is a key cell responsible for the tissue contraction in Dupuytren's disease. In vitro models have been developed to study the underlying cellular basis of myofibroblast differentiation and contraction. Studies suggest that the growth factor TGF-beta 1 combined with mechanical stress can promote the differentiation of fibroblasts into myofibroblasts. Agonists, such as LPA and thrombin, can promote the contraction of myofibroblasts through specific intracellular signaling pathways that regulate levels of phosphorylated myosin light chain. Agents that can affect these intracellular signaling pathways hold promise as a means to decrease contraction of the myofibroblast and of the palmar fascia in Dupuytren's disease. Finally, the recent finding that IFN-gamma can suppress both the differentiation of the myofibroblast and the generation of contractile force, together with preliminary clinical results using IFN-gamma, suggest the potential use of IFN-gamma for nonsurgical therapy of Dupuytren's disease. Future studies into the cellular basis of tissue contraction should provide alternative methods to improve management of Dupuytren's contracture.

The generation of tension in granulation tissue undergoing contraction is believed to be a cell-mediated event. In this study we used attached collagen lattices as a model system for studying the cellular mechanisms of tension generation by fibroblasts in an extracellular matrix. Fibroblasts in attached collagen lattices developed stress fibers, surface associated fibronectin fibrils, and a fibronexus-like transmembrane association interconnecting the two structural components. Release of the attached collagen lattice from its points of attachment resulted in a rapid, symmetrical contraction of the collagen lattice. Rapid contraction occurred within the first 10 minutes after release of the lattice from the substratum, with greater than 70% of the contraction occurring within the first 2 minutes. Rapid contraction resulted in a shortening of the elongate fibroblasts and compaction of the stress fibers with their subsequent disappearance from the cell. Cytochalasin D treatment prior to release disrupted the actin cytoskeleton and completely inhibited rapid contraction. The removal of serum prior to release inhibited rapid contraction, while the re-addition of serum restored rapid contraction. These results demonstrate that fibroblasts can develop tension in an attached collagen lattice and that upon release of tension the fibroblasts undergo contraction resulting in a rapid contraction of the collagen lattice. Fibroblast contraction is dependent upon an organized actin cytoskeleton and is promoted by the presence of serum.