In Vitro Development of a Mucocutaneous Junction for Lip Reconstruction.

PURPOSE: We present a straightforward and reproducible technique to create, in vitro, a construct containing a mucocutaneous junction (MCJ) with a transitional zone (vermilion) for fabrication of a microvascular prelaminated flap for use in lip reconstruction. MATERIALS AND METHODS: Cultured primary human skin keratinocytes and oral mucosal epithelial cells at premixed ratios of 50% skin cells to 50% oral cells, 25% skin cells to 75% oral cells, and 75% skin cells to 25% oral cells were grown on an AlloDerm dermal equivalent (LifeCell, Branchburg, NJ) to create an MCJ equivalent with a lip or transitional zone (vermilion) using a novel 3-dimensional (3D) culture device with a barrier to separate co-cultured skin and oral cells. The 3 different cell ratios were compared by staining for the following specific differentiation markers to define the different areas of skin and mucosal keratinocytes: filaggrin, cytokeratin 10, cytokeratin 19, and small proline-rich protein 3. RESULTS: Immunohistochemical results showed that MCJ equivalents seeded with premixed cells were similar to the differentiation patterns of tissue-engineered 3D cultures using 100% oral mucosal epithelial cells or skin keratinocytes. The engineered MCJ-equivalent constructs, grown in the 3D device specifically constructed with a cell-free gap at the barrier site, formed a transitional zone (vermilion) at the barrier site with intermingling of the skin and oral keratinocytes. The results showed different and unique expression patterns of filaggrin, cytokeratin 10, cytokeratin 19, and small proline-rich protein 3 by those cells migrating into the gap, which were similar to those seen in human lip tissue. This pattern was not seen in MCJ equivalents created using premixed skin and oral cells. CONCLUSIONS: Using a device to separately co-culture human oral and skin keratinocytes to allow the cells to migrate into a cell-free zone resulted in phenotypic expression closer to what is seen in native tissue, in comparison to premixing the skin and oral cells before seeding.

Hypoxia induces an undifferentiated phenotype of oral keratinocytes in vitro.

The aim of this study was to determine the effects of hypoxia on the proliferating potential and phenotype of primary human oral keratinocytes cultured at ambient oxygen tension (20%) or at different levels of hypoxia (2 and 0.5% O2). The effects of oxygen tensions on cellular metabolic activity, cell proliferation, clonogenicity and proliferation heterogeneity were measured. Cell cycle profiles were analyzed by a fluorescent-activated cell sorter, and p21(WAF1/CIP1) expression in the G0/G1 phase was also concomitantly quantitated. The expression levels of cell cycle regulatory proteins were examined by immunoblotting, and the cellular senescence was assessed by senescence-associated ß-galactosidase staining. Basal and suprabasal keratinocyte phenotypes were determined by the expression levels of 14-3-3σ, p75(NTR) and α6 integrin. Despite having a lower metabolism, the proliferation rate and clonogenic potential were remarkably enhanced in hypoxic cells. The significantly higher percentage of cells in the G0/G1 phase under hypoxia and the expression patterns of cell cycle regulatory proteins in hypoxic cells were indicative of a state of cell cycle arrest in hypoxia. Furthermore, a decrease in the expression of p21(WAF1/CIP1) and p16(INK4A) and fewer ß-galactosidase-positive cells suggested a quiescent phenotype rather than a senescent one in hypoxic cells. Compared with normoxic cells, the differential expression patterns of keratinocyte phenotypic markers suggest that hypoxic cells that generate minimal reactive oxygen species, suppress the mammalian target of rapamycin activity and express hypoxia-inducible factor-1α favor a basal cell phenotype. Thus, regardless of the predisposition to the state of cell cycle arrest, hypoxic conditions can maintain oral keratinocytes in vitro in an undifferentiated and quiescent state.

Tuft cells are required for a rhinovirus-induced asthma phenotype in immature mice.

Infection of immature mice with rhinovirus (RV) induces an asthma-like phenotype consisting of type 2 inflammation, mucous metaplasia, eosinophilic inflammation, and airway hyperresponsiveness that is dependent on IL-25 and type 2 innate lymphoid cells (ILC2s). Doublecortin-like kinase 1-positive (DCLK1+) tuft cells are a major source of IL-25. We sought to determine the requirement of tuft cells for the RV-induced asthma phenotype in wild-type mice and mice deficient in Pou2f3, a transcription factor required for tuft cell development. C57BL/6J mice infected with RV-A1B on day 6 of life and RV-A2 on day 13 of life showed increased DCLK1+ tuft cells in the large airways. Compared with wild-type mice, RV-infected Pou2f3-/- mice showed reductions in IL-25 mRNA and protein expression, ILC2 expansion, type 2 cytokine expression, mucous metaplasia, lung eosinophils, and airway methacholine responsiveness. We conclude that airway tuft cells are required for the asthma phenotype observed in immature mice undergoing repeated RV infections. Furthermore, RV-induced tuft cell development provides a mechanism by which early-life viral infections could potentiate type 2 inflammatory responses to future infections.

M2 Macrophages promote IL-33 expression, ILC2 expansion and mucous metaplasia in response to early life rhinovirus infections.

Wheezing-associated rhinovirus (RV) infections are associated with asthma development. We have shown that infection of immature mice with RV induces type 2 cytokine production and mucous metaplasia which is dependent on IL-33 and type 2 innate lymphoid cells (ILC2s) and intensified by a second heterologous RV infection. We hypothesize that M2a macrophages are required for the exaggerated inflammation and mucous metaplasia in response to heterologous RV infection. Wild-type C57Bl/6J mice and LysMCre IL4Rα KO mice lacking M2a macrophages were treated as follows: (1) sham infection on day 6 of life plus sham on day 13 of life, (2) RV-A1B on day 6 plus sham on day 13, (3) sham on day 6 and RV-A2 on day 13, or (4) RV-A1B on day 6 and RV-A2 on day 13. Lungs were harvested one or seven days after the second infection. Wild-type mice infected with RV-A1B at day 6 showed an increased number of Arg1- and Retnla-expressing lung macrophages, indicative of M2a polarization. Compared to wild-type mice infected with RV on day 6 and 13 of life, the lungs of LysMCre IL4Rα KO mice undergoing heterologous RV infection showed decreased protein abundance of the epithelial-derived innate cytokines IL-33, IL-25 and TSLP, decreased ILC2s, decreased mRNA expression of IL-13 and IL-5, and decreased PAS staining. Finally, mRNA analysis and immunofluorescence microscopy of double-infected LysMCre IL4Rα KO mice showed reduced airway epithelial cell IL-33 expression, and treatment with IL-33 restored the exaggerated muco-inflammatory phenotype. Conclusion: Early-life RV infection alters the macrophage response to subsequent heterologous infection, permitting enhanced IL-33 expression, ILC2 expansion and intensified airway inflammation and mucous metaplasia.

Noninvasive Optical Assessment of Implanted Tissue-Engineered Construct Success In Situ.

Quantitative diffuse reflectance spectroscopy (DRS) was developed for label-free, noninvasive, and real-time assessment of implanted tissue-engineered devices manufactured from primary human oral keratinocytes (six batches in two 5-patient cohorts). Constructs were implanted in a murine model for 1 and 3 weeks. DRS evaluated construct success in situ using optical absorption (hemoglobin concentration and oxygenation, attributed to revascularization) and optical scattering (attributed to cellular density and layer thickness). Destructive pre- and postimplantation histology distinguished experimental control from stressed constructs, whereas noninvasive preimplantation measures of keratinocyte glucose consumption and residual glucose in spent culture media did not. In constructs implanted for 1 week, DRS distinguished control due to stressed and compromised from healthy constructs. In constructs implanted for 3 weeks, DRS identified constructs with higher postimplantation success. These results suggest that quantitative DRS is a promising, clinically compatible technology for rapid, noninvasive, and localized tissue assessment to characterize tissue-engineered construct success in vivo. Impact statement Despite the recent advance in tissue engineering and regenerative medicine, there is still a lack of nondestructive tools to longitudinally monitor the implanted tissue-engineered devices. In this study, we demonstrate the potential of quantitative diffuse reflectance spectroscopy as a clinically viable technique for noninvasive, label-free, and rapid characterization of graft success in situ.

Optical Metric Assessed Engineered Tissues Over a Range of Viability States.

Many conventional methods to assess engineered tissue morphology and viability are destructive techniques with limited utility for tissue constructs intended for implantation in patients. Sterile label-free optical molecular imaging methods analyzed tissue endogenous fluorophores without staining, noninvasively and quantitatively assessing engineered tissue, in lieu of destructive assessment methods. The objective of this study is to further investigate label-free optical metrics and their correlation with destructive methods. Tissue-engineered constructs (n = 33 constructs) fabricated with primary human oral keratinocytes (n = 10 patients) under control, thermal stress, and rapamycin treatment manufacturing conditions exhibited a range of tissue viability states, as evaluated by quantitative histology scoring, WST-1 assay, Ki-67 immunostaining imaging, and label-free optical molecular imaging methods. Both histology sections of fixed tissues and cross-sectioned label-free optical images of living tissues provided quantitative spatially selective information on local tissue morphology, but optical methods noninvasively characterized both local tissue morphology and cellular viability at the same living tissue site. Furthermore, optical metrics noninvasively assessed living tissue viability with a statistical significance consistent with the destructive tissue assays WST-1 and histology. Over the range of cell viability states created experimentally, optical metrics noninvasively and quantitatively characterized living tissue viability and correlated with the destructive WST-1 tissue assay. By providing, under sterile conditions, noninvasive metrics that were comparable with conventional destructive tissue assays, label-free optical molecular imaging has the potential to monitor and assess engineered tissue construct viability before surgical implantation.

Production of progenitor cells from primary human epithelial cell monolayer cultures.

Primary keratinocytes derived from human epidermis are widely used in tissue engineering and regenerative medicine. An important aspect in clinical applications is the preservation of human skin keratinocyte stem cells. However, it is difficult to expand the number of human skin keratinocyte stem cells, which are undifferentiated and highly proliferative in culture by using standard cell culture methods. It is even more difficult to identify them, since universal specific markers for human skin keratinocyte stem cells have not been identified. In this paper, we show a method to produce a large number of primary progenitor human skin keratinocytes by using our novel culture techniques. Primary human skin keratinocyte monolayers are cultured using twice the volume of medium without serum and lacking essential fatty acids. Once the cells reach 70-80% confluence, they begin to float up into the overlying medium and are called "epithelial pop-up keratinocytes (ePUKs)" allowing the cells to be passaged without the use of trypsin. We analyzed the properties of ePUKs by cell size, cell viability, immunocytofluorescence biomarker staining, and cell cycle phase distribution by fluorescence-activated cell sorting (FACS). Our results showed that these ePUKs appear to be progenitor epithelial cells, which are small in size, undifferentiated, and have a high proliferative capacity. We believe that ePUKs are suitable for use in medical applications requiring a large number of primary human progenitor skin keratinocytes.

Utilisation of a Bioreactor for Culture and Expansion of Epithelial Cells without the use of Trypsin or Enzymes.

OBJECTIVE: To develop a bioreactor for automated culture, maintenance, and collection of normal human keratinocytes using an enzyme-free propagation method. METHODS: The culture of normal human epithelial keratinocytes was compared in two culture methods - a study team-developed automated bioreactor utilising an enzyme-free passage method, and a manual culture method. Cell size, glucose utilisation, and the proliferative capacity of the two cultures were evaluated. RESULTS: An automated bioreactor, not using enzymes for passage, but instead using the novel Epithelial Pop Up Keratinocytes (ePUK)1 culture technique, resulted in an extended culture longevity and proliferative capacity in normal primary human keratinocytes. Daughter cells were collected up to three times per day utilising the bioreactor. The daughter cells produced by the bioreactor were smaller than daughter cells produced by the manual culture method. The proliferative capacity and health of the parent monolayer within both the bioreactor and the manual culture flask was dependent upon sufficient glucose availability. Due to the contact inhibition nature of epithelial keratinocytes, the bioreactor enabled the study of an adherent cell type soon after cytokinesis and before the cell has integrated as part of an adherent matrix. CONCLUSION: The study demonstrates that increasing the number of media changes per day as necessary, based on glucose utilisation, is necessary for prolonged longevity and functional productivity of ePUK cultures.

Comparison of two decellularized dermal equivalents.

Immunologically inert allogeneic acellular dermal scaffolds provide a matrix with molecular architecture close to native tissues, which synthetic scaffolds cannot. Not all nature-derived scaffolds possess the same biological and physical properties. The different properties of scaffolds supporting cellular growth used for manufacturing tissue engineered grafts could lead to different implantation results. The scaffold properties should be carefully considered in order to meet the expected outcomes of tissue engineered grafts. In this report, we evaluated the cellular growth on AlloDerm® and Allopatch, 2 acellular scaffolds derived from human cadaver skin, using a fabricated 3D organotypic culture with primary human oral keratinocytes to produce an ex vivo produced oral mucosa equivalent (EVPOME). A well stratified epithelium could be constructed on both scaffolds. AlloDerm® and Allopatch EVPOMEs were also implanted into severe combined immunodeficiency mice to compare the ingrowth of blood vessels into the dermal component of the two EVPOMEs. Blood vessel counts were 3.3 times higher (p = .01) within Allopatch EVPOMEs than within AlloDerm® EVPOMEs. An oral and skin keratinocyte co-culture, separated by a physical barrier to create a cell-free zone, was used to evaluate cell migration on AlloDerm® and Allopatch. Slower cell migration was observed on Allopatch than on AlloDerm®.

Noninvasive Optical Assessment of Implanted Engineered Tissues Correlates with Cytokine Secretion.

Fluorescence lifetime sensing has been shown to noninvasively characterize the preimplantation health and viability of engineered tissue constructs. However, current practices to monitor postimplantation construct integration are either qualitative (visual assessment) or destructive (tissue histology). We employed label-free fluorescence lifetime spectroscopy for quantitative, noninvasive optical assessment of engineered tissue constructs that were implanted into a murine model. The portable system was designed to be suitable for intravital measurements and included a handheld probe to precisely and rapidly acquire data at multiple sites per construct. Our model tissue constructs were manufactured from primary human cells to simulate patient variability based on a standard protocol, and half of the manufactured constructs were stressed to create a range of health states. Secreted amounts of three cytokines that relate to cellular viability were measured in vitro to assess preimplantation construct health: interleukin-8 (IL-8), human ß-defensin 1 (hBD-1), and vascular endothelial growth factor (VEGF). Preimplantation cytokine secretion ranged from 1.5 to 33.5 pg/mL for IL-8, from 3.4 to 195.0 pg/mL for hBD-1, and from 0.1 to 154.3 pg/mL for VEGF. In vivo optical sensing assessed constructs at 1 and 3 weeks postimplantation. We found that at 1 week postimplantation, in vivo optical parameters correlated with in vitro preimplantation secretion levels of all three cytokines (p < 0.05). This correlation was not observed in optical measurements at 3 weeks postimplantation when histology showed that the constructs had re-epithelialized, independent of preimplantation health state, supporting the lack of a correlation. These results suggest that clinical optical diagnostic tools based on label-free fluorescence lifetime sensing of endogenous tissue fluorophores could noninvasively monitor postimplantation integration of engineered tissues.

A new scrambled gene in the ciliate Uroleptus.

In the germline micronucleus of spirotrichous ciliates, the gene segments, or macronuclear destined sequences (MDSs), that give rise to the somatic macronucleus are interrupted by internal eliminated sequences (IESs). For some genes, the MDSs are not arranged sequentially, but rather are scrambled, in the micronucleus. Three scrambled genes have been extensively studied in many species: actin I, alpha-telomere binding protein, and DNA polymerase alpha. However, in the past decade, no new scrambled genes have been reported, and the prevalence of scrambled genes is still an important question. To screen for scrambled genes, we completely sequenced 11 macronuclear chromosomes in the spirotrich Uroleptus sp., and then pursued their micronuclear organization. This allowed us to identify new scrambled genes, which also display novel features. In this study we describe one of these newly discovered scrambled genes. This gene, tentatively named USG1 (Unknown Scrambled Gene 1), encodes a putative protein of 1016 aa. While the function of this protein product is not clear, dN/dS calculated from the two alleles suggests the encoded protein is under purifying selection. USG1 consists of 16 germline MDSs, of which 14 are located on one locus. The other locus, which is at least 3 kb away from the main locus, contains two scrambled MDSs separated by a nonscrambled IES. Curiously, one MDS and its outgoing (3') pointer (direct repeat) overlap intron splice sites, indicating that these DNA sequences may be under dual (or multiple) constraints. Our findings identify a new scrambled gene in the micronuclear genome of a spirotrichous ciliate, and suggest that even more complicated structures may be present.

Proteomics Characterization of Primary Human Oral Epithelial Cells Using a Novel Culture Technique for Use in Tissue Regeneration.

Oral mucosa keratinocytes are widely used in regenerative medicine. The unique cultured cell population "Epithelial-derived Pop-Up Keratinocytes (ePUKs)" was previously reported as undifferentiated cells. Gravity Assisted Cell Sorting (GACS) was used to isolate a small-sized population of undifferentiated cells enriched ePUKs. LC/MS/MS analysis was performed to define the cellular profile of ePUKs of primary human oral mucosa keratinocytes. Small sized ePUKs which showed increased expression of Dickkopf WNT signaling pathway inhibitor 1 (DKK1), serpin peptidase inhibitor, clade E (nexin, plasminogen activator inhibitor type 1), member 1 (SERPINE1), follistatin and tenascin-C were verified by Western blots. These proteins are involved in the regulation of cellular movement, hair follicle development and the maintenance of its stem cell niche. The fabrication of a tissue-engineered oral mucosa, ex vivo produced oral mucosa equivalent (EVPOME), using ePUKs showed increased abundance of these verified proteins. These findings indicate that the specific phenotype of ePUKs and their ability to influence wound healing promotion are implicated by highly expressed cellular movement regulatory proteins. Therefore, ePUKs may be a useful cell source for use in regenerative medicine.

Human oral mucosa tissue-engineered constructs monitored by Raman fiber-optic probe.

In maxillofacial and oral surgery, there is a need for the development of tissue-engineered constructs. They are used for reconstructions due to trauma, dental implants, congenital defects, or oral cancer. A noninvasive monitoring of the fabrication of tissue-engineered constructs at the production and implantation stages done in real time is extremely important for predicting the success of tissue-engineered grafts. We demonstrated a Raman spectroscopic probe system, its design and application, for real-time ex vivo produced oral mucosa equivalent (EVPOME) constructs noninvasive monitoring. We performed in vivo studies to find Raman spectroscopic indicators for postimplanted EVPOME failure and determined that Raman spectra of EVPOMEs preexposed to thermal stress during manufacturing procedures displayed correlation of the band height ratio of CH2 deformation to phenylalanine ring breathing modes, giving a Raman metric to distinguish between healthy and compromised postimplanted constructs. This study is the step toward our ultimate goal to develop a stand-alone system, to be used in a clinical setting, where the data collection and analysis are conducted on the basis of these spectroscopic indicators with minimal user intervention.

High-frequency ultrasonic imaging of growth and development in manufactured engineered oral mucosal tissue surfaces.

This study uses high-resolution ultrasound to examine the growth and development of engineered oral mucosal tissues manufactured under aseptic conditions. The specimens are a commercially available natural tissue scaffold, AlloDerm, and oral keratinocytes seeded onto AlloDerm to form an ex vivo-produced oral mucosal equivalent (EVPOME) suitable for intra-oral grafting. The seeded cells produce a keratinized protective upper layer that smooths out any remaining surface irregularities on the underlying AlloDerm. Two-dimensional acoustic imaging of unseeded AlloDerm and developing EVPOMEs was performed on each day of their growth and development, each tissue specimen being imaged under aseptic conditions (total time from seeding to maturation: 11 d). Ultrasonic monitoring offers us the ability to determine the constituents of the EVPOME that are responsible for changes in its mechanical behavior during the manufacturing process. Ultrasonic monitoring affords us an opportunity to non-invasively assess, in real time, tissue-engineered constructs before release for use in patient care.

The potential of label-free nonlinear optical molecular microscopy to non-invasively characterize the viability of engineered human tissue constructs.

Nonlinear optical molecular imaging and quantitative analytic methods were developed to non-invasively assess the viability of tissue-engineered constructs manufactured from primary human cells. Label-free optical measures of local tissue structure and biochemistry characterized morphologic and functional differences between controls and stressed constructs. Rigorous statistical analysis accounted for variability between human patients. Fluorescence intensity-based spatial assessment and metabolic sensing differentiated controls from thermally-stressed and from metabolically-stressed constructs. Fluorescence lifetime-based sensing differentiated controls from thermally-stressed constructs. Unlike traditional histological (found to be generally reliable, but destructive) and biochemical (non-invasive, but found to be unreliable) tissue analyses, label-free optical assessments had the advantages of being both non-invasive and reliable. Thus, such optical measures could serve as reliable manufacturing release criteria for cell-based tissue-engineered constructs prior to human implantation, thereby addressing a critical regulatory need in regenerative medicine.

Characterizing morphology and nonlinear elastic properties of normal and thermally stressed engineered oral mucosal tissues using scanning acoustic microscopy.

This study examines the use of high-resolution ultrasound to monitor changes in the morphology and nonlinear elastic properties of engineered oral mucosal tissues under normal and thermally stressed culture conditions. Nonlinear elastic properties were determined by first developing strain maps from acoustic ultrasound, followed by fitting of nonlinear stress-strain data to a 1-term Ogden model. Testing examined a clinically developed ex vivo produced oral mucosa equivalent (EVPOME). As seeded cells proliferate on an EVPOME surface, they produce a keratinized protective upper layer that fills in and smoothens out surface irregularities. These transformations can also alter the nonlinear stress/strain parameters as EVPOME cells differentiate. This EVPOME behavior is similar to those of natural oral mucosal tissues and in contrast to an unseeded scaffold. If ultrasonic monitoring could be developed, then tissue cultivation could be adjusted in-process to account for biological variations in their development of the stratified cellular layer. In addition to ultrasonic testing, an in-house-built compression system capable of accurate measurements on small (∼1.0-1.5 cm(2)) tissue samples is presented. Results showed a near 2.5-fold difference in the stiffness properties between the unstressed EVPOME and the noncell-seeded acellular scaffold (AlloDerm(®)). There were also 4×greater differences in root mean square values of the thickness in the unseeded AlloDerm compared to the mature unstressed EVPOME; this is a strong indicator for quantifying surface roughness.

Tissue-engineered constructs of human oral mucosa examined by Raman spectroscopy.

A noninvasive quality monitoring of tissue-engineered constructs is a required component of any successful tissue-engineering technique. During a 2-week production period, ex vivo produced oral mucosa-equivalent constructs (EVPOMEs) may encounter adverse culturing conditions that might compromise their quality and render them ineffective. We demonstrate the application of near-infrared Raman spectroscopy to in vitro monitoring of EVPOMEs during their manufacturing process, with the ultimate goal of applying this technology in situ to monitor the grafted EVPOMEs. We identify Raman spectroscopic failure indicators for less-than optimal EVPOMEs that are stressed by higher temperature and exposure to higher than normal concentration of calcium ions. Raman spectra of EVPOMEs exposed to thermal and calcium stress showed correlation of the band height ratio of CH(2) deformation to phenylalanine ring breathing modes, providing a Raman metric to distinguish between viable and nonviable constructs. We compared these results to histology and glucose consumption measurements, demonstrating that Raman spectroscopy is more sensitive and specific to changes in proteins' secondary structure not visible by H&E histology. We also exposed the EVPOMEs to rapamycin, a cell growth inhibitor and cell proliferation capacity preserver, and distinguished between EVPOMEs pretreated with 2 nM rapamycin and controls, using the ratio of the Amide III envelope to the phenylalanine band as an indicator.

Characterization of squamous cell carcinoma in an organotypic culture via subsurface non-linear optical molecular imaging.

Tristetraprolin (TTP) is an RNA-binding protein which downregulates multiple cytokines that mediate progression of head and neck squamous cell carcinoma (HNSCC). We previously showed that HNSCC cells with shRNA-mediated knockdown of TTP are more invasive than controls. In this study, we use control and TTP-deficient cells to present a novel subsurface non-linear optical molecular imaging method using a three-dimensional (3D) organotypic construct, and compare the live cell imaging data to histology of fixed tissue specimens. This manuscript describes how to prepare and image the novel organotypic system that closely mimics HNSCC in a clinical setting. The oral cancer equivalent (OCE) system allows HNSCC cells to stratify and invade beyond the basement membrane into underlying connective tissue prepared from decellularized human dermal tissue. The OCE model was inspired by tissue engineering strategies to prepare autologous transplants from human keratinocytes. Advantages of this method over previously used in vitro cancer models include the simulation of the basement membrane and complex connective tissue in the construct, in addition to the ability to track the 3D movement of live invading cells and quantify matrix destruction over time. The OCE model and novel live cell imaging strategy may be applied to study other types of 3D tissue constructs.

Inactivation or loss of TTP promotes invasion in head and neck cancer via transcript stabilization and secretion of MMP9, MMP2, and IL-6.

PURPOSE: Invasion is the critical step in progression of a precancerous lesion to squamous cell carcinoma of the head and neck (HNSCC). Invasion is regulated by multiple proinflammatory mediators. Tristetraprolin (TTP) is an mRNA-degrading protein that regulates multiple proinflammatory mediators. TTP may serve as an excellent treatment target. Rap1 is a ras-like oncoprotein that induces critical signaling pathways. In this study, the role of rap1 in TTP-mediated invasion was investigated. EXPERIMENTAL DESIGN: Using complementary approaches, we modulated TTP and altered expression of interleukin (IL)-6 and matrix metalloproteinase (MMP) 2/9, which were quantified by ELISA and zymogram. Invasion was evaluated in vitro using the oral-cancer-equivalent (OCE) three-dimensional model and in vivo in the chick chorioallantoic membrane (CAM). The role of rap1 and p38 were established using knockdown strategies. RESULTS: Downregulation of TTP significantly increased invasion via secretion of MMP9/2 and IL-6. In the novel OCE and CAM invasion models of HNSCC, cells with downregulated TTP destroyed the basement membrane to invade the underlying connective tissue. Rap1 induces p38 mitogen-activated protein kinase (p38)-mediated inactivation of TTP. Inactive TTP enhances transcript stability via binding to the 3'-untranslated region (UTR). High IL-6 and MMP9 are prognostic for poor clinical outcomes in patients with HNSCC. CONCLUSIONS: Targeting the rap1-p38-TTP cascade is an attractive novel treatment strategy in HNSCC to concurrently suppress multiple mediators of invasion.

Acoustic microscopy analyses to determine good vs. failed tissue engineered oral mucosa under normal or thermally stressed culture conditions.

This study uses scanning acoustic microscopy (SAM) ultrasonic profilometry to determine acceptable vs. failed tissue engineered oral mucosa. Specifically, ex vivo-produced oral mucosal equivalents (EVPOMEs) under normal or thermally stressed culture conditions were scanned with the SAM operator blinded to the culture conditions. As seeded cells proliferate, they fill in and smooth out the surface irregularities; they then stratify and produce a keratinized protective upper layer. Some of these transformations could alter backscatter of ultrasonic signals and in the case of the thermally stressed cells, produce backscatter similar to an unseeded device. If non-invasive ultrasonic monitoring could be developed, then tissue cultivation could be adjusted to measure biological variations in the stratified surface. To create an EVPOME device, oral mucosa keratinocytes were seeded onto acellular cadaveric dermis. Two sets of EVPOMEs were cultured: one at physiological temperature 37 °C and the other at 43 °C. The specimens were imaged with SAM consisting of a single-element transducer: 61 MHz center frequency, 32 MHz bandwidth, 1.52 f#. Profilometry for the stressed and unseeded specimens showed higher surface irregularities compared to unstressed specimens. Elevated thermal stress retards cellular differentiation, increasing root mean square values; these results show that SAM can potentially monitor cell/tissue development.