Brain Mechanisms of Virtual Reality Breathing Versus Traditional Mindful Breathing in Pain Modulation: Observational Functional Near-infrared Spectroscopy Study.

BACKGROUND: Pain is a complex experience that involves sensory-discriminative and cognitive-emotional neuronal processes. It has long been known across cultures that pain can be relieved by mindful breathing (MB). There is a common assumption that MB exerts its analgesic effect through interoception. Interoception refers to consciously refocusing the mind's attention to the physical sensation of internal organ function. OBJECTIVE: In this study, we dissect the cortical analgesic processes by imaging the brains of healthy subjects exposed to traditional MB (TMB) and compare them with another group for which we augmented MB to an outside sensory experience via virtual reality breathing (VRB). METHODS: The VRB protocol involved in-house-developed virtual reality 3D lungs that synchronized with the participants' breathing cycles in real time, providing them with an immersive visual-auditory exteroception of their breathing. RESULTS: We found that both breathing interventions led to a significant increase in pain thresholds after week-long practices, as measured by a thermal quantitative sensory test. However, the underlying analgesic brain mechanisms were opposite, as revealed by functional near-infrared spectroscopy data. In the TMB practice, the anterior prefrontal cortex uniquely modulated the premotor cortex. This increased its functional connection with the primary somatosensory cortex (S1), thereby facilitating the S1-based sensory-interoceptive processing of breathing but inhibiting its other role in sensory-discriminative pain processing. In contrast, virtual reality induced an immersive 3D exteroception with augmented visual-auditory cortical activations, which diminished the functional connection with the S1 and consequently weakened the pain processing function of the S1. CONCLUSIONS: In summary, our study suggested two analgesic neuromechanisms of VRB and TMB practices-exteroception and interoception-that distinctively modulated the S1 processing of the ascending noxious inputs. This is in line with the concept of dualism (Yin and Yang).

Examining spatiotemporal variability of urban particulate matter and application of high-time resolution data from a network of low-cost air pollution sensors.

Traditional air monitoring approaches using regulatory monitors have historically been used to assess regional-scale trends in air pollutants across large geographical areas. Recent advances in air pollution sensor technologies could provide additional information about nearby sources, support the siting of regulatory monitoring stations, and improve our knowledge of finer-scale spatiotemporal variation of ambient air pollutants and their associated health effects. Sensors are now being developed that are much smaller and lower cost than traditional ambient air monitoring systems and are capable of being deployed as a network to provide greater coverage of a given area. The CitySpace project conducted by the US EPA and the Shelby County Health Department included the deployment of a network of 17 sensor pods using Alphasense OPC-N2 particulate matter (PM) sensors integrated with meteorological sensors in Memphis, TN for six months. Sensor pods were collocated with a federal equivalent method (FEM) tapered element oscillating microbalance (TEOM) monitor both before and after the primary study period. Six of the sensor pods were found to meet the data quality objective (DQO) of coefficient of determination (R2) greater than 0.5 when collocated with the TEOM. Seven pods were decommissioned before the end of the study due to mechanical failure. The six pods meeting the DQO were used to examine the spatiotemporal variability of fine PM (PM2.5) across the Memphis area. One site was found to have higher relative PM2.5 concentrations when compared to the other sites in the network. The 1-min data from this sensor pod were evaluated to quantify the regional urban background and local-scale contributions to PM2.5 at that monitoring location. This method found that approximately 20% of the PM2.5 was attributed to local sources at this location, compared to 9% at a local regulatory monitoring site. Additionally, the 1-min data were combined with 1-min wind speed and wind direction data to examine potential sources in the area using the nonparametric trajectory analysis (NTA) technique. This method geographically identified local source areas that contributed to the measured concentrations at the high reading sensor location throughout the course of the study.

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.

Reconstruction of functional human lips utilizing a prelaminated flap.

Lips form a structure that are difficult to reconstruct after a traumatic avulsion injury or cancer ablative surgery secondary to loss of volumetric muscle mass. Traditional tissue engineering approaches of in vitro fabrication of mature tissue constructs can supply an alternative to the current surgical standard of care for functional lip reconstruction. We demonstrate a hybrid approach that combines the advantages of in situ muscle flap prefabrication with in vitro fabrication of an autogenous mucocutaneous construct as the laminate for prelamination to form a designer microvascular muscle free flap for lip reconstruction.

Characterization of cultured epithelial cells using a novel technique not requiring enzymatic digestion for subculturing.

Our laboratory had developed a methodology to expand epithelial cells in culture by growing keratinocyte monolayers, under large volumes of medium that produces large numbers of keratinocytes that leave the monolayer and move into suspension. The cells have been defined as epithelial Pop Up Keratinocytes or ePUKs cells and appear to be highly suitable for clinical applications. In this publication we extend the characterization of the cells with a detailed analysis of the capabilities of the monolayer of a single culture flask to produce, over time, ePUK cells. The cells were characterized using standard epithelial markers for proliferation and differentiation. Analysis of morphology of the monolayer formed and total number of cells produced is presented for a variety of human epithelial cell strains. These keratinocytes provide an additional controlled human cell system for investigation of the mechanisms regulating epithelia cell growth and differentiation and since they are produced in large numbers, they are highly suitable for use in epithelial cell banking.

Characterization of a unique technique for culturing primary adult human epithelial progenitor/"stem cells".

BACKGROUND: Primary keratinocytes derived from epidermis, oral mucosa, and urothelium are used in construction of cell based wound healing devices and in regenerative medicine. This study presents in vitro technology that rapidly expands keratinocytes in culture by growing monolayers under large volumes of serum-free, essential fatty acid free, low calcium medium that is replaced every 24 hrs. METHODS: Primary cell cultures were produced from epidermal skin, oral mucosa and ureter by trypsinization of tissue. Cells were grown using Epilife medium with growth factors under high medium volumes. Once densely confluent, the keratinocyte monolayer produced cells in suspension in the overlying medium that can be harvested every 24 hrs. over a 7-10 day period. The cell suspension (approximately 8 X 105 cells/ml) is poured into a new flask to form another confluent monolayer over 2-4 days. This new culture, in turn produced additional cell suspensions that when serially passed expand the cell strain over 2-3 months, without the use of enzymes to split the cultures. The cell suspension, called epithelial Pop Up Keratinocytes (ePUKs) were analyzed for culture expansion, cell size and glucose utilization, attachment to carrier beads, micro-spheroid formation, induction of keratinocyte differentiation, and characterized by immunohistochemistry. RESULTS: The ePUKs expanded greatly in culture, attached to carrier beads, did not form micro-spheroids, used approximately 50% of medium glucose over 24 hrs., contained a greater portion of smaller diameter cells (8-10 microns), reverted to classical appearing cultures when returned to routine feeding schedules (48 hrs. and 15 ml/T-75 flask) and can be differentiated by either adding 1.2 mM medium calcium, or essential fatty acids. The ePUK cells are identified as cycling (Ki67 expressing) basal cells (p63, K14 expressing). CONCLUSIONS: Using this primary culture technique, large quantities of epithelial cells can be generated without the use of the enzyme trypsin to split the cultures. The cells are small in diameter and have basal cell progenitor/"stem" (P/SC) cell characteristics induced by daily feeding with larger than normal medium volumes. The ePUK epithelial cells have the potential to be used in regenerative medicine and for basic studies of epithelia P/SC phenotype.

Synthesis and characterization of cytocompatible sulfonated polyanilines.

We report here that by good design, polyaniline (PANI) can be cytocompatible and formed into usable scaffolds for bio-medical applications. By adjusting the ratio of two monomers, aniline (AN) and metanilic acid (MA), a series of copolymers with different sulfonation degrees have been synthesized. Four-probe conductivity measurements showed that as the sulfonation degree increased, the conductivity decreased. XPS analysis was used to determine the sulfur/nitrogen ratio. In vitro cell culture study was conducted with human osteosarcoma (HOS) cells. Microscopic observation did not show abnormal cellular behavior when sulfonated polyaniline (SPAN) was put in direct contact with HOS cells. Cells growing on the non-transparent dark green SPAN films were observed with fluorescence by laser scanning cytometry (LSC). In proliferation studies more than 70% of cells were found viable on SPAN compared to 88% for poly(L-lactic acid) with the number of cells growing on glass as a control, indicating generally good biocompatibility. We expect these polymers would have great potential in biological applications of conducting polymers as we determine that a variety of physical and chemical properties can be controlled through synthesis.

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.

Living cell-based regenerative medicine technologies for periodontal soft tissue augmentation.

The cultivation of human living cells into scaffolding matrices has progressively gained popularity in the field of periodontal wound healing and regeneration. Living cellular constructs based on fibroblasts, keratinocytes alone or in combination have been developed and used as alternatives to autogenous soft tissue grafts in keratinized tissue augmentation and in root coverage procedures. Their promising advantages include reduced patient morbidity, unlimited graft availability, and comparable esthetics. This manuscript reviews soft tissue augmentation and root coverage procedures using bioengineered living cellular therapy and highlights their expected clinical, esthetic, and patient-related outcomes.

Biologics-based regenerative technologies for periodontal soft tissue engineering.

This manuscript provides a state-of-the-art review on the efficacy of biologics in root coverage procedures, including enamel matrix derivative, platelet-derived growth factor, platelet concentrates, and fibroblast-growth factor-2. The mechanism of action and the rationale for using biologics in periodontal plastic surgery, as well as their anticipated benefits when compared with conventional approaches are discussed. Although the clinical significance is still under investigation, preclinical data and histologic evidence demonstrate that biologic-based techniques are able to promote periodontal regeneration coupled with the provision of tooth root coverage.

Autogenous soft tissue grafting for periodontal and peri-implant plastic surgical reconstruction.

This state-of-the-art review presents the latest evidence and the current status of autogenous soft tissue grafting for soft tissue augmentation and recession coverage at teeth and dental implant sites. The indications and predictability of the free gingival graft and connective tissue graft (CTG) techniques are highlighted, together with their expected clinical and esthetic outcomes. CTGs can be harvested from the maxillary tuberosity or from palate with different approaches that can have an impact on graft quality and patient morbidity. The influence of CTGs on soft tissue thickness and keratinized tissue width are also discussed.

Extracellular matrix-based scaffolding technologies for periodontal and peri-implant soft tissue regeneration.

The present article focuses on the properties and indications of scaffold-based extracellular matrix (ECM) technologies as alternatives to autogenous soft tissue grafts for periodontal and peri-implant plastic surgical reconstruction. The different processing methods for the creation of cell-free constructs resulting in preservation of the extracellular matrices influence the characteristics and behavior of scaffolding biomaterials. The aim of this review is to discuss the properties, clinical application, and limitations of ECM-based scaffold technologies in periodontal and peri-implant soft tissue augmentation when used as alternatives to autogenous soft tissue grafts.

Constitutive release of cytokines by human oral keratinocytes in an organotypic culture.

PURPOSE: The Food and Drug Administration requires an accurate determination of the dose and potency of tissue-engineered or combination products as is required for drugs. This needs to be done as a rapid, quantitative, and noninvasive measurement of biologic function/activity in a way so as not to perturb the tissue-engineered product being developed. The aim of this study was to correlate constitutive release of cytokine(s) from unstimulated cells, at different stages of development, within a 3-dimensional (3D) organotypic ex vivo produced oral mucosa equivalent (EVPOME) to be used for intraoral grafting, with oral keratinocyte cell viability of the EVPOME. MATERIALS AND METHODS: Tissue culture medium was assayed with an enzyme-linked immunosorbent assay from monolayer culture of oral keratinocytes and a 3D EVPOME to determine the constitutive release of interleukin (IL) 1alpha, IL-6, IL-8, and vascular endothelial growth factor (VEGF). VEGF messenger ribonucleic acid expression by oral keratinocytes within the 3D EVPOME was detected by in situ hybridization at days 4, 7, and 11. The number of viable oral keratinocytes within the EVPOME was extrapolated from VEGF release by use of a modified MTT assay. RESULTS: Both VEGF release level and the number of viable cells in the monolayer cultures and 3D EVPOME as measured by MTT assay significantly increased in a time-dependent manner (P < .001, r = 0.743). CONCLUSION: These results suggest that the increasing detectable levels of VEGF associated with the increasing number of viable cells in the EVPOME may provide a useful noninvasive/nondestructive means of assessing both cellular viability (dose) and biologic function/activity (potency) of a combination cell-based device such as the EVPOME.

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.

The Kansas City Transportation and Local-Scale Air Quality Study (KC-TRAQS): Integration of Low-Cost Sensors and Reference Grade Monitoring in a Complex Metropolitan Area. Part 1: Overview of the Project.

Emissions from transportation sources can impact local air quality and contribute to adverse health effects. The Kansas City Transportation and Local-Scale Air Quality Study (KC-TRAQS), conducted over a 1-year period, researched emissions source characterization in the Argentine, Turner, and Armourdale, Kansas (KS) neighborhoods and the broader southeast Kansas City, KS area. This area is characterized as a near-source environment with impacts from large railyard operations, major roadways, and commercial and industrial facilities. The spatial and meteorological effects of particulate matter less than 2.5 µm (PM2.5), and black carbon (BC) pollutants on potential population exposures were evaluated at multiple sites using a combination of regulatory grade methods and instrumentation, low-cost sensors, citizen science, and mobile monitoring. The initial analysis of a subset of these data showed that mean reference grade PM2.5 concentrations (gravimetric) across all sites ranged from 7.92 to 9.34 µg/m3. Mean PM2.5 concentrations from low-cost sensors ranged from 3.30 to 5.94 µg/m3 (raw, uncorrected data). Pollution wind rose plots suggest that the sites are impacted by higher PM2.5 and BC concentrations when the winds originate near known source locations. Initial data analysis indicated that the observed PM2.5 and BC concentrations are driven by multiple air pollutant sources and meteorological effects. The KC-TRAQS overview and preliminary data analysis presented will provide a framework for forthcoming papers that will further characterize emission source attributions and estimate near-source exposures. This information will ultimately inform and clarify the extent and impact of air pollutants in the Kansas City area.

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.

Phenotypic markers of oral keratinocytes seeded on two distinct 3D oral mucosa models.

This study validates the use of a full-thickness oral mucosa model for in vitro studies with a collagen type I matrix, by comparison of this model with two other 3D oral mucosa models: human-sourced and porcine acellular dermal matrices (AlloDerm®/Strattice®, respectively). For the collagen matrix model, gingival fibroblasts were seeded either onto the dermal side of the AlloDerm® and Strattice® matrices or within the collagen matrices in complete culture medium (DMEM). For all scaffolds, DMEM was replaced every 24 h up to 72 h. For the full-thickness oral mucosa models, 72 h after fibroblast seeding, oral keratinocytes were seeded on the epidermal sides of AlloDerm® and Strattice® matrices or collagen matrices. All matrices and models were subjected to histological analysis, complementing phenotypic characterization by evaluation of glucose consumption, cell proliferation, gene expression and synthesis of growth factors. A higher fibroblast ratio was observed for the collagen matrix, in which the distribution of gingival fibroblasts was also more homogeneous. Metabolism, proliferation, and gene expression and synthesis of VEGF of these cells were also increased for the collagen matrix. All matrices provided a suitable substrate for oral keratinocytes adhesion, proliferation, and phenotypic expression; however, higher proliferation, stratification, and differentiation were noted when oral keratinocytes were seeded on the dermal matrices.

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®.