Ex Vivo Produced Oral Mucosa Equivalent Research Articles

In-process monitoring of a tissue-engineered oral mucosa fabricated on a micropatterned collagen scaffold: use of optical coherence tomography for quality control

BackgroundWe previously reported a novel technique for fabricating dermo-epidermal junction (DEJ)-like micropatterned collagen scaffolds to manufacture an ex vivo produced oral mucosa equivalent (EVPOME) for clinical translation; however, more biomimetic micropatterns are required to promote oral keratinocyte-based tissue engineering/regenerative medicine. In addition, in-process monitoring for quality control of tissue-engineered products is key to successful clinical outcomes. However, evaluating three-dimensional tissue-engineered constructs such as EVPOME is challenging. This study aimed to update our technique to fabricate a more biomimetic DEJ structure of oral mucosa and to investigate the efficacy of optical coherence tomography (OCT) in combination with deep learning for non-invasive EVPOME monitoring. MethodsA picosecond laser-textured microstructure mimicking DEJ on stainless steel was used as a negative mould to fabricate the micropatterned collagen scaffold. During EVPOME manufacturing, OCT was applied twice to monitor the EVPOME and evaluate its epithelial thickness. FindingsOur moulding system resulted in successful micropattern replication on the curved collagen scaffold. OCT imaging visualised the epithelial layer and the underlying micropatterned scaffold in EVPOME, enabling to non-invasively detect specific defects not found before the histological examination. Additionally, a gradual increase in epithelial thickness was observed over time. ConclusionThese findings demonstrate the feasibility of using a stainless-steel negative mould to create a more biomimetic micropattern on collagen scaffolds and the potential of OCT imaging for quality control in oral keratinocyte-based tissue engineering/regenerative medicine.

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®.

LLLT Effects on Oral Keratinocytes in an Organotypic 3D Model.

Several invitro studies evaluated the cellular and molecular events related to interactions between phototherapy and target tissues, including oral keratinocytes and fibroblasts, providing elucidative data about phototherapy-induced healing. However, these interactions were limited to the application of a bidimensional cell culture model of oral mucosal cells. Thus, thisstudy evaluated the use of an organotypic oral epithelium model to elucidate the morphological and phenotypic responses of cells subjected to low-level laser therapy (LLLT). Oral keratinocytes were seeded in the exvivo-produced oral mucosal equivalent (EVPOME) model, with a porcine acellular dermal matrix. LLLT was applied by means of the LaserTABLE device (780nm, 25mW) at 0.5, 1.5 and 3Jcm-2 . After three irradiations, morphology, proliferation and gene expression of growth factors were assessed. LLLT and control groups presented similar morphological features, characterized by the formation of a stratified, differentiated and keratinized epithelium. LLLT enhanced the cell proliferation and gene expression of keratinocytes (hKGF) as well as epidermal (hEGF) growth factors. In general, analysis of these data shows that the three-dimensional cell culture model can be applied for phototherapy studies and that the positive effects of LLLT were confirmed by the use of an organotypic model.

Development of an oral mucosa equivalent using a porcine dermal matrix

We evaluated the suitability of a porcine acellular dermal matrix for the development of a 3-dimensional oral mucosal equivalent using an ex vivo-produced oral mucosal equivalent (EVPOME). Oral keratinocytes were seeded in a submerged model, and then in an air-liquid interphase model, using Transwell® inserts. EVPOME showed good cell viability and increased glucose consumption over time. Histological evaluation showed that stratified differentiated epithelium had formed in all matrices.

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.

Remodeling of the basement membrane during the healing process after intraoral grafting of tissue-engineered human oral mucosa in athymic mice

Background and objective: An ex vivo-produced oral mucosa equivalent (EVPOME) is composed of a stratified layer of human oral keratinocytes seeded onto a human acellular dermis, AlloDerm®. Our preliminary clinical study showed the EVPOME grafts had enhanced maturation of the underlying submucosal layer associated with rapid epithelial coverage. Results of our animal study suggested that grafting of the EVPOME onto an oral wound promotes re-epithlialization of oral mucosa in the healing process. The objective of the present study was to investigate the change in the basement membrane of EVPOME during the healing process after intraoral grafting in athymic mice.

Intraoral Grafting of Tissue-Engineered Human Oral Mucosa

The primary objective of this study was to evaluate the safety of a tissue-engineered human ex vivo-produced oral mucosa equivalent (EVPOME) in intraoral grafting procedures. The secondary objective was to assess the efficacy of the grafted EVPOME in producing a keratinized mucosal surface epithelium. Five patients who met the inclusion criteria of having one mucogingival defect or a lack of keratinized gingiva on a nonmolar tooth, along with radiographic evidence of sufficient interdental bone height, were recruited as subjects to increase the width of keratinized gingiva at the defect site. A punch biopsy specimen of the hard palate was taken to acquire oral keratinocytes, which were expanded, seeded, and cultured on an acellular dermal matrix for fabrication of an EVPOME. EVPOME grafts were applied directly over an intact periosteal bed and secured in place. At baseline (biopsy specimen retrieval) and at 7, 14, 30, 90, and 180 days postsurgery, Plaque Index and Gingival Index were recorded for each subject. In addition, probing depths, keratinized gingival width, and keratinized gingival thickness were recorded at baseline, 30, 90, and 180 days. No complications or adverse reactions to EVPOME were observed in any subjects during the study. The mean gain in keratinized gingival width was 3 mm (range, 3 to 4 mm). The mean gain in keratinized gingival thickness was 1 mm (range, 1 to 2 mm). No significant changes in probing depths were observed. Based on these findings, it can be concluded that EVPOME is safe for intraoral use and has the ability to augment keratinized tissue around teeth. Future clinical trials are needed to further explore this potential.

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.

Comparison of Scanning Acoustic Microscopy and Histology Images in Characterizing Surface Irregularities Among Engineered Human Oral Mucosal Tissues

Acoustic microscopy was used to monitor an ex vivo produced oral mucosal equivalent (EVPOME) developed on acellular cadaveric dermis (AlloDerm ®). As seeded cells adhered and grew, they filled in and smoothed out the surface irregularities, followed by the production of a keratinized protective outermost layer. If noninvasive in vitro ultrasonic monitoring of these cellular changes could be developed, then tissue cultivation could be adjusted in-process to account for biologic variations in the development of these stratified cell layers. Cultured keratinocytes (from freshly obtained oral mucosa) were harvested and seeded onto AlloDerm ® coated with human type IV collagen and cultured 11 days. EVPOMEs were imaged on the 11th day post-seeding using a scanning acoustic microscope (SAM) that consists of a single-element transducer: 61 MHz center frequency, 32 MHz bandwidth, 1.52 f-number. The specimen surface was determined by thresholding the magnitude of the signal at the first axial incidence of a value safely above noise: 20–40 dB above the signal for the water and 2-dimensional (2-D) ultrasonic images were created using confocal image reconstruction. A known area from each micrograph was divided into 12–40 even segments and examined for surface irregularities. These irregularities were quantified and one-way analysis of variance (ANOVA) and linear regression analysis were performed to correlate the surface profiles for both the AlloDerm ® and EVPOME specimens imaged by SAM. Histology micrographs of the AlloDerm ® and EVPOME specimens were also prepared and examined for surface irregularities. Unseeded AlloDerm ® averaged seven to nine surface changes per 400 μm. The number of changes in surface irregularities decreased to two to three per 400 μm on the mature EVPOMEs. The numbers of surface irregularities between the unseeded AlloDerm ® vs. developing EVPOME are similar for both histology and SAM 2-D B-scan images. For the EVPOME 2-D B-scan micrographs produced by SAM, the decrease in surface irregularities is indicative of the stratified epithelium formed by seeded oral keratinocytes; verified in the histology images between the AlloDerm ® and EVPOME. A near 1:1 linear correlation shows the similarities between the two imaging modalities. SAM demonstrates its ability to discern the cell development and differentiation occurring on the EVPOME devices. Unlike histology, SAM measurements are noninvasive and can be used to monitor tissue graft development without damaging any cells/tissues.

Keratinocytes of Tissue-Engineered Human Oral Mucosa Promote Re-Epithelialization After Intraoral Grafting in Athymic Mice

The objective of this study was to investigate the role of grafted oral keratinocytes in a transplanted ex vivo-produced oral mucosa equivalent (EVPOME) in the regeneration and/or healing process of the oral mucosa at the recipient site. The EVPOME was developed in a serum-free defined culture system without a feeder layer. EVPOME is composed of a stratified layer of human oral keratinocytes that are seeded onto a human cadaveric dermis, AlloDerm (LifeCell, Branchburg, NJ). Intraorally grafted EVPOMEs in athymic mice (BALB/c) were excised, contiguous with the surrounding oral mucosa, on days 5, 7, 14, and 21 after grafting. Serial sections were stained with hematoxylin-eosin and immunohistochemically analyzed for cytokeratin 17 (CK17) expression to distinguish the human-cultured EVPOME epithelial keratinocytes from murine oral keratinocytes. All EVPOME epithelial cells showed intense immunoreactivity for CK17, whereas mouse buccal mucosal epithelial cells did not show CK17 immunoreactivity. The grafted EVPOME maintained a stratified epithelial layer for up to 5 days after grafting. By day 7 after grafting, a portion of the EVPOME epithelial layer peeled away from the AlloDerm, and a thin, CK17-immunonegative epithelial layer extended from the adjacent thick epithelial layer of the mouse and contacted the CK17-immunopositive EVPOME epithelium. From days 14 to 21 after grafting, the stratification of the CK17-immunonegative continuous mouse epithelium increased compared with earlier time points and showed a similar appearance to the epithelium of the adjacent mouse mucosa. In contrast, no epithelial coverage of the AlloDerm that was grafted without keratinocytes was observed for up to 21 days after grafting. The grafted AlloDerm without cells resulted in tissue necrosis that was accompanied by a dramatic infiltration of inflammatory cells by day 14. These findings suggest that grafting of EVPOME with viable oral keratinocytes onto an intraoral mucosal wound plays an active role in promotion of re-epithelialization of the oral wound during the subsequent healing process.

Ex vivo produced oral mucosa equivalent preliminary report: a technical note

The aim of this study was to produce of ex vivo oral mucosal equivalent. The continuous stratified layer of human oral keratinocytes was grown on a cadaveric human dermal matrix in a defined culture medium without a feeder layer. Materials and methods: Human oral keratinocytes from a keratinized oral mucosa biopsy specimen were enzymatically dissociated and cultured in a serum-free defined culture medium, EpiLife (Cascade Biologics, Portland, OR, USA), containing a low calcium concentration of 0.06 mM. Oral keratinocytes were expanded one passage. Once a sufficient population of keratinocytes was reached, they were seeded onto the type IV collagen coated acellular nonimmunogenic cadaveric human dermis (AlloDerm), at cell densities of 1.8 × 105. To form a continuous epithelial monolayer, and to enhance keratinocyte differentiation, oral keratinocyte-AlloDerm composites were cultured while submerged in a high calcium concentration of 1.2 mM medium for 4 days. After 4 days, AlloDerm-keratinocyte composites were raised to an air-liquid interface to encourage stratification of the epithelial monolayer. After additional 7 days, they were taken for histologic examination at 11 days postseeding of the keratinocytes on the AlloDerms. Results: Histologically, on day 11, EVPOME's development showed multilayered epithelium comprising basal, suprabasal, and parakeratinized layers. Conclusion: Histologically, an ex vivo produced oral mucosa equivalent (EVPOME) that consisted of a stratified epidermis on a dermal matrix was successfully developed by the method used.

Development of an in vitro model for radiation-induced effects on oral keratinocytes

Changes in epithelial cell activity and the production of pro-inflammatory cytokines were examined utilizing an organotypic culture system as an in vitro model to study the effects of radiation on oral keratinocytes to simulate what is thought to occur in radiation-induced oral mucositis. Monolayer cultures of oral keratinocyte were irradiated by varying the dose. Cell injury was assessed using a colony forming efficiency (CFE) assay. Third passage oral keratinocytes were seeded onto AlloDerm to form a 3D construct of an ex vivo produced oral mucosa equivalent (EVPOME) which was irradiated with 0, 1, 3 and 8Gy. Formalin-fixed sections of the EVPOME were used for histology and immunohistochemistry to examine proliferative capacity. Epithelial cell viability of EVPOME was measured by MTT assay. Spent culture medium was used to determine post-radiation pro-inflammatory cytokine production. Basal cells became more swollen and pyknotic as radiation increased, implying loss of cell viability also determined by MTT assay. The number of Ki-67 immunopositive cells and CFE showed negative correlation with radiation, indicating loss of cell proliferative capacity. The production of pro-inflammatory cytokines, IL-1alpha and IL-8, tended to increase in a radiation dose dependent manner. The EVPOME lacking submucosal cellular components was a useful model.

Constitutive Release of Cytokines by Human Oral Keratinocytes in an Organotypic Culture

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. 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. 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). 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.

The expression and production of vascular endothelial growth factor in oral mucosa equivalents

Angiogenesis is anticipated during wound healing. Vascular endothelial growth factor (VEGF) is a potent direct angiogenic factor that stimulates endothelial cell migration and proliferation in vitro and angiogenesis in vivo. Ex vivo produced oral mucosa equivalent (EVPOME) grafts have been reported to promote re-vascularization in the underlying tissue after grafting. The aim of this study was to evaluating the following: VEGF mRNA and its protein expression in EVPOME grafts, the protein levels in conditioned media produced by EVPOMEs, and the ability of VEGF to stimulate the growth of microvascular endothelial cells. VEGF mRNA expression and immunoreaction were found in basal and suprabasal layers. VEGF secreted by EVPOME was detected throughout the period of manufacture of the grafts, and became elevated for the first week at an air–liquid interface. Both EVPOME-conditioned media and a medium containing 10 ng/mL recombinant human VEGF induced endothelial cell proliferation, while neutralization of VEGF by an antibody blocked cell growth. These results suggest that VEGF secreted by EVPOME grafts might assist initial vascularization after grafting, which is critical to subsequent graft survival.

Development and Characterization of a Human Tissue-Engineered Oral Mucosa

Open wounds in the mouth should be covered by a graft to prevent microbial infection, excessive fluid loss, foreign material contamination, or relapse (secondary to wound contracture). Currently oral mucosal or skin grafts are used for this purpose; however, both of these grafts require a second surgical procedure and have disadvantages in intraoral use. Split thickness skin grafts may contain adnexal structures, and express a different pattern of surface keratinization. The elective nature of the majority of oral and maxillofacial surgical procedures should allow the flexibility and timing to develop a tissue-engineered oral mucosa. As a tissue-engineered skin has been developed to treat patients suffering burn wounds, chronic ulcers, etc, there is a similar need for the development of a tissue-engineered oral mucosa suitable for intraoral grafting procedures. This graft would be used after major trauma, surgical resections, and for maxillofacial prosthetic surgery. Our research team has been successful in developing an ex vivo produced oral mucosal equivalent (EVPOME) fabricated in an environment free of serum, transformed irradiated feeder cells and pituitary extract. Our EVPOME consists of autologous oral mucosa keratinocytes and an acellular, non-immunogenic dermal equivalent, AlloDerm. The EVPOME was cultured at an air-liquid interface for up to two weeks, resulting in a well-differentiated, parakeratinized epithelial layer similar to native oral mucosa. The expression pattern of differentiation and proliferation markers showed an active and hyperproliferative state. The ultrastructure of EVPOME demonstrated hemi-desmosome-like structures were formed at the interface (basement membrane) of basal keratinocytes and a dermal layer by day 7 at an air-liquid interface. Tissue-engineered cell-based products usually fall under the auspices of the Center for Biological Evaluation and Research (CBER). They are considered a combination product by the Food and Drug Administration (FDA), and need to comply with the regulatory standards for determining safety and efficacy. Animal models are important to determine for conventional safety and efficacy. The SCID mice transplantation model (small animal) allowed us to investigate how EVPOME behaves in an in vivo environment and to determine the optimal stage of the EVPOME for clinical use. A large animal model is also required by the FDA prior to human clinical use; thus we developed a canine EVPOME fabrication protocol that was identical to our human EVPOME protocol. Our canine model enables us to evaluate the fate of EVPOME grafts in situ. Furthermore, the guidelines of CBER require us to demonstrate “dose and potency” of cell-based products, as is seen with drugs. Dose and potency appear to be representative of viable cell numbers and relevant biological function/activity of EVPOME, which provides a quantitative measurement of product quality. These quantitative measurements are challenging and specific for each cell-based product and need to be addressed.

Poster 113: A Comparison of Wound Healing Processes Between Ex Vivo Produced Oral Mucosa Equivalent Transplantation and Artificial Dermis Transplantation: The Influence of Keratinocytes on Biological Events in the Lamina Propria

For clinical application we fabricated ex vivo produced oral mucosa equivalent (EVPOME) from patient oral keratinocytes, completely excluding both heterogenic protein interaction and xenoplantation. This study was aimed at comparing artificial dermis and EVPOME transplantations on wound healing mechanisms. 1Analyses of fibroblast dynamics (1), mast cell expression (2), nerve regeneration (3), and revascularization (4) of EVPOME and artificial dermis transplantations were carried out at the Department of Oral and Maxillofacial Surgery, Kobe University Hospital.2Using an acute tongue wound model on rats, the relation between keratinocyte regeneration and biological events in the lamina propria were immunohistochemically analyzed. 1Immunohistochemistry2Clinical investigation3Statistical analysis 1The keratinocyte-induced neuromucosal system was involved in the promotion of wound healing after EVPOME transplantation and induced faster regeneration of wounds than after artificial dermis transplantation.2Although the period of time for complete epithelialization by artificial dermis did not differ from that by EVPOME, monitoring of nerve and vascular regeneration demonstrated that tissues of the transplanted artificial dermis were incomplete and still in the process of regeneration.3With EVPOME, fibroblast migration was inhibited because of the dense structure of the acellular dermal matrix, and the wound healing process by the keratinocyte-derived cytokine system was manifest predominantly in the early stages after transplantation. The keratinocytes and the acellular dermal matrix of EVPOME are involved in a variety of biological events and are considered to induce faster wound healing, a feature that is useful in clinical applications.

Isolation of a Progenitor/Stem Cell From Oral Mucosa

Statement of the Problem: Our long-term goal is to produce a “smart” transduced oral mucosal graft that will be used for reconstruction of major oral defects secondary to oncologic resection, traumatic events or developmental disturbances. The graft would act both as a material for reconstruction and as a repository for in situ transmucosal delivery of recombinant growth factors or cytokines. Several barriers presently exist that prevent this technology from moving into the clinical arena. First, is the ability to fabricate these “smart” grafts in a more efficient manner, i.e. develop a more highly proliferative and expanding cell population, and, second, the ability to isolate a stem/progenitor cell. Our objective of this study was to isolate using physical criterion, size, a progenitor/stem cell from oral mucosa to regenerate an ex vivo produced oral mucosa equivalent (EVPOME) for human clinical trials.

Development and characterization of a canine oral mucosa equivalent in a serum-free environment.

The objectives of this study were to develop a serum-free system for culturing canine oral keratinocytes, the construction and characterization of a canine ex vivo produced oral mucosa equivalent (EVPOME), and transduction green fluorescent protein (GFP) into keratinocytes as a post-grafting tracking marker. Dissociated canine buccal mucosa keratinocytes were cultured in a chemically defined serum-free medium, Epilife trade mark. First-passage keratinocytes were transfected with the GFP gene using a lentiviral vector, sorted by flow cytometer and seeded onto a dermal equivalent, AlloDerm(R) to form EVPOMEs. The EVPOME was characterized by histology and immunohistochemistry, for p63, Ki-67, and involucrin. Laser confocal microscopy was used to locate GFP-transfected keratinocytes within the EVPOME. Cultured canine oral keratinocytes grew rapidly over the first three passages and then the proliferative rate decreased. The canine EVPOME formed a well-stratified epithelial layer. The majority of p63 and Ki-67 immunopositive cells were located in the basal layer whereas cytoplasmic involucrin expression was seen in the suprabasal layers, similar to native canine buccal mucosa. Under laser confocal microscopy, significant green fluorescence was observed throughout the EVPOME. In conclusion, canine EVPOMEs were successfully fabricated in a defined serum-free system with similar characteristics to native buccal mucosa. GFP-transfected canine oral keratinocytes could be identified within the EVPOME.