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1.
Tissue Eng Part C Methods ; 27(5): 287-295, 2021 05.
Article in English | MEDLINE | ID: mdl-33726570

ABSTRACT

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.


Subject(s)
Tissue Engineering , Tissue Scaffolds , Animals , Cell Count , Humans , Keratinocytes , Mice
2.
Tissue Eng Part C Methods ; 25(5): 305-313, 2019 05.
Article in English | MEDLINE | ID: mdl-30973066

ABSTRACT

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.


Subject(s)
Optical Imaging , Tissue Engineering/methods , Tissue Survival , Cell Survival , Humans , Keratinocytes/cytology , Molecular Imaging , Staining and Labeling , Tissue Scaffolds/chemistry
3.
Tissue Eng Part C Methods ; 24(4): 214-221, 2018 04.
Article in English | MEDLINE | ID: mdl-29448894

ABSTRACT

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.


Subject(s)
Cytokines/metabolism , Image Processing, Computer-Assisted/methods , Keratinocytes/transplantation , Microscopy, Fluorescence/methods , Mouth Mucosa/transplantation , Tissue Engineering/methods , Animals , Cell Survival , Female , Humans , Keratinocytes/cytology , Keratinocytes/metabolism , Mice, SCID , Mouth Mucosa/cytology , Mouth Mucosa/metabolism , Tissue Scaffolds , Transplantation, Heterologous
4.
J Tissue Eng Regen Med ; 12(4): 983-990, 2018 04.
Article in English | MEDLINE | ID: mdl-28752668

ABSTRACT

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®.


Subject(s)
Dermis/chemistry , Keratinocytes/metabolism , Mouth Mucosa/chemistry , Tissue Engineering , Tissue Scaffolds/chemistry , Animals , Humans , Keratinocytes/cytology , Mice , Mice, SCID
5.
J Oral Maxillofac Surg ; 74(11): 2317-2326, 2016 Nov.
Article in English | MEDLINE | ID: mdl-27160362

ABSTRACT

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.


Subject(s)
Lip/surgery , Plastic Surgery Procedures/methods , Tissue Engineering/methods , Cell Differentiation , Cells, Cultured , Coculture Techniques , Collagen , Filaggrin Proteins , Humans , In Vitro Techniques , Keratinocytes , Mouth Mucosa/cytology , Tissue Scaffolds
6.
Tissue Eng Part C Methods ; 21(9): 872-80, 2015 Sep.
Article in English | MEDLINE | ID: mdl-25760802

ABSTRACT

The soft tissue reconstruction of significant avulsed and/or surgically created tissue defects requires the ability to manufacture substantial soft tissue constructs for repair of the resulting wounds. In this study, we detail the issues that need to be addressed in upsizing the manufacture of larger tissue-engineered devices (ex vivo-produced oral mucosa equivalent [EVPOME]) in vitro from a methodology previously used for smaller constructs. The larger-sized EVPOME, consisting of autologous human oral keratinocytes and a dermal substitute, AlloDerm(®), was fabricated for the purpose of reconstructing large clinical defects. Regulated as an autologous somatic cell therapy product, the fabrication process abided by current Good Manufacturing Practices and current Good Tissue Practices as required by the Center for Biologics Evaluation and Research (CBER) of the United States Food and Drug Administration (FDA). Successful fabrication of large EVPOMEs utilized a higher cell seeding density (5.3×10(5) cells/cm(2)) with a relatively thinner AlloDerm, ranging from 356.6 to 508.0 µm in thickness. During the air-liquid interface culture, the thickness of the scaffold affected the medium diffusion rate, which, in turn, resulted in changes of epithelial stratification. Histologically, keratinocyte progenitor (p63), proliferation (Ki-67), and late differentiation marker (filaggrin) expression showed differences correlating with the expression of glucose transporter-1 (GLUT1) in the EVPOMEs from the thickest (550-1020 µm) to the thinnest (228.6-330.2 µm) AlloDerm scaffold. Glucose consumption and 2-deoxyglucose (2DG) uptake showed direct correlation with scaffold thickness. The scaffold size and thickness have an impact on the cellular phenotype and epithelial maturation in the manufacturing process of the EVPOME due to the glucose accessibility influenced by the diffusion rate. These outcomes provide basic strategies to manufacture a large-sized, healthy EVPOME graft for reconstructing large mucosa defects.


Subject(s)
Mouth Mucosa/physiology , Tissue Engineering/methods , Adult , Cell Count , Deoxyglucose/metabolism , Diffusion , Epithelium/metabolism , Female , Filaggrin Proteins , Humans , Keratinocytes/cytology , Male , Staining and Labeling , Tissue Culture Techniques
7.
Article in English | MEDLINE | ID: mdl-27042699

ABSTRACT

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.

8.
Tissue Eng Part C Methods ; 21(1): 46-51, 2015 Jan.
Article in English | MEDLINE | ID: mdl-24826804

ABSTRACT

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.


Subject(s)
Fiber Optic Technology/methods , Mouth Mucosa/physiology , Spectrum Analysis, Raman/methods , Tissue Engineering/methods , Tissue Scaffolds/chemistry , Animals , Humans , Immunohistochemistry , Implants, Experimental , Mice, SCID , Phenylalanine/analysis
9.
Stem Cells Dev ; 23(23): 2883-94, 2014 Dec 01.
Article in English | MEDLINE | ID: mdl-25027178

ABSTRACT

The dentin sialophosphoprotein (dspp) transcript is expressed during tooth development as a DSPP precursor protein, which then undergoes cleavage to form mature dentin sialoprotein (DSP) and phosphophoryn (PP) proteins. Previous studies using DSPP-knockout (KO) mice have reported that these animals have hypomineralized teeth, thin dentin, and a large dental pulp chamber, similar to those from patients with dentinogenesis imperfecta III. However, there is no information about factors that regulate dental pulp stem cell lineage fate, a critical early event in the odontoblast-dentin mineralization scheme. To reveal the role of DSPP in odontoblast lineage differentiation during tooth development, we systematically examined teeth from wild-type (wt) and DSPP-KO C57BL/6 mice between the ages of postnatal day 1 and 3 months. We found developmental abnormalities not previously reported, such as circular dentin formation within dental pulp cells and altered odontoblast differentiation in DSPP-KO mice, even as early as 1 day after birth. Surprisingly, we also identified chondrocyte-like cells in the dental pulp from KO-mice teeth. Thus, these studies that compare wt and DSPP-KO mice suggest that the expression of DSPP precursor protein is required for normal odontoblast lineage differentiation and that the absence of DSPP allows dental pulp cells to differentiate into chondrocyte-like cells, which could negatively impact pulpal wound healing and tissue regeneration.


Subject(s)
Cell Differentiation/physiology , Dental Pulp/metabolism , Dentin/metabolism , Extracellular Matrix Proteins/metabolism , Odontoblasts/metabolism , Phosphoproteins/metabolism , Sialoglycoproteins/metabolism , Stem Cells/metabolism , Animals , Dental Pulp/cytology , Dentin/cytology , Extracellular Matrix Proteins/genetics , Mice , Mice, Knockout , Odontoblasts/cytology , Phosphoproteins/genetics , Sialoglycoproteins/genetics , Stem Cells/cytology
10.
Biomaterials ; 35(25): 6667-76, 2014 Aug.
Article in English | MEDLINE | ID: mdl-24854093

ABSTRACT

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.


Subject(s)
Microscopy, Fluorescence, Multiphoton/methods , Tissue Engineering , Cell Differentiation , Cell Survival , Cross-Sectional Studies , Humans , Image Processing, Computer-Assisted , Keratinocytes/chemistry , Mouth Mucosa/chemistry , Mouth Mucosa/cytology , Tissue Scaffolds/chemistry
11.
Methods Mol Biol ; 989: 293-303, 2013.
Article in English | MEDLINE | ID: mdl-23483404

ABSTRACT

The isolation of human oral mucosa/skin keratinocytes progenitor/stem cells is clinically important to regenerate epithelial tissues for the treatment of oral mucosa/skin defects. Researchers have attempted to isolate a keratinocyte progenitor/stem cell population using cell markers, rapid adherence to collagen type IV, and other methods. In this regard, one of the specific characteristics of keratinocyte progenitor/stem cells is that these cells have a smaller diameter than differentiated cells. This chapter describes methods used in our laboratory to set up primary human oral mucosa and skin keratinocytes in a chemically defined culture system devoid of animal derived products. We utilized the cells in a FDA-approved human clinical trial that involved the intraoral grafting of an ex vivo produced oral mucosa equivalent to increase keratinized tissue around teeth. We also provide two protocols on how to sort keratinocytes using physical criterion, cell size, using a cell sorter and a serial filtration system.


Subject(s)
Keratinocytes/cytology , Mouth Mucosa/cytology , Stem Cells/cytology , Cell Culture Techniques , Cells, Cultured , Flow Cytometry , Humans
12.
Tissue Eng Part C Methods ; 19(4): 299-306, 2013 Apr.
Article in English | MEDLINE | ID: mdl-22992065

ABSTRACT

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.


Subject(s)
Mouth Mucosa , Spectrum Analysis, Raman/methods , Tissue Engineering , Calcium/metabolism , Glucose/metabolism , Humans , Sirolimus/pharmacology
13.
Arch Dermatol Res ; 305(2): 163-71, 2013 Mar.
Article in English | MEDLINE | ID: mdl-22960739

ABSTRACT

In this manuscript, we report observations of the effects of rapamycin in an organotypic culture of human skin explants. The tissues were cultured for 5 days at the air-liquid interface or in submersed conditions with media with and without rapamycin at 2 nM concentration. Histological analysis of tissue sections indicated that rapamycin-treated samples maintained a better epidermal structure in the upper layers of the tissue than untreated samples, mostly evident when skin was cultured in submersed conditions. A significant decrease in the number of positive proliferative cells using the Ki67 antigen was observed when specimens were treated with rapamycin, in both air-liquid and submersed conditions but apoptosis differences between treated and untreated specimens, as seen by cleaved caspase-3 positive cells, were only observed in submersed specimens. Finally, a decrease and variability in the location in the expression of the differentiation marker involucrin and in E-cadherin were also evident in submersed samples. These results suggest that the development of topical applications containing rapamycin, instead of systemic delivery, may be a useful tool in the treatment of skin diseases that require reduction of proliferation and modulation or control of keratinocyte differentiation.


Subject(s)
Keratinocytes/drug effects , Sirolimus/administration & dosage , Skin Diseases/drug therapy , Skin/drug effects , Administration, Topical , Apoptosis/drug effects , Cadherins/metabolism , Caspase 3/metabolism , Cell Differentiation/drug effects , Cell Proliferation/drug effects , Humans , Keratinocytes/pathology , Ki-67 Antigen/metabolism , Organ Culture Techniques , Protein Precursors/metabolism , Skin/pathology , Skin Transplantation , Tissue Engineering
14.
Cell Tissue Bank ; 14(3): 423-35, 2013 Sep.
Article in English | MEDLINE | ID: mdl-23149549

ABSTRACT

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.


Subject(s)
Cell Culture Techniques/methods , Enzymes/metabolism , Epithelial Cells/cytology , Adult , Cell Count , Cell Differentiation , Cell Proliferation , Cell Shape , Cells, Cultured , Humans , Immunohistochemistry , Keratinocytes/cytology , Male , Time Factors
15.
BMC Dermatol ; 12: 8, 2012 Jun 24.
Article in English | MEDLINE | ID: mdl-22726819

ABSTRACT

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.


Subject(s)
Cell Culture Techniques/methods , Keratinocytes/cytology , Stem Cells/cytology , Adult , Epidermal Cells , Glucose/metabolism , Humans , Immunohistochemistry , Keratinocytes/metabolism , Mouth Mucosa/cytology , Regenerative Medicine/methods , Urothelium/cytology
16.
Tissue Eng Part C Methods ; 18(4): 273-82, 2012 Apr.
Article in English | MEDLINE | ID: mdl-22067042

ABSTRACT

We report for the first time the fabrication of a three-dimensional tissue structure containing, in a continuous layer, the morphological features of a lip: epidermal skin, vermillion, and oral mucosa. This tissue engineered muco-cutaneous (M/C) equivalent was manufactured using human oral and skin keratinocytes grown on an acellular, nonimmunogenic dermal equivalent (AlloDerm(®)) to produce a tissue equivalent with similar anatomic and handling properties as native human lips. Confirmation of the structural composition of the construct was performed using routine histology and immunohistochemistry by identification of epithelial markers that are differentially expressed in separate anatomic areas of the lips. These full-thickness human lip skin equivalents can be used in surgical lip reconstruction in individuals suffering from lip loss from cancer, congenital deformations, and injuries after accidents. We propose this technique can be used as a general basis for tissue engineering of M/C junctions in other parts of the body, such as anus and vagina.


Subject(s)
Lip/physiology , Mouth Mucosa/anatomy & histology , Plastic Surgery Procedures/methods , Skin/anatomy & histology , Tissue Engineering/methods , Tissue Scaffolds/chemistry , Cell Movement , Cell Proliferation , Humans , Immunohistochemistry , Keratins/metabolism
17.
J Dermatol Sci ; 64(2): 92-8, 2011 Nov.
Article in English | MEDLINE | ID: mdl-21908176

ABSTRACT

BACKGROUND: Mammalian hair development and tooth development are controlled by a series of reciprocal epithelial-mesenchymal interactions. Similar growth factors and transcription factors, such as fibroblast growth factor (FGF), sonic hedgehog homolog (SHH), bone morphogenetic proteins (BMPs) and Wnt10a, were reported to be involved in both of these interactions. Dentin sialoprotein (DSP) and phosphophoryn (PP) are the two major non-collagenous proteins secreted by odontoblasts that participate in dentin mineralization during tooth development. Because of striking similarities between tooth development and hair follicle development, we investigated whether DSP and/or PP proteins may also play a role in hair follicle development. OBJECTIVE: In this study, we examined the presence and location of DSP/PP proteins during hair follicle development. METHODS: Rat PP proteins were detected using immunohistochemical/immunofluorescent staining. DSP-PP mRNAs were detected by in situ hybridization with riboprobes. LacZ expression was detected in mouse tissues using a DSP-PP promoter-driven LUC in transgenic mice. RESULTS: We found that PP proteins and DSP-PP mRNAs are present in rat hair follicles. We also demonstrate that an 8 kb DSP-PP promoter is able to drive lacZ expression in hair follicles. CONCLUSION: We have firmly established the presence of DSP/PP in mouse and rat hair follicles by immunohistochemical/immunofluorescent staining, in situ hybridization with riboprobes and transgenic mice studies. The expression of DSP/PP in hair follicles is the first demonstration that major mineralization proteins likely may also contribute to soft tissue development. This finding opens a new avenue for future investigations into the molecular-genetic management of soft tissue development.


Subject(s)
Extracellular Matrix Proteins/physiology , Hair Follicle/growth & development , Phosphoproteins/physiology , Sialoglycoproteins/physiology , Animals , Extracellular Matrix Proteins/analysis , Extracellular Matrix Proteins/genetics , Hair Follicle/chemistry , In Situ Hybridization , Integrin-Binding Sialoprotein/analysis , Mice , Mice, Transgenic , Osteopontin/analysis , Phosphoproteins/analysis , Phosphoproteins/genetics , Phosphorylation , Promoter Regions, Genetic , RNA, Messenger/analysis , Rats , Rats, Long-Evans , Rats, Sprague-Dawley , Sialoglycoproteins/analysis , Sialoglycoproteins/genetics
18.
Artif Organs ; 34(2): E27-33, 2010 Feb.
Article in English | MEDLINE | ID: mdl-20420587

ABSTRACT

We have developed an in vitro culture system composed of organotypic human skin explants interfaced with titanium rods attached to a fluid pump. This device was designed to mimic the process of natural mucosa delivery at the point where a rigid, permanent object penetrates living skin. Full thickness human breast skin explants discarded from surgeries were cultured at different time points at the air-liquid interface. The skin specimens were punctured to fit at the bottom of hollow cylindrical titanium rods. Sodium lauryl sulfate (SLS) was delivered continuously to the specimens through the rods by using an attached fluid pump. Histological analysis of the skin explants as well as no-pump controls was then performed. Our results show substantial differences between controls, where no material was pumped at the interface of rod-skin, and specimens treated with SLS, indicating that the technique of pumping the material is effective in producing observable epithelial changes. These results suggest that an adaptation of this type of device may be useful for the treatment of complications arising from the contact between tissues and percutaneous devices in vivo.


Subject(s)
Biocompatible Materials , Skin , Tissue Culture Techniques , Cell Differentiation , Humans , Immunohistochemistry , Tissue Scaffolds
19.
Ann Biomed Eng ; 38(6): 2013-31, 2010 Jun.
Article in English | MEDLINE | ID: mdl-20140520

ABSTRACT

This discussion and review article focuses on the possible use of regenerative techniques applied to the interfaces between skin and medical implants. As is widely known, the area of contact between an implant and the skin--the skin-implant interface--is prone to recurrent and persistent problems originated from the lack of integration between the material of the implant and the skin. Producing a long-term successful biointerface between skin and the implanted device is still an unsolved problem. These complications have prevented the development of advanced prosthetics and the evolution of biointegrated devices with new technologies. While previous techniques addressing these issues have relied mostly on the coating of the implants or the modification of the topology of the devices, recent in vitro developed techniques have shown that is possible to introduce biocompatible and possibly regenerative materials at the skin-device interface. These techniques have also shown that the process of delivering the materials has biological effects on the skin surrounding the implant, thus converting bioinert into bioactive, dynamic interfaces. Given that the best clinical outcome is the long-term stabilization and integration of the soft tissue around the implant, this article presents the basis for the selection of regenerative materials and therapies for long-term use at the skin-device interface, with focus on the use of natural biopolymers and skin cell transplantation.


Subject(s)
Biocompatible Materials/chemistry , Biocompatible Materials/chemical synthesis , Prostheses and Implants , Regeneration/physiology , Skin Physiological Phenomena , Skin, Artificial , Skin/chemistry , Animals , Humans , Prosthesis Design , Regenerative Medicine/instrumentation , Regenerative Medicine/trends , Surface Properties
20.
Ann Biomed Eng ; 38(3): 1098-110, 2010 Mar.
Article in English | MEDLINE | ID: mdl-20020211

ABSTRACT

Cellular apoptosis and proliferation was analyzed in an in vitro culture system of organotypic human skin explants in the presence or absence of external fixator pins. The effect on the tissues of a mixture of hyaluronic acid and dermatan sulfate (HA+DS) delivered at the skin-pin interface was also studied. After 2 weeks in culture, skin specimens interfaced with fixator pins showed increased keratinocyte apoptosis and proliferation compared to specimens without fixator pins. Simultaneously, a relative reduction of apoptosis and proliferation was observed in specimens treated with the HA+DS mixture, regardless of fixation pin presence. In addition, the HA+DS mixture appeared to help in the preservation of the epidermal basal membrane. It is concluded that in this in vitro model, fixator pins induce keratinocyte apoptosis and hyperproliferation, which are reduced in the presence of the HA+DS mixture. These methods may be useful for a better maintenance of the soft tissue surrounding percutaneous devices in vivo.


Subject(s)
Dermatan Sulfate/pharmacology , External Fixators , Hyaluronic Acid/pharmacology , Organ Culture Techniques/methods , Organ Preservation Solutions/pharmacology , Skin Transplantation/methods , Skin/drug effects , Humans , Skin Transplantation/instrumentation
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