Jonathan A. Garlick

Biofunctionalized electrospun silk mats as a topical bioactive dressing for accelerated wound healing

Materials able to deliver topically bioactive molecules represent a new generation of biomaterials. In this article, we describe the use of silk mats, made of electrospun nanoscale silk fibers containing epidermal growth factor (EGF), for the promotion of wound healing processes. In our experiments, we demonstrated that EGF is incorporated into the silk mats and slowly released in a time-dependent manner (25% EGF release in 170h). We tested these materials using a new model of wounded human skin-equivalents displaying the same structure as human skin and able to heal using the same molecular and cellular mechanisms found in vivo. This human three-dimensional model allows us to demonstrate that the biofunctionalized silk mats, when placed on the wounds as a dressing, aid the healing by increasing the time of wound closure by the epidermal tongue by 90%. The preservation of the structure of the mats during the healing period as demonstrated by electronic microscopy, the biological action of the dressing, as well as the biocompatibility of the silk demonstrate that this biomaterial is a new and very promising material for medical applications, especially for patients suffering from chronic wounds.

Self-Assembling Peptide Nanofiber Scaffolds Accelerate Wound Healing

Cutaneous wound repair regenerates skin integrity, but a chronic failure to heal results in compromised tissue function and increased morbidity. To address this, we have used an integrated approach, using nanobiotechnology to augment the rate of wound reepithelialization by combining self-assembling peptide (SAP) nanofiber scaffold and Epidermal Growth Factor (EGF). This SAP bioscaffold was tested in a bioengineered Human Skin Equivalent (HSE) tissue model that enabled wound reepithelialization to be monitored in a tissue that recapitulates molecular and cellular mechanisms of repair known to occur in human skin. We found that SAP underwent molecular self-assembly to form unique 3D structures that stably covered the surface of the wound, suggesting that this scaffold may serve as a viable wound dressing. We measured the rates of release of EGF from the SAP scaffold and determined that EGF was only released when the scaffold was in direct contact with the HSE. By measuring the length of the epithelial tongue during wound reepithelialization, we found that SAP scaffolds containing EGF accelerated the rate of wound coverage by 5 fold when compared to controls without scaffolds and by 3.5 fold when compared to the scaffold without EGF. In conclusion, our experiments demonstrated that biomaterials composed of a biofunctionalized peptidic scaffold have many properties that are well-suited for the treatment of cutaneous wounds including wound coverage, functionalization with bioactive molecules, localized growth factor release and activation of wound repair.

Defining the cellular precursors to human breast cancer

Human breast cancers are broadly classified based on their gene-expression profiles into luminal- and basal-type tumors. These two major tumor subtypes express markers corresponding to the major differentiation states of epithelial cells in the breast: luminal (EpCAM+) and basal/myoepithelial (CD10+). However, there are also rare types of breast cancers, such as metaplastic carcinomas, where tumor cells exhibit features of alternate cell types that no longer resemble breast epithelium. Until now, it has been difficult to identify the cell type(s) in the human breast that gives rise to these various forms of breast cancer. Here we report that transformation of EpCAM+ epithelial cells results in the formation of common forms of human breast cancer, including estrogen receptor-positive and estrogen receptor-negative tumors with luminal and basal-like characteristics, respectively, whereas transformation of CD10+ cells results in the development of rare metaplastic tumors reminiscent of the claudin-low subtype. We also demonstrate the existence of CD10+ breast cells with metaplastic traits that can give rise to skin and epidermal tissues. Furthermore, we show that the development of metaplastic breast cancer is attributable, in part, to the transformation of these metaplastic breast epithelial cells. These findings identify normal cellular precursors to human breast cancers and reveal the existence of a population of cells with epidermal progenitor activity within adult human breast tissues.

Tumor progression of skin carcinoma cells in vivo promoted by clonal selection, mutagenesis, and autocrine growth regulation by granulocyte colony-stimulating factor and granulocyte-macrophage colony-stimulating factor

Tumor microenvironment is crucial for cancer growth and progression as evidenced by reports on the significance of tumor angiogenesis and stromal cells. Using the HaCaT/HaCaT-ras human skin carcinogenesis model, we studied tumor progression from benign tumors to highly malignant squamous cell carcinomas. Progression of tumorigenic HaCaT-ras clones to more aggressive and eventually metastatic phenotypes was reproducibly achieved by their in vivo growth as subcutaneous tumors in nude mice. Their enhanced malignant phenotype was stably maintained in recultured tumor cells that represented, identified by chromosomal analysis, a distinct subpopulation of the parental line. Additional mutagenic effects were apparent in genetic alterations involving chromosomes 11 and 2, and in amplification and overexpression of the H-ras oncogene. Importantly, in vitro clonal selection of benign and malignant cell lines never resulted in late-stage malignant clones, indicating the importance of the in vivo environment in promoting an enhanced malignant phenotype. Independently of their H-ras status, all in vivo-progressed tumor cell lines (five of five) exhibited a constitutive and stable expression of the hematopoietic growth factors granulocyte colony-stimulating factor and granulocyte-macrophage colony-stimulating factor, which may function as autocrine/paracrine mediators of tumor progression in vivo. Thus, malignant progression favored by the in vivo microenvironment requires both clonal selection of subpopulations adapted to in vivo growth and mutational events leading to stable functional alterations.

E-cadherin Suppression Accelerates Squamous Cell Carcinoma Progression in Three-Dimensional, Human Tissue Constructs

We studied the link between loss of E-cadherin-mediated adhesion and acquisition of malignant properties in three-dimensional, human tissue constructs that mimicked the initial stages of squamous cell cancer progression. Suppression of E-cadherin expression in early-stage, skin-derived tumor cells (HaCaT-II-4) was induced by cytoplasmic sequestration of beta-catenin upon stable expression of a dominant-negative E-cadherin fusion protein (H-2Kd-Ecad). In monolayer cultures, expression of H-2Kd-Ecad resulted in decreased levels of E-cadherin, redistribution of beta-catenin to the cytoplasm, and complete loss of intercellular adhesion when compared with control II-4 cells. This was accompanied by a 7-fold decrease in beta-catenin-mediated transcription and a 12-fold increase in cell migration. In three-dimensional constructs, E-cadherin-deficient tissues showed disruption of architecture, loss of adherens junctional proteins from cell contacts, and focal tumor cell invasion. Invasion was linked to activation of matrix metalloproteinase (MMP)-mediated degradation of basement membrane in H-2Kd-Ecad-expressing tissue constructs that was blocked by MMP inhibition (GM6001). Quantitative reverse transcription-PCR showed a 2.5-fold increase in MMP-2 and an 8-fold increase in MMP-9 in cells expressing the H-2Kd-Ecad fusion protein when compared with controls, and gel zymography showed increased MMP protein levels. Following surface transplantation of three-dimensional tissues, suppression of E-cadherin expression greatly accelerated tumorigenesis in vivo by inducing a switch to high-grade carcinomas that resulted in a 5-fold increase in tumor size after 4 weeks. Suppression of E-cadherin expression and loss of its function fundamentally modified squamous cell carcinoma progression by activating a highly invasive, aggressive tumor phenotype, whereas maintenance of E-cadherin prevented invasion in vitro and limited tumor progression in vivo.

Human papillomavirus infection of the oral mucosa.

This article reviews the lesions of oral mucosa that contain human papillomavirus (HPV). These HPV-associated lesions can be classified into two broad types on the basis of their biologic behavior, benign lesions and premalignant malignant or malignant lesions. Benign oral lesions include squamous cell papilloma (SCP), verruca vulgaris (VV), condyloma acuminatum (CA), and focal epithelial hyperplasia (FEH). Of these entities, VV, CA, and FEH demonstrate characteristic HPV-induced cytopathic effects, whereas SCP infrequently shows such changes. All of these lesions show a clear association with HPV. Premalignant and malignant oral lesions include leukoplakia and squamous cell carcinoma. The etiologic role of HPV in these lesions is still unclear. Koilocytosis is the most common cytopathic effect seen in both groups of lesions. Even though it is sometimes difficult to distinguish between hyperplastic lesions such as SCP, VV, and CA, clinical and certain histologic features can facilitate the diagnosis. Although exceptions do exist, each of the two classes of lesions is most commonly associated with particular HPV types. The benign oral lesions are associated with HPV 2, 4, 6, 11, 13, and 32; the malignant oral lesions are associated with HPV 16 and 18. No preferential association has been demonstrated between specific HPV types and a particular oral lesion.

In vitro 3D Full-Thickness Skin-Equivalent Tissue Model Using Silk and Collagen Biomaterials

Current approaches to skin equivalents often only include the epidermis and dermis. Here, a full-thickness skin equivalent is described including epidermis, dermis, and hypodermis, that could serve as an in vitro model for studying skin biology or as a platform for consumer product testing. The construct is easy to handle and is maintained for >14 d while expressing physiological morphologies of the epidermis and dermis, seen by keratin 10, collagens I and IV expression. The skin equivalent produces glycerol and leptin, markers of adipose metabolism. This work serves as a foundation for understanding a few necessary factors needed to develop a stable, functional model of full-thickness skin.

Re-epithelialization of Human Oral Keratinocytes in vitro

Re-epithelialization involves interactions between keratinocytes and the extracellular matrix upon which these cells move. It is hypothesized that keratinocytes are activated when wounded, and the resultant phenotypic change directs re-epithelialization. We have adapted organotypic cultures, in which oral gingival keratinocytes are fully differentiated, to study re-epithelialization following wounding. To elucidate keratinocyte behavior and phenotype during re-epithelialization, we have investigated this process in the presence and absence of the growth factor TGF-β1 and have monitored expression of MMP-1 (Type I collagenase) mRNA by in situ hybridization. In addition, we have followed proliferation and migration of wound keratinocytes by genetically marking these cells with a retroviral vector and by measuring their proliferative index. We found that keratinocytes grown without TGF-β1 were hyperproliferative in response to wounding, and re-epithelialization was complete by 24 h. However, 2.5 ng/mL TGF-β1 induced a transient delay in re-epithelialization, a reduction in proliferation, and fewer clusters of genetically marked cells. Keratinocytes expressed MMP-1 mRNA only when they covered the wounded surface, suggesting that the cells acquire a collagenolytic phenotype during re-epithelializaation and that contact with different ECM components may modulate keratinocyte expression of MMP-1. We conclude that the phenotype of oral keratinocytes is altered during re-epithelialization in vitro and that this process is modulated by TGF-pl. Reepithelialization occurs as keratinocytes are activated to move over the wound bed. Understanding the phenotype of wounded keratinocytes may facilitate treatment of chronic oral wounds and periodontal disease.

Three‐Dimensional Tissue Models of Normal and Diseased Skin

Over the last decade, the development of in vitro, human, three‐dimensional (3D) tissue models, known as human skin equivalents (HSEs), has furthered understanding of epidermal cell biology and provided novel experimental systems. Signaling pathways that mediate the linkage between growth and differentiation function optimally when cells are spatially organized to display the architectural features seen in vivo, but are uncoupled and lost in two‐dimensional culture systems. HSEs consist of a stratified squamous epithelium grown at an air‐liquid interface on a collagen matrix populated with dermal fibroblasts. These 3D tissues demonstrate in vivo–like epithelial differentiation and morphology, and rates of cell division, similar to those found in human skin. This unit describes fabrication of HSEs, allowing the generation of human tissues that mimic the morphology, differentiation, and growth of human skin, as well as disease processes of cancer and wound re‐epithelialization, providing powerful new tools for the study of diseases in humans. Curr. Protoc. Cell Biol. 41:19.9.1‐19.9.17.

E-cadherin loss promotes the initiation of squamous cell carcinoma invasion through modulation of integrin-mediated adhesion.

Much remains to be learned about how cell-cell and cell-matrix interactions are coordinated to influence the earliest development of neoplasia. We used novel 3D human tissue reconstructs that mimic premalignant disease in normal epidermis, to directly investigate how loss of E-cadherin function directs conversion to malignant disease. We used a genetically tagged variant of Ha-Ras-transformed human keratinocytes (II-4) expressing dominant-interfering E-cadherin fusion protein (H-2kd-Ecad). These cells were admixed with normal human keratinocytes and tumor cell fate was monitored in 3D reconstructed epidermis upon transplantation to immunodeficient mice. Tumor initiation was suppressed in tissues harboring control- and mock-infected II-4 cells that lost contact with the stromal interface. By contrast, H-2kd-Ecad-expressing cells persisted at this interface, thus enabling incipient tumor cell invasion upon in vivo transplantation. Loss of intercellular adhesion was linked to elevated cell surface expression of α2, α3 and β1 integrins and increased adhesion to laminin-1 and Types I and IV collagen that was blocked with β1-integrin antibodies, suggesting that invasion was linked to initial II-4 cell attachment at the stromal interface. Collectively, these results outline a novel aspect to loss of E-cadherin function that is linked to the mutually interdependent regulation of cell-cell and cell-matrix adhesion and has significant consequences for the conversion of premalignancy to cancer.