Doron J. Aframian

The Growth and Morphological Behavior of Salivary Epithelial Cells on Matrix Protein-Coated Biodegradable Substrata

The purpose of this study was to examine the growth and morphology of a salivary epithelial cell line (HSG) in vitro on several biodegradable substrata as an important step toward developing an artificial salivary gland. The substrates examined were poly-L-lactic acid (PLLA), polyglycolic acid (PGA), and two co-polymers, 85% and 50% PLGA, respectively. The substrates were formed into 20- to 25-mm disks, and the cells were seeded directly onto the polymers or onto polymers coated with specific extracellular matrix proteins. The two copolymer substrates became friable over time in aqueous media and proved not useful for these experiments. The purified matrix proteins examined included fibronectin (FN), laminin (LN), collagen I, collagen IV, and gelatin. In the absence of preadsorbed proteins, HSG cells did not attach to the polymer disks. The cells, in general, behaved similarly on both PLLA and PGA, although optimal results were obtained consistently in PLLA. On FN-coated PLLA disks, HSG cells were able to form a uniform monolayer, which was dependent on time and FN concentration. Coating of disks with LN, collagen I, and gelatin also promoted monolayer growth. This study defines the conditions necessary for establishing a monolayer organization of salivary epithelial cells with rapid proliferation on a biodegradable substrate useful for tissue engineering.

Absence of Tight Junction Formation in an Allogeneic Graft Cell Line Used for Developing an Engineered Artificial Salivary Gland

An essential structural feature of fluid-secreting epithelial tissues is the presence of tight junctions. To develop a tissue-engineered organ capable of fluid secretion, the cellular component must establish these structures. As part of efforts to create an engineered artificial salivary gland, we have examined the ability of a candidate allogeneic graft cell line, HSG, to produce several key tight junction proteins, as well as to exhibit functional activities consistent with effective tight junction strand formation. In contrast to results obtained with a control kidney cell line, MDCK-II, HSG cells were unable to synthesize four important tight junction-associated proteins: ZO-1, occludin, claudin-1, and claudin-2. In addition, unlike MDCK-II cells, HSG cell monolayers could not restrict paracellular permeability. HSG cells were, thus, unable to generate significant transepithelial electrical resistance or serve as an effective barrier to osmotically imposed fluid movement. Furthermore, these two functional activities could not be reconstituted via the stable transfection of HSG cells with cDNAs encoding either claudin-1 or claudin-2. We conclude that because of their inability to form tight junctions, HSG cells are unsuitable for use as an allogeneic graft cell in an artificial salivary fluid secretory device. These studies also emphasize the importance of graft cell selection in artificial organ development, as certain required characteristics may be difficult to reengineer.

Tissue Compatibility of Two Biodegradable Tubular Scaffolds Implanted Adjacent to Skin or Buccal Mucosa in Mice

Radiation therapy for cancer in the head and neck region leads to a marked loss of salivary gland parenchyma, resulting in a severe reduction of salivary secretions. Currently, there is no satisfactory treatment for these patients. To address this problem, we are using both tissue engineering and gene transfer principles to develop an orally implantable, artificial fluid-secreting device. In the present study, we examined the tissue compatibility of two biodegradable substrata potentially useful in fabricating such a device. We implanted in Balb/c mice tubular scaffolds of poly-L-lactic acid (PLLA), poly-glycolic acid coated with PLLA (PGA/PLLA), or nothing (sham-operated controls) either beneath the skin on the back, a site widely used in earlier toxicity and biocompatibility studies, or adjacent to the buccal mucosa, a site quite different functionally and immunologically. At 1, 3, 7, 14, and 28 days postimplantation, implant sites were examined histologically, and systemic responses were assessed by conventional clinical chemistry and hematology analyses. Inflammatory responses in the connective tissue were similar regardless of site or type of polymer implant used. However, inflammatory reactions were shorter and without epithelioid and giant cells in sham-operated controls. Also, biodegradation proceeded more slowly with the PLLA tubules than with the PGA/PLLA tubules. No significant changes in clinical chemistry and hematology were seen due to the implantation of tubular scaffolds. These results indicate that the tissue responses to PLLA and PGA/PLLA scaffolds are generally similar in areas subjacent to skin in the back and oral cavity. However, these studies also identified several potentially significant concerns that must be addressed prior to initiating any clinical applications of this device.

Characterization of Murine Autologous Salivary Gland Graft Cells: A Model for Use with an Artificial Salivary Gland

The purpose of this study was to examine the growth and key functional abilities of primary cultures of salivary epithelial cells toward developing an artificial salivary gland. Cultures of epithelial cells originating from submandibular glands of BALB/c mice were established. Parenchymal cells were isolated by a Percoll gradient technique and thereafter seeded on irradiated NIH 3T3 fibroblasts serving as a feeder layer. The isolated cells were termed autologous salivary gland epithelial (ASGE) cells and could be cultivated for at least five passages (time limit of experiments). ASGE cells presented the typical organizational behavior of epithelial cells and electron microscopy, as well as immunostaining for cytokeratins, confirmed their epithelial origin. Furthermore, measurements of transepithelial resistance and water permeability indicated the ability of the ASGE cells to form a functional epithelial barrier. This study suggests that primary salivary epithelial cells can be obtained that exhibit critical characteristics needed for use with an artificial secretory device.

Using HSV-Thymidine Kinase for Safety in an Allogeneic Salivary Graft Cell Line

Extreme salivary hypofunction is a result of tissue damage caused by irradiation therapy for cancer in the head and neck region. Unfortunately, there is no currently satisfactory treatment for this condition that affects up to 40,000 people in the United States every year. As a novel approach to managing this problem, we are attempting to develop an orally implantable, fluid-secreting device (an artificial salivary gland). We are using the well-studied HSG salivary cell line as a potential allogeneic graft cell for this device. One drawback of using a cell line is the potential for malignant transformation. If such an untoward response occurred, the device could be removed. However, in the event that any HSG cells escaped, we wished to provide additional patient protection. Accordingly, we have engineered HSG cells with a hybrid adeno-retroviral vector, AdLTR.CMV-tk, to express the herpes simplex virus thymidine kinase (HSV-tk) suicide gene as a novel safety factor. Cells were grown on plastic plates or on poly-L-lactic acid disks and then transduced with different multiplicities of infection (MOIs) of the hybrid vector. Thereafter, various concentrations of ganciclovir (GCV) were added, and cell viability was tested. Transduced HSG cells expressed HSV-tk and were sensitive to GCV treatment. Maximal effects were seen at a MOI of 10 with 50 microM of GCV, achieving 95% cell killing on the poly-L-lactic acid substrate. These results suggest that engineering the expression of a suicide gene in an allogeneic graft cell may provide additional safety for use in an artificial salivary gland device.

Salivary glands as a model for craniofacial applications of gene transfer.

The potential applications of gene transfer technology to all branches of medicine are increasing. It is quite likely that within the next 10-20 years surgical practice routinely will utilize gene transfer, at least adjunctively. The purpose of this review is to familiarize the oral and maxillofacial surgeon with this technology. Studies performed with salivary glands in animal models are presented as examples of proof of concept.