Faculty Bibliography

We have used DD-PCR (differential display-polymerase chain reaction) to identify new genes that are over- or underexpressed during wound repair. DD-PCR performed on excisional wounds identified the expression of rat c49a. Cloning and sequence analysis of the rat c49a gene revealed high homology to a novel human melanoma differentiation associated gene, mda-7. The human mda-7gene isolated from melanoma cell lines, has been linked with human melanoma differentiation, and growth suppression. Moreover, transfection of human mda-7 constructs into human tumor cells suppresses the growth and colony formation of tumor cells from diverse origins. To confirm and relatively quantitate expression of rat c49a gene during repair, specific primer, reduced cycle RT-PCR (reverse transcription-PCR) was performed. RT-PCR showed an approximately 9 to 12-fold elevation of rat c49a mRNA at 12 h to 5 days above nonwounded controls that gradually decreased to approximately 1.5 to 3-fold by day 14. Cloning and sequence analysis of the entire 1200 base pair c49a gene product showed 78% nucleotide homology to human mda-7. Immunohistochemistry studies localized rat C49A expression primarily to fibroblast-like cells at the wound edge and base. The marked up-regulation of rat c49a transcripts during the inflammatory and early granulation tissue phases of wound repair where cellular processes such as re-epithelialization, angiogenesis, and fibroplasia predominate--suggest that c49a is associated with proliferation of fibroblasts in wound healing

Surgical correction of unilateral coronal synostosis offers a unique opportunity to examine the molecular differences between an abnormal and a normal cranial suture. We isolated and identified a cDNA fragment whose expression was up-regulated in the premature fusing and fused coronal sutures, as compared with normal coronal sutures. The nucleotide sequence of the full-length cDNA of this gene, human NELL-1, has approximately 61% homology with the chicken Nel gene. Both chicken Nel and human NELL-1 are comprised of six epidermal growth factor-like repeats. The human NELL-1 messages were localized primarily in the mesenchymal cells and osteoblasts at the osteogenic front, along the parasutural bone margins, and within the condensing mesenchymal cells of newly formed bone in sites of premature sutural fusion. Human multiorgan tissue mRNA blot showed that NELL-1 was specifically expressed in fetal brain but not in fetal kidney, liver, or lung. We also showed that Nell-1 was expressed in rat calvarial osteoprogenitor cells and was largely absent in rat tibiae and fibroblast cell cultures. In conclusion, our data suggest that the NELL-1 gene is preferentially expressed in cranial intramembranous bone and neural tissue (both of neural crest cell origin) and is up-regulated during unilateral premature closure of the coronal suture. The precise role of this gene is unknown

PURPOSE: The purpose of this study was to evaluate the effect of chitosan on lingual hemostasis in rabbits whose coagulation pathway had been impaired by administration of intravenous heparin. MATERIALS AND METHODS: Bleeding times were measured for bilateral (15 mm x 2 mm) tongue incisions in 10 New Zealand white rabbits. Using a randomized, blinded experimental design, one incision in each animal was treated with chitosan, and the other was treated with the control vehicle without chitosan. Activated coagulation times and extraoral bleeding times were measured for each animal before, during, and after heparinization. RESULTS: Intravenous infusion of heparin more than tripled the mean activated coagulation time and increased mean systemic bleeding time by 40%. In this heparinized animal model, lingual incisions receiving the experimental substance showed a 43% improvement in bleeding time as compared with lingual incisions receiving the control solution (P< or =.001). Chitosan treatment brought bleeding time of the lingual incision for heparinized animals within the normal range. Scanning electron microscopic evaluation of the incisions treated with chitosan showed an altered red blood cell morphology and an unusual affinity between erythrocytes. CONCLUSIONS: Topical application of chitosan to lingual incisions effectively decreased intraoral bleeding time in a therapeutically anticoagulated (heparinized) rabbit model. Chitosan facilitated lingual hemostasis, possibly through interaction with erythrocytes, linking them together to establish a cellular clot or hemostatic plug

This study examined the effect of exogenous TGF -beta1 on platelet derived growth factor alpha and beta (PDGF-alpha, beta) receptor expression in human dermal fibroblasts derived from both normal cutaneous tissues (normal skin [NSk]) and (normal scar [NSc]) and abnormal scar (keloid). TGF-beta and PDGF are present in the early phases of wound healing and are implicated in tissue fibrosis. In this study, replicate samples of NSk, NSc and keloid fibroblasts were grown to subconfluency in DMEM/10% FBS followed by replacement of media with DMEM/0.1%FBS for 24 hrs. One group of cells (NSk, NSc and keloid) were exposed to 10 ng/mL of exogenous TGF-beta1 for 24 hours, while the other group was used as control with no exposure to exogenous TGF-beta1. RadioImmunoBinding assays, Western and Northern blot analysis were performed to examine both PDGF-alpha and PDGF-beta receptor expression at the transcriptional and post-transcriptional levels. cDNA receptor probes were synthesized using polymerase chain reaction (PCR) with selected primer sets derived from published sequences. Beta-actin probe was used as a control to confirm that the same quantity of RNA was used for each experimental condition. TGF-beta1 was found to upregulate the expression of PDGF-alpha receptor for keloid fibroblasts but not for NSk or NSc fibroblasts. No effect was observed for TGF-beta 1 on PDGF-beta receptor expression for any of the cell lines examined