Porcine allograft mandible revitalization using autologous adipose-derived stem cells, bone morphogenetic protein-2, and periosteum.

BACKGROUND: Critical defects of the craniomaxillofacial region are often treated with vascularized osteocutaneous free flaps. These lengthy operations may be associated with considerable donor-site morbidity and suboptimal functional and aesthetic results. To overcome these issues, this study investigates an engineered vascularized bone flap using allograft bone, adipose-derived stem cells, and recombinant human bone morphogenic protein (rhBMP)-2 and compares two alternative blood supplies. METHODS: Edentulous porcine hemimandibles were commercially sterilized, packed with rhBMP-2-soaked absorbable collagen sponge and autologous, culture-expanded adipose-derived stem cells, and implanted into two locations within 10 pigs: (1) an intercostal-based periosteal envelope (thoracic) and (2) within the rectus abdominis muscle with insertion of the superficial inferior epigastric vascular pedicle into the medullary cavity (abdominal). The constructs were incubated in vivo for 7 to 8 weeks and harvested to assess de novo bone formation. RESULTS: Radiographic, micro-computed tomographic, and histologic assessments of harvested constructs were performed. Abdominal constructs had a thin rim of new, cancellous bone surrounding a fibrotic core with little allograft remaining. Thoracic allografts were absorbed completely and replaced with new, full-thickness, cancellous bone. Calcitic tissue content was significantly higher in thoracic (474.16 +/- 75.93 ml) compared with abdominal (143.20 +/- 46.39 ml) constructs (p < 0.006). New bone in both groups contained Haversian systems, but only thoracic constructs contained marrow elements and blood vessels resembling normal bone. CONCLUSIONS: These data demonstrate revitalization of large-volume allograft bone, and have positive implications for bone tissue engineering. Allograft revitalization in thoracic but not abdominal constructs reinforces the critical role of the periosteum in the process.

Retinoblastoma family proteins have distinct functions in pulmonary epithelial cells in vivo critical for suppressing cell growth and tumorigenesis.

Lung cancer is the leading cause of cancer deaths, accounting for more deaths than breast, colon, and prostate cancer combined. The retinoblastoma (Rb)/p16 tumor suppressive pathway is deregulated in most cancers. Loss of p16 occurs more frequently than Rb loss, suggesting that p16 suppresses cancer by regulating Rb as well as the related proteins p107 and p130. However, direct evidence demonstrating that p130 or p107 cooperate with Rb to suppress epithelial cancers associated with p16 loss is currently lacking. Moreover, the roles of p130 and p107 in lung cancer are not clear. In the present studies, Rb ablation was targeted to the lung epithelium in wild-type, p107, or p130 null mice to determine unique and overlapping Rb family functions critical in tumor suppression. Rb ablation during development resulted in marked epithelial abnormalities despite p107 upregulation. In contrast, p130 and p107 were not required during development but had distinct functions in the Rb-deficient epithelium: p107 was required to suppress proliferation, whereas a novel proapoptotic function was identified for p130. Adult Rb-ablated lungs lacked the epithelial phenotype seen at birth and showed compensatory p107 upregulation and p16 induction in epithelial cell lineages that share phenotypic characteristics with human non-small cell lung cancers (NSCLC) that frequently show p16 loss. Importantly, Rb/p107-deficient, but not Rb/p130-deficient, lungs developed tumors resembling NSCLC. Taken together, these studies identify distinct Rb family functions critical in controlling epithelial cell growth, and provide direct evidence that p107 cooperates with Rb to protect against a common adult cancer.

Overexpression of the cellular DEK protein promotes epithelial transformation in vitro and in vivo.

High levels of expression of the human DEK gene have been correlated with numerous human malignancies. Intracellular DEK functions have been described in vitro and include DNA supercoiling, DNA replication, RNA splicing, and transcription. We have shown that DEK also suppresses cellular senescence, apoptosis, and differentiation, thus promoting cell growth and survival in monolayer and organotypic epithelial raft models. Such functions are likely to contribute to cancer, but direct evidence to implicate DEK as an oncogene has remained elusive. Here, we show that in line with an early role in tumorigenesis, murine papilloma formation in a classical chemical carcinogenesis model was reduced in DEK knockout mice. Additionally, human papillomavirus E6/E7, hRas, and DEK cooperated in the transformation of keratinocytes in soft agar and xenograft establishment, thus also implicating DEK in tumor promotion at later stages. Finally, adenoviral DEK depletion via short hairpin RNA expression resulted in cell death in human tumor cells in vitro and in vivo, but did not significantly affect differentiated epithelial cells. Taken together, our data uncover oncogenic DEK activities as postulated from its frequent up-regulation in human malignancies, and suggest that the targeted suppression of DEK may become a strategic approach to the treatment of cancer.