We have developed a novel panel of cell-surface markers for the isolation and study of all major cell types of the human pancreas. Hybridomas were selected after subtractive immunization of Balb/C mice with intact or dissociated human islets and assessed for cell-type specificity and cell-surface reactivity by immunohistochemistry and flow cytometry. Antibodies were identified by specific binding of surface antigens on islet (panendocrine or alpha-specific) and nonislet pancreatic cell subsets (exocrine and duct). These antibodies were used individually or in combination to isolate populations of alpha, beta, exocrine, or duct cells from primary human pancreas by FACS and to characterize the detailed cell composition of human islet preparations. They were also employed to show that human islet expansion cultures originated from nonendocrine cells and that insulin expression levels could be increased to up to 1% of normal islet cells by subpopulation sorting and overexpression of the transcription factors Pdx-1 and ngn3, an improvement over previous results with this culture system. These methods permit the analysis and isolation of functionally distinct pancreatic cell populations with potential for cell therapy.

The biology of progenitor activation in the liver is of considerable medical and scientific interest. The powerful genetic tools available for the mouse make it an ideal model system to study this complex process involving many different cell types. However, reagents for the isolation and study of distinct hepatic subpopulations have been quite limited compared to those available for hematopoietic cells. To produce cell surface reactive reagents more specific for the oval cell response, we generated

Although bone regeneration is typically a reliable process, type 2 diabetes is associated with impaired or delayed healing processes. In addition, angiogenesis, a crucial step in bone regeneration, is often altered in the diabetic state. In this study, different stages of bone regeneration were characterized in an unicortical bone defect model comparing transgenic type 2 diabetic (db-/db-) and wild type (WT) mice in vivo. We investigated angiogenesis, callus formation and bone remodeling at early, intermediate and late time points by means of histomorphometry as well as protein level analyses. In order to enhance bone regeneration, defects were locally treated with recombinant FGF-9 or VEGFA. Histomorphometry of aniline blue stained sections indicated that bone regeneration is significantly decreased in db-/db- as opposed to WT mice at intermediate (5 days post operation) and late stages (7 days post operation) of bone regeneration. Moreover, immunohistochemical analysis revealed significantly decreased levels of RUNX-2, PCNA, Osteocalcin and PECAM-1 in db-/db- defects. In addition, osteoclastogenesis is impaired in db-/db- indicating altered bone remodeling. These results indicate significant impairments in angiogenesis and osteogenesis in type 2 diabetic bones. Importantly, angiogenesis, osteogenesis and bone remodeling could be reconstituted by application of recombinant FGF-9 and, in part, by VEGFA application. In conclusion, our study demonstrates that type 2 diabetes affects angiogenesis, osteogenesis and subsequently bone remodeling, which in turn leads to decreased bone regeneration. These effects could be reversed by local application of FGF-9 and to a lesser degree VEGFA. These data could serve as a basis for future therapeutic applications aiming at improving bone regeneration in the type 2 diabetic patient population.

Scaphoid bones have a high prevalence for non‐union. Even with adequate treatment, bone regeneration may not occur in certain instances. Although this condition is well described, the molecular pathology of scaphoid non‐unions is still poorly defined. In this study, gene expression of osteogenic and angiogenic growth and transcription factors as well as inflammatory mediators were analysed in human scaphoid non‐unions and intraindividually compared to adjacent autologous cancellous bone from the distal radius. In addition, histology and immunohistochemical stainings were performed to verify qRT‐PCR data. Gene expression analysis revealed a significant up‐regulation of RANKL, ALP, CYCLIN D1, MMP‐13, OPG, NFATc1, TGF‐β and WNT5A in scaphoid non‐unions. Interestingly, RANKL and NFATc1, both markers for osteoclastogenesis, were significantly induced in non‐unions. Moreover, WNT5A was highly up‐regulated in all non‐union samples. TRAP staining confirmed the observation of induced osteoclastogenesis in non‐unions. With respect to genes related to osteogenesis, alkaline phosphatase was significantly up‐regulated in scaphoid non‐unions. No differences were detectable for other osteogenic genes such as RUNX‐2 or BMP‐2. Importantly, we did not detect differences in angiogenesis between scaphoid non‐unions and controls in both gene expression and immunohistochemistry. Summarized, our data indicate increased osteoclast activity in scaphoid non‐unions possibly as a result of the alterations in RANKL, TGF‐β and WNT5A expression levels. These data increase our understanding for the reduced bone regeneration capacity present in scaphoid non‐unions and may translate into the identification of new therapeutic targets to avoid secondary damages and prevent occurrence of non‐unions to scaphoid bones.