Transient embolization with microspheres of polyhydroxyalkanoate renders efficient adenoviral transduction of pancreatic capillary in vivo.

BACKGROUND: Our previous study showed an efficient targeting of islets of Langerhans by adenoviral injection via the celiac trunk. Unexpectedly, none of the endothelial cells was infected given the direct contact between adenoviruses and the capillary wall. The present study intended to provide an efficient approach for adenoviral targeting of the microcapillary endothelial cells in the pancreas. METHODS: We prepared microspheres of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx) with a size comparable to the diameter of capillary (5-10 µm). Scanning electron microscopy was applied to verify that adenoviruses carrying a green fluorescence protein gene were complexed with PHBHHx-microspheres after 30 min of co-incubation. The complexes were then injected into the pancreas of mice via the celiac trunk. RESULTS: Approximately 40% of endothelial cells in the pancreas were labeled 5 days after surgery. Islet cells were labeled occasionally, whereas labeling of the acinar and ductal tissues was barely detectable. Endothelium targeting was inefficient in other internal organs. Consistent with the reported superior tissue compatibility of PHBHHx, no discernable microspheres were found in all of the organs examined. Furthermore, splenocyte activation was dampened when adenoviruses were complexed with the microspheres. CONCLUSIONS: The present study has established an approach for efficient pancreatic capillary targeting by using microsphere-adenoviral complexes. This procedure could be invaluable for the treatment of capillary-related diseases.

Induced apoptosis of osteoblasts proliferating on polyhydroxyalkanoates.

The mechanism study on behaviors of cells influenced by biomaterial surface properties can provide profound guidances for functional tissue engineering scaffolds design. In this study, regulation of integrin-mediated cell-substrate interactions using rat osteoblasts incubated on PHA films was investigated. Compared with tissue culture plate (TCP), poly-3-hydroxybutyrate (PHB), copolymer of 3-hydroxybutyrate and 3-hydroxyvalerate (PHBV) and copolymer of 3-hydroxybutyrate and 3-hydroxyhexanoate (PHBHHx), osteoblasts inoculated on a terpolymer of 3-hydroxybutyrate, 3-hydroxyvalerate and 3-hydroxyhexanoate (PHBVHHx) were found to have higher apoptosis rates. Several integrin subunits in osteoblasts grown on PHBVHHx showed altered expressions. Simultaneously, extracellular matrics (ECM) were also remodeled on the material surface. Osteoblasts showed a higher expression of integrin subunit ß3 and αv on PHBVHHx films compared with that on TCP. On the other hand, less vitronectin, osteopontin and fibronectin, the main ligands for integrin ß3 were expressed and deposited in ECM. The unligated integrin ß3 could recruit caspase-8 to the membrane and activate its downstream signaling which was proven by the caspase-8 activation assay. It was therefore concluded that the induced apoptosis of osteoblasts on PHBVHHx was regulated by recruitment of caspase-8 to the unligated integrin ß3.

An assessment of the risks of carcinogenicity associated with polyhydroxyalkanoates through an analysis of DNA aneuploid and telomerase activity.

Polyhydroxyalkanoates (PHA) are aliphatic polyesters synthesized by many bacteria. Because of their flexible mechanical strengths, superior elastic property, biodegradability and biocompatibility, PHA have been developed for applications as medical implants, drug delivery matrices, and devices to support cell growth. Lots of studies showed that PHA matrices improved cell proliferation and tissue regeneration. However, the possibility of whether rapid cell proliferation on PHA matrices will induce tumor formation is unclear. Here we confirmed that proliferating rat osteoblasts grown on films of various PHA including PHB, PHBV, P3HB4HB, PHBHHx and PHBVHHx did not lead to cancer induction at least for p8th. Cell proliferation was evaluated by the incorporation of 5-bromodeoxyuridine (BrdU), the transcript expression of cancer related genes Ki67, p53 and c-Fos was monitored by quantitative Real-time PCR, the results showed the cells proliferating on the PHA films were under normal cell cycle regulation. Moreover, DNA aneuploid and telomerase activity were only detected in the positive control UMR-108 cells; compared with cells grown on films, UMR-108 cells had longer telomeres, further demonstrated the normal status of cells proliferating on the PHA films. It indicated that the above PHA family members could be used to support cell growth without indication of susceptibility to tumor induction. These results will be important for promoting the application of PHA as new members of biomaterials.

Nicotinic acid inhibits glucose-stimulated insulin secretion via the G protein-coupled receptor PUMA-G in murine islet ß cells.

OBJECTIVES: Chronic administration of nicotinic acid (NA), a potent antilipidemic compound, aggravates glycemic control in diabetic patients. It is not known if NA has direct effects on islet ß cells. METHODS: Real-time reverse transcriptase-polymerase chain reaction, in situ hybridization, and immunofluorescence techniques were used to examine the expression of NA receptor PUMA-G, a member of the G protein-coupled receptor (G-PCR) family, in murine islet ß cells. Calcium transient was measured using confocal microscopy, whereas the intracellular cyclic adenosine monophosphate and glucose-stimulated insulin secretion (GSIS) from isolated islets were determined by the enzyme-linked immunosorbent assay. RESULTS: High levels of PUMA-G transcripts and protein were detected in all ß cells, and about 40% of α cells. PUMA-G transcripts increased more than 3-fold in islets incubated with interferon γ. Cyclic adenosine monophosphate accumulation, induced by IBMX/forskolin, was inhibited by NA; however, the inhibition was completely abolished by pretreatment of the culture with pertussis toxin. No calcium transient was detected in islet cells in the presence of NA. Static incubation of islets with NA led to an approximately 30% reduction of GSIS. CONCLUSIONS: The results indicated that PUMA-G stimulation by NA in islet ß cells inhibited GSIS likely via activation of the Gi signaling pathway.

The use of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) scaffolds for tarsal repair in eyelid reconstruction in the rat.

Tarsal repair is an important part for eyelid reconstruction. Presently traditional clinic treatments do not produce satisfactory repair effects. The key is to find a proper tarsal repair material. Microbial poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx) was studied for application as tarsal substitute in this study. PHBHHx scaffolds were implanted into tarsal defects of Sprague-Dawley rats. Eyelid samples of implanted materials and blank defect controls were collected for histological examination at weekly intervals post surgery. Results were compared among PHBHHx scaffolds, commercial acellular dermal matrices (ADM) and blank defect controls. Both PHBHHx scaffolds and ADM provided satisfactory repair results compared with the blank controls even though the implanted PHBHHx scaffolds showed a 2 weeks inflammation. Fibrous encapsulation and scaffold degradation were observed for the PHBHHx implants. Combined with its strong, elastic mechanical properties, the tissue compatible and biodegradable PHBHHx was proven to be a suitable candidate for tarsal repair.

The behaviour of neural stem cells on polyhydroxyalkanoate nanofiber scaffolds.

Polyhydroxyalkanoates (PHA) have demonstrated their potentials as medical implant biomaterials. Neural stem cells (NSCs) grown on/in PHA scaffolds may be useful for repairing central nervous system (CNS) injury. To investigate this possibility, nanofiber matrices (scaffolds) prepared from several PHA via a novel phase separation process were studied to mimic natural extracellular matrix (ECM), and rat-derived NSCs grown in the PHA matrices were characterized regarding their in vitro differentiation behaviors. All three PHA materials including poly(3-hydroxybutyrate) (PHB), copolymer of 3-hydroxybutyrate and 4-hydroxybutyrate (P3HB4HB), and copolymer of 3-hydroxybutyrate and 3-hydroxyhexanoate (PHBHHx) supported NSC growth and differentiation both on their 2D films and 3D matrices. Among three PHA nanofiber matrices, PHBHHx one showed the strongest potentials to promote NSC differentiation into neurons which is beneficial for CNS repair. Compared to the 2D films, 3D nanofiber matrices appeared to be more suitable for NSC attachment, synaptic outgrowth and synaptogenesis. It was suggested that PHBHHx nanofiber scaffolds (matrices) that promote NSC growth and differentiation, can be developed for treating central nervous system injury.

Heterogeneity in mitotic activity and telomere length implies an important role of young islets in the maintenance of islet mass in the adult pancreas.

Understanding the mechanisms of beta-cell dynamics in postnatal animals is central to cure diabetes. A major obstacle in evaluating the status of pancreatic cells is the lack of surface markers. Here we performed quantitative measurements of two internal markers to follow the developmental history of islets. One marker, cell-cycle activity, was established by measuring expression of Ki67 and the incorporation of 5-bromodeoxyuridine. The other marker, the aging process, was delineated by the determination of telomere length. Moreover, islet neogenesis, possibly from ductal precursors, was monitored by pancreatic duct labeling with an enhanced green fluorescence protein (EGFP) transgene. We found that islets from younger animals, on average, expressed higher Ki67 transcripts, displayed elevated 5-bromodeoxyuridine incorporation, and had longer telomeres. However, significant heterogeneity of these parameters was observed among islets from the same mouse. In contrast, the levels of proinsulin-1 transcripts in islets of different ages did not change significantly. Moreover, mitotic activities correlated significantly with telomere lengths of individual islets. Lastly, after 5.5 d pancreatic duct labeling, a few EGFP-positive islets could be identified in neonatal but not from adult pancreases. Compared with unlabeled control islets, EGFP-positive islets had higher mitotic activities and longer telomeres. The results suggest that islets are born at different time points during the embryonic and neonatal stages and imply that young islets might play an important role in the maintenance of islet mass in the adult pancreas.