PURION® processed human amnion chorion membrane allografts retain material and biological properties supportive of soft tissue repair.

The reparative properties of amniotic membrane allografts are well-suited for a broad spectrum of specialties. Further enhancement of their utility can be achieved by designing to the needs of each application through the development of novel processing techniques and tissue configurations. As such, this study evaluated the material characteristics and biological properties of two PURION® processed amniotic membrane products, a lyophilized human amnion, intermediate layer, and chorion membrane (LHACM) and a dehydrated human amnion, chorion membrane (DHACM). LHACM is thicker; therefore, its handling properties are ideal for deep, soft tissue deficits; whereas DHACM is more similar to a film-like overlay and may be used for shallow defects or surgical on-lays. Characterization of the similarities and differences between LHACM and DHACM was conducted through a series of in vitro and in vivo studies relevant to the healing cascade. Compositional analysis was performed through histological staining along with assessment of barrier membrane properties through equilibrium dialysis. In vitro cellular response was assessed in fibroblasts and endothelial cells using cell proliferation, migration, and metabolic assays. The in vivo cellular response was assessed in an athymic nude mouse subcutaneous implantation model. The results indicated the PURION® process preserved the native membrane structure, nonviable cells and collagen distributed in the individual layers of both products. Although, LHACM is thicker than DHACM, a similar composition of growth factors, cytokines, chemokines and proteases is retained and consequently elicit comparable in vitro and in vivo cellular responses. In culture, both treatments behaved as potent mitogens, chemoattractants and stimulants, which translated to the promotion of cellular infiltration, neocollagen deposition and angiogenesis in a murine model. PURION® processed LHACM and DHACM differ in physical properties but possess similar in vitro and in vivo activities highlighting the impact of processing method on the versatility of clinical use of amniotic membrane allografts.

Dehydrated human amniotic membrane modulates canonical Wnt signaling in multiple cell types in vitro.

Canonical Wnt signaling is a major pathway known to regulate diverse physiological processes in multicellular organisms. Signaling is tightly regulated by feedback mechanisms; however, persistent dysregulation of this pathway is implicated in the progression of multiple disease states. In this study, proteomic analysis identified endogenous Wnt antagonists in micronized dehydrated human amnion/chorion membrane (µdHACM); thereby, prompting a study to further characterize the intrinsic properties of µdHACM as it relates to Wnt activity, in vitro. A TCF/LEF reporter cell line demonstrated the general ability of µdHACM to inhibit ß-catenin induced transcription activity. Furthermore, in vitro systems, modeling elevated Wnt signaling, were developed in relevant cell types including tenocytes, synoviocytes, and human dermal fibroblasts (HDFs). Stimulation of these cells with Wnt3A resulted in translocation of ß-catenin to the nucleus and increased expression of Wnt related genes. The subsequent addition of µdHACM, in the continued presence of Wnt-stimulus, mitigated the downstream effects of Wnt3A in tenocytes, synoviocytes, and HDFs. Nuclear localization of ß-catenin was abated with corresponding reduction of Wnt related gene expression. These data demonstrate the in vitro regulation of canonical Wnt signaling as an inherent property of µdHACM and a novel mechanism of action.

Development and characterization of a tissue engineered pancreatic substitute based on recombinant intestinal endocrine L-cells.

A tissue engineered pancreatic substitute (TEPS) consisting of insulin-producing cells appropriately designed and encapsulated to support cellular function and prevent interaction with the host may provide physiological blood glucose regulation for the treatment of insulin dependent diabetes (IDD). The performance of agarose-based constructs which contained either a single cell suspension of GLUTag-INS cells, a suspension of pre-aggregated GLUTag-INS spheroids, or GLUTag-INS cells on small intestinal submucosa (SIS), was evaluated in vitro for total cell number, weekly glucose consumption and insulin secretion rates (GCR and ISR), and induced insulin secretion function. The three types of TEPS studied displayed similar number of cells, GCR, and ISR throughout 4 weeks of culture. However, the TEPS, which incorporated SIS as a substrate for the GLUTag-INS cells, was the only type of TEPS tested which was able to retain the induced insulin secretion function of non-encapsulated GLUTag-INS cells. Though improvements in the expression level of GLUTag-INS cells and/or the number of viable cells contained within the TEPS are needed for successful treatment of a murine model of IDD, this study has revealed a potential method for promoting proper cellular function of recombinant L-cells upon incorporation into an implantable three-dimensional TEPS.

A cell-based approach for diabetes treatment using engineered non-beta cells.

BACKGROUND: Implantation of insulin-secreting cells has the potential to provide tight glycemic regulation in diabetic subjects. Implantation of cadaveric human islets in immunosuppressed human patients is currently applied at a very small scale. To overcome the limitations of tissue availability and recipient immunosuppression, encapsulation of nonautologous cells and use of potentially autologous nonislet cells, the latter engineered for insulin secretion, are being pursued. This article reports on recent findings with the implantation of tissue constructs containing enteroendocrine cells stably expressing recombinant insulin in diabetic mice. The concept of a dual recombinant hepatic and enteroendocrine cell system, which may better approximate the secretory response of islets, is discussed. METHODS: Mouse GLUTag-INS cells engineered to secrete human insulin were developed and incorporated in tissue constructs as reported previously. Constructs were implanted intraperitoneally in diabetic mice, and blood glucose levels, animal weights, and plasma insulin levels were measured at various time points. RESULTS: GLUTag-INS-containing tissue constructs secreted insulin preimplantation and postexplantation, and human insulin was detected in the plasma of diabetic mice. However, normoglycemia was not restored. CONCLUSIONS: A variety of cell types and of encapsulation methods to enhance immune acceptance of insulin-secreting grafts are being pursued. Recombinant enteroendocrine cells show promise, but it is likely that they need to be combined with recombinant hepatic cells to achieve glycemic normalization.

Insulin-secreting L-cells for the treatment of insulin-dependent diabetes.

Cell-based treatments for insulin-dependent diabetes (IDD) may provide more physiologic regulation of blood glucose levels than daily insulin injections, thereby reducing the occurrence of secondary complications associated with diabetes. An autologous cell source is especially attractive for regulatory and ethical reasons in addition to eliminating the need for immunosuppression. This study uses non-beta-cells, genetically modified for physiologic insulin secretion. Enteroendocrine L-cells, exhibit regulated secretion in response to physiologic stimuli and their endogenous products are fully compatible with prandial metabolism. Murine GLUTag L-cells were transfected with a plasmid co-expressing human insulin and neomycin resistance and the stable cell line, GLUTag-INS, was established. Secretion properties of GLUTag-INS cells were investigated in vitro through induced secretion tests using meat hydrolysate or 3-isobutyl-1-methylxanthine and forskolin as secretagogues. GLUTag-INS cells rapidly co-secreted recombinant insulin and endogenous glucagon-like peptide in response to metabolic cues from the surrounding medium and demonstrated efficient processing of proinsulin to insulin.