In recent studies, we showed that exogenous hyaluronic acid oligomers (HA-o) stimulate functional endothelialization, though native long-chain HA is more bioinert and possibly more biocompatible. Thus, in this study, hydrogels containing high molecular weight (HMW) HA (1 x 10(6) Da) and HA-o mixtures (HA-o: 0.75-10 kDa) were created by crosslinking with divinyl sulfone (DVS). The incorporation of HA-o was found to compromise the physical and mechanical properties of the gels (rheology, apparent crosslinking density, swelling ratio, degradation) and to very mildly enhance inflammatory cell recruitment in vivo; increasing the DVS crosslinker content within the gels in general, had the opposite effect, though the relatively high concentration of DVS within these gels (necessary to create a solid gel) also stimulated a mild subcutaneous inflammatory response in vivo and VCAM-1 expression by endothelial cells (ECs) cultured atop; ICAM-expression levels remained very low irrespective extent of DVS crosslinking or HA-o content. The greatest EC attachment and proliferation (MTT assay) was observed on gels that contained the highest amount of HA-o. The study shows that the beneficial EC response to HA-o and biocompatibility of HA is mostly unaltered by their chemical derivatization and crosslinking into a hydrogel. However, the study also demonstrates that the relatively high concentrations of DVS, necessary to create solid gels, compromise their biocompatibility. Moreover, the poor mechanics of even these heavily crosslinked gels, in the context of vascular implantation, necessitates the investigation of other, more appropriate crosslinking agents. Alternately, the outcomes of this study may be used to guide an approach based on chemical immobilization and controlled surface-presentation of both bioactive HA-o and more biocompatible HMW HA on synthetic or tissue engineered grafts already in use, without the use of a crosslinker, so that improved, predictable, and functional endothelialization can be achieved, and the need to create a mechanically compliant biomaterial for standalone use, circumvented.

Angiogenesis represents the outgrowth of new blood vessels from existing ones, a physiologic process that is vital to supply nourishment to newly forming tissues during development and tissue remodeling and repair (wound healing). Regulation of angiogenesis in the healthy body occurs through a fine balance of angiogenesis-stimulating factors and angiogenesis inhibitors. When this balance is disturbed, excessive or deficient angiogenesis can result and contribute to development of a wide variety of pathological conditions. The therapeutic stimulation or suppression of angiogenesis could be the key to abrogating these diseases. In recent years, tissue engineering has emerged as a promising technology for regenerating tissues or organs that are diseased beyond repair. Among the critical challenges that deter the practical realization of the vision of regenerating functional tissues for clinical implantation, is how tissues of finite size can be regenerated and maintained viable in the long-term. Since the diffusion of nutrients and essential gases to cells, and removal of metabolic wastes is typically limited to a depth of 150-250 microm from a capillary (3-10 cells thick), tissue constructs must mandatorily permit in-growth of a blood capillary network to nourish and sustain the viability of cells within. The purpose of this article is to provide an overview of the role and significance of hyaluronan (HA), a glycosaminoglycan (GAG) component of connective tissues, in physiologic and pathological angiogenesis, its applicability as a therapeutic to stimulate or suppress angiogenesis in situ within necrotic tissues in vivo, and the factors determining its potential utility as a pro-angiogenic stimulus that will enable tissue engineering of neo-vascularized and functional tissue constructs for clinical use.

Current vascular implant materials insufficiently recruit endothelial cells (ECs) to form a normally functional and confluent endothelium, a key challenge to reinstating vascular homeostasis at the surgical site. Recent studies indicate that hyaluronan (HA), a connective tissue GAG whose biological effects are often dictated by its fragment size, may inherently stimulate endothelialization. We previously showed that ECs respond poorly to large HA fragments (10 kDa < MW < 1 MDa), therefore, we currently sought to comprehensively study the effects of exogenous high molecular weight (> 1000 kDa) and oligomeric (0.75-10 kDa) ranges on various phenotypic and functional aspects of cultured ECs. HA-1500 (1500 kDa) was enzymatically digested into oligomers under iteratively defined conditions, until a mixture (HA-o) containing a maximal yield of HA-6-mer and 12-mers (33.3 +/- 2.44% and 39.2 +/- 2.68% w/w, respectively) was obtained. The effects of HA-1500, HA-o and pure HA-6-mers on rat aortic ECs were compared. DNA and tube formation assays revealed HA-o and HA-6-mers to stimulate EC proliferation and EC tube formation (angiogenesis) much more than non-HA controls, while HA-1500 had a significant but more modest effect. Both HA-o and HA-6-mers attenuated platelet adhesion and activation on EC layers, while HA-1500 drastically inhibited the same relative to controls. However, flow cytometry and cytokine array studies found that HA-o incited increased expression levels of EC activation markers (ICAM-1, VCAM-1) and promoted the release of select inflammatory cytokines to a greater degree than HA-1500. These results suggest that HA-o and HA-1500 both provide benefits, although frequently of different kinds, to endothelial cell sustenance, proliferation and normal functionality. Thus, tissue engineering scaffolds containing both these cues in optimized ratios could potentially serve as excellent materials for vascular EC regeneration. This forms the basis of our ongoing studies. Copyright (c) 2008 John Wiley & Sons, Ltd.

Current vascular implant materials poorly interact with smooth muscle cells (SMCs) of the media allowing the permanent loss of vascular elastin, eliminated by trauma or disease, a crucial element in maintaining the natural biomechanics of the blood vessel and overall vascular homeostasis. In addition, these materials insufficiently recruit vascular endothelial cells (ECs) to form a normally functional, confluent endothelium that acts as the interface between the blood and vascular tissue and regulates numerous vital vascular processes. As a result, the restenosis, or re-occlusion, rate within these devices has remained fairly high stimulating the investigation of numerous new materials capable of providing the necessary stimuli for the regeneration of vascular tissue. Our lab has reported extensively on hyaluronic acid (HA) as a vascular regenerative agent. We found it beneficially impact two key aspects of vascular regeneration, namely functional endothelialization and elastin matrix repair/ regeneration, and that the biologic impact is dependant on HA molecular weight. This project was initiated to investigate the sizespecific ability of exogenous HA to promote endothelialization, and then attempted to utilize this, and previously acquired information on the HA-dependant upregulation of elastogenesis of vascular SMCs, for the development of HA biomaterials, in the form of surface coatings and hydrogels, for vascular tissue engineering. Exogenous supplementation of a HA digest (D2) containing a concentrated mixture of HA oligomers (0.75–10 kDa) promoted EC proliferation and tube formation, but also enhanced platelet attachment, CAM expression, and cytokine release.

PURPOSE:We evaluated the effectiveness of placing a skin tube in the subcutaneous plane to manage refractory ascites by draining the ascitic fluid from the peritoneal cavity into the long saphenous vein.METHODS:Twenty patients with refractory ascites underwent this technique which was performed in two stages. In the first stage, a thin piece of partial thickness skin graft was rolled into a tube and implanted in the subcutaneous plane of the lower abdomen and the upper thigh near and parallel to the upper segment of the long saphenous vein. In the second stage, which was done 3 months later, we anastomosed the upper end of the skin tube to the peritoneal cavity and the lower end of the skin tube to the long saphenous vein. The follow-up period was 4 years.RESULTS:There was no mortality. The complications consisted of hematoma formation in two patients, wound infection in three, and ascitic fluid leakage from the upper anastomosis in three. All these complications were managed conservatively.CONCLUSION:These findings show that creating a saphenoperitoneal shunt with a skin graft tube interposition is a novel, safe, and cost-effective technique of resolving the problem of refractory ascites.

Crosslinked gels (hylans) containing long-chain (MW>1×106Da) hyaluronan (HA), a connective tissue GAG, show exceptional biocompatibility for vascular implantation but poorly interact with vascular endothelial cells (ECs). Previous studies showed in situ fragmentation of HA by UV light to bioactivate hylan gels and elicit enhanced EC responses. Since fragmented HA can be pro-inflammatory, it is important to define an optimal size distribution of HA fragments on the hylan surface that will recruit and support normally functional ECs and limit ulterior responses. Related studies have shown that exogenous models of HA do not necessarily replicate cell responses to HA scaffolds. Since scaffolds cannot be created based on fragmented HA alone, we sought to determine size-specific responses of ECs to HA substrates of defined fragment sizes by creation of HA-tethered culture surfaces. HA (1000, 200, 20kDa) and an oligomer mixture were tethered onto an aminosilane (APTMS)-treated glass surfaces using a carbodiimide reaction. MALDI–TOF showed the HA digests to contain HA 4–8mers with a 75±0.4% w/w of 4mers. Immuno-fluorescence, SEM, AFM and XPS analysis revealed homogeneous amine and HA surfaces. An amine s-SDTB assay and HA fluorophore-assisted carbohydrate electrophoresis (FACE) indicated surface densities of 9±3 amine groups/nm2 and 0.57±0.44μg/cm2, respectively. HA/HA fragments/oligomers were stable over 21 days of incubation in serum-free culture media. EC proliferation on these surfaces resulted was limited, a possible effect of smooth surface topography, high anionicity, and in case of 4mers, non-interaction with primary HA cell–surface receptors (CD44). This work is significant in that it allows testing of cell responses to substrates composed of single-sized fragments of HA that cannot by themselves be cross-linked into a gel. Future work in our lab will use this model to assess the effects of other HA oligomer sizes on EC behavior.

Molded components for the electrolysis module have been procured. This allows consolidation of parts and cost reduction. Membrane fabrication hardware has been installed at the Teledyne Energy Systems Inc. (TESI) facility. Membrane samples have been successfully produced and preliminary membrane testing in an electrolysis stack has been performed. The Aerovironment Power supply DFMA study has been completed. Fabrication of the single-cell high-pressure module has been completed. The design has been hydrostatically tested up to 1,500 psig. The Benchtop I system (pressure control), has been tested. Pressure control logic and hardware has been finalized for use in Benchtop II. Hazards and Operability Analysis (HAZOP) on Benchtop II has been completed and system design has been modified for safety. Benchtop II (sub-scale electrolysis system) has been fabricated and is now ready for debugging and testing. • •