Development of tissue-engineered devices may be enhanced by combining cells with porous absorbable polymeric scaffolds before implantation. The cells are seeded throughout the scaffolds and allowed to proliferate in vitro for a predetermined amount of time. The distribution of cells throughout the porous material is one critical component determining success or failure of the tissue-engineered device. This can influence both the successful integration of the device with the host tissue as well as the development of a vascularized network throughout the entire scaffold volume. This research sought to compare different seeding and proliferation methods to select an ideal method for a polyglycolide/aortic endothelial cell system. Two seeding environments, static and dynamic, and three proliferation environments, static, dynamic, and bioreactor, were analyzed, for a total of six possible methods. The six seeding and proliferation combinations were analyzed following a 1-week total culture time. It was determined that for this specific system, dynamic seeding followed by a dynamic proliferation phase is the least promising method and dynamic seeding followed by a bioreactor proliferation phase is the most promising.

Soft tissue reconstruction using tissue-engineered constructs requires the development of materials that are biocompatible and support cell adhesion and growth. The objective of this study was to evaluate the use of macroporous hydrogel fragments that were formed using either unmodified alginate or alginate covalently linked with the fibronectin cell adhesion peptide RGD (alginate-RGD). These materials were injected into the subcutaneous space of adult, domesticated female sheep and harvested for histological comparisons at 1 and 3 months. In addition, the alginate-RGD porous fragments were seeded with autologous sheep preadipocytes isolated from the omentum, and these cell-based constructs were also implanted. The results from this study indicate that both the alginate and alginate-RGD subcutaneous implants supported tissue and vascular ingrowth. Furthermore, at all time points of the experiment, a minimal inflammatory response and capsule formation surrounding the implant were observed. The implanted materials also maintained their sizes over the 3-month study period. In addition, the alginate-RGD fragments supported the adhesion and proliferation of sheep preadipocytes, and adipose tissue was present within the transplant site of these cellular constructs, which was not present within the biomaterial control sites.

There are many clinical situations in which a large tissue mass is required to replace tissue lost to surgical resection (e.g., mastectomy). It is possible that autologous cell transplantation on biodegradable polymer matrices may provide a new therapy to engineer large tissue which can be used to treat these patients. A number of challenges must be met to engineer a large soft tissue mass. These include the design of (1) a structural framework to maintain a space for tissue development, (2) a space-filling matrix which provides for localization of transplanted cells, and (3) a strategy to enhance vascularization of the forming tissue. In this paper we provide an overview of several technologies which are under development to address these issues. Specifically, support matrices to maintain a space for tissue development have been fabricated from polymers of lactide and glycolide. The ability of these structures to resist compressive forces was regulated by the ratio of lactide to glycolide in the polymer. Smooth muscle cell seeding onto polyglycolide fiber-based matrices has been optimized to allow formation of new tissues in vitro and in vivo. Finally, polymer microsphere drug delivery technology is being developed to release vascular endothelial growth factor (VEGF), a potent angiogenic molecule, at the site of tissue formation. This strategy, which combines several different technologies, may ultimately allow for the engineering of large soft tissues.

Tissue engineered biomaterial constructs are needed for plastic and reconstructive applications. To successfully form a space-filling tissue, the construct should induce a minimal inflammatory response, create minimal or no fibrotic capsule, and establish a vascular bed within the first few days after implantation to ensure survival of the implanted cells. In addition, the biomaterial should support cellular adhesion and induce tissue ingrowth. A macroporous hydrogel bead using sodium alginate covalently coupled with an arginine, glycine, and aspartic acid-containing peptide was created. A 6-month subcutaneous rat model study was performed to determine if the implanted material induced tissue ingrowth throughout the implantation area and maintained a three-dimensional vascular bed. The implanted materials produced a vascular bed, minimal inflammation and capsule formation, and good tissue ingrowth throughout the experiment. The material retained its bulking capacity by demonstration of no significant change of the cross-sectional area as measured from the center of the implants after the 2-week time point. In addition, the granulation tissue formed around the implant was loosely organized, and the surrounding tissue had integrated well with the implant. These results indicate that this material has the desired properties for the development of soft-tissue-engineering constructs.

The liver is likely exposed to high levels of H2S from endogenous hepatic synthesis and exogenous sources from the gastrointestinal tract. Little is known about the consequence of H2S exposure on the liver or hepatic regulation of H2S levels. We hypothesized that the liver has a high capacity to metabolize H2S and that H2S oxidation is decreased during sepsis; a condition in which hepatic O2 is limited and H2S synthesis is increased. Using a non-recirculating isolated and perfused liver system, we demonstrated rapid hepatic H2S metabolism up to an infusion concentration 200μM H2S. H2S metabolism was associated with an increase in O2 consumption from a baseline 96.7 ± 7.6 μmoles O2/min/kg to 109 ± 7.4 μmoles O2/min/kg at an infusion concentration of 150 μM H2S (P<0.001). Removal of O2 from the perfusate decreased H2S clearance from a maximal 97% to only 23%. Livers isolated from rats subjected to cecal ligation and puncture (CLP) did not differ significantly from control livers in their capacity to metabolize H2S suggesting that H2S oxidation remains a priority during sepsis. To test whether H2S induces O2 consumption in vivo, intravital microscopy was utilized to monitor the oxygen content in the hepatic microenvironment. Infusion of H2S increased the NADH/NAD+ ratio (645 grey scale unit increase, P=0.035) and decreased hepatic O2 availability visualized with Ru(Phen)32+ (439 grey scale unit increase, P=0.040). We conclude that the liver has a high hepatic capacity for H2S metabolism. Moreover, H2S oxidation consumes available oxygen and may exacerbate the tissue hypoxia associated with sepsis.

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Absorbable biomaterials have been recently incorporated into the field of tissue engineering. Little work has been performed, even with the clinically acceptable absorbables, concerning their tissue promoting capability or lack, thereof. Furthermore, the relative attractions of cells to these implants may be largely disguised by the presence of serum. This research involved the development of an adhesion assay to compare the adhesion behavior of two cell types to two different polylactides in a serum free environment. The results showed that the attachment behavior depends not only on the cell or the polymer but a combination of the two.

Background Exposure to alcohol and its metabolites can initiate hepatic injury and fibrogenesis. Fibrosis is mediated through HSC activation, leading to global changes in mRNA and microRNA (miR) expression. miRs are expressed in cells or shuttled to exosomes which can be detected in tissue culture media and biological fluids. The mechanisms and function underlying the differential expression and processing of miRs and their downstream effects during hepatic injury remain poorly understood. Methods Expression of pri-miR17-92 and individual members of this cluster, miR17a, 18a, 19a, 20a, 19b and 92 were examined in primary HSCs and human LX2 cells exposed to alcohol-conditioned media (CM), liver tissue from a rodent model of alcoholic injury, and in exosomes from tissue culture media and plasma of rodent models and patients with ALD. miR expression was examined in HSCs transduced with an AAV2 vector carrying GFP-miR19b or GFP-control transgene under the collagen promoter. Results Pro-fibrotic markers were enhanced in primary HSCs and LX2 cells exposed to alcohol-CM, concomitant with decreased miR19b expression and a significant increase in pri-miR17-92. Increased miR17-92 was confirmed in a rodent model of alcohol-induced liver injury. Individual members of the cluster were inversely proportionate in cells and exosomes. AAV2-mediated miR19b overexpression inhibited miR17-92 and altered expression of individual cluster members in cells and exosomes. Expression of individual miR17-92 cluster members in plasma exosomes isolated from patients with ALD were similar to those seen in a rodent model of alcoholic injury and in vitro. Conclusions Reintroduction of miR19b inhibits HSC activation and modulates expression of pri-miR17-92 and the inverse expression of individual cluster members in cells and exosomes. Better understanding of miR17-92 processing may provide mechanistic insights to the role of individual miRs and exosomes during hepatic injury, revealing new therapeutic targets.