Pro-angiogenic character of endothelial cells and gingival fibroblasts cocultures in perfused degradable polyurethane scaffolds.

Gingival atrophy manifests as exposure of the tooth root surface because of recession of the gingiva, a condition that affects >20% of adults and leads to increased root sensitivity and ultimately, tooth loss. Tissue engineering approaches that employ novel synthetic polymeric scaffolds are being considered for rebuilding the gingival lamina propria lost in the atrophic process. Specifically, polyurethane hydrogels (degradable/polar/hydrophobic/ionic polyurethane [D-PHI]) can enhance the proliferation of human gingival fibroblasts (HGFs) and collagen production in a perfusion system. However, few studies have assessed the potential of synthetic block copolyurethanes to initiate blood vessel formation in an in vitro bioreactor system. As the gingival lamina propria is highly vascular, a coculture system of human umbilical vein endothelial cells (HUVECs) with HGFs was used in perfused D-PHI scaffolds to determine the feasibility of initiating vascularization. Culture conditions were optimized for driving cocultures toward the desired tissue-engineered construct. HUVEC-HGF coculture in perfused D-PHI scaffolds with a cell seeding density of at least 80,000 cells/scaffold in a 50/50 mix of HUVEC and HGF media (by volume) exhibited enhanced cell growth and increased vascular endothelial growth factor and fibroblast growth factor (FGF)-2 production, as well as reduced myofibroblast differentiation. A greater fibroblast proportion (seeding ratio of 1:2) in the coculture resulted in HUVEC cluster formations and increased transforming growth factor-ß1 and FGF-2 production. The combined pro-angiogenic effects provided by these culture conditions are anticipated to be important in the development of a highly vascularized tissue-engineered construct for regenerating the gingival lamina propria and possibly other soft tissues.

Establishing a gingival fibroblast phenotype in a perfused degradable polyurethane scaffold: mediation by TGF-ß1, FGF-2, ß1-integrin, and focal adhesion kinase.

Medium perfusion has been shown to enhance cell proliferation and matrix protein production. In more recent work, under perfusion, a degradable/polar/hydrophobic/ionic polyurethane (D-PHI) scaffold was shown to enhance growth and production of collagen by human gingival fibroblasts (HGFs). However, the nature of the HGFs cultured in the perfused D-PHI scaffolds, and the mechanisms by which medium perfusion activates these cells to facilitate proliferation and collagen production are not defined. The current study sought to investigate HGF interaction within the D-PHI scaffolds under perfusion by examining the production and the spatial distribution of α-smooth muscle actin (α-SMA) and type I collagen (Col I), the secretion of transforming growth factor (TGF)-ß1 and basic fibroblast growth factor (FGF-2) in the conditioned medium, with a goal of defining the mechanistic pathways affecting the production of these markers in the dynamic culture. It was found that the perfused D-PHI scaffold shifted the HGF phenotype from myofibroblast-like (upregulation of α-SMA) to fibroblast-like (downregulation of α-SMA) over the course of 28 days. Both TGF-ß1 and FGF-2 were significantly greater in the dynamic vs. static culture at day 1. Although TGF-ß1 has been often reported to increase α-SMA and collagen expression, the D-PHI material and significant high level of FGF-2 at day 1 of dynamic culture appear to play a role in regulating α-SMA production while allowing HGFs to increase Col I production. ß1-integrin production was increased and focal adhesion kinase (FAK) were activated 2 h after HGFs were exposed to medium perfusion, which may have in part promoted cell growth, α-SMA and Col I production in the early dynamic culture. Consequently, the D-PHI material and medium perfusion has modulated fibroblast phenotype, and enhanced cell growth and Col I production through the coordinated actions of TGF-ß1, FGF-2, ß1-integrin and FAK.

Biomaterials in co-culture systems: towards optimizing tissue integration and cell signaling within scaffolds.

Most natural tissues consist of multi-cellular systems made up of two or more cell types. However, some of these tissues may not regenerate themselves following tissue injury or disease without some form of intervention, such as from the use of tissue engineered constructs. Recent studies have increasingly used co-cultures in tissue engineering applications as these systems better model the natural tissues, both physically and biologically. This review aims to identify the challenges of using co-culture systems and to highlight different approaches with respect to the use of biomaterials in the use of such systems. The application of co-culture systems to stimulate a desired biological response and examples of studies within particular tissue engineering disciplines are summarized. A description of different analytical co-culture systems is also discussed and the role of biomaterials in the future of co-culture research are elaborated on. Understanding the complex cell-cell and cell-biomaterial interactions involved in co-culture systems will ultimately lead the field towards biomaterial concepts and designs with specific biochemical, electrical, and mechanical characteristics that are tailored towards the needs of distinct co-culture systems.

Perfused culture of gingival fibroblasts in a degradable/polar/hydrophobic/ionic polyurethane (D-PHI) scaffold leads to enhanced proliferation and metabolic activity.

Periodontal diseases cause the breakdown of the tooth-supporting gingival tissue. In treatments aimed at gingival tissue regeneration, tissue engineering is preferred over the common treatments such as scaling. Perfused (dynamic) culture has been shown to increase cell growth in tissue-engineered scaffolds. Since gingival tissues are highly vascularized, it was desired to investigate the influence of perfusion on the function of human gingival fibroblasts (HGF) when cultured in a degradable/polar/hydrophobic/ionic polyurethane scaffold during the early culture phase (4weeks) of engineering gingival tissues. It was observed that the growth of HGF was continuous over 28days in dynamic culture (3-fold increase, p<0.05), while it was reduced after 14days in static culture (i.e. no flow condition). Cell metabolic activity, as measured by a WST-1 assay, and total protein production show that HGF were in different metabolic states in the dynamic vs. static cultures. Observations from scanning electron microscopy and type I collagen (Col I) production measured by Western blotting suggest that medium perfusion significantly promoted collagen production in HGF after the first 4weeks of culture (p<0.05). The different proliferative and metabolic states for HGF in the perfused scaffolds suggest a different cell phenotype which may favour tissue regeneration.

Immuno-atomic force microscopy characterization of adsorbed fibronectin.

The fibronectin (Fn) binding conformation on mica and ultraflat poly(D,L-lactide-co-glycolide) (UPLGA) was characterized using atomic force microscopy (AFM). AFM topographic images showed that Fn was in an extended form on mica and in a compact structure on UPLGA. With immuno-AFM, an antibody (Ab(hep)) was used to characterize the Fn binding conformation. When Fn opens its binding site for an antibody upon adsorption to a surface, the resulting Fn-antibody complex creates an additional peak in the sample's height distribution. Immuno-AFM uses this change to detect antigen-antibody binding. In this letter, height histograms (distributions) were generated using the mean true height of molecules, which was measured by examining the histogram for each individual molecule and subtracting the mica background. Mean true height values were obtained from the histograms and showed that Fn and Ab(hep) formed complexes on mica, signifying that one of the heparin binding sites on Fn was open when Fn was adsorbed to mica. The mean true height of the Fn-antibody complex from the histogram is greater than expected, suggesting that the antibody had pulled the extended "arms" of Fn together and caused an Fn conformation change upon binding. The height histograms can illustrate the Fn binding conformation and other antigen-antibody binding.