Joanne E. McBane

Biodegradation and in vivo biocompatibility of a degradable, polar/hydrophobic/ionic polyurethane for tissue engineering applications

A degradable, polar/hydrophobic/ionic polyurethane (D-PHI) scaffold was optimized in in vitro studies to yield mechanical properties appropriate to replicate vascular graft tissue while eliciting a more wound-healing phenotype macrophage when compared to established materials. The objectives of this study were to characterize the biodegradation (in vitro and in vivo) and assess the in vivo biocompatibility of D-PHI, comparing it to a well-established, commercially-available scaffold biomaterial, polylactic glycolic acid (PLGA), recognized as being degradable, non-cytotoxic, and showing good biocompatibility. PLGA and D-PHI were formed into 6 mm diameter disk-shaped scaffolds (2 mm thick) of similar porosity (∼82%) and implanted subcutaneously in rats. Both PLGA and D-PHI scaffolds were well-tolerated at the 7 d time point in vivo. In vitro D-PHI scaffolds degraded slowly (only 12 wt% in PBS in vitro after 120 d at 37 °C). In vivo, D-PHI scaffolds degraded at a more controlled rate (7 wt% loss over the acute 7 d implant phase and subsequently a linear profile of degradation leading to a 21 wt% mass loss by 100 d (chronic period)) than PLGA scaffolds which showed an initial more rapid degradation (14 wt% over 7 d), followed by minimal change between 7 and 30 d, and then a very rapid breakdown of the scaffold over the next 60 d. Histological examination of D-PHI scaffolds showed tissue ingrowth into the pores increased with time whereas PLGA scaffolds excluded cells/tissue from its porous structure as it degraded. The results of this study suggest that D-PHI has promising qualities for use as an elastomeric scaffold material for soft TE applications yielding well integrated tissue within the scaffold and a controlled rate of degradation stabilizing the form and shape of the implant.

Effect of polyurethane chemistry and protein coating on monocyte differentiation towards a wound healing phenotype macrophage.

Tissue regeneration alternatives for peripheral vascular disease are actively being investigated; however, few studies in this area have probed the role of the wound healing monocyte-derived macrophage (MDM). Inflammatory MDMs transition to wound healing MDMs as the relative levels of tumor necrosis factor-alpha (TNF-alpha) decrease and IL-10 increase. TNF-alpha has been linked to the regulation of HMGB1 (high mobility group box 1 protein), a nuclear protein that upon macrophage stimulation can be secreted and act as a pro-inflammatory cytokine. This study investigated the influence of a degradable polar hydrophobic ionic polyurethane (D-PHI) on MDM cell expression of pro- versus anti-inflammatory markers, when the material was uncoated or pre-coated with collagen prior to cell studies. Effects were compared to similar groups on tissue culture polystyrene (TCPS). Collagen coated TCPS and D-PHI had significantly more DNA than the uncoated TCPS after 7d (p=0.001 and p=0.006 respectively); however, there was significantly less esterase activity from cells on D-PHI (+/-collagen) than for cells on TCPS after 7d (p=0.002, p=0.0003 respectively). No significant differences in esterase activity were observed between collagen coated and non-coated D-PHI surfaces. Analyses of pro-inflammatory cytokines (TNF-alpha, IL-1beta and HMGB1) secreted from differentiating monocytes adherent to D-PHI demonstrated a decrease whereas anti-inflammatory IL-10 increased over time when compared to TCPS, suggesting that D-PHI was less inflammatory than TCPS. Since D-PHI maintains cell attachment while aiding in the transition of MDM to a wound healing phenotype, this material has qualities suitable to be used in tissue engineering applications where MDM play a key role in tissue regeneration.

Glyoxalase-1 overexpression in bone marrow cells reverses defective neovascularization in STZ-induced diabetic mice

AIMS Methylglyoxal (MG) accumulates in diabetes and impairs neovascularization. This study assessed whether overexpressing the MG-metabolizing enzyme glyoxalase-1 (GLO1) in only bone marrow cells (BMCs) could restore neovascularization in ischaemic tissue of streptozotocin-induced diabetic mice. METHODS AND RESULTS After 24 h of hyperglycaemic and hypoxic culture, BMCs from GLO1 overexpressing and wild-type (WT) diabetic mice were compared for migratory potential, viability, and mRNA expression of anti-apoptotic genes (Bcl-2 and Bcl-XL). In vivo, BMCs from enhanced green fluorescent protein (eGFP) mice that overexpress GLO1 were used to reconstitute the BM of diabetic mice (GLO1-diabetics). Diabetic and non-diabetic recipients of WT GFP(+) BM served as controls (WT-diabetics and non-diabetics, respectively). Following hindlimb ischaemia, the mobilization of BMCs was measured by flow cytometry. In hindlimbs, the presence of BM-derived angiogenic (GFP(+)CXCR4(+)) and endothelial (GFP(+)vWF(+)) cells and also arteriole density were determined by immunohistochemistry. Hindlimb perfusion was measured using laser Doppler. GLO1-BMCs had superior migratory potential, increased viability, and greater Bcl-2 and Bcl-XL expression, compared with WT BMCs. In vivo, the mobilization of pro-angiogenic BMCs (CXCR4(+), c-kit(+), and Flk(+)) was enhanced post-ischaemia in GLO1-diabetics compared to WT-diabetics. A greater number of GFP(+)CXCR4(+) and GFP(+)vWF(+) BMCs incorporated into the hindlimb tissue of GLO1-diabetics and non-diabetics than in WT-diabetics. Arteriole and capillary density and perfusion were also greater in GLO1-diabetics and non-diabetics. CONCLUSION This study demonstrates that protection from MG uniquely in BM is sufficient to restore BMC function and neovascularization of ischaemic tissue in diabetes and identifies GLO1 as a potential therapeutic target.

Differentiation of monocytes on a degradable, polar, hydrophobic, ionic polyurethane: Two-dimensional films vs. three-dimensional scaffolds

A degradable, polar, hydrophobic, ionic polyurethane (D-PHI), with physical properties comparable to those of peripheral arterial vascular tissue, was evaluated for monocyte interactions with two different physical forms: two-dimensional films and three-dimensional porous scaffolds. Monocytes, isolated from human whole blood, were seeded onto D-PHI films and scaffolds, and differentiated to monocyte-derived macrophages (MDM) for up to 28 days. The effect of surface structure on the MDM phenotype was assessed by assaying: cell attachment (DNA), activation (intracellular protein expression, esterase and acid phosphatase (AP) activity) as well as pro- and anti-inflammatory cytokines (TNF-α and IL-10, respectively). The cells on scaffolds exhibited an initial peak in total protein synthesized per DNA at 3 days; however, both substrates generated similar protein levels per DNA at all other time points. While scaffolds generated more esterase and AP per cell than for films, the cells on films expressed significantly more of these two proteins relative to their total protein produced. At day 7 (acute phase of monocyte activation), cells on films were significantly more activated than monocytes on the scaffolds as assessed by cell morphology and tumor necrosis factor-α and interleukin-10 levels. Histological analysis of scaffolds showed that cells were able to migrate throughout the three-dimensional matrix. By inducing a low inflammatory, high wound-healing phenotype monocyte, the negative effects of the foreign body reaction in vivo may be controlled in a manner possible to direct the vascular tissue cells into the appropriate functional phenotypes necessary for successful tissue engineering.

Evaluation of a Collagen-Chitosan Hydrogel for Potential Use as a Pro-Angiogenic Site for Islet Transplantation

Islet transplantation to treat type 1 diabetes (T1D) has shown varied long-term success, due in part to insufficient blood supply to maintain the islets. In the current study, collagen and collagen:chitosan (10:1) hydrogels, +/- circulating angiogenic cells (CACs), were compared for their ability to produce a pro-angiogenic environment in a streptozotocin-induced mouse model of T1D. Initial characterization showed that collagen-chitosan gels were mechanically stronger than the collagen gels (0.7kPa vs. 0.4kPa elastic modulus, respectively), had more cross-links (9.2 vs. 7.4/µm2), and were degraded more slowly by collagenase. After gelation with CACs, live/dead staining showed greater CAC viability in the collagen-chitosan gels after 18h compared to collagen (79% vs. 69%). In vivo, collagen-chitosan gels, subcutaneously implanted for up to 6 weeks in a T1D mouse, showed increased levels of pro-angiogenic cytokines over time. By 6 weeks, anti-islet cytokine levels were decreased in all matrix formulations ± CACs. The 6-week implants demonstrated increased expression of VCAM-1 in collagen-chitosan implants. Despite this, infiltrating vWF+ and CXCR4+ angiogenic cell numbers were not different between the implant types, which may be due to a delayed and reduced cytokine response in a T1D versus non-diabetic setting. The mechanical, degradation and cytokine data all suggest that the collagen-chitosan gel may be a suitable candidate for use as a pro-angiogenic ectopic islet transplant site.

Tissue Engineering a Small Diameter Vessel Substitute: Engineering Constructs with Select Biomaterials and Cells

Cardiovascular disease (CVD) is a leading cause of death and hospitalization worldwide. The need for small caliber vessels ( < 6mm) to treat CVD patients has grown; however the availability of autologous vessels in cardiac and peripheral bypass candidates is limited. The search for an alternative vessel source is widespread with both natural and synthetic tissue engineered materials being investigated as scaffolds. Despite decades of exhaustive studies with decellularized extracellular matrices (ECM) and synthetic graft materials, the field remains in search of a commercially viable biomaterial construct substitute. While the previous materials have been assessed by evaluating their compatibility with fibroblasts, smooth muscle cells and endothelial cells, current materials are being conceived based on their interactions with stem cells, progenitor cells and monocytes, as the latter may hold the key to repair and regeneration. The grafts ability to recruit and maintain these cells has become a major research focus. The successful tissue engineering of a small caliber vessel graft requires the use of optimal material chemistry and biological function to promote cell recruitment into the graft while maintaining each functional phenotype during vessel tissue maturation. The discussion of these significant research challenges constitutes the focus of this review.

Co-culturing monocytes with smooth muscle cells improves cell distribution within a degradable polyurethane scaffold and reduces inflammatory cytokines.

Activated monocytes can promote inflammation or wound repair, depending on the nature of the implant environment. Recent work showed that a degradable, polar-hydrophobic-ionic polyurethane (D-PHI) induced an anti-inflammatory monocyte phenotype. In the current study it is hypothesized that wound-healing phenotype monocytes (activated by D-PHI material chemistry) will promote human vascular smooth muscle cells (hVSMC) to attach and migrate into porous D-PHI scaffolds. hVSMC migration is necessary for hVSMC population of the scaffold and tissue formation to occur, and then, once tissue formation is complete, the monocyte should promote contractile phenotype markers in the hVSMC. hVSMC were cultured for up to 28 days with or without monocytes and analyzed for cell viability, attachment (DNA) and migration. Lysates were analyzed for the hVSMC contractile phenotype markers calponin and α-smooth muscle actin (α-SMA) as well as urokinase plasminogen activator (uPA; pro-migration marker) using immunoblotting analysis. Histological staining showed that hVSMC alone remained around the perimeter of the scaffold, whereas co-culture samples had co-localization of monocytes with hVSMC in the pores, a more even cell distribution throughout the scaffold and increased total cell attachment (P<0.05). Co-culture samples had higher cell numbers and more DNA than the addition of both single cell cultures. The water-soluble tetrazolium-1 data suggested that cells were not dying over the 28 day culture period. Calponin, also linked to cell motility, was maintained up to 28 days in the co-culture and hVSMC alone, whereas α-SMA disappeared after 7 days. Co-cultures on D-PHI showed that monocytes were activated to a wound-healing phenotype (low TNF-α, elevated IL-10), while promoting uPA expression. In summary, this study showed that, by co-culturing monocytes with hVSMC, the latter showed increased total cell attachment and infiltration into the D-PHI scaffold compared with hVSMC alone, suggesting that monocytes may promote hVSMC migration, a condition necessary for ultimately achieving uniform tissue formation in porous scaffolds.

Is cell culture stressful? Effects of degradable and nondegradable culture surfaces on U937 cell function.

In vitro cell culture has become one of the most widely used techniques in biological and health sciences research, with the most common culture supports being either tissue culture grade polystyrene (TCPS) or polydimethylsiloxane (PDMS). It has previously been shown that monocyte-derived macrophages (MDMs) respond to material surface chemistry, synthesizing and releasing degradative activities that could produce products, which alter the cells response. In this study, functional parameters of differentiated U937 macrophage-like cells were compared when cultured on nondegradable standard control surfaces versus models of biomaterials (polycarbonate-based polyurethanes) used in the manufacture of medical devices previously shown to degrade and/or elicit pathways of inflammation. Although the influence of PDMS and TCPS on cell function is often underappreciated by investigators, both surfaces elicited enzyme markers of inflammation. Cells on TCPS had the highest intracellular and released esterase activities and protein levels. Cells on PDMS had the most released acid phosphatase activity and protein (P < 0.001), as well as de novo 57- and 59-kDa released proteins. The criteria for defining an activated cell phenotype become critically important when materials such as PDMS and TCPS are used as standard control surfaces whether in experiments for research in elucidating metabolic pathways or in screening drugs and materials for therapeutic uses.

Differences in protein binding and cytokine release from monocytes on commercially sourced tissue culture polystyrene.

Tissue culture polystyrene (TCPS) is a ubiquitous substrate used by many researchers in the biomedical and biological sciences. Different parameters involved in the production of TCPS, including the treatment time and the use of reactive gases and chemical agents, can have a significant influence on the ultimate surface properties achieved. The assumption that they will all yield a consistent and controlled product has not proven to be true. To provide a better insight into the bioactivity differences in TCPS supplied by different manufacturers, TCPS from three different companies (Sarstedt, Wisent Corp., and Becton Dickinson (BD)) were analyzed for their surface properties, protein adsorption characteristics, and interactions with human monocytes. Marked differences were observed in terms of surface wettability and surface chemistry. Furthermore, Wisent TCPS adsorbed more than twice the amount of serum proteins compared with BD and Sarstedt TCPS. Sarstedt showed significantly more cell retention (more DNA) compared with both BD and Wisent TCPS brands over a 7 day culture period. Cytokine release from monocytes adherent on the three different TCPS also differed significantly, suggesting that the differences in the surface properties were sufficient to differentially mediate monocyte activation. These results have important implications for TCPS research use, in terms of appreciating the interpretation of the data when TCPS is used as a control substrate as well as when it is used where a pre-conditioned state would influence the outcome of the study.

Effect of Phorbol Esters on the Macrophage-Mediated Biodegradation of Polyurethanes via Protein Kinase C Activation and Other Pathways

It was previously found that re-seeding monocyte-derived macrophages (MDM) on polycarbonate-based polyurethanes (PCNUs) in the presence of the protein kinase C (PKC) activator phorbol myristate acetate (PMA) inhibited MDM-mediated degradation of PCNUs synthesized with 1,6-hexane diisocyanate (HDI), as well as esterase activity and monocyte-specific esterase (MSE) protein. However, no effect on the degradation of a 4,4′-methylene bisphenyl (MDI)-derived PCNU (MDI321) occurred. This finding suggested that oxidation, a process linked to the PKC pathway, was not activated in the same manner for all PCNUs. In the current study MDM were re-seeded onto the above PCNU surfaces with PMA, PKC-inactive 4αPMA and the PKC inhibitor bisindolylmaleimide I hydrochloride (BIM) for 48 h before assaying for PCNU degradation, esterase activity, MSE protein, DNA, cell viability and cell morphology. 4αPMA did not alter MDM-mediated HDI PCNU degradation but MDI321 degradation increased in this condition. BIM alone had no effect on any parameter; however, when BIM and PMA were added together, the PMA inhibition of biodegradation, esterase activity and MSE protein was partially reversed for MDM on HDI PCNUs only. Adding PMA to MDM on HDI PCNUs increased intercellular connections, whereas 4αPMA or BIM+PMA increased cell size. Although this study demonstrated a role for oxidation via a PKC-activated pathway in MDM-mediated PCNU degradation, phorbol esters appear to also activate non-PKC pathways that have roles in biodegradation. Moreover, the sensitivity to material surface chemistry in the MDM response to each PCNU dictates a multi-factorial degradative process involving alternate material specific oxidative and hydrolytic mechanisms.