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 graft's 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.

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.

Functional characterization of human coronary artery smooth muscle cells under cyclic mechanical strain in a degradable polyurethane scaffold.

There are few synthetic elastomeric biomaterials that simultaneously provide the required biological conditioning and the ability to translate biomechanical stimuli to vascular smooth muscle cells (VSMCs). Biomechanical stresses are important physiological elements that regulate VSMC function, and polyurethane elastomers are a class of materials capable of facilitating the translation of stress induced biomechanics. In this study, human coronary artery smooth muscle cells (hCASMCs), which were seeded into a porous degradable polar/hydrophobic/ionic (D-PHI) polyurethane scaffold, were subjected to uniaxial cyclic mechanical strain (CMS) over a span of four weeks using a customized bioreactor. The distribution, proliferation and contractile protein expression of hCASMCs in the scaffold were then analyzed and compared to those grown under static conditions. Four weeks of CMS, applied to the elastomeric scaffold, resulted in statistically greater DNA mass, more cell area coverage and a better distribution of cells deeper within the scaffold construct. Furthermore, CMS samples demonstrated improved tensile mechanical properties following four weeks of culture, suggesting the generation of more extracellular matrix within the polyurethane constructs. The expression of smooth muscle α-actin, calponin and smooth muscle myosin heavy chain and the absence of Ki-67+ cells in both static and CMS cultures, throughout the 4 weeks, suggest that hCASMCs retained their contractile character on these biomaterials. The study highlights the importance of implementing physiologically-relevant biomechanical stimuli in the development of synthetic elastomeric tissue engineering scaffolds.

The effect of degradable polymer surfaces on co-cultures of monocytes and smooth muscle cells.

Strategies to optimize biomaterial chemistry for applications in vascular tissue engineering attempt to promote endothelial and smooth muscle cell recruitment into porous material constructs. The primary objective is to facilitate relevant tissue formation in a wound healing versus pro-inflammatory manner. The present work investigated the interactive co-cellular response of human monocytes and human vascular smooth muscle cells (VSMCs) with a novel degradable, polar/hydrophobic/ionic (D-PHI) polyurethane and compared it to a commercially available biomaterial, poly-lactic-glycolic acid (PLGA) as well as tissue culture polystyrene (TCPS). D-PHI triggered a smaller pro-inflammatory response (acid phosphatase, esterase, tumor necrosis factor-α) at later time points (>14 d) than PLGA suggesting that monocytes may be transitioning to a more wound-healing phenotype on the D-PHI surface. When D-PHI was coated with collagen, monocyte cell attachment did not differ with the native D-PHI; however, PLGA showed significant differences between collagen coated versus uncoated surfaces. There were more VSMCs and monocytes attached in co-culture to D-PHI when compared to PLGA. Co-cultures on D-PHI released more IL-10 (anti-inflammatory) than monocytes cultured alone, while the VSMCs retained the expression of its marker protein calponin. Together the above data suggest that co-culturing monocytes with VSMCs may aid in stimulating the attachment of VSMCs to D-PHI while eliciting the desired functional phenotypes for both monocytes (i.e. low inflammation based on IL-10 values) and VSMCs (expressing calponin, a marker of contractility). Moreover, the results of this study demonstrated that D-PHI performed equally or better to PLGA in terms of the assayed biological parameters.

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.

A study of vascular smooth muscle cell function under cyclic mechanical loading in a polyurethane scaffold with optimized porosity.

High porosity and pore interconnectivity are important features of a successful tissue engineering scaffold. The objective of this work was to optimize the pore interconnectivity and to increase the porosity of an elastomeric degradable/polar/hydrophobic/ionic (D-PHI) polyurethane porous scaffold while maintaining its mechanical integrity in order to allow for the transfer of mechanical stimulus to vascular smooth muscle cells (VSMCs) seeded onto the scaffold. The effect of varying porogen (sodium bicarbonate (salt) and polyethylene glycol (PEG)) composition and concentration on the mechanical properties, degree of swelling and porosity of the scaffolds was investigated. It was found that the use of 10 wt.% PEG and 65 wt.% salt in scaffold fabrication (D-PHI-75T) resulted in micropore (1-5 µm) formation, a high porosity (79 ± 3%) and mechanical properties (elastic modulus=0.16 ± 0.03 MPa, elongation-at-yield = 31 ± 5% and tensile strength=0.04 ± 0.01 MPa) required to withstand the physiologically relevant mechanical strain experienced by VMSCs in vivo. This study also investigated the influence of cyclic mechanical strain (CMS) on select molecular markers of A10 VSMCs when seeded into the optimized D-PHI scaffold. To study the interaction of A10 cells with the optimized D-PHI-75T scaffold in the presence of uniaxial strain (10%, 1 Hz), a CMS bioreactor was designed and constructed. Molecular marker studies showed a statistical increase in DNA mass and calponin expression after 3 and 7 days of CMS when compared to static samples, indicating that the translation of mechanical loading from the novel polyurethane elastomeric scaffold onto VSMCs will be important to consider with regard to modulating cell phenotype.

Synthesis and characterization of degradable polar hydrophobic ionic polyurethane scaffolds for vascular tissue engineering applications.

In tissue engineering, the ability to manipulate scaffold design characteristics is important to achieve functional tissue regeneration. In this study, degradable polar hydrophobic ionic polyurethane (D-PHI) porous scaffolds were synthesized using a lysine-based divinyl oligomer (DVO). Optimization studies on the DVO and D-PHI scaffold synthesis were conducted to maximize isocyanate and methacrylate monomer conversion, respectively. D-PHI scaffold properties were manipulated through the introduction of a lysine-based cross-linker. Specifically, increasing D-PHI cross-linker concentration resulted in an increase of the elastic modulus (0.5-21 MPa), a decrease of the elongation-at-yield (45-5%) and a reduction of scaffold swelling (170-100%). Based on a preliminary study with A10 vascular smooth muscle cells, D-PHI scaffolds demonstrated the ability to support cell adhesion and growth during 2 weeks of culture, suggesting their potential suitability for longer term vascular tissue engineering. The versatility of the D-PHI properties may allow for the tailoring of cell-material interaction and ultimately functional tissue regeneration.

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.

In vitro release of a water-soluble agent from low viscosity biodegradable, injectable oligomers.

Low-molecular-weight poly(epsilon-caprolactone-co-1,3-trimethylene carbonate) and poly(1,3-trimethylene carbonate) are potential vehicles for the regio-specific delivery of water-soluble agents. In this paper, the characteristics and the mechanism governing the in vitro release of a model water-soluble drug, vitamin B12, from these polymer vehicles were determined. The loading of vitamin B12 was kept to 1 w/w%. The oligomers examined ranged from amorphous, high viscosity to crystalline but low viscosity. The oligomers did not degrade appreciably in vitro. The total fraction of vitamin B12 released increased as the crystallinity of the oligomers decreased, reaching nearly total release only for the completely amorphous oligomers. The rate of release was fastest for the amorphous oligomers and dependent on their viscosity. Inclusion of a more osmotically active agent, trehalose, into the vitamin B12 particles through co-lyophilization resulted in enhanced total fraction released and a faster release rate. The results are consistent with an osmotically driven release mechanism.

Initiator structure influence on thermal and rheological properties of oligo(epsilon-caprolactone).

Biodegradable thermoplastic oligomers have potential as biomaterials for tissue augmentation and drug-delivery applications. One means of obtaining such a biomaterial is through the ring-opening polymerization of epsilon-caprolactone using an alcohol initiator. In this paper we continue to examine the influence of the structure of the initiator used on the thermal and rheological properties of oligo(epsilon-caprolactone). Specifically, primary and secondary pentanol, and cis- and trans-pentenol were studied as initiators in the preparation of oligomers of constant molecular weight. In agreement with previous work, the secondary conformer yielded higher melt viscosities, lower degrees of crystallinity and lower glass transition temperatures. The cis conformer produced the lowest melt viscosity; however, the activation energy for flow was higher than obtained previously with oleyl alcohol. This result was attributed to the alkane chain lengths on either side of the cis double bond in the initiator. The order of melt viscosity increased with initiator conformer as follows: cis, trans, primary and secondary. The results were explained in terms of oligomer chain flexibility in the melt.