Use of a pedicled omental flap to reduce inflammation and vascularize an abdominal wall patch.

BACKGROUND: Although a variety of synthetic materials have been used to reconstruct tissue defects, these materials are associated with complications such as seromas, fistulas, chronic patient discomfort, and surgical site infection. While alternative, degradable materials that facilitate tissue growth have been examined. These materials can still trigger a foreign body inflammatory response that can lead to complications and discomfort. MATERIALS AND METHODS: In this report, our objective was to determine the effect of placing a pedicled omental flap under a biodegradable, microfibrous polyurethane scaffold serving as a full-wall thickness replacement of the rat abdominal wall. It was hypothesized that the presence of the omental tissue would stimulate greater vascularization of the scaffold and act to reduce markers of elevated inflammation in the patch vicinity. For control purposes, a polydimethylsiloxane sheet was placed as a barrier between the omental tissue and the overlying microfibrous scaffold. Both groups were sacrificed 8 wk after the implantation, and immunohistological and reverse transcription polymerase chain reaction (RT-PCR) assessments were performed. RESULTS: The data showed omental tissue placement to be associated with increased vascularization, a greater local M2/M1 macrophage phenotype response, and mRNA levels reduced for inflammatory markers but increased for angiogenic and antiinflammatory factors. CONCLUSIONS: From a clinical perspective, the familiarity with utilizing omental flaps for an improved healing response and infection resistance should naturally be considered as new tissue engineering approaches that are translated to tissue beds where omental flap application is practical. This report provides data in support of this concept in a small animal model.

Skeletal muscle derived stem cells microintegrated into a biodegradable elastomer for reconstruction of the abdominal wall.

A variety of techniques have been applied to generate tissue engineered constructs, where cells are combined with degradable scaffolds followed by a period of in vitro culture or direct implantation. In the current study, a cellularized scaffold was generated by concurrent deposition of electrospun biodegradable elastomer (poly(ester urethane)urea, PEUU) and electrosprayed culture medium + skeletal muscle-derived stem cells (MDSCs) or electrosprayed culture medium alone as a control. MDSCs were obtained from green fluorescent protein (GFP) transgenic rats. The created scaffolds were implanted into allogenic strain-matched rats to replace a full thickness abdominal wall defect. Both control and MDSC-integrated scaffolds showed extensive cellular infiltration at 4 and 8 wk. The number of blood vessels was higher, the area of residual scaffold was lower, number of multinucleated giant cells was lower and area of connective tissue was lower in MDSC-integrated scaffolds (p < 0.05). GFP + cells co-stained positive for VEGF. Bi-axial mechanical properties of the MDSC-microintegrated constructs better approximated the anisotropic behavior of the native abdominal wall. GFP + cells were observed throughout the scaffold at ∼5% of the cell population at 4 and 8 wk. RNA expression at 4 wk showed higher expression of early myogenic marker Pax7, and b-FGF in the MDSC group. Also, higher expression of myogenin and VEGF were seen in the MDSC group at both 4 and 8 wk time points. The paracrine effect of donor cells on host cells likely contributed to the differences found in vivo between the groups. This approach for the rapid creation of highly-cellularized constructs with soft tissue like mechanics offers an attractive methodology to impart cell-derived bioactivity into scaffolds providing mechanical support during the healing process and might find application in a variety of settings.

Large strain stimulation promotes extracellular matrix production and stiffness in an elastomeric scaffold model.

Mechanical conditioning of engineered tissue constructs is widely recognized as one of the most relevant methods to enhance tissue accretion and microstructure, leading to improved mechanical behaviors. The understanding of the underlying mechanisms remains rather limited, restricting the development of in silico models of these phenomena, and the translation of engineered tissues into clinical application. In the present study, we examined the role of large strip-biaxial strains (up to 50%) on ECM synthesis by vascular smooth muscle cells (VSMCs) micro-integrated into electrospun polyester urethane urea (PEUU) constructs over the course of 3 weeks. Experimental results indicated that VSMC biosynthetic behavior was quite sensitive to tissue strain maximum level, and that collagen was the primary ECM component synthesized. Moreover, we found that while a 30% peak strain level achieved maximum ECM synthesis rate, further increases in strain level lead to a reduction in ECM biosynthesis. Subsequent mechanical analysis of the formed collagen fiber network was performed by removing the scaffold mechanical responses using a strain-energy based approach, showing that the denovo collagen also demonstrated mechanical behaviors substantially better than previously obtained with small strain training and comparable to mature collagenous tissues. We conclude that the application of large deformations can play a critical role not only in the quantity of ECM synthesis (i.e. the rate of mass production), but also on the modulation of the stiffness of the newly formed ECM constituents. The improved understanding of the process of growth and development of ECM in these mechano-sensitive cell-scaffold systems will lead to more rational design and manufacturing of engineered tissues operating under highly demanding mechanical environments.

Nanometer-sized extracellular matrix coating on polymer-based scaffold for tissue engineering applications.

Surface modification can play a crucial role in enhancing cell adhesion to synthetic polymer-based scaffolds in tissue engineering applications. Here, we report a novel approach for layer-by-layer (LbL) fabrication of nanometer-size fibronectin and gelatin (FN-G) layers on electrospun fibrous poly(carbonate urethane)urea (PCUU) scaffolds. Alternate immersions into the solutions of fibronectin and gelatin provided thickness-controlled FN-G nano-layers (PCUU(FN-G) ) which maintained the scaffold's 3D structure and width of fibrous bundle of PCUU as evidenced by scanning electron miscroscopy. The PCUU(FN-G) scaffold improved cell adhesion and proliferation of bladder smooth muscles (BSMCs) when compared to uncoated PCUU. The high affinity of PCUU(FN-G) for cells was further demonstrated by migration of adherent BSMCs from culture plates to the scaffold. Moreover, the culture of UROtsa cells, human urothelium-derived cell line, on PCUU(FN-G) resulted in an 11-15 µm thick multilayered cell structure with cell-to-cell contacts although many UROtsa cells died without forming cell connections on PCUU. Together these results indicate that this approach will aid in advancing the technology for engineering bladder tissues in vitro. Because FN-G nano-layers formation is based on nonspecific physical adsorption of fibronectin onto polymer and its subsequent interactions with gelatin, this technique may be applicable to other polymer-based scaffold systems for various tissue engineering/regenerative medicine applications.

Abdominal wall reconstruction by a regionally distinct biocomposite of extracellular matrix digest and a biodegradable elastomer.

Current extracellular matrix (ECM) derived scaffolds offer promising regenerative responses in many settings, however in some applications there may be a desire for more robust and long lasting mechanical properties. A biohybrid composite material that offers both strength and bioactivity for optimal healing towards native tissue behavior may offer a solution to this problem. A regionally distinct biocomposite scaffold composed of a biodegradable elastomer (poly(ester urethane)urea) and porcine dermal ECM gel was generated to meet this need by a concurrent polymer electrospinning/ECM gel electrospraying technique where the electrosprayed component was varied temporally during the processing. A sandwich structure was achieved with polymer fiber rich upper and lower layers for structural support and an ECM-rich inner layer to encourage cell ingrowth. Increasing the upper and lower layer fiber content predictably increased tensile strength. In a rat full thickness abdominal wall defect model, the sandwich scaffold design maintained its thickness whereas control biohybrid scaffolds lacking the upper and lower fiber-rich regions failed at 8 weeks. Sandwich scaffold implants also showed higher collagen content 4 and 8 weeks after implantation, exhibited an increased M2 macrophage phenotype response at later times and developed biaxial mechanical properties better approximating native tissue. By employing a processing approach that creates a sheet-form scaffold with regionally distinct zones, it was possible to improve biological outcomes in body wall repair and provide the means for further tuning scaffold mechanical parameters when targeting other applications. Copyright © 2013 John Wiley & Sons, Ltd.

Fabrication of elastomeric scaffolds with curvilinear fibrous structures for heart valve leaflet engineering.

Native semi-lunar heart valves are composed of a dense fibrous network that generally follows a curvilinear path along the width of the leaflet. Recent models of engineered valve leaflets have predicted that such curvilinear fiber orientations would homogenize the strain field and reduce stress concentrations at the commissure. In the present work, a method was developed to reproduce this curvilinear fiber alignment in electrospun scaffolds by varying the geometry of the collecting mandrel. Elastomeric poly(ester urethane)urea was electrospun onto rotating conical mandrels of varying angles to produce fibrous scaffolds where the angle of fiber alignment varied linearly over scaffold length. By matching the radius of the conical mandrel to the radius of curvature for the native pulmonary valve, the electrospun constructs exhibited a curvilinear fiber structure similar to the native leaflet. Moreover, the constructs had local mechanical properties comparable to conventional scaffolds and native heart valves. In agreement with prior modeling results, it was found under quasi-static loading that curvilinear fiber microstructures reduced strain concentrations compared to scaffolds generated on a conventional cylindrical mandrels. Thus, this simple technique offers an attractive means for fabricating scaffolds where key microstructural features of the native leaflet are imitated for heart valve tissue engineering.

Polypropylene surgical mesh coated with extracellular matrix mitigates the host foreign body response.

Surgical mesh devices composed of synthetic materials are commonly used for ventral hernia repair. These materials provide robust mechanical strength and are quickly incorporated into host tissue; factors that contribute to reduced hernia recurrence rates. However, such mesh devices cause a foreign body response with the associated complications of fibrosis and patient discomfort. In contrast, surgical mesh devices composed of naturally occurring extracellular matrix (ECM) are associated with constructive tissue remodeling, but lack the mechanical strength of synthetic materials. A method for applying a porcine dermal ECM hydrogel coating to a polypropylene mesh is described herein with the associated effects upon the host tissue response and biaxial mechanical behavior. Uncoated and ECM coated heavy-weight BARD™ Mesh were compared to the light-weight ULTRAPRO™ and BARD™ Soft Mesh devices in a rat partial thickness abdominal defect overlay model. The ECM coated mesh attenuated the pro-inflammatory response compared to all other devices, with a reduced cell accumulation and fewer foreign body giant cells. The ECM coating degraded by 35 days, and was replaced with loose connective tissue compared to the dense collagenous tissue associated with the uncoated polypropylene mesh device. Biaxial mechanical characterization showed that all of the mesh devices were of similar isotropic stiffness. Upon explanation, the light-weight mesh devices were more compliant than the coated or uncoated heavy-weight devices. This study shows that an ECM coating alters the default host response to a polypropylene mesh, but not the mechanical properties in an acute in vivo abdominal repair model.

Non-invasive characterization of polyurethane-based tissue constructs in a rat abdominal repair model using high frequency ultrasound elasticity imaging.

The evaluation of candidate materials and designs for soft tissue scaffolds would benefit from the ability to monitor the mechanical remodeling of the implant site without the need for periodic animal sacrifice and explant analysis. Toward this end, the ability of non-invasive ultrasound elasticity imaging (UEI) to assess temporal mechanical property changes in three different types of porous, biodegradable polyurethane scaffolds was evaluated in a rat abdominal wall repair model. The polymers utilized were salt-leached scaffolds of poly(carbonate urethane) urea, poly(ester urethane) urea and poly(ether ester urethane) urea at 85% porosity. A total of 60 scaffolds (20 each type) were implanted in a full thickness muscle wall replacement in the abdomens of 30 rats. The constructs were ultrasonically scanned every 2 weeks and harvested at weeks 4, 8 and 12 for compression testing or histological analysis. UEI demonstrated different temporal stiffness trends among the different scaffold types, while the stiffness of the surrounding native tissue remained unchanged. The changes in average normalized strains developed in the constructs from UEI compared well with the changes of mean compliance from compression tests and histology. The average normalized strains and the compliance for the same sample exhibited a strong linear relationship. The ability of UEI to identify herniation and to characterize the distribution of local tissue in-growth with high resolution was also investigated. In summary, the reported data indicate that UEI may allow tissue engineers to sequentially evaluate the progress of tissue construct mechanical behavior in vivo and in some cases may reduce the need for interim time point animal sacrifice.

Microstructural manipulation of electrospun scaffolds for specific bending stiffness for heart valve tissue engineering.

Biodegradable thermoplastic elastomers are attractive for application in cardiovascular tissue construct development due to their amenability to a wide range of physical property tuning. For heart valve leaflets, while low flexural stiffness is a key design feature, control of this parameter has been largely neglected in the scaffold literature where electrospinning is being utilized. This study evaluated the effect of processing variables and secondary fiber populations on the microstructure, tensile and bending mechanics of electrospun biodegradable polyurethane scaffolds for heart valve tissue engineering. Scaffolds were fabricated from poly(ester urethane) urea (PEUU) and the deposition mandrel was translated at varying rates in order to modify fiber intersection density. Scaffolds were also fabricated in conjunction with secondary fiber populations designed either for mechanical reinforcement or to be selectively removed following fabrication. It was determined that increasing fiber intersection densities within PEUU scaffolds was associated with lower bending moduli. Further, constructs fabricated with stiff secondary fiber populations had higher bending moduli whereas constructs with secondary fiber populations which were selectively removed had noticeably lower bending moduli. Insights gained from this work will be directly applicable to the fabrication of soft tissue constructs, specifically in the development of cardiac valve tissue constructs.

An elastomeric patch electrospun from a blended solution of dermal extracellular matrix and biodegradable polyurethane for rat abdominal wall repair.

A biodegradable elastomeric scaffold was created by electrospinning a mixed solution of poly(ester urethane)urea (PEUU) and porcine dermal extracellular matrix (dECM) digest, with PEUU included to provide elasticity, flexibility, and mechanical support and dECM used to enhance bioactivity and biocompatibility. Micrographs and differential scanning calorimetry demonstrated partial miscibility between PEUU and dECM. With greater dECM content, scaffolds were found to possess lower breaking strains and suture retention strength, although initial modulus was greater with higher dECM concentrations. The hybrid scaffolds containing 0% to 50% dECM had tensile strengths of 5 to 7 MPa, breaking strains of 138% to 611%, initial moduli of 3 to 11 Mpa, and suture retention strengths of 35 to 59 MPa. When hydrated, scaffolds were found to contract markedly with 50% dECM content. When used in a rat full-thickness abdominal wall replacement model, no herniation, infection, or tissue adhesion was observed after 4 and 8 weeks with a scaffold containing 25% dECM or a control 100% PEUU scaffold. Scaffolds incorporating dECM were significantly thicker at the time of explant, with greater numbers of associated smooth muscle actin-positive staining cells than in the control, but minimal cellular infiltration and remodeling of the scaffold were detected regardless of dECM addition. The processing of dECM and PEUU from a mixed solution thus provided a scaffold with evidence of better bioactivity and with mechanical properties not achievable with digested dECM alone.

Mechanical properties and in vivo behavior of a biodegradable synthetic polymer microfiber-extracellular matrix hydrogel biohybrid scaffold.

A biohybrid composite consisting of extracellular matrix (ECM) gel from porcine dermal tissue and biodegradable elastomeric fibers was generated and evaluated for soft tissue applications. ECM gel possesses attractive biocompatibility and bioactivity with weak mechanical properties and rapid degradation, while electrospun biodegradable poly(ester urethane)urea (PEUU) has good mechanical properties but limited cellular infiltration and tissue integration. A concurrent gel electrospray/polymer electrospinning method was employed to create ECM gel/PEUU fiber composites with attractive mechanical properties, including high flexibility and strength. Electron microscopy revealed a structure of interconnected fibrous layers embedded in ECM gel. Tensile mechanical properties could be tuned by altering the PEUU/ECM weight ratio. Scaffold tensile strengths for PEUU/ECM ratios of 67/33, 72/28 and 80/20 ranged from 80 to 187 kPa in the longitudinal axis (parallel to the collecting mandrel axis) and 41-91 kPa in the circumferential axis with 645-938% breaking strains. The 72/28 biohybrid composite and a control scaffold generated from electrospun PEUU alone were implanted into Lewis rats, replacing a full-thickness abdominal wall defect. At 4 wk, no infection or herniation was found at the implant site. Histological staining showed extensive cellular infiltration into the biohybrid scaffold with the newly developed tissue well integrated with the native periphery, while minimal cellular ingress into the electrospun PEUU scaffold was observed. Mechanical testing of explanted constructs showed evidence of substantial remodeling, with composite scaffolds adopting properties more comparable to the native abdominal wall. The described elastic biohybrid material imparts features of ECM gel bioactivity with PEUU strength and handling to provide a promising composite biomaterial for soft tissue repair and replacement.

Morphological and mechanical characteristics of the reconstructed rat abdominal wall following use of a wet electrospun biodegradable polyurethane elastomer scaffold.

Although a variety of materials are currently used for abdominal wall repair, general complications encountered include herniation, infection, and mechanical mismatch with native tissue. An approach wherein a degradable synthetic material is ultimately replaced by tissue mechanically approximating the native state could obviate these complications. We report here on the generation of biodegradable scaffolds for abdominal wall replacement using a wet electrospinning technique in which fibers of a biodegradable elastomer, poly(ester urethane)urea (PEUU), were concurrently deposited with electrosprayed serum-based culture medium. Wet electrospun PEUU (wet ePEUU) was found to exhibit markedly different mechanical behavior and to possess an altered microstructure relative to dry processed ePEUU. In a rat model for abdominal wall replacement, wet ePEUU scaffolds (1x2.5 cm) provided a healing result that developed toward approximating physiologic mechanical behavior at 8 weeks. An extensive cellular infiltrate possessing contractile smooth muscle markers was observed together with extensive extracellular matrix (collagens, elastin) elaboration. Control implants of dry ePEUU and expanded polytetrafluoroethylene did not experience substantial cellular infiltration and did not take on the native mechanical anisotropy of the rat abdominal wall. These results illustrate the markedly different in vivo behavior observed with this newly reported wet electrospinning process, offering a potentially useful refinement of an increasingly common biomaterial processing technique.