Michael J. McClure, Ph.D.

Vanderbilt University Undergraduate Research Initiative Award

2004

Decellularized muscle supports new muscle fibers and improves function following volumetric injury

2018-02-12

Current strategies to treat volumetric muscle loss use primarily pedicle or free muscle transfers, but these grafts fail to adequately regenerate functional tissue. Decellularized soft tissue grafts possess physical and chemical cues to promote muscle regeneration, suggesting their potential for use in large muscle defects. In this study, we developed a decellularized muscle matrix (DMM) graft using rat gastrocnemius. Anisotropy and chemical components of the extracellular matrix were retained, including laminin, fibronectin, and collagen. We compared the ability of DMM, autologous muscle grafts (clinical standard), and type I collagen plugs (negative control) to support muscle regeneration. DMM supported regeneration over a 56-day period in 1x1cm and 1.5x1cm gastrocnemius defects in rats. Muscle function tests demonstrated improved muscle recovery in rats with DMM grafts when compared to collagen. Histological sections were assessed using morphometrics and immunostaining. DMM supported muscle regeneration with less fibrosis and more de novo neuromuscular receptors than either autograft or collagen. Overall, our results indicate that DMM may be used as a muscle replacement graft based on its ability to improve muscle function recovery, promote muscle regeneration, and support new neuromuscular junctions.

Regulation of extracellular matrix vesicles via rapid responses to steroid hormones during endochondral bone formation

2017-12-30

Endochondral bone formation is a precise and highly ordered process whose exact regulatory framework is still being elucidated. Multiple regulatory pathways are known to be involved. In some cases, regulation impacts gene expression, resulting in changes in chondrocyte phenotypic expression and extracellular matrix synthesis. Rapid regulatory mechanisms are also involved, resulting in release of enzymes, factors and micro RNAs stored in extracellular matrisomes called matrix vesicles. Vitamin D metabolites modulate endochondral development via both genomic and rapid membrane-associated signaling pathways. 1α,25-dihydroxyvitamin D3 [1α,25(OH)2D3] acts through the vitamin D receptor (VDR) and a membrane associated receptor, protein disulfide isomerase A3 (PDIA3). 24R,25-dihydroxyvitamin D3 [24R,25(OH)2D3] affects primarily chondrocytes in the resting zone (RC) of the growth plate, whereas 1α,25(OH)2D3 affects cells in the prehypertrophic and upper hypertrophic cell zones (GC). This includes genomically directing the cells to produce matrix vesicles with zone specific characteristics. In addition, vitamin D metabolites produced by the cells interact directly with the matrix vesicle membrane via rapid signal transduction pathways, modulating their activity in the matrix. The matrix vesicle payload is able to rapidly impact the extracellular matrix via matrix processing enzymes as well as providing a feedback mechanism to the cells themselves via the contained micro RNAs.

Galectin-1 promotes an M2 macrophage response to polydioxanone scaffolds

2017-06-15

Regulating soft tissue repair to prevent fibrosis and promote regeneration is central to creating a microenvironment conducive to soft tissue development. Macrophages play an important role in this process. The macrophage response can be modulated using biomaterials, altering cytokine and growth factor secretion to promote regeneration. Electrospun polydioxanone (PDO) fiber scaffolds promoted an M2 phenotype when macrophages were cultured on large diameter, highly porous scaffolds, but an M1 phenotype on smaller diameter fibers. In this study, we investigated whether incorporation of galectin-1, an immunosuppressive protein that enhances muscle regeneration, could promote the M2 response. Galectin-1 was incorporated into large and small fiber PDO scaffolds during electrospinning. Galectin-1 incorporation increased arginase-1 and reduced iNOS and IL-6 production in mouse bone-marrow derived macrophages compared with PDO alone for both scaffold types. Inhibition of ERK mitogen-activated protein kinase did not alter galectin-1 effects on arginase-1 and iNOS expression, but reversed IL-6 suppression, indicating that IL-6 is mediated by a different mechanism. Our results suggest that galectin-1 can be used to modulate macrophage commitment to a pro-regenerative M2 phenotype, which may positively impact tissue regeneration when using small diameter PDO scaffolds.

Role of Integrin α7β1 Signaling in Myoblast Differentiation on Aligned Polydioxanone Scaffolds

2016-07-15

The aligned structural environment in skeletal muscle is believed to be a crucial component in functional muscle regeneration. Myotube formation is increased on aligned biomaterials, but we do not fully understand the mechanisms that direct this enhanced fusion. Previous studies indicate that the α7 integrin subunit is upregulated during myoblast differentiation, suggesting that signaling via α7β1 mediates the effect of alignment. To test this hypothesis, we took advantage of an in vitro model using random and aligned polydioxanone (PDO) matrices and C2C12 myoblasts.

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The Influence of Platelet Rich Plasma on Myogenic Differentiation.

2016-04-01

The ability to expand and direct both precursor and stem cells towards a differential fate is considered extremely advantageous in tissue engineering. Platelet-rich plasma (PRP) possesses a milieu of growth factors and cytokines, which have the potential to have either a differentiative or proliferative influence on the cell type tested. Here, we investigated the effect of PRP on C2C12 myoblasts. A range of PRP concentrations in differentiation media was used to determine whether a concentration dependence existed, while PRP embedded in fibres of aligned electrospun polydioxanone and polycaprolactone was used to determine whether this presence of fibres would cause any differences in response.

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Electrospinning collagen/chitosan/poly( L -lactic acid- co -ϵ-caprolactone) to form a vascular graft: Mechanical and biological characterization

2013-05-01

For blood vessel tissue engineering, an ideal vascular graft should possess excellent biocompatibility and mechanical properties. For this study, a elastic material of poly (L-lactic acid-co-ϵ-caprolactone) (P(LLA-CL)), collagen and chitosan blended scaffold at different ratios were fabricated by electrospinning. Upon fabrication, the scaffolds were evaluated to determine the tensile strength, burst pressure, and dynamic compliance.

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Tri-layered vascular grafts composed of polycaprolactone, elastin, and silk: Optimization of graft properties.

2012-06-01

The purpose of this study was to create seamless, acellular, small diameter bioresorbable arterial grafts that attempt to mimic the extracellular matrix and mechanical properties of native artery using synthetic and natural polymers. Silk fibroin, collagen, elastin, and polycaprolactone (PCL) were electrospun to create a tri-layered structure for evaluation.

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The use of air-flow impedance to control fiber deposition during electrospinning.

2011-11-01

Electrospun non-woven structures have the potential to form bioresorbable vascular grafts that promote tissue regeneration in situ as they degrade and are replaced by autologous tissue. Current bioresorbable grafts lack appropriate regeneration potential since they do not have optimal architecture, and their fabrication must be altered by the manipulation of process parameters, especially enhancing porosity. We describe here an air-impedance process where the solid mandrel is replaced with a porous mandrel that has pressurized air exiting the pores to impede fiber deposition.

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Bioengineered Vascular Grafts: Improving Vascular Tissue Engineering Through Scaffold Design.

2011-12-01

Arteriosclerosis has accounted for three quarters of the deaths related to cardiovascular disease (CVD). Arteriosclerosis is a vascular disease that is characterized by a thickening of the arterial wall and subsequent decrease in the arterial lumen, eventually causing loss of circulation distal to the site of disease.

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Preliminary Investigation of Airgap Electrospun Silk-Fibroin-Based Structures for Ligament Analogue Engineering.

2010-07-01

The process of electrospinning has proven to be highly beneficial for use in a number of tissue-engineering applications due to its ease of use, flexibility and tailorable properties. There have been many publications on the creation of aligned fibrous structures created through various forms of electrospinning, most involving the use of a metal target rotating at high speeds. This work focuses on the use of a variation known as airgap electrospinning, which does not use a metal collecting target but rather a pair of grounded electrodes equidistant from the charged polymer solution to create highly aligned 3D structures.

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Tri-layered electrospinning to mimic arterial architecture using polycaprolactone, elastin, and collagen: A preliminary study.

2011-01-04

Throughout native artery, collagen and elastin play an important role, providing a mechanical backbone, preventing vessel rupture, and promoting recovery under pulsatile deformations. The goal of this study was to mimic the structure of native artery by fabricating a multi-layered electrospun conduit composed of poly(caprolactone) (PCL) with the addition of elastin and collagen with blends of 45-45-10, 55-35-10, and 65-25-10 PCL-ELAS-COL to demonstrate mechanical properties indicative of native arterial tissue, while remaining conducive to tissue regeneration.

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A three-layered electrospun matrix to mimic native arterial architecture using polycaprolactone, elastin, and collagen: A preliminary study.

2010-07-01

Throughout native artery, collagen, and elastin play an important role, providing a mechanical backbone, preventing vessel rupture, and promoting recovery under pulsatile deformations. The goal of this study was to mimic the structure of native artery by fabricating a multi-layered electrospun conduit composed of poly(caprolactone) (PCL) with the addition of elastin and collagen with blends of 45–45–10, 55–35–10, and 65–25–10 PCL–ELAS–COL to demonstrate mechanical properties indicative of native arterial tissue, while remaining conducive to tissue regeneration.

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Electrospinning-Aligned and Random Polydioxanone-Polycaprolactone-Silk Fibroin Scaffolds: Geometry for a Vascular Matrix.

2009-10-01

Extracellular matrices are arranged with a specific geometry based on tissue type and mechanical stimulus. For blood vessels in the body, preferential alignment of fibers is in the direction of repetitive force. Electrospinning is a controllable process which can result in fiber alignment and randomization depending on the parameters utilized. In this study, arterial grafts composed of polycaprolactone (PCL), polydioxanone (PDO) and silk fibroin in blends of 100:0 and 50:50 for both PCL:silk and PDO:silk were investigated to determine if fibers could be controllably aligned using a mandrel rotational speed ranging from 500 to 8000 revolutions per minute (RPM).

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Electrospun polydioxanone-elastin blends: potential for bioresorbable vascular grafts.

2006-06-01

An electrospun cardiovascular graft composed of polydioxanone (PDO) and elastin has been designed and fabricated with mechanical properties to more closely match those of native arterial tissue, while remaining conducive to tissue regeneration. PDO was chosen to provide mechanical integrity to the prosthetic, while elastin provides elasticity and bioactivity (to promote regeneration in vitro/in situ). It is the elastic nature of elastin that dominates the low-strain mechanical response of the vessel to blood flow and prevents pulsatile energy from being dissipated as heat. Uniaxial tensile and suture retention tests were performed on the electrospun grafts to demonstrate the similarities of the mechanical properties between the grafts and native vessel. Dynamic compliance measurements produced values that ranged from 1.2 to 5.6%/100 mmHg for a set of three different mean arterial pressures. Results showed the 50:50 ratio to closely mimic the compliance of native femoral artery, while grafts that contained less elastin exceeded the suture retention strength of native vessel. Preliminary cell culture studies showed the elastin-containing grafts to be bioactive as cells migrated through their full thickness within 7 days, but failed to migrate into pure PDO scaffolds. Electrospinning of the PDO and elastin-blended composite into a conduit for use as a small diameter vascular graft has extreme potential and warrants further investigation as it thus far compares favorably to native vessel.

Electrospinning of Collagen / Biopolymers for Regenerative Medicine and Cardiovascular Tissue Engineering

2009-10-05

The process of electrospinning has seen a resurgence of interest in the last few decades which has led to a rapid increase in the amount of research devoted to its use in tissue engineering applications. Of this research, the area of cardiovascular tissue engineering makes up a large percentage, with substantial resources going towards the creation of bioresorbable vascular grafts composed of electrospun nanofibers of collagen and other biopolymers. These bioresorbable grafts have compositions that allow for the in situ remodeling of the structure, with the eventual replacement of the graft with completely autologous tissue. This review will highlight some of the work done in the field of electrospinning for cardiovascular applications, with an emphasis on the use of biopolymers such as collagens, elastin, gelatin, fibrinogen, and silk fibroin, as well as biopolymers used in combination with resorbable synthetic polymers.

Cross-linking methods of electrospun fibrinogen scaffolds for tissue engineering applications.

2008-12-01

The purpose of this study was to enhance the mechanical properties and slow the degradation of an electrospun fibrinogen scaffold, while maintaining the scaffold's high level of bioactivity. Three different cross-linkers were used to achieve this goal: glutaraldehyde vapour, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) in ethanol and genipin in ethanol. Scaffolds with a fibrinogen concentration of 120 mg ml(-1) were electrospun and cross-linked with one of the aforementioned cross-linkers. Mechanical properties were determined through uniaxial tensile testing performed on scaffolds incubated under standard culture conditions for 1 day, 7 days and 14 days. Cross-linked scaffolds were seeded with human foreskin fibroblasts (BJ-GFP-hTERT) and cultured for 7, 14 and 21 days, with histology and scanning electron microscopy performed upon completion of the time course. Mechanical testing revealed significantly increased peak stress and modulus values for the EDC and genipin cross-linked scaffolds, with significantly slowed degradation. However, cross-linking with EDC and genipin was shown to have some negative effect on the bioactivity of the scaffolds as cell migration throughout the thickness of the scaffold was slowed.

Suture-reinforced electrospun polydioxanone-elastin small-diameter tubes for use in vascular tissue engineering: a feasibility study

2008-01-01

This study characterizes the cross-linking of electrospun elastin and the mechanical properties of suture-reinforced 1.5mm internal diameter electrospun tubes composed of blended polydioxanone (PDO) and soluble elastin. Several tube configurations were tested to assess the effects of reinforcement on tube mechanical properties. Between the electrospun layers of each double-layered prosthetic, zero, one or two 6-0 sutures were wound, maintaining 1mm spacing with a pitch of 9 degrees . Single-layered tubes without suture were also examined. Samples were cross-linked and tested for compliance and burst strength. Compliance decreased significantly (p

Polydioxanone, Elastin, and Collagen Vascular Scaffolds: Uniaxial Cyclic Distension

2009-06-01

The development of vascular grafts requires the matching of material and viscoelastic properties to those of native artery. The hypothesis of this study was to subject electrospun tissue engineering scaffolds composed of polydioxanone, elastin, and collagen to cyclic loading in order to quantify the hysteretic properties, uniaxial tensile mechanical properties of conditioned scaffolds, and stress relaxation properties over a period of 400 cycles when compared to ePTFE, one of the most popular vascular prosthetic materials, and decellularized pig artery. In the electrospun graft, polydioxanone would provide a mechanical backbone, providing tensile support and preventing vessel rupture; while the elastin would provide elasticity and collagen would provide bioactivity (promote regeneration in vitro/in situ).

Cross-linking Electrospun Polydioxanone-Soluble Elastin Blends: Material Characterization

2008-03-01

The purpose of this study was to establish whether material properties of elastin co-electrospun with polydioxanone (PDO) would change over time in both the uncross-linked state and the cross-linked state. First, uncross-linked scaffolds were placed in phosphate buffered saline (PBS) for three separate time periods: 15 minutes, 1 hour, and 24 hours, and subsequently tested using uniaxial materials testing. Several cross-linking reagents were then investigated to verify their ability to crosslink elastin: 1-ethyl-3-(dimethylaminopropyl)-carbodiimide (EDC), ethylene glycol diglycidyl ether (EGDE), and genipin. Uniaxial tensile testing was performed on scaffolds cross-linked with EDC and genipin, yielding results that warranted further investigation for PDO-elastin blends. Material properties of the cross-linked scaffolds were then found within range of both pig femoral artery and human femoral artery. These results demonstrate PDO-elastin blends could potentially be favorable as vascular grafts, thus warranting future in vitro and in vivo studies.

Thermal and mechanical properties of electrospun PMMA, PVC, Nylon 6, and Nylon 6,6

2007-08-07

Poly(methyl methacrylate) (PMMA), poly(vinyl chloride) (PVC), Nylon 6, and Nylon 6,6 have been electrospun successfully. The nanofibers have been characterized by scanning electron microscopy (SEM), confirming the presence of bead free and fiber-bead free morphologies. Thermogravimetric analysis (TGA) indicated differences between the thermal stability of PMMA nanofibers and PMMA powder. However, no significant differences were observed between the starting physical form (powder or pellet) of PVC, Nylon 6 and Nylon 6,6, and their corresponding electrospun nanofibers. Differential scanning calorimetry (DSC) demonstrated a lower glass transition temperature (Tg) and water absorption for PMMA electrospun nanofibers. Furthermore, electrospun Nylon 6 and Nylon 6,6 had a slight decrease in crystallinity. Tensile testing was performed on the electrospun nanofibers to obtain the Young modulus, peak stress, strain at break, and energy to break, revealing that the non-woven mats obtained had modest mechanical properties that need to be enhanced.

Extracellular matrix regenerated: tissue engineering via electrospun biomimetic nanofibers.

2007-09-10

While electrospinning had seen intermittent use in the textile industry from the early twentieth century, it took the explosion of the field of tissue engineering, and its pursuit of biomimetic extracellular matrix (ECM) structures, to create an electrospinning renaissance. Over the past decade, a growing number of researchers in the tissue engineering community have embraced electrospinning as a polymer processing technique that effectively and routinely produces non-woven structures of nanoscale fibers (sizes of 80 nm to 1.5 µm). These nanofibers are of physiological significance as they closely resemble the structure and size scale of the native ECM (fiber diameters of 50 to 500 nm). Attempts to replicate the many roles of native ECM have led to the electrospinning of a wide array of polymers, both synthetic (poly(glycolic acid), poly(lactic acid), polydioxanone, polycaprolactone, etc.) and natural (collagen, fibrinogen, elastin, etc.) in origin, for a multitude of different tissue applications. With various compositions, fiber dimensions and fiber orientations, the biological, chemical and mechanical properties of the electrospun materials can be tailored. In this review we highlight the role of electrospinning in the engineering of different tissues and applications (skin/wound healing, cartilage, bone, vascular tissue, urological tissues, nerve, and ligament), and discuss its potential role in future work.

Polymer scaffold architecture is a key determinant in mast cell inflammatory and angiogenic responses

Daniel Abebayehu, Andrew J Spence, Michael J McClure, Tamara T Haque, Kevin O Rivera, John J Ryan

2019-04-01

Implanted polymer scaffolds can induce inflammation leading to the foreign body response (FBR), fibrosis, and implant failure. Thus, it is important to understand how immune cells interact with scaffolds to mitigate inflammation and promote a regenerative response. We previously demonstrated that macrophage phenotype is modulated by fiber and pore diameters of an electrospun scaffold. However, it is unclear if this effect is consistent among other innate immune cells. Mast cells are inflammatory sentinels that play a vital role in the FBR of implanted biomaterials, as well as angiogenesis. We determined if altering electrospun scaffold architecture modulates mast cell responses, with the goal of promoting regenerative cell-scaffold interactions. Polydioxanone (PDO) scaffolds were made from 60 mg/mL or 140 mg/mL PDO solutions, yielding structures with divergent fiber and pore diameters. Mouse mast cells plated on these scaffolds were activated with IL-33 or lipopolysaccharide (LPS). Relative to the 60 mg/mL scaffold, 140 mg/mL scaffolds yielded less IL-6 and TNF, and greater VEGF secretion. Pores >4-6 μm elicited less IL-6 and TNF secretion. IL-33-induced VEGF regulation was more complex, showing effects of both pore size and fiber diameter. These data indicate parameters that can predict mast cell responses to scaffolds, informing biomaterial design to increase wound healing and diminish implant rejection.