Reduced skeletal loading associated with many conditions, such as neuromuscular injuries, can lea... more Reduced skeletal loading associated with many conditions, such as neuromuscular injuries, can lead to bone fragility and may threaten the success of implant therapy. Our group has developed a botulinum toxin A (botox) injection model to imitate disease-reduced skeletal loading and reported that botox dramatically impaired the bone formation and osseointegration of titanium implants. Semaphorin 3A (sema3A) is an osteoprotective factor that increases bone formation and inhibits bone resorption, indicating its potential therapeutic role in improving osseointegration in vivo. We first evaluated the sema3A effect on whole bone morphology following botox injections by delivering sema3A via injection. We then evaluated the sema3A effect on the osseointegration of titanium implants with two different surface topographies by delivering sema3A to cortical bone defect sites prepared for implant insertion and above the implants after insertion using a copper-free click hydrogel that polymerizes...
Transfection of chondrocytes with microRNA-451(miR-451), present in growth zone cartilage of the ... more Transfection of chondrocytes with microRNA-451(miR-451), present in growth zone cartilage of the growth plate, upregulates production of enzymes association with extracellular matrix degradation. miR-451 is also present in articular cartilage and exacerbates IL-1β effects in articular chondrocytes. Moreover, when osteoarthritis (OA) was induced in Sprague Dawley rats via bilateral anterior cruciate ligament transection (ACLT), miR-451 expression was increased in OA cartilage compared to control, suggesting its inhibition might be used to prevent or treat OA. To examine the prophylactic and therapeutic potential of inhibiting miR-451, we evaluated treatment with miR-451 power inhibitor (451-PI) at the onset of joint trauma and treatment after OA had developed. The prophylactic animal cohort received twice-weekly intra-articular injections of either 451-PI or a negative control (NC-PI) beginning on post-surgical day 3. OA was allowed to develop for 24 days in the therapeutic cohort be...
Volumetric muscle loss (VML) is the acute loss of muscle mass due to trauma. Such injuries occur ... more Volumetric muscle loss (VML) is the acute loss of muscle mass due to trauma. Such injuries occur primarily in the extremities and are debilitating, as there is no clinical treatment to restore muscle function. Pro-inflammatory advanced glycation end-products (AGEs) and the soluble receptor for advanced glycation end-products (RAGE) are known to increase in acute trauma patient’s serum and are correlated with increased injury severity. However, it is unclear whether AGEs and RAGE increase in muscle post-trauma. To test this, we used decellularized muscle matrix (DMM), a pro-myogenic, non-immunogenic extracellular matrix biomaterial derived from skeletal muscle. We delivered adipose-derived stromal cells (ASCs) and primary myoblasts to support myogenesis and immunomodulation (N = 8 rats/group). DMM non-seeded and seeded grafts were compared to empty defect and sham controls. Then, 56 days after surgery muscle force was assessed, histology characterized, and protein levels for AGEs, RA...
Journal of biomedical materials research. Part A, Jan 22, 2017
Regulating soft tissue repair to prevent fibrosis and promote regeneration is central to creating... more 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. Their 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. Here 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 to PDO alone for both scaffold types. Inhibition of ERK mitogen activated protein kinase did...
Decellularized tissues are biocompatible materials that engraft well, but the age of their source... more Decellularized tissues are biocompatible materials that engraft well, but the age of their source has not been explored for clinical translation. Advanced glycation end products (AGEs) are chemical cross-links that accrue on skeletal muscle collagen in old age, stiffening the matrix and increasing inflammation. Whether decellularized biomaterials derived from aged muscle would suffer from increased AGE collagen cross-links is unknown. We characterized gastrocnemii of 1-, 2-, and 20-month-old C57BL/6J mice before and after decellularization to determine age-dependent changes to collagen stiffness and AGE cross-linking. Total and soluble collagen was measured to assess if age-dependent increases in collagen and cross-linking persisted in decellularized muscle matrix (DMM). Stiffness of aged DMM was determined using atomic force microscopy. AGE levels and the effect of an AGE cross-link breaker, ALT-711, were tested in DMM samples. Our results show that age-dependent increases in colla...
148 Introduction 149 Materials and Methods 152 Results 159 Discussion 182 Conclusion 197 6 Integr... more 148 Introduction 149 Materials and Methods 152 Results 159 Discussion 182 Conclusion 197 6 Integrin binding analysis of electrospun polycaprolactone, collagen, elastin, and silk 199 Abstract 199 Introduction 200 Materials and Methods 204 Results 210 Discussion 219 Conclusion 224 Conclusion and Future Studies 225 Literature Cited 227 Appendices 249 A SEM of intimal, medial, and adventitial layers of the 45-45-10 (A-F), 5535-10 (G-L), and 65-25-10 (M-S) PCL-Elastin-Collagen graft after cross-linking with 50 mM EDC 249
Advanced age causes skeletal muscle to undergo deleterious changes including muscle atrophy, fast... more Advanced age causes skeletal muscle to undergo deleterious changes including muscle atrophy, fast-to-slow muscle fiber transition, and an increase in collagenous material that culminates in the age-dependent muscle wasting disease known as sarcopenia. Advanced glycation end-products (AGEs) non-enzymatically accumulate on the muscular collagens in old age via the Maillard reaction, potentiating the accumulation of intramuscular collagen and stiffening the microenvironment through collagen cross-linking. This review contextualizes known aspects of skeletal muscle extracellular matrix (ECM) aging, especially the role of collagens and AGE cross-linking, and underpins the motor nerve’s role in this aging process. Specific directions for future research are also discussed, with the understudied role of AGEs in skeletal muscle aging highlighted. Despite more than a half century of research, the role that intramuscular collagen aggregation and cross-linking plays in sarcopenia is well accep...
Products developed for skeletal muscle regeneration frequently incorporate allogeneic and xenogen... more Products developed for skeletal muscle regeneration frequently incorporate allogeneic and xenogeneic materials to elicit a regenerative response to heal skeletal muscle wounds. To avoid graft rejection in preclinical studies, immunodeficient rodents are used. Whether the immunodeficiency alters the host response to the material in skeletal muscle has not been studied. In this study, we hypothesized that an allogeneic acellular skeletal muscle grafts implanted in an immunodeficient rat (RNU, Foxn1-deficient) would exhibit better new muscle fiber formation compared to grafts implanted in immunocompetent Sprague Dawley (SD) rats. Decellularized SD skeletal muscle matrix (DMM) was implanted in the gastrocnemius (N = 8 rats/group). 56 days after surgery, animal gait was examined and animals were euthanized. Muscle force was assessed and fiber number as well as immune cell infiltrate was measured by histomorphometry and immunohistochemistry. Animal gait and percent recovery of muscle forc...
American Journal of Physiology-Cell Physiology, 2019
Regenerative medicine treatments for severe skeletal muscle injuries are limited, resulting in pe... more Regenerative medicine treatments for severe skeletal muscle injuries are limited, resulting in persistent functional deficits. Clinical options include neglecting the wound with the expectation that fibrosis will develop or using an autologous muscle graft with minimal functional improvement. A regenerative matrix can be used, but muscle fiber development on these matrices remains a challenge in vivo. Here, we explored the fundamental mechanisms that mediate cell-substrate signaling and its effect on cell-cell communication during myoblast fusion and tube formation to improve outcomes following implantation of matrices used to stimulate muscle regeneration. We previously reported that integrin-α7 was increased on anisotropic biomaterials, suggesting a role for α7β1 signaling in myoblast communication via connexin 43 and M-cadherin. Our results demonstrated that α7 silencing blocked expression of myogenic differentiation factor 1 (Myod), myogenin (Myog), myogenic factor 6 (Myf6), myo...
The purpose of this study was to establish whether material properties of elastin co-electrospun ... more 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 vascu...
The aligned structural environment in skeletal muscle is believed to be a crucial component in fu... more 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. We measured expression and production of myoblast markers: paired box-7 (Pax7), myogenic differentiation factor-1 (MyoD), myogenin (MyoG), myogenic factor-6 (Myf6), and myosin heavy chain (MyHC). To examine the role of α7β1 signaling, we measured expression and production of α7, α5, and β1 and myoblast markers in wild type cells and in cells silenced for α7 and assessed effects of silencing on myogenic differentiation. ...
ASME 2011 Summer Bioengineering Conference, Parts A and B, 2011
ABSTRACT The architecture of the vascular wall is highly intricate and requires unique biomechani... more ABSTRACT The architecture of the vascular wall is highly intricate and requires unique biomechanical properties in order to function properly. Native artery is composed of a mix of collagen, elastin, endothelial cells (ECs), smooth muscle cells (SMC), fibroblasts, and proteoglycans arranged into three distinct layers: the intima, media, and adventitia. Throughout artery, collagen and elastin play an important role, providing a mechanical backbone, preventing vessel rupture, and promoting recovery while undergoing pulsatile deformations [1]. The low-strain mechanical response of artery to blood flow is dominated by the elastic behavior of elastin which prevents pulsatile energy from being dissipated as heat [2]. Previous work has shown the ability to fabricate multi-layered electrospun scaffolds composed of polycaprolactone (PCL), elastin (ELAS), and collagen (COL), and their associated mechanical advantages. PCL was chosen, in this case, to provide mechanical integrity and elasticity, while elastin and collagen would provide further elasticity and bioactivity [3,4]. However, when the grafts were implanted in the descending aorta of a rat, cellular results were not as desirable as predicted. Therefore, further graft optimization was required. The hypothesis of this study was that blended polymers and biopolymers would be conducive for cellular attachment through specific integrin binding sites. To test this hypothesis, human umbilical artery smooth muscle cells (hUASMC) were seeded on electrospun PCL, COL, and ELAS blends for evaluation in a cell adhesion inhibition experiment.
Journal of Biomaterials Science, Polymer Edition, 2011
The process of electrospinning has proven to be highly beneficial for use in a number of tissue-e... more 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. This study involved a preliminary investigation and comparison of traditionally and airgap electrospun silk-fibroin-based ligament constructs. Structures were characterized with SEM and alignment FFT, and underwent porosity, permeability, and mechanical anisotropy evaluation. Preliminary cell culture with human dermal fibroblasts was performed to determine the degree of cellular orientation and penetration. Results showed airgap electrospun structures to be anisotropic with significantly increased porosity and cellular penetration compared to their traditionally electrospun counterparts.
The purpose of this study was to enhance the mechanical properties and slow the degradation of an... more 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.
An electrospun cardiovascular graft composed of polydioxanone (PDO) and elastin has been designed... more 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.
Chondrocytes at different maturation states in the growth plate produce matrix vesicles (MVs), me... more Chondrocytes at different maturation states in the growth plate produce matrix vesicles (MVs), membrane organelles found in the extracellular matrix, with a wide range of contents, such as matrix processing enzymes and receptors for hormones. We have shown that MVs harvested from growth zone (GC) chondrocyte cultures contain abundant small RNAs, including miRNAs. Here, we determined whether RNA also exists in MVs produced by less mature resting zone (RC) chondrocytes and, if so, whether it differs from the RNA in MVs produced by GC cells. Our results showed that RNA, small RNA specifically, was present in RC-MVs, and it was well-protected from RNase by the phospholipid membrane. A group of miRNAs was enriched in RC-MVs compared RC-cells, suggesting that miRNAs are selectively packaged into MVs. High throughput array and RNA sequencing showed that ~39% miRNAs were differentially expressed between RC-MVs and GC-MVs. Individual RT-qPCR also confirmed that miR-122-5p and miR-150-5p were...
Reduced skeletal loading associated with many conditions, such as neuromuscular injuries, can lea... more Reduced skeletal loading associated with many conditions, such as neuromuscular injuries, can lead to bone fragility and may threaten the success of implant therapy. Our group has developed a botulinum toxin A (botox) injection model to imitate disease-reduced skeletal loading and reported that botox dramatically impaired the bone formation and osseointegration of titanium implants. Semaphorin 3A (sema3A) is an osteoprotective factor that increases bone formation and inhibits bone resorption, indicating its potential therapeutic role in improving osseointegration in vivo. We first evaluated the sema3A effect on whole bone morphology following botox injections by delivering sema3A via injection. We then evaluated the sema3A effect on the osseointegration of titanium implants with two different surface topographies by delivering sema3A to cortical bone defect sites prepared for implant insertion and above the implants after insertion using a copper-free click hydrogel that polymerizes...
Transfection of chondrocytes with microRNA-451(miR-451), present in growth zone cartilage of the ... more Transfection of chondrocytes with microRNA-451(miR-451), present in growth zone cartilage of the growth plate, upregulates production of enzymes association with extracellular matrix degradation. miR-451 is also present in articular cartilage and exacerbates IL-1β effects in articular chondrocytes. Moreover, when osteoarthritis (OA) was induced in Sprague Dawley rats via bilateral anterior cruciate ligament transection (ACLT), miR-451 expression was increased in OA cartilage compared to control, suggesting its inhibition might be used to prevent or treat OA. To examine the prophylactic and therapeutic potential of inhibiting miR-451, we evaluated treatment with miR-451 power inhibitor (451-PI) at the onset of joint trauma and treatment after OA had developed. The prophylactic animal cohort received twice-weekly intra-articular injections of either 451-PI or a negative control (NC-PI) beginning on post-surgical day 3. OA was allowed to develop for 24 days in the therapeutic cohort be...
Volumetric muscle loss (VML) is the acute loss of muscle mass due to trauma. Such injuries occur ... more Volumetric muscle loss (VML) is the acute loss of muscle mass due to trauma. Such injuries occur primarily in the extremities and are debilitating, as there is no clinical treatment to restore muscle function. Pro-inflammatory advanced glycation end-products (AGEs) and the soluble receptor for advanced glycation end-products (RAGE) are known to increase in acute trauma patient’s serum and are correlated with increased injury severity. However, it is unclear whether AGEs and RAGE increase in muscle post-trauma. To test this, we used decellularized muscle matrix (DMM), a pro-myogenic, non-immunogenic extracellular matrix biomaterial derived from skeletal muscle. We delivered adipose-derived stromal cells (ASCs) and primary myoblasts to support myogenesis and immunomodulation (N = 8 rats/group). DMM non-seeded and seeded grafts were compared to empty defect and sham controls. Then, 56 days after surgery muscle force was assessed, histology characterized, and protein levels for AGEs, RA...
Journal of biomedical materials research. Part A, Jan 22, 2017
Regulating soft tissue repair to prevent fibrosis and promote regeneration is central to creating... more 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. Their 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. Here 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 to PDO alone for both scaffold types. Inhibition of ERK mitogen activated protein kinase did...
Decellularized tissues are biocompatible materials that engraft well, but the age of their source... more Decellularized tissues are biocompatible materials that engraft well, but the age of their source has not been explored for clinical translation. Advanced glycation end products (AGEs) are chemical cross-links that accrue on skeletal muscle collagen in old age, stiffening the matrix and increasing inflammation. Whether decellularized biomaterials derived from aged muscle would suffer from increased AGE collagen cross-links is unknown. We characterized gastrocnemii of 1-, 2-, and 20-month-old C57BL/6J mice before and after decellularization to determine age-dependent changes to collagen stiffness and AGE cross-linking. Total and soluble collagen was measured to assess if age-dependent increases in collagen and cross-linking persisted in decellularized muscle matrix (DMM). Stiffness of aged DMM was determined using atomic force microscopy. AGE levels and the effect of an AGE cross-link breaker, ALT-711, were tested in DMM samples. Our results show that age-dependent increases in colla...
148 Introduction 149 Materials and Methods 152 Results 159 Discussion 182 Conclusion 197 6 Integr... more 148 Introduction 149 Materials and Methods 152 Results 159 Discussion 182 Conclusion 197 6 Integrin binding analysis of electrospun polycaprolactone, collagen, elastin, and silk 199 Abstract 199 Introduction 200 Materials and Methods 204 Results 210 Discussion 219 Conclusion 224 Conclusion and Future Studies 225 Literature Cited 227 Appendices 249 A SEM of intimal, medial, and adventitial layers of the 45-45-10 (A-F), 5535-10 (G-L), and 65-25-10 (M-S) PCL-Elastin-Collagen graft after cross-linking with 50 mM EDC 249
Advanced age causes skeletal muscle to undergo deleterious changes including muscle atrophy, fast... more Advanced age causes skeletal muscle to undergo deleterious changes including muscle atrophy, fast-to-slow muscle fiber transition, and an increase in collagenous material that culminates in the age-dependent muscle wasting disease known as sarcopenia. Advanced glycation end-products (AGEs) non-enzymatically accumulate on the muscular collagens in old age via the Maillard reaction, potentiating the accumulation of intramuscular collagen and stiffening the microenvironment through collagen cross-linking. This review contextualizes known aspects of skeletal muscle extracellular matrix (ECM) aging, especially the role of collagens and AGE cross-linking, and underpins the motor nerve’s role in this aging process. Specific directions for future research are also discussed, with the understudied role of AGEs in skeletal muscle aging highlighted. Despite more than a half century of research, the role that intramuscular collagen aggregation and cross-linking plays in sarcopenia is well accep...
Products developed for skeletal muscle regeneration frequently incorporate allogeneic and xenogen... more Products developed for skeletal muscle regeneration frequently incorporate allogeneic and xenogeneic materials to elicit a regenerative response to heal skeletal muscle wounds. To avoid graft rejection in preclinical studies, immunodeficient rodents are used. Whether the immunodeficiency alters the host response to the material in skeletal muscle has not been studied. In this study, we hypothesized that an allogeneic acellular skeletal muscle grafts implanted in an immunodeficient rat (RNU, Foxn1-deficient) would exhibit better new muscle fiber formation compared to grafts implanted in immunocompetent Sprague Dawley (SD) rats. Decellularized SD skeletal muscle matrix (DMM) was implanted in the gastrocnemius (N = 8 rats/group). 56 days after surgery, animal gait was examined and animals were euthanized. Muscle force was assessed and fiber number as well as immune cell infiltrate was measured by histomorphometry and immunohistochemistry. Animal gait and percent recovery of muscle forc...
American Journal of Physiology-Cell Physiology, 2019
Regenerative medicine treatments for severe skeletal muscle injuries are limited, resulting in pe... more Regenerative medicine treatments for severe skeletal muscle injuries are limited, resulting in persistent functional deficits. Clinical options include neglecting the wound with the expectation that fibrosis will develop or using an autologous muscle graft with minimal functional improvement. A regenerative matrix can be used, but muscle fiber development on these matrices remains a challenge in vivo. Here, we explored the fundamental mechanisms that mediate cell-substrate signaling and its effect on cell-cell communication during myoblast fusion and tube formation to improve outcomes following implantation of matrices used to stimulate muscle regeneration. We previously reported that integrin-α7 was increased on anisotropic biomaterials, suggesting a role for α7β1 signaling in myoblast communication via connexin 43 and M-cadherin. Our results demonstrated that α7 silencing blocked expression of myogenic differentiation factor 1 (Myod), myogenin (Myog), myogenic factor 6 (Myf6), myo...
The purpose of this study was to establish whether material properties of elastin co-electrospun ... more 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 vascu...
The aligned structural environment in skeletal muscle is believed to be a crucial component in fu... more 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. We measured expression and production of myoblast markers: paired box-7 (Pax7), myogenic differentiation factor-1 (MyoD), myogenin (MyoG), myogenic factor-6 (Myf6), and myosin heavy chain (MyHC). To examine the role of α7β1 signaling, we measured expression and production of α7, α5, and β1 and myoblast markers in wild type cells and in cells silenced for α7 and assessed effects of silencing on myogenic differentiation. ...
ASME 2011 Summer Bioengineering Conference, Parts A and B, 2011
ABSTRACT The architecture of the vascular wall is highly intricate and requires unique biomechani... more ABSTRACT The architecture of the vascular wall is highly intricate and requires unique biomechanical properties in order to function properly. Native artery is composed of a mix of collagen, elastin, endothelial cells (ECs), smooth muscle cells (SMC), fibroblasts, and proteoglycans arranged into three distinct layers: the intima, media, and adventitia. Throughout artery, collagen and elastin play an important role, providing a mechanical backbone, preventing vessel rupture, and promoting recovery while undergoing pulsatile deformations [1]. The low-strain mechanical response of artery to blood flow is dominated by the elastic behavior of elastin which prevents pulsatile energy from being dissipated as heat [2]. Previous work has shown the ability to fabricate multi-layered electrospun scaffolds composed of polycaprolactone (PCL), elastin (ELAS), and collagen (COL), and their associated mechanical advantages. PCL was chosen, in this case, to provide mechanical integrity and elasticity, while elastin and collagen would provide further elasticity and bioactivity [3,4]. However, when the grafts were implanted in the descending aorta of a rat, cellular results were not as desirable as predicted. Therefore, further graft optimization was required. The hypothesis of this study was that blended polymers and biopolymers would be conducive for cellular attachment through specific integrin binding sites. To test this hypothesis, human umbilical artery smooth muscle cells (hUASMC) were seeded on electrospun PCL, COL, and ELAS blends for evaluation in a cell adhesion inhibition experiment.
Journal of Biomaterials Science, Polymer Edition, 2011
The process of electrospinning has proven to be highly beneficial for use in a number of tissue-e... more 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. This study involved a preliminary investigation and comparison of traditionally and airgap electrospun silk-fibroin-based ligament constructs. Structures were characterized with SEM and alignment FFT, and underwent porosity, permeability, and mechanical anisotropy evaluation. Preliminary cell culture with human dermal fibroblasts was performed to determine the degree of cellular orientation and penetration. Results showed airgap electrospun structures to be anisotropic with significantly increased porosity and cellular penetration compared to their traditionally electrospun counterparts.
The purpose of this study was to enhance the mechanical properties and slow the degradation of an... more 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.
An electrospun cardiovascular graft composed of polydioxanone (PDO) and elastin has been designed... more 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.
Chondrocytes at different maturation states in the growth plate produce matrix vesicles (MVs), me... more Chondrocytes at different maturation states in the growth plate produce matrix vesicles (MVs), membrane organelles found in the extracellular matrix, with a wide range of contents, such as matrix processing enzymes and receptors for hormones. We have shown that MVs harvested from growth zone (GC) chondrocyte cultures contain abundant small RNAs, including miRNAs. Here, we determined whether RNA also exists in MVs produced by less mature resting zone (RC) chondrocytes and, if so, whether it differs from the RNA in MVs produced by GC cells. Our results showed that RNA, small RNA specifically, was present in RC-MVs, and it was well-protected from RNase by the phospholipid membrane. A group of miRNAs was enriched in RC-MVs compared RC-cells, suggesting that miRNAs are selectively packaged into MVs. High throughput array and RNA sequencing showed that ~39% miRNAs were differentially expressed between RC-MVs and GC-MVs. Individual RT-qPCR also confirmed that miR-122-5p and miR-150-5p were...
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Papers by Michael McClure