Skin Tissue Engineering for the Infected Wound Site: Biodegradable PLA Nanofibers and a Novel Approach for Silver Ion Release Evaluated in a 3D Coculture System of Keratinocytes and Staphylococcus aureus

Wound infection presents a challenging and growing problem. With the increased prevalence and growth of multidrug-resistant bacteria, there is a mounting need to reduce and eliminate wound infections using methodologies that limit the ability of bacteria to evolve into further drug-resistant strains. A well-known strategy for combating bacterial infection and preventing wound sepsis is through the delivery of silver ions to the wound site. High surface area silver nanoparticles (AgNPs) allowing extensive silver ion release have therefore been explored in different wound dressings and/or skin substitutes. However, it has been recently shown that AgNPs can penetrate into the stratum corneum of skin or diffuse into the cellular plasma membrane, and may interfere with a variety of cellular mechanisms. The goal of this study was to introduce and evaluate a new type of high surface area metallic silver in the form of highly porous silver microparticles (AgMPs). Polylactic acid (PLA) nanofibers were successfully loaded with either highly porous AgMPs or AgNPs and the antimicrobial efficacy and cytotoxicity of the two silver-based wound dressings were assessed and compared. To better mimic the physiological environment in vivo where both human cells and bacteria are present, a novel coculture system combining human epidermal keratinocytes and Staphylococcus aureus bacteria was designed to simultaneously evaluate human skin cell cytotoxicity with antimicrobial efficacy in a three-dimensional environment. We found that highly porous AgMPs could be successfully incorporated in nanofibrous wound dressings, and exhibited comparable antimicrobial efficacy and cytotoxicity to AgNPs. Further, PLA nanofibers containing highly porous AgMPs exhibited steady silver ion release, at a greater rate of release, than nanofibers containing AgNPs. The replacement of AgNPs with the newly introduced AgMPs overcomes concerns regarding the use of nanoparticles and holds great promise as skin substitutes or wound dressings for infected wound sites

Evaluation of Silver Ion-Releasing Scaffolds in a 3D Coculture System of MRSA and Human Adipose-Derived Stem Cells for Their Potential Use in Treatment or Prevention of Osteomyelitis

Bone infection, also called osteomyelitis, can result when bacteria invade a bone. Treatment of osteomyelitis usually requires surgical debridement and prolonged antimicrobial therapy. The rising incidence of infection with multidrug-resistant bacteria, in particular methicillin-resistant staphylococcus aureus (MRSA), however, limits the antimicrobial treatment options available. Silver is well known for its antimicrobial properties and is highly toxic to a wide range of microorganisms. We previously reported our development of biocompatible, biodegradable, nanofibrous scaffolds that released silver ions in a controlled manner. The objective of this study was to determine the efficacy of these scaffolds in treating or preventing osteomyelitis. To achieve this objective, antimicrobial efficacy was determined using a 3D coculture system of human adipose-derived stem cells (hASC) and MRSA. Human ASC were seeded on the scaffolds and induced to undergo osteogenic differentiation in both the absence and presence of MRSA. Our results indicated that the silver ion-releasing scaffolds not only inhibited biofilm formation, but also supported osteogenesis of hASC. Our findings suggest that these biocompatible, degradable, silver ion-releasing scaffolds can be used at an infection site to treat osteomyelitis and/or to coat bone implants as a preventative measure against infection postsurgery

Electrospun nanofibrous scaffolds increase the efficacy of stem cell-mediated therapy of surgically resected glioblastoma

Engineered stem cell (SC)-based therapy holds enormous promise for treating the incurable brain cancer glioblastoma (GBM). Retaining the cytotoxic SCs in the surgical cavity after GBM resection is one of the greatest challenges to this approach. Here, we describe a biocompatible electrospun nanofibrous scaffold (bENS) implant capable of delivering and retaining tumor-homing cytotoxic stem cells that suppress recurrence of post-surgical GBM. As a new approach to GBM therapy, we created poly(l-lactic acid) (PLA) bENS bearing drug-releasing human mesenchymal stem cells (hMSCs). We discovered that bENS-based implant increased hMSC retention in the surgical cavity 5-fold and prolonged persistence 3-fold compared to standard direct injection using our mouse model of GBM surgical resection/recurrence. Time-lapse imaging showed cytotoxic hMSC/bENS treatment killed co-cultured human GBM cells, and allowed hMSCs to rapidly migrate off the scaffolds as they homed to GBMs. In vivo, bENS loaded with hMSCs releasing the anti-tumor protein TRAIL (bENSsTR) reduced the volume of established GBM xenografts 3-fold. Mimicking clinical GBM patient therapy, lining the post-operative GBM surgical cavity with bENSsTR implants inhibited the re-growth of residual GBM foci 2.3-fold and prolonged post-surgical median survival from 13.5 to 31 days in mice. These results suggest that nanofibrous-based SC therapies could be an innovative new approach to improve the outcomes of patients suffering from terminal brain cancer

Fabrication and Evaluation of Electrospun, 3D-Bioplotted, and Combination of Electrospun/3D-Bioplotted Scaffolds for Tissue Engineering Applications

Electrospun scaffolds provide a dense framework of nanofibers with pore sizes and fiber diameters that closely resemble the architecture of native extracellular matrix. However, it generates limited three-dimensional structures of relevant physiological thicknesses. 3D printing allows digitally controlled fabrication of three-dimensional single/multimaterial constructs with precisely ordered fiber and pore architecture in a single build. However, this approach generally lacks the ability to achieve submicron resolution features to mimic native tissue. The goal of this study was to fabricate and evaluate 3D printed, electrospun, and combination of 3D printed/electrospun scaffolds to mimic the native architecture of heterogeneous tissue. We assessed their ability to support viability and proliferation of human adipose derived stem cells (hASC). Cells had increased proliferation and high viability over 21 days on all scaffolds. We further tested implantation of stacked-electrospun scaffold versus combined electrospun/3D scaffold on a cadaveric pig knee model and found that stacked-electrospun scaffold easily delaminated during implantation while the combined scaffold was easier to implant. Our approach combining these two commonly used scaffold fabrication technologies allows for the creation of a scaffold with more close resemblance to heterogeneous tissue architecture, holding great potential for tissue engineering and regenerative medicine applications of osteochondral tissue and other heterogeneous tissues

Extracellular Calcium Modulates Chondrogenic and Osteogenic Differentiation of Human Adipose-Derived Stem Cells: A Novel Approach for Osteochondral Tissue Engineering Using a Single Stem Cell Source

We have previously shown that elevating extracellular calcium from a concentration of 1.8 to 8 mM accelerates and increases human adipose-derived stem cell (hASC) osteogenic differentiation and cell-mediated calcium accretion, even in the absence of any other soluble osteogenic factors in the culture medium. However, the effects of elevated calcium on hASC chondrogenic differentiation have not been reported. The goal of this study was to determine the effects of varied calcium concentrations on chondrogenic differentiation of hASC. We hypothesized that exposure to elevated extracellular calcium (8 mM concentration) in a chondrogenic differentiation medium (CDM) would inhibit chondrogenesis of hASC when compared to basal calcium (1.8 mM concentration) controls. We further hypothesized that a full osteochondral construct could be engineered by controlling local release of calcium to induce site-specific chondrogenesis and osteogenesis using only hASC as the cell source. Human ASC was cultured as micromass pellets in CDM containing transforming growth factor-β1 and bone morphogenetic protein 6 for 28 days at extracellular calcium concentrations of either 1.8 mM (basal) or 8 mM (elevated). Our findings indicated that elevated calcium induced osteogenesis and inhibited chondrogenesis in hASC. Based on these findings, stacked polylactic acid nanofibrous scaffolds containing either 0% or 20% tricalcium phosphate (TCP) nanoparticles were electrospun and tested for site-specific chondrogenesis and osteogenesis. Histological assays confirmed that human ASC differentiated locally to generate calcified tissue in layers containing 20% TCP, and cartilage in the layers with no TCP when cultured in CDM. This is the first study to report the effects of elevated calcium on chondrogenic differentiation of hASC, and to develop osteochondral nanofibrous scaffolds using a single cell source and controlled calcium release to induce site-specific differentiation. This approach holds great promise for osteochondral tissue engineering using a single cell source (hASC) and single scaffold

Fabrication and Evaluation of Electrospun, 3D-Bioplotted, and Combination of Electrospun/3D-Bioplotted Scaffolds for Tissue Engineering Applications

Electrospun scaffolds provide a dense framework of nanofibers with pore sizes and fiber diameters that closely resemble the architecture of native extracellular matrix. However, it generates limited three-dimensional structures of relevant physiological thicknesses. 3D printing allows digitally controlled fabrication of three-dimensional single/multimaterial constructs with precisely ordered fiber and pore architecture in a single build. However, this approach generally lacks the ability to achieve submicron resolution features to mimic native tissue. The goal of this study was to fabricate and evaluate 3D printed, electrospun, and combination of 3D printed/electrospun scaffolds to mimic the native architecture of heterogeneous tissue. We assessed their ability to support viability and proliferation of human adipose derived stem cells (hASC). Cells had increased proliferation and high viability over 21 days on all scaffolds. We further tested implantation of stacked-electrospun scaffold versus combined electrospun/3D scaffold on a cadaveric pig knee model and found that stacked-electrospun scaffold easily delaminated during implantation while the combined scaffold was easier to implant. Our approach combining these two commonly used scaffold fabrication technologies allows for the creation of a scaffold with more close resemblance to heterogeneous tissue architecture, holding great potential for tissue engineering and regenerative medicine applications of osteochondral tissue and other heterogeneous tissues

Skin Tissue Engineering for the Infected Wound Site: Biodegradable PLA Nanofibers and a Novel Approach for Silver Ion Release Evaluated in a 3D Coculture System of Keratinocytes and Staphylococcus aureus

Wound infection presents a challenging and growing problem. With the increased prevalence and growth of multidrug-resistant bacteria, there is a mounting need to reduce and eliminate wound infections using methodologies that limit the ability of bacteria to evolve into further drug-resistant strains. A well-known strategy for combating bacterial infection and preventing wound sepsis is through the delivery of silver ions to the wound site. High surface area silver nanoparticles (AgNPs) allowing extensive silver ion release have therefore been explored in different wound dressings and/or skin substitutes. However, it has been recently shown that AgNPs can penetrate into the stratum corneum of skin or diffuse into the cellular plasma membrane, and may interfere with a variety of cellular mechanisms. The goal of this study was to introduce and evaluate a new type of high surface area metallic silver in the form of highly porous silver microparticles (AgMPs). Polylactic acid (PLA) nanofibers were successfully loaded with either highly porous AgMPs or AgNPs and the antimicrobial efficacy and cytotoxicity of the two silver-based wound dressings were assessed and compared. To better mimic the physiological environment in vivo where both human cells and bacteria are present, a novel coculture system combining human epidermal keratinocytes and Staphylococcus aureus bacteria was designed to simultaneously evaluate human skin cell cytotoxicity with antimicrobial efficacy in a three-dimensional environment. We found that highly porous AgMPs could be successfully incorporated in nanofibrous wound dressings, and exhibited comparable antimicrobial efficacy and cytotoxicity to AgNPs. Further, PLA nanofibers containing highly porous AgMPs exhibited steady silver ion release, at a greater rate of release, than nanofibers containing AgNPs. The replacement of AgNPs with the newly introduced AgMPs overcomes concerns regarding the use of nanoparticles and holds great promise as skin substitutes or wound dressings for infected wound sites

Evaluation of Silver Ion-Releasing Scaffolds in a 3D Coculture System of MRSA and Human Adipose-Derived Stem Cells for Their Potential Use in Treatment or Prevention of Osteomyelitis

Bone infection, also called osteomyelitis, can result when bacteria invade a bone. Treatment of osteomyelitis usually requires surgical debridement and prolonged antimicrobial therapy. The rising incidence of infection with multidrug-resistant bacteria, in particular methicillin-resistant staphylococcus aureus (MRSA), however, limits the antimicrobial treatment options available. Silver is well known for its antimicrobial properties and is highly toxic to a wide range of microorganisms. We previously reported our development of biocompatible, biodegradable, nanofibrous scaffolds that released silver ions in a controlled manner. The objective of this study was to determine the efficacy of these scaffolds in treating or preventing osteomyelitis. To achieve this objective, antimicrobial efficacy was determined using a 3D coculture system of human adipose-derived stem cells (hASC) and MRSA. Human ASC were seeded on the scaffolds and induced to undergo osteogenic differentiation in both the absence and presence of MRSA. Our results indicated that the silver ion-releasing scaffolds not only inhibited biofilm formation, but also supported osteogenesis of hASC. Our findings suggest that these biocompatible, degradable, silver ion-releasing scaffolds can be used at an infection site to treat osteomyelitis and/or to coat bone implants as a preventative measure against infection postsurgery

Electrospun nanofibrous scaffolds increase the efficacy of stem cell-mediated therapy of surgically resected glioblastoma

Engineered stem cell (SC)-based therapy holds enormous promise for treating the incurable brain cancer glioblastoma (GBM). Retaining the cytotoxic SCs in the surgical cavity after GBM resection is one of the greatest challenges to this approach. Here, we describe a biocompatible electrospun nanofibrous scaffold (bENS) implant capable of delivering and retaining tumor-homing cytotoxic stem cells that suppress recurrence of post-surgical GBM. As a new approach to GBM therapy, we created poly(l-lactic acid) (PLA) bENS bearing drug-releasing human mesenchymal stem cells (hMSCs). We discovered that bENS-based implant increased hMSC retention in the surgical cavity 5-fold and prolonged persistence 3-fold compared to standard direct injection using our mouse model of GBM surgical resection/recurrence. Time-lapse imaging showed cytotoxic hMSC/bENS treatment killed co-cultured human GBM cells, and allowed hMSCs to rapidly migrate off the scaffolds as they homed to GBMs. In vivo, bENS loaded with hMSCs releasing the anti-tumor protein TRAIL (bENS(sTR)) reduced the volume of established GBM xenografts 3-fold. Mimicking clinical GBM patient therapy, lining the post-operative GBM surgical cavity with bENS(sTR) implants inhibited the re-growth of residual GBM foci 2.3-fold and prolonged post-surgical median survival from 13.5 to 31 days in mice. These results suggest that nanofibrous-based SC therapies could be an innovative new approach to improve the outcomes of patients suffering from terminal brain cancer