Josephine B. Allen

The blood and vascular cell compatibility of heparin-modified ePTFE vascular grafts

Prosthetic vascular grafts do not mimic the antithrombogenic properties of native blood vessels and therefore have higher rates of complications that involve thrombosis and restenosis. We developed an approach for grafting bioactive heparin, a potent anticoagulant glycosaminoglycan, to the lumen of ePTFE vascular grafts to improve their interactions with blood and vascular cells. Heparin was bound to aminated poly(1,8-octanediol-co-citrate) (POC) via its carboxyl functional groups onto POC-modified ePTFE grafts. The bioactivity and stability of the POC-immobilized heparin (POC-Heparin) were characterized via platelet adhesion and clotting assays. The effects of POC-Heparin on the adhesion, viability and phenotype of primary endothelial cells (EC), blood outgrowth endothelial cells (BOECs) obtained from endothelial progenitor cells (EPCs) isolated from human peripheral blood, and smooth muscle cells were also investigated. POC-Heparin grafts maintained bioactivity under physiologically relevant conditions in vitro for at least one month. Specifically, POC-Heparin-coated ePTFE grafts significantly reduced platelet adhesion and inhibited whole blood clotting kinetics. POC-Heparin supported EC and BOEC adhesion, viability, proliferation, NO production, and expression of endothelial cell-specific markers von Willebrand factor (vWF) and vascular endothelial-cadherin (VE-cadherin). Smooth muscle cells cultured on POC-Heparin showed increased expression of α-actin and decreased cell proliferation. This approach can be easily adapted to modify other blood contacting devices such as stents where antithrombogenicity and improved endothelialization are desirable properties.

Toward engineering a human neoendothelium with circulating progenitor cells.

Tissue‐engineered vascular grafts may one day provide a solution to many of the limitations associated with using synthetic vascular grafts. However, identifying a suitable cell source and polymer scaffold to recreate the properties of a native blood vessel remains a challenge. In this work, we assess the feasibility of using endothelial progenitor cells (EPCs) found in circulating blood to generate a functional endothelium on poly(1,8‐octanediol‐co‐citrate) (POC), a biodegradable elastomeric polyester. EPCs were isolated from human blood and biochemically differentiated into endothelial‐like cells (HE‐like) in vitro. The differentiated cell phenotype and function was confirmed by the appearance of the characteristic endothelial cell (EC) cobblestone morphology and positive staining for EC markers, von Willebrand factor, vascular endothelial cadherin, flk‐1, and CD31. In addition, HE‐like cells cultured on POC express endothelial nitric oxide synthase at levels comparable to aortic ECs. Furthermore, as with mature endothelial cells, HE‐like cell populations show negligible expression of tissue factor. Similarly, HE‐like cells produce and secrete prostacyclin and tissue plasminogen activator at levels comparable to venous and aortic ECs. When compared to fibroblast cells, HE‐like cells cultured on POC show a decrease in the rate of plasma and whole‐blood clot formation as well as a decrease in platelet adhesion. Finally, the data show that HE‐like cells can withstand physiological shear stress of 10 dynes/cm2 when cultured on POC‐modified expanded poly(tetrafluoroethylene) vascular grafts. Collectively, these data are the foundation for future clinical studies in the creation of an autologous endothelial cell‐seeded vascular graft. STEM CELLS 2010;28:318–328

Characterization of Porcine Circulating Progenitor Cells: Toward a Functional Endothelium

The lack of available healthy vessels, significant patient morbidity, and high costs hinders the successful clinical utilization of autologous endothelial cells (ECs). Herein we assess the feasibility of using endothelial progenitor cells (EPC) found in circulating blood to engineer a functional endothelium on poly(1,8-octanediol-co-citrate) (POC), a hemocompatible and biodegradable elastomer used in vascular tissue engineering. EPCs were isolated from porcine blood and biochemically differentiated into porcine endothelial (PE)-like cells in vitro. Once differentiated, EC phenotype and function on POC were assessed according to the presence of the EC-specific markers von Willebrand factor, platelet EC adhesion molecule, and vascular endothelial cadherin; metabolism of acetylated low-density lipoprotein; secretion of the anti-thrombogenic factors nitric oxide and prostacyclin; and inhibition of platelet adhesion and clotting processes in vitro. The effects of PE-like cells on porcine aortic smooth muscle cells (PASMCs) were also investigated via co-culture. PE-like cells on POC had phenotype, function, and clotting responses similar to those of primary aortic ECs. The presence of PE-like cells resulted in a 71 +/- 20% decrease in PASMC proliferation; a 52 +/- 2% decrease in the protein:deoxyribonucleic acid ratio; and an elongated, spindle-shaped morphology indicative of a shift from the proliferative to the contractile phenotype. These data suggest that EPCs and POC can provide the basis for a functional tissue-engineered endothelium.

A study of a biodegradable Mg–3Sc–3Y alloy and the effect of self-passivation on the in vitro degradation☆

Magnesium and its alloys have been investigated for their potential application as biodegradable implant materials. Although properties of magnesium such as biocompatibility and susceptibility to dissolution are desirable for biodegradable implant applications, its high degradation rate and low strength pose a significant challenge. A potential way to reduce the initial degradation rate is to form a self-passivating protective layer on the surface of the alloy. Oxides with a low enthalpy of formation result in a strong thermodynamic driving force to produce oxide surfaces that are more stable than the native oxide (MgO), and possibly reduce the initial degradation rate in these alloys. In the present study a ternary Mg-3wt.% Sc-3wt.% Y alloy was investigated and its oxidation behavior studied. The effect of surface passivation on the in vitro degradation rate was studied and the degradation products identified. The results show that the oxide provided an initial degradation barrier and 24h oxidation resulted in a negligible degradation rate for up to 23 days. Furthermore, the degradation products of the alloy showed no significant toxicity to osteoblastic cells, and cell proliferation studies confirmed cell attachment and proliferation on the surface of the oxidized alloy.

Peri-implant tissue response and biodegradation performance of a Mg-1.0Ca-0.5Sr alloy in rat tibia.

Biodegradable magnesium (Mg) alloys combine the advantages of traditional metallic implants and biodegradable polymers, having high strength, low density, and a stiffness ideal for bone fracture fixation. A recently developed Mg-Ca-Sr alloy potentially possesses advantageous characteristics over other Mg alloys, such as slower degradation rates and minimal toxicity. In this study, the biocompatibility of this Mg-Ca-Sr alloy was investigated in a rat pin-placement model. Cylindrical pins were inserted in the proximal tibial metaphyses in pre-drilled holes orthogonal to the tibial axis. Implant and bone morphologies were investigated using μCT at 1, 3, and 6 weeks after implant placement. At the same time points, the surrounding tissue was evaluated using H&E, TRAP and Goldners trichrome staining. Although gas bubbles were observed around the degrading implant at early time points, the bone remained intact with no evidence of microfracture. Principle findings also include new bone formation in the area of the implant, suggesting that the alloy is a promising candidate for biodegradable orthopedic implants.

Advanced nanocomposites for bone regeneration

The field of orthopedic tissue engineering is quickly expanding with the development of novel materials and strategies designed for rapid bone regeneration. While autologous bone grafts continue to be the standard of care, drawbacks include donor-site morbidity and short tissue supplies. Herein we report a novel nanocomposite sponge composed of poly(1,8-octanediol-co-citrate) (POC) and the bioactive ceramic β-tricalcium phosphate (TCP). We show that these nanocomposite sponges can be used as a depot for bone-producing (a.k.a. osteogenic) growth factors. In vitro bioactivity is demonstrated by significant upregulation of osteogenic genes, osteopontin (∼3 fold increase), osteocalcin (∼22 fold increase), alkaline phosphatase (∼10 fold increase), and transcription factor, RUNX2 (∼5 fold increase) over basal expression levels in mesenchymal stem cells. In vivo osteogenicity and biocompatibility is demonstrated in a standard subcutaneous implant model in rat. Results show that the nanocomposite sponge supports complete cell infiltration, minimal adverse foreign body response, positive cellular proliferation, and cellular expression of osteogenic markers in subcutaneous tissue. The results shown herein are encouraging and support the use of this sponge for future bone tissue engineering efforts.

Light-scattering fingerprinting for characterization of smooth-muscle cell proliferation

Accelerated proliferation of smooth muscle cells (SMC) is known to play an integral role in atherosclerotic lesion formation. Thus, there has been significant interest in defining both positive and negative regulators of SMC growth. We have applied a novel optical technique referred to as four-dimensional light scattering fingerprinting (4D-ELF) that enables non-invasive assessment of living cells. 4D-ELF can serve for highly sensitive detection of slight alterations in cellular and subcellular microstructure. Using 4D-ELF, we characterized the proliferation of SMC grown on two different substrates: laminin and fibronectin. Fibronectin-grown SMC have been previously shown to be more proliferative. Our results indicate that light scattering can be used to monitor the changes in the intracellular structure caused by the cell-substrate interaction and differentiate between more and less proliferative SMCs. Thus, light scattering fingerprinting may potentially provide a quick, inexpensive, and accurate means to noninvasively characterize the proliferation of living cells as well as cell-biomaterial interaction.

The effect of Mg–Ca–Sr alloy degradation products on human mesenchymal stem cells

Abstract Biodegradable Mg alloys have the potential to replace currently used metallic medical implant devices, likely eliminating toxicity concerns and the need for secondary surgeries, while also providing a potentially stimulating environment for tissue growth. A recently developed Mg–Ca–Sr alloy possesses advantageous characteristics over other Mg alloys, having a good combination of strength and degradation behavior, while also displaying potentially osteogenic properties. To better understand the effect of alloy degradation products on cellular mechanisms, in vitro studies using human bone marrow‐derived mesenchymal stem cells were conducted. Ionic products of alloy dissolution were found to be nontoxic but changed the proliferation profile of stem cells. Furthermore, their presence changed the progress of osteogenic development, while concentrations of Mg in particular appeared to induce stem cell differentiation. The work presented herein provides a foundation for future alloy design where structures can be tailored to obtain specific implant performance. These potentially bioactive implants would reduce the risks for patients by shortening their healing time, minimizing discomfort and toxicity concerns, while reducing hospital costs.

Development of poly (1,8 octanediol-co-citrate) and poly (acrylic acid) nanofibrous scaffolds for wound healing applications

Wound care is one of the leading health care problems in the United States costing billions of dollars yearly. Annually, millions of acute wounds occur due to surgical procedures or traumas such as burns and abrasions, and these wounds can become non-healing due to bacterial infection or underlying pathologies. Current wound care treatments include the use of bioinert constructs combined with topical administration of anti-bacterial agents and growth factors. However, there is a growing need for the development of bioactive wound dressing materials that are able to promote wound healing and the regeneration of healthy tissue. In this work, we evaluate and report the use of a novel electrospun polymeric scaffold consisting of poly (1,8 octanediol-co-citrate) and poly (acrylic acid) for wound healing applications. The scaffold exhibits intrinsic antibacterial activity, hydrogel-like water uptake abilities, and the ability to deliver physiologically relevant concentrations of growth factor. Additionally, the scaffold shows antibacterial function when tested with bacteria relevant to wound healing applications. Biological characterization of the electrospun scaffold shows excellent cellular adhesion, low cytotoxicity, and enhanced proliferation of skin fibroblasts. This work has potential towards the development of novel bioactive scaffolds for prevention of bacterial infiltration into the wound bed and enhanced healing.

Fabrication of a Free Radical Scavenging Nanocomposite Scaffold for Bone Tissue Regeneration

Oxidative stresses have become a large influence on bone tissue regeneration. Increased by trauma and fracture, reactive oxygen species (ROS) negatively impact the remodeling function of osteoblasts by damaging DNA and cellular structures while triggering apoptosis. This greatly hinders the efficacy of bone grafts to facilitate bone remodeling. Cerium oxide nanoparticles (CNPs) have been utilized to reduce ROS and have made an impact in biological applications. In this study, we fabricated bioactive, nanocomposite scaffolds incorporating cerium oxide nanoparticles. The architectural, chemical, and mechanical properties of the scaffolds were characterized using techniques such as scanning electron microscopy, energy-dispersive X-ray spectroscopy, X-ray diffraction, and compression testing. Biological assessments were completed to gauge the pro-osteogenic nature of the scaffolds through the attachment, viability, and mineralization of a pre-osteoblast cell line. Finally, free radical scavenging (FRS) function of the scaffolds was tested by measuring the decomposition of hydrogen peroxide over time and quantifying the cytotoxicity of cells on scaffolds after inducing oxidative stress. Through these assessments, it was determined that the nanocomposites contained the desired porous architectural and chemical properties. Scaffolds exhibited biocompatibility by supporting cell attachment, viability, and initiation of mineralization in the absence of supplemental mineralization-promoting factors. FRS behavior was displayed via a statistically significant reduction in scaffold-mediated hydrogen peroxide concentration and functional protection of cells from induced oxidative stress. In this work, we show that the successful incorporation of CNPs into nanocomposite scaffolds was able to decrease free radical damage to cells while providing a suitable environment for pre-osteoblast cells.Lay SummaryThe function of bone-forming cells, osteoblasts, in the bone remodeling cycle is hindered by oxidative stress created by an increase of reactive oxygen species. This is often seen at sites of injury and surgery, where bone grafts are often utilized. Our group investigated incorporating cerium oxide nanoparticles into a bioactive polymer-ceramic nanocomposite scaffolds for bone grafting applications. By incorporating the nanoparticles into our system, we are able to create a bioactive scaffold that can reduce reactive oxygen species and support osteogenic cell growth, both requirements for bone tissue formation. After initial in vitro testing, we would like to expand our investigation of the nanocomposite system in various in vivo models. To begin, an ectopic study in mice to determine biocompatibility, immune response, and mineralization. Additional studies would follow including critical size defect models in larger animal models.