Human placenta-derived matrix with cancellous autograft and demineralized bone matrix for large segmental long-bone defects in two dogs with septic nonunion.

OBJECTIVE: To report the successful treatment of septic nonunion in two dogs with large segmental defects secondary to long-bone fractures by using a novel human placenta-derived matrix (hPM) as adjunct to fixation. ANIMALS: One 3-kg 9-year-old neutered male Yorkshire terrier with a distal antebrachial fracture and one 6-kg 4-year-old spayed female miniature pinscher with a distal humeral fracture. STUDY DESIGN: Short case series. METHODS: Both dogs presented for septic nonunion after internal fixation of Gustilo type II open diaphyseal fractures from dog bite injuries. During revision, debridement of nonviable bone resulted in segmental defects of 32% and 20% of the bone length for the antebrachial and humeral fractures, respectively. The antebrachial fracture was stabilized with a circular external fixator, and the humeral fracture was stabilized with biaxial bone plating. The fracture sites were not collapsed, and full length was maintained with the fixation. Autogenous cancellous bone graft and canine demineralized bone allograft were packed into the defects, and hPM was injected into the graft sites after closure. RESULTS: Radiographic union was documented at 8 weeks and 6 weeks for the antebrachial and humeral fractures, respectively. Both dogs became fully weight bearing on the affected limbs and returned to full activity. CONCLUSION: Augmenting fixation with grafts and hPM led to a relatively rapid union in both dogs reported here.

Approaches to improve integration and regeneration of an ex vivo derived temporomandibular joint disc scaffold with variable matrix composition.

Due to their natural biochemical and biomechanical characteristics, using ex vivo tissues as platforms for guided tissue regeneration has become widely accepted, however subsequent attachment and integration of these constructs in vivo is often overlooked. A decellularized porcine temporomandibular joint (TMJ) disc has shown promise as a scaffold to guide disc regeneration and preliminary work has shown the efficacy of surfactant (SDS) treatment within the fibrocartilaginous disc to remove cellular components. The majority of studies focus on the intermediate region of the disc (or disc proper). Using this approach, inherent attachment tissues can be maintained to improve construct stability and integration within the joint. Unlike human disc attachment tissue, the porcine attachment tissues have high lipid content which would require a different processing approach to remove immunogenic components. In order to examine the effect of delipidation on the attachment tissue properties, SDS and two organic solvent mixtures (acetone/ethanol and chloroform/methanol) were compared. Lipid and cellular solubilization, ECM alteration, and seeded human mesenchymal stem cell (MSC) morphology and viability were assessed. Quantitative analysis showed SDS treatments did not effectively delipidate the attachment tissues and cytotoxicity was noted toward MSC in these regions. Acetone/ethanol removed cellular material but not all lipids, while chloroform/methanol removed all visible lipid deposits but residual porcine cells were observed in histological sections. When a combination of approaches was used, no residual lipid or cytotoxicity was noted. Preparing a whole TMJ graft with a combined approach has the potential to improve disc integration within the native joint environment.

Pilot assessment of a human extracellular matrix-based vascular graft in a rabbit model.

BACKGROUND: Herein we describe a small-diameter vascular graft constructed from rolled human amniotic membrane (hAM), with in vitro evaluation and subsequent in vivo assessment of its mechanical and initial biologic viability in the early postimplantation period. This approach for graft construction allows customization of graft dimensions, with wide-ranging potential clinical applicability as a nonautologous, allogeneic, cell-free graft material. METHODS: Acellular hAMs were rolled into layered conduits (3.2-mm diameter) that were bound with fibrin and lyophilized. Constructs were seeded with human smooth muscle cells and cultured under controlled arterial hemodynamic conditions in vitro. Additionally, the acellular hAM conduits were surgically implanted as arterial interposition grafts into the carotid arteries of immunocompetent rabbits. RESULTS: On in vitro analysis, smooth muscle cells were shown to adhere to, proliferate within, and remodel the scaffold during a 4-week culture period. At the end of the culture period, there was histologic and biomechanical evidence of graft wall layer coalescence. In vivo analysis demonstrated graft patency after 4 weeks (n = 3), with no hyperacute rejection or thrombotic occlusion. Explants displayed histologic evidence of active cellular remodeling, with endogenous cell repopulation of the graft wall concurrent with degradation of initial graft material. Cells were shown to align circumferentially to resemble a vascular medial layer. CONCLUSIONS: The vascular grafts were shown to provide a supportive scaffold allowing cellular infiltration and remodeling by host cell populations in vivo. By use of this approach, "off-the-shelf" vascular grafts can be created with specified diameters and wall thicknesses to satisfy specific anatomic requirements in diverse populations of patients.

Engineered microporosity: enhancing the early regenerative potential of decellularized temporomandibular joint discs.

The temporomandibular joint (TMJ) disc is susceptible to numerous pathologies that may lead to structural degradation and jaw dysfunction. The limited treatment options and debilitating nature of severe temporomandibular disorders has been the primary driving force for the introduction and development of TMJ disc tissue engineering as an approach to alleviate this important clinical issue. This study aimed to evaluate the efficacy of laser micropatterning (LMP) ex vivo-derived TMJ disc scaffolds to enhance cellular integration, a major limitation to the development of whole tissue implant technology. LMP was incorporated into the decellularized extracellular matrix scaffold structure using a 40 W CO2 laser ablation system to drill an 8×16 pattern with a bore diameter of 120 µm through the scaffold thickness. Disc scaffolds were seeded with human neonatal-derived umbilical cord mesenchymal stem cells differentiated into chondrocytes at a density of 900 cells per mm(2) and then assessed on days 1, 7, 14, and 21 of culture. Results derived from histology, PicoGreen DNA quantification, and cellular metabolism assays indicate that the LMP scaffolds improve cellular remodeling compared to the unworked scaffold over the 21-day culture period. Mechanical analysis further supports the use of the LMP showing the compressive properties of the LMP constructs closely represent native disc mechanics. The addition of an artificial path of infiltration by LMP culminated in improved chondrocyte adhesion, dispersion, and migration after extended culture aiding in recapitulating the native TMJ disc characteristics.

The effect of terminal sterilization on structural and biophysical properties of a decellularized collagen-based scaffold; implications for stem cell adhesion.

Terminal sterilization induces physical and chemical changes in the extracellular matrix (ECM) of ex vivo-derived biomaterials due to their aggressive mechanism of action. Prior studies have focused on how sterilization affects the mechanical integrity of tissue-based biomaterials but have rarely characterized effects on early cellular interaction, which is indicative of the biological response. Using a model fibrocartilage disc scaffold, these investigations compare the effect of three common sterilization methods [peracetic acid (PAA), gamma irradiation (GI), and ethylene oxide (EtO)] on a range of material properties and characterized early cellular interactions. GI and EtO produced unfavorable structural damage that contributed to inferior cell adhesion. Conversely, exposure to PAA resulted in limited structural alterations while inducing chemical modifications that favored cell attachment. Results suggest that the sterilization approach can be selected to modulate biomaterial properties to favor cellular adhesion and has relevance in tissue engineering and regenerative medicine applications. Furthermore, the study of cellular interactions with modified biomaterials in vitro provides information of how materials may react in subsequent clinical applications.

Mechanical and in vitro investigation of a porous PEEK foam for medical device implants.

PURPOSE: Implantable-grade polyetheretherketone (PEEK-OPTIMA®) is a high-performance thermoplastic that has been used in implant devices such as spinal-fusion cages since its introduction in 1999. Here, a new porous PEEK version was investigated. METHODS: Porous PEEK was fabricated using industrial scale relevant methods of compounding with porogen filler, extrusion, and subsequent extraction with water at supercritical temperatures and pressures. Mechanical properties were assessed according to ISO standards. Marrow stromal cells were cultured on porous PEEK samples and in vitro cytocompatibility was assessed by total DNA, alkaline phosphatase activity, osteopontin, calcium, and cell morphology to indicate stages of proliferation, differentiation, and mineralization. Compressive strength was assessed statically on 21 day cell cultures and media-soaked samples and dynamically within a medical device application specific context for interbody fusion cages (ASTM F2077). RESULTS: Manufacturing resulted in a biomaterial with ~50% porosity and a mean pore size of 100 microns. The porous PEEK was found to have: tensile strength (14.5MPa), strain at break (3.5%), impact strength (3.6 kJ/m2), flexural strength (21.6MPa), and flexural modulus (0.8GPa). Production of extracellular mineralized matrix occurred very early in the culture period, indicating a preferred surface for differentiation. SEM images revealed polygonal cell morphology supporting a differentiated osteoblastic-like phenotype. EDS analysis detected levels of carbon, phosphorus, and calcium coinciding with assay results for the proliferation and differentiation stages. CONCLUSION: Previous observations of cytocompatibility and calcification on the PEEK biomaterial could be carried through to this new porous form of the PEEK biomaterial. This helps porous PEEK to potentially offer more design options for implant devices requiring reduced modulus and/or increased tissue ingrowth aspects at the surface.

The influence of early-phase remodeling events on the biomechanical properties of engineered vascular tissues.

OBJECTIVES: During the last decade, the use of ex vivo-derived materials designed as implant scaffolds has increased significantly. This is particularly so in the area of regenerative medicine, or tissue engineering, where the natural chemical and biomechanical properties have been shown to be advantageous. By focusing on detailed events that occur during early-phase remodeling processes, our objective was to detail progressive changes in graft biomechanics to further our understanding of these processes. METHODS: A perfusion bioreactor system and acellular human umbilical veins were used as a model three-dimensional vascular scaffold on which human myofibroblasts were seeded and cultured under static or defined pulsatile conditions. Cell function in relation to graft mechanical properties was assessed. RESULTS: Cells doubled in density from approximately 1 × 10(6) to 2 ± 0.4 × 10(6) cells/cm ringlet, whereas static cultures remained unchanged. The material's compressive stiffness and ultimate tensile strength remained unchanged in both static and dynamic systems. However the Young's modulus values increased significantly in the physiologic range, whereas in the failure range, a significant reduction (66%) was shown under dynamic conditions. CONCLUSIONS: As pulse and flow conditions are modulated, complex mechanical changes are occurring that modify the elastic modulus differentially in both physiologic and failure ranges. Mechanical properties play an important role in graft patency, and a dynamic relationship between structure and function occurs during graft remodeling. These investigations have shown that as cells migrate into this ex vivo scaffold model, significant variation in material elasticity occurs that may have important implications in our understanding of early-stage vascular remodeling events.

Enzyme prodrug therapy designed to target L-methioninase to the tumor vasculature.

A new approach for enzyme prodrug therapy for cancer was tested using human endothelial cells and two breast cancer cell lines in vitro. The concept is to use the human annexin V protein to selectively target the enzyme L-methioninase to the tumor vasculature. The major finding was that enzyme prodrug treatment using the L-methioninase-annexin V fusion protein and selenomethionine as the prodrug over 3 days was shown to be lethal to the endothelial cells and the cancer cells, while having little or no effect with the prodrug but with no fusion protein present. Thus, this new approach appears promising.

Inverted human umbilical arteries with tunable wall thicknesses for nerve regeneration.

Tubular nerve guides have shown a potential to bridge nerve defects, by directing neuronal elongation, localizing growth factors, and inhibiting fibrotic cellular ingrowth. These investigations describe a novel acellular scaffold derived from the human umbilical cord artery that aims to enhance nerve regeneration by presenting a unique mechanical and chemical environment to the damaged nerve ends. A rapid, semiautomated dissection technique is described that isolates the human umbilical artery (HUA) from the umbilical cord, after which the vessel is decellularized using sodium dodecyl sulfate (SDS). The artery is turned inside out to produce a 3D scaffold, that unlike previous vessels for nerve repair, is more resistant to collapse. The scaffold has the potential as either an acellular bridge-implant, or for in vitro nerve regeneration. Stress-strain relationships and suture retention were assessed to determine whether the material had similar mechanical properties to native nerves. A dual process-flow perfusion bioreactor was developed to assess glucose mass transfer, and to investigate the culture of neuronal-like PC12 cells within the scaffold. These investigations have shown the automated dissecting method yields a smooth tubular scaffold, where wall thickness can be tuned to alter the mechanical behavior of the scaffold. Inverting the scaffold prevents collapse, with the decellularized iHUA having comparable mechanical properties to native nerves. Bioreactor cultures with PC12 cells seeded within iHUA lumenal void were shown to adhere and migrate into the preexisting ECM after 11 days of culture. These investigations show the potential of the iHUA as a unique 3D scaffold that may enhance nerve regeneration.

Modification of single walled carbon nanotube surface chemistry to improve aqueous solubility and enhance cellular interactions.

Single walled carbon nanotubes (SWNTs) continue to demonstrate the potential of nanoscaled materials in a wide range of applications. The ability to modulate the mechanical or electrical properties of a material by varying the SWNT component may result in diverse "application tunable" materials. Similarly, biomaterials used in tissue engineering applications may benefit from these characteristics by varying electrical and mechanical properties to enhance or direct tissue specific regeneration. The interactions between SWNTs and cellular systems need to be optimized to integrate these highly hydrophobic nanoparticles within an aqueous environment while maintaining their unique properties. We assessed solubility, conductance, and cellular interactions between four different SWNT preparations (unrefined, refined, and SWNT with either albumin or human plasma adsorbed). Initial interactions between cells and SWNTs were assessed within a 3D environment using a red blood cell lysis model, with longer-term interactions assessing the effects on PC12 and 3T3 fibroblast function when cultured on SWNT-collagen composite hydrogels. After SWNT purification, the lytic effect on red blood cells (RBCs) is significantly reduced from 11% to 0.7%, indicating manufacturing contaminants play a significant role in undesirable cell interactions. Nanotubes with either human plasma or albumin physisorbed onto the nanotube surface were significantly more hydrophilic than either unrefined or refined preparations and displayed improved RBC interactions. Despite improved dispersion, purification, and adsorption of either plasma or albumin, SWNTs caused a significant reduction in conductance. Although the molecular interactions occurring at the cell membrane remain unclear, these investigations have identified two main factors contributing to membrane failure: manufacturing impurities and to a lesser extend the material's innate hydrophobicity. Although purification is a critical step to remove toxic manufacturing contaminants, care must be taken to ensure improved aqueous dispersion does not compromise desirable mechanical and electrical attributes.

Vascular tissue engineering: bioreactor design considerations for extended culture of primary human vascular smooth muscle cells.

The influence of mechanical stimulation on cell populations not only helps maintain the specific cellular phenotype but also plays a significant role during differentiation and maturation of plastic cells. This is particularly true of tissue-engineered vascular tissue, where in vivo shear forces at the blood interface help maintain the function of the endothelium. Considerable effort has gone into the design and implementation of functional bioreactors that mimic the chemical and mechanical forces associated with the in vivo environment. Using a decellularized ex vivo porcine carotid artery as a model scaffold, we describe a number of important design criteria used to develop a vascular perfusion bioreactor and its supporting process-flow. The results of a comparative analysis of primary human vascular smooth muscle cells cultured under traditional"static conditions" and "dynamic loading" are described, where the expression of MMP-2 and 9 and cathepsin-L were assessed. Continued design improvements to perfusion bioreactors may improve cellular interactions, leading to constructs with improved biological function.

Polypyrrole thin films formed by admicellar polymerization support the osteogenic differentiation of mesenchymal stem cells.

The objective of this study was to evaluate the attachment, proliferation, and differentiation of rat mesenchymal stem cells (MSC) toward the osteoblastic phenotype seeded on polypyrrole (PPy) thin films made by admicellar polymerization. Three different concentrations of pyrrole (Py) monomer (20, 35, and 50 x 10(-3) M) were used with the PPy films deposited on tissue culture polystyrene dishes (TCP). Regular TCP dishes and PPy polymerized on TCP by chemical polymerization without surfactant using 5 x 10(-3) M Py, were used as controls. Rat MSC were seeded on these surfaces and cultured for up to 20 d in osteogenic media. Surface topography was characterized by atomic force microscopy, X-ray photoelectron spectroscopy, and static contact angle. Cell attachment, proliferation, alkaline phosphatase (ALP) activity, and calcium content were measured to evaluate the ability of MSC to adhere and differentiate on PPy-coated TCP. Increased monomer concentrations resulted in PPy films of increased thickness and surface roughness. PPy films generated by different monomer concentrations induced drastically different cellular events. A wide spectrum of cell attachment characteristics (from excellent cell attachment to the complete inability to adhere) were obtained by varying the monomer concentration from 20 m to 50 x 10(-3) M. In particular the 20 x 10(-3) M PPy thin films demonstrated superior induction of MSC osteogenicity, which was comparable to standard TCP dishes, unlike PPy films of similar thickness prepared by chemical polymerization without surfactant. Adhesion of mesenchymal stem cells on tissue culture plates (TCP) coated with polypyrrole thin films made by admicellar polymerization.