Peter I. Lelkes

Mechanical properties and biomineralization of multifunctional nanodiamond-PLLA composites for bone tissue engineering.

Multifunctional bone scaffold materials have been produced from a biodegradable polymer, poly(L-lactic acid) (PLLA), and 1-10% wt of octadecylamine-functionalized nanodiamond (ND-ODA) via solution casting followed by compression molding. By comparison to pure PLLA, the addition of 10% wt of ND-ODA resulted in a significant improvement of the mechanical properties of the composite matrix, including a 280% increase in the strain at failure and a 310% increase in fracture energy in tensile tests. The biomimetic process of bonelike apatite growth on the ND-ODA/PLLA scaffolds was studied using microscopic and spectroscopic techniques. The enhanced mechanical properties and the increased mineralization capability with higher ND-ODA concentration suggest that these biodegradable composites may potentially be useful for a variety of biomedical applications, including scaffolds for orthopedic regenerative engineering.

Biomechanical and biochemical remodeling of stromal extracellular matrix in cancer.

The extracellular matrix (ECM) provides structural and biochemical signals that regulate cell function. A well-controlled balance between cells and surroundings (i.e., dynamic reciprocity) is crucial for regulating ECM architecture. During cancer progression, epithelial cells undergo genetic alterations which, together with stromal changes including ECM remodeling, disturb the homeostatic dynamics of the epithelium. A parallel organization of stromal ECM fibrils is associated with tumorigenic responses. In an emerging paradigm, continuous and progressive regulation via mechanical forces and aberrant signaling are believed to be responsible for tumor-associated ECM remodeling. In this review we discuss the discrete biomechanical and biochemical mechanisms that underlie these architectural changes and highlight their particular relevance to the regulation of the alignment of ECM in the mesenchymal stroma.

Textile-templated electrospun anisotropic scaffolds for regenerative cardiac tissue engineering

For patients with end-stage heart disease, the access to heart transplantation is limited due to the shortage of donor organs and to the potential for rejection of the donated organ. Therefore, current studies focus on bioengineering approaches for creating biomimetic cardiac patches that will assist in restoring cardiac function, by repairing and/or regenerating the intrinsically anisotropic myocardium. In this paper we present a simplified, straightforward approach for creating bioactive anisotropic cardiac patches, based on a combination of bioengineering and textile-manufacturing techniques in concert with nano-biotechnology based tissue-engineering stratagems. Using knitted conventional textiles, made of cotton or polyester yarns as template targets, we successfully electrospun anisotropic three-dimensional scaffolds from poly(lactic-co-glycolic) acid (PLGA), and thermoplastic polycarbonate-urethane (PCU, Bionate(®)). The surface topography and mechanical properties of textile-templated anisotropic scaffolds significantly differed from those of scaffolds electrospun from the same materials onto conventional 2-D flat-target electrospun scaffolds. Anisotropic textile-templated scaffolds electrospun from both PLGA and PCU, supported the adhesion and proliferation of H9C2 cardiac myoblasts cell line, and guided the cardiac tissue-like anisotropic organization of these cells in vitro. All cell-seeded PCU scaffolds exhibited mechanical properties comparable to those of a human heart, but only the cells on the polyester-templated scaffolds exhibited prolonged spontaneous synchronous contractility on the entire engineered construct for 10 days in vitro at a near physiologic frequency of ∼120 bpm. Taken together, the methods described here take advantage of straightforward established textile manufacturing strategies as an efficient and cost-effective approach to engineering 3D anisotropic, elastomeric PCU scaffolds that can serve as a cardiac patch.

Neural stem cells: therapeutic potential for neurodegenerative diseases.

INTRODUCTIONnNeural stem cells (NSCs) from specific brain areas or developed from progenitors of different sources are of therapeutic potential for neurodegenerative diseases.nnnSOURCES OF DATAnTreatment strategies involve the (i) transplantation of exogenous NSCs; (ii) pharmacological modulations of endogenous NSCs and (iii) modulation of endogenous NSCs via the transplantation of exogenous NSCs.nnnAREAS OF AGREEMENTnThere is a consensus about the therapeutic potential of transplanted NSCs. The ability of NSCs to home into areas of central nervous system injury allows their delivery by intravenous injection. There is also a general agreement about the neuroprotective mechanisms of NSCs involving a bystander effect.nnnAREAS OF CONTROVERSYnIndividual laboratories may be using phenotypically diverse NSCs, since these cells have been differentiated by a variety of neurotrophins and/or cultured on different ECM proteins, therefore differing in the expression of neuronal markers.nnnGROWING POINTSnOptimization of the dose, delivery route, timing of administration of NSCs, their interactions with the immune system and combination therapies in conjunction with tissue engineered neural prostheses are under investigation.nnnAREAS TIMELY FOR DEVELOPING RESEARCHnIn-depth understanding of the biological properties of NSCs, including mechanisms of therapy, safety, efficacy and elimination from the organism. These areas are central for further use in cell therapy. CAUTIONARY NOTE: As long as critical safety and efficacy issues are not resolved, we need to be careful in translating NSC therapy from animal models to patients.

Biocompatibility and biodegradation studies of PCL/β-TCP bone tissue scaffold fabricated by structural porogen method

Three-dimensional printer (3DP) (Z-Corp) is a solid freeform fabrication system capable of generating sub-millimeter physical features required for tissue engineering scaffolds. By using plaster composite materials, 3DP can fabricate a universal porogen which can be injected with a wide range of high melting temperature biomaterials. Here we report results toward the manufacture of either pure polycaprolactone (PCL) or homogeneous composites of 90/10 or 80/20 (w/w) PCL/beta-tricalcium phosphate (β-TCP) by injection molding into plaster composite porogens fabricated by 3DP. The resolution of printed plaster porogens and produced scaffolds was studied by scanning electron microscopy. Cytotoxicity test on scaffold extracts and biocompatibility test on the scaffolds as a matrix supporting murine osteoblast (7F2) and endothelial hybridoma (EAhy 926) cells growth for up to 4xa0days showed that the porogens removal process had only negligible effects on cell proliferation. The biodegradation tests of pure PCL and PCL/β-TCP composites were performed in DMEM with 10xa0% (v/v) FBS for up to 6xa0weeks. The PCL/β-TCP composites show faster degradation rate than that of pure PCL due to the addition of β-TCP, and the strength of 80/20 PCL/β-TCP composite is still suitable for human cancellous bone healing support after 6xa0weeks degradation. Combining precisely controlled porogen fabrication structure, good biocompatibility, and suitable mechanical properties after biodegradation, PCL/β-TCP scaffolds fabricated by 3DP porogen method provide essential capability for bone tissue engineering.

A new, cryoprecipitate based coating for improved endothelial cell attachment and growth on medical grade artificial surfaces.

Monoprotein coatings of biomaterials with either natural adhesion molecules or genetically designed analogs have been used to facilitate attachment and spreading of endothelial cells. However, such treatments were found insufficient to maintain the integrity of the endothelial surface under turbulent flow conditions. In addition, when brought into contact with blood, these coatings were susceptible to plasma and cell proteinases that could readily destroy their structure and weaken cell adherence to the surface. In addressing these problems, we developed a cryoprecipitate-based coating that can firmly bind to any nonporous, prosthetic surface and interact with endothelial cells. The primary structure of the coating consisted of an autologous fibrin meshwork. It was refined by various compositions of the fibrinogen containing mixture and secured to polystyrene or polyurethane surfaces by dry-heat treatment. Further modulation of the coating was achieved by physically immobilizing various doses of heparin and insulin into the three dimensional matrix of the meshwork. Endothelial cells attached and grew much better on polyurethanes coated with this autologous protein complex than on a polystyrene tissue culture surface. With proper use of its capacity to mimic the properties of basal membrane, and absence of immunologic complications, the resulting coating may become an ideal multifunctional interface between cells and prosthetic materials.

Enhanced reseeding of decellularized rodent lungs with mouse embryonic stem cells

Repopulation of decellularized lung scaffolds (DLS) is limited due to alterations in the repertoire and ratios of the residual extracellular matrix (ECM) proteins, characterized by e.g., the retention of type I collagen and loss of glycoproteins. We hypothesized that pre-treatment of decellularized matrices with defined ECM proteins, which match the repertoire of integrin receptors expressed by the cells to be seeded (e.g., embryonic stem cells) can increase the efficacy of the reseeding process. To test this hypothesis, we first determined the integrin receptors profile of mouse embryonic stem cells (mESCs). Mouse ESCs express α3, α5, α6, α9 and β1, but not α1, α2 and α4 integrin subunits, as established by Western blotting and adhesion to laminin and fibronectin, but not to collagens type I and IV. Reseeding of DLS with mESCs was inefficient (6.9 ± 0.5%), but was significantly enhanced (2.3 ± 0.1 fold) by pre-treating the scaffolds with media conditioned by A549 human lung adenocarcinoma cells, which we found to contain ∼5 μg/ml laminin. Furthermore, pre-treatment with A549-conditioned media resulted in a significantly more uniform distribution of the seeded mESCs throughout the engineered organ as compared to untreated DLS. Our study may advance whole lung engineering by stressing the importance of matching the integrin receptor repertoire of the seeded cells and the cell binding motifs of DLS.

Electrospun Rapamycin-Eluting Polyurethane Fibers for Vascular Grafts

ABSTRACTPurposeTo develop rapamycin-eluting electrospun polyurethane (PU) vascular grafts that could effectively suppress local smooth muscle cell (SMC) proliferation.MethodsRapamycin (RM) was incorporated in PU fibers by blend electrospinning using three distinct blending methods. The drug release profiles and the bioavailability of RM-containing PU fibers in the form of fibrous mats and vascular grafts were evaluated up to 77xa0days in vitro.ResultsRM-contained PU fibers generated by the three distinct blending methods exhibited significantly different fiber diameters (200–500xa0nm) and distinct RM release kinetics. Young’s moduli of the electrospun fibrous mats increased with higher RM contents and decreased with larger fiber diameters. For all blending methods, RM release kinetics was characteristic of a Fickian diffusion for at least 77xa0days in vitro. RM-PU fibers generated via powder blending showed the highest encapsulation efficiency. The RM in grafts made of these fibers remained bioactive and was still able to inhibit smooth muscle cell proliferation after 77xa0days of continual in vitro release.ConclusionsElectrospun RM-containing PU fibers can serve as effective drug carriers for the local suppression of SMC proliferation and could be used as RM-eluting scaffolds for vascular grafts.

Revascularization of decellularized lung scaffolds: principles and progress.

There is a clear unmet clinical need for novel biotechnology-based therapeutic approaches to lung repair and/or replacement, such as tissue engineering of whole bioengineered lungs. Recent studies have demonstrated the feasibility of decellularizing the whole organ by removal of all its cellular components, thus leaving behind the extracellular matrix as a complex three-dimensional (3D) biomimetic scaffold. Implantation of decellularized lung scaffolds (DLS), which were recellularized with patient-specific lung (progenitor) cells, is deemed the ultimate alternative to lung transplantation. Preclinical studies demonstrated that, upon implantation in rodent models, bioengineered lungs that were recellularized with airway and vascular cells were capable of gas exchange for up to 14 days. However, the long-term applicability of this concept is thwarted in part by the failure of current approaches to reconstruct a physiologically functional, quiescent endothelium lining the entire vascular tree of reseeded lung scaffolds, as inferred from the occurrence of hemorrhage into the airway compartment and thrombosis in the vasculature in vivo. In this review, we explore the idea that successful whole lung bioengineering will critically depend on 1) preserving and/or reestablishing the integrity of the subendothelial basement membrane, especially of the ultrathin respiratory membrane separating airways and capillaries, during and following decellularization and 2) restoring vascular physiological functionality including the barrier function and quiescence of the endothelial lining following reseeding of the vascular compartment. We posit that physiological reconstitution of the pulmonary vascular tree in its entirety will significantly promote the clinical translation of the next generation of bioengineered whole lungs.

Endothelial cell–lined skeletal muscle ventricles in circulation

Skeletal muscle ventricles were constructed from the latissimus dorsi in six dogs by wrapping the muscle around a polypropylene mandrel. Jugular vein endothelial cells were harvested enzymatically and grown in tissue culture. After 3 weeks of vascular delay and 4 weeks of electrical conditioning, five skeletal muscle ventricles were seeded with 5 to 8 x 10(6) autologous endothelial cells by percutaneous injection of a cellular suspension into the lumen of the skeletal muscle ventricle; one skeletal muscle ventricle was injected with culture medium alone as an unseeded control. The autologous endothelial cells were all prelabeled with a lipid-bound cellular marker, PKH-26. After an additional 4 weeks of electrical conditioning, the mandrels were removed and the skeletal muscle ventricles were connected to the descending thoracic aorta and activated to contract during cardiac diastole at a 1:2 ratio with the heart. After 3 hours of continuous pumping, mean diastolic pressure was increased by 35% (58 +/- 7 versus 78 +/- 6 mm Hg, p < 0.05). At this time, the skeletal muscle ventricles were excised for histologic examination. Sections stained with hematoxylin and eosin revealed a continuous cellular layer lining the skeletal muscle ventricle; no cells were present on the lumen of the control skeletal muscle ventricle. All seeded skeletal muscle ventricles exhibited fluorescence as a result of the PKH-26 cellular marker. Immunofluorescent staining with antibodies to von Willebrand factor and ultrastructural analysis with an electron microscope confirmed the endothelial character of these cells lining the lumen of the skeletal muscle ventricles. The ability to create endothelial cell-lined muscular pumping chambers holds important implications for the resolution of thrombotic events in cardiac assist devices as well as toward the clinical application of skeletal muscle ventricles.