Development of a slow non-viral DNA release system from PDLLA scaffolds fabricated using a supercritical CO2 technique.

Polyamidoamine polymers (PAA) comprising methylene-bisacrylamide/dimethylethylene-diamine monomers were synthesized, complexed with DNA and incorporated into porous P(DL)LA scaffolds by using a supercritical CO(2) (scCO(2)) technique. Scaffolds were made in a dry state consequently there was a need to lyophilize the complexes. A statistically significant reduction of the transfection efficiency was observed in the absence of trehalose when compared to the original complex after freeze-drying. Increasing concentrations (0-10% w/v) of trehalose were added to the complex prior to freeze-drying. Structure dependent differences in DNA binding were evaluated by gel electrophoresis and thermal transition analysis. TEM and PCS showed aggregate formation after freeze-drying without trehalose. Scaffolds were characterized by pore sizes of 173 +/- 73 microm and a porosity of 71%. The transfection potential of the released DNA was investigated by seeding scaffolds with A549 cells and following firefly luciferase as a marker gene after 48 h exposure. Low but continuous levels of transfection were observed for PAA complexes during a 60-day study. Complexes made with Lipofectaminetrade mark gave initially higher levels of DNA release but no further expression was seen after 40 days. Uncomplexed DNA showed background levels of transfection. Culturing cells on 3D scaffolds showed a benefit in retention of transfection activity with time compared to 2D controls. Transfection levels could be increased when cells were grown in OptiMEM. This study demonstrated that PAA/DNA complexes incorporated into a P(DL)LA scaffold made by using scCO(2) processing exhibited a slow release and extended gene expression profile.

Gene therapy used for tissue engineering applications.

This review highlights the advances at the interface between tissue engineering and gene therapy. There are a large number of reports on gene therapy in tissue engineering, and these cover a huge range of different engineered tissues, different vectors, scaffolds and methodology. The review considers separately in-vitro and in-vivo gene transfer methods. The in-vivo gene transfer method is described first, using either viral or non-viral vectors to repair various tissues with and without the use of scaffolds. The use of a scaffold can overcome some of the challenges associated with delivery by direct injection. The ex-vivo method is described in the second half of the review. Attempts have been made to use this therapy for bone, cartilage, wound, urothelial, nerve tissue regeneration and for treating diabetes using viral or non-viral vectors. Again porous polymers can be used as scaffolds for cell transplantation. There are as yet few comparisons between these many different variables to show which is the best for any particular application. With few exceptions, all of the results were positive in showing some gene expression and some consequent effect on tissue growth and remodelling. Some of the principal advantages and disadvantages of various methods are discussed.

Taking tissue-engineering principles into theater: augmentation of impacted allograft with human bone marrow stromal cells.

Human bone marrow contains bone progenitor cells that arise from multipotent mesenchymal stem cells. Seeding bone progenitor cells onto a scaffold can produce a 3D living composite with significant mechanical and biological potential. This article details laboratory and clinical findings from two clinical cases, where different proximal femoral conditions were treated using impacted allograft augmented with marrow-derived autogenous progenitor cells. Autologous bone marrow was seeded onto highly washed morselized allograft and impacted. Samples of the impacted graft were also taken for ex vivo analysis. Both patients made an uncomplicated clinical recovery. Imaging confirmed defect filling with encouraging initial graft incorporation. Histochemical and alkaline phosphatase staining demonstrated that a live composite graft with osteogenic activity had been introduced into the defects. These studies demonstrate that marrow-derived cells can adhere to highly washed morselized allograft, survive the impaction process and proliferate with an osteoblastic phenotype, thus creating a living composite.

Biological and mechanical enhancement of impacted allograft seeded with human bone marrow stromal cells: potential clinical role in impaction bone grafting.

With the demographics of an aging population the incidence of revision surgery is rapidly increasing. Clinical imperatives to augment skeletal tissue loss have brought mesenchymal stem cells to the fore in combination with the emerging discipline of tissue engineering. Impaction bone grafting for revision hip surgery is a recognized technique to reconstitute bone, the success of which relies on a combination of mechanical and biological factors. The use of morsellized allograft is currently the accepted clinical standard providing a good mechanical scaffold with little osteoinductive biological potential. We propose that applying the principles of a tissue engineering paradigm, the combination of human bone marrow stromal cells (hBMSCs) with allograft to produce a living composite, offers a biological and mechanical advantage over the current gold standard of allograft alone. This study demonstrates that hBMSCs combined with allograft can withstand the forces equivalent to a standard femoral impaction and continue to differentiate and proliferate along the bony lineage. In addition, the living composite provides a biomechanical advantage, with increased interparticulate cohesion and shear strength when compared with allograft alone.

Gene delivery in bone tissue engineering: progress and prospects using viral and nonviral strategies.

Bone tissue loss as a consequence of the natural aging process or as a result of trauma and degenerative disease has led to the need for procedures to generate cartilage and bone for a variety of orthopedic applications. The ability to transfer genes into multipotential mesenchymal stem cells, while still in its infancy, offers considerable therapeutic hope in a variety of musculoskeletal disorders. However, the choice of gene delivery method is key. This review examines the various techniques and methods currently available to enable gene transfer into a target population from viral methods (transduction) to nonviral (transfection) methods and the limitations associated with each method. The potential applications and current understanding of each method are presented. Given the demographic challenge of an aging population, the ultimate goal remains the development of simple, safe, and reproducible strategies for gene delivery that will address the pressing orthopedic clinical imperatives of many.

Induction of human osteoprogenitor chemotaxis, proliferation, differentiation, and bone formation by osteoblast stimulating factor-1/pleiotrophin: osteoconductive biomimetic scaffolds for tissue engineering.

The process of bone growth, regeneration, and remodeling is mediated, in part, by the immediate cell-matrix environment. Osteoblast stimulating factor-1 (OSF-1), more commonly known as pleiotrophin (PTN), is an extracellular matrix-associated protein, present in matrices, which act as targets for the deposition of new bone. However, the actions of PTN on human bone progenitor cells remain unknown. We examined the effects of PTN on primary human bone marrow stromal cells chemotaxis, differentiation, and colony formation (colony forming unit-fibroblastic) in vitro, and in particular, growth and differentiation on three-dimensional biodegradable porous scaffolds adsorbed with PTN in vivo. Primary human bone marrow cells were cultured on tissue culture plastic or poly(DL-lactic acid-co-glycolic acid) (PLGA; 75:25) porous scaffolds with or without addition of recombinant human PTN (1 pg-50 ng/ml) in basal and osteogenic conditions. Negligible cellular growth was observed on PLGA scaffold alone, generated using a super-critical fluid mixing method. PTN (50 microg/ml) was chemotactic to human osteoprogenitors and stimulated total colony formation, alkaline phosphatase-positive colony formation, and alkaline phosphatase-specific activity at concentrations as low as 10 pg/ml compared with control cultures. The effects were time-dependent. On three-dimensional scaffolds adsorbed with PTN, alkaline phosphatase activity, type I collagen formation, and synthesis of cbfa-1, osteocalcin, and PTN were observed by immunocytochemistry and PTN expression by in situ hybridization. PTN-adsorbed constructs showed morphologic evidence of new bone matrix and cartilage formation after subcutaneous implantation as well as within diffusion chambers implanted into athymic mice. In summary, PTN has the ability to promote adhesion, migration, expansion, and differentiation of human osteoprogenitor cells, and these results indicate the potential to develop protocols for de novo bone formation for skeletal repair that exploit cell-matrix interactions.

Immunoselection and adenoviral genetic modulation of human osteoprogenitors: in vivo bone formation on PLA scaffold.

The aim of this study was to examine the potential of immunoselected genetically modified human osteoprogenitors to form bone in vivo on porous PLA scaffolds. Human osteoprogenitors from bone marrow were selected using the antibody STRO-1 utilising a magnetically activated cell separation system. The STRO-1(+) fraction isolated 7% of nucleated marrow cells and increased fibroblastic colony formation by 300% and alkaline phosphatase activity by 190% over unselected marrow cell cultures. To engineer bone tissue, STRO-1(+) culture-expanded cells were transduced with AxCAOBMP-2, an adenovirus carrying the human BMP-2 gene, injected into diffusion chambers containing porous PLA scaffolds, and implanted in vivo. After 11 weeks the presence of bone mineral was observed by X-ray analysis and confirmed for mineral by von Kossa, as well as bone matrix composition by Sirius red staining, birefringence, and type I collagen immunohistochemistry. Bone formation in vivo indicates the potential of using immunoselected progenitor cells and ex vivo gene transfer with biodegradable scaffolds, for the development of protocols for the treatment of a wide variety of musculo-skeletal disorders.

Interaction of Neisseria meningitidis with human meningeal cells induces the secretion of a distinct group of chemotactic, proinflammatory, and growth-factor cytokines.

The interactions of Neisseria meningitidis with cells of the leptomeninges are pivotal events in the progression of bacterial leptomeningitis. An in vitro model based on the culture of human meningioma cells was used to investigate the role of the leptomeninges in the inflammatory response. Following challenge with meningococci, meningioma cells secreted specifically the proinflammatory cytokine interleukin-6 (IL-6), the CXC chemokine IL-8, the CC chemokines monocyte chemoattractant protein 1 (MCP-1) and regulated-upon-activation, normal-T-cell expressed and secreted protein (RANTES), and the cytokine growth factor granulocyte-macrophage colony-stimulating factor (GM-CSF). A temporal pattern of cytokine production was observed, with early secretion of IL-6, IL-8, and MCP-1 followed by later increases in RANTES and GM-CSF levels. IL-6 was induced equally by the interactions of piliated and nonpiliated meningococci, whereas lipopolysaccharide (LPS) had a minimal effect, suggesting that other, possibly secreted, bacterial components were responsible. Induction of IL-8 and MCP-1 also did not require adherence of bacteria to meningeal cells, but LPS was implicated. In contrast, efficient stimulation of RANTES by intact meningococci required pilus-mediated adherence, which served to deliver increased local concentrations of LPS onto the surface of meningeal cells. Secretion of GM-CSF was induced by pilus-mediated interactions but did not involve LPS. In addition, capsule expression had a specific inhibitory effect on GM-CSF secretion, which was not observed with IL-6, IL-8, MCP-1, or RANTES. Thus, the data demonstrate that cells of the leptomeninges are not inert but are active participants in the innate host response during leptomeningitis and that there is a complex relationship between expression of meningococcal components and cytokine induction.

Adenoviral BMP-2 gene transfer in mesenchymal stem cells: in vitro and in vivo bone formation on biodegradable polymer scaffolds.

The aim of this study was to determine the feasibility of adenoviral gene transfer into primary human bone marrow osteoprogenitor cells in combination with biodegradeable scaffolds to tissue-engineer bone. Osteoprogenitors were infected with AxCAOBMP-2, a vector carrying the human BMP-2 gene. Alkaline phosphatase activity was induced in C2C12 cells following culture with conditioned media from BMP-2 expressing cells, confirming successful secretion of active BMP-2. Expression of alkaline phosphatase activity, type I collagen and mineralisation confirmed bone cell differentiation and maintenance of the osteoblast phenotype in extended culture for up to 6 weeks on PLGA porous scaffolds. In vivo implantation of adenoviral osteoprogenitor constructs on PLGA biodegradeable scaffolds, using diffusion chambers, also demonstrated bone cell differentiation and production of bone tissue. The maintenance of the osteoblast phenotype in extended culture and generation of mineralised 3-D scaffolds containing such constructs indicate the potential of such bone tissue engineering approaches in bone repair.