Gene delivery of bone morphogenetic protein-2 plasmid DNA promotes bone formation in a large animal model.

In the field of bone regeneration, BMP-2 is considered one of the most important growth factors because of its strong osteogenic activity, and is therefore extensively used in clinical practice. However, the short half-life of BMP-2 protein necessitates the use of supraphysiological doses, leading to severe side-effects. This study investigated the efficiency of bone formation at ectopic and orthotopic sites as a result of a low-cost, prolonged presence of BMP-2 in a large animal model. Constructs consisting of alginate hydrogel and BMP-2 cDNA, together acting as a non-viral gene-activated matrix, were combined with goat multipotent stromal cells (gMSCs) and implanted in spinal cassettes or, together with ceramic granules, intramuscularly in goats, both for 16 weeks. Bone formation occurred in all cell-seeded ectopic constructs, but the constructs containing both gMSCs and BMP-2 plasmid DNA showed higher collagen I and bone levels, indicating an osteogenic effect of the BMP-2 plasmid DNA. This was not seen in unseeded constructs, even though transfected, BMP-2-producing cells were detected in all constructs containing plasmid DNA. Orthotopic constructs showed mainly bone formation in the unseeded groups. Besides bone, calcified alginate was present in these groups, acting as a surface for new bone formation. In conclusion, transfection of seeded or resident cells from this DNA delivery system led to stable expression of BMP-2 during 16 weeks, and promoted osteogenic differentiation and subsequent bone formation in cell-seeded constructs at an ectopic location and in cell-free constructs at an orthotopic location in a large animal model.

Bone morphogenetic protein-2 plasmid DNA as a substitute for bone morphogenetic protein-2 protein in bone tissue engineering.

Bone regeneration is one of the focus points in the field of regenerative medicine. A well-known stimulus of bone formation is bone morphogenetic protein-2 (BMP-2), which has already been extensively used in clinical applications. However, due to a short half-life, supraphysiological doses are applied resulting in severe side effects such as ectopic bone formation or even loss of bone. We compared the effectivity of transient BMP-2 gene delivery with the BMP-2 protein at clinical (high) and physiological (low) doses by subcutaneous implantation of alginate-based constructs in mice. After 6 weeks of implantation, both the protein laden constructs and BMP-2 plasmid DNA-based constructs showed similar early bone onset and elevated bone formation compared to controls without any BMP-2 added. We found no differences in efficiency by using BMP-2 plasmid DNA or any of the BMP-2 protein dosages. Therefore, we conclude that BMP-2 plasmid DNA-based gene therapy in alginate is a promising new strategy for BMP-2 administration for bone (re)generation.

Porous bioprinted constructs in BMP-2 non-viral gene therapy for bone tissue engineering.

A well-known osteogenic agent in the field of regenerative medicine is bone morphogenetic protein-2 (BMP-2). Non-viral delivery of a plasmid containing the gene encoding BMP-2 has shown to induce bone formation in vivo. In order to develop gene activated matrices into larger constructs, we created porosity in a hydrogel using bioprinting technology, thereby allowing better diffusion and blood vessel ingrowth. We were able to produce 3D constructs that were accurate and reproducible in size, shape and pore geometry. Constructs consisting of alginate supplemented with multipotent stromal cells (MSCs) and calcium phosphate particles were printed either in a porous or a non-porous/solid fashion. The plasmid DNA encoding BMP-2 was included in the constructs. Porous constructs were reproducibly bioprinted and remained intact for at least 14 days in culture. Cells were efficiently transfected by the plasmid DNA, and differentiated towards the osteogenic lineage as shown by elevated BMP-2 and ALP production. Porous constructs performed in the first week were better in producing BMP-2 than solid constructs. However, after implantation for six weeks subcutaneously in nude mice, no bone formation was seen, which calls for optimization of the biomaterials used. In conclusion, we show for the first time a model in which 3D printing and non-viral gene therapy can be combined.

Non-viral gene therapy for bone tissue engineering.

The possibilities of using gene therapy for bone regeneration have been extensively investigated. Improvements in the design of new transfection agents, combining vectors and delivery/release systems to diminish cytotoxicity and increase transfection efficiencies have led to several successful in vitro, ex vivo and in vivo strategies. These include growth factor or short interfering ribonucleic acid (siRNA) delivery, or even enzyme replacement therapies, and have led to increased osteogenic differentiation and bone formation in vivo. These results provide optimism to consider use in humans with some of these gene-delivery strategies in the near future.

Influence of endothelial progenitor cells and platelet gel on tissue-engineered bone ectopically in goats.

For the development of functional large bone tissue constructs, optimal oxygen and nutrients supply of seeded multipotent stromal cells (MSCs) is likely dependent on vascularization. The introduction of endothelial progenitor cells (EPCs) to MSC cultures might enhance vascularization and therefore increase bone formation. In this study we cocultured MSCs and EPCs and investigated performance and bone formation both in vitro and in vivo. The EPCs used were characterized by uptake of acetylated low-density lipoproteins, binding of isolectin B4 and expression of platelet endothelial cell adhesion molecule. EPC/MSC in vitro coculture showed that both cell types exerted a positive effect on proliferation of the other. For the in vivo studies, we applied platelet-leukocyte gel (PLG), containing several growth factors, as a means to further induce vascularization and thereby enhance bone formation. Cocultures and monocultures were combined with either PLG or plasma, seeded on ceramic scaffolds, and implanted intramuscularly in nine goats. After 16 weeks of implantation, it turned out that seeding MSCs and EPCs both resulted in significant more bone lining the scaffold than the unseeded controls, and MSCs and cocultures with highest MSC/EPC ratio were most competent. Cocultures did not show a higher bone content than the monoculture of MSCs. Fluorochrome incorporation results showed that the presence of seeded cells, either MSCs or EPCs, in the constructs accelerated bone formation. Finally, the addition of PLG instead of plasma did have a positive influence on the quantity of incorporated bone.