Adenovirus-Based Gene Therapy for Bone Regeneration: A Comparative Analysis of In Vivo and Ex Vivo BMP2 Gene Delivery.

Adenovirus-mediated gene therapy is a promising tool in bone regenerative medicine. In this work, gene-activated matrices (GAMs) composed of (1) polylactide granules (PLA), which serve as a depot for genetic constructs or matrices for cell attachment, (2) a PRP-based fibrin clot, which is a source of growth factors and a binding gel, and (3) a BMP2 gene providing osteoinductive properties were studied. The study aims to compare the effectiveness of in vivo and ex vivo gene therapy based on adenoviral constructs with the BMP2 gene, PLA particles, and a fibrin clot for bone defect healing. GAMs with Ad-BMP2 and MSC(Ad-BMP2) show osteoinductive properties both in vitro and in vivo. However, MSCs incubated with GAMs containing transduced cells showed a more significant increase in osteopontin gene expression, protein production, Alpl activity, and matrix mineralization. Implantation of the studied matrices into critical-size calvarial defects after 56 days promotes the formation of young bone. The efficiency of neoosteogenesis and the volume fraction of newly formed bone tissue are higher with PLA/PRP-MSC(Ad-BMP2) implantation (33%) than PLA/PRP-Ad-BMP2 (28%). Thus, ex vivo adenoviral gene therapy with the BMP2 gene has proven to be a more effective approach than the in vivo delivery of gene constructs for bone regeneration.

Comparative Efficiency of Gene-Activated Matrices Based on Chitosan Hydrogel and PRP Impregnated with BMP2 Polyplexes for Bone Regeneration.

Gene therapy is one of the most promising approaches in regenerative medicine. Gene-activated matrices provide stable gene expression and the production of osteogenic proteins in situ to stimulate osteogenesis and bone repair. In this study, we developed new gene-activated matrices based on polylactide granules (PLA) impregnated with BMP2 polyplexes and included in chitosan hydrogel or PRP-based fibrin hydrogel. The matrices showed high biocompatibility both in vitro with mesenchymal stem cells and in vivo when implanted intramuscularly in rats. The use of porous PLA granules allowed the inclusion of a high concentration of polyplexes, and the introduction of the granules into hydrogel provided the gradual release of the plasmid constructs. All gene-activated matrices showed transfecting ability and ensured long-term gene expression and the production of target proteins in vitro. At the same time, the achieved concentration of BMP-2 was sufficient to induce osteogenic differentiation of MSCs. When implanted into critical-size calvarial defects in rats, all matrices with BMP2 polyplexes led to new bone formation. The most significant effect on osteoinduction was observed for the PLA/PRP matrices. Thus, the developed gene-activated matrices were shown to be safe and effective osteoplastic materials. PLA granules and PRP-based fibrin hydrogel containing BMP2 polyplexes were shown to be the most promising for future applications in bone regeneration.

Osteoinductive Moldable and Curable Bone Substitutes Based on Collagen, BMP-2 and Highly Porous Polylactide Granules, or a Mix of HAP/ß-TCP.

In dentistry, maxillofacial surgery, traumatology, and orthopedics, there is a need to use osteoplastic materials that have not only osteoinductive and osteoconductive properties but are also convenient for use. In the study, compositions based on collagen hydrogel were developed. Polylactide granules (PLA) or a traditional bone graft, a mixture of hydroxyapatite and ß-tricalcium phosphate (HAP/ß-TCP), were used for gel filling to improve mechanical osteoconductive properties of compositions. The mechanical tests showed that collagen hydrogels filled with 12 wt% highly porous PLA granules (elastic modulus 373 ± 55 kPa) or 35 wt% HAP/ß-TCP granules (elastic modulus 451 ± 32 kPa) had optimal manipulative properties. All composite components were cytocompatible. The cell's viability was above 90%, and the components' structure facilitated the cell's surface adhesion. The bone morphogenetic protein-2 (BMP-2) provided osteoinductive composition properties. It was impregnated directly into the collagen hydrogel with the addition of fibronectin or inside porous PLA granules. The implantation of a collagen hydrogel with BMP-2 and PLA granules into a critical-size calvarial defect in rats led to the formation of the most significant volume of bone tissue: 61 ± 15%. It was almost 2.5 times more than in the groups where a collagen-fibronectin hydrogel with a mixture of HAP/ß-TCP (25 ± 7%) or a fibronectin-free composition with porous PLA granules impregnated with BMP-2 (23 ± 8%) were used. Subcutaneous implantation of the compositions also showed their high biocompatibility and osteogenic potential in the absence of a bone environment. Thus, the collagen-fibronectin hydrogel with BMP-2 and PLA granules has optimal biocompatibility, osteogenic, and manipulative properties.

Influence of the Degree of Deacetylation of Chitosan and BMP-2 Concentration on Biocompatibility and Osteogenic Properties of BMP-2/PLA Granule-Loaded Chitosan/ß-Glycerophosphate Hydrogels.

Compositions based on chitosan/ß-glycerophosphate hydrogels with highly porous polylactide granules can be used to obtain moldable bone graft materials that have osteoinductive and osteoconductive properties. To eliminate the influence of such characteristics as chain length, degree of purification, and molecular weight on a designed material, the one-stock chitosan sample was reacetylated to degrees of deacetylation (DD%) of 19.5, 39, 49, 55, and 56. A study of the chitosan/ß-glycerophosphate hydrogel with chitosan of a reduced DD% showed that a low degree of deacetylation increased the MSCs (multipotent stromal cells) viability rate in vitro and reduced the leukocyte infiltration in subcutaneous implantation to Wistar rats in vivo. The addition of 12 wt% polylactide granules resulted in optimal composite mechanical and moldable properties, and increased the modulus of elasticity of the hydrogel-based material by approximately 100 times. Excessive filling of the material with PLA (polylactide) granules (more than 20%) led to material destruction at a ~10% strain. Osteoinductive and osteoconductive properties of the chitosan hydrogel-based material with reacetylated chitosan (39 DD%) and highly porous polylactide granules impregnated with BMP-2 (bone morphogenetic protein-2) have been demonstrated in models of orthotopic and ectopic bone formation. When implanted into a critical-size calvarial defect in rats, the optimal concentration of BMP-2 was 10 µg/mL: bone tissue areas filled the entire material's thickness. Implantation of the material with 50 µg/mL BMP-2 was accompanied with excessive growth of bone tissue and material displacement beyond the defect. Significant osteoinductive and osteoconductive properties of the material with 10 µg/mL of BMP-2 were also shown in subcutaneous implantation.