Preoperative and Postoperative Imaging and Outcome Scores for Osteochondral Lesion Repair of the Ankle.

Cartilage lesions to the ankle joint are common and can result in pain and functional limitations. Surgical treatment aims to restore the damaged cartilage's integrity and quality. However, the current evidence for establishing best practices in ankle cartilage repair is characterized by limited quality and a low level of evidence. One of the contributing factors is the lack of standardized preoperative and postoperative assessment methods to evaluate treatment effectiveness and visualize repaired cartilage. This review article seeks to examine the importance of preoperative imaging, classification systems, patient-reported outcome measures, and radiological evaluation techniques for cartilage repair surgeries.

BMP-2 gene delivery in cell-loaded and cell-free constructs for bone regeneration.

To induce osteogenicity in bone graft substitutes, plasmid-based expression of BMP-2 (pBMP-2) has been successfully applied in gene activated matrices based on alginate polymer constructs. Here, we investigated whether cell seeding is necessary for non-viral BMP-2 gene expression in vivo. Furthermore, to gain insight in the role of BMP-producing cells, we compared inclusion of bone progenitor cells with non-osteogenic target cells in gene delivery constructs. Plasmid DNA encoding GFP (pGFP) was used to trace transfection of host tissue cells and seeded cells in a rat model. Transgene expression was followed in both cell-free alginate-ceramic constructs as well as constructs seeded with syngeneic fibroblasts or multipotent mesenchymal stromal cells (MSCs). Titration of pGFP revealed that the highest pGFP dose resulted in frequent presence of positive host cells in the constructs. Both cell-loaded groups were associated with transgene expression, most effectively in the MSC-loaded constructs. Subsequently, we investigated effectiveness of cell-free and cell-loaded alginate-ceramic constructs with pBMP-2 to induce bone formation. Local BMP-2 production was found in all groups containing BMP-2 plasmid DNA, and was most pronounced in the groups with MSCs transfected with high concentration pBMP-2. Bone formation was only apparent in the recombinant protein BMP-2 group. In conclusion, we show that non-viral gene delivery of BMP-2 is a potentially effective way to induce transgene expression in vivo, both in cell-seeded as well as cell-free conditions. However, alginate-based gene delivery of BMP-2 to host cells or seeded cells did not result in protein levels adequate for bone formation in this setting, calling for more reliable scaffold compatible transfection methods.

The Osteoinductive Effect of Controlled Bone Morphogenic Protein 2 Release Is Location Dependent.

IMPACT STATEMENT: The main challenge in bone morphogenic protein 2 (BMP-2)-based application lies in finding strategies to prolong its biologic activity as it has a short biological half-life. The present study uses a phosphate-modified oligo[(polyethylene glycol) fumarate] hydrogel that can be tuned to achieve differential release profiles of biologically active BMP-2 release. We demonstrate that this platform outperforms Infuse®, currently used in the clinic and that the osteoinductive effect of BMP-2 is location dependent. Altogether, this study stresses the importance of evaluating efficacy of bone tissue engineering strategies at an orthotopic location rather than subcutaneously. Even more so, it emphasizes the role of biomaterials as a scaffold to achieve proper bone tissue engineering.

Osteoinduction by Ex Vivo Nonviral Bone Morphogenetic Protein Gene Delivery Is Independent of Cell Type.

Ex vivo nonviral gene delivery of bone inductive factors has the potential to heal bone defects. Due to their inherent role in new bone formation, multipotent stromal cells (MSCs) have been studied as the primary target cell for gene delivery in a preclinical setting. The relative contribution of autocrine and paracrine mechanisms, and the need of osteogenic cells, remains unclear. This study investigates the contribution of MSCs as producer of transgenic bone morphogenetic proteins (BMPs) and to what extent the seeded MSCs participate in actual osteogenesis. Rat-derived MSCs or fibroblasts (FBs) were cotransfected with pBMP-2 and pBMP-6 or pBMP-7 via nucleofection. The bioactivity of BMP products was shown through in vitro osteogenic differentiation assays. To investigate their role in new bone formation, transfected cells were seeded on ceramic scaffolds and implanted subcutaneously in rats. Bone formation was assessed by histomorphometry after 8 weeks. As a proof of principle, we also investigated the suitability of bone marrow-derived mononuclear cells and the stromal vascular fraction isolated from adipose tissue for a one-stage gene delivery strategy. Bone formation was induced in all conditions containing cells overexpressing BMP heterodimers. Constructs seeded with FBs transfected with BMP-2/6 and MSCs transfected with BMP-2/6 showed comparable bone volumes, both significantly higher than controls. Single-stage gene delivery proved possible and resulted in some bone formation. We conclude that bone formation as a result of ex vivo BMP gene delivery can be achieved even without direct osteogenic potential of the transfected cell type, suggesting that transfected cells mainly function as a production facility for osteoinductive proteins. In addition, single-stage transfection and reimplantation of cells appeared feasible, thus facilitating future clinical translation of the method.

Bone morphogenetic protein-2 nonviral gene therapy in a goat iliac crest model for bone formation.

Treatment and reconstruction of large bone defects, delayed unions, and nonunions is challenging and has resulted in an ongoing search for novel tissue-engineered therapies. Bone morphogenetic protein-2 (BMP-2) gene therapy is a promising strategy to provide sustained production of BMP-2 locally. Alginate polymer-based nonviral gene therapy with BMP-2 plasmid DNA (pBMP-2) in constructs with multipotent mesenchymal stromal cells (MSCs) has resulted in prolonged gene expression and bone formation in vivo. To further translate this technology toward larger animal models, important issues remain to be investigated, such as the necessity of seeded cells as a target for gene therapy. For that purpose, a large animal-screening model in an orthotopic location, with fully separated chambers, was investigated. Four cylinder-shaped implants were placed in the iliac crests of ten goats. Polycaprolactone tubes around each implant allowed bone ingrowth from the underlying bone and bone marrow and ensured separation of the experimental conditions. An empty tube showed low levels of spontaneous bone ingrowth, and implantation of autologous bone indicated proper bone function with respect to remodeling and resorption. Control ceramic scaffolds were compared to scaffolds containing pBMP-2 either or not combined with seeded MSCs. Fluorochrome incorporation evaluated at 3, 6, and 9 weeks and histomorphometry at 12 weeks after implantation revealed clear differences between the groups, with pBMP-2 combined with MSCs being the most effective. The BMP-2 was demonstrated in a variety of bone-residing cells through immunohistochemistry. Further analysis indicated that multinucleated giant cells might have an important role in transgene expression. Taken together, this work introduces a large animal model for studying bone formation at multiple sites simultaneously in an orthotopic location. The model appeared robust, showed no neighboring effects, and demonstrated effectivity of combined cell and gene therapy.

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.