Bone morphogenetic proteins (BMPs) have been widely investigated for treating non-healing fractures. They participate in bone reconstruction by inducing osteoblast differentiation, and osteoid matrix production.¹ The human recombinant protein of BMP-7 was among the first growth factors approved for clinical use. Despite achieving comparable results to autologous bone grafting, severe side effects have been associated with its use.² Furthermore, BMP-7 was removed from the market.³ These complications are related to the high doses used (1.5-40 miligrams per surgery)² compared to the physiological concentration of BMP in fracture healing (in the nanogram to picogram per milliliter range).⁴ In this study, we use transcript therapy to deliver chemically modified mRNA (cmRNA) encoding BMP-7. Compared to direct use of proteins, transcript therapy allows the sustained synthesis of proteins with native conformation and true post-translational modifications using doses comparable to the physiological ones.⁵ Moreover, cmRNA technology overcomes the safety and affordability limitations of standard gene therapy i.e. pDNA.⁶ BMP-7 cmRNA was delivered using Lipofectamine™ MessengerMAX™ to human mesenchymal stromal cells (hMSCs). We assessed protein expression and osteogenic capacity of hMSCs in monolayer culture and in a house-made, collagen hydroxyapatite scaffold. Using fluorescently-labelled cmRNA we observed an even distribution after loading complexes into the scaffold and a complete release after 3 days. For both monolayer and 3D culture, BMP-7 production peaked at 24 hours post-transfection, however cells transfected in scaffolds showed a sustained expression. BMP-7 transfected hMSCs yielded significantly higher ALP activity and Alizarin red staining at later timepoints compared to the untransfected group. Interestingly, BMP-7 cmRNA treatment triggered expression of osteogenic genes like OSX, RUNX-2 and OPN, which was also reflected in immunostainings. This work highlights the relevance of cmRNA technology that may overcome the shortcomings of protein delivery while circumventing issues of traditional pDNA-based gene therapy for bone regeneration.

Acknowledgement: This work has been performed as part of the cmRNAbone project and has received funding from the European Union's Horizon 2020 research and innovation programme under the Grant Agreement No 874790.

Chronic osteomyelitis (OM) is a progressive, inflammatory infection of bone caused predominantly by S. aureus and requires treatment through surgical debridement and systemic antibiotic administration. We have previously reported the fabrication of an antibiotic-eluting scaffold which is responsive to microbial activity for the treatment of OM. Herein, we ventured to assess the capacity of this antibiotic-eluting scaffold to treat infection in a rabbit model of chronic OM. Infections were established in the radii of New Zealand White rabbits using inoculations of 2×10⁶ CFUs S. aureus JAR 060131 over a period of 4 weeks. Following surgical debridement (6mm), rabbits underwent treatment for a period of 8 weeks until euthanasia. The treatment groups were; 1) empty, 2) antibiotic-eluting scaffold (collagen/hydroxyapatite scaffold loaded with vancomycin) and 3) commercially available antibiotic-eluting fleece (Septocoll E®, collagen fleece loaded with gentamicin). During the treatment period, all groups received systemic antibiotics (Cefazolin 25mg/kg) administered subcutaneously twice daily for 4 weeks. Inoculation of the radius resulted in the development of a sequestrum containing S. aureus, demonstrating the successful establishment of OM. After the 8-week treatment period, 4/5 rabbits in the empty group were still infected, indicating that systemic antibiotic administration following debridement was insufficient to treat the infection. Fewer rabbits in both the antibiotic-eluting scaffold group (2/4) and the antibiotic-eluting fleece group (1/3) were infected. This work demonstrates that the implantation of an antibiotic-eluting biomaterial into a defect following debridement enhances bacterial clearance in conditions of chronic OM.

Background

Articular cartilage has poor repair properties and poses a significant challenge in orthopaedics. Damage as a result of disease or injury frequently leads to formation of an osteochondral defect. Conventional repair methods, including allograft, autograft and microfracture, have a number of disadvantages in terms of cost, associated technical challenges and the requirement for multiple operations. A novel tri-layered scaffold developed in our lab, addresses this issue as it closely matches the structure and composition of osteochondral tissue.

Background

The gradient structure of osteochondral tissue, with bone, calcified and cartilage regions, challenges the design of biomaterials for defect repair. A novel biomimetic tri-layered collagen-based scaffold, designed to replicate these 3 anatomical layers, has been developed within our group and has shown success as an off-the-shelf product in treatment of focal defects in several animal models by recruiting host cells and directing them to form bone and cartilage in the requisite layers. This study aimed to elucidate the mechanism by which the extracellular matrix macromolecules in the scaffold directed stem cell differentiation in each layer.