Sustained delivery of the bone morphogenetic proteins BMP-2 and BMP-7 for cartilage repair and regeneration in osteoarthritis.

Objective: Attempts to utilise growth factors (GF) such as bone morphogenic proteins (BMPs) to treat osteoarthritis (OA) in the clinic have not secured widespread adoption. However, the novel crystalline GF formulation called PODS offers new perspectives. This study investigated the hypothesis that Polyhedrin Delivery System (PODS) BMP-2 and PODS BMP-7, compared with conventional BMP-2 and BMP-7 increase capacity for cartilage repair. Design: Sustained release from PODS BMP-2 and PODS BMP-7 and their effects on OA patient-derived cells as well as a chondrocyte cell line were first assessed in vitro. Here, extra cellular matrix (ECM) protein gene expression and actual ECM deposition were measured and compared to the effect achieved with conventional, soluble BMPs. Subsequently, in an established murine model of cartilage regeneration of the knee joint, changes were traced over 8 weeks and scored with two metrics, modified Pineda and Mankin. Results: Both crystalline PODS BMP formulations strongly induced proliferation in primary as well as immortal cell line chondrocytes, outperforming conventional soluble BMP-2 and BMP-7. Furthermore, ECM-producing genes were upregulated and the production of ECM could be demonstrated. In the murine cartilage regeneration model, both PODS BMP-2 and PODS-BMP-7 improved cartilage repair assessed with both histological scoring methods. Conclusions: This study showed that the sustained release of GF from PODS BMPs is effective in promoting chondrogenesis in vitro. The small animal data suggests that this novel approach of delivering therapeutic proteins sustainably and locally to the knee has promise for developing future disease-modifying therapies of OA.

Sustained Neurotrophin Release from Protein Nanoparticles Mediated by Matrix Metalloproteinases Induces the Alignment and Differentiation of Nerve Cells.

The spatial and temporal availability of cytokines, and the microenvironments this creates, is critical to tissue development and homeostasis. Creating concentration gradients in vitro using soluble proteins is challenging as they do not provide a self-sustainable source. To mimic the sustained cytokine secretion seen in vivo from the extracellular matrix (ECM), we encapsulated a cargo protein into insect virus-derived proteins to form nanoparticle co-crystals and studied the release of this cargo protein mediated by matrix metalloproteinase-2 (MMP-2) and MMP-8. Specifically, when nerve growth factor (NGF), a neurotrophin, was encapsulated into nanoparticles, its release was promoted by MMPs secreted by a PC12 neuronal cell line. When these NGF nanoparticles were spotted onto a cover slip to create a uniform circular field, movement and alignment of PC12 cells via their extended axons along the periphery of the NGF nanoparticle field was observed. Neural cell differentiation was confirmed by the expression of specific markers of tau, neurofilament, and GAP-43. Connections between the extended axons and the growth cones were also observed, and expression of connexin 43 was consistent with the formation of gap junctions. Extensions and connection of very fine filopodia occurred between growth cones. Our studies indicate that crystalline protein nanoparticles can be utilized to generate a highly stable cytokine gradient microenvironment that regulates the alignment and differentiation of nerve cells. This technique greatly simplifies the creation of protein concentration gradients and may lead to therapies for neuronal injuries and disease.

The regulation of sclerostin by cathepsin K in periodontal ligament cells.

Sclerostin is a clinically important protein with key functions in the musculoskeletal system playing a key role in bone formation and remodelling. Whilst a wide range of mechanisms have been identified which regulate sclerostin expression, little is known about the degradation of the protein. The aim of this study was to identify enzymes capable of degrading sclerostin in peridontal ligament (PDL) fibroblasts cells in vitro and to investigate the functionality of these enzymes. We have demonstrated that cathepsin K cleaves sclerostin in vitro in PDL. We have shown that cathepsin K and sclerostin are co-localised in PDL cells and, using cathepsin K knockdown experiments, we have shown that cathepsin K actively controls sclerostin levels in these cells, through a lysosomal mechanism that is affected by hypoxia. These results are the first description of the degradative control of sclerostin in musculoskeletally derived cells in vitro and suggest that degradation of the protein may well play an important role in the control of bone formation and remodelling.

New insights into the location and form of sclerostin.

Sclerostin is widely reported to be a monomeric osteocyte specific protein. In this study we have investigated whether sclerostin is produced in different forms and in which cell and tissue types they are produced. We have demonstrated that recombinant sclerostin is composed of monomers and dimers, and that these, and other forms, notably 46 and 70 kDa forms, are found widely throughout the musculo-skeletal system. We have shown that 'dimeric' sclerostin is highly resistant to reduction, implying the presence of highly stable, non-reducible covalent bonds. We have also demonstrated that the form of sclerostin is not associated with the mineralisation state of the tissue or cell. Sclerostin was secreted by bone explants as high molecular weight forms that were reducible to the dimeric form. This dimeric form was detected in sera and in non-skeletal soft tissues specifically kidney, live, heart and lung. We therefore hypothesise: (a) sclerostin exists in multiple forms not associated with the mineralised state of the cell/tissue and (b) circulating sclerostin is dimeric, as is the sclerostin found in non-musculoskeletal soft tissues. These observations may have significant implications for the therapeutic modulation of sclerostin.