The current ‘gold’ standard surgical intervention for critical size bone defect repair involves autologous bone grafting, that risks inadequate graft containment and soft tissue invasion. Here, a new regenerative strategy was explored, that uses a barrier membrane to contain bone graft. The membrane is designed to prevent soft tissue ingrowth, whilst supporting periosteal regrowth, an important component to bone regeneration. This study shows the development of a collagen-based barrier membrane supportive of periosteal-derived mesenchymal stem cell (P-MSC) growth. P-MSC-homing barrier membranes were successfully obtained with nonaligned fibres, via free-surface electrospinning using type I collagen and poly(E-caprolactone) in 1,1,1,3,3,3-Hexafluoro-2-propanol. Introduction of collagen in the electrospinning mixture was correlated with decreased mean fibre diameter (d: 319 nm) and pore size (p: 0.2–0.6 μm), with respect to collagen-free membrane controls (d: 372 nm; p: 1–2 μm). Consequently, as the average MSC diameter is 20 μm, this provides convincing evidence of the creation of a MSC containment membrane. SEM-EDX confirmed Nitrogen and therefore collagen fibre localisation. Quantification of collagen content, using Picro Sirius Red dye, showed a 50% reduction after 24 hours (PBS, 37 °C), followed by a drop to 25% at week 3. The collagen-based membrane has a significantly higher elastic modulus compared to collagen-free control membranes. P-MSCs attached and proliferated when grown onto collagen-based membranes, imaged using confocal microscopy over 3 weeks. A modified transwell cell migration assay was developed, using MINUSHEET® tissue carriers to assess barrier functionality. In line with the matrix architecture, the collagen-based membrane proved to prevent cell migration (via confocal microscopy) in comparison to the migration facilitating positive control. The aforementioned results obtained at molecular, cellular and macroscopic scales, highlight the applicability of this barrier membrane in a new ‘hybrid graft’ regenerative approach for the surgical treatment of critical size bone defects

Critical size bone defects are frequently caused by accidental trauma, oncologic surgery, and infection. Distraction osteogenesis (DO) is a useful technique to promote the repair of critical size bone defects. However, DO is usually a lengthy treatment, therefore accompanied with increased risks of complications such as infections and delayed union. Herein, we developed an innovative intramedullary biodegradable magnesium (Mg) nail to accelerate bone regeneration in critical size bone defect repair during DO. We observed that Mg nail induced almost 4-fold increase of new bone formation and over 5-fold of new vessel formation at 2 weeks after distraction. Mg nail upregulated the expression of calcitonin gene-related peptide (CGRP) in the new bone as compared with the DO alone group. We further revealed that blockade of the sensory nerve by overdose capsaicin blunted Mg nail enhanced critical size bone defect repair during the DO process. Moreover, inhibitors/antagonist of CGRP receptor, FAK, and VEGF receptor blocked the Mg nail stimulated vessel and bone formation. In summary, we revealed, for the first time, a CGRP-FAK-VEGF signaling axis linking sensory nerve and endothelial cells, which may be the main mechanism underlying Mg-enhanced critical size bone defect repair when combined with DO, suggesting a great potential of Mg implants in reducing DO treatment time for clinical applications

Stem cell therapy is an effective means to address the repair of large segmental bone defects. However, the intense inflammatory response triggered by the implants severely impairs stem cell differentiation and tissue regeneration. High-dose transforming growth factor β1 (TGF-β1), the most locally expressed cytokine in implants, inhibits osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) and promotes tissue fibrosis, severely compromising the efficacy of stem cell therapy. Small molecule inhibitors of TGF-β1 can be used to ameliorate the osteogenic disorders caused by high concentrations of TGF-β1, but systemic inhibition of TGF-β1 function will cause strong adverse effects. How to find safe and reliable molecular targets to antagonize TGF-β1 remains to be elucidated. Orphan nuclear receptor Nr4a1, an endogenous inhibitory molecule of TGF-β1, suppresses tissue fibrosis, but its role in BMSC osteogenesis is unclear. We found that TGF-β1 inhibited Nr4a1 expression through HDAC4. Overexpression of Nr4a1 in BMSCs reversed osteogenic differentiation inhibited by high levels of TGF- β1. Mechanistically, RNA sequencing showed that Nr4a1 activated the ECM-receptor interaction and Hippo signaling pathway, which in turn promoted BMSC osteogenesis. In bone defect repair and fracture healing models, transplantation of Nr4a1-overexpressing BMSCs into C57BL/6J mice or treatment with the Nr4a1 agonist Csn-B significantly ameliorated inflammation-induced bone regeneration disorders. In summary, our findings confirm the endogenous inhibitory effect of Nr4a1 on TGF- β1 and uncover the effectiveness of Nr4a1 agonists as a therapeutic tool to improve bone regeneration, which provides a new solution strategy for the treatment of clinical bone defects and inflammatory skeletal diseases

Autologous cancellous bone graft is the gold standard in large bone defect repair. However, studies using autologous bone grafting in rats are rare and donor sites as well as harvesting techniques vary. The aim of this study was to determine the feasibility of autologous cancellous bone graft harvest from 5 different anatomical sites in rats and compare their suitability as donor sites for autologous bone graft. 13 freshly euthanised rats were used to describe the surgical approaches for autologous bone graft harvest from the humerus, iliac crest, femur, tibia and tail vertebrae (n=4), determine the cancellous bone volume and microstructure of those five donor sites using µCT (n=5), and compare their cancellous bone collected qualitatively by looking at cell outgrowth and osteogenic differentiation using an ALP assay and Alizarin Red S staining (n=4). It was feasible to harvest cancellous bone graft from all 5 anatomical sites with the humerus and tail being more surgically challenging. The microstructural analysis showed a significantly lower bone volume fraction, bone mineral density, and trabecular thickness of the humerus and iliac crest compared to the femur, tibia, and tail vertebrae. The harvested volume did not differ between the donor sites. All donor sites apart from the femur yielded primary osteogenic cells confirmed by the presence of ALP and Alizarin Red S stain. Bone samples from the iliac crest showed the most consistent outgrowth of osteoprogenitor cells. The tibia and iliac crest may be the most favourable donor sites considering the surgical approach. However, due to the differences in microstructure of the cancellous bone and the consistency of outgrowth of osteoprogenitor cells, the donor sites may have different healing properties, that need further investigation in an in vivo study

Harnessing the potential of mesenchymal stem cell (MSC) mediated endochondral ossification for the repair of large bone defects represents a promising avenue of investigation as an alternative option to autologous bone transplantation. To date, it has been shown that undifferentiated MSCs are somewhat immune-privileged. In order to induce bone formation from MSCs by endochondral ossification it is usually necessary to first differentiate these cells chondrogenically. However, the status of differentiated cells is less clear than that of undifferentiated MSCs. Furthermore, the fate of implanted bone forming constructs in an allogeneic setting is not known. The potential to use allogeneic MSCs for large bone defect repair would offer opportunities to researchers to develop new therapies using more potent MSC sources and in a more readily available manner with regard to the patient. I will present our research investigating the interactions between chondrogenically primed MSCs and immune cell subsets, namely T cells and dendritic cells. Furthermore, I will discuss the ability of human paediatric MSCs to form bone in the in vivo allogeneic setting

In a rabbit model we investigated the efficacy of a silk fibroin/hydroxyapatite (SF/HA) composite on the repair of a segmental bone defect. Four types of porous SF/HA composites (SF/HA-1, SF/HA-2, SF/HA-3, SF/HA-4) with different material ratios, pore sizes, porosity and additives were implanted subcutaneously into Sprague-Dawley rats to observe biodegradation. SF/HA-3, which had characteristics more suitable for a bone substitite based on strength and resorption was selected as a scaffold and co-cultured with rabbit bone-marrow stromal cells (BMSCs). A segmental bone defect was created in the rabbit radius. The animals were randomised into group 1 (SF/HA-3 combined with BMSCs implanted into the bone defect), group 2 (SF/HA implanted alone) and group 3 (nothing implanted). They were killed at four, eight and 12 weeks for visual, radiological and histological study.

The bone defects had complete union for group 1 and partial union in group 2, 12 weeks after operation. There was no formation of new bone in group 3. We conclude that SF/HA-3 combined with BMSCs supports bone healing and offers potential as a bone-graft substitute.