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

Addressing bone defects is a complex medical challenge that involves dealing with various skeletal conditions, including fractures, osteoporosis (OP), bone tumours, and bone infection defects. Despite the availability of multiple conventional treatments for these skeletal conditions, numerous limitations and unresolved issues persist. As a solution, advancements in biomedical materials have recently resulted in novel therapeutic concepts. As an emerging biomaterial for bone defect treatment, graphene oxide (GO) in particular has gained substantial attention from researchers due to its potential applications and prospects. In other words, GO scaffolds have demonstrated remarkable potential for bone defect treatment. Furthermore, GO-loaded biomaterials can promote osteoblast adhesion, proliferation, and differentiation while stimulating bone matrix deposition and formation. Given their favourable biocompatibility and osteoinductive capabilities, these materials offer a novel therapeutic avenue for bone tissue regeneration and repair. This comprehensive review systematically outlines GO scaffolds’ diverse roles and potential applications in bone defect treatment.

Cite this article: Bone Joint Res 2024;13(12):725–740.

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

Bone regeneration includes a well-orchestrated series of biological events of bone induction and conduction. Among them, the Wnt/β-catenin signaling pathway is critical for bone regeneration. Being involved in several developmental processes, Wnt/β-catenin signaling must be safely targeted. There are currently only few specific therapeutic agents which are FDA-approved and already entered clinical trials. A published work has shown that Tideglusib, a selective and irreversible small molecule non-ATP-competitive glycogen synthase kinase 3-β(GSK-3β) inhibitor currently in trial for Alzheimer's patients, can promote tooth growth and repair cavities. [1]Despite some differences, they are some similarities between bone and tooth formation and we hypothesise that this new drug could represent a new avenue to stimulate bone healing. In this work, we locally delivered Tideglusib (GSK3β inhibitor) in the repair of femoral cortical window defects and investigated bone regeneration. A biodegradable FDA-approved collagen sponge was soaked in GSK-3βinhibitor solution or vehicle only (DMSO) and was implanted in 1 × 2 mm unicortical defects created in femora of 35 adult wild-type male mice. Bone defect repair on control and experimental (GSK-3βinhibitor) groups was assessed after 1 week (n=22), 2 weeks (n=24) and 4 weeks (n=24) with microCT and histological analysis foralkaline phosphatase (ALP, osteoblast activity), tartrate resistant acid phosphatase (TRAP, osteoclasts), and immunohistochemistry to confirm the activation of the Wnt/β-catenin pathway. Our results showed that Tideglusib significantly enhanced cortical bone bridging (20.6 ±2.3) when compared with the control (12.7 ±1.9, p=0.001). Activity of GSK-3β was effectively downregulated at day 7 and 14 resulting in a higher accumulation of active β-catenin at day 14 in experimental group (2.5±0.3) compared to the control (1.1±0.2, p=0.03). Furthermore, the onset of ALP activity appears earlier in the experimental group (day 14, 1.79±0.28), a level of activity never reached at any end-point by the control defects. At 4 weeks treatment, we observed a significant drop in ALP in the experimental group (0.47±0.05) compared to the control (1.01±0.19, p=0.02) and a decrease in osteoclast (experimental=1.32±0.36, control=2.23±0.67, p=0.04). Local downregulation of GSK-3β by tideglusib during bone defect repair resulted in significant increase in amount of new bone formation. The early upregulation of osteoblast activity is one explanation of bone healing augmentation. This is likely the effect of upregulation of β-catenin following pharmaceutical inhibition of GSK-3β since β-catenin activation is known to positively regulate osteoblasts, once committed to the osteoblast lineage. As a GSK-3β inhibitor, Tideglusib demonstrates a different mechanism of action compared with other GSK-3β antagonists as treatment was started immediately upon injury and did not interfere with precursor cells recruitment and commitment. This indicates that tideglusib could be used at the fracture site during the initial intraoperative internal fixation without the need for further surgery. This safe and FDA-approved drug could be used in prevention of non-union in patients presenting with high risk for fracture-healing complications

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

Cite this article: Bone Joint Res 2023;12(5):311–312.

Autogenous bone grafting limitations have motivated the development of Tissue-Engineered (TE) biomaterials that offer an alternative as bone void fillers. However, the lack of a blood supply within implanted constructs may result in avascular necrosis and construct failure. 1. The aim of this project was to investigate the potential of novel TE constructs to promote vascularisation and bone defect repair using two distinct approaches. In Study 1, we investigated the potential of a mesenchymal stem cell (MSC) and endothelial cell (EC) co-culture to stimulate pre-vascularisation of biomaterials prior to in vivo implantation. 2. In Study 2, we investigated the potential of TE hypertrophic cartilage to promote the release of angiogenic factors such as VEGF, vascular invasion and subsequent endochondral bone formation in an in vivo model. Collagen-only (Coll), collagen-glycosaminoglycan (CG) and collagen-hydroxyapatite (CHA) scaffolds were fabricated by freeze-drying. 3. , seeded with cells and implanted into critical-sized calvarial and femoral defects in immunocompetent rats. In Study 1, Coll and CG scaffolds were initially seeded with ECs, allowed to form capillary-like networks before the delayed addition of MSCs and continued culture prior to calvarial implantation. In Study 2, CG and CHA scaffolds were seeded with MSCs and cultured under chondrogenic and subsequent hypertrophic conditions to form a cartilage pre-cursor prior to calvarial and femoral implantation in vivo. MicroCT and histomorphometry quantification demonstrated the ability of both systems to support increased bone formation compared to controls. Moreover, the greatest levels of bone formation were observed in the CG groups, notably in those containing cartilage tissue (Study 2). Assessment of the immune response suggests the addition of MSCs promotes the polarisation of macrophages away from inflammation (M1) towards a pro-remodelling phenotype (M2). We have developed distinct collagen-based systems that promote vascularisation and ultimately enhance bone formation, confirming their potential as advanced strategies for bone repair applications

Aims

Several artificial bone grafts have been developed but fail to achieve anticipated osteogenesis due to their insufficient neovascularization capacity and periosteum support. This study aimed to develop a vascularized bone-periosteum construct (VBPC) to provide better angiogenesis and osteogenesis for bone regeneration.

Large bone defect repair has always presented a difficult treatment problem. Marrow-derived osteogenic progenitor cells combined with hydroxyapatite (HA) were used for segmental bone reconstruction. The validity of this model has been shown for the repair of bone defects of critical size in large animal models. We used this cell-based therapeutic approach to treat three patients with large bone defects. The patients were 41, 22 and 16 years old and had large tibial, ulnar and humeral diaphyseal gaps that ranged in size from 3.0 to 28.3 cm. 3. Marrow samples were harvested from the iliac crest and osteogenic progenitors isolated and expanded “ex vivo”. The expanded cells were then combined with a highly macroporous bioceramic scaffold whose size and shape reflected each individual bony defect. The cell/bioceramic composites were implanted at the lesion sites. External fixation was used to stabilise the grafts. At present all patients have been followed up for 4–5 years. Already after the first month after surgery an initial integration at the bone/implant interface was evident. Bone formation in the implants, assessed by X-ray, progressed steadily in the follow-up period. Two patients achieved full functional recovery at 6 months after surgery, one patient at 12 months after surgery. The present report shows that large segmental bone reconstruction can be achieved in humans using osteoprogenitor cells. This technique can be improved by a more biodegradable and more biomechanically resistant scaffold use

Aims

In contrast to operations performed for other fractures, there is a high incidence rate of surgical site infection (SSI) post-open reduction and internal fixation (ORIF) done for tibial plateau fractures (TPFs). This study investigates the effect of induced membrane technique combined with internal fixation for managing SSI in TPF patients who underwent ORIF.

Aims

The use of 3D-printed titanium implant (DT) can effectively guide bone regeneration. DT triggers a continuous host immune reaction, including macrophage type 1 polarization, that resists osseointegration. Interleukin 4 (IL4) is a specific cytokine modulating osteogenic capability that switches macrophage polarization type 1 to type 2, and this switch favours bone regeneration.

Objective. In the present study, we aimed to assess whether gelatin/β-tricalcium phosphate (β-TCP) composite porous scaffolds could be used as a local controlled release system for vancomycin. We also investigated the efficiency of the scaffolds in eliminating infections and repairing osteomyelitis defects in rabbits. Methods. The gelatin scaffolds containing differing amounts of of β-TCP (0%, 10%, 30% and 50%) were prepared for controlled release of vancomycin and were labelled G-TCP0, G-TCP1, G-TCP3 and G-TCP5, respectively. The Kirby-Bauer method was used to examine the release profile. Chronic osteomyelitis models of rabbits were established. After thorough debridement, the osteomyelitis defects were implanted with the scaffolds. Radiographs and histological examinations were carried out to investigate the efficiency of eliminating infections and repairing bone defects. Results. The prepared gelatin/β-TCP scaffolds exhibited a homogeneously interconnected 3D porous structure. The G-TCP0 scaffold exhibited the longest duration of vancomycin release with a release duration of eight weeks. With the increase of β-TCP contents, the release duration of the β-TCP-containing composite scaffolds was decreased. The complete release of vancomycin from the G-TCP5 scaffold was achieved within three weeks. In the treatment of osteomyelitis defects in rabbits, the G-TCP3 scaffold showed the most efficacious performance in eliminating infections and repairing bone defects. Conclusions. The composite scaffolds could achieve local therapeutic drug levels over an extended duration. The G-TCP3 scaffold possessed the optimal porosity, interconnection and controlled release performance. Therefore, this scaffold could potentially be used in the treatment of chronic osteomyelitis defects. Cite this article: J. Zhou, X. G. Zhou, J. W. Wang, H. Zhou, J. Dong. Treatment of osteomyelitis defects by a vancomycin-loaded gelatin/β-tricalcium phosphate composite scaffold. Bone Joint Res 2018;7:46–57. DOI: 10.1302/2046-3758.71.BJR-2017-0129.R2

Large bone defects remain a tremendous clinical challenge. There is growing evidence in support of treatment strategies that direct defect repair through an endochondral route, involving a cartilage intermediate. While culture-expanded stem/progenitor cells are being evaluated for this purpose, these cells would compete with endogenous repair cells for limited oxygen and nutrients within ischaemic defects. Alternatively, it may be possible to employ extracellular vesicles (EVs) secreted by culture-expanded cells for overcoming key bottlenecks to endochondral repair, such as defect vascularization, chondrogenesis, and osseous remodelling. While mesenchymal stromal/stem cells are a promising source of therapeutic EVs, other donor cells should also be considered. The efficacy of an EV-based therapeutic will likely depend on the design of companion scaffolds for controlled delivery to specific target cells. Ultimately, the knowledge gained from studies of EVs could one day inform the long-term development of synthetic, engineered nanovesicles. In the meantime, EVs harnessed from in vitro cell culture have near-term promise for use in bone regenerative medicine. This narrative review presents a rationale for using EVs to improve the repair of large bone defects, highlights promising cell sources and likely therapeutic targets for directing repair through an endochondral pathway, and discusses current barriers to clinical translation.

Cite this article: E. Ferreira, R. M. Porter. Harnessing extracellular vesicles to direct endochondral repair of large bone defects. Bone Joint Res 2018;7:263–273. DOI: 10.1302/2046-3758.74.BJR-2018-0006.

Objectives

Long bone defects often require surgical intervention for functional restoration. The ‘gold standard’ treatment is autologous bone graft (ABG), usually from the patient’s iliac crest. However, autograft is plagued by complications including limited supply, donor site morbidity, and the need for an additional surgery. Thus, alternative therapies are being actively investigated. Autologous bone marrow (BM) is considered as a candidate due to the presence of both endogenous reparative cells and growth factors. We aimed to compare the therapeutic potentials of autologous bone marrow aspirate (BMA) and ABG, which has not previously been done.

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.