VEGF with AMD3100 Endogenously Mobilizes Mesenchymal Stem Cells and Improves Fracture Healing

A significant number of fractures develop non‐union. Mesenchymal stem cell (MSC) therapy may be beneficial, however, this requires cell acquisition, culture and delivery. Endogenous mobilization of stem cells offers a non‐invasive alternative. The hypothesis was administration of VEGF and the CXCR4 antagonist AMD3100 would increase the circulating pool of available MSCs and improve fracture healing. Ex‐breeder female wistar rats received VEGF followed by AMD3100, or sham PBS. Blood prepared for culture and colonies were counted. P3 cells were analyzed by flow cytometry, bi‐differentiation. The effect of mobilization on fracture healing was evaluated with 1.5 mm femoral osteotomy stabilized with an external fixator in 12–14 week old female Wistars. The mobilized group had significantly greater number of cfus/ml compared to controls, p = 0.029. The isolated cells expressed 1.8% CD34, 35% CD45, 61% CD29, 78% CD90, and differentiated into osteoblasts but not into adipocytes. The fracture gap in animals treated with VEGF and AMD3100 showed increased bone volume; 5.22 ± 1.7 µm3 and trabecular thickness 0.05 ± 0.01 µm compared with control animals (4.3 ± 3.1 µm3, 0.04 ± 0.01 µm, respectively). Radiographic scores quantifying fracture healing (RUST) showed that the animals in the mobilization group had a higher healing score compared to controls (9.6 vs. 7.7). Histologically, mobilization resulted in significantly lower group variability in bone formation (p = 0.032) and greater amounts of bone and less fibrous tissue than the control group. Clinical significance: This pre‐clinical study demonstrates a beneficial effect of endogenous MSC mobilization on fracture healing, which may have translation potential to prevent or treat clinical fractures at risk of delayed or non‐union fractures

The <i>in vivo</i> effect of a porous titanium alloy flange with hydroxyapatite, silver and fibronectin coatings on soft-tissue integration of intraosseous transcutaneous amputation prostheses

AIMS: The Intraosseous Transcutaneous Amputation Prosthesis (ITAP) may improve quality of life for amputees by avoiding soft-tissue complications associated with socket prostheses and by improving sensory feedback and function. It relies on the formation of a seal between the soft tissues and the implant and currently has a flange with drilled holes to promote dermal attachment. Despite this, infection remains a significant risk. This study explored alternative strategies to enhance soft-tissue integration. MATERIALS AND METHODS: The effect of ITAP pins with a fully porous titanium alloy flange with interconnected pores on soft-tissue integration was investigated. The flanges were coated with fibronectin-functionalised hydroxyapatite and silver coatings, which have been shown to have an antibacterial effect, while also promoting viable fibroblast growth in vitro. The ITAP pins were implanted along the length of ovine tibias, and histological assessment was undertaken four weeks post-operatively. RESULTS: The porous titanium alloy flange reduced epithelial downgrowth and increased soft-tissue integration compared with the current drilled flange. The addition of coatings did not enhance these effects. CONCLUSION: These results indicate that a fully porous titanium alloy flange has the potential to increase the soft-tissue seal around ITAP and reduce susceptibility to infection compared with the current design

Prosthetic glenoid fixation: lateralisation of the centre of rotation of a fixed-fulcrum total shoulder replacement is not associated with suboptimal glenoid bone formation during a functionally-relevant loading protocol

Prosthetic glenoid fixation: lateralisation of the centre of rotation of a fixed-fulcrum total shoulder replacement is not associated with suboptimal glenoid bone formation during a functionally-relevant loading protocol

CXCR4 Antagonism to Treat Delayed Fracture Healing

A significant number of fractures develop non-union. Stem cell homing is regulated through SDF-1 and its receptor CXCR4. Stem/progenitor cell populations can be endogenously mobilised by administering growth factors with a pharmacological antagonist of CXCR4, AMD3100, which may be a means to improve fracture healing. Methods: A 1.5mm femoral osteotomy in Wistar rats was stabilised with an external fixator. Rats were pre-treated with PBS(P), VEGF(V), IGF-1(I) or GCSF(G) prior to AMD3100. A control group (C) did not receive growth factors or AMD3100. Bone formation after five weeks was analysed. Results: Group P had a significant increase in total bone volume (p=0.01) and group I in % bone in the fracture gap (p=0.035). Group G showed a decrease in bone volume. All treated groups had an increase in trabecular thickness. Histology showed decreased cartilage tissue associated with increased bone in groups with improved healing, and increased fibrous tissue in poorly performing groups. Conclusion: Antagonism of SDF1-CXCR4 axis can boost impaired fracture healing. AMD3100 given alone was the most effective means to boost healing whilst pre-treatment with GCSF reduced healing. AMD3100 is likely mobilizing stem cells into the blood stream that home to the fracture site enhancing healing

The effect of temperature on the viability of human mesenchymal stem cells

Introduction Impaction allograft with cement is a common technique used in revision hip surgeries for the last 20 years. However, its clinical results are inconsistent. Recent studies have shown that mesenchymal stem cells (MSCs) seeded onto allograft can enhance bone formation. This in vitro study investigates whether the increase in temperature related to the polymerisation of bone cement will affect the viability of human MSCs. Methods The viability of human MSCs was measured after incubating them at temperatures of 38°C, 48°C and 58°C; durations 45 seconds, 80 seconds and 150 seconds. A control group was kept at 37°C and 5% carbon dioxide for the duration of the investigation (7 days). During the course of the study the human MSCs were analysed for cell metabolic activity using the alamarBlue™ assay, cell viability using both Trypan Blue dye exclusion and calcein staining under fluorescent microscopy, and necrosis and apoptosis using Annexin V and propidium iodide for flow cytometric analysis. A one-way analysis of variance with a priori Dunnett’s test was used to indicate the differences between the treatment groups, when analysed against the control. This identified conditions with a significant difference in cell metabolic activity (alamarBlue™) and cell viability (Trypan Blue). Results Results showed that cell metabolism was not severely affected up to 48°C/150 seconds, while cells in the 58°C group died. Similar results were shown using Trypan Blue and calcein analysis for cell viability. No significant difference in apoptosis and necrosis of the cells was observed when human MSCs treated at 48°C/150 seconds were compared with the control group. Conclusions The study suggests that human MSCs seeded onto allograft can be exposed to temperatures up to 48°C for 150 seconds. Exposure to this temperature for this time period is unlikely to occur during impaction allograft surgery when cement is used. Therefore, in many situations, the addition of human MSCs to cemented impaction grafting may be carried out without detrimental effects to the cells. Furthermore, previous studies have shown that this can enhance new bone formation and repair the defects in revision situations

Mesenchymal stem cells with increased stromal cell-derived factor 1 expression enhanced fracture healing

Treatment of critical size bone defects pose a challenge in orthopedics. Stem cell therapy together with cytokines has the potential to improve bone repair as they cause the migration and homing of stem cells to the defect site. However, the engraftment, participation, and recruitment of other cells within the regenerating tissue are important. To enhance stem cell involvement, this study investigated overexpression of stem cells with stromal cell-derived factor 1 (SDF-1) using an adenovirus. We hypothesized that these engineered cells would effectively increase the migration of native cells to the site of fracture, enhancing bone repair. Before implantation, we showed that SDF-1 secreted by transfected cells increased the migration of nontransfected cells. In a rat defect bone model, bone marrow mesenchymal stem cells overexpressing SDF-1 showed significantly (p=0.003) more new bone formation within the gap and less bone mineral loss at the area adjacent to the defect site during the early bone healing stage. In conclusion, SDF-1 was shown to play an important role in accelerating fracture repair and contributing to bone repair in rat models, by recruiting more host stem cells to the defect site and encouraging osteogenic differentiation and production of bone

Intrinsic Osteoinductivity of Porous Titanium Scaffold for Bone Tissue Engineering

Large bone defects and nonunions are serious complications that are caused by extensive trauma or tumour. As traditional therapies fail to repair these critical-sized defects, tissue engineering scaffolds can be used to regenerate the damaged tissue. Highly porous titanium scaffolds, produced by selective laser sintering with mechanical properties in range of trabecular bone (compressive strength 35 MPa and modulus 73 MPa), can be used in these orthopaedic applications, if a stable mechanical fixation is provided. Hydroxyapatite coatings are generally considered essential and/or beneficial for bone formation; however, debonding of the coatings is one of the main concerns. We hypothesised that the titanium scaffolds have an intrinsic potential to induce bone formation without the need for a hydroxyapatite coating. In this paper, titanium scaffolds coated with hydroxyapatite using electrochemical method were fabricated and osteoinductivity of coated and noncoated scaffolds was compared in vitro. Alizarin Red quantification confirmed osteogenesis independent of coating. Bone formation and ingrowth into the titanium scaffolds were evaluated in sheep stifle joints. The examinations after 3 months revealed 70% bone ingrowth into the scaffold confirming its osteoinductive capacity. It is shown that the developed titanium scaffold has an intrinsic capacity for bone formation and is a suitable scaffold for bone tissue engineering