Time course changes to structural, mechanical and material properties of bone in rats after complete spinal cord injury.

OBJECTIVE: Characterise the spatiotemporal trabecular and cortical bone responses to complete spinal cord injury (SCI) in young rats. METHODS: 8-week-old male Wistar rats received T9-transection SCI and were euthanised 2-, 6-, 10- or 16-weeks post-surgery. Outcome measures were assessed using micro-computed tomography, mechanical testing, serum markers and Fourier-transform infrared spectroscopy. RESULTS: The trabecular and cortical bone responses to SCI are site-specific. Metaphyseal trabecular BV/TV was 59% lower, characterised by fewer and thinner trabeculae at 2-weeks post-SCI, while epiphyseal BV/TV was 23% lower with maintained connectivity. At later-time points, metaphyseal BV/TV remained unchanged, while epiphyseal BV/TV increased. The total area of metaphyseal and mid-diaphyseal cortical bone were lower from 2-weeks and between 6- and 10-weeks post-SCI, respectively. This suggested that SCI-induced bone changes observed in the rat model were not solely attributable to bone loss, but also to suppressed bone growth. No tissue mineral density differences were observed at any time-point, suggesting that decreased whole-bone mechanical properties were primarily the result of changes to the spatial distribution of bone. CONCLUSION: Young SCI rat trabecular bone changes resemble those observed clinically in adult and paediatric SCI, while cortical bone changes resemble paediatric SCI only.

Nanovibrational Stimulation of Mesenchymal Stem Cells Induces Therapeutic Reactive Oxygen Species and Inflammation for Three-Dimensional Bone Tissue Engineering.

There is a pressing clinical need to develop cell-based bone therapies due to a lack of viable, autologous bone grafts and a growing demand for bone grafts in musculoskeletal surgery. Such therapies can be tissue engineered and cellular, such as osteoblasts, combined with a material scaffold. Because mesenchymal stem cells (MSCs) are both available and fast growing compared to mature osteoblasts, therapies that utilize these progenitor cells are particularly promising. We have developed a nanovibrational bioreactor that can convert MSCs into bone-forming osteoblasts in two- and three-dimensional, but the mechanisms involved in this osteoinduction process remain unclear. Here, to elucidate this mechanism, we use increasing vibrational amplitude, from 30 nm (N30) to 90 nm (N90) amplitudes at 1000 Hz and assess MSC metabolite, gene, and protein changes. These approaches reveal that dose-dependent changes occur in MSCs' responses to increased vibrational amplitude, particularly in adhesion and mechanosensitive ion channel expression and that energetic metabolic pathways are activated, leading to low-level reactive oxygen species (ROS) production and to low-level inflammation as well as to ROS- and inflammation-balancing pathways. These events are analogous to those that occur in the natural bone-healing processes. We have also developed a tissue engineered MSC-laden scaffold designed using cells' mechanical memory, driven by the stronger N90 stimulation. These mechanistic insights and cell-scaffold design are underpinned by a process that is free of inductive chemicals.

Hybrid core-shell scaffolds for bone tissue engineering.

The tissue engineering applications of coaxial electrospinning are growing due to the potential increased functionality of the fibres compared to basic electrospinning. Previous studies of core and shell scaffolds have placed the active elements in the core, however, the surface response to a biomaterial affects the subsequent behaviour, thus here hydroxyapatite (HA) was added to the shell. Coaxial electrospun polycaprolactone (PCL)-polylactic acid (PLA)/HA (core-shell) scaffolds were produced in 2D sheets using a plate collector, or 3D tubes for bone tissue engineering using a rotating needle collector. The scaffolds include high hydroxyapatite content while retaining their structural and mechanical integrity. The effect of the collector type on fibre diameter, fibre alignment and mechanical properties have been evaluated, and the impact of HA incorporation on bioactivity, BMP-2 release, cell behaviour and mechanical properties for up to 12 weeks degradation were assessed. Fibre uniformity in coaxial electrospinning depends on the relative flow rate of the core and shell solutions. Using a rotating needle collector increased fibre alignment compared to a stationary collector, without affecting fibre diameter significantly, while HA content increased fibre non-uniformity. Coaxial PCL-PLA/HA fibres exhibited significantly higher bioactivity compared to PCL-PLA scaffolds due to the surface exposure of the HA particles. Apatite formation increased with increasing SBF immersion time. Coaxial tubular scaffolds with and without HA incorporation showed gradual reductions in their mechanical properties over 12 weeks in PBS or SBF but still retained their structural integrity. Coaxial scaffolds with and without HA exhibited gradual and sustained BMP-2 release and supported MSCs proliferation and differentiation with no significant difference between the two scaffolds types. These materials therefore show potential applications as bone tissue engineering scaffolds.

Calcium Sulphate/Hydroxyapatite Carrier for Bone Formation in the Femoral Neck of Osteoporotic Rats.

This study investigated bone regeneration in the femoral neck canal of osteoporotic rats using a novel animal model. A calcium sulphate (CS)/hydroxyapatite (HA) carrier was used to deliver a bisphosphonate, zoledronic acid (ZA), locally, with or without added recombinant human bone morphogenic protein-2 (rhBMP-2). Twenty-eight-week-old ovariectomized Sprague-Dawley rats were used. A 1 mm diameter and 8 mm long defect was created in the femoral neck by drilling from the lateral cortex in the axis of the femoral neck, leaving the surrounding cortex intact. Three treatment groups and one control group were used: (1) CS/HA alone, (2) CS/HA + ZA (10 µg) (3) CS/HA + ZA (10 µg) + rhBMP-2 (4 µg), and (4) empty defect (control). The bone formation was assessed at 4 weeks post surgery using in vivo micro computed tomography (micro-CT). At 8 weeks post surgery, the animals were sacrificed, and both defect and contralateral femurs were subjected to micro-CT, mechanical testing, and histology. Micro-CT results showed that the combination of CS/HA with ZA or ZA + rhBMP-2 increased the bone formation in the defect when compared to the other groups and to the contralateral hips. Evidence of new dense bone formation in CS/HA + ZA and CS/HA + ZA + rhBMP-2 groups was seen histologically. Mechanical testing results showed no differences in the load to fracture between the treatments in either of the treated or contralateral legs. The CS/HA biomaterial can be used as a carrier for ZA and rhBMP-2 to regenerate bone in the femoral neck canal of osteoporotic rats.

Patient-specific bone mineral density distribution in the tibia of individuals with chronic spinal cord injury, derived from multi-slice peripheral Quantitative Computed Tomography (pQCT) - A cross-sectional study.

BACKGROUND: The high risk of fracture associated with chronic spinal cord injury (SCI) is attributed to extensive disuse-related bone loss in previously weight-bearing long bones. Changes in bone mineral density (BMD) after SCI have been documented extensively for the epiphyses of the tibia and femur, fracture-prone sites in this patient group. Less attention has been given to patterns of cortical bone loss in the diaphyses, but variability in BMD distributions throughout the long bones may contribute to some patients' increased susceptibility to shaft fractures in chronic SCI. AIM: A cross-sectional study was carried out to determine whether BMD distributions along the tibia differ between individuals with chronic SCI and healthy able-bodied (AB) controls, in both the trabecular and cortical bone compartments. The effects of time post-injury and gender on BMD distribution were also explored. METHODS: Individuals with chronic (≥6months post-injury) motor-complete SCI were recruited from the Queen Elizabeth National Spinal Injuries Unit (Glasgow, UK). AB control subjects were recruited to achieve similar age and gender profiles for the SCI and control groups. Multi-slice pQCT (XCT3000, Stratec) was performed along the length of the tibia (2mm thickness, 0.5mm voxel size), at 1% intervals in the epiphyses and 5% intervals in the diaphysis (34 slices in total). These were used to reconstruct full 3-D subject-specific models (Mimics, Materialise) of BMD distribution, by interpolating between slices. Subjects with chronic SCI were subdivided into 'early' (<4years post-injury) and 'established' SCI (≥4years post-injury). Subject-specific BMD distribution was described according to new parameters determined from the 3-D patient-specific models, quantifying descriptors of the trabecular and cortical BMD regions separately (volume, peak BMD, half-peak width, area under the curve). These were compared between sub-groups (using independent-samples t-tests or Mann-Whitney tests, significance level of 5%). RESULTS: 11 men (age range 17-59years old; mean 35.7±10.6) and 3 post-menopausal women (age range 56-58years old; mean 56.7±1.2years) with motor-complete SCI (ranging from 6months to 27years post-injury) were recruited; 6 men (age range 20-56years old; 33.0±12.7years) and 1 post-menopausal woman (56years) formed the AB control group. Overall, SCI resulted in lower BMD at both trabecular and cortical regions of the tibia. In men, longer time since injury resulted in greater BMD differences when compared to AB, throughout the tibia. For the post-menopausal women, differences in BMD between SCI and AB were greater in cortical bone than in trabecular bone. From the models, individual BMD distribution curves showed healthy double-peaks in AB subjects: one trabecular peak (around 200-300mg/cm3) and the other cortical (around 1000-1100mg/cm3). In most subjects with established SCI, trabecular peaks were exaggerated whilst the cortical peaks were barely discernible, with crucially some individuals already exhibiting a diminishing cortical BMD peak even <4years post-injury. CONCLUSIONS: These findings may have implications for determining the fracture susceptibility of the long bones in individual patients with SCI. Epiphyseal fractures associated with low trabecular BMD are well characterised, but our data show that some individuals with SCI may also be at higher risk of shaft fractures. The proposed BMD distribution description parameters, determined from patient-specific models, could be used to identify patients with a weakened diaphysis who may be susceptible to fractures of the tibial shaft, but this requires validation.

Radiological assessment of bioengineered bone in a muscle flap for the reconstruction of critical-size mandibular defect.

This study presents a comprehensive radiographic evaluation of bone regeneration within a pedicled muscle flap for the reconstruction of critical size mandibular defect. The surgical defect (20 mm × 15 mm) was created in the mandible of ten experimental rabbits. The masseter muscle was adapted to fill the surgical defect, a combination of calcium sulphate/hydroxyapatite cement (CERAMENT™ |SPINE SUPPORT), BMP-7 and rabbit mesenchymal stromal cells (rMSCs) was injected inside the muscle tissue. Radiographic assessment was carried out on the day of surgery and at 4, 8, and 12 weeks postoperatively. At 12 weeks, the animals were sacrificed and cone beam computerized tomography (CBCT) scanning and micro-computed tomography (µ-CT) were carried out. Clinically, a clear layer of bone tissue was identified closely adherent to the border of the surgical defect. Sporadic radio-opaque areas within the surgical defect were detected radiographically. In comparison with the opposite non operated control side, the estimated quantitative scoring of the radio-opacity was 46.6% ± 15, the mean volume of the radio-opaque areas was 63.4% ± 20. Areas of a bone density higher than that of the mandibular bone (+35% ± 25%) were detected at the borders of the surgical defect. The micro-CT analysis revealed thinner trabeculae of the regenerated bone with a more condensed trabecular pattern than the surrounding native bone. These findings suggest a rapid deposition rate of the mineralised tissue and an active remodelling process of the newly regenerated bone within the muscle flap. The novel surgical model of this study has potential clinical application; the assessment of bone regeneration using the presented radiolographic protocol is descriptive and comprehensive. The findings of this research confirm the remarkable potential of local muscle flaps as local bioreactors to induce bone formation for reconstruction of maxillofacial bony defects.

Prediction of risk of fracture in the tibia due to altered bone mineral density distribution resulting from disuse: a finite element study.

The disuse-related bone loss that results from immobilisation following injury shares characteristics with osteoporosis in post-menopausal women and the aged, with decreases in bone mineral density leading to weakening of the bone and increased risk of fracture. The aim of this study was to use the finite element method to: (i) calculate the mechanical response of the tibia under mechanical load and (ii) estimate of the risk of fracture; comparing between two groups, an able-bodied group and spinal cord injury patients group suffering from varying degrees of bone loss. The tibiae of eight male subjects with chronic spinal cord injury and those of four able-bodied age-matched controls were scanned using multi-slice peripheral quantitative computed tomography. Images were used to develop full three-dimensional models of the tibiae in Mimics (Materialise) and exported into Abaqus (Simulia) for calculation of stress distribution and fracture risk in response to specified loading conditions - compression, bending and torsion. The percentage of elements that exceeded a calculated value of the ultimate stress provided an estimate of the risk of fracture for each subject, which differed between spinal cord injury subjects and their controls. The differences in bone mineral density distribution along the tibia in different subjects resulted in different regions of the bone being at high risk of fracture under set loading conditions, illustrating the benefit of creating individual material distribution models. A predictive tool can be developed based on these models, to enable clinicians to estimate the amount of loading that can be safely allowed onto the skeletal frame of individual patients who suffer from extensive musculoskeletal degeneration (including spinal cord injury, multiple sclerosis and the ageing population). The ultimate aim is to reduce fracture occurrence in these vulnerable groups.

Lactic acid based PEU/HA and PEU/BCP composites: Dynamic mechanical characterization of hydrolysis.

Lactic acid based poly(ester-urethane) (PEU-BDI) and its composites with 20 and 40 vol.% bioceramic filler were characterized prior to their use as biocompatible and bioabsorbable artificial bone materials. Morphological, dynamic mechanical properties, and degradation of these either hydroxyapatite or biphasic calcium phosphate containing composites were determined. Addition of particulate bioactive filler increased the composite stiffness and the glass transition temperature, indicating strong interactions between the filler and matrix. Materials were sterilized by gamma-irradiation, which reduced the average molecular weights by 30-40%. However, dynamic mechanical properties were not significantly affected by irradiation. Specimens were immersed in 0.85 w/v saline at 37 degrees C for 5 weeks, and changes in molecular weights, mass, water absorption, and dynamic mechanical properties were recorded. All the composite materials showed promising dynamic mechanical performance over the 5 weeks of hydrolysis. Average molecular weights of PEU-BDI and its composites did not change substantially during the test period. PEU-BDI retained its modulus values relatively well, and although the moduli of the composite materials were much higher, especially at high filler content, they exhibited faster loss of mechanical integrity.