Physiologically engineered porous titanium/brushite scaffolds for critical-size bone defects: A design and manufacturing study.

Repairing critical-size bone defects still represents a critical clinical challenge in the field of trauma surgery. This study focuses on a physiological design and manufacturing of porous composite scaffold (titanium Ti with 10 % mole iron doped brushite DCPD-Fe3+) which can mimic the biomechanical properties of natural cortical bone, specifically for the purpose of repairing critical-size defects. To achieve this, the principle of design of experiments (DOE) was applied for investigating the impact of sintering temperature, mineral ratio, and volume fraction of porosity on the mechanical properties of the fabricated scaffolds. The fabricated scaffolds had open porosity up to 60 %, with pore size approximately between 100 µm and 850 µm. The stiffness of the porous composite scaffolds varied between 3.30 GPa and 20.50 GPa, while the compressive strength ranged from approximately 130 MPa-165 MPa at sintering temperatures equal to or exceeding 1000 °C. Scaffolds with higher porosity and mineral content demonstrated lower stiffness values, resembling natural bone. Numerical simulation was employed by Ansys Workbench to investigate the stress and strain distribution of a critical size defect in mid-shaft femur which was designed to be replaced with the fabricated scaffold. The fabricated scaffolds showed flexible biomechanical behaviour at the bone/scaffold interface, generating lower stress levels and indicating a better match with the femoral shaft stiffness. The experimental and numerical findings demonstrated promising applications for manufacturing a patient-specific bone scaffold for critical and potentially large defects for reducing stress shielding and minimizing non-union risk.

Dicalcium Phosphate Dihydrate Mineral Loaded Freeze-Dried Scaffolds for Potential Synthetic Bone Applications.

Dicalcium Phosphate Dihydrate (DCPD) mineral scaffolds alone do not possess the mechanical flexibility, ease of physicochemical properties' tuneability or suitable porosity required for regenerative bone scaffolds. Herein, we fabricated highly porous freeze-dried chitosan scaffolds embedded with different concentrations of Dicalcium Phosphate Dihydrate (DCPD) minerals, i.e., 0, 20, 30, 40 and 50 (wt)%. Increasing DCPD mineral concentration led to increased scaffold crystallinity, where the % crystallinity for CH, 20, 30, 40, and 50-DCPD scaffolds was determined to be 0.1, 20.6, 29.4, 38.8 and 69.9%, respectively. Reduction in scaffold pore size distributions was observed with increasing DCPD concentrations of 0 to 40 (wt)%; coalescence and close-ended pore formation were observed for 50-DCPD scaffolds. 50-DCPD scaffolds presented five times greater mechanical strength than the DCPD mineral-free scaffolds (CH). DCPD mineral enhanced cell proliferation for the 20, 30 and 40-DCPD scaffolds. 50-DCPD scaffolds presented reduced pore interconnectivity due to the coalescence of many pores in addition to the creation of closed-ended pores, which were found to hinder osteoblast cell proliferation.

Interrelationships between the structural, spectroscopic, and antibacterial properties of nanoscale (< 50 nm) cerium oxides.

Bone healing is a complex process, and if not managed successfully, it can lead to non-union, metal-work failure, bacterial infections, physical and psychological patient impairment. Due to the growing urgency to minimise antibiotic dependency, alternative treatment strategies, including the use of nanoparticles, have attracted significant attention. In the present study, cerium oxide nanoparticles (Ce4+, Ce3+) have been selected due to their unique antibacterial redox capability. We found the processing routes affected the agglomeration tendency, particle size distribution, antibacterial potential, and ratio of Ce3+:Ce4+ valence states of the cerium oxide nanoparticles. The antibacterial efficacy of the nanoparticles in the concentration range of 50-200 µg/ml is demonstrated against Escherichia coli, Staphylococcus epidermis, and Pseudomonas aeruginosa by determining the half-maximal inhibitory concentration (IC50). Cerium oxide nanoparticles containing a more significant amount of Ce3+ ions, i.e., FRNP, exhibited 8.5 ± 1.2%, 10.5 ± 4.4%, and 13.8 ± 5.8% increased antibacterial efficacy compared with nanoparticles consisting mainly of Ce4+ ions, i.e., nanoparticles calcined at 815 °C.

Ex-vivo recellularisation and stem cell differentiation of a decellularised rat dental pulp matrix.

Implementing the principles of tissue engineering within the clinical management of non-vital immature permanent teeth is of clinical interest. However, the ideal scaffold remains elusive. The aim of this work was to assess the feasibility of decellularising rat dental pulp tissue and evaluate the ability of such scaffold to support stem cell repopulation. Rat dental pulps were retrieved and divided into control and decellularised groups. The decellularisation protocol incorporated a low detergent concentration and hypotonic buffers. After decellularisation, the scaffolds were characterised histologically, immunohistochemistry and the residual DNA content quantified. Surface topography was also viewed under scanning electron microscopy. Biocompatibility was evaluated using cytotoxicity assays utilising L-929 cell line. Decellularised scaffolds were recellularised with human dental pulp stem cells up to 14 days in vitro. Cellular viability was assessed using LIVE/DEAD stain kit and the recellularised scaffolds were further assessed histologically and immunolabelled using makers for odontoblastic differentiation, cytoskeleton components and growth factors. Analysis of the decellularised scaffolds revealed an acellular matrix with histological preservation of structural components. Decellularised scaffolds were biocompatible and able to support stem cell survival following recellularisation. Immunolabelling of the recellularised scaffolds demonstrated positive cellular expression against the tested markers in culture. This study has demonstrated the feasibility of developing a biocompatible decellularised dental pulp scaffold, which is able to support dental pulp stem cell repopulation. Clinically, decellularised pulp tissue could possibly be a suitable scaffold for use within regenerative (reparative) endodontic techniques.

Analysis of the osteogenic and mechanical characteristics of iron (Fe2+/Fe3+)-doped ß­calcium pyrophosphate.

The calcium phosphate is the main mineral constituent of bone. Although there has been significant amount of research on finding ideal synthetic bone, no suitable scaffold material has yet been found. In this investigation, the iron doped brushite (CaHPO4·2H2O) has been investigated for osteogenic potential and mechanical properties. The synthesis of iron-oxide doping in the form of Fe2+,3+-ions were carried out using the solution based method in which the ammonium hydrogen phosphate and calcium nitrate solutions were used in stoichiometric ratio for synthesizing CaHPO4·2H2O, with doping concentrations of Fe2+,3+-ions between 5 mol% and 30 mol%. The synthesized powders were analysed using X-ray powder diffraction, FTIR, SEM and Raman spectroscopic techniques. The heat treatment of synthesized powder was carried out at 1000 °C in air for 5 h, and it was found that the dominant crystalline phase in samples with <20 mol% was ß-CPP, which also formed an iron-rich solid solution phase. Increasing the concentrations of Fe2+,3+-ions enhances the phase fraction of FePO4 and amorphous phase. Amongst the Fe2+,3+-doped ß-CPP minerals, it was found that the 10 mol% Fe2+,3+-doped ß-CPP offers the best combination of bio-mechanical and osteogenic properties as a scaffold for bone tissue regenerative engineering.

Mutations in C4orf26, encoding a peptide with in vitro hydroxyapatite crystal nucleation and growth activity, cause amelogenesis imperfecta.

Autozygosity mapping and clonal sequencing of an Omani family identified mutations in the uncharacterized gene, C4orf26, as a cause of recessive hypomineralized amelogenesis imperfecta (AI), a disease in which the formation of tooth enamel fails. Screening of a panel of 57 autosomal-recessive AI-affected families identified eight further families with loss-of-function mutations in C4orf26. C4orf26 encodes a putative extracellular matrix acidic phosphoprotein expressed in the enamel organ. A mineral nucleation assay showed that the protein's phosphorylated C terminus has the capacity to promote nucleation of hydroxyapatite, suggesting a possible function in enamel mineralization during amelogenesis.

Effect of cyclic tensile load on the regulation of the expression of matrix metalloproteases (MMPs -1, -3) and structural components in synovial cells.

Synovial cells are reported to colonize synthetic ligament scaffolds following anterior cruciate ligament (ACL) reconstruction but the process leading to ligamentization is poorly understood. The present study investigated the effect of cyclic tensile strain on the expression of genes involved in matrix remodelling in bovine synovial cells seeded onto an artificial ligament scaffold. Synovial cells were seeded and cultured on polyester scaffolds for 3 weeks and subsequently subjected to cyclic tensile strain of 4.5% for 1 hr at frequency of 1 Hz. Changes in the levels of expression of genes for major ligament components (type I and type III collagen) and also metalloproteinases (MMP-1 and MMP-3), and TIMP-1 were examined using RT-PCR. Additionally, metalloproteinase activity was measured using both zymography and collagenase assays. The gene expression of MMP-3 transcripts in the loaded group was almost 3-fold that observed in control group but no differences were observed in other transcripts. Consistent with these findings, MMP-3 activity increased by 85% under mechanical stimulus, and MMP-1 activity showed no changes. Over expression of MMP-3 under cyclic tensile load may mediate the proteolysis of certain substrates surrounding the ligament scaffold. This will play a critical role in facilitating cell migration, proliferation and tissue remodelling by breaking down the provisional tissue formed by the synovium, and by generating factors that induce angiogenesis and chemotactic cell migration.

Cyclic straining of cell-seeded synthetic ligament scaffolds: development of apparatus and methodology.

Cyclic tensile strains acting along a ligament implant are known to stimulate cells that colonize it to proliferate and to synthesize an extracellular matrix (ECM), which will then remodel and form a new ligament structure. However, this process of tissue induction is poorly understood. As a first step toward elucidating this process, we aimed to investigate the effect of cyclic tensile strain on the proliferation of, and possible ECM synthesis by, cells colonizing ligament scaffolds. Because there was no commercially available apparatus to undertake such investigation the objectives of this study were to develop an apparatus for the application of cyclic tensile strains on cell-seeded synthetic ligament scaffolds and to develop and validate (through preliminary data obtained using the apparatus) methodology for studying the effect of cyclic strain on cell proliferation. We designed a multi-station test apparatus that operated inside an incubator. It allowed the application of tensile cyclic strains of between 0.5% and 5% at a frequency of 1 Hz on cell-seeded polyester ligament scaffolds immersed in culture medium. Test stations with windows in their bases could be easily de-coupled from the apparatus. This allowed monitoring of cell proliferation and morphology, with inverted light microscopy, through the transparent glass bases of the culture wells. Preliminary experiments lasting for 1 day or 9 weeks examined the effect of selected aspects of the cyclic strain on proliferation of cells seeded onto ligament scaffolds. Tests lasting for 1 day showed that the application of cyclic tensile strain of 5% for 4 h increased cell proliferation 24% above that observed in unstrained controls (p < .05). Scanning electron microscopy data from tests lasting for 9 weeks demonstrated further the dependency of cell proliferation and possible ECM synthesis on the magnitude of the strain. The larger the amplitude, the greater was the coverage of the scaffold with cells and ECM. Transmission electron microscopy of the ECM observed at 9 weeks showed evidence of collagen fibrils aligned in the direction of load in strained scaffolds, whereas the tissue on the control scaffolds was random.

Effect of cyclic tensile strain on proliferation of synovial cells seeded onto synthetic ligament scaffolds--an in vitro simulation.

OBJECTIVE: Cyclic tensile strain is pivotal to the remodeling of tissue induced in implants used in reconstruction of anterior cruciate ligaments whether these implants were of autogenous tissues or synthetic materials. However, this process is poorly understood. The objective of this study was to investigate the short and medium-term effect of cyclic tensile strain on the proliferation of synovial cells seeded on ligament scaffolds. METHODS: 206 ligament scaffolds made from plasma treated polyester with an ultimate tensile strength of 320 N were used in this study. Synovial cells were obtained from the metatarsophalangeal joints of 2 years old bovines. After expansion of these, they were seeded onto the scaffolds which were subjected, in a specialized apparatus, to a cyclic tensile strain of 4.5% at a frequency of 1 Hz. Initially, the strain was applied for a period of 4 h, which was subsequently reduced in further experiments to 1.0 h and 0.5 h. In further tests, strains of approximately 2.5%, 1% and 0.6% were applied for 1 h at the same frequency. In all the above tests, which were short-term tests (lasting for approximately 1 day), cell proliferation was investigated by the uptake of thymidine with which cells were labeled according to prescribed protocols. Cell proliferation was further examined with light microscopy after 5 weeks and the degree of fill of inter-yarn spaces was quantified for strain amplitudes of 1, 2.5 and 4.5%. Equal number of control (not strained) specimens was used at each time point. RESULTS: In the 1-day experiments, for all durations of application of cyclic strain (exercise), the effect of strain on cell proliferation was inhibitory during the period of exercise and up to 18 h from its commencement, but was stimulatory 22-24 h afterwards. This stimulatory effect was maximal at an exercise period of 1 h. The study has also shown that there is a threshold for the amplitude of the strain (1%), at and below which cell proliferation was not significantly different from that observed in control specimens (P was <0.05 for all data). After 5 weeks of cyclic strain application, it was shown that the higher the amplitude of strain the larger was the area occupied by cells of the intra-yarn space. CONCLUSION: Both the amplitude of cyclic strain and duration of its application affect the proliferation of synovial cells seeded on ligament scaffolds. The data should be useful when selecting or designing an implant, and when prescribing a postoperative exercise regime.