An interpenetrating network composite for a regenerative spinal disc application.

Severe degeneration of the intervertebral disc has an immensely debilitating effect on quality of life that has become a serious health and economic burden throughout the world. The disc plays an integral role in biomechanical movement and support within the spine. The emergence of tissue engineering endeavours to restore the structural characteristics and functionality of the native tissue. Hydrogels have been widely investigated as a candidate for regeneration of the gelatinous nucleus pulposus due to its architectural resemblance and fluid retention characteristics. However, hydrogels are often limited due to small compressive stiffness and tear resistance, leading to extrusion complications. Reinforcement of the hydrogel network using polymeric scaffolds may address these issues of inadequate mechanical properties and implant instability. This study investigates the potential of a carrageenan gel-infused polycaprolactone scaffold for nucleus pulposus tissue engineering. Mechanical properties were characterised using viscoelastic and poroelastic frameworks via microindentation. The incorporation of polymeric reinforcement within the gels increased material stiffness to that comparable to the native nucleus pulposus, however permeability was significantly greater than native values. A preliminary cell evaluation culturing NIH 3T3s over 21 days suggested the incorporation of polymeric networks also enhanced cellular proliferation compared to gels alone.

Osteoblast attachment to hydroxyapatite micro-tube scaffolds.

Tissue engineering offers a novel route for repairing damaged or diseased tissue by incorporating the patient's own healthy cells or donated cells into temporary scaffolds that act as a matrix for cell cultivation. Tissue scaffolds that are biocompatible and are porous with interconnected porous channels for cell ingrowth with a suitable degradation rate would be advantageous. In this study hydroxyapatite micro-tubes produced using the biomimetic coating technique will be pressed into a tissue scaffold. A compaction and sintering study will be done to observe appropriate pressure and heat treatment to produce a mechanically stable scaffold material. The ideal pressure was found to be 2.5 MPa where the tube-like structure was maintained, high porosity was achieved and suitable strength was possible. Sintering between 1,000 and 1,100 °C was found to produce good results. The average porosity for the chosen pressure of 2.5 MPa was 68%. The scaffold was observed with SEM, micro tomography (micro-CT), chemical analysis and degradation testing. Porous channels were established using micro-CT where the porous channels were roughly 100 µm. Chemical analysis showed constant release of calcium and phosphorous, and far below toxic levels of heavy metals from the die. Degradation testing showed high degradation compared to tested commercially available materials. Cell culturing was done on the scaffold to characterise the biological performance of the scaffolds. Cell culturing was done in a 7 and 24 day cell culture to examine cell morphology and cell ingrowth. The results showed cell ingrowth into a micro-tube and cell orientation in a longitudinal direction. SEM, confocal microscopy and histology were employed as characterisation tools for observing cell ingrowth.

Spatial control of bone formation using a porous polymer scaffold co-delivering anabolic rhBMP-2 and anti-resorptive agents.

Current clinical delivery of recombinant human bone morphogenetic proteins (rhBMPs) utilises freeze-dried collagen. Despite effective new bone generation, rhBMP via collagen can be limited by significant complications due to inflammation and uncontrolled bone formation. This study aimed to produce an alternative rhBMP local delivery system to permit more controllable and superior rhBMP-induced bone formation. Cylindrical porous poly(lactic-co-glycolic acid) (PLGA) scaffolds were manufactured by thermally-induced phase separation. Scaffolds were encapsulated with anabolic rhBMP-2 (20 µg) ± anti-resorptive agents: zoledronic acid (5 µg ZA), ZA pre-adsorbed onto hydroxyapatite microparticles, (5 µg ZA/2% HA) or IkappaB kinase (IKK) inhibitor (10 µg PS-1145). Scaffolds were inserted in a 6-mm critical-sized femoral defect in Wistar rats, and compared against rhBMP-2 via collagen. The regenerate region was examined at 6 weeks by 3D microCT and descriptive histology. MicroCT and histology revealed rhBMP-induced bone was more restricted in the PLGA scaffolds than collagen scaffolds (-92.3% TV, p < 0.01). The regenerate formed by PLGA + rhBMP-2/ZA/HA showed comparable bone volume to rhBMP-2 via collagen, and bone mineral density was +9.1% higher (p < 0.01). Local adjunct ZA/HA or PS-1145 significantly enhanced PLGA + rhBMP-induced bone formation by +78.2% and +52.0%, respectively (p ≤ 0.01). Mechanistically, MG-63 human osteoblast-like cells showed cellular invasion and proliferation within PLGA scaffolds. In conclusion, PLGA scaffolds enabled superior spatial control of rhBMP-induced bone formation over clinically-used collagen. The PLGA scaffold has the potential to avoid uncontrollable bone formation-related safety issues and to customise bone shape by scaffold design. Moreover, local treatment with anti-resorptive agents incorporated within the scaffold further augmented rhBMP-induced bone formation.

The influence of surface chemistry and topography on the contact guidance of MG63 osteoblast cells.

The purpose of the present study was to determine in vitro the effects of different surface topographies and chemistries of commercially pure titanium (cpTi) and diamond-like carbon (DLC) surfaces on osteoblast growth and attachment. Microgrooves (widths of 2, 4, 8 and 10 microm and a depth of 1.5-2 microm) were patterned onto silicon (Si) substrates using microlithography and reactive ion etching. The Si substrates were subsequently vapor coated with either cpTi or DLC coatings. All surfaces were characterized using atomic force microscopy (AFM), scanning electron microscopy (SEM), X-ray photoelectron spectroscopy (XPS) and contact angle measurements. Using the MG63 Osteoblast-Like cell line, we determined cell viability, adhesion, and morphology on different substrates over a 3 day culture period. The results showed cpTi surfaces to be significantly more hydrophilic than DLC for groove sizes larger than 2 microm. Cell contact guidance was observed for all grooved samples in comparison to the unpatterned controls. The cell viability tests indicated a significantly greater cell number for 8 and 10 microm grooves on cpTi surfaces compared to other groove sizes. The cell adhesion study showed that the smaller groove sizes, as well as the unpatterned control groups, displayed better cell adhesion to the substrate.

Calcium phosphate fibres synthesized from a simulated body fluid.

The biomimetic coating method was used for fabricating calcium phosphate fibres for biomedical applications such as bone defect fillers. Natural cotton substrate was pre-treated with phosphorylation and a Ca(OH)2 saturated solution. The pre-treated samples were then soaked in simulated body fluid (SBF) of two different concentrations, 1.5 times and 5.0 times the ion concentration of blood plasma. The cotton was then burnt out via sintering of the ceramic coating at 950 degrees C, 1050 degrees C, 1150 degrees C, and 1250 degrees C. The results demonstrated that osteoblastic cells were able to cover the entire surface cotton fibres, and the cell coverage appeared to be independent of surface roughness and Ca/P ratio of fibres.

Precipitation of hydroxyapatite nanoparticles: effects of precipitation method on electrophoretic deposition.

Electrophoretic deposition is a low-cost, simple, and flexible coating method for producing hydroxyapatite (HA) coatings on metal implants with a broad range of thicknesses, from < 1 microm to > 500 microm. As for many other HA coating techniques, densification of electrophoretically deposited coatings involves heating the coated metal to temperatures above 1000 degrees C. Metal substrates tend to react with HA coatings at such temperatures inducing decomposition at temperatures below 1050 degrees C (decomposition for pure HA normally occurs above 1300 degrees C). Therefore, densification of these coatings needs to be conducted at temperatures lower than 1050 degrees C, and this necessitates the use of high-surface-area HA nano-precipitates, rather than commercially available pre-calcined powders, which densify at temperatures typically higher than 1200 degrees C. HA nano-precipitates were prepared by three methods and deposited on metal substrates by electrophoresis: (1) the acid base method, which produced plate-like nano-particles with a 2.5:1 aspect ratio, and severely cracked coatings; (2) the calcium acetate method, which produced needle-like nano-particles with a 10:1 aspect ratio, and slightly cracked coatings; (3) the metathesis method, which produced rounded nano-particles with a 2:1 aspect ratio, and high-quality crack-free coatings. The results suggested that the less equiaxed the nano-particles, the more cracked the coatings obtained by the electrophoretic deposition technique.

Hydroxyapatite-coated metals: interfacial reactions during sintering.

Electrophoretic deposition (EPD) is a low cost flexible process for producing HA coatings on metal implants. Its main limitation is that it requires heating the coated implant in order to densify the HA. HA typically sinters at a temperature below 1150 degrees C, but metal implants are degraded above 1000 degrees C. Further, the metal induces the decomposition of the HA coating upon sintering. Recent developments have enabled EPD of metathesis-synthesised uncalcined HA which sinters at approximately 1000 degrees C. The effects of temperature on HA-coated Ti, Ti6Al4V, and 316L stainless steel were investigated for dual coatings of metathesis HA sintered at 1000 degrees C. The use of dual HA coatings (coat, sinter, coat, sinter) enabled decomposition to be confined to the "undercoat" (HA layer 1), with the surface coating decomposition free. The tensile strength of the three metals was not significantly affected by the high sintering temperatures (925 degrees C < T < 1000 degrees C). XRD/SEM/EDS analyses of the interfacial zones revealed that 316L had a negligible HA:metal interfacial zone (approximately 1 microm) while HA:Ti and HA:Ti6Al4V had large interfacial zones (>10 microm) comprising a TiO2 oxidation zone and a CaTiO2 reaction zone.

Solution ripening of hydroxyapatite nanoparticles: effects on electrophoretic deposition.

Electrophoretic deposition is a low-cost, simple, and flexible coating method for producing hydroxyapatite (Hap) coatings on metal implants. However, densification requires heating the coated metal to high temperatures, which, for commercial HAp powders, generally means at least 1200 degrees C. At such temperatures, the metal tends to react with the HAp coating, inducing decomposition, and the strength of titanium and stainless steel implants is severely degraded. With the use of raw uncalcined nanoparticulate Hap, densification can occur at 900 degrees -1050 degrees C; however, such coatings are prone to cracking due to the high drying shrinkage. This problem was solved by precipitating nanoparticulate HAp by the metathesis process [10Ca(NO3)2 + 6NH4H2PO4 + 8NH4OH] and optimizing the approximately 30 nm of nanoprecipitates by an Ostwald ripening approach, that is, by boiling and/or ambient aging in the mother liquor. While the as-precipitated nanoparticles produced severely cracked coatings, 2 h of boiling or 10 days of ambient aging ripened the "gel-like" mass into unagglomerated nanoparticles, which produced crack-free coatings. Since boiling enhanced particle size but ambient aging did not, crack elimination probably was due to the transition from the highly agglomerated gel-like state to the dispersed nanoparticulate state rather than to particle growth. Furthermore, boiling only reduced the amount of cracking whereas aging completely eliminated cracking.

Interfacial bond strength of electrophoretically deposited hydroxyapatite coatings on metals.

Hydroxyapatite (HAp) coatings were deposited onto substrates of metal biomaterials (Ti, Ti6Al4V, and 316L stainless steel) by electrophoretic deposition (EPD). Only ultra-high surface area HAp powder, prepared by the metathesis method 10Ca(NO3)2 + 6(NH4)2HPO4 + 8NH4OH), could produce dense coatings when sintered at 875-1000degreesC. Single EPD coatings cracked during sintering owing to the 15-18% sintering shrinkage, but the HAp did not decompose. The use of dual coatings (coat, sinter, coat, sinter) resolved the cracking problem. Scanning electron microscopy/energy dispersive spectroscopy (SEM/EDS) inspection revealed that the second coating filled in the "valleys" in the cracks of the first coating. The interfacial shear strength of the dual coatings was found, by ASTM F1044-87, to be approximately 12 MPa on a titanium substrate and approximately 22 MPa on 316L stainless steel, comparing quite favorably with the 34 MPa benchmark (the shear strength of bovine cortical bone was found to be 34 MPa). Stainless steel gave the better result since -316L (20.5 microm mK(-1)) > alpha-HAp (approximately 14 microm mK(-1)), resulting in residual compressive stresses in the coating, whereas alpha-titanium (approximately 10.3 microm mK(-1)) < alpha-HAp, resulting in residual tensile stresses in the coating.

Sintering effects on the strength of hydroxyapatite.

Mechanisms underlying temperature-strength interrelations for dense (> 95% dense, pores closed) hydroxyapatite (HAp) were investigated by comparative assessment of temperature effects on tensile strength, Weibull modulus, apparent density, decomposition (HAp:tricalcium phosphate ratio), dehydroxylation and microstructure. Significant dehydroxylation occurred above approximately 800 degrees C. Strength peaked at approximately 80 MPa just before the attainment of closed porosity (approximately 95% dense). For higher temperatures (closed porosity), the strength dropped sharply to approximately 60 MPa due to the closure of dehydroxylation pathways, and then stabilized at approximately 60 MPa. At very high temperatures (> 1350 degrees C), the strength dropped catastrophically to approximately 10 MPa corresponding to the decomposition of HAp to tricalcium phosphate and the associated sudden release of the remaining bonded water.