Synthetic Hydroxyapatite Particles Research Articles

Systemically administered zoledronic acid activates locally implanted synthetic hydroxyapatite particles enhancing peri-implant bone formation: A regenerative medicine approach to improve fracture fixation

Fracture fixation in an ageing population is challenging and fixation failure increases mortality and societal costs. We report a novel fracture fixation treatment by applying a hydroxyapatite (HA) based biomaterial at the bone-implant interface and biologically activating the biomaterial by systemic administration of a bisphosphonate (zoledronic acid, ZA). We first used an animal model of implant integration and applied a calcium sulphate (CaS)/HA biomaterial around a metallic screw in the tibia of osteoporotic rats. Using systemic ZA administration at 2-weeks post-surgery, we demonstrated that the implant surrounded by HA particles showed significantly higher peri‑implant bone formation compared to the unaugmented implants at 6-weeks. We then evaluated the optimal timing (day 1, 3, 7 and 14) of ZA administration to achieve a robust effect on peri‑implant bone formation. Using fluorescent ZA, we demonstrated that the uptake of ZA in the CaS/HA material was the highest at 3- and 7-days post-implantation and the uptake kinetics had a profound effect on the eventual peri‑implant bone formation. We furthered our concept in a feasibility study on trochanteric fracture patients randomized to either CaS/HA augmentation or no augmentation followed by systemic ZA treatment. Radiographically, the CaS/HA group showed signs of increased peri‑implant bone formation compared with the controls. Finally, apart from HA, we demonstrated that the concept of biologically activating a ceramic material by ZA could also be applied to β-tricalcium phosphate. This novel approach for fracture treatment that enhances immediate and long-term fracture fixation in osteoporotic bone could potentially reduce reoperations, morbidity and mortality. Statement of significance• Fracture fixation in an ageing population is challenging. Biomaterial-based augmentation of fracture fixation devices has been attempted but lack of satisfactory biological response limits their widespread use. • We report the biological activation of locally implanted microparticulate hydroxyapatite (HA) particles placed around an implant by systemic administration of the bisphosphonate zoledronic acid (ZA). The biological activation of HA by ZA enhances peri‑implant bone formation. •Timing of ZA administration after HA implantation is critical for optimal ZA uptake and consequently determines the extent of peri‑implant bone formation. • We translate the developed concept from small animal models of implant integration to a proof-of-concept clinical study on osteoporotic trochanteric fracture patients. • ZA based biological activation can also be applied to other calcium phosphate biomaterials.

Direct monitoring of single-cell response to biomaterials by Raman spectroscopy

There is continued focus on the development of new biomaterials and associated biological testing methods needed to reduce the time taken for their entry to clinical use. The application of Raman spectroscopy to the study of individual cells that have been in contact with biomaterials offers enhanced in vitro information in a potentially non-destructive testing regime. The work presented here reports the Raman spectral analysis of discreet U-2 OS bone cells after exposure to hydroxyapatite (HA) coated titanium (Ti) substrates in both the as-deposited and thermally annealed states. These data show that cells that were in contact with the bioactive HA surface for 7 days had spectral markers similar to those cultured on the Ti substrate control for the same period. However, the spectral features for those cells that were in contact with the annealed HA surface had indicators of significant differentiation at day 21 while cells on the as-deposited surface did not show these Raman changes until day 28. The cells adhered to pristine Ti control surface showed no spectral changes at any of the timepoints studied. The validity of these spectroscopic results has been confirmed using data from standard in vitro cell viability, adhesion, and proliferation assays over the same 28-day culture period. In this case, cell maturation was evidenced by the formation of natural bone apatite, which precipitated intracellularly for cells exposed to both types of HA-coated Ti at 21 and 28 days, respectively. The properties of the intracellular apatite were markedly different from that of the synthetic HA used to coat the Ti substrate with an average particle size of 230 nm, a crystalline-like shape and Ca/P ratio of 1.63 ± 0.5 as determined by SEM-EDX analysis. By comparison, the synthetic HA particles used as a control had an average size of 372 nm and were more-rounded in shape with a Ca/P ratio of 0.8 by XPS analysis and 1.28 by SEM-EDX analysis. This study shows that Raman spectroscopy can be employed to monitor single U-2 OS cell response to biomaterials that promote cell maturation towards de novo bone thereby offering a label-free in vitro testing method that allows for non-destructive analyses.

Synthetic hydroxyapatite: a recruiting platform for biologically active molecules

Background and purpose — Targeted delivery of drugs is important to achieve efficient local concentrations and reduce systemic side effects. We hypothesized that locally implanted synthetic hydroxyapatite (HA) particles can act as a recruiting moiety for systemically administered drugs, leading to targeted drug accretion.Methods — Synthetic HA particles were implanted ectopically in a muscle pouch in rats, and the binding of systemically circulating drugs such as zoledronic acid (ZA), tetracycline and 18F-fluoride (18F) was studied. The local biological effect was verified in an implant integration model in rats, wherein a hollow implant was filled with synthetic HA particles and the animals were given systemic ZA, 2-weeks post-implantation. The effect of HA particle size on drug binding and the possibility of reloading HA particles were also evaluated in the muscle pouch.Results — The systemically administered biomolecules (ZA, tetracycline and 18F) all sought the HA moiety placed in the muscle pouch. Statistically significant higher peri-implant bone volume and peak force were observed in the implant containing HA particles compared with the empty implant. After a single injection of ZA at 2 weeks, micro HA particles showed a tendency to accumulate more 14C-zoledronic acid (14C-ZA) than nano-HA particles in the muscle pouch. HA particles could be reloaded when ZA was given again at 4 weeks, showing increased 14C-ZA accretion by 73% in microparticles and 77% in nanoparticles.Interpretation — We describe a novel method of systemic drug loading resulting in targeted accretion in locally implanted particulate HA, thereby biologically activating the material.

Friday, September 28, 2018 4:05 PM–5:05 PM abstracts: basic science of spinal fusion: 236. Composition of hyperelastic bone composite scaffolds affects de novo bone formation

BACKGROUND CONTEXT Bone graft extenders for spine fusion include demineralized bone matrix (DBM) and ceramic-based products, but their efficacy is insufficient to qualify them as bona fide bone graft substitutes (BGS). We previously developed a unique 3D-printable scaffold composed of synthetic hydroxyapatite particles (HA) and poly lactic-co-glycolic acid (PLGA). PURPOSE The purpose of this study is to improve the efficacy of this hyperelastic “bone” composite (HBC) by incorporating osteoinductive DBM particles. We evaluated the ability of various iterations of these HA-DBM HBC scaffolds to promote bone regeneration and spine fusion in a rat posterolateral spine fusion (PLF) model. METHODS Female Sprague Dawley rats underwent L4-L5 PLF utilizing HBC scaffolds composed of 30vol.% PLGA and a 70vol.% particles. For each group, the particle components consisted of the following vol.% ratios of HA:DBM: 1:0, 3:1, 1:1, 1:3, and 0:1. Eight weeks postoperative, spine fusion was evaluated via radiography and manual palpation. Synchrotron microCT was used to qualitatively assess bone formation within the struts of the scaffolds. Histology (Hematoxylin/Orange G/Alcian Blue and Masson's Trichrome) was used to characterize the bone growing within and around the struts and the bone-scaffold interface. RESULTS 3:1 HA:DBM HBC scaffolds had the highest fusion rate (92%), although this was only significantly higher than the 0:1 group (42% fusion; p CONCLUSIONS This study evaluated the use of 3D printed HBC scaffolds containing both HA and DBM particles as a potential BGS. Varying the proportions of HA:DBM within each scaffold resulted in only slight differences in fusion scores and rates. However, microCT showed that de novo bone spicule formation was only present within scaffold struts that contained DBM particles. We hypothesize that (1) the presence of osteoinductive DBM is key to de novo bone formation, and (2) the hydroxyapatite provides the calcium and phosphate necessary to re-mineralize the DBM particles. Future studies will aim to further improve the efficacy of this HBC as a BGS for spine fusion.

Crystallinity of hydroxyapatite drives myofibroblastic activation and calcification in aortic valves

Calcific aortic valve disease (CAVD) is an inexorably degenerative pathology characterized by progressive calcific lesion formation on the valve leaflets. The interaction of valvular cells in advanced lesion environments is not well understood yet highly relevant as clinically detectable CAVD exhibits calcifications composed of non-stoichiometric hydroxyapatite (HA). In this study, Fourier transform infrared spectroscopic imaging was used to spatially analyze mineral properties as a function of disease progression. Crystallinity (size and perfection) increased with increased valve calcification. To study the relationship between crystallinity and cellular behavior in CAVD, valve cells were seeded into 3D mineral-rich collagen gels containing synthetic HA particles, which had varying crystallinities. Lower crystallinity HA drove myofibroblastic activation in both valve interstitial and endothelial cells, as well as osteoblastic differentiation in interstitial cells. Additionally, calcium accumulation within gels depended on crystallinity, and apoptosis was insufficient to explain differences in HA-driven cellular activity. The protective nature of endothelial cells against interstitial cell activation and calcium accumulation was completely inhibited in the presence of less crystalline HA particles. Elucidating valve cellular behavior post-calcification is of vital importance to better predict and treat clinical pathogenesis, and mineral-containing hydrogel models provide a unique 3D platform to evaluate valve cell responses to a later stage of valve disease. Statement of SignificanceWe implement a 3D in vitro platform with embedded hydroxyapatite (HA) nanoparticles to investigate the interaction between valve interstitial cells, valve endothelial cells, and a mineral-rich extracellular environment. HA nanoparticles were synthesized based on analysis of the mineral properties of calcific regions of diseased human aortic valves. Our findings indicate that crystallinity of HA drives activation and differentiation in interstitial and endothelial cells. We also show that a mineralized environment blocks endothelial protection against interstitial cell calcification. Our HA-containing hydrogel model provides a unique 3D platform to evaluate valve cell responses to a mineralized ECM. This study additionally lays the groundwork to capture the diversity of mineral properties in calcified valves, and link these properties to progression of the disease.

Interfaces in graded coatings on titanium‐based implants

Graded bilayered glass-ceramic composite coatings on Ti6Al4V substrates were fabricated using an enameling technique. The layers consisted of a mixture of glasses in the CaO-MgO-Na(2)O-K(2)O-P(2)O(5) system with different amounts of calcium phosphates (CPs). Optimum firing conditions have been determined for the fabrication of coatings having good adhesion to the metal, while avoiding deleterious reactions between the glass and the ceramic particles. The final coatings do not crack or delaminate. The use of high-silica layers (>60 wt % SiO(2)) in contact with the alloy promotes long-term stability of the coating; glass-metal adhesion is achieved through the formation of a nanostructured Ti(5)Si(3) layer. A surface layer containing a mixture of a low-silica glass ( approximately 53 wt % SiO(2)) and synthetic hydroxyapatite particles promotes the precipitation of new apatite during tests in vitro. The in vitro behavior of the coatings in simulated body fluid depends both on the composition of the glass matrix and the CP particles, and is strongly affected by the coating design and the firing conditions.

Adsorption of myoglobin onto various synthetic hydroxyapatite particles

The adsorption of myoglobin (MGB) onto various kinds of colloidal synthetic hydroxyapatite [X10(PO4)6(OH)2, noted CaHap, SrHap and CaSrHap for X=Ca, Sr and Ca+Sr, respectively] particles was investigated at 15°C in a 1×10−4 mol dm−3 KCl solution of pH 6.0. The adsorption rate of MGB onto CaHap was comparable to that of lysozyme (LSZ) and was faster than that of bovine serum albumin (BSA). This distinction was explained by the difference in the molecular mass of these proteins (MGB: 17800 Da, LSZ: 14600 Da, BSA: 67200 Da); the diffusion rates of the smaller MGB and LSZ molecules to the CaHap surface are faster than that of the larger BSA. The adsorption isotherms of MGB onto these synthetic hydroxyapatites (Haps) exhibited the Langmuir type and the surface charge of the MGB-covered Haps showed almost constant negative value and was independent of the MGB concentration for all the Haps. The values of the saturated amounts of adsorbed MGB for CaHap were independent of the Ca:PO4 molar ratio of the materials, supporting less importance of an electrostatic interaction between MGB and Haps because MGB molecules are electrostatically neutral at pH 6. It was presumed that MGB molecules are adsorbed onto phosphate ions exposed on ac and bc crystal faces by the van der Waals attractive force without taking advantage of the electrostatic interaction.

Hydroxyapatite-polyethylene composites for bone substitution: effects of ceramic particle size and morphology

Synthetic hydroxyapatite particles of two median sizes and different morphologies have been used to manufacture hydroxyapatite reinforced high density polyethylene composites (HAPEX TM) for medical applications. The effects of hydroxyapatite particle size on properties of the resultant composites were investigated using various techniques. It was found that composites with smaller hydroxyapatite particles had higher torsional modulus, tensile modulus and tensile strength, but lower strain to failure. Examination of fracture surfaces revealed that only a mechanical bond existed between the filler and the matrix. It was shown that dynamic mechanical analysis is useful in studying the viscoelastic behaviour of the composite.

A morphological study on the response between primary periosteum cultures and synthetic hydroxyapatite particles.

A morphological study on the response between primary periosteum cultures and synthetic hydroxyapatite particles.

Effects of culture supernatants of leukocytes from periodontal patients on monocyte-mediated hydroxyapatite resorption.

In this study, the effects of culture supernatants on various activities of the monocyte, as a bone-resorbing cell, were compared between peripheral blood leukocyte (PBL) cultures from patients with periodontal disease and those from subjects with a clinically healthy periodontium. We have reported that normal human monocytes in vitro induce the release of calcium from synthetic hydroxyapatite particles and that the activity is enhanced by supernatants from cultures of stimulated or non-stimulated peripheral blood leukocytes. Monocytes from both patients and healthy subjects induced the release of calcium from hydroxyapatite particles (HA) to an equal degree. This activity of monocytes from healthy subjects showed a statistically significant increase by addition of supernatants from stimulated or unstimulated cultures of peripheral blood leukocytes from periodontitis patients. This increase was greater than that seen with supernatants from cells of healthy controls. The Nitro Blue Tetrazolium reduction activity and [3H]-thymidine incorporation of monocytes were also increased by addition of the supernatants from leukocyte cultures from either patients or healthy controls, but no significant difference was noted in the increase. These results suggest that the HA-resorbing activity of monocytes was enhanced by factors from cultured leukocytes. Furthermore, these studies showed that production of these factors by peripheral mononuclear cells from patients with periodontal disease was greater than that seen with cells from normal subjects.