Effect of copper substitution on the local chemical structure and dissolution property of copper-doped ß-tricalcium phosphate.

Substitution of inorganic ions into ß-tricalcium phosphate (ß-TCP) is a well-known approach for facilitating biological functions of bioceramics. However, the dissolution mechanism of those ß-TCPs is still under intensive debates. In the present study, the effect of copper substitution into ß-TCP crystal structure on the local chemical structure and dissolution property of the copper-doped ß-TCP (CuTCP) was investigated to clarify the dissolution mechanism of ß-TCP. A copper-dependent decrease in the dissolution rate of CuTCP with time was observed. The 1H → 31P nuclear magnetic resonance (NMR) spectra of 10 mol% copper-doped ß-TCP after the dissolution test demonstrated an amorphous hydrated layer on the surface of ß-TCP core particles, which contained hydroxyapatite and dicalcium phosphate dihydrate and anhydrate. As such, all the dissolution curves could be curve-fitted by a heterogeneous dissolution model composing of fast and slow dissolution components. Overall, dissolution mechanism could be proposed as follows: the CuTCP particles initially dissolved by hydrolysis based on the fast dissolution component. Subsequently, the amorphous hydrated layers were formed on their surface, and caused the diffusion-controlled dissolution. As the result, the slow dissolution component would be dominant, and led to the decreased dissolution rate. STATEMENT OF SIGNIFICANCE: Understanding the dissolution mechanism of copper doped ß-tricalcium phosphate (CuTCP) is crucial for designing an angiogenetic controlled copper release CuTCP for therapeutic biomaterials. However, dissolution mechanism of ß-TCP or CuTCP is still under intensive debates. This study demonstrated for the first time, that amorphous hydrated layers were formed on the CuTCP particle surface during its dissolution process, which caused a diffusion-controlled dissolution, and decreased the dissolution rate of CuTCP. This work not only provided a novel dissolution mechanism of ß-TCP or CuTCP, but also a new finding for designing an angiogenetic controlled copper release CuTCP for therapeutic biomaterials.

Fabrication of chelate-setting α-tricalcium phosphate cement using sodium citrate and sodium alginate as mixing solution and its in vivo osteoconductivity.

Moldable and injectable calcium-phosphate cements (CPCs) are material candidates for bone replacement applications. In the present study, we examined the effectiveness of sodium alginate and sodium citrate additives to the liquid phase of CPC, in improving its handling property as well as mechanical strength. The use of these additives enhanced the handling property significantly, in terms of consistency as compared to CPC without additives due to the liquefying effect caused by the adsorption of citrate ions on the cement particles. Sodium alginate and sodium citrate were added to CPC, which was set by the chelate-bonding capability of inositol phosphate, and was composed of mainly α-tricalcium phosphate (α-TCP) phase (>90%). The compressive strength of the CPC containing sodium alginate and sodium citrate was 3.4 ± 0.3 MPa, which was significantly higher than cement without additives. Furthermore, this cement exhibited favorable osteoconductivity and bioresorbability, and remained the α-TCP phase after 4-week implantation in a pig tibiae model. These results suggested that the cement is a potential candidate as a bioresorbable paste-like artificial bone. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 2361-2370, 2018.

Preparation of chitosan-hydroxyapatite composite mono-fiber using coagulation method and their mechanical properties.

Autograft has been carried out for anterior cruciate ligament (ACL) reconstruction surgery. However, it has negative aspect because patients lose their healthy ligaments from other part. We focus on a chitosan-hydroxyapatite (HAp) composite fiber as a scaffold of ligament regeneration. Chitosan- HAp composite fiber was made by using coagulation method. Chitosan-NaH2PO4 solution was coagulated with coagulation bath including calcium ion to get the mono-fiber and then treated with sodium hydroxide solution to form HAp in fiber matrix. The mechanical property of the fiber was improved by the stretching of the wet one because of the orientation of chitosan molecule and the interaction between chitosan and HAp. Maximum stress was improved with increasing of sodium dihydrogen phosphate until 0.03M. The swelling ratio of the fiber was inhibited by composited with HAp. Additionally, bone-bonding ability was confirmed by SBF soaking tests.

Effects of Adding Polysaccharides and Citric Acid into Sodium Dihydrogen Phosphate Mixing Solution on the Material Properties of Gelatin-Hybridized Calcium-Phosphate Cement.

We have succeeded in improving the material properties of a chelate-setting calcium-phosphate cement (CPC), which is composed of hydroxyapatite (HAp) the surface of which has been modified with inositol hexaphosphate (IP6) by adding α-tricalcium phosphate (α-TCP) powder. In order to create a novel chelate-setting CPC with sufficient bioresorbability, gelatin particles were added into the IP6-HAp/α-TCP cement system to modify the material properties. The effects of adding polysaccharides (chitosan, chondroitin sulfate, and sodium alginate) into the sodium dihydrogen phosphate mixing solution on the material properties of the gelatin-hybridized CPC were evaluated. The results of mechanical testing revealed that chondroitin sulfate would be the most suitable for fabricating the hybridized CPC with higher compressive strength. Moreover, further addition of an appropriate amount of citric acid could improve the anti-washout capability of the cement paste. In summary, a gelatin-hybridized IP6-HAp/α-TCP cement system prepared with a mixing solution containing chondroitin sulfate and citric acid is expected to be a beneficial CPC, with sufficient bioresorbability and material properties.

In-vivo evaluation of subcutaneously implanted cell-loaded apatite microcarriers for osteogenic potency.

Cell-loaded apatite microcarriers present as potential scaffolds for direct in-vivo delivery of cells post-expansion to promote bone regeneration. The objective of this study was to evaluate the osteogenic potency of human foetal mesenchymal stem cells (hfMSC)-loaded apatite microcarriers when implanted subcutaneously in a mouse model. This was done by examining for ectopic bone formation at 2 weeks, 1 month and 2 months, which were intended to coincide with the inflammation, healing and remodelling phases, respectively. Three histological examinations including haematoxylin and eosin staining to examine general tissue morphology, Masson's trichrome staining to identify tissue type, and Von Kossa staining to examine extent of tissue mineralisation were performed. In addition, immunohistochemistry assay of osteopontin was conducted to confirm active bone formation by the seeded hfMSCs. Results showed a high level of tissue organisation and new bone formation, with active bone remodelling being observed at the end of 2 months, and an increase in tissue density, organisation, and mineralisation could also be observed for hfMSC-loaded apatite microcarriers. Various cell morphology resembling that of osteoblasts and osteoclasts could be seen on the surfaces of the hfMSC-loaded apatite microcarriers, with presence of woven bone tissue formation being observed at the intergranular space. These observations were consistent with evidence of ectopic bone formation, which were absent in group containing apatite microcarriers only. Overall, results suggested that hfMSC-loaded apatite microcarriers retained their osteogenic potency after implantation, and provided an effective platform for bone tissue regeneration.

Injectable chelate-setting hydroxyapatite cement prepared by using chitosan solution: Fabrication, material properties, biocompatibility, and osteoconductivity.

An injectable chelate-setting hydroxyapatite cement (IP6-HAp), formed by chelate-bonding capability of inositol phosphate (IP6), was developed. The effects of ball-milling duration of starting HAp powder and IP6 concentration on the material properties such as injectability and mechanical strength of the cement were examined. The cement powder was prepared by ball-milling the as-synthesized HAp powder for 5 min using ZrO2 beads with a diameter of 10 mm, followed by another 60 min with ZrO2 beads with a diameter of 2 mm, and thereafter surface-modified with 5000 ppm of IP6 solution. Injectable cement was then fabricated with this HAp powder and 2.5 mass% chitosan as a mixing solution, with a setting time of 36.3 ± 4.7 min and a compressive strength of 19.0 ± 2.1 MPa. The IP6-HAp cements prepared with chitosan showed favorable biocompatibility in vitro using an osteoblast cell model, and osteoconductivity in vivo using a pig tibia model.

Nanomaterial scaffolds to regenerate musculoskeletal tissue: signals from within for neovessel formation.

Current treatments for musculoskeletal disease and injury are restricted with the usage of autografts and allografts. Tissue engineering that applies the principles of biology and engineering to develop functional substitutes has potential promise of therapeutic regeneration for musculoskeletal tissues. However, engineering sizable tissues needs a vascular network to supply cells with nutrients, oxygen and signals after implantation. For this purpose, recent developments on therapeutic nanomaterials have been explored in delivering different vessel-inductive growth factors, small biomolecules and ions for scalable engineering into vascularizable scaffolds. Here, we provide an overview on the current efforts, and propose future perspectives for precise regulation on vascularization processes and musculoskeletal tissue functionality.

A multi-material coating containing chemically-modified apatites for combined enhanced bioactivity and reduced infection via a drop-on-demand micro-dispensing technique.

Prevention of infection and enhanced osseointegration are closely related, and required for a successful orthopaedic implant, which necessitate implant designs to consider both criteria in tandem. A multi-material coating containing 1:1 ratio of silicon-substituted hydroxyapatite and silver-substituted hydroxyapatite as the top functional layer, and hydroxyapatite as the base layer, was produced via the drop-on-demand micro-dispensing technique, as a strategic approach in the fight against infection along with the promotion of bone tissue regeneration. The homogeneous distribution of silicon-substituted hydroxyapatite and silver-substituted hydroxyapatite micro-droplets at alternate position in silicon-substituted hydroxyapatite-silver-substituted hydroxyapatite/hydroxyapatite coating delayed the exponential growth of Staphylococcus aureus for up to 24 h, and gave rise to up-regulated expression of alkaline phosphatase activity, type I collagen and osteocalcin as compared to hydroxyapatite and silver-substituted hydroxyapatite coatings. Despite containing reduced amounts of silicon-substituted hydroxyapatite and silver-substituted hydroxyapatite micro-droplets over the coated area than silicon-substituted hydroxyapatite and silver-substituted hydroxyapatite coatings, silicon-substituted hydroxyapatite-silver-substituted hydroxyapatite/hydroxyapatite coating exhibited effective antibacterial property with enhanced bioactivity. By exhibiting good controllability of distributing silicon-substituted hydroxyapatite, silver-substituted hydroxyapatite and hydroxyapatite micro-droplets, it was demonstrated that drop-on-demand micro-dispensing technique was capable in harnessing the advantages of silver-substituted hydroxyapatite, silicon-substituted hydroxyapatite and hydroxyapatite to produce a multi-material coating along with enhanced bioactivity and reduced infection.

In vitro and in vivo antimicrobial properties of silver-containing hydroxyapatite prepared via ultrasonic spray pyrolysis route.

Hydroxyapatite (HAp), with its high biocompatibility and osteoconductivity, readily absorbs proteins, amino acids and other substances, which in turn favor the adsorption and colonization of bacteria. To prevent bacterial growth and biofilm formation on HAp discs, silver-containing (1-20 mol%) HAp (Ag-HAp) powders were synthesized using an ultrasonic spray pyrolysis (USSP) technique. The X-ray diffraction (XRD) peaks were very broad, indicating low crystallinity, and this induced the release of Ag(+) ions from Ag-HAp powders. In addition, a gradual increase in Ca(2+) ion release was observed. These results suggest that dissolution of Ca(2+) ion in Ag-HAp triggered the release of Ag(+) ions. The antimicrobial efficacy of Ag-HAp disc was tested against Staphylococcus aureus. Samples with Ag contents of more than 5 mol% were found to be highly effective against bacterial colonization and biofilm formation in vitro. In vivo antibacterial tests using bioluminescent strains also showed reductions in the viability of bacteria with Ag-HAp (5 mol%) discs. Biocompatibility tests using a modified Transwell® insert method showed that Ag-HAp (5 mol%) discs have negative effects on osteoblast proliferation. These results indicate that Ag-HAp (5 mol%) has effective antibacterial activity and good biocompatibility both in vitro and in vivo together with good biocompatibility, thus confirming its utility as a bactericidal material.

Biodegradable ß-tricalcium phosphate cement with anti-washout property based on chelate-setting mechanism of inositol phosphate.

Novel biodegradable ß-tricalcium phosphate (ß-TCP) cements with anti-washout properties were created on the basis of chelate-setting mechanism of inositol phosphate (IP6) using ß-TCP powders. The ß-TCP powders were ball-milled using ZrO2 beads for 0-6 h in the IP6 solutions with concentrations from 0 to 10,000 ppm. The chelate-setting ß-TCP cement with anti-washout property was successfully fabricated by mixing the ß-TCP powder ball-milled in 3,000 ppm IP6 solution for 3 h and 2.5 mass% Na2HPO4 solution, and compressive strength of the cement was 13.4 ± 0.8 MPa. An in vivo study revealed that the above cement was directly in contact with host and newly formed bones without fibrous tissue layers, and was resorbed by osteoclast-like cells on the surface of the cement. The chelate-setting ß-TCP cement with anti-washout property is promising for application as a novel injectable artificial bone with both biodegradability and osteoconductivity.

Development of a,b-plane-oriented hydroxyapatite ceramics as models for living bones and their cell adhesion behavior.

In vertebrate bones and tooth enamel surfaces, the respective a,b-planes and c-planes of hydroxyapatite (HAp) crystals are preferentially exposed. However, the reason why the HAp crystals show different orientations depending on the type of hard tissues is not yet understood. To clarify this question, appropriate ceramic models with highly preferred orientation are necessary. In the present study, dense HAp ceramic models which have the same orientation as living bones were fabricated using composite powders of c-axis-oriented single-crystal apatite fibers (AF) and wet-synthesized apatite gels (AG). The results of crystalline identification and ultrastructural observation showed that the resulting HAp ceramics maintained the c-axis orientation of the AF particles, and their high a,b-plane orientation degrees could be maintained with small additive amounts of AG; however, when the AG content was over 30 mass%, this value decreased. The influence of orientation degree on the surface characteristics was investigated by evaluating the surface zeta-potential and wettability. These results show that increasing the a,b-plane orientation degree shifted the surface charge from negative to positive, and decreased the surface wettability. Initial cell-attachment assays were performed on these resulting ceramics using MC3T3-E1 cells as models of osteoblasts. The results show that the cell-attachment efficiency decreased with increasing a,b-plane orientation degree.

Novel chelate-setting calcium-phosphate cements fabricated with wet-synthesized hydroxyapatite powder.

The hydroxyapatite (HAp) powder preparation process was optimized to fabricate inositol phosphate-HAp (IP6-HAp) cement with enhanced mechanical properties. Starting HAp powders were synthesized via a wet chemical process. The effect of the powder preparation process on the morphology, crystallinity, median particle size, and specific surface area (SSA) of the cement powders was examined, together with the mechanical properties of the resulting cement specimens. The smallest crystallite and median particle sizes, and the highest SSA were obtained from ball-milling of as-synthesized HAp powder under wet conditions and then freeze-drying. IP6-HAp cement fabricated with this powder had a maximum compressive strength of 23.1 ± 2.1 MPa. In vivo histological studies using rabbit models revealed that the IP6-HAp cements were directly in contact with newly formed and host bones. Thus, the present chelate-setting HAp cement is promising for application as a novel paste-like artificial bone.

Enhanced early osteogenic differentiation by silicon-substituted hydroxyapatite ceramics fabricated via ultrasonic spray pyrolysis route.

The influence of silicon-substituted hydroxyapatite (Si-HAp) on osteogenic differentiation was assessed by biological analysis. Si-HAp was prepared by ultrasonic spray pyrolysis (USSP) technique using various amounts of Si (0, 0.8, and 1.6 mass%). Chemical analysis revealed that Si was incorporated into the hydroxyapatite (HAp) lattice with no other crystalline phase and which caused the change of crystal structure. Biological analyses showed that the Si contents affected the cell proliferation and morphology, suggesting that there is an optimal Si content for cell culture. As for differentiation, alkaline phosphatase activity and osteocalcin production of Si-HAp were higher than those of HAp. Gene expression profiles also revealed that substitution of Si (0.8 mass%) up-regulated the expression levels of osteocalcin and especially Runx2, a master gene for osteoblast development. These results suggest that incorporating Si into the HAp lattice may enhance the bioactivity, particularly during early osteoblast development.