Influence of Culture Period on Osteoblast Differentiation of Tissue-Engineered Bone Constructed by Apatite-Fiber Scaffolds Using Radial-Flow Bioreactor.

With the limitation of autografts, the development of alternative treatments for bone diseases to alleviate autograft-related complications is highly demanded. In this study, a tissue-engineered bone was formed by culturing rat bone marrow cells (RBMCs) onto porous apatite-fiber scaffolds (AFSs) with three-dimensional (3D) interconnected pores using a radial-flow bioreactor (RFB). Using the optimized flow rate, the effect of different culturing periods on the development of tissue-engineered bone was investigated. The 3D cell culture using RFB was performed for 0, 1 or 2 weeks in a standard medium followed by 0, 1 or 2 weeks in a differentiation medium. Osteoblast differentiation in the tissue-engineered bone was examined by alkaline phosphatase (ALP) and osteocalcin (OC) assays. Furthermore, the tissue-engineered bone was histologically examined by hematoxylin and eosin and alizarin red S stains. We found that the ALP activity and OC content of calcified cells tended to increase with the culture period, and the differentiation of tissue-engineered bone could be controlled by varying the culture period. In addition, the employment of RFB and AFSs provided a favorable 3D environment for cell growth and differentiation. Overall, these results provide valuable insights into the design of tissue-engineered bone for clinical applications.

Silver, silicon co-substituted hydroxyapatite modulates bacteria-cell competition for enhanced osteogenic function.

Combating bacteria while promoting tissue regeneration is an aim of highest priority for employing biomaterials in orthopedics that often embroiled with pre-operative contamination. Through simulating a surgical site infection environment and an infected implant site, we showcase the ability of a functionally modified hydroxyapatite, Ag,Si-HA that permits preferential adhesion of human bone marrow derived mesenchymal stem cells (BMSCs) over co-cultured bacterial pathogen,Pseudomonas aeruginosa, by displaying immediate suppression and killing of the bacteria present with minimum cytotoxicity for 28 d. And, at the same time, Ag,Si-HA stimulates BMSCs towards osteogenic differentiation despite being within the contaminated milieu. These findings provide well-defined requirements for incorporating antibacterial properties to biomaterials in managing pre-operative contamination. In addition, it highlights the dual positive attributes of Ag,Si-HA as an effective antibacterial biomaterial and at the same time, promotes bone tissue regeneration.

Regulating size of silver nanoparticles on calcium carbonate via ultrasonic spray for effective antibacterial efficacy and sustained release.

Calcium carbonate is used as bone-filling material due to its good biocompatibility, bioactivity, and bioabsorbability, but the prevalence of infectious complications associated with calcium carbonate has created a persisting challenge in the treatment of bone defect. Therefore, this greatly necessitate the need to endow calcium carbonate with antibacterial properties. In this study, calcium carbonate powders loaded with silver nanoparticles (Ag-CaCO3) were prepared in attempt to serve as a novel antibacterial inorganic filler material. This objective was achieved using ultrasonic spray-pyrolysis (USSP) route to produce Ag-CaCO3 with 1, 5 and 10 mol% silver. The size of silver nanoparticles on CaCO3 microspheres could be regulated by adjusting silver concentration to facilitate effective release of Ag+ ions. This was demonstrated in Ag-CaCO3 (1), where the lowest silver content at 1 mol% achieved the highest Ag+ ions release over 28 days. This in turn gave rise to effective antibacterial efficiency against Staphylococcus aureus and Escherichia coli. Furthermore, CaCO3 (1) could also support osteoblast-like cells (MG-63) at a cell viability of 80%. Overall, this work extends the capabilities in employing USSP to produce inorganic filler materials with sustained antibacterial properties, bringing one step closer to the development of antibacterial products.

Silicon-incorporated nanohydroxyapatite-reinforced poly(ε-caprolactone) film to enhance osteogenesis for bone tissue engineering applications.

Biomaterials composed of polymers and bioceramics have great prospects to repair large and complicated bone defects. Here, we developed a composite film consisting of poly(ε-caprolactone) (PCL) and silicon-substituted hydroxyapatite (Si-HA) nanoparticles to enhance the osteogenic effects of the scaffold for bone tissue engineering applications. The results showed that the Si-HA nanoparticles obtained an even distribution in the PCL matrix, resulting in a homogeneous composite film. Compared to HA-incorporated PCL film, the addition of silicon did not cause hydrophilic alterations to the film surface. With the seeding of mouse calvarial preosteoblasts (MC3T3-E1), the cells exhibited the good behaviors of adhesion and growth on the PCL/Si-HA film. Compared to the PCL/HA films, incorporation of Si-HA nanoparticles in PCL/Si-HA films showed the increased production of alkaline phosphatase (ALP) and calcium content by MC3T3-E1 cells. These results suggested the suitability of the PCL/Si-HA composite film to elicit cellular growth and functional differentiation with the potential for bone tissue engineering applications.

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.

Beta-cyclodextrin modified mesoporous bioactive glass nanoparticles/silk fibroin hybrid nanofibers as an implantable estradiol delivery system for the potential treatment of osteoporosis.

Osteoporosis, a systemic skeletal disease prevalent in elderly women, is associated with post-menopausal estrogen deficiency. Although systemic administration of exogenous estradiol (E2) reduced fragility fractures, the treatment has adverse effects. Localized delivery technologies of E2 could be utilized to circumvent the systemic adverse effects of systemic administration. In this study, a localized E2 delivery system is developed. Mesoporous bioactive glass nanoparticles (MBGNPs) with inherent osteogenic properties are modified with ß-cyclodextrin (CD-MBGNPs) to enhance their affinity for E2. To ensure mechanical stability and integrity, E2 loaded CD-MBGNPs are further electrospun with silk fibroin (SF) to produce a nanofibrous mesh (E2@CD-MBGNPs/SF). The incorporation of MBGNPs in SF enhances in vitro apatite formation and sustains the constant release of E2. Moreover, osteoblast proliferation and differentiation markers such as alkaline phosphatase activity, collagen 1 and osteocalcin expression of MC3T3-E1 are augmented in CD-MBGNPs/SF and E2@CD-MBGNPs/SF as compared to SF nanofibers. On the other hand, osteoclast DNA, tartrate resistant acid phosphatase activity and multinucleated cell formation are reduced in E2@CD-MBGNPs/SF as compared to CD-MBGNPs/SF and SF. Hence the presence of CD-MBGNPs in SF stimulates osteoblast function whereas E2 incorporation in CD-MBGNPs/SF reduces osteoclast activity. This is the first report to develop CD-MBGNPs/SF as a localized delivery system for hydrophobic molecules such as estradiol to treat osteoporosis.

Estradiol-Loaded Poly(ε-caprolactone)/Silk Fibroin Electrospun Microfibers Decrease Osteoclast Activity and Retain Osteoblast Function.

Estrogen, a steroid hormone, plays an important role in modulating osteoclast proliferation and development. Estrogen deficiency boosts osteoclast activity, leading to osteoporosis in elderly women. In this study, 17-ß estradiol (E2)-loaded poly(ε-caprolactone) (PCL)/silk fibroin (SF) electrospun microfibers were developed as a proposed localized E2 delivery system to treat osteoporotic fractures. PCL is a synthetic polymer known for its biocompatibility and excellent mechanical properties. The bioactivity of PCL was enhanced by mixing it with a natural SF polymer that has low immunogenicity and inherent bioactivity. Different ratios of PCL/SF blends were electrospun and characterized by scanning electron microscopy, Fourier transform infrared spectroscopy, and water contact angle measurement. PCL and SF at a ratio of 50:50 (PCL50/SF50) augmented cell proliferation of murine preosteoblast MC3T3-E1 cells and murine preosteoclast RAW 264.7 cells. Hence, PCL50/SF50 was selected and mixed with three concentrations of E2 to produce electrospun fiber mesh (0.1% E2@PCL/SF, 1% E2@PCL/SF, and 5% E2@PCL/SF). Sustained release of E2 was obtained for about 3 weeks at higher E2 concentration 5% E2@PCL/SF. An E2-loaded PCL50/SF50 elecrospun microfiber (1% E2@PCL/SF and 5% E2@PCL/SF) reduced tartrate-resistant acid phosphate activity, total DNA, and multinucleated cell formation of osteoclasts. On the other hand, the alkaline phosphatase activity and collagen I expression of osteoblasts were retained on all E2-loaded electrospun microfibers. The E2@PCL/SF system shows potential to be used for localized E2 delivery for the treatment of osteoporotic fractures.

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.

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.

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.

Uniformly-dispersed nanohydroxapatite-reinforced poly(ε-caprolactone) composite films for tendon tissue engineering application.

Regeneration of injuries at tendon-to-bone interface (TBI) remains a challenging issue due to the complex tissue composition involving both soft tendon tissues and relatively hard bone tissues. Tissue engineering using polymeric/ceramic composites has been of great interest to generate scaffolds for tissue's healing at TBI. Herein, we presented a novel method to blend polymers and bioceramics for tendon tissue engineering application. A homogeneous composite comprising of nanohydroxyapatite (nHA) particles in poly(ε-caprolactone) (PCL) matrix was obtained using a combination of solvent and mechanical blending process. X-ray diffraction analysis showed that the as-fabricated PCL/nHA composite film retained phase-pure apatite and semi-crystalline properties of PCL. Infrared spectroscopy spectra confirmed that the PCL/nHA composite film exhibited the characteristics functional groups of PCL and nHA, without alteration to the chemical properties of the composite. The incorporation of nHA resulted in PCL/nHA composite film with improved mechanical properties such as Young's Modulus and ultimate tensile stress, which were comparable to that of the native human rotator tendon. Seeding with human tenocytes, cells attached on the PCL/nHA composite film, and after 14days of culturing, these cells could acquire elongated morphology without induced cytotoxicity. PCL/nHA composite film could also result in increased cell metabolism with prolonged culturing, which was comparable to that of the PCL group and higher than that of the nHA group. All these results demonstrated that the developed technique of combining solvent and mechanical blending could be applied to fabricate composite films with potential for tendon tissue engineering applications.

Development of nanosized silver-substituted apatite for biomedical applications: A review.

The favorable biocompatibility of hydroxyapatite (HA) makes it a popular bone graft material as well as a coating layer on metallic implant. To reduce implant-related infections, silver ions were either incorporated into the apatite during co-precipitation process (AgHA-CP) or underwent ion-exchange with the calcium ions in the apatite (AgHA-IE). However, the distribution of silver ions in AgHA-CP and AgHA-IE was different, thus affecting the antibacterial action. Several studies reported that nanosized AgHA-CP containing 0.5 wt.% of silver provided an optimal trade-off between antibacterial properties and cytotoxicity. Nevertheless, nanosized AgHA and AgHA nanocoatings could not function ideally due to the compromise in the bone differentiation of mesenchymal stem cells, as evidenced in the reduced alkaline phosphatase, type I collagen and osteocalcin. Preliminary studies showed that biological responses of nanosized AgHA and AgHA nanocoatings could be improved with the addition of silicon. This review will discuss on nanosized AgHA and AgHA nanocoatings. FROM THE CLINICAL EDITOR: In many patients needing bone graft material, hydroxyapatite (HA) has proven to be a popular choice. Nonetheless, implant-related infections remain a major concern. Hence, effective preventive measures are needed. In this review article, the authors discussed the application of incorporating silver nanoparticles in HA and its use as bone graft biomaterials together with the addition of silica.

The effects of silver, silicon-containing apatite towards bacteria and cell responses.

An integrated approach is proposed to incorporate silicon and silver into hydroxyapatite (HA) to enhance the biological response and reduce implant-related infection in bone substitutes. This study examined the responses of Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli) bacteria to silver, silicon-containing apatite (Ag,Si-HA). Scanning electron microscopy images revealed significant reduction in adherence of S. aureus and E. coli bacteria on Ag,Si-HA as compared to HA. The antibacterial property of Ag,Si-HA was shown from a 7-log reduction of S. aureus population in the test solution and on the sample's surface as compared to HA at day 7. Rapid inhibition of the adherent bacteria suggested that the antibacterial action of Ag incorporated in Ag,Si-HA could be attributed to the Ag(+) ions on the crystal surface rather than the released Ag(+) ions. Presence of Ag may influence the biological response of HA and as such, the long-term interaction between human adipose-derived mesenchymal stem cells and Ag,Si-HA was evaluated in-vitro. An alamarBlue™ assay showed higher cell proliferation for Ag,Si-HA as compared to HA from day 3 onwards. Immunofluorescence staining revealed well-spread actin cytoskeletons on Ag,Si-HA. In addition, signs of extracellular matrix secretion and biomineralization were observed on Ag,Si-HA at day 14 onwards. Results demonstrated enhanced bone differentiation on Ag,Si-HA, as indicated by a higher level of protein expressions (type 1 collagen and osteocalcin) from day 14 to 21. Therefore, the incorporation of Ag and Si complement each other by endowing HA with antibacterial property, and concurrently promoting biological performance of the cells.

Effect of silver content on the antibacterial and bioactive properties of silver-substituted hydroxyapatite.

The long-term success of a biomaterial used during surgery may be compromised by infection. A possible effective solution is to make the biomaterial osteoconductive and antibacterial. A range of silver-substituted hydroxyapatite (AgHA) of up to 1.1 wt. % of Ag was synthesized. AgHA displayed a rod-like morphology of dimensions ~50 nm in length and ~15 nm in width. Phase-pure AgHA was demonstrated in the X-ray diffraction patterns and Fourier transform infrared spectroscopy spectra. Comparing with hydroxyaptite (HA), 0.5AgHA exhibited a 3-log reduction in the number of bacteria. Diffusion of the entrapped Ag(+) ions towards the crystal structure surface was revealed by an increase of 6 at. % Ag in the X-ray photoelectron spectroscopy results. Furthermore, less than 0.5 ppm of Ag(+) ions being released from 0.5AgHA into the deionized water medium was evidenced from the inductively coupled plasma mass spectrometry results. AgHA produced by co-precipitation gave rise to minimal release of Ag(+) ions. It was hypothesized that the diffused surface Ag(+) ions damaged the bacteria cell membrane and impede its replication. With the culturing time, significant increase in the number of human mesenchymal stem cells (p < 0.05) was demonstrated on 0.5AgHA.

Synthesis and characterization of silver/silicon-cosubstituted nanohydroxyapatite.

Favorable cell-material interaction and the absence of undesirable reaction from the host body defence system play a critical role in determining the success and long-term survival of the implants. Substitution of various elements into hydroxyapatite (HA) has been done to alter its chemical composition, thereby mimicking that of the bone mineral. In this study, a cosubstituted nanosized apatite (Ag/Si-HA) containing Ag (0.3 wt %) and Si (0.8 wt %) was synthesized by an aqueous precipitation technique. The synthesized Ag/Si-HA displayed a rod-like morphology of dimensions ~50 nm in length and ~15 nm in width, as observed from the transmission electron microscope image. With an increase in temperature, the aspect ratio of nanosized Ag/Si-HA decreased, whilst the size increased. Autoclaving was used to achieve sufficient crystallinity while maintaining the rod-like morphology and size that were comparable to that of the bone apatite. A pure Ag/Si-HA was produced without any undesirable secondary phases, as evidenced from the X-ray diffraction and thermal gravimetric results. The Ag/Si cosubstitution affected the lattice cell parameters, in particularly the a- and c- axes which further led to an expansion of the unit cell volume. In addition, the relative intensity of the hydroxyl vibrational bands was reduced. These results demonstrated that a stable phase-pure Ag/Si-HA was produced using an aqueous precipitation reaction.