Key Engineering Materials Vol. 631

Abstract: In the present study, we used an apatite-fiber scaffold (AFS) to culture P19.CL6 cells three-dimensionally. AFS was originally developed to use for the bone substitute and has high porosity and complex pore structure. The highly porous structure of AFS suggests it may be compatible for the in vitro reorganization of soft tissue. We previously showed the formation of small cell aggregates in AFS, and that culture in AFS increased the expression of cardiac-specific gene markers without the need for any inducing agent such as DMSO. However, it is difficult to evaluate the physiological function of cells in three-dimensional culture. In this study, we transformed P19.CL6 cells with the pTnnt2::GCaMP5G vector and observed the cells by fluorescence microscopy. pTnnt2::GCaMP5G consists of green fluorescent protein (GFP) fused with the Ca²⁺-sensitive domain of calmodulin; its expression is driven by a cardiomyocyte-specific promoter. We observed that the blinking of green fluorescence was synchronized to the beating of cardiomyocytes when the P19.CL6 cells were cultured in a dish, but blinking was not observed when the cells were cultured in AFS, even after 16 days. The expression of connexin 43 (Cx43) and enhanced Green Fluorescence Protein (EGFP) was examined by reverse transcriptase-polymerase chain reaction (RT-PCR). Cx43 is a gap junction protein expressed in cardiomyocytes that mediates cell-to-cell coupling. Although the expression of Cx43 and EGFP in transformed cells cultured in AFS was evident, fluorescence blinking of the cells was not observed. The results demonstrate that P19.CL6 cells cultured in AFS rapidly differentiated into early stage cardiomyocytes; however, additional modifications or developments are needed for further differentiation.

Abstract: Natural bone ECM is a hierarchical nanocomposite made of an inorganic phase deposited within an organic matrix. In order to mimic the bone highly organized hybrid structure and functionality, strategies that allow assembling ceramic and polymer phase can be applied. To this aim, we investigated an in situ growth method able to nucleate a nanoHydroxyapatite (nHAp) phase into and around the interconnected porous structure of chitosan sponges. By increasing the calcium and phosphate concentration in the meta-stable solution used for the nHAp nucleation, the inorganic phase raised proportionally, in the range 10%-30% wt. In order to be compared with nHAp loaded scaffolds, pure chitosan samples have been produced by cross-linking biopolymer with arginine. Moreover, nHAp loaded samples, containing the 20 % wt of inorganic phase have been prepared by simply mixing low crystalline nHAp powders with the chitosan gel. The in situ nucleation method highlighted evident advantages in terms of nanophase distribution and mechanical performances with respect to a merely mixing procedure.

Abstract: This study investigated the feasibility of manufacturing hydroxyapatite (HA)-based scaffolds using 3D printing technology by incorporating different binding additives, such as maltodextrin and polyvinyl alcohol (PVOH), into the powder formulation. Different grades of PVOH were evaluated in terms of their impact on the printing quality. Results showed that scaffolds with high architectural accuracy in terms of the design and excellent green compressive strength were obtained when the PVOH (high viscosity) was used as the binding additive for HA.

Abstract: Strontium is known for efficient actions on bone formation and resorption. Strontium ranelate (SrR) is a commercial drug which maintains this balance during bone turnover, reducing the risks of vertebral fractures in the patients. Calcium phosphate bioceramics associated with alginate matrices containing strontium (Sr) could improve bone regeneration due to gradual Sr release. In this report, the strontium ranelate was incorporated on microspheres of alginate (ALG)/β-tricalcium phosphate in a single route of the production. Energy dispersive spectroscopy showed that strontium was incorporated on the surface of the microspheres produced. The dissolution behaviour into a buffer solution at pH 4.0 and at 7.4 was evaluated, measuring Sr content on the microspheres after in vitro assays by atomic absorption spectrometry. Dissolution tests showed a rapid strontium release in both assays, however, it was more pronounced at pH 4.0. Fourier transform infrared spectra indicated the presence of a new precipitated phase at pH 7.4 up after 14 days. Scanning electron microscopy of the microspheres submitted to in vitro revealed that the microspheres at pH 4.0 buffer underwent erosion up to 7 days, while the ones in pH 7.4 buffer, eroded in 48h. This behaviour is due to the high swelling rate of the microspheres in neutral pH. The solubility of the microspheres favors its use as a great material for a local strontium release and remodeling bone.

Abstract: The aim of the present study was the in vitro investigation of a synthetic bone graft substitute loaded with individual antibiotics for the treatment of osteomyelitis and infectious bone disease. The elution of gentamicin, an aminoglycoside antibiotic, from the NanoBone^® products NanoBone^® S granules (NBG) and lyophilized NanoBone^® (NBP) putty was tested over a period of one week. An indirect photometrically-based detection system was used to measure the released antibiotic concentration. Both materials showed very different release behaviour. After one day lyophilized NanoBone^® putty delivered 100% of the gentamicin value, whereas NanoBone^® S granules released one-fifth of the used gentamicin level.

Abstract: When either the pH or temperature of simulated body fluid (SBF) are raised, fine particles of calcium phosphate are precipitated. We found that these fine particles actively induce hydroxyapatite formation from body fluid or SBF and named the particles Apatite Nuclei. In this study, we fabricated hollow hydroxyapatite microcapsules by using Apatite Nuclei. We inserted vitamin B₁₂ in the hollow microcapsule and examined the sustained-release properties.

Abstract: The release property of osteoprotegerin (OPG) from hydroxyapatite / chondroitin sulphate (HAp/ChS) microparticles was described in this paper. The formulation was conducted by subsequent addition of zinc chloride solution into the mixture of HAp/ChS microparticles as a ceramic carrier and OPG solution. The release of OPG from the microparticles in PBS was positively correlated with soaking time without initial burst. The release rate of OPG was decreased with the increase of the amounts of zinc cation, which was not dependent of ChS wt% in the microparticles. The controlled release of OPG would provide the improvement of drug effect and the reduction of adverse effects. These findings reveal further investigation of the other antiresortive agents as a potential safer medication for osteoporosis.

Abstract: This work revealed the ability of silver,silicon co-substituted apatite (Ag,Si-HA) to inhibit the growth of Staphylococcus aureus (S. aureus), with a 7-log reduction of adherent bacteria on Ag,Si-HA as compared to HA. Furthermore, it demonstrated that surface-bound Ag⁺ ions was responsible for the antibacterial action since there was not much Ag⁺ ions being released.

Abstract: Hydroxiapatite (HA), one of the most widely employed bioceramic bone substitutes, when applied on its nanostructured form (nHA) may contribute to achieve a crystalline structure which is closer to the size and morphology of biological apatite. Furthermore, HA might also be doped with several different cations with biological effects including Sr2+. Therefore, a biomaterial based on nanostructured HA containing 1% Strontium (nSrHA) could present interesting biological properties, as strontium is described as a modulator of both osteoblast and osteoclast activities, presenting an important regulatory role on bone resorption. However, such modifications may also affect the biocompatibility of this material, which should be accessed initially by in vitro methods. Therefore, the present work aimed to evaluate the in vitro biocompatibility of 1% nSrHA discs with human primary osteoblasts through a multiparametric assay which assesses simultaneously metabolic activity (XTT assay), membrane integrity (NR test) and cell density (CVDE). Extracts of nSrHA, latex fragments (positive control), polystyrene beads (negative control) and nHA (for comparison) were prepared and exposed to 104 cells for 24h at 37°C/5% CO₂ on test plates, according to ISO 10993-5:2009, on quintuplicates. Cells exposed to unconditioned media were used as experimental control. After exposure, cells were tested for viability with a commercial multiparametric kit (In Cytotox, Xenometrix, Germany). The positive and negative controls presented the expected results, validating the assay. Both nHA and SrnHA were considered biocompatible, since the presented a cell viability after exposure statistically similar to the experimental control. In conclusion, the synthesized nSrHA discs are cytocompatible and, consequently, adequate for further in vitro tests on cell adhesion, proliferation and differentiation.

Abstract: Synthetic hydroxyapatite (HA) is a widely used ceramic biomaterial due to its well described biocompatibility. Some modifications in HA surface can be made to increase surface porosity. Likewise, HA can be modified by the coating with proteins, which may impact on biocompatibility. In this work, we aimed to evaluate the impact of two surface modifications – coating with albumin, a major serum protein, and augmented porosity - over osteoblast adhesion on stoichiometric HA discs. Dense HA discs were obtained by pressing HA powder at 30 KN and sinterization at 1000°C, while porous HA was molded after the addition of alginate (15:1), followed by thermal treatment. Protein adsorption was attained by incubation on 0.5mg/mL bovine serum albumin (BSA) for 24 h at 37°C. MC3T3 mouse preosteoblasts were seeded over both protein-coated and uncoated dense or porous tablets, and cell viability after 24 h was estimated by XTT and Neutral Red assays. Cell density was quantified by fluorescence microscopy. While both dense and porous discs presented altered surfaces after protein treatment, as observed by scanning electron microscopy, porous HA tablets presented significantly higher levels of adsorbed protein. There was a decrease in the concentration of calcium ions in all samples analyzed. Porous HA treated with protein presented significant higher mitochondrial dehydrogenase activity (XTT) than non treated tablets (p<0.001). Although the BSA adsorption didn`t affect cell adhesion, the results obtained in fluorescence quantification suggests that de dense surface was best for cellular adhesion and spread than the porous one. We conclude that differences in the topography of a biomaterial can directly influence their ability to adsorb proteins, while the dense surface was more favorable for both the adhesion and the spreading of pre-osteoblasts.