# Novel antimicrobial phosphate-free glass–ceramic scaffolds for bone tissue regeneration

Aug 21, 2020

### Manufacturing of highly porous scaffolds

In this study, a glass powder chemically composed of 40.3 SiO2, 8.5 B2O3, 18.8 Na2O, 19.2 CaO, 0.57 K2O, 11.7 Al2O3, 0.12 Fe2O3 and 0.81 of minor oxides (wt%), with a particle size of d50 = 13.05 ± 0.1 µm determined by using a particle size analyzer (LS Particle Size Analyzer 13 320, Beckman Coulter, Indianapolis, IN, USA) was used as starting material. The scaffolds were fabricated using a three-step methodology involving slurry preparation, induction of porosity by a surfactant-assisted foaming, freeze-drying and sintering of porous green scaffolds. For this, scaffolds with 60 and 70 wt% of solid content were prepared by adding a percentage (0.2–0.4 wt%) of surfactant agent (Tritón X 100, Panreac, Spain) and 10 wt% of binder (Duramax B1000, Rohm & Haas, USA) to 30 g of the slurry. Air bubbles were incorporated by continuous stirring with a mechanical shaker for 5 min. The homogeneous glass slurries incorporating uniformly-distributed bubbles throughout their volume were freeze-dried, thus obtaining green scaffolds. The treatment consisted of freezing at -19 °C for 24 h, followed by extraction of the ice crystals in a lyophilizer (CryoDos, Telstar, Spain) under vacuum (0.1 mbar) and held to -50 °C for 72 h. Glass–ceramic scaffolds were obtained by sintering at 750 °C for 1 h with a heating rate of 5 °C·min-1.The reference material Repros (BCP) (JRI Orthopaedics Ltd., Sheffield, UK) is composed of 60% hydroxyapatite and 40% β-tricalcium phosphate.

### Physicochemical characterization of the scaffolds

#### Porosity

The apparent density of the studied scaffolds was determined by using the Archimedes principle. Previously, 30 min vacuum treatment was used to remove the air inside of pores. Then the penetration of water inside the pores of the scaffold was forced. After this, the apparent porosity was calculated using the Eq. (1).

$$AP% = frac{{w_{2} – w_{1} }}{{w_{2} – w_{3} }} 100$$

(1)

where w2 is the mass of the saturated sample weighed in air, w1 is the mass of the dried sample and w3 is the apparent mass of the saturated sample weighed in liquid.

#### X-ray diffraction (XRD)

Phases and crystallinity of scaffolds were characterized by X-ray diffraction (XRD, AXS D8 Advance, Bruker, UK), by using Cu-Kα radiation (λ = 0.15406 nm) in the range from 5° to 70°. The step was 0.02° and the step time 0.5 s. The measurement conditions used were copper anticathode water cooled with an intensity of 40 mA and a current of 30 kV. The identification of the crystalline phases was realized by using diffraction pattern files provided by JCPDS (International Centre for Diffraction Data).

The assessment of in vitro degradation of scaffolds was carried out by immersing 0.05 g of scaffold in 10 mL of buffer solution (Tris–HCl). The buffer solution was prepared according to EN ISO 10,993–14:2001. The Tris–HCl solution was prepared by dissolving tris-hydroxymethylaminomethane (ACS reagent, ≥ 99.8%, Sigma-Aldrich, Spain) in water (grade 2) with buffering at pH 7.4 ± 0.1 by 1 mol/L hydrochloric acid (ACS reagent, 37%, Sigma-Aldrich, Spain) at 37 °C. The bioactivity of the samples was studied in simulated body fluid (SBF) at 37 °C, 120 rpm by immersing 0.05 g of scaffold in 10 mL of SBF. Simulated body fluid (SBF) was prepared as described by Kokubo and Takadama37. For degradation and bioactivity test six samples were collected after 2, 6, 72, 168, 336 and 672 h soaking at 37 °C and 120 rpm, respectively. At each time point, the samples were taken out, rinsed with deionized water and dried in an oven at 120 °C for 24 h. The element concentrations in the immersion solution at different time were analyzed by inductively coupled plasma-mass spectroscopy (ICP-MS 7700x, Agilent, US).

#### Morphology

The microstructural characterization of the scaffolds was performed by Scanning Electron Microscopy (SEM, Hitachi TM 3000, Japan). The pore size and its distribution are also calculated from SEM images using ImageJ software (ImageJ, U.S. National Institutes of Health, Bethesda, Maryland, USA). Chemical composition was analyzed by Elemental Dispersive Spectroscopy (EDS, Quantax 70).

#### Mechanical properties

Uniaxial compression testing was conducted to investigate the mechanical properties of glass–ceramic and Repros (BCP) scaffolds by using a universal mechanical testing machine (Shimadzu-Serie AGS-IX, Japan). Briefly, the scaffolds with nominal dimensions: 10 × 10 × 10 mm3 were fixed on the testing platen. The compressive strength was determined from the maximum load of the obtained stress–strain curve obtained from the load–displacement measurements using a 10 kN load cell at a crosshead speed of 0.5 mm/min. The stress (σ) was evaluated using the Eq. (2). Ten samples (n = 10) were used for each scaffold type and data were presented as mean ± standard deviation (SD).

$$sigma = frac{4F}{{d^{2} }}$$

(2)

where F is the load under the compressive test and d is the length of the scaffold.

### Biological interactions in vitro

#### Antimicrobial activity of Glass–ceramic scaffolds

The potential of the new glass–ceramic scaffolds to inhibit the growth of both gram-negative and gram-positive strains is assessed by using Escherichia coli DH10B (ATCC 207151) and Staphylococcus aureus (ATCC 25923) as model microorganisms. Two additional tests are conducted to evaluate the antibacterial activity due to direct contact and/or to dissolution products. Bacteria are grown in Luria Bertani (LB) broth (Difco, BD Diagnostics, Sparks, MD, USA) up to approx. 1.0 × 106 CFU ml−1. On one side, an inoculum of bacterial suspension (5 µl) is pipetted into the scaffolds (n = 3), and samples incubated at 37 °C in a humid atmosphere for 24 h. Microorganisms are then extracted from the inorganic constructs by shaking in phosphate-buffered saline solution (PBS, Sigma-Aldrich, Spain) (10 ml).On the other side, scaffolds (n = 3) are immersed into the bacterial suspension (200 µl), and incubated in agitation at 37 °C for 24 h. In both experiments, the number of survivors is determined by serial dilution plating.

The adsorption of model proteins on scaffold specimens was compared. Sterilized scaffolds (n = 9) were incubated in PBS (10 mM, pH 7.4) containing bovine serum albumin (BSA; 350 μg ml−1) or fetal bovine serum (FBS; 1 vol.%) at 37 °C for 60 min38. After incubation, the scaffolds were PBS-rinsed twice and the proteins extracted with a 2.0 wt% sodium dodecylsulfate (SDS, Sigma Aldrich, Spain) solution for 24 h. The total protein concentration was quantified with a microplate reader (BIO-RAD, Model 680, US) at λ = 530 nm with a commercial protein assay kit (BCA, Thermo Scientific Pierce, Spain) following the manufacturer’s instructions. Protein quantities are reported as μg of proteins per mm3 of scaffold measured from a BSA calibration standard (BSA, Thermo Scientific Pierce, Spain).

### ADSC response to ceramic scaffolds

#### Ethics statements

Adipose Derived Stem Cells (ADSCs) were obtained by the Central University Hospital of Asturias (HUCA, Spain) from discarded tissues after a biopsy by ethically approved protocols. Informed consent was waved by Internal Review board. All methods were performed in compliance with the Declaration of Helsinki.

#### Cell culture

ADSCs are harvested from omentum of informed donors during surgery at the Central University Hospital of Asturias (HUCA, Spain). Cellular isolating is made using high-glucose DMEM-Dulbecco’s Modified Eagle Medium (GIBCO, Thermo Scientific, Spain) with 10% fetal bovine serum (FBS, PAA Laboratories GmbH, Austria) and 1% antibiotics (HyClone, 10 U mL−1 of penicillin and 10 mg mL−1 of streptomycin). Non-adherent cells are removed by medium exchange after 24 h and, afterwards every 2–3 days. Cells are maintained to 70–80% confluence at 37 °C in a balanced 5% CO2 atmosphere. Biological assays are carried out with cells at fourth passage in order to avoid old-age signs.

Cellular attachment was analyzed by SEM (Hitachi TM 3000, Japan) 24 and 48 h after seeding (104 cells ml-1). At each study time, those ADSCs adhered at scaffolds were fixed with 2.5% paraformaldehyde (PFA, Sigma Aldrich, Spain), dehydrated within an Et-OH gradient, and dried in a chamber with silica gel.

Cell proliferation was evaluated 3 and 7 days after plating (104 cells ml−1) by measuring the cytoplasmic lactate dehydrogenase (LDH) activity with the Cytotoxicity Detection KitPLUS (Roche Diagnostics, Merck, Spain) as per manufacturer’s instructions. Protein extraction was made with the Mammalian Protein Extraction Reagent (M-PER, Thermo Scientific, Spain). The reduction of tetrazolium salts into formazan dye, a reaction coupled to LDH activity, was spectrophotometrically measured at 492 nm using a microplate reader (BIO-RAD, Model 680, US). The greater the absorbance value, the greater the number of proliferating cells into the scaffold. Results were normalized with the corresponding value of total protein determined with the Piece BCA Protein Assay Kit (BCA, Thermo Scientific Pierce, Spain). The percent cell viability was calculated according to Eq. (3):

$$% Viability = 100 times frac{{Abs_{sample} }}{{Abs_{blank} }}$$

(3)

#### Osteoblastic differentiation and mineralization

Osteoblastic differentiation starts with a matrix maturation phase that is characterized by a maximal expression of certain markers, such as alkaline phosphatase (ALP). This was quantified 7 and 14 days after seeding (104 cells ml-1) with the SensoLyte pNPP Alkaline Phosphatase Assay Kit (AnaSpec Inc.). Protein extraction was made with the Mammalian Protein Extraction Reagent (M-PER, Thermo Scientific, Spain). Cellular extracts were incubated for 15 min at room temperature with the colorimetric substrate, pNPP, and then spectrophotometrically evaluated at 492 nm with microplate reader (BIO-RAD, Model 680, US). Results were also normalized with the corresponding value of total protein determined with the Piece BCA Protein Assay Kit (BCA, Thermo Scientific Pierce, Spain).

Alizarin Red Solution (ARS, Merck, Germany) staining is used to evaluate extracellular calcium-rich deposits in the investigated scaffolds after 21 days, which are an indication of in vitro bone formation. After 21 days in cell culture, specimens were PBS-rinsed with, fixed in cold Et-OH (70% v/v) for 1 h, and then stained for 10–12 min with 2% ARS solution prepared as indicated by Gregory et al.39. The unincorporated dye was removed with distilled water. The calcium deposits are specifically stained bright orange-red with ARS staining.

Osteogenic factors (50 μg ml-1 of ascorbic acid, 10 mM β-glycerol phosphate and 100 mM dexamethasone (all reagents from Sigma-Aldrich, USA) were added from the third day onwards.