Design and Manufacture of Bone Cements Based on Calcium Sulfate Hemihydrate and Mg, Sr-Doped Bioactive Glass

Citation: Moazeni, N.; Hesaraki, S.; Behnamghader, A.; Esmaeilzadeh, J.; Orive, G.; Dolatshahi-Pirouz, A.; Borhan, S. Design and Manufacture of Bone Cements Based on Calcium Sulfate Hemihydrate and Mg, Sr-Doped Bioactive Glass. Biomedicines 2023,11, 2833. https://doi.org/10.3390/ biomedicines11102833 Academic Editor: Viviana Di Giacomo Received: 11 September 2023 Revised: 4 October 2023 Accepted: 16 October 2023 Published: 18 October 2023 Copyright: © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). biomedicines Article Design and Manufacture of Bone Cements Based on Calcium Sulfate Hemihydrate and Mg, Sr-Doped Bioactive Glass Nazanin Moazeni 1, Saeed Hesaraki 1,*, Aliasghar Behnamghader 1, Javad Esmaeilzadeh 2, Gorka Orive 3,4, Alireza Dolatshahi-Pirouz 5and Shokoufeh Borhan 6 1Nanotechnology and Advanced Materials Department, Materials and Energy Research Center, Karaj 31779-83634, Alborz, Iran; [email protected] (N.M.); [email protected] (A.B.) 2Department of Materials and Chemical Engineering, Esfarayen University of Technology, Esfarayen 96619-98195, North Khorasan, Iran; [email protected] 3NanoBioCel Research Group, School of Pharmacy, University of the Basque Country (UPV/EHU), 01006 Vitoria-Gasteiz, Spain; [email protected] 4Biomedical Research Networking Centre in Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN), Institute of Health Carlos III, 28029 Madrid, Spain 5Department of Health Technology, Technical University of Denmark, 2800 Lyngby, Denmark; [email protected] 6Department of Materials, Chemical and Polymer Engineering, Buein Zahra Technical University, Buein Zahra 34518-66391, Qazvin, Iran; [email protected] *Correspondence: [email protected]; Tel.: +98-263-6204-1314 Abstract: In the present study, a novel composite bone cement based on calcium sulfate hemihydrate (CSH) and Mg, Sr-containing bioactive glass (BG) as solid phase, and solution of chitosan as liquid phase were developed. The phase composition, morphology, setting time, injectability, viscosity, and cellular responses of the composites with various contents of BG (0, 10, 20, and 30 wt.%) were investigated. The pure calcium sulfate cement was set at approximately 180 min, whereas the setting time was drastically decreased to 6 min by replacing 30 wt.% glass powder for CSH in the cement solid phase. BG changed the microscopic morphology of the set cement and decreased the size and compaction of the precipitated gypsum phase. Replacing the CSH phase with BG increased injection force of the produced cement; however, all the cements were injected at a nearly constant force, lower than 20 N. The viscosity measurements in oscillatory mode determined the shear-thinning behavior of the pastes. Although the viscosity of the pastes increased with increasing BG content, it was influenced by the frequency extent. Pure calcium sulfate cement exhibited some transient cytotoxicity on human-derived bone mesenchymal stem cells and it was compensated by introducing BG phase. Moreover, BG improved the cell proliferation and mineralization of extracellular matrix as shown by calcein measurements. The results indicate the injectable composite cement comprising 70 wt.% CSH and 30 wt.% Mg, Sr-doped BG has better setting, mechanical and cellular behaviors and hence, is a potential candidate for bone repair, however more animal and human clinical evaluations are essential. Keywords: calcium sulfate; bioactive glass; rheology; bone cement; chitosan; Sr-doped biomaterials 1. Introduction It is the standard procedure for surgeons to use donor bone or synthetic materials to fill up osseous defects left by procedures like arthroplasty, bone tumor removal, infection, fracture, or even trauma. Alternatives to allografts and autografts have been available for many decades in the form of synthetic polymers, ceramics, and polymer/ceramics composites. These bone substitutes and fillers are available in different shapes, including bulk pieces, injectable non-cement pastes, and moldable cement-type formulations [1–6]. Bone substitutes cements (BSCs) are composed of powder and appropriate solution phases and by mixing, they give a shapeable and self-setting paste. Therefore, these BSC Biomedicines 2023,11, 2833. https://doi.org/10.3390/biomedicines11102833 https://www.mdpi.com/journal/biomedicines Biomedicines 2023,11, 2833 2 of 17 biomaterials must have good rheological and injectable properties prior to self-setting to allow the surgeon to deliver bone cements paste through a needle or cannula into bone defects with irregular shapes and without proper access during the surgical process. Calcium sulfate (CS), tri-calcium phosphate, and hydroxyapatite are some typical forms of bone fillers used therapeutically [ 7 , 8 ]. Among bone cements, calcium sulfate cement is known as a well-tolerated, fast setting, rapidly and completely bioresorbable material which not only is able to be used as bone filler, but can also be used as local delivery media of therapeutic agents [ 9 ]. Despite the favorable properties, calcium sulfate suffers from some weak points such as the rapid resorption rate and disability in chemical bonding with adjacent tissues (bioacivity) and weak mechanical strength [ 10 , 11 ]. Allthe above-mentioned points remarkably limit the clinical applications of CS cements. To overcome the above-mentioned challenges, researchers have engineered calcium sulfate composite with more suitable properties using different types of additives such as natural polymers (e.g., sodium alginate, gelatin, collagen) and bioactive inorganic materials such as hydroxyapatite and bioactive glass (BG) [ 12 – 16 ]. It has been shown that incorporation of some polymers into CS cement matrix could enhance the injectability, cohesion, and mechanical properties [17]. Beside the polymeric additives, the presence of inorganic nanoparticles such as hydroxyapatite and BGs can offer the physical and mechanical properties and regulate the degradation rate and the bioactivity of CS cements, owing to their dissolution products, which act as triggering osteoprogenitor cells at the genetic level. In one study [ 18 ], the addition of titanium-doped nano-hydroxyapatite in CS-based composites enhanced the setting time, mechanical strength, and bioactivity. Another study examined cement pastes composed of three biopolymers of gelatin/alginate/chondroitin sulfate and α -CSH and calcium-deficient hydroxyapatite (CDHA) [ 17 ]. Their results demonstrated the composite pastes exhibited optimal rheological behaviors, good handling properties as well as appropriate anti-washout characteristics. BGs in combination with various polymers have also been introduced into the CS cement; the biological, physicochemical, and mechanical features of the acquired cement pastes were investigated [ 9 , 17 , 19 ]. BG can form a chemical bond with living tissues and promotes bone formation in areas distant from the implant site [20,21]. Chitosan is an appropriate natural polymer with distinctive advantages that can be incorporated with cements to provide adequate physical and biological properties. It is hemostatic, fungistatic, and biodegradable, and exhibits antitumor, immunoadjuvant characteristics. It can bind to human and microbial cells, and accelerates the generation of osteoblast, which is responsible for bone formation [ 22 ]. In addition, chitosan shows antimicrobial property against Gram-positive and negative bacteria. The antibacterial activity exhibits a considerable dependence on both the cationic charge and the molecular weight [ 23 ]. These diverse features of chitosan make it easy for them to be used in scaffolds, nanoparticles, beads, microparticles, nanofibers, membranes, and other forms [22]. One of the important characteristics of CS-based cements is injectability. Injectability provides non-invasive transfer of cementitious material into the bone cavities through a needle or cannula, followed by quick in situ setting of the filling paste [ 24 ]. Since flow behavior is a key factor in injectable systems, the rheological characteristics of the cement pastes should be carefully assessed. Zima et al. [ 25 ] conducted research on the rheology of CS cements, emphasizing the influence of the ion presence on multiple inflections in the viscosity-time diagram. In the present study, a novel bone cement based on calcium sulfate, Mg, Sr-containing bioactive glass, and chitosan was developed. A series of cements were prepared by mixing various amounts of sol-gel derived Mg, Sr-doped BG particles, and α -CS as powder phase and chitosan solution as liquid phase. The rheological behavior, injectability, and the correlation between injection force and viscosity were determined. The phase composition, morphology, setting time, and finally cellular cytocompatibility were also assessed. Biomedicines 2023,11, 2833 3 of 17 2. Materials and Methods 2.1. Initial Powders Preparation Calcium sulfate hemihydrate, CSH (CaSO 4· 0.5H 2 O, CAS-No. 10034-76-1, product No. 12090) was purchased from Merck. Citric acid and medium molecular weight chitosan (CAS-No. 9012-76-4, product No. 448877), C 12 H 24 N 2 O 9 , with M W : 200,000 g/mol, Deacetylation ≥ 75%, and purity > 98%, were prepared from Sigma-Aldrich, Taufkirchen, Germany. Mg, Sr-doped bioactive glass (BG) was also synthesized according to the previously described method [ 26 ] in 64SiO 2 -30CaO-5P 2 O 5 system, in which the concentration of Sr and Mg dopants was 0.5 wt.% of CaO component. 2.2. Cement Pastes Preparation The CS–BG cements were made by a combination of calcium sulfate hemihydrate and BG powders in various mass ratios, and chitosan and citric acid solution with a powder to liquid ratio of 1.5. Table 1shows the composition of several CS–BG cements made at a constant P/L of 1.5 g/mL. Table 1. Composition various CS–BG cements. Code Powder Phase Liquid Phase CSH Solution of 19.2% citric acid and 2% chitosan (w/v) CS100-BG0 100 0 CS90-BG10 90 10 CS80-BG20 80 20 CS70-BG30 70 30 2.3. Setting Time, Porosity and Compressive Strength The initial and final setting times of the CS–BG pastes were determined using a standard test with Gillmore needles, as described in ASTM C266–99 [27]. To determine the compressive strength, the cylindrical specimens were fabricated with a diameter of 6 mm and a height of 12 mm. At 24 h after setting, the specimens were transferred to a mechanical testing device (SANTAM STM-20) and the compression tests were conducted with at a loading speed of 1 mm/min. The total porosity (P) of the cements was calculated on the cylindrical specimens, Archimedes method, where ethanol was used as medium [28]. 2.4. Phase Composition The phase composition of as-set cements was determined using X-ray diffractometry, XRD, (Philips PW 3710, Amsterdam, Netherlands) with Cu-K α radiation and a 1.54050 nm X-ray wavelength, which was operated at 40 kV and 40 mA with a step size of 0.02 and a count duration of 2 s/step. 2.5. SEM Observation The morphological and chemical characteristics of CS–BG cements were investigated using scanning electron microscope (SEM) (TESCAN-XMU, VEGA II, Brno–Kohoutovice, Czech Republic) equipped with an energy dispersive X-ray spectroscopy (EDS) analysis unit. The map of S, Si, and N elements also determined the elemental image analysis. Moreover, the particle size analysis of the microstructure was performed using Image J software version 1.0.8_112 and the graphs were drawn by ORIGIN PRO 2022. 2.6. Injectability An extrusion test was used to assess the paste injectability [ 29 ]. After 1-min mixing procedure, the obtained paste was placed into a commercial 3 mL syringe (AvaPezeshk Co., Tehran, Iran) with cannula length and tip diameter of 10 mm and 2 mm, respectively. Biomedicines 2023,11, 2833 4 of 17 The paste was exerted in syringe tube (the length of filled paste in the tube was 30 mm) and extruded from the syringe (held rigidly in a fixture) at predetermined times after the mixing process was completed by applying force using a universal testing machine (STM-20, Santam Co., Tehran, Iran) at a crosshead speed of 30 mm/min. The injection curve was plotted as injection force vs. plunger displacement. The injectability coefficient (I) was calculated as I = [(M0 −M)/M0] ×100 (1) where M0 is the initial mass of the composite in the syringe and M is the mass left inside the syringe after extrusion. 2.7. The Viscosity Measurements The Rheometer (Anton Paar, Physica MC-R301, Graz, Austria) with plate–plate measuring geometry was used to examine the rheological properties of calcium sulfate-bioactive glass pastes (plate diameter of 25 mm). For this measurement, a homogeneous paste was made during 1 min and the resultant paste was subsequently applied to the lower plate’s center at predetermined intervals. The rheological test began 2 min after mixing the powder and liquid phases at both oscillatory and rotation modes. A modest (low) amplitude sinusoidal oscillation strain was firstly applied to the paste in a dynamic oscillation test. The complex viscosity can be stated using the following formula due to the phase difference between two sinusoidal waves. G* = G0+ iG00 (2) Biomedicines 2023, 11, 2833 4 of 17 2.6. Injectability An extrusion test was used to assess the paste injectability [29]. After 1-min mixing procedure, the obtained paste was placed into a commercial 3 mL syringe (AvaPezeshk Co., Tehran, Iran) with cannula length and tip diameter of 10 mm and 2 mm, respectively. The paste was exerted in syringe tube (the length of filled paste in the tube was 30 mm) and extruded from the syringe (held rigidly in a fixture) at predetermined times after the mixing process was completed by applying force using a universal testing machine (STM20, Santam Co., Tehran, Iran) at a crosshead speed of 30 mm/min. The injection curve was plotted as injection force vs. plunger displacement. The injectability coefficient (I) was calculated as I = [(M0 − M)/M0] × 100 (1) where M0 is the initial mass of the composite in the syringe and M is the mass left inside the syringe after extrusion. 2.7. The Viscosity Measurements The Rheometer (Anton Paar, Physica MC-R301, Graz, Austria) with plate–plate measuring geometry was used to examine the rheological properties of calcium sulfate-bioactive glass pastes (plate diameter of 25 mm). For this measurement, a homogeneous paste was made during 1 min and the resultant paste was subsequently applied to the lower plate’s center at predetermined intervals. The rheological test began 2 min after mixing the powder and liquid phases at both oscillatory and rotation modes. A modest (low) amplitude sinusoidal oscillation strain was firstly applied to the paste in a dynamic oscillation test. The complex viscosity can be stated using the following formula due to the phase difference between two sinusoidal waves. G* = G′ + iG″ (2) ղ* = ղ′ − iղ″ (3) ղ’ = G″/ω (4) ղ” = G′/ω (5) where ω is the frequency in rads−1, G′ is shear storage modulus, G″ is shear loss modulus, G* is complex shear modulus, ղ″ is out-of-phase viscosity, ղ′ is dynamic (absolute) viscosity, and ղ* is complex viscosity. The real part of the complex viscosity (dynamic viscosity) (ղ′) is a criterion of force required to make a fluid flow at a certain speed [30,31]. Firstly, strain sweep mode measurement was performed to identify the linear viscoelastic region (LVR). Then, the dynamic frequency sweep test was performed while maintaining constant strain (1%) and temperature (25 °C). In rotatory mode, the viscosity–time curve was also drawn at a constant shear rate of 1 s−1 to identify the variation of the paste viscosity as a function of time. 2.8. The Cell Studies 2.8.1. Cell Survival, Viability, and Growth For in vitro cellular studies, the samples were immersed in an antibiotic solution for a duration of 30 min; afterwards, they were rinsed in phosphate-buffered saline (PBS), and subjected to ultraviolet irradiation (UV) for a period of 15 min. The cytotoxic effect of cements on human bone mesenchymal stem cells (hMSCs) was studied by 3-(4,5-dimethylthiazol-2)-2,5-diphenyltetrazolium bromide assay (MTT; Sigma-Aldrich, Taufkirchen, Germany). The cells were seeded in 100 μL of the medium at a density of 104 cells/well in 96-well culture plate and were incubated at 37 °C in a 95% * = Biomedicines 2023, 11, 2833 4 of 17 2.6. Injectability An extrusion test was used to assess the paste injectability [29]. After 1-min mixing procedure, the obtained paste was placed into a commercial 3 mL syringe (AvaPezeshk Co., Tehran, Iran) with cannula length and tip diameter of 10 mm and 2 mm, respectively. The paste was exerted in syringe tube (the length of filled paste in the tube was 30 mm) and extruded from the syringe (held rigidly in a fixture) at predetermined times after the mixing process was completed by applying force using a universal testing machine (STM20, Santam Co., Tehran, Iran) at a crosshead speed of 30 mm/min. The injection curve was plotted as injection force vs. plunger displacement. The injectability coefficient (I) was calculated as I = [(M0 − M)/M0] × 100 (1) where M0 is the initial mass of the composite in the syringe and M is the mass left inside the syringe after extrusion. 2.7. The Viscosity Measurements The Rheometer (Anton Paar, Physica MC-R301, Graz, Austria) with plate–plate measuring geometry was used to examine the rheological properties of calcium sulfate-bioactive glass pastes (plate diameter of 25 mm). For this measurement, a homogeneous paste was made during 1 min and the resultant paste was subsequently applied to the lower plate’s center at predetermined intervals. The rheological test began 2 min after mixing the powder and liquid phases at both oscillatory and rotation modes. A modest (low) amplitude sinusoidal oscillation strain was firstly applied to the paste in a dynamic oscillation test. The complex viscosity can be stated using the following formula due to the phase difference between two sinusoidal waves. G* = G′ + iG″ (2) ղ* = ղ′ − iղ″ (3) ղ’ = G″/ω (4) ղ” = G′/ω (5) where ω is the frequency in rads−1, G′ is shear storage modulus, G″ is shear loss modulus, G* is complex shear modulus, ղ″ is out-of-phase viscosity, ղ′ is dynamic (absolute) viscosity, and ղ* is complex viscosity. The real part of the complex viscosity (dynamic viscosity) (ղ′) is a criterion of force required to make a fluid flow at a certain speed [30,31]. Firstly, strain sweep mode measurement was performed to identify the linear viscoelastic region (LVR). Then, the dynamic frequency sweep test was performed while maintaining constant strain (1%) and temperature (25 °C). In rotatory mode, the viscosity–time curve was also drawn at a constant shear rate of 1 s−1 to identify the variation of the paste viscosity as a function of time. 2.8. The Cell Studies 2.8.1. Cell Survival, Viability, and Growth For in vitro cellular studies, the samples were immersed in an antibiotic solution for a duration of 30 min; afterwards, they were rinsed in phosphate-buffered saline (PBS), and subjected to ultraviolet irradiation (UV) for a period of 15 min. The cytotoxic effect of cements on human bone mesenchymal stem cells (hMSCs) was studied by 3-(4,5-dimethylthiazol-2)-2,5-diphenyltetrazolium bromide assay (MTT; Sigma-Aldrich, Taufkirchen, Germany). The cells were seeded in 100 μL of the medium at a density of 104 cells/well in 96-well culture plate and were incubated at 37 °C in a 95% 0−i Biomedicines 2023, 11, 2833 4 of 17 2.6. Injectability An extrusion test was used to assess the paste injectability [29]. After 1-min mixing procedure, the obtained paste was placed into a commercial 3 mL syringe (AvaPezeshk Co., Tehran, Iran) with cannula length and tip diameter of 10 mm and 2 mm, respectively. The paste was exerted in syringe tube (the length of filled paste in the tube was 30 mm) and extruded from the syringe (held rigidly in a fixture) at predetermined times after the mixing process was completed by applying force using a universal testing machine (STM20, Santam Co., Tehran, Iran) at a crosshead speed of 30 mm/min. The injection curve was plotted as injection force vs. plunger displacement. The injectability coefficient (I) was calculated as I = [(M0 − M)/M0] × 100 (1) where M0 is the initial mass of the composite in the syringe and M is the mass left inside the syringe after extrusion. 2.7. The Viscosity Measurements The Rheometer (Anton Paar, Physica MC-R301, Graz, Austria) with plate–plate measuring geometry was used to examine the rheological properties of calcium sulfate-bioactive glass pastes (plate diameter of 25 mm). For this measurement, a homogeneous paste was made during 1 min and the resultant paste was subsequently applied to the lower plate’s center at predetermined intervals. The rheological test began 2 min after mixing the powder and liquid phases at both oscillatory and rotation modes. A modest (low) amplitude sinusoidal oscillation strain was firstly applied to the paste in a dynamic oscillation test. The complex viscosity can be stated using the following formula due to the phase difference between two sinusoidal waves. G* = G′ + iG″ (2) ղ* = ղ′ − iղ″ (3) ղ’ = G″/ω (4) ղ” = G′/ω (5) where ω is the frequency in rads−1, G′ is shear storage modulus, G″ is shear loss modulus, G* is complex shear modulus, ղ″ is out-of-phase viscosity, ղ′ is dynamic (absolute) viscosity, and ղ* is complex viscosity. The real part of the complex viscosity (dynamic viscosity) (ղ′) is a criterion of force required to make a fluid flow at a certain speed [30,31]. Firstly, strain sweep mode measurement was performed to identify the linear viscoelastic region (LVR). Then, the dynamic frequency sweep test was performed while maintaining constant strain (1%) and temperature (25 °C). In rotatory mode, the viscosity–time curve was also drawn at a constant shear rate of 1 s−1 to identify the variation of the paste viscosity as a function of time. 2.8. The Cell Studies 2.8.1. Cell Survival, Viability, and Growth For in vitro cellular studies, the samples were immersed in an antibiotic solution for a duration of 30 min; afterwards, they were rinsed in phosphate-buffered saline (PBS), and subjected to ultraviolet irradiation (UV) for a period of 15 min. The cytotoxic effect of cements on human bone mesenchymal stem cells (hMSCs) was studied by 3-(4,5-dimethylthiazol-2)-2,5-diphenyltetrazolium bromide assay (MTT; Sigma-Aldrich, Taufkirchen, Germany). The cells were seeded in 100 μL of the medium at a density of 104 cells/well in 96-well culture plate and were incubated at 37 °C in a 95% 00 (3) Biomedicines 2023, 11, 2833 4 of 17 2.6. Injectability An extrusion test was used to assess the paste injectability [29]. After 1-min mixing procedure, the obtained paste was placed into a commercial 3 mL syringe (AvaPezeshk Co., Tehran, Iran) with cannula length and tip diameter of 10 mm and 2 mm, respectively. The paste was exerted in syringe tube (the length of filled paste in the tube was 30 mm) and extruded from the syringe (held rigidly in a fixture) at predetermined times after the mixing process was completed by applying force using a universal testing machine (STM20, Santam Co., Tehran, Iran) at a crosshead speed of 30 mm/min. The injection curve was plotted as injection force vs. plunger displacement. The injectability coefficient (I) was calculated as I = [(M0 − M)/M0] × 100 (1) where M0 is the initial mass of the composite in the syringe and M is the mass left inside the syringe after extrusion. 2.7. The Viscosity Measurements The Rheometer (Anton Paar, Physica MC-R301, Graz, Austria) with plate–plate measuring geometry was used to examine the rheological properties of calcium sulfate-bioactive glass pastes (plate diameter of 25 mm). For this measurement, a homogeneous paste was made during 1 min and the resultant paste was subsequently applied to the lower plate’s center at predetermined intervals. The rheological test began 2 min after mixing the powder and liquid phases at both oscillatory and rotation modes. A modest (low) amplitude sinusoidal oscillation strain was firstly applied to the paste in a dynamic oscillation test. The complex viscosity can be stated using the following formula due to the phase difference between two sinusoidal waves. G* = G′ + iG″ (2) ղ* = ղ′ − iղ″ (3) ղ’ = G″/ω (4) ղ” = G′/ω (5) where ω is the frequency in rads−1, G′ is shear storage modulus, G″ is shear loss modulus, G* is complex shear modulus, ղ″ is out-of-phase viscosity, ղ′ is dynamic (absolute) viscosity, and ղ* is complex viscosity. The real part of the complex viscosity (dynamic viscosity) (ղ′) is a criterion of force required to make a fluid flow at a certain speed [30,31]. Firstly, strain sweep mode measurement was performed to identify the linear viscoelastic region (LVR). Then, the dynamic frequency sweep test was performed while maintaining constant strain (1%) and temperature (25 °C). In rotatory mode, the viscosity–time curve was also drawn at a constant shear rate of 1 s−1 to identify the variation of the paste viscosity as a function of time. 2.8. The Cell Studies 2.8.1. Cell Survival, Viability, and Growth For in vitro cellular studies, the samples were immersed in an antibiotic solution for a duration of 30 min; afterwards, they were rinsed in phosphate-buffered saline (PBS), and subjected to ultraviolet irradiation (UV) for a period of 15 min. The cytotoxic effect of cements on human bone mesenchymal stem cells (hMSCs) was studied by 3-(4,5-dimethylthiazol-2)-2,5-diphenyltetrazolium bromide assay (MTT; Sigma-Aldrich, Taufkirchen, Germany). The cells were seeded in 100 μL of the medium at a density of 104 cells/well in 96-well culture plate and were incubated at 37 °C in a 95% 0= G00/ω(4) Biomedicines 2023, 11, 2833 4 of 17 2.6. Injectability An extrusion test was used to assess the paste injectability [29]. After 1-min mixing procedure, the obtained paste was placed into a commercial 3 mL syringe (AvaPezeshk Co., Tehran, Iran) with cannula length and tip diameter of 10 mm and 2 mm, respectively. The paste was exerted in syringe tube (the length of filled paste in the tube was 30 mm) and extruded from the syringe (held rigidly in a fixture) at predetermined times after the mixing process was completed by applying force using a universal testing machine (STM20, Santam Co., Tehran, Iran) at a crosshead speed of 30 mm/min. The injection curve was plotted as injection force vs. plunger displacement. The injectability coefficient (I) was calculated as I = [(M0 − M)/M0] × 100 (1) where M0 is the initial mass of the composite in the syringe and M is the mass left inside the syringe after extrusion. 2.7. The Viscosity Measurements The Rheometer (Anton Paar, Physica MC-R301, Graz, Austria) with plate–plate measuring geometry was used to examine the rheological properties of calcium sulfate-bioactive glass pastes (plate diameter of 25 mm). For this measurement, a homogeneous paste was made during 1 min and the resultant paste was subsequently applied to the lower plate’s center at predetermined intervals. The rheological test began 2 min after mixing the powder and liquid phases at both oscillatory and rotation modes. A modest (low) amplitude sinusoidal oscillation strain was firstly applied to the paste in a dynamic oscillation test. The complex viscosity can be stated using the following formula due to the phase difference between two sinusoidal waves. G* = G′ + iG″ (2) ղ* = ղ′ − iղ″ (3) ղ’ = G″/ω (4) ղ” = G′/ω (5) where ω is the frequency in rads−1, G′ is shear storage modulus, G″ is shear loss modulus, G* is complex shear modulus, ղ″ is out-of-phase viscosity, ղ′ is dynamic (absolute) viscosity, and ղ* is complex viscosity. The real part of the complex viscosity (dynamic viscosity) (ղ′) is a criterion of force required to make a fluid flow at a certain speed [30,31]. Firstly, strain sweep mode measurement was performed to identify the linear viscoelastic region (LVR). Then, the dynamic frequency sweep test was performed while maintaining constant strain (1%) and temperature (25 °C). In rotatory mode, the viscosity–time curve was also drawn at a constant shear rate of 1 s−1 to identify the variation of the paste viscosity as a function of time. 2.8. The Cell Studies 2.8.1. Cell Survival, Viability, and Growth For in vitro cellular studies, the samples were immersed in an antibiotic solution for a duration of 30 min; afterwards, they were rinsed in phosphate-buffered saline (PBS), and subjected to ultraviolet irradiation (UV) for a period of 15 min. The cytotoxic effect of cements on human bone mesenchymal stem cells (hMSCs) was studied by 3-(4,5-dimethylthiazol-2)-2,5-diphenyltetrazolium bromide assay (MTT; Sigma-Aldrich, Taufkirchen, Germany). The cells were seeded in 100 μL of the medium at a density of 104 cells/well in 96-well culture plate and were incubated at 37 °C in a 95% 00 = G0/ω(5) where ω is the frequency in rads −1 , G 0 is shear storage modulus, G 00 is shear loss modulus, G* is complex shear modulus, Biomedicines 2023, 11, 2833 4 of 17 2.6. Injectability An extrusion test was used to assess the paste injectability [29]. After 1-min mixing procedure, the obtained paste was placed into a commercial 3 mL syringe (AvaPezeshk Co., Tehran, Iran) with cannula length and tip diameter of 10 mm and 2 mm, respectively. The paste was exerted in syringe tube (the length of filled paste in the tube was 30 mm) and extruded from the syringe (held rigidly in a fixture) at predetermined times after the mixing process was completed by applying force using a universal testing machine (STM20, Santam Co., Tehran, Iran) at a crosshead speed of 30 mm/min. The injection curve was plotted as injection force vs. plunger displacement. The injectability coefficient (I) was calculated as I = [(M0 − M)/M0] × 100 (1) where M0 is the initial mass of the composite in the syringe and M is the mass left inside the syringe after extrusion. 2.7. The Viscosity Measurements The Rheometer (Anton Paar, Physica MC-R301, Graz, Austria) with plate–plate measuring geometry was used to examine the rheological properties of calcium sulfate-bioactive glass pastes (plate diameter of 25 mm). For this measurement, a homogeneous paste was made during 1 min and the resultant paste was subsequently applied to the lower plate’s center at predetermined intervals. The rheological test began 2 min after mixing the powder and liquid phases at both oscillatory and rotation modes. A modest (low) amplitude sinusoidal oscillation strain was firstly applied to the paste in a dynamic oscillation test. The complex viscosity can be stated using the following formula due to the phase difference between two sinusoidal waves. G* = G′ + iG″ (2) ղ* = ղ′ − iղ″ (3) ղ’ = G″/ω (4) ղ” = G′/ω (5) where ω is the frequency in rads−1, G′ is shear storage modulus, G″ is shear loss modulus, G* is complex shear modulus, ղ″ is out-of-phase viscosity, ղ′ is dynamic (absolute) viscosity, and ղ* is complex viscosity. The real part of the complex viscosity (dynamic viscosity) (ղ′) is a criterion of force required to make a fluid flow at a certain speed [30,31]. Firstly, strain sweep mode measurement was performed to identify the linear viscoelastic region (LVR). Then, the dynamic frequency sweep test was performed while maintaining constant strain (1%) and temperature (25 °C). In rotatory mode, the viscosity–time curve was also drawn at a constant shear rate of 1 s−1 to identify the variation of the paste viscosity as a function of time. 2.8. The Cell Studies 2.8.1. Cell Survival, Viability, and Growth For in vitro cellular studies, the samples were immersed in an antibiotic solution for a duration of 30 min; afterwards, they were rinsed in phosphate-buffered saline (PBS), and subjected to ultraviolet irradiation (UV) for a period of 15 min. The cytotoxic effect of cements on human bone mesenchymal stem cells (hMSCs) was studied by 3-(4,5-dimethylthiazol-2)-2,5-diphenyltetrazolium bromide assay (MTT; Sigma-Aldrich, Taufkirchen, Germany). The cells were seeded in 100 μL of the medium at a density of 104 cells/well in 96-well culture plate and were incubated at 37 °C in a 95% 00 is out-of-phase viscosity, Biomedicines 2023, 11, 2833 4 of 17 2.6. Injectability An extrusion test was used to assess the paste injectability [29]. After 1-min mixing procedure, the obtained paste was placed into a commercial 3 mL syringe (AvaPezeshk Co., Tehran, Iran) with cannula length and tip diameter of 10 mm and 2 mm, respectively. The paste was exerted in syringe tube (the length of filled paste in the tube was 30 mm) and extruded from the syringe (held rigidly in a fixture) at predetermined times after the mixing process was completed by applying force using a universal testing machine (STM20, Santam Co., Tehran, Iran) at a crosshead speed of 30 mm/min. The injection curve was plotted as injection force vs. plunger displacement. The injectability coefficient (I) was calculated as I = [(M0 − M)/M0] × 100 (1) where M0 is the initial mass of the composite in the syringe and M is the mass left inside the syringe after extrusion. 2.7. The Viscosity Measurements The Rheometer (Anton Paar, Physica MC-R301, Graz, Austria) with plate–plate measuring geometry was used to examine the rheological properties of calcium sulfate-bioactive glass pastes (plate diameter of 25 mm). For this measurement, a homogeneous paste was made during 1 min and the resultant paste was subsequently applied to the lower plate’s center at predetermined intervals. The rheological test began 2 min after mixing the powder and liquid phases at both oscillatory and rotation modes. A modest (low) amplitude sinusoidal oscillation strain was firstly applied to the paste in a dynamic oscillation test. The complex viscosity can be stated using the following formula due to the phase difference between two sinusoidal waves. G* = G′ + iG″ (2) ղ* = ղ′ − iղ″ (3) ղ’ = G″/ω (4) ղ” = G′/ω (5) where ω is the frequency in rads−1, G′ is shear storage modulus, G″ is shear loss modulus, G* is complex shear modulus, ղ″ is out-of-phase viscosity, ղ′ is dynamic (absolute) viscosity, and ղ* is complex viscosity. The real part of the complex viscosity (dynamic viscosity) (ղ′) is a criterion of force required to make a fluid flow at a certain speed [30,31]. Firstly, strain sweep mode measurement was performed to identify the linear viscoelastic region (LVR). Then, the dynamic frequency sweep test was performed while maintaining constant strain (1%) and temperature (25 °C). In rotatory mode, the viscosity–time curve was also drawn at a constant shear rate of 1 s−1 to identify the variation of the paste viscosity as a function of time. 2.8. The Cell Studies 2.8.1. Cell Survival, Viability, and Growth For in vitro cellular studies, the samples were immersed in an antibiotic solution for a duration of 30 min; afterwards, they were rinsed in phosphate-buffered saline (PBS), and subjected to ultraviolet irradiation (UV) for a period of 15 min. The cytotoxic effect of cements on human bone mesenchymal stem cells (hMSCs) was studied by 3-(4,5-dimethylthiazol-2)-2,5-diphenyltetrazolium bromide assay (MTT; Sigma-Aldrich, Taufkirchen, Germany). The cells were seeded in 100 μL of the medium at a density of 104 cells/well in 96-well culture plate and were incubated at 37 °C in a 95% 0 is dynamic (absolute) viscosity, and Biomedicines 2023, 11, 2833 4 of 17 2.6. Injectability An extrusion test was used to assess the paste injectability [29]. After 1-min mixing procedure, the obtained paste was placed into a commercial 3 mL syringe (AvaPezeshk Co., Tehran, Iran) with cannula length and tip diameter of 10 mm and 2 mm, respectively. The paste was exerted in syringe tube (the length of filled paste in the tube was 30 mm) and extruded from the syringe (held rigidly in a fixture) at predetermined times after the mixing process was completed by applying force using a universal testing machine (STM20, Santam Co., Tehran, Iran) at a crosshead speed of 30 mm/min. The injection curve was plotted as injection force vs. plunger displacement. The injectability coefficient (I) was calculated as I = [(M0 − M)/M0] × 100 (1) where M0 is the initial mass of the composite in the syringe and M is the mass left inside the syringe after extrusion. 2.7. The Viscosity Measurements The Rheometer (Anton Paar, Physica MC-R301, Graz, Austria) with plate–plate measuring geometry was used to examine the rheological properties of calcium sulfate-bioactive glass pastes (plate diameter of 25 mm). For this measurement, a homogeneous paste was made during 1 min and the resultant paste was subsequently applied to the lower plate’s center at predetermined intervals. The rheological test began 2 min after mixing the powder and liquid phases at both oscillatory and rotation modes. A modest (low) amplitude sinusoidal oscillation strain was firstly applied to the paste in a dynamic oscillation test. The complex viscosity can be stated using the following formula due to the phase difference between two sinusoidal waves. G* = G′ + iG″ (2) ղ* = ղ′ − iղ″ (3) ղ’ = G″/ω (4) ղ” = G′/ω (5) where ω is the frequency in rads−1, G′ is shear storage modulus, G″ is shear loss modulus, G* is complex shear modulus, ղ″ is out-of-phase viscosity, ղ′ is dynamic (absolute) viscosity, and ղ* is complex viscosity. The real part of the complex viscosity (dynamic viscosity) (ղ′) is a criterion of force required to make a fluid flow at a certain speed [30,31]. Firstly, strain sweep mode measurement was performed to identify the linear viscoelastic region (LVR). Then, the dynamic frequency sweep test was performed while maintaining constant strain (1%) and temperature (25 °C). In rotatory mode, the viscosity–time curve was also drawn at a constant shear rate of 1 s−1 to identify the variation of the paste viscosity as a function of time. 2.8. The Cell Studies 2.8.1. Cell Survival, Viability, and Growth For in vitro cellular studies, the samples were immersed in an antibiotic solution for a duration of 30 min; afterwards, they were rinsed in phosphate-buffered saline (PBS), and subjected to ultraviolet irradiation (UV) for a period of 15 min. The cytotoxic effect of cements on human bone mesenchymal stem cells (hMSCs) was studied by 3-(4,5-dimethylthiazol-2)-2,5-diphenyltetrazolium bromide assay (MTT; Sigma-Aldrich, Taufkirchen, Germany). The cells were seeded in 100 μL of the medium at a density of 104 cells/well in 96-well culture plate and were incubated at 37 °C in a 95% * is complex viscosity. The real part of the complex viscosity (dynamic viscosity) ( Biomedicines 2023, 11, 2833 4 of 17 2.6. Injectability An extrusion test was used to assess the paste injectability [29]. After 1-min mixing procedure, the obtained paste was placed into a commercial 3 mL syringe (AvaPezeshk Co., Tehran, Iran) with cannula length and tip diameter of 10 mm and 2 mm, respectively. The paste was exerted in syringe tube (the length of filled paste in the tube was 30 mm) and extruded from the syringe (held rigidly in a fixture) at predetermined times after the mixing process was completed by applying force using a universal testing machine (STM20, Santam Co., Tehran, Iran) at a crosshead speed of 30 mm/min. The injection curve was plotted as injection force vs. plunger displacement. The injectability coefficient (I) was calculated as I = [(M0 − M)/M0] × 100 (1) where M0 is the initial mass of the composite in the syringe and M is the mass left inside the syringe after extrusion. 2.7. The Viscosity Measurements The Rheometer (Anton Paar, Physica MC-R301, Graz, Austria) with plate–plate measuring geometry was used to examine the rheological properties of calcium sulfate-bioactive glass pastes (plate diameter of 25 mm). For this measurement, a homogeneous paste was made during 1 min and the resultant paste was subsequently applied to the lower plate’s center at predetermined intervals. The rheological test began 2 min after mixing the powder and liquid phases at both oscillatory and rotation modes. A modest (low) amplitude sinusoidal oscillation strain was firstly applied to the paste in a dynamic oscillation test. The complex viscosity can be stated using the following formula due to the phase difference between two sinusoidal waves. G* = G′ + iG″ (2) ղ* = ղ′ − iղ″ (3) ղ’ = G″/ω (4) ղ” = G′/ω (5) where ω is the frequency in rads−1, G′ is shear storage modulus, G″ is shear loss modulus, G* is complex shear modulus, ղ″ is out-of-phase viscosity, ղ′ is dynamic (absolute) viscosity, and ղ* is complex viscosity. The real part of the complex viscosity (dynamic viscosity) (ղ′) is a criterion of force required to make a fluid flow at a certain speed [30,31]. Firstly, strain sweep mode measurement was performed to identify the linear viscoelastic region (LVR). Then, the dynamic frequency sweep test was performed while maintaining constant strain (1%) and temperature (25 °C). In rotatory mode, the viscosity–time curve was also drawn at a constant shear rate of 1 s−1 to identify the variation of the paste viscosity as a function of time. 2.8. The Cell Studies 2.8.1. Cell Survival, Viability, and Growth For in vitro cellular studies, the samples were immersed in an antibiotic solution for a duration of 30 min; afterwards, they were rinsed in phosphate-buffered saline (PBS), and subjected to ultraviolet irradiation (UV) for a period of 15 min. The cytotoxic effect of cements on human bone mesenchymal stem cells (hMSCs) was studied by 3-(4,5-dimethylthiazol-2)-2,5-diphenyltetrazolium bromide assay (MTT; Sigma-Aldrich, Taufkirchen, Germany). The cells were seeded in 100 μL of the medium at a density of 104 cells/well in 96-well culture plate and were incubated at 37 °C in a 95% 0 ) is a criterion of force required to make a fluid flow at a certain speed [ 30 , 31 ]. Firstly, strain sweep mode measurement was performed to identify the linear viscoelastic region (LVR). Then, the dynamic frequency sweep test was performed while maintaining constant strain (1%) and temperature (25 ◦C). In rotatory mode, the viscosity–time curve was also drawn at a constant shear rate of 1 s−1to identify the variation of the paste viscosity as a function of time. 2.8. The Cell Studies 2.8.1. Cell Survival, Viability, and Growth For in vitro cellular studies, the samples were immersed in an antibiotic solution for a duration of 30 min; afterwards, they were rinsed in phosphate-buffered saline (PBS), and subjected to ultraviolet irradiation (UV) for a period of 15 min. The cytotoxic effect of cements on human bone mesenchymal stem cells (hMSCs) was studied by 3-(4,5-dimethylthiazol-2)-2,5-diphenyltetrazolium bromide assay (MTT; Sigma-Aldrich, Taufkirchen, Germany). The cells were seeded in 100 µ L of the medium at a density of 10 4 cells/well in 96-well culture plate and were incubated at 37 ◦ C in a 95% humidified atmosphere of 95% air and 5% CO 2 , for 24 h. Subsequently, spherical specimens (with the same shape and surface area) were positioned in the cell culture medium and incubated for 24, 48, and 72 h. The sample-free culture plate was used as control group. After incubation, the medium was evacuated and 100 µ L of serum free MTT-containing culture medium (0.5 mg/mL) was added to each well, followed by a 4-h incubation at 37 ◦ C. Finally, 100 µ L of dimethyl sulfoxide (Sigma, Germany) was added to each well, and cell viability was determined using a microplate reader (BioTek ELx800, Winooski, Biomedicines 2023,11, 2833 5 of 17 VT, USA) at 570 nm. The percentage of viable cells (compared to the control group) were evaluated and reported. 2.8.2. DAPI Staining DAPI (4 0 ,6-diamidino-2-phenylindole) is a fluorescent stain that binds strongly to adenine–thymine rich regions in DNA and allows for observing the health or death of the cell. For this purpose, the hBMSCs was cultured on the cements, and on days of 1, 3, and 7, it was taken out of the incubator for staining, the complete culture medium was removed and washed once, then fixed with 4% (w/v) paraformaldehyde for 30 min. The paraformaldehyde was removed, the DAPI solution (D9542-1MG SIGMA) with a concentration of 300 nM was poured on the cements and after 5 min, it was observed under the fluorescent microscope NIB-100F (NOVEL Co, Ningbo, China). 2.8.3. Calcein Staining Cells were cultured on the cement according to the standard procedure. On the desired day of study (days 14 and 21), cements containing cells were washed to the assay with 1000 µ L of a phosphate buffer to remove any serum esterase activity that may be present in the growth media. Calcein powder (L468415 MERCK) was dissolved in DMSO and a working solution with a final concentration of 2 µ M was prepared. Next, about 200 microliters was added to the samples, so it was covered and incubated at temperature 25 ◦ C for 30 min. After the passage of time, it was observed with a fluorescent microscope NIB-100F (NOVEL Co. Ningbo, China Co.) 2.9. Statistical Analysis The data were collected from at least three separate experiments. The one-way ANOVA was performed for the statistical analysis, and pvalue < 0.05 was considered statistically significant (* p< 0.05, ** p< 0.01, *** p< 0.001). 3. Results 3.1. Phase Composition Figure 1illustrates the XRD patterns of various CS–BG cements as well as starting materials (chitosan, CSH and BG) presented for comparison. The x-ray diffraction patterns of chitosan and bioactive glass powder reveal characteristics of an amorphous phase. In the patterns of set cement CS100-BG0, the unreacted CSH is the predominant phase and some minor gypsum product is observed (PDF-Number 33-0311). The same phase composition can also be seen in the diffraction patterns of BG-containing cements, except an amorphous background seen due to the presence of BG phase. The amorphous-like background in the XRD patterns of these samples may also be related to the formation of amorphous chelate (complex) produced from the chemical reaction of Ca ions (delivered from BG and CSH) and citric acid and/or chitosan molecules. Biomedicines 2023,11, 2833 6 of 17 Biomedicines 2023, 11, 2833 6 of 17 Figure 1. XRD pattern of initial powders and CS–BG cements. 3.2. Setting Time The initial and final setting times, compressive strength and porosity of the cements are given in Table 2. From here, we can see that while pure calcium sulfate hemihydrate combined with pure distilled water sets at about 35 min, the chitosan-containing calcium sulfate cement exhibits a very long setting time. The C100-BG0 is also weak in terms of compressive strength. A significant decrease in setting time was observed when BG was included into the composition. Here, we observed a significant difference between the setting times of the various compositions, especially for CS70-BG30. The compressive strength is drastically improved in high contents of BG. Also, the porosity is in the range of 30–60% and except for CS90-BG10, adding BG decreases the total pore content. Table 2. Initial and final setting times, compressive strength, and total porosity of calcium sulfate cements containing different amounts of BG. Sample Initial Setting Time (min) Final Setting Time (min) Compressive Strength (MPa) Porosity (%) Cement made of pure CS and distilled water 25 ± 3 35 ± 3 - - Cement made of pure CS and citric acid 180 ± 15 240 ± 20 - - CS100-BG0 >300 >600 0.29 ± 0.02 50 ± 2 CS90-BG10 60 ± 5 150 ± 20 0.20 ± 0.07 61 ± 3 CS80-BG20 32 ± 5 120 ± 10 1.25 ± 0.11 38 ± 1 CS70-BG30 6 ± 1 20 ± 4 4.25 ± 0.85 36 ± 2 3.3. SEM Observation Microstructures of investigated CS–BG cements are shown in Figures 2 and 3. Figure 2 (A1–D3) depicts the SEM images and related EDX analysis of CS–BG cements. The results of particle size distribution performed by Image J software are shown in the inset of each related image. CS100-BG0 has porous structure morphology consisting of interlocking hexagonal flake-like crystals. Ca, S, and O elements are found in the related EDXA image of CS100-BG0. A mono-modal particle size distribution with average size of 7.9 μm is observed. By adding BG to calcium sulfate, the following changes in the microstructure are observed: (i) Fine glass particles embedded within a monolithic-shaped phase that covered the large block-like calcium sulfate crystals are seen; (ii) The morphology and shape of flakes changes from regular hexagonal to relatively shapeless blocks particles Figure 1. XRD pattern of initial powders and CS–BG cements. 3.2. Setting Time The initial and final setting times, compressive strength and porosity of the cements are given in Table 2. From here, we can see that while pure calcium sulfate hemihydrate combined with pure distilled water sets at about 35 min, the chitosan-containing calcium sulfate cement exhibits a very long setting time. The C100-BG0 is also weak in terms of compressive strength. A significant decrease in setting time was observed when BG was included into the composition. Here, we observed a significant difference between the setting times of the various compositions, especially for CS70-BG30. The compressive strength is drastically improved in high contents of BG. Also, the porosity is in the range of 30–60% and except for CS90-BG10, adding BG decreases the total pore content. Table 2. Initial and final setting times, compressive strength, and total porosity of calcium sulfate cements containing different amounts of BG. Sample Initial Setting Time (min) Final Setting Time (min) Compressive Strength (MPa) Porosity (%) Cement made of pure CS and distilled water 25 ±3 35 ±3 - - Cement made of pure CS and citric acid 180 ±15 240 ±20 - - CS100-BG0 >300 >600 0.29 ±0.02 50 ±2 CS90-BG10 60 ±5 150 ±20 0.20 ±0.07 61 ±3 CS80-BG20 32 ±5 120 ±10 1.25 ±0.11 38 ±1 CS70-BG30 6 ±1 20 ±4 4.25 ±0.85 36 ±2 3.3. SEM Observation Microstructures of investigated CS–BG cements are shown in Figures 2and 3. Figure 2 (A1–D3) depicts the SEM images and related EDX analysis of CS–BG cements. The results of particle size distribution performed by Image J software are shown in the inset of each related image. CS100-BG0 has porous structure morphology consisting of interlocking hexagonal flake-like crystals. Ca, S, and O elements are found in the related EDXA image of CS100-BG0. A mono-modal particle size distribution with average size of 7.9 µ m is observed. By adding BG to calcium sulfate, the following changes in the microstructure are observed: (i) Fine glass particles embedded within a monolithic-shaped phase that covered Biomedicines 2023,11, 2833 7 of 17 the large block-like calcium sulfate crystals are seen; (ii) The morphology and shape of flakes changes from regular hexagonal to relatively shapeless blocks particles (particularly for CS90-BG10 and CS80-BG20); (iii) In CS70-BG30, a flake-like morphology with reduced thickness and particle size is observed. The particle size distribution determines that when BG is added, a bimodal size distribution is found. In the authors’ opinion, the smaller average size relates to remaining BG that did not react with chitosan molecules, whereas the larger average size is attributed to calcium sulfate crystals. By increasing the BG content, the average size of calcium sulfate crystals decreases, so that in CS70-BG30, the bimodal graph tends to a mono-modal. In the EDXA patterns of BG-added CS, in addition to Ca, S, and O, the P and Si elements are also found. The peaks of Sr overlap with P and Si, meanwhile the detection of Mg and Sr peaks is impossible because of the very low concentration of them in the glass composition. It should be noted that the EDXA is not a quantitative analysis and the content of the elements should not be judged by the intensity of the peaks. Biomedicines 2023, 11, 2833 7 of 17 (particularly for CS90-BG10 and CS80-BG20); (iii) In CS70-BG30, a flake-like morphology with reduced thickness and particle size is observed. The particle size distribution determines that when BG is added, a bimodal size distribution is found. In the authors’ opinion, the smaller average size relates to remaining BG that did not react with chitosan molecules, whereas the larger average size is attributed to calcium sulfate crystals. By increasing the BG content, the average size of calcium sulfate crystals decreases, so that in CS70BG30, the bimodal graph tends to a mono-modal. In the EDXA patterns of BG-added CS, in addition to Ca, S, and O, the P and Si elements are also found. The peaks of Sr overlap with P and Si, meanwhile the detection of Mg and Sr peaks is impossible because of the very low concentration of them in the glass composition. It should be noted that the EDXA is not a quantitative analysis and the content of the elements should not be judged by the intensity of the peaks. Figure 2. SEM and the corresponding EDS images of CS100-BG0 (A1–A3), CS90-BG10 (B1–B3), CS80-BG20 (C1–C3), and CS70-BG30 (D1–D3) cements. Figure 2. SEM and the corresponding EDS images of CS100-BG0 ( A1 – A3 ), CS90-BG10 ( B1 – B3 ), CS80-BG20 (C1–C3), and CS70-BG30 (D1–D3) cements. Biomedicines 2023,11, 2833 8 of 17 Biomedicines 2023, 11, 2833 8 of 17 Figure 3 depicts the SEM elemental mapping of CS–BG samples for elemental distribution. Blue spots indicate the presence of Si as the principal element in the BG structure. Green dots represent S (Sulfur) as representative of CaSO4, whereas yellow dots symbolize N (nitrogen) as ingredient of chitosan. The SEM of the surfaces of the samples (provided in the left side of the map images) is in agreement with those shown in Figure 2. The addition of BG to calcium sulfate alters the microstructure, and when the percentage of bioactive glass reaches 30 percent, the microstructure of calcium sulfate-bioactive glass changes generally. The surface of CS–BGs shows a significant difference when the BG content increased. In CS90-BG10 and CS80-BG20, large dark areas are observed and the Si distribution seems heterogeneous. It can be related to the inhomogeneity of the surface created by the large pores. In the case of CS70-BG30, with a higher percent of BG, the microstructure was changed and fine flakes along with the remaining CSH reactants are observed. Also, the SEM image reveal that the microstructure of the CS70-BG30 contains a finer particle, more reduced porosity, denser and smoother surfaces. Therefore, the distribution of blue dots seems more homogenous. Figure 3. Surface morphology and elemental distribution of S, Si, and N. 3.4. Injectability Figure 4A shows the injection curves of cements with different contents of BG. At the beginning point of test, a sudden increase in extruding force is observed for all cements. This was also observed in other research. In a pilot test, the force-displacement diagram of empty syringe was checked. In this situation, no overshoot was observed at the start of the test. It determines that the syringe wall-plunger friction is negligible and the beginning Figure 3. Surface morphology and elemental distribution of S, Si, and N. Figure 3depicts the SEM elemental mapping of CS–BG samples for elemental distribution. Blue spots indicate the presence of Si as the principal element in the BG structure. Green dots represent S (Sulfur) as representative of CaSO 4 , whereas yellow dots symbolize N (nitrogen) as ingredient of chitosan. The SEM of the surfaces of the samples (provided in the left side of the map images) is in agreement with those shown in Figure 2. The addition of BG to calcium sulfate alters the microstructure, and when the percentage of bioactive glass reaches 30 percent, the microstructure of calcium sulfate-bioactive glass changes generally. The surface of CS–BGs shows a significant difference when the BG content increased. In CS90-BG10 and CS80-BG20, large dark areas are observed and the Si distribution seems heterogeneous. It can be related to the inhomogeneity of the surface created by the large pores. In the case of CS70-BG30, with a higher percent of BG, the microstructure was changed and fine flakes along with the remaining CSH reactants are observed. Also, the SEM image reveal that the microstructure of the CS70-BG30 contains a finer particle, more reduced porosity, denser and smoother surfaces. Therefore, the distribution of blue dots seems more homogenous. 3.4. Injectability Figure 4A shows the injection curves of cements with different contents of BG. At the beginning point of test, a sudden increase in extruding force is observed for all cements. This was also observed in other research. In a pilot test, the force-displacement diagram of empty syringe was checked. In this situation, no overshoot was observed at the start of the test. It determines that the syringe wall-plunger friction is negligible and the Biomedicines 2023,11, 2833 9 of 17 beginning overshoot corresponds to the force required to overcome the paste-syringe wall friction caused by hydraulic pressure inside the syringe [ 29 , 32 ]. After that, all the cements are injected with a nearly constant force, lower than 20 N. Overall, it is found that the composites with higher BG content exhibit higher injection force. No filter-pressing phenomenon is observed during the injection. It seems that the extrusion force decreased after 5 mm of displacement. It was found that the paste was coherent and slippery due the presence of chitosan. The authors suggest, when a volume of the paste is extruded, lower force is required to extrude the remaining paste. Thus, a decreased profile is seen for the extrusion by syringe displacement. Biomedicines 2023, 11, 2833 9 of 17 overshoot corresponds to the force required to overcome the paste-syringe wall friction caused by hydraulic pressure inside the syringe [29,32]. After that, all the cements are injected with a nearly constant force, lower than 20 N. Overall, it is found that the composites with higher BG content exhibit higher injection force. No filter-pressing phenomenon is observed during the injection. It seems that the extrusion force decreased after 5 mm of displacement. It was found that the paste was coherent and slippery due the presence of chitosan. The authors suggest, when a volume of the paste is extruded, lower force is required to extrude the remaining paste. Thus, a decreased profile is seen for the extrusion by syringe displacement. Since in the injectability test, the paste is extruded at a nearly constant rate, and the force required for the extrusion is evaluated, it may be possible to create an equivalence between the real part of the viscosity (ղ’) and the force required for injection. It means that ղ’ can be considered a criterion of the force required for injection. Figure 4B shows the real part of the complex viscosity (ղ’) of various cements as a function of ω calculated from Equation (4). In other words, there is a direct correlation between the injection force and (ղ’). It can be seen that the starting points of ղ’ increase with introducing considerable amount of BG, which is consistent with the injectability curve of cements. It means that a higher injection force is required. Moreover, at low frequencies range (ω = 1 − 8 s−1), with increasing ω, a sharp decrease in the real part of viscosity is observed (especially CS70-BG30) and the force required the injection to be continued decreases. Figure 4. (A) The injection curves of different cements shown as applied force vs. plunger displacement. (B) The real part of the complex viscosity (ղ’) of various cements as a function of ω. 3.5. Viscosity Figure 5 shows the complex viscosity (ղ*) of the paste as a function of ω. The complex viscosity of cements with higher percent of bioactive glass is more sensitive to angular frequency. The angular frequency value of 10 s−1 is the intersection point of ղ*-ω curves. In other words, at ω < 10 s−1, cements with higher BG content have a higher ղ* value and the cement CS70-BG30 exhibits the highest complex viscosity. At frequencies higher than 10 s−1, the viscosity behaves completely different and samples with more bioactive glass content have a lower viscosity. In this case, the highest viscosity relates to the glass-free cement. In fact, more internal structures can be formed in pastes containing bioactive glass, and when the frequency exceeds 10 s−1, these structures are broken leading to a sudden decrease of viscosity. In the case of these cements, with an increase in frequency (more than 100 s−1), the viscosity increases again and shear thickening behavior is observed. It means that the destroyed internal structures (links) are reformed due to the intensive stresses. Figure 4. ( A ) The injection curves of different cements shown as applied force vs. plunger displacement. (B) The real part of the complex viscosity ( Biomedicines 2023, 11, 2833 4 of 17 2.6. Injectability An extrusion test was used to assess the paste injectability [29]. After 1-min mixing procedure, the obtained paste was placed into a commercial 3 mL syringe (AvaPezeshk Co., Tehran, Iran) with cannula length and tip diameter of 10 mm and 2 mm, respectively. The paste was exerted in syringe tube (the length of filled paste in the tube was 30 mm) and extruded from the syringe (held rigidly in a fixture) at predetermined times after the mixing process was completed by applying force using a universal testing machine (STM20, Santam Co., Tehran, Iran) at a crosshead speed of 30 mm/min. The injection curve was plotted as injection force vs. plunger displacement. The injectability coefficient (I) was calculated as I = [(M0 − M)/M0] × 100 (1) where M0 is the initial mass of the composite in the syringe and M is the mass left inside the syringe after extrusion. 2.7. The Viscosity Measurements The Rheometer (Anton Paar, Physica MC-R301, Graz, Austria) with plate–plate measuring geometry was used to examine the rheological properties of calcium sulfate-bioactive glass pastes (plate diameter of 25 mm). For this measurement, a homogeneous paste was made during 1 min and the resultant paste was subsequently applied to the lower plate’s center at predetermined intervals. The rheological test began 2 min after mixing the powder and liquid phases at both oscillatory and rotation modes. A modest (low) amplitude sinusoidal oscillation strain was firstly applied to the paste in a dynamic oscillation test. The complex viscosity can be stated using the following formula due to the phase difference between two sinusoidal waves. G* = G′ + iG″ (2) ղ* = ղ′ − iղ″ (3) ղ’ = G″/ω (4) ղ” = G′/ω (5) where ω is the frequency in rads−1, G′ is shear storage modulus, G″ is shear loss modulus, G* is complex shear modulus, ղ″ is out-of-phase viscosity, ղ′ is dynamic (absolute) viscosity, and ղ* is complex viscosity. The real part of the complex viscosity (dynamic viscosity) (ղ′) is a criterion of force required to make a fluid flow at a certain speed [30,31]. Firstly, strain sweep mode measurement was performed to identify the linear viscoelastic region (LVR). Then, the dynamic frequency sweep test was performed while maintaining constant strain (1%) and temperature (25 °C). In rotatory mode, the viscosity–time curve was also drawn at a constant shear rate of 1 s−1 to identify the variation of the paste viscosity as a function of time. 2.8. The Cell Studies 2.8.1. Cell Survival, Viability, and Growth For in vitro cellular studies, the samples were immersed in an antibiotic solution for a duration of 30 min; afterwards, they were rinsed in phosphate-buffered saline (PBS), and subjected to ultraviolet irradiation (UV) for a period of 15 min. The cytotoxic effect of cements on human bone mesenchymal stem cells (hMSCs) was studied by 3-(4,5-dimethylthiazol-2)-2,5-diphenyltetrazolium bromide assay (MTT; Sigma-Aldrich, Taufkirchen, Germany). The cells were seeded in 100 μL of the medium at a density of 104 cells/well in 96-well culture plate and were incubated at 37 °C in a 95% ’) of various cements as a function of ω. Since in the injectability test, the paste is extruded at a nearly constant rate, and the force required for the extrusion is evaluated, it may be possible to create an equivalence between the real part of the viscosity ( Biomedicines 2023, 11, 2833 4 of 17 2.6. Injectability An extrusion test was used to assess the paste injectability [29]. After 1-min mixing procedure, the obtained paste was placed into a commercial 3 mL syringe (AvaPezeshk Co., Tehran, Iran) with cannula length and tip diameter of 10 mm and 2 mm, respectively. The paste was exerted in syringe tube (the length of filled paste in the tube was 30 mm) and extruded from the syringe (held rigidly in a fixture) at predetermined times after the mixing process was completed by applying force using a universal testing machine (STM20, Santam Co., Tehran, Iran) at a crosshead speed of 30 mm/min. The injection curve was plotted as injection force vs. plunger displacement. The injectability coefficient (I) was calculated as I = [(M0 − M)/M0] × 100 (1) where M0 is the initial mass of the composite in the syringe and M is the mass left inside the syringe after extrusion. 2.7. The Viscosity Measurements The Rheometer (Anton Paar, Physica MC-R301, Graz, Austria) with plate–plate measuring geometry was used to examine the rheological properties of calcium sulfate-bioactive glass pastes (plate diameter of 25 mm). For this measurement, a homogeneous paste was made during 1 min and the resultant paste was subsequently applied to the lower plate’s center at predetermined intervals. The rheological test began 2 min after mixing the powder and liquid phases at both oscillatory and rotation modes. A modest (low) amplitude sinusoidal oscillation strain was firstly applied to the paste in a dynamic oscillation test. The complex viscosity can be stated using the following formula due to the phase difference between two sinusoidal waves. G* = G′ + iG″ (2) ղ* = ղ′ − iղ″ (3) ղ’ = G″/ω (4) ղ” = G′/ω (5) where ω is the frequency in rads−1, G′ is shear storage modulus, G″ is shear loss modulus, G* is complex shear modulus, ղ″ is out-of-phase viscosity, ղ′ is dynamic (absolute) viscosity, and ղ* is complex viscosity. The real part of the complex viscosity (dynamic viscosity) (ղ′) is a criterion of force required to make a fluid flow at a certain speed [30,31]. Firstly, strain sweep mode measurement was performed to identify the linear viscoelastic region (LVR). Then, the dynamic frequency sweep test was performed while maintaining constant strain (1%) and temperature (25 °C). In rotatory mode, the viscosity–time curve was also drawn at a constant shear rate of 1 s−1 to identify the variation of the paste viscosity as a function of time. 2.8. The Cell Studies 2.8.1. Cell Survival, Viability, and Growth For in vitro cellular studies, the samples were immersed in an antibiotic solution for a duration of 30 min; afterwards, they were rinsed in phosphate-buffered saline (PBS), and subjected to ultraviolet irradiation (UV) for a period of 15 min. The cytotoxic effect of cements on human bone mesenchymal stem cells (hMSCs) was studied by 3-(4,5-dimethylthiazol-2)-2,5-diphenyltetrazolium bromide assay (MTT; Sigma-Aldrich, Taufkirchen, Germany). The cells were seeded in 100 μL of the medium at a density of 104 cells/well in 96-well culture plate and were incubated at 37 °C in a 95% ’) and the force required for injection. It means that Biomedicines 2023, 11, 2833 4 of 17 2.6. Injectability An extrusion test was used to assess the paste injectability [29]. After 1-min mixing procedure, the obtained paste was placed into a commercial 3 mL syringe (AvaPezeshk Co., Tehran, Iran) with cannula length and tip diameter of 10 mm and 2 mm, respectively. The paste was exerted in syringe tube (the length of filled paste in the tube was 30 mm) and extruded from the syringe (held rigidly in a fixture) at predetermined times after the mixing process was completed by applying force using a universal testing machine (STM20, Santam Co., Tehran, Iran) at a crosshead speed of 30 mm/min. The injection curve was plotted as injection force vs. plunger displacement. The injectability coefficient (I) was calculated as I = [(M0 − M)/M0] × 100 (1) where M0 is the initial mass of the composite in the syringe and M is the mass left inside the syringe after extrusion. 2.7. The Viscosity Measurements The Rheometer (Anton Paar, Physica MC-R301, Graz, Austria) with plate–plate measuring geometry was used to examine the rheological properties of calcium sulfate-bioactive glass pastes (plate diameter of 25 mm). For this measurement, a homogeneous paste was made during 1 min and the resultant paste was subsequently applied to the lower plate’s center at predetermined intervals. The rheological test began 2 min after mixing the powder and liquid phases at both oscillatory and rotation modes. A modest (low) amplitude sinusoidal oscillation strain was firstly applied to the paste in a dynamic oscillation test. The complex viscosity can be stated using the following formula due to the phase difference between two sinusoidal waves. G* = G′ + iG″ (2) ղ* = ղ′ − iղ″ (3) ղ’ = G″/ω (4) ղ” = G′/ω (5) where ω is the frequency in rads−1, G′ is shear storage modulus, G″ is shear loss modulus, G* is complex shear modulus, ղ″ is out-of-phase viscosity, ղ′ is dynamic (absolute) viscosity, and ղ* is complex viscosity. The real part of the complex viscosity (dynamic viscosity) (ղ′) is a criterion of force required to make a fluid flow at a certain speed [30,31]. Firstly, strain sweep mode measurement was performed to identify the linear viscoelastic region (LVR). Then, the dynamic frequency sweep test was performed while maintaining constant strain (1%) and temperature (25 °C). In rotatory mode, the viscosity–time curve was also drawn at a constant shear rate of 1 s−1 to identify the variation of the paste viscosity as a function of time. 2.8. The Cell Studies 2.8.1. Cell Survival, Viability, and Growth For in vitro cellular studies, the samples were immersed in an antibiotic solution for a duration of 30 min; afterwards, they were rinsed in phosphate-buffered saline (PBS), and subjected to ultraviolet irradiation (UV) for a period of 15 min. The cytotoxic effect of cements on human bone mesenchymal stem cells (hMSCs) was studied by 3-(4,5-dimethylthiazol-2)-2,5-diphenyltetrazolium bromide assay (MTT; Sigma-Aldrich, Taufkirchen, Germany). The cells were seeded in 100 μL of the medium at a density of 104 cells/well in 96-well culture plate and were incubated at 37 °C in a 95% ’ can be considered a criterion of the force required for injection. Figure 4B shows the real part of the complex viscosity ( Biomedicines 2023, 11, 2833 4 of 17 2.6. Injectability An extrusion test was used to assess the paste injectability [29]. After 1-min mixing procedure, the obtained paste was placed into a commercial 3 mL syringe (AvaPezeshk Co., Tehran, Iran) with cannula length and tip diameter of 10 mm and 2 mm, respectively. The paste was exerted in syringe tube (the length of filled paste in the tube was 30 mm) and extruded from the syringe (held rigidly in a fixture) at predetermined times after the mixing process was completed by applying force using a universal testing machine (STM20, Santam Co., Tehran, Iran) at a crosshead speed of 30 mm/min. The injection curve was plotted as injection force vs. plunger displacement. The injectability coefficient (I) was calculated as I = [(M0 − M)/M0] × 100 (1) where M0 is the initial mass of the composite in the syringe and M is the mass left inside the syringe after extrusion. 2.7. The Viscosity Measurements The Rheometer (Anton Paar, Physica MC-R301, Graz, Austria) with plate–plate measuring geometry was used to examine the rheological properties of calcium sulfate-bioactive glass pastes (plate diameter of 25 mm). For this measurement, a homogeneous paste was made during 1 min and the resultant paste was subsequently applied to the lower plate’s center at predetermined intervals. The rheological test began 2 min after mixing the powder and liquid phases at both oscillatory and rotation modes. A modest (low) amplitude sinusoidal oscillation strain was firstly applied to the paste in a dynamic oscillation test. The complex viscosity can be stated using the following formula due to the phase difference between two sinusoidal waves. G* = G′ + iG″ (2) ղ* = ղ′ − iղ″ (3) ղ’ = G″/ω (4) ղ” = G′/ω (5) where ω is the frequency in rads−1, G′ is shear storage modulus, G″ is shear loss modulus, G* is complex shear modulus, ղ″ is out-of-phase viscosity, ղ′ is dynamic (absolute) viscosity, and ղ* is complex viscosity. The real part of the complex viscosity (dynamic viscosity) (ղ′) is a criterion of force required to make a fluid flow at a certain speed [30,31]. Firstly, strain sweep mode measurement was performed to identify the linear viscoelastic region (LVR). Then, the dynamic frequency sweep test was performed while maintaining constant strain (1%) and temperature (25 °C). In rotatory mode, the viscosity–time curve was also drawn at a constant shear rate of 1 s−1 to identify the variation of the paste viscosity as a function of time. 2.8. The Cell Studies 2.8.1. Cell Survival, Viability, and Growth For in vitro cellular studies, the samples were immersed in an antibiotic solution for a duration of 30 min; afterwards, they were rinsed in phosphate-buffered saline (PBS), and subjected to ultraviolet irradiation (UV) for a period of 15 min. The cytotoxic effect of cements on human bone mesenchymal stem cells (hMSCs) was studied by 3-(4,5-dimethylthiazol-2)-2,5-diphenyltetrazolium bromide assay (MTT; Sigma-Aldrich, Taufkirchen, Germany). The cells were seeded in 100 μL of the medium at a density of 104 cells/well in 96-well culture plate and were incubated at 37 °C in a 95% ’) of various cements as a function of ω calculated from Equation (4). In other words, there is a direct correlation between the injection force and ( Biomedicines 2023, 11, 2833 4 of 17 2.6. Injectability An extrusion test was used to assess the paste injectability [29]. After 1-min mixing procedure, the obtained paste was placed into a commercial 3 mL syringe (AvaPezeshk Co., Tehran, Iran) with cannula length and tip diameter of 10 mm and 2 mm, respectively. The paste was exerted in syringe tube (the length of filled paste in the tube was 30 mm) and extruded from the syringe (held rigidly in a fixture) at predetermined times after the mixing process was completed by applying force using a universal testing machine (STM20, Santam Co., Tehran, Iran) at a crosshead speed of 30 mm/min. The injection curve was plotted as injection force vs. plunger displacement. The injectability coefficient (I) was calculated as I = [(M0 − M)/M0] × 100 (1) where M0 is the initial mass of the composite in the syringe and M is the mass left inside the syringe after extrusion. 2.7. The Viscosity Measurements The Rheometer (Anton Paar, Physica MC-R301, Graz, Austria) with plate–plate measuring geometry was used to examine the rheological properties of calcium sulfate-bioactive glass pastes (plate diameter of 25 mm). For this measurement, a homogeneous paste was made during 1 min and the resultant paste was subsequently applied to the lower plate’s center at predetermined intervals. The rheological test began 2 min after mixing the powder and liquid phases at both oscillatory and rotation modes. A modest (low) amplitude sinusoidal oscillation strain was firstly applied to the paste in a dynamic oscillation test. The complex viscosity can be stated using the following formula due to the phase difference between two sinusoidal waves. G* = G′ + iG″ (2) ղ* = ղ′ − iղ″ (3) ղ’ = G″/ω (4) ղ” = G′/ω (5) where ω is the frequency in rads−1, G′ is shear storage modulus, G″ is shear loss modulus, G* is complex shear modulus, ղ″ is out-of-phase viscosity, ղ′ is dynamic (absolute) viscosity, and ղ* is complex viscosity. The real part of the complex viscosity (dynamic viscosity) (ղ′) is a criterion of force required to make a fluid flow at a certain speed [30,31]. Firstly, strain sweep mode measurement was performed to identify the linear viscoelastic region (LVR). Then, the dynamic frequency sweep test was performed while maintaining constant strain (1%) and temperature (25 °C). In rotatory mode, the viscosity–time curve was also drawn at a constant shear rate of 1 s−1 to identify the variation of the paste viscosity as a function of time. 2.8. The Cell Studies 2.8.1. Cell Survival, Viability, and Growth For in vitro cellular studies, the samples were immersed in an antibiotic solution for a duration of 30 min; afterwards, they were rinsed in phosphate-buffered saline (PBS), and subjected to ultraviolet irradiation (UV) for a period of 15 min. The cytotoxic effect of cements on human bone mesenchymal stem cells (hMSCs) was studied by 3-(4,5-dimethylthiazol-2)-2,5-diphenyltetrazolium bromide assay (MTT; Sigma-Aldrich, Taufkirchen, Germany). The cells were seeded in 100 μL of the medium at a density of 104 cells/well in 96-well culture plate and were incubated at 37 °C in a 95% ’). It can be seen that the starting points of Biomedicines 2023, 11, 2833 4 of 17 2.6. Injectability An extrusion test was used to assess the paste injectability [29]. After 1-min mixing procedure, the obtained paste was placed into a commercial 3 mL syringe (AvaPezeshk Co., Tehran, Iran) with cannula length and tip diameter of 10 mm and 2 mm, respectively. The paste was exerted in syringe tube (the length of filled paste in the tube was 30 mm) and extruded from the syringe (held rigidly in a fixture) at predetermined times after the mixing process was completed by applying force using a universal testing machine (STM20, Santam Co., Tehran, Iran) at a crosshead speed of 30 mm/min. The injection curve was plotted as injection force vs. plunger displacement. The injectability coefficient (I) was calculated as I = [(M0 − M)/M0] × 100 (1) where M0 is the initial mass of the composite in the syringe and M is the mass left inside the syringe after extrusion. 2.7. The Viscosity Measurements The Rheometer (Anton Paar, Physica MC-R301, Graz, Austria) with plate–plate measuring geometry was used to examine the rheological properties of calcium sulfate-bioactive glass pastes (plate diameter of 25 mm). For this measurement, a homogeneous paste was made during 1 min and the resultant paste was subsequently applied to the lower plate’s center at predetermined intervals. The rheological test began 2 min after mixing the powder and liquid phases at both oscillatory and rotation modes. A modest (low) amplitude sinusoidal oscillation strain was firstly applied to the paste in a dynamic oscillation test. The complex viscosity can be stated using the following formula due to the phase difference between two sinusoidal waves. G* = G′ + iG″ (2) ղ* = ղ′ − iղ″ (3) ղ’ = G″/ω (4) ղ” = G′/ω (5) where ω is the frequency in rads−1, G′ is shear storage modulus, G″ is shear loss modulus, G* is complex shear modulus, ղ″ is out-of-phase viscosity, ղ′ is dynamic (absolute) viscosity, and ղ* is complex viscosity. The real part of the complex viscosity (dynamic viscosity) (ղ′) is a criterion of force required to make a fluid flow at a certain speed [30,31]. Firstly, strain sweep mode measurement was performed to identify the linear viscoelastic region (LVR). Then, the dynamic frequency sweep test was performed while maintaining constant strain (1%) and temperature (25 °C). In rotatory mode, the viscosity–time curve was also drawn at a constant shear rate of 1 s−1 to identify the variation of the paste viscosity as a function of time. 2.8. The Cell Studies 2.8.1. Cell Survival, Viability, and Growth For in vitro cellular studies, the samples were immersed in an antibiotic solution for a duration of 30 min; afterwards, they were rinsed in phosphate-buffered saline (PBS), and subjected to ultraviolet irradiation (UV) for a period of 15 min. The cytotoxic effect of cements on human bone mesenchymal stem cells (hMSCs) was studied by 3-(4,5-dimethylthiazol-2)-2,5-diphenyltetrazolium bromide assay (MTT; Sigma-Aldrich, Taufkirchen, Germany). The cells were seeded in 100 μL of the medium at a density of 104 cells/well in 96-well culture plate and were incubated at 37 °C in a 95% ’ increase with introducing considerable amount of BG, which is consistent with the injectability curve of cements. It means that a higher injection force is required. Moreover, at low frequencies range ( ω = 1 − 8 s −1 ), with increasing ω , a sharp decrease in the real part of viscosity is observed (especially CS70-BG30) and the force required the injection to be continued decreases. 3.5. Viscosity Figure 5shows the complex viscosity ( Biomedicines 2023, 11, 2833 4 of 17 2.6. Injectability An extrusion test was used to assess the paste injectability [29]. After 1-min mixing procedure, the obtained paste was placed into a commercial 3 mL syringe (AvaPezeshk Co., Tehran, Iran) with cannula length and tip diameter of 10 mm and 2 mm, respectively. The paste was exerted in syringe tube (the length of filled paste in the tube was 30 mm) and extruded from the syringe (held rigidly in a fixture) at predetermined times after the mixing process was completed by applying force using a universal testing machine (STM20, Santam Co., Tehran, Iran) at a crosshead speed of 30 mm/min. The injection curve was plotted as injection force vs. plunger displacement. The injectability coefficient (I) was calculated as I = [(M0 − M)/M0] × 100 (1) where M0 is the initial mass of the composite in the syringe and M is the mass left inside the syringe after extrusion. 2.7. The Viscosity Measurements The Rheometer (Anton Paar, Physica MC-R301, Graz, Austria) with plate–plate measuring geometry was used to examine the rheological properties of calcium sulfate-bioactive glass pastes (plate diameter of 25 mm). For this measurement, a homogeneous paste was made during 1 min and the resultant paste was subsequently applied to the lower plate’s center at predetermined intervals. The rheological test began 2 min after mixing the powder and liquid phases at both oscillatory and rotation modes. A modest (low) amplitude sinusoidal oscillation strain was firstly applied to the paste in a dynamic oscillation test. The complex viscosity can be stated using the following formula due to the phase difference between two sinusoidal waves. G* = G′ + iG″ (2) ղ* = ղ′ − iղ″ (3) ղ’ = G″/ω (4) ղ” = G′/ω (5) where ω is the frequency in rads−1, G′ is shear storage modulus, G″ is shear loss modulus, G* is complex shear modulus, ղ″ is out-of-phase viscosity, ղ′ is dynamic (absolute) viscosity, and ղ* is complex viscosity. The real part of the complex viscosity (dynamic viscosity) (ղ′) is a criterion of force required to make a fluid flow at a certain speed [30,31]. Firstly, strain sweep mode measurement was performed to identify the linear viscoelastic region (LVR). Then, the dynamic frequency sweep test was performed while maintaining constant strain (1%) and temperature (25 °C). In rotatory mode, the viscosity–time curve was also drawn at a constant shear rate of 1 s−1 to identify the variation of the paste viscosity as a function of time. 2.8. The Cell Studies 2.8.1. Cell Survival, Viability, and Growth For in vitro cellular studies, the samples were immersed in an antibiotic solution for a duration of 30 min; afterwards, they were rinsed in phosphate-buffered saline (PBS), and subjected to ultraviolet irradiation (UV) for a period of 15 min. The cytotoxic effect of cements on human bone mesenchymal stem cells (hMSCs) was studied by 3-(4,5-dimethylthiazol-2)-2,5-diphenyltetrazolium bromide assay (MTT; Sigma-Aldrich, Taufkirchen, Germany). The cells were seeded in 100 μL of the medium at a density of 104 cells/well in 96-well culture plate and were incubated at 37 °C in a 95% *) of the paste as a function of ω . The complex viscosity of cements with higher percent of bioactive glass is more sensitive to angular frequency. The angular frequency value of 10 s −1 is the intersection point of Biomedicines 2023, 11, 2833 4 of 17 2.6. Injectability An extrusion test was used to assess the paste injectability [29]. After 1-min mixing procedure, the obtained paste was placed into a commercial 3 mL syringe (AvaPezeshk Co., Tehran, Iran) with cannula length and tip diameter of 10 mm and 2 mm, respectively. The paste was exerted in syringe tube (the length of filled paste in the tube was 30 mm) and extruded from the syringe (held rigidly in a fixture) at predetermined times after the mixing process was completed by applying force using a universal testing machine (STM20, Santam Co., Tehran, Iran) at a crosshead speed of 30 mm/min. The injection curve was plotted as injection force vs. plunger displacement. The injectability coefficient (I) was calculated as I = [(M0 − M)/M0] × 100 (1) where M0 is the initial mass of the composite in the syringe and M is the mass left inside the syringe after extrusion. 2.7. The Viscosity Measurements The Rheometer (Anton Paar, Physica MC-R301, Graz, Austria) with plate–plate measuring geometry was used to examine the rheological properties of calcium sulfate-bioactive glass pastes (plate diameter of 25 mm). For this measurement, a homogeneous paste was made during 1 min and the resultant paste was subsequently applied to the lower plate’s center at predetermined intervals. The rheological test began 2 min after mixing the powder and liquid phases at both oscillatory and rotation modes. A modest (low) amplitude sinusoidal oscillation strain was firstly applied to the paste in a dynamic oscillation test. The complex viscosity can be stated using the following formula due to the phase difference between two sinusoidal waves. G* = G′ + iG″ (2) ղ* = ղ′ − iղ″ (3) ղ’ = G″/ω (4) ղ” = G′/ω (5) where ω is the frequency in rads−1, G′ is shear storage modulus, G″ is shear loss modulus, G* is complex shear modulus, ղ″ is out-of-phase viscosity, ղ′ is dynamic (absolute) viscosity, and ղ* is complex viscosity. The real part of the complex viscosity (dynamic viscosity) (ղ′) is a criterion of force required to make a fluid flow at a certain speed [30,31]. Firstly, strain sweep mode measurement was performed to identify the linear viscoelastic region (LVR). Then, the dynamic frequency sweep test was performed while maintaining constant strain (1%) and temperature (25 °C). In rotatory mode, the viscosity–time curve was also drawn at a constant shear rate of 1 s−1 to identify the variation of the paste viscosity as a function of time. 2.8. The Cell Studies 2.8.1. Cell Survival, Viability, and Growth For in vitro cellular studies, the samples were immersed in an antibiotic solution for a duration of 30 min; afterwards, they were rinsed in phosphate-buffered saline (PBS), and subjected to ultraviolet irradiation (UV) for a period of 15 min. The cytotoxic effect of cements on human bone mesenchymal stem cells (hMSCs) was studied by 3-(4,5-dimethylthiazol-2)-2,5-diphenyltetrazolium bromide assay (MTT; Sigma-Aldrich, Taufkirchen, Germany). The cells were seeded in 100 μL of the medium at a density of 104 cells/well in 96-well culture plate and were incubated at 37 °C in a 95% *- ω curves. In other words, at ω < 10 s −1 , cements with higher BG content have a higher Biomedicines 2023, 11, 2833 4 of 17 2.6. Injectability An extrusion test was used to assess the paste injectability [29]. After 1-min mixing procedure, the obtained paste was placed into a commercial 3 mL syringe (AvaPezeshk Co., Tehran, Iran) with cannula length and tip diameter of 10 mm and 2 mm, respectively. The paste was exerted in syringe tube (the length of filled paste in the tube was 30 mm) and extruded from the syringe (held rigidly in a fixture) at predetermined times after the mixing process was completed by applying force using a universal testing machine (STM20, Santam Co., Tehran, Iran) at a crosshead speed of 30 mm/min. The injection curve was plotted as injection force vs. plunger displacement. The injectability coefficient (I) was calculated as I = [(M0 − M)/M0] × 100 (1) where M0 is the initial mass of the composite in the syringe and M is the mass left inside the syringe after extrusion. 2.7. The Viscosity Measurements The Rheometer (Anton Paar, Physica MC-R301, Graz, Austria) with plate–plate measuring geometry was used to examine the rheological properties of calcium sulfate-bioactive glass pastes (plate diameter of 25 mm). For this measurement, a homogeneous paste was made during 1 min and the resultant paste was subsequently applied to the lower plate’s center at predetermined intervals. The rheological test began 2 min after mixing the powder and liquid phases at both oscillatory and rotation modes. A modest (low) amplitude sinusoidal oscillation strain was firstly applied to the paste in a dynamic oscillation test. The complex viscosity can be stated using the following formula due to the phase difference between two sinusoidal waves. G* = G′ + iG″ (2) ղ* = ղ′ − iղ″ (3) ղ’ = G″/ω (4) ղ” = G′/ω (5) where ω is the frequency in rads−1, G′ is shear storage modulus, G″ is shear loss modulus, G* is complex shear modulus, ղ″ is out-of-phase viscosity, ղ′ is dynamic (absolute) viscosity, and ղ* is complex viscosity. The real part of the complex viscosity (dynamic viscosity) (ղ′) is a criterion of force required to make a fluid flow at a certain speed [30,31]. Firstly, strain sweep mode measurement was performed to identify the linear viscoelastic region (LVR). Then, the dynamic frequency sweep test was performed while maintaining constant strain (1%) and temperature (25 °C). In rotatory mode, the viscosity–time curve was also drawn at a constant shear rate of 1 s−1 to identify the variation of the paste viscosity as a function of time. 2.8. The Cell Studies 2.8.1. Cell Survival, Viability, and Growth For in vitro cellular studies, the samples were immersed in an antibiotic solution for a duration of 30 min; afterwards, they were rinsed in phosphate-buffered saline (PBS), and subjected to ultraviolet irradiation (UV) for a period of 15 min. The cytotoxic effect of cements on human bone mesenchymal stem cells (hMSCs) was studied by 3-(4,5-dimethylthiazol-2)-2,5-diphenyltetrazolium bromide assay (MTT; Sigma-Aldrich, Taufkirchen, Germany). The cells were seeded in 100 μL of the medium at a density of 104 cells/well in 96-well culture plate and were incubated at 37 °C in a 95% * value and the cement CS70-BG30 exhibits the highest complex viscosity. At frequencies higher than 10 s −1 , the viscosity behaves completely different and samples with more bioactive glass content have a lower viscosity. In this case, the highest viscosity relates to the glass-free cement. In fact, more internal structures can be formed in pastes containing bioactive glass, and when the frequency exceeds 10 s −1 , these structures are broken leading to a sudden decrease of viscosity. In the case of these cements, with an increase in frequency (more than 100 s −1 ), the viscosity increases again and shear thickening behavior is observed. It means that the destroyed internal structures (links) are reformed due to the intensive stresses. Biomedicines 2023,11, 2833 16 of 17 8. Jensen, T.; Dolatshahi-Pirouz, A.; Foss, M.; Baas, J.; Lovmand, J.; Duch, M.; Pedersen, F.S.; Kassem, M.; Bünger, C.; Søballe, K.; et al. Interaction of human mesenchymal stem cells with osteopontin coated hydroxyapatite surfaces. Colloids Surf. B Biointerfaces 2010,75, 186–193. [CrossRef] 9. Morley, R.; Lopez, F.; Webb, F. Calcium sulphate as a drug delivery system in a deep diabetic foot infection. Foot 2016 ,27, 36–40. [CrossRef] 10. Stubbs, D.; Deakin, M.; Chapman-Sheath, P.; Bruce, W.; Debes, J.; Gillies, R.M.; Walsh, W.R. In vivo evaluation of resorbable bone graft substitutes in a rabbit tibial defect model. Biomaterials 2004,25, 5037–5044. [CrossRef] 11. Thomas, M.V.; Puleo, D.A.; Al-Sabbagh, M. Calcium sulfate: A review. J. Long-Term Eff. Med. Implant. 2005 ,15, 599–607. [CrossRef] [PubMed] 12. Beenken, K.E.; Smith, J.K.; Skinner, R.A.; Mclaren, S.G.; Bellamy, W.; Gruenwald, M.J.; Spencer, H.J.; Jennings, J.A.; Haggard, W.O.; Smeltzer, M.S. Chitosan coating to enhance the therapeutic efficacy of calcium sulfate-based antibiotic therapy in the treatment of chronic osteomyelitis. J. Biomater. Appl. 2014,29, 514–523. [CrossRef] [PubMed] 13. Chen, Z.; Liu, H.; Liu, X.; Cui, F.Z. Injectable calcium sulfate/mineralized collagen-based bone repair materials with regulable self-setting properties. J. Biomed. Mater. Res. Part A 2011,99, 554–563. [CrossRef] 14. Shuai, C.; Zhou, J.; Wu, P.; Gao, C.; Feng, P.; Xiao, T.; Deng, Y.; Peng, S. Enhanced stability of calcium sulfate scaffolds with 45S5 bioglass for bone repair. Materials 2015,8, 7498–7510. [CrossRef] [PubMed] 15. Hsu, P.Y.; Kuo, H.C.; Tuan, W.H.; Shih, S.J.; Naito, M.; Lai, P.L. Manipulation of the degradation behavior of calcium sulfate by the addition of bioglass. Prog. Biomater. 2019,8, 115–125. [CrossRef] 16. Shams, M.; Nezafati, N.; Poormoghadam, D.; Zavareh, S.; Zamanian, A.; Salimi, A. Synthesis and characterization of electrospun bioactive glass nanofibers-reinforced calcium sulfate bone cement and its cell biological response. Ceram. Int. 2020 ,46, 10029–10039. [CrossRef] 17. Chamansara, A.; Behnamghader, A.; Zamanian, A. Preparation and characterization of injectable gelatin/alginate/chondroitin sulfate/ α -calcium sulfate hemihydrate composite paste for bone repair application. J. Biomater. Appl. 2022 ,36, 1758–1774. [CrossRef] 18. Czechowska, J.; Zima, A.; Siek, D.; ´ Slósarczyk, A. The importance of chitosan and nano-TiHA in cement-type composites on the basis of calcium sulfate. Ceram. Int. 2016,42, 15559–15567. [CrossRef] 19. Zeimaran, E.; Pourshahrestani, S.; Fathi, A.; bin Abd Razak, N.A.; Kadri, N.A.; Sheikhi, A.; Baino, F. Advances in bioactive glass-containing injectable hydrogel biomaterials for tissue regeneration. Acta Biomater. 2021,136, 1–36. [CrossRef] 20. Jones, J.R. Review of bioactive glass: From Hench to hybrids. Acta Biomater. 2013,9, 4457–4486. [CrossRef] 21. Ruan, Z.; Yao, D.; Xu, Q.; Liu, L.; Tian, Z.; Zhu, Y. Effects of mesoporous bioglass on physicochemical and biological properties of calcium sulfate bone cements. Appl. Mater. Today 2017,9, 612–621. [CrossRef] 22. Jennings, J.A.; Bumgardner, J.D. (Eds.) Chitosan Based Biomaterials Volume 1: Fundamentals; Woodhead Publishing: Cambridge, UK, 2016. 23. Patel, S.K.; Kim, J.H.; Kalia, V.C.; Lee, J.K. Antimicrobial activity of amino-derivatized cationic polysaccharides. Indian J. Microbiol. 2019,59, 96–99. [CrossRef] [PubMed] 24. Demir-O˘guz, Ö.; Boccaccini, A.R.; Loca, D. Injectable bone cements: What benefits the combination of calcium phosphates and bioactive glasses could bring? Bioact. Mater. 2023,19, 217–236. [CrossRef] [PubMed] 25. Zima, A.; Czechowska, J.; Siek, D.; ´ Slósarczyk, A. Influence of magnesium and silver ions on rheological properties of hydroxyapatite/chitosan/calcium sulphate based bone cements. Ceram. Int. 2017,43, 16196–16203. [CrossRef] 26. Sharifianjazi, F.; Moradi, M.; Abouchenari, A.; Pakseresht, A.H.; Esmaeilkhanian, A.; Shokouhimehr, M.; Asl, M.S. Effects of Sr and Mg dopants on biological and mechanical properties of SiO 2 –CaO–P 2 O 5 bioactive glass. Ceram. Int. 2020 ,46, 22674–22682. [CrossRef] 27. ASTM C266-08; Standard Test Method for Time of Setting of Hydraulic Cement Paste by Gilmore Needles. ASTM International: West Conshohocken, PA, USA, 2009. 28. Zou, Y.; Malzbender, J. Development and optimization of porosity measurement techniques. Ceram. Int. 2016 ,42, 2861–2870. [CrossRef] 29. Borhan, S.; Hesaraki, S.; Behnamghader, A.A.; Ghasemi, E. Rheological evaluations and in vitro studies of injectable bioactive glass–polycaprolactone–sodium alginate composites. J. Mater. Sci. Mater. Med. 2016,27, 137. [CrossRef] 30. Kaffashi, B.; Rezapour-Sarabi, M. Polymers Rheology; Boroon Sepehr Publishing: Washington, DC, USA, 2022; p. 141. 31. Fatimi, A.; Tassin, J.F.; Bosco, J.; Deterre, R.; Axelos, M.A.; Weiss, P. Injection of calcium phosphate pastes: Prediction of injection force and comparison with experiments. J. Mater. Sci. Mater. Med. 2012,23, 1593–1603. [CrossRef] 32. Medrano-David, D.; Lopera, A.M.; Londoño, M.E.; Araque-Marín, P. Formulation and characterization of a new injectable bone substitute composed PVA/borax/CaCO3and Demineralized Bone Matrix. J. Funct. Biomater. 2021,12, 46. [CrossRef] 33. Sarda, S.; Fernández, E.; Llorens, J.; Martinez, S.; Nilsson, M.; Planell, J.A. Rheological properties of an apatitic bone cement during initial setting. J. Mater. Sci. Mater. Med. 2001,12, 905–909. [CrossRef] 34. Le Gouellec, Y.A.; Elimelech, M. Calcium sulfate (gypsum) scaling in nanofiltration of agricultural drainage water. J. Membr. Sci. 2002,205, 279–291. [CrossRef] Biomedicines 2023,11, 2833 17 of 17 35. Shamsi, M.; Karimi, M.; Ghollasi, M.; Nezafati, N.; Shahrousvand, M.; Kamali, M.; Salimi, A. In vitro proliferation and differentiation of human bone marrow mesenchymal stem cells into osteoblasts on nanocomposite scaffolds based on bioactive glass (64SiO 2 -31CaO-5P 2 O 5 )-poly-l-lactic acid nanofibers fabricated by electrospinning method. Mater. Sci. Eng. C 2017 ,78, 114–123. [CrossRef] 36. Barreto, M.E.; Medeiros, R.P.; Shearer, A.; Fook, M.V.; Montazerian, M.; Mauro, J.C. Gelatin and Bioactive Glass Composites for Tissue Engineering: A Review. J. Funct. Biomater. 2022,14, 23. [CrossRef] [PubMed] 37. Hesaraki, S.; Moztarzadeh, F.; Nezafati, N. Evaluation of a bioceramic-based nanocomposite material for controlled delivery of a non-steroidal anti-inflammatory drug. Med. Eng. Phys. 2009,31, 1205–1213. [CrossRef] 38. Sony, S.; Suresh Babu, S.; Nishad, K.V.; Varma, H.; Komath, M. Development of an injectable bioactive bone filler cement with hydrogen orthophosphate incorporated calcium sulfate. J. Mater. Sci. Mater. Med. 2015,26, 31. [CrossRef] [PubMed] 39. Hesaraki, S.; Zamanian, A.; Moztarzadeh, F. The influence of the acidic component of the gas-foaming porogen used in preparing an injectable porous calcium phosphate cement on its properties: Acetic acid versus citric acid. J. Biomed. Mater. Res. Part B Appl. Biomater. 2008,86, 208–216. [CrossRef] 40. Yokoyama, A.; Yamamoto, S.; Kawasaki, T.; Kohgo, T.; Nakasu, M. Development of calcium phosphate cement using chitosan and citric acid for bone substitute materials. Biomaterials 2002,23, 1091–1101. [CrossRef] 41. Zhao, Y.; Jia, L.; Liu, K.; Gao, P.; Ge, H.; Fu, L. Inhibition of calcium sulfate scale by poly (citric acid). Desalination 2016 ,392, 1–7. [CrossRef] 42. Muzzarelli, R.A. Natural chelating polymers. Alginic acid, chitin and chitosan. In International Series of Monographs in Analytical Chemistry; Pergamon Press: Oxford, UK, 1973. 43. Krajewska, B. Diffusion of metal ions through gel chitosan membranes. React. Funct. Polym. 2001,47, 37–47. [CrossRef] 44. Hesaraki, S.; Moztarzadeh, F.; Nemati, R.; Nezafati, N. Preparation and characterization of calcium sulfate–biomimetic apatite nanocomposites for controlled release of antibiotics. J. Biomed. Mater. Res. Part B Appl. Biomater. 2009,91, 651–661. [CrossRef] 45. Zhang, X.; Jia, W.; Gu, Y.; Xiao, W.; Liu, X.; Wang, D.; Zhang, C.; Huang, W.; Rahaman, M.N.; Day, D.E.; et al. Teicoplanin-loaded borate bioactive glass implants for treating chronic bone infection in a rabbit tibia osteomyelitis model. Biomaterials 2010 ,31, 5865–5874. [CrossRef] [PubMed] 46. Rauschmann, M.A.; Wichelhaus, T.A.; Stirnal, V.; Dingeldein, E.; Zichner, L.; Schnettler, R.; Alt, V. Nanocrystalline hydroxyapatite and calcium sulphate as biodegradable composite carrier material for local delivery of antibiotics in bone infections. Biomaterials 2005,26, 2677–2684. [CrossRef] [PubMed] 47. Bernardo, M.P.; da Silva, B.C.; Hamouda, A.E.; de Toledo, M.A.; Schalla, C.; Rütten, S.; Goetzke, R.; Mattoso, L.H.; Zenke, M.; Sechi, A. PLA/Hydroxyapatite scaffolds exhibit in vitro immunological inertness and promote robust osteogenic differentiation of human mesenchymal stem cells without osteogenic stimuli. Sci. Rep. 2022,12, 2333. [CrossRef] 48. Liang, H.; Xu, X.; Feng, X.; Ma, L.; Deng, X.; Wu, S.; Liu, X.; Yang, C. Gold nanoparticles-loaded hydroxyapatite composites guide osteogenic differentiation of human mesenchymal stem cells through Wnt/ β -catenin signaling pathway. Int. J. Nanomed. 2019 ,14, 6151–6163. [CrossRef] [PubMed] Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.