Production of nano hydroxyapatite and Mg-Whitlockite from biowaste-derived products via continuous flow hydrothermal synthesis : a step towards circular economy

Biowastes from agriculture, sewage, household wastes, and industries comprise promising resources to produce biomaterials while reducing adverse environmental effects. This study focused on utilising waste-derived materials (i.e., eggshells as a calcium source, struvite as a phosphate source, and CH3COOH as dissolution media) to produce value-added products (i.e., calcium phosphates (CaPs) derived from biomaterials) using a continuous flow hydrothermal synthesis route. The prepared materials were characterised via XRD, FEG-SEM, EDX, FTIR, and TEM analysis. Magnesium whitlockite (Mg-WH) and hydroxyapatite (HA) were produced by single-phase or biphasic CaPs by reacting struvite with either calcium nitrate tetrahydrate or an eggshell solution at 200 °C and 350 °C. Rhombohedral-shaped Mg-WH (23–720 nm) along with tube (50–290 nm diameter, 20–71 nm thickness) and/or ellipsoidal morphologies of HA (273–522 nm width) were observed at 350 °C using HNO3 or CH3COOH to prepare the eggshell and struvite solutions, and NH4OH was used as the pH buffer. The Ca/P (atomic%) ratios obtained ranged between 1.3 and 1.7, indicating the formation of Mg-WH and HA. This study showed that eggshells and struvite usage, along with CH3COOH, are promising resources as potential sustainable precursors and dissolution media, respectively, to produce CaPs with varying morphologies

Production of Nano Hydroxyapatite and Mg-Whitlockite from Biowaste-Derived Products via Continuous Flow Hydrothermal Synthesis: A Step towards Circular Economy

Biowastes from agriculture, sewage, household wastes, and industries comprise promising resources to produce biomaterials while reducing adverse environmental effects. This study focused on utilising waste-derived materials (i.e., eggshells as a calcium source, struvite as a phosphate source, and CH3COOH as dissolution media) to produce value-added products (i.e., calcium phosphates (CaPs) derived from biomaterials) using a continuous flow hydrothermal synthesis route. The prepared materials were characterised via XRD, FEG-SEM, EDX, FTIR, and TEM analysis. Magnesium whitlockite (Mg-WH) and hydroxyapatite (HA) were produced by single-phase or biphasic CaPs by reacting struvite with either calcium nitrate tetrahydrate or an eggshell solution at 200 °C and 350 °C. Rhombohedral-shaped Mg-WH (23–720 nm) along with tube (50–290 nm diameter, 20–71 nm thickness) and/or ellipsoidal morphologies of HA (273–522 nm width) were observed at 350 °C using HNO3 or CH3COOH to prepare the eggshell and struvite solutions, and NH4OH was used as the pH buffer. The Ca/P (atomic%) ratios obtained ranged between 1.3 and 1.7, indicating the formation of Mg-WH and HA. This study showed that eggshells and struvite usage, along with CH3COOH, are promising resources as potential sustainable precursors and dissolution media, respectively, to produce CaPs with varying morphologies

Development of calcium phosphates from waste resources and fabrication of calcium phosphate-chitosan composites

Biowastes from agriculture, sewage, household wastes and industries comprise promising resources to produce biomaterials whilst reducing environmental impact. This study focused on utilising waste-derived materials (i.e., eggshells as calcium source, struvite as phosphate source and CH3COOH as dissolution media) to produce value-added products (i.e., calcium phosphate (CaP) derived biomaterials) using three different routes such as continuous flow hydrothermal synthesis, ball milling followed by sintering and flame spheroidisaion. Fabrication of composites (scaffolds and films) using commercial chitosan (Ch) and CaPs prepared using eggshells and struvite via three different methods mentioned above was also explored. The prepared materials were characterised via XRD, TGA/DSC, FEG-SEM, EDX, FTIR and TEM analysis. Nanocrystalline hydroxyapatite (HA) formation was observed by reacting eggshell solution (prepared using HNO3) and diammonium hydrogen phosphate solution whereas magnesium whitlockite (Mg-WH - the second most abundant bone mineral), and HA were produced as a single phase or biphasic CaPs by reacting struvite with either calcium nitrate tetrahydrate or eggshell solution at 200 °C and 350 °C via continuous flow hydrothermal synthesis, where waste-derived precursors solutions were prepared using either HNO3 or CH3COOH. Structural characterisation and compositional analysis confirmed the sheet-like morphology of nano HA formation (with crystallite size ranges between 29 - 81 nm) at 200 °C. However, HA tubes (32-212 nm inner diameter and 15-49 nm wall thickness) and rod morphologies (32 – 86 nm) were observed at 350 °C using NH4OH and NaOH as pH buffer, respectively. Rhombohedral-shaped Mg-WH (23 – 720 nm) along with tube (50 – 290 nm in diameter with 20 – 71 nm wall thickness) and/or elliptical morphologies of HA (273 – 522 nm in width) were observed at 350 °C when using both dissolution media (HNO3 and CH3COOH) to prepare the eggshell and struvite solutions where NH4OH was used as the pH buffer. The Ca/P (atomic%) ratio of the samples produced ranged between 1.3 and 1.7, indicating formation of non-stoichiometric HA and Mg-WH. A single phase HA along with brucite [Mg(OH)2] was formed by simple ball milling of calcined eggshell powder and struvite , while biphasic CaPs (i.e. HA and Mg-WH) alongside MgO was produced at higher sintering temperatures (i.e. 900 and 1150 oC) using either raw or calcined eggshells and struvite. The ranges of crystallite size of HA and Mg-WH were found to be 30.5 – 80.6 and 28.45 – 82.5 nm, respectively. The CaPs microspheres were manufactured successfully from ball-milled raw/calcined eggshell-struvite of two granules size ranges (63 – 100 and 100 – 160 µm) via flame spheroidisation process. Single-phase of α-TCP was formed for the ball milled raw eggshell -struvite samples (smaller size granules), whereas biphasic CaPs (i.e. α-TCP and HA) was formed when calcined eggshell was used with struvite for ball milling. The Ca/P ratio of microspheres were found to be within the range of 1.5 – 1.65. CaP-Ch composites (i.e. scaffolds and film) were fabricated successfully via a freeze drying and casting method. Resonance acoustic mixing (RAM), a newly developed mixing method, was used to prepare a homogeneous composite suspension prior to freeze drying. Crosslinking of the scaffolds was also performed by using genipin, a naturally-derived crosslinker. Morphological, structural, compositional, swelling properties, mass loss and mechanical properties were evaluated for the composites. Interconnected porous structure was observed for all the scaffolds. However, the reduction in porous structure and mass loss and no significant change in mechanical properties for the cross-linked scaffolds compared to bare scaffolds was observed. This was due to the partial crosslinking of scaffolds by genipin, confirmed via FTIR. Films of the same formulations (except using CaPs derived via hydrothermal synthesis) were also fabricated using casting method and their properties were evaluated. The films containing commercial HA exhibited highly interconnected porous structure. However, a film comprising ball-milled CaPs showed a moderate range of swelling percentage (211 %) with highest mass loss (about 34 % after 96 hrs) in comparison to other formulations. This study shows that use of eggshells and struvite offer a promising resource as potential sustainable precursors, along with acetic acid as sustainable dissolution media to produce calcium phosphates with varying morphologies, microspheres and their composites with chitosan for biomedical applications

Development of calcium phosphates from waste resources and fabrication of calcium phosphate-chitosan composites

Biowastes from agriculture, sewage, household wastes and industries comprise promising resources to produce biomaterials whilst reducing environmental impact. This study focused on utilising waste-derived materials (i.e., eggshells as calcium source, struvite as phosphate source and CH3COOH as dissolution media) to produce value-added products (i.e., calcium phosphate (CaP) derived biomaterials) using three different routes such as continuous flow hydrothermal synthesis, ball milling followed by sintering and flame spheroidisaion. Fabrication of composites (scaffolds and films) using commercial chitosan (Ch) and CaPs prepared using eggshells and struvite via three different methods mentioned above was also explored. The prepared materials were characterised via XRD, TGA/DSC, FEG-SEM, EDX, FTIR and TEM analysis. Nanocrystalline hydroxyapatite (HA) formation was observed by reacting eggshell solution (prepared using HNO3) and diammonium hydrogen phosphate solution whereas magnesium whitlockite (Mg-WH - the second most abundant bone mineral), and HA were produced as a single phase or biphasic CaPs by reacting struvite with either calcium nitrate tetrahydrate or eggshell solution at 200 °C and 350 °C via continuous flow hydrothermal synthesis, where waste-derived precursors solutions were prepared using either HNO3 or CH3COOH. Structural characterisation and compositional analysis confirmed the sheet-like morphology of nano HA formation (with crystallite size ranges between 29 - 81 nm) at 200 °C. However, HA tubes (32-212 nm inner diameter and 15-49 nm wall thickness) and rod morphologies (32 – 86 nm) were observed at 350 °C using NH4OH and NaOH as pH buffer, respectively. Rhombohedral-shaped Mg-WH (23 – 720 nm) along with tube (50 – 290 nm in diameter with 20 – 71 nm wall thickness) and/or elliptical morphologies of HA (273 – 522 nm in width) were observed at 350 °C when using both dissolution media (HNO3 and CH3COOH) to prepare the eggshell and struvite solutions where NH4OH was used as the pH buffer. The Ca/P (atomic%) ratio of the samples produced ranged between 1.3 and 1.7, indicating formation of non-stoichiometric HA and Mg-WH. A single phase HA along with brucite [Mg(OH)2] was formed by simple ball milling of calcined eggshell powder and struvite , while biphasic CaPs (i.e. HA and Mg-WH) alongside MgO was produced at higher sintering temperatures (i.e. 900 and 1150 oC) using either raw or calcined eggshells and struvite. The ranges of crystallite size of HA and Mg-WH were found to be 30.5 – 80.6 and 28.45 – 82.5 nm, respectively. The CaPs microspheres were manufactured successfully from ball-milled raw/calcined eggshell-struvite of two granules size ranges (63 – 100 and 100 – 160 µm) via flame spheroidisation process. Single-phase of α-TCP was formed for the ball milled raw eggshell -struvite samples (smaller size granules), whereas biphasic CaPs (i.e. α-TCP and HA) was formed when calcined eggshell was used with struvite for ball milling. The Ca/P ratio of microspheres were found to be within the range of 1.5 – 1.65. CaP-Ch composites (i.e. scaffolds and film) were fabricated successfully via a freeze drying and casting method. Resonance acoustic mixing (RAM), a newly developed mixing method, was used to prepare a homogeneous composite suspension prior to freeze drying. Crosslinking of the scaffolds was also performed by using genipin, a naturally-derived crosslinker. Morphological, structural, compositional, swelling properties, mass loss and mechanical properties were evaluated for the composites. Interconnected porous structure was observed for all the scaffolds. However, the reduction in porous structure and mass loss and no significant change in mechanical properties for the cross-linked scaffolds compared to bare scaffolds was observed. This was due to the partial crosslinking of scaffolds by genipin, confirmed via FTIR. Films of the same formulations (except using CaPs derived via hydrothermal synthesis) were also fabricated using casting method and their properties were evaluated. The films containing commercial HA exhibited highly interconnected porous structure. However, a film comprising ball-milled CaPs showed a moderate range of swelling percentage (211 %) with highest mass loss (about 34 % after 96 hrs) in comparison to other formulations. This study shows that use of eggshells and struvite offer a promising resource as potential sustainable precursors, along with acetic acid as sustainable dissolution media to produce calcium phosphates with varying morphologies, microspheres and their composites with chitosan for biomedical applications

Evolution of silicate bioglass particles as porous microspheres with a view towards orthobiologics

Although FDA approved and clinically utilised, research on 45S5 Bioglass® and S53P4 including other bioactive glasses continues in order to advance their applicability for a range of alternate applications. For example, rendering these particles porous would enable incorporation of varying biological payloads (i.e. cells, drugs and growth factors) and making them spherical would enhance their flow properties enabling delivery to target sites via minimally invasive injection procedures. This paper reports on the manufacture of solid (non-porous; SGMS) and highly porous microspheres (PGMS) with large external pores and fully interconnected porosity from bioactive silicate glass formulations (45S5 and S53P4) via a single stage flame spheroidisation process and their physicochemical properties including in vitro biological response. Morphological and physical characterisation of the SGMS and PGMS revealed interconnected porosity up to 65 ± 5%. Mass loss studies comparing between SGMS and PGMS revealed 1.5 times higher mass loss for the PGMS over 28 days. Also, in vitro bioactivity studies using simulated body fluid (SBF) revealed hydroxyapatite (HA) formation at earlier time point for PGMS compared to their SGMS counterparts (i.e day 1 for PGMS and day 3 for SGMS of 45S5). In addition, HA layers were also formed in cell culture media, with the exception of SGMS of 45S5, which revealed CaP formation with a ratio of 1.52–1.78. Direct cell seeding and indirect cell culture studies (via incubation with microsphere degradation products) revealed mouse 3T3 cells were able to grow and undergo osteogenic differentiation in vitro, confirming cytocompatibility of both 45S5 and S53P4 SGMS and PGMS. More importantly and especially for orthobiologic applications, cells were observed to have migrated within the pores of the PGMS. As such, the PGMS developed from these bioactive silicate glasses are highly promising candidate materials for orthobiologics and alternate applications requiring delivery of biologic payloads