In Vitro Osteoinductivity Assay of Hydroxylapatite Scaffolds, Obtained with Biomorphic Transformation Processes, Assessed Using Human Adipose Stem Cell Cultures

Abstract

In this study, the in vitro biocompatibility and osteoinductive ability of a recently developed biomorphic hydroxylapatite ceramic scaffold (B-HA) derived from transformation of wood structures were analyzed using human adipose stem cells (hASCs). Cell viability and metabolic activity were evaluated in hASCs, parental cells and in recombinant genetically engineered hASC-eGFP cells expressing the green fluorescence protein. B-HA osteoinductivity properties, such as differentially expressed genes (DEG) involved in the skeletal development pathway, osteocalcin (OCN) protein expression and mineral matrix deposition in hASCs, were evaluated. In vitro induction of osteoblastic genes, such as Alkaline phosphatase (ALPL), Bone gamma-carboxyglutamate (gla) protein (BGLAP), SMAD family member 3 (SMAD3), Sp7 transcription factor (SP7) and Transforming growth factor, beta 3 (TGFB3) and Tumor necrosis factor (ligand) superfamily, member 11 (TNFSF11)/Receptor activator of NF-κB (RANK) ligand (RANKL), involved in osteoclast differentiation, was undertaken in cells grown on B-HA. Chondrogenic transcription factor SRY (sex determining region Y)-box 9 (SOX9), tested up-regulated in hASCs grown on the B-HA scaffold. Gene expression enhancement in the skeletal development pathway was detected in hASCs using B-HA compared to sintered hydroxylapatite (S-HA). OCN protein expression and calcium deposition were increased in hASCs grown on B-HA in comparison with the control. This study demonstrates the biocompatibility of the novel biomorphic B-HA scaffold and its potential use in osteogenic differentiation for hASCs. Our data highlight the relevance of B-HA for bone regeneration purposes.

Evaluation of Human Bone Marrow Mesenchymal Stromal Cell (MSC) Functions on a Biomorphic Rattan-Wood-Derived Scaffold: A Comparison between Cultured and Uncultured MSCs

“…In contrast, the current study focused on patient-derived uncultured BMSCs, as these cells most closely reflect the bone marrow in vivo. Furthermore, their initial work used dynamic seeding, and their most recent work explored the gene expression of osteogenic transcripts in osteogenic media [ 36 ]. In contrast, the principal aim of this work was to investigate the feasibility of loading the B-HA scaffolds with unprocessed BM aspirates, as happens in the operating theatre, and to study the attachment, survival and gene expression of BMSCs on B-HA in comparison to that of cMSCs.…”

Stem Cell Fate and Immunomodulation Promote Bone Regeneration via Composite Bio-Oss®/AviteneTM Biomaterial

“…Primary hASC cultures were maintained 1) on the Bio-Oss ® /Avitene biomaterial and 2) in tissue culture polystyrene (TCPS) vessels (24-well plates, Ø 10 mm), as control ( Mazzoni et al, 2019 ), at a density of 5,000 cells/well. Osteogenic conditions (OC) were obtained utilizing differentiation Bullekit osteogenic medium (Lonza, Milan, Italy), which included osteogenic basal medium (Lonza, Milan, Italy) and osteogenic SigleQuotes, which included dexamethasone, ascorbate, mesenchymal cell growth supplement, l -glutamine, and -glycerophosphate (Lonza, Milan, Italy) ( Iaquinta et al, 2021b ). The scaffolds were separately arranged in 24-well plates (Ø, 10 mm) to cover the surface area.…”

“…The AlamarBlue assay (ThermoFisher Scientific, Milan, Italy) was used to assess the proliferation rate of hASCs grown on the scaffolds ( Iaquinta et al, 2021b ). As previously reported, the experiment was done to determine the metabolic activity of hASCs when attached (day 0) and cultured on Bio-Oss ® /Avitene biomaterial and TCPS at day 3, 6, and 9 ( Iaquinta et al, 2021b ).…”

“…Bio-Oss ® plays a decisive role in controlling bone regeneration ( Gong et al, 2018 ; Lee et al, 2019 ; Kosinski et al, 2020 ). Our previous studies showed that hASCs are an excellent in vitro cellular model to assay the proprieties of scaffolds ( Mazzoni et al, 2017 , Mazzoni et al, 2019 ; Iaquinta et al, 2021b ). For this reason, in our work hASCs were grown on a Bio-Oss ® /Avitene microfibrillar Collagen scaffold in order to verify how the bone biomaterial can modulate osteoinductivity and immune response.…”

Genetics and Epigenetics of Bone Remodeling and Metabolic Bone Diseases

“…Bone matrix is considered to form the intercellular substance of bone tissue and the inorganic phase. The extracellular matrix (ECM) consists primarily of type I collagen (COL1), which is the most abundant protein in bone tissue, complexed with a crystalline inorganic component composed of hydroxylapatite (HA; Ca 10 (PO 4 ) 6 (OH) 2 ) with citrate, carbonate, and ions, such as F − , K + , Sr 2+ , Pb 2+ , Zn 2+ , Cu 2+ , Mg 2+ , and Fe 2+ [15][16][17]. The mechanical quality of the bone matrix is influenced by non-collagenous proteins [18].…”