Enhanced osteogenic proliferation and differentiation of human adipose-derived stem cells on a porous n-HA/PGS-M composite scaffold

Abstract

This study explored the applicability, cellular efficacy, and osteogenic activities of porous nano-hydroxyapatite/Poly (glycerol sebacate)-grafted maleic anhydride (n-HA/PGS-g-M) composite scaffolds. Nuclear magnetic resonance (NMR) analyses indicated that approximately 43% of the hydroxide radicals in PGS were displaced by maleic anhydride. Resonance bands at 1036 cm −1 occurred in scaffolds containing nHA powders, and peak areas increased when n-HA weight increased in PGS-M-n-HA-0.4, PGS-M-n-HA-0.5, and PGS-M-n-HA-0.6 scaffolds. The n-HA/PGS-g-M composite scaffolds exhibited porous microstructure with average pore size of 150–300 µm in scanning electron microscopy (SEM) analysis. Differential scanning calorimetry (DSC) identified the glass transition temperature (Tg) as −25–30 °C, indicative of quality resilience. The modulus of compressibility increased when n-HA content increased. Interestingly, viability of human adipose-derived stem cells (hADSCs) in vitro and expression of the osteogenic related genes RUNX2 , OCN , and COL1A1 was enhanced in the n-HA/PGS-g-M composite scaffolds compared to those factors observed in PGS-g-M scaffolds. Finally, simulated body fluid (SBF) tests indicated more apatite deposits on the surface of n-HA/PGS-g-M scaffolds compared to PGS-g-M scaffolds. Overall, porous n-HA/PGS-g-M composite scaffolds possessed acceptable biocompatibility and mechanical properties, and they stimulated hADSC cell proliferation and differentiation. Given these qualities, the composite scaffolds have potential applications in bone tissue engineering.

Translating Material Science into Bone Regenerative Medicine Applications: State-of-The Art Methods and Protocols

“…Fluorescent live/dead assays are able to discriminate between live and dead cells by evaluating plasma membrane integrity and the activity of the esterase enzyme, both maintained only in viable cells [ 54 , 55 , 60 , 62 , 70 , 74 , 75 , 77 , 78 , 79 , 80 , 81 , 82 , 83 , 84 ]. Other commercial colorimetric assays for the evaluation of cell viability are available on the market and, among the most widely used are the cell counting kit-8 (CCK-8) assay that allows a sensitive colorimetric measure of viable cells, using a water-soluble tetrazolium salt that produces, in presence of active dehydrogenases in living cells, an orange formazan product, and the amount of formazan produced is directly proportional to the number of viable cells [ 55 , 57 , 58 , 62 , 73 , 76 , 80 , 81 , 85 , 86 , 87 , 88 , 89 ]. Moreover, the Alamar Blue assay method is frequently used to assess the metabolic activity of proliferating cells: the active resazurin compound, upon entering living cells, is reduced to resorufin, a red fluorescent molecule that can be quantified [ 14 , 59 , 64 , 72 , 75 , 90 , 91 ].…”

“…MSCs isolated from bone marrow are regarded as the gold standard [ 14 , 53 , 55 , 57 , 67 , 68 , 73 , 76 , 77 , 79 , 83 , 91 , 94 , 98 , 107 , 108 , 109 ]. MSC-like cells isolated from the stromal vascular fraction of the adipose tissue, namely, adipose-derived stem cells, ASC, are seldom used in selected applications [ 2 , 65 , 72 , 82 , 89 , 104 , 109 , 110 , 111 , 112 ]. MSCs isolated from the dental pulp also find a wide range of applications, but the application of this cell type is beyond the scope of this review.…”

“…In the vast majority of papers in the literature, the expression levels of one or more genes associated with osteoblast differentiation are usually evaluated by quantitative real-time PCR (qRT-PCR) and/or Western blot and/or immunofluorescence analysis and/or ELISA assay [ 14 , 55 , 57 , 58 , 60 , 62 , 64 , 65 , 68 , 69 , 70 , 72 , 73 , 76 , 77 , 79 , 80 , 82 , 83 , 84 , 86 , 87 , 89 , 90 , 91 , 94 , 96 , 98 , 100 , 108 , 109 , 113 ]. Table 3 reports the most representative osteogenic genes usually analyzed.…”

“…Given that hydroxyapatite is like the mineral phase of bone tissue (bone-like apatite) that is key for enabling the attachment, growth, and proliferation of precursor cells on scaffolds, this test provides an easy-to-use and low cost tool to qualitatively estimate the bone-bonding and potential mineralization abilities of a scaffold [ 117 ]. The superficial film developed on the biomaterial’s surface is usually evaluated with SEM [ 62 , 77 , 79 , 83 , 89 , 91 , 95 , 97 , 103 , 118 , 119 , 120 , 121 , 122 ]. This method can also be used to assess the biomaterial biodegradation: the scaffold is immersed in a SBF solution and the scaffold weight loss can be measured [ 91 , 118 ].…”

Biocompatibility of Polylactic Acid as a Bone Substitute: An In Vitro Study

“…According to previous studies, the polymers used exhibit low elasticity and bone resorption. (Wang et al, 2019).…”