Biomimetic Composite Scaffold With Phosphoserine Signaling for Bone Tissue Engineering Application

Abstract

In guided bone tissue engineering, successful ingrowth of MSCs depends primarily on the nature of the scaffold. It is well-known that only seconds after implantation, biomaterials are coated by a layer of adsorbed proteins/peptides which modulates the subsequent cell/scaffold interactions, especially at early times after implantation. In this work, nanohydroxyapatite and collagen based composite materials (Coll/nanoHA) were modified with phosphorylated amino acid (O-phospho-L-serine–OPS) to mimic bone tissue, and induce cell differentiation. The choice for this phosphorylated amino acid is due to the fact that osteopontin is a serine-rich glycol-phosphoprotein and has been associated to the early stages of bone formation, and regeneration. Several concentrations of OPS were added to the Coll/nanoHA scaffold and physico-chemical, mechanical, and in vitro cell behavior were evaluated. Afterwards, the composite scaffold with stronger mechanical and best cellular behavior was tested in vivo, with or without previous in vitro culture of human MSC's (bone tissue engineering). The OPS signaling of the biocomposite scaffolds showed similar cellular adhesion and proliferation, but higher ALP enzyme activity (HBMSC). In vivo bone ectopic formation studies allowed for a thorough evaluation of the materials for MSC's osteogenic differentiation. The OPS-scaffolds results showed that the material could modulated mesenchymal cells behavior in favor of osteogenic differentiation into late osteoblasts that gave raised to their ECM with human bone proteins (osteopontin) and calcium deposits. Finally, OPS-modified scaffolds enhanced cell survival, engraftment, migration, and spatial distribution within the 3D matrix that could be used as a cell-loaded scaffold for tissue engineering applications and accelerate bone regeneration processes.

Clarifying the Tooth-Derived Stem Cells Behavior in a 3D Biomimetic Scaffold for Bone Tissue Engineering Applications

“…These scaffolds under osteoinduction medium were characterized by DNA concentration, ALP activity and osteogenic gene expression to compare with the results for basic medium, although the results showed no statistically significant difference between the culture mediums ( Supplementary Figure 6 ). In a previous work ( Salgado et al, 2019 ) and according to the literature ( Scotti et al, 2013 ; Stüdle et al, 2018 ), the in vivo experiment was performed with 3D cultures in the presence of the osteoinductive medium. Dynamic culture conditions were chosen, as they enhanced the dental follicle MSCs migration into the inner part of the scaffold, favoring mass transfer and oxygenation, avoiding the hypoxia-necrosis effect of in vitro engineered tissues.…”

“…This type of scaffolds has shown osteoconductive properties to MSCs which expressed osteoblast-like gene markers ( McCafferty et al, 2014 ). Also, recent studies showed that nanohydroxyapatite integrated in scaffolds was capable to recruit bone marrow MSCs and promote their osteogenic differentiation ( Rodrigues et al, 2013 ; Salgado et al, 2016 , 2019 ). In this work, the results illustrated the ability of dental MSCs cultured in biocomposite cryogels to differentiate into osteoblastic cells, with high ALP enzyme activity and bone phenotype gene expression.…”

“…Even in intra-bone critical defects, implants did not show the formation of mature and organized trabecular bone structure after the 8 weeks of implantation. Usually, in ectopic bone formation models, the bone tissue growth should be observed after 12 weeks ( Scotti et al, 2013 ), but in a previous work, evidence of mineralized tissue was observed in a study after only 4 weeks post-implantation, suggesting that the employed scaffold had an osteoinductive effect and promoted bone tissue ingrowth ( Salgado et al, 2019 ). To track the transplanted cells, immunostaining for human nucleus was performed, allowing to observe a few human dental MSCs at the periphery of the scaffold, which showed some migration capacity toward the animal surrounding tissue ( Figures 8F,L ).…”

“…Collagen, Nanohydroxyapatite and Phosphoserine (Coll-nanoHA/OPS) by a Cryogelation Method Cryogels were produced as previously described (Rodrigues et al, 2013;Salgado et al, 2016Salgado et al, , 2019. Briefly, bovine collagen Type I (Sigma-Aldrich, Germany) was homogenized (Ultra Turrax T25, IKA) at 10000 rpm, in 5 mM HCl (36.5-38% grade, Sigma-Aldrich, Germany) at a concentration of 2% (w/v).…”

“…These composite materials could have surface modifications in order to improve cell behavior, favor mesenchymal stem cells adhesion and control cell differentiation into the desired cell types, allowing the regeneration of the host tissue/organ. Phosphoserine (O-phospho-L-serine) is a phosphorylated amino-acid (OPS) and shall be able to mimic typical osteopontin functionalities, such as the regulation of cell response, such as mitosis (proliferation), signaling, differentiation ( D’Ambrosio et al, 2007 ), and also provide osteoconductive properties ( Salgado et al, 2019 ).…”