Manipulation of miRNA activity accelerates osteogenic differentiation of hMSCs in engineered 3D scaffolds.
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ABSTRACT:
SUBMITTER: Mariner PD
PROVIDER: S-EPMC3184319 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Human platelet lysate (hPL) has been considered as the preferred supplement for the xeno-free stem cell culture for many years. However, the biological effect of hPL on the proliferation and differentiation of dental stem cells combined with the use of medical grade synthetic biomaterial is still under investigation. Thus, the optimal scaffold composition, cell type and specific growth conditions, yet need to be formulated. In this study, we aimed to investigate the regenerative potential of dental stem cells seeded on synthetic scaffolds and maintained in osteogenic media supplemented with either hPL or xeno-derived fetal bovine serum (FBS). Two types of dental stem cells were isolated from human impacted third molars and intact teeth; stem cells of apical papilla (SCAP) and periodontal ligament stem cells (PDLSCs). Cells were expanded in cell culture media supplemented with either hPL or FBS. Consequently, proliferative capacity, immunophenotypic characteristics and multilineage differentiation potential of the derived cells were evaluated on monolayer culture (2D) and on synthetic scaffolds fabricated from poly 'lactic-co-glycolic' acid (PLGA) (3D). The functionality of the induced cells was examined by measuring the concentration of osteogenic markers ALP, OCN and OPN at different time points. Our results indicate that the isolated dental stem cells showed similar mesenchymal characteristics when cultured on hPL or FBS-containing culture media. Scanning electron microscopy (SEM) and H&E staining revealed the proper adherence of the derived cells on the 3D scaffold cultures. Moreover, the increase in the concentration of osteogenic markers proved that hPL was able to produce functional osteoblasts in both culture conditions (2D and 3D), in a way similar to FBS culture. These results reveal that hPL provides a suitable substitute to the animal-derived serum, for the growth and functionality of both SCAP and PDLSCs. Thus the use of hPL, in combination with PLGA scaffolds, can be useful in future clinical trials for dental regeneration.
| S-EPMC6504042 | biostudies-literature
Project description:BackgroundThe poor regenerative capability and structural complexity make the reconstruction of meniscus particularly challenging in clinic. 3D printing of polymer scaffolds holds the promise of precisely constructing complex tissue architecture, however the resultant scaffolds usually lack of sufficient bioactivity to effectively generate new tissue.ResultsHerein, 3D printing-based strategy via the cryo-printing technology was employed to fabricate customized polyurethane (PU) porous scaffolds that mimic native meniscus. In order to enhance scaffold bioactivity for human mesenchymal stem cells (hMSCs) culture, scaffold surface modification through the physical absorption of collagen I and fibronectin (FN) were investigated by cell live/dead staining and cell viability assays. The results indicated that coating with fibronectin outperformed coating with collagen I in promoting multiple-aspect stem cell functions, and fibronectin favors long-term culture required for chondrogenesis on scaffolds. In situ chondrogenic differentiation of hMSCs resulted in a time-dependent upregulation of SOX9 and extracellular matrix (ECM) assessed by qRT-PCR analysis, and enhanced deposition of collagen II and aggrecan confirmed by immunostaining and western blot analysis. Gene expression data also revealed 3D porous scaffolds coupled with surface functionalization greatly facilitated chondrogenesis of hMSCs. In addition, the subcutaneous implantation of 3D porous PU scaffolds on SD rats did not induce local inflammation and integrated well with surrounding tissues, suggesting good in vivo biocompatibility.ConclusionsOverall, this study presents an approach to fabricate biocompatible meniscus constructs that not only recapitulate the architecture and mechanical property of native meniscus, but also have desired bioactivity for hMSCs culture and cartilage regeneration. The generated 3D meniscus-mimicking scaffolds incorporated with hMSCs offer great promise in tissue engineering strategies for meniscus regeneration.
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Project description:Biomineralized collagen with intrafibrillar calcium phosphate mineral provides an excellent mimic of the composition and structure of the extracellular matrix of bone, from nano- to micro-scale. Scaffolds prepared from this material have the potential to become the next-generation of synthetic bone graft substitutes, as their unique properties make them closer to the native tissue than synthetic alternatives currently available to clinicians. To understand the interaction between biomineralized collagen and cells that are relevant in the context of bone regeneration, we studied the growth and osteogenic differentiation of bone marrow derived human mesenchymal stromal cells (hMSCs) cultured on biomineralized collagen membranes, and compared it to the cell behavior on collagen membranes without mineral. Cells proliferated normally on both biomimetic membranes, and were more triggered to differentiate toward the osteogenic lineage by the biomineralized collagen. This was shown by the elevated mRNA levels of RUNX2, SPP1, ENPP1, and OCN after 3 days of culture, and COL1A1 after 14 days of culture on mineralized collagen. The mRNA levels of the tested markers of osteogenesis were lower on collagen membranes without mineral, with the exception of OCN, which was more highly expressed on collagen than on biomineralized collagen membranes. Expression by hMSCs of OPG, a gene involved in inhibition of osteoclastogenesis, was higher on biomineralized collagen at day 3, while M-CSF, involved in osteoblast-osteoclast communication, was upregulated on both membranes at day 3 and 14 of culture. Alkaline phosphatase activity of hMSCs was high on both biomimetic membranes when compared with cells cultured on tissue culture plastic. Cell-induced mineralization was observed on collagen membranes, while the high mineral content of the biomineralized membranes prohibited a reliable analysis of cell-induced mineralization on these membranes. In conclusion, we have identified that both collagen and biomineralized collagen support proliferation, osteogenic differentiation and mineralization of hMSCs, with biomineralized membranes having a more pronounced positive effect. These findings support the existing evidence that biomineralized collagen is a promising material in the field of bone regeneration.
| S-EPMC7644787 | biostudies-literature
Project description:With advances in bone tissue engineering, various materials and methods have been explored to find a better scaffold that can help in improving bone growth and regeneration. Three-dimensional (3D) printing by fused deposition modeling can produce customized scaffolds from biodegradable polyesters such as polycaprolactone (PCL). Although the fabricated PCL scaffolds exhibited a lack of bioactivity and poor cell attachment on their surfaces, herein, using a simple postfabrication modification method with hydroxyapatite (HA) and bioglasses (BGs), we obtained better cell proliferation and attachment. Biological behavior and osteosupportive capacity of the 3D-printed scaffolds including PCL, PCL/HA, PCL/BG, and PCL/HA/BG were evaluated in this study, while human adipose tissue-derived mesenchymal stem cells (hADSCs) were cultured on the scaffolds. The cell morphology, attachment, and proliferation were investigated using scanning electron microscopy (SEM), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay, and 4',6-diamidino-2-phenylindole (DAPI) staining. In the next step, the ability of stem cells to differentiate into osteoblasts was evaluated by measuring alkaline phosphatase (ALP) activity, calcium deposition, and bone-related gene and protein expression. In the end, the expression levels of miR-20a, miR-125a, and their target genes were also investigated as positive and negative regulators in osteogenesis pathways. The results showed that the coated scaffolds with bioceramics present a more appropriate surface for cell adhesion and proliferation, as well as efficient potential in inducing osteoconduction and osteointegration compared to PCL alone and control. The PCL/HA/BG scaffold exhibited higher in vitro cell viability and bone formation compared to the other groups, which can be due to the synergistic effect of HA and BG. On the whole, this tricomponent 3D-printing scaffold has a promising prospect for bone tissue engineering applications.
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Project description:The morphological clues of scaffolds can determine cell behavior and, therefore, the patterning of electroactive polymers can be a suitable strategy for bone tissue engineering. In this way, this work reports on the influence of poly(vinylidene fluoride-co-trifluoroethylene) (P(VDF-TrFE)) electroactive micropatterned scaffolds on the proliferation and differentiation of bone cells. For that, micropatterned P(VDF-TrFE) scaffolds were produced by lithography in the form of arrays of lines and hexagons and then tested for cell proliferation and differentiation of pre-osteoblast cell line. Results show that more anisotropic surface microstructures promote bone differentiation without the need of further biochemical stimulation. Thus, the combination of specific patterns with the inherent electroactivity of materials provides a promising platform for bone regeneration.
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Project description:Bone defect is a noteworthy health problem and is the second most transplanted tissue after blood. Numerous bone grafts are designed and applied in clinics. Limitations, however, from different aspects still exist, including limited supply, mechanical strength, and bioactivity. In this study, two biomimetic peptides (P2 and P6) are incorporated into a composite bioactive xeno hybrid bone graft named SmartBonePep®, with the aim to increase the bioactivity of the bone graft. The results, which include cytotoxicity, proliferation rate, confocal microscopy, gene expression, and protein qualification, successfully prove that the SmartBonePep® has multi-modal biological effects on human mesenchymal stem cells from bone marrow. The effective physical entrapment of P6 into a composite xeno-hybrid bone graft, withstanding manufacturing processes including exposure to strong organic solvents and ethylene oxide sterilization, increases the osteogenic potential of the stem cells as well as cell attachment and proliferation. P2 and P6 both show a strong biological potential and may be future candidates for enhancing the clinical performance of bone grafts.
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Project description:The apparent rise of bone disorders demands advanced treatment protocols involving tissue engineering. Here, we describe self-assembling tetrapeptide scaffolds for the growth and osteogenic differentiation of human mesenchymal stem cells (hMSCs). The rationally designed peptides are synthetic amphiphilic self-assembling peptides composed of four amino acids that are nontoxic. These tetrapeptides can quickly solidify to nanofibrous hydrogels that resemble the extracellular matrix and provide a three-dimensional (3D) environment for cells with suitable mechanical properties. Furthermore, we can easily tune the stiffness of these peptide hydrogels by just increasing the peptide concentration, thus providing a wide range of peptide hydrogels with different stiffnesses for 3D cell culture applications. Since successful bone regeneration requires both osteogenesis and vascularization, our scaffold was found to be able to promote angiogenesis of human umbilical vein endothelial cells (HUVECs) in vitro. The results presented suggest that ultrashort peptide hydrogels are promising candidates for applications in bone tissue engineering.
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Project description:Polyetheretherketone (PEEK) titanium composite (PTC) is a novel interbody fusion device that combines a PEEK core with titanium alloy (Ti6Al4V) endplates. The present study aimed to investigate the in vitro biological reactivity of human bone-marrow-derived mesenchymal stem cells (hBM-MSCs) to micro- and nanotopographies produced by an acid-etching process on the surface of 3D-printed PTC endplates. Optical profilometer and scanning electron microscopy were used to assess the surface roughness and identify the nano-features of etched or unetched PTC endplates, respectively. The viability, morphology and the expression of specific osteogenic markers were examined after 7 days of culture in the seeded cells. Haralick texture analysis was carried out on the unseeded endplates to correlate surface texture features to the biological data. The acid-etching process modified the surface roughness of the 3D-printed PTC endplates, creating micro- and nano-scale structures that significantly contributed to sustaining the viability of hBM-MSCs and triggering the expression of early osteogenic markers, such as alkaline phosphatase activity and bone-ECM protein production. Finally, the topography of 3D-printed PTC endplates influenced Haralick's features, which in turn correlated with the expression of two osteogenic markers, osteopontin and osteocalcin. Overall, these data demonstrate that the acid-etching process of PTC endplates created a favourable environment for osteogenic differentiation of hBM-MSCs and may potentially have clinical benefit.
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Project description:3D-printing technology can be used to construct personalized bone substitutes with customized shapes, but it cannot regulate the topological morphology of the scaffold surface, which plays a vital role in regulating the biological behaviors of stem cells. In addition, stem cells are able to sense the topographical and mechanical cues of surface of scaffolds by mechanosensing and mechanotransduction. In our study, we fabricated a 3D-printed poly(ε-caprolactone) (PCL) scaffold with a nanotopographical surface and loaded it with urine-derived stem cells (USCs) for application of bone regeneration. The topological 3D-printed PCL scaffolds (TPS) fabricated by surface epiphytic crystallization, possessed uniformly patterned nanoridges, of which the element composition and functional groups of nanoridges were the same as PCL. Compared with bare 3D-printed PCL scaffolds (BPS), TPS have a higher ability for protein adsorption and mineralization in vitro. The proliferation, cell length, and osteogenic gene expression of USCs on the surface of TPS were significantly higher than that of BPS. In addition, the TPS loaded with USCs exhibited a good ability for bone regeneration in cranial bone defects. Our study demonstrated that nanotopographical 3D-printed scaffolds loaded with USCs are a safe and effective therapeutic strategy for bone regeneration.
| S-EPMC9317219 | biostudies-literature
Project description:Exposure to Pulsed Electromagnetic Field (PEMF) has been shown to affect proliferation and differentiation of human mesenchymal stem cells derived from bone marrow stroma (BM-hMSC). These cells offer considerable promise in the field of regenerative medicine, but their clinical application is hampered by major limitations such as poor availability and the time required to differentiate up to a stage suitable for implantation. For this reason, several research efforts are focusing on identifying strategies to speed up the differentiation process. In this work we investigated the in vitro effect of PEMF on Ca(2+)-related mechanisms promoting the osteogenic differentiation of BM-hMSC. Cells were daily exposed to PEMF while subjected to osteogenic differentiation and various Ca(2+)-related mechanisms were monitored using multiple approaches for identifying functional and structural modifications related to this process. The results indicate that PEMF exposure promotes chemically induced osteogenesis by mechanisms that mainly interfere with some of the calcium-related osteogenic pathways, such as permeation and regulation of cytosolic concentration, leaving others, such as extracellular deposition, unaffected. The PEMF effect is primarily associated to early enhancement of intracellular calcium concentration, which is proposed here as a reliable hallmark of the osteogenic developmental stage.
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