Regular ArticleA Chemically Defined Medium Supports in Vitro Proliferation and Maintains the Osteochondral Potential of Rat Marrow-Derived Mesenchymal Stem Cells
Abstract
Among the stromal elements in mammalian and avian bone marrow there exists a pluripotent subset of cells which we refer to as mesenchymal stem cells (MSCs). These cells can be isolated and will proliferate in culture. When such subcultured cells are introduced into porous tricalcium phosphate-hydroxyapatite ceramic cubes and implanted subcutaneously into syngeneic or immunocompromised hosts, the passaged MSCs are observed to differentiate into bone and cartilage. Heretofore, those assays have been conducted with MSCs which had been maintained in vitro in serum-containing medium. A serum-free medium (RDM-F), which consists of insulin, 5 μg/ml, linoleic acid-bovine serum albumin, 0.1%, platelet-derived growth factor-BB, 10 ng/ml, and basic fibroblast growth factor, 1 ng/ml in a base medium of 60% Dulbecco's modified Eagle's medium with low glucose and 40% MCDB-201, has been developed for rat marrow-derived MSCs. Proliferation rates of MSCs maintained in RDM-F equal those of cells maintained in serum-containing medium through Day 4 following subculturing and continue at up to 80% of the rate of the latter through Day 8 of subculture. When tested in the in vivo ceramic cube assay, MSCs cultured in RDM-F retain their osteochondral potential and differentiate into bone and cartilage in a manner indistinguishable from those cultivated in serum-containing medium. Utilization of this serum-free medium will facilitate analysis of the effects of other growth factors and cytokines on the proliferation and differentiation of MSCs, without the complexity of exogenous serum.
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The biology of mesenchymal stem/stromal cells in the treatment of osteoarthritis
2022, Journal of Cartilage and Joint PreservationOsteoarthritis affects the whole joint and is usually treated using pain relief for many years followed by arthroplasty. Mesenchymal stem/stromal cells have the potential to form cartilage and bone and have been investigated for their capacity to repair these tissues, but until recently there has been no strong rationale for their use in the treatment of age-related, idiopathic osteoarthritis.
The aim of this review is to explore the origins of cell therapy for joint diseases and how the early work in cartilage repair has built toward the possibility of an injectable mesenchymal cell approach for osteoarthritis.
A broad selection of publications has been identified relating to cartilage repair, mesenchymal cell biology, meniscal cartilage repair, and osteoarthritis therapeutics. Primary studies as well as several systematic reviews and meta-analyses have been included.
Cell therapies for cartilage lesions have been shown to be successful for traumatic injury but will be difficult to adapt for the treatment of idiopathic osteoarthritis. However the biological understanding of mesenchymal cells as a reservoir for trophic factors has led to their use as an injectable therapy. These studies have provided good evidence that sustained pain reduction can be achieved by injecting mesenchymal cells into the osteoarthritic joint, with some evidence also for functional improvement. Exosomes derived from mesenchymal may provide a scalable alternative to the cell therapy approach in future.
Mesenchymal cells have potential as a possible injectable cell therapy for idiopathic osteoarthritis and should be further explored through larger-scale, carefully designed clinical trials.
Mesenchymal stem cells are a tool in cell therapies but demand a large cell number per treatment, for that, suitable culture media is required which contains fetal bovine serum (FBS). However, for cell-based therapy applications, the use of FBS is problematic. Several alternatives to FBS have been explored, including human derivatives from platelet-rich plasma (hD-PRP). Although various studies have evaluated the impact of hD-PRP on MSC proliferation and differentiation, few of them have assessed their influence on processes, such as metabolism and gene expression. Here, we cultured human adipose-derived MSCs (hAD-MSCs) in media supplemented with either 10% hD-PRP (hD-PRP-SM) or 10% FBS (FBS-SM) in order to characterize them and evaluate the effect of hD-PRP on cell metabolism, gene expression of associated regenerative factors, as well as chromosome stability during cell expansion. We found that hAD-MSCs cultured in hD-PRP-SM have a greater cell elongation but express similar surface markers; in addition, hD-PRP-SM promoted a significant osteogenic differentiation in the absence of differentiation medium and increased the growth rate, maintaining chromosomal stability. In terms of cell metabolic profile, hAD-MSC behavior did not reveal any differences between both culture conditions. Conversely, significant differences in collagen I and angiopoietin 2 expression were observed between both conditions. The present results suggest that hD-PRP may influence hAD-MSC behavior.
Kindlin-3 mutation in mesenchymal stem cells results in enhanced chondrogenesis
2021, Experimental Cell ResearchIdentifying patient mutations driving skeletal development disorders has driven our understanding of bone development. Integrin adhesion deficiency disease is caused by a Kindlin-3 (fermitin family member 3) mutation, and its inactivation results in bleeding disorders and osteopenia. In this study, we uncover a role for Kindlin-3 in the differentiation of bone marrow mesenchymal stem cells (BMSCs) down the chondrogenic lineage. Kindlin-3 expression increased with chondrogenic differentiation, similar to RUNX2. BMSCs isolated from a Kindlin-3 deficient patient expressed chondrocyte markers, including SOX9, under basal conditions, which were further enhanced with chondrogenic differentiation. Rescue of integrin activation by a constitutively activated β3 integrin construct increased adhesion to multiple extracellular matrices and reduced SOX9 expression to basal levels. Growth plates from mice expressing a mutated Kindlin-3 with the integrin binding site ablated demonstrated alterations in chondrocyte maturation similar to that seen with the human Kindlin-3 deficient BMSCs. These findings suggest that Kindlin-3 expression mirrors RUNX2 during chondrogenesis.
Peptide decorated demineralized dentin matrix with enhanced bioactivity, osteogenic differentiation via carboxymethyl chitosan
2021, Dental MaterialsCitation Excerpt :DDM composites were prepared using FITC-conjugated carboxyl-terminated modified peptides and observed using a fluorescence microscope (Naning, China) to identify whether they were successfully grafted onto the DDM. Primary rBMSCs were cultured as described previously [28]. Briefly, the femurs and tibias of Sprague Dawley (SD) rats (male, 100−150 g, 2–4 weeks old) were exposed.
To improve the biocompatibility and osteogenic activity of demineralized dentin matrix (DDM) by grafting peptides on its surface.
DDM was obtained by pulverizing extracted human teeth that had been systematically demineralized and dried. Four groups of materials were evaluated: DDM, DDM/carboxymethyl chitosan (CMC), DDM/CMC/bone forming peptide-1 (BFP-1), and blank. X-ray photoelectron spectroscopy (XPS), Fourier transform infrared spectroscopy (FT-IR) and fluorescence localization were used to characterize the surface of the DDM materials. Cell viability was assessed using a CCK8 assay, scanning electron microscopy (SEM) and in vitro osteogenesis was analyzed using real-time RT-PCR (RT-qPCR) and Alizarin red and alkaline phosphatase staining. Three different materials were implanted into mandibular bone defects in rats. After 8 weeks, bone regeneration was assessed by histomorphometry of HE-stained slides.
FT-IR, XPS, and fluorescence microscopy demonstrated that the DDM surfaces were successfully modified with BFP-1. The CCK8 assay indicated that the proliferation of cells is higher on the DDM/CMC/BFP-1 material than on DDM or DDM/CMC (P < 0.05). Cells were more likely to adhere to DDM/CMC/BFP-1, as observed by SEM. Greater in vitro osteogenesis was observed in the DDM/CMC/BFP-1 group which displayed stronger alkaline phosphatase activity, more alizarin red-stained nodules, and higher target gene expression, as detected by RT-qPCR (P＜0.05). HE staining of in vivo explants indicated that greater quantities of new bone had formed in the DDM/CMC/BFP-1 group.
Compared with DDM, DDM/CMC/BFP-1 exhibited superior biocompatibility and osteogenesis, using a method of surface modification that has great potential for future clinical use.
The use of animal serum in culture medium brings safety concerns and batch-to-batch variability, and thus may restrict the clinical use of ex vivo expanded mesenchymal stromal cells (MSCs). Clinically compliant MSCs should be developed in adherence to serum-free, xeno-free and chemically defined medium (S&XFM-CD). In this study, we develop a S&XFM-CD by replacing all serum components with synthetic alternatives for the derivation of clinical-grade umbilical cord-derived MSCs (UCMSCs). The critical aspects including characterization, safety concerns, potency and exogenous factors contamination risk of UCMSCs in S&XFM-CD are compared with serum-containing medium (SCM). UCMSCs in S&XFM-CD retain fibroblastic-like morphology and immunophenotype of MSCs, and exhibit superior clone efficiency, proliferation capacity, and osteogenic and chondrogenic differentiation potential compared with SCM. Moreover, UCMSCs in S&XFM-CD retain similar immunosuppressive potential, and exhibit superior secretion levels of bFGF, PDGF-BB and IGF-1 compared with SCM. In addition, UCMSCs in S&XFM-CD do not undergo transformation, preserve the normal karyotypes and genomic stability, and are less prone to senescence process after long-term in vitro culture, which conforms to the current guidance of international and national evaluation standard. The S&XFM-CD developed here may serve as a GMP-grade production platform of UCMSCs for future clinical application.
Aligned biomimetic scaffolds based on carbon nanotubes-reinforced polymeric nanofibers for knee meniscus tissue engineering
2020, Materials LettersMany strategies in tissue engineering have been developed in order to provide potential treatments for Knee meniscal injuries since it is a high incidence lesion and without effective treatment available in clinical practice. The success of this approach is directly related to the creation of a scaffold able to reproduce the complex extracellular matrix of the native meniscus with adequate mechanical properties. Here we fabricated an aligned biomimetic scaffold, based on carbon nanotube (CNT)-reinforced polymeric nanofibers. For that, scaffolds with both circumferentially and radially aligned polycaprolactone fibers were developed from a specific electrospinning setup and incorporated with two different concentrations of CNT: 0.05% and 0.10%. Characterization by scanning electron microscopy confirmed the spatial distribution and the alignment of the electrospun nanofibers as well as a decrease in average fiber size with the addition of CNT (the presence of CNT was confirmed by transmission electron microscopy). Dynamic mechanical analysis and biological assays showed an improvement in the mechanical properties related to increased CNT content without influence in mesenchymal stem cells survival. These findings suggest a potential applicability of this nanocomposite for knee meniscus tissue engineering.