Mesenchymal Stem Cells as a Potent Cell Source for Bone Regeneration

While small bone defects heal spontaneously, large bone defects need surgical intervention for bone transplantation. Autologous bone grafts are the best and safest strategy for bone repair. An alternative method is to use allogenic bone graft. Both methods have limitations, particularly when bone defects are of a critical size. In these cases, bone constructs created by tissue engineering technologies are of utmost importance. Cells are one main component in the manufacture of bone construct. A few cell types, including embryonic stem cells (ESCs), adult osteoblast, and adult stem cells, can be used for this purpose. Mesenchymal stem cells (MSCs), as adult stem cells, possess characteristics that make them good candidate for bone repair. This paper discusses different aspects of MSCs that render them an appropriate cell type for clinical use to promote bone regeneration

Effect of chitosan grafted polyethylenimine nanoparticles as a gene carrier on mesenchymal stem cells viability

      This study discusses the effect of complexes of chitosan grafted polyethylenimine(Ch-PEI) with plasmid DNA on viability of mesenchymal stem cells(MSCs) derived from human marrow. Ch-PEI/pDNA nanoparticles were synthesized through the complex coacervation method using pIRES plasmid containing Green Fluorescent Protein (GFP) gene. To confirm the complexation, samples were run through an agarose gel. Human bone marrow mesenchymal stem cells were studied for the cytotoxicity of the nanoparticles by MTT assay. MTT results indicated Ch-PEI does not have any significant cytotoxicity compared with PEI and Lipofectamine2000 leading to 40% cytotoxicity. According to the results it seems that grafting chitosan with PEI improves the MSCs viability

Ex vivo Expansion and Differentiation of Mesenchymal Stem Cells from Goat Bone Marrow

Objective(s)Mesenchymal stem cells (MSCs) from large animals as goat which is genetically more closely related tohuman have rarely been gained attentions. The present study tried to isolate and characterize MSCs fromgoat bone marrow.Materials and MethodsFibroblastic cells appeared in goat marrow cell culture were expanded through several subcultures.Passaged-3 cells were then differentiated among the osteogenic, adipogenic and chondrogenic cell lineagesto determine their MSC nature. Differentiations were determined by RT-PCR analysis of related geneexpression. To identify the best culture conditions for propagation, passage-3 cells were plated either atvarying cell densities or different fetal bovine serum (FBS) concentrations for a week, at the end of whichthe cultures were statistically compared with respect to the cell proliferation. In this study, we alsodetermined goat MSC population doubling time (PDT) as the index of their in vitro expansion rate.ResultsPassage-3 fibroblastic cells tended to differentiate into skeletal cell lineages. This was evident in bothspecific staining as well as the specific gene expression profile. Moreover, there appeared to be moreexpansion when the cultures were initiated at 100 cells/cm2 in a medium supplemented with 15% FBS. Arelatively short PDT (24.94±2.67 hr) was a reflection of the goat MSC rapid rate of expansion.ConclusionTaken together, fibroblastic cells developed at goat marrow cell culture are able to differentiate into skeletalcell lineages. They undergo extensive proliferation when being plated at low cell density in 15% FBSconcentration.Keywords: Adipogenesis, Bovine serum, Cell seeding density, Chondrogenesis, Goat mesenchymal stemcells, Osteogenesi

Development of PLGA-coated β-TCP scaffolds containing VEGF for bone tissue engineering

Bone tissue engineering is sought to apply strategies for bone defects healing without limitations and short-comings of using either bone autografts or allografts and xenografts. The aim of this study was to fabricate a thin layer poly(lactic-co-glycolic) acid (PLGA) coated beta-tricalcium phosphate (β-TCP) scaffold with sustained release of vascular endothelial growth factor (VEGF). PLGA coating increased compressive strength of the β-TCP scaffolds significantly. For in vitro evaluations, canine mesenchymal stem cells (cMSCs) and canine endothelial progenitor cells (cEPCs) were isolated and characterized. Cell proliferation and attachment were demonstrated and the rate of cells proliferation on the VEGF released scaffold was significantly more than compared to the scaffolds with no VEGF loading. A significant increase in expression of COL1 and RUNX2 was indicated in the scaffolds loaded with VEGF and MSCs compared to the other groups. Consequently, PLGA coated β-TCP scaffold with sustained and localized release of VEGF showed favourable results for bone regeneration in vitro, and this scaffold has the potential to use as a drug delivery device in the future

Extra virgin olive oil in maternal diet increases osteogenic genes expression, but high amounts have deleterious effects on bones in mice offspring at adolescence

Objective(s): Maternal high-fat diet has been shown to have deleterious effects on the offspring bones. However, there is no study to assess the effects of type and amount of maternal dietary oil in an isocaloric diet, with focus on extra virgin olive oil (EVOO). The objective of the current study was to test the hypothesis that type of maternal dietary oil has more effects than its amount in an isocaloric diet during gestation and lactation on bone genes expression in offspring in adolescence. Materials and Methods: Virgin female C57BL/6 mice were impregnated and fed either the AIN 93G diet (received 16% of calories as soybean oil, as a control diet, or EVOO) or a high fat AIN 93G diet (received 45% of calories as soybean oil or EVOO) from the time of vaginal plug confirmation until offspring’s weaning. Results: After adjusting for the amount of oils, osteoprotegerin/ receptor activator of nuclear factor NF-κB ligand (OPG/RANK-L) and OPG expressions were 6.1- and 2.8-folds higher in offspring born to EVOO compared with soybean oil-fed mothers. OPG, beta-catenin, and OPG/RANK-L expression were 88%, 94%, and 70% lower in offspring born to the 45% oil-fed mothers compared with the 16% group. In contrast, peroxisome proliferator-activated receptor gamma-2 (PPARγ2) gene expression was higher in the 45% oil group, adjusted for the types of oil. Conclusion: Maternal EVOO consumption, but not soybean oil increased osteoblastic gene expression, and high amounts of both oils decreased osteoblastic and increased adipogenic genes expression in adolescent offspring

Dental Pulp Polyps Contain Stem Cells Comparable to the Normal Dental Pulps

Objectives: Few studies investigated the isolation of stem cells from pathologically injured dental tissues. The aim of this study was to assess the possibility of isolation of stem cells from pulp polyps (chronic hyperplastic pulpitis), a pathological tissue produced in an inflammatory proliferative response within a tooth. Study design: Pulp polyp tissues were enzymatically digested and the harvested single cells were cultured. Cultured cells underwent differentiation to adipocytes and osteoblasts as well as flowcytometric analysis for markers such as: CD90, CD73, CD105, CD45, and CD14. In addition we tried to compare other characteristics (including colonigenic efficacy, population doubling time and the cell surface antigen panels) of these cells to that of healthy dental pulp stem cells (DPSCs). Results: Cells isolated from pulp polyps displayed spindle shape morphology and differentiated into adipocytes and osteoblasts successfully. These cells expressed CD90, CD73, and CD105 while were negative for CD45, CD14. Number of colonies among 104 tissue cells was higher in the normal pulp tissue derived cells than the pulp polyps (P=0.016); but as polyp tissues are larger and contain more cells (P=0.004), the total number of the stem cell in a sample tissue was higher in polyps but not significantly (P=0.073). Conclusions: The cells isolated from pulp polyps fulfill minimal criteria needed for MSC definition; hence, it can be concluded that pulp polyps contain stem cells. Although pulp polyps are rare tissues in daily practice but when they are present, may serve as a possible new non-invasively acquired tissue resource of stem cells for affected patients. List of abbreviations: APC = allophycocyanin, BM = Bone Marrow, CFU-F = Colony Forming Unit Fibroblast, DPSC = Dental Pulp Stem Cell, FITC = fluorescein isothiocyanate, MNC = mononuclear cells, MSC = Multipotent Mesenchymal Stromal Cell, PE = Phycoerythrin, PerCP = Peridinin chlorophyll protein, PPSC = Pulp Polyp Stem Cell

Functional differences of Toll-like receptor 4 in osteogenesis, adipogenesis and chondrogenesis in human bone marrow- derived mesenchymal stem cells

Multipotent human bone marrow-derived mesenchymal stem cells (hMSCs) are promising candidates for bone and cartilage regeneration. Toll-like receptor 4 (TLR4) is expressed by hMSCs and is a receptor for both exogenous and endogenous danger signals. TLRs have been shown to possess functional differences based on the species (human or mouse) they are isolated from therefore, the effects of knockdown of TLR4 were evaluated in humans during the differentiation of MSCs into bone, fat and chondrocyte cells in vitro. We investigated the expression profile of TLR4 during the differentiation of hMSCs into three different lineages on days 7, 14 and 21 and assessed the differentiation potential of the cells in the presence of lipopolysaccharide (LPS, as an exogenous agonist) and fibronectin fragment III-1c (FnIII-1c, as an endogenous agonist). TLR4 expression increased following the induction of hMSC differentiation into all three lineages. Alkaline phosphatase activity revealed that FnIII-1c accelerated calcium deposition on day 7, whereas LPS increased calcium deposition on day 14. Chondrogenesis increased in the presence of LPS; however, FnIII-1c acted as a reducer in the late stage. TLR4 silencing led to decreased osteogenesis and increased adipogenesis. Furthermore, Wnt5a expression was inversely related to chondrogenesis during the late stage of differentiation. We suggest that understanding the functionality of TLR4 (in the presence of pathogen or stress signal) during the differentiation of hMSCs into three lineages would be useful for MSC-based treatments

Critical-Sized Bone Defects Regeneration Using a Bone-Inspired 3D Bilayer Collagen Membrane in Combination with Leukocyte and Platelet-Rich Fibrin Membrane (L-PRF): An \u3cem\u3eIn Vivo\u3c/em\u3e Study

Objectives We aim to develop a 3D-bilayer collagen (COL) membrane reinforced with nano beta-tricalcium-phosphate (nβ-TCP) particles and to evaluate its bone regeneration in combination with leukocyte-platelet-rich fibrin (L-PRF) in vivo. Background data L-PRF has exhibited promising results as a cell carrier in bone regeneration in a number of clinical studies, however there are some studies that did not confirm the positive results of L-PRF application. Methods Mechanical & physiochemical characteristics of the COL/nβ-TCP membrane (1/2 & 1/4) were tested. Proliferation and osteogenic differentiation of seeded cells on bilayer collagen/nβ-TCP thick membrane was examined. Then, critical-sized calvarial defects in 8 white New Zealand rabbits were filled with either Col, Col/nβ-TCP, Col/nβ-TCP combined with L-PRF membrane, or left empty. New bone formation (NBF) was measured histomorphometrically 4 & 8 weeks postoperatively. Results Compressive modulus increases while porosity decreases with higher β-TCP concentrations. Mechanical properties improve, with 89 % porosity (pore size ∼100 μm) in the bilayer-collagen/nβ-TCP membrane. The bilayer design also enhances the proliferation and ALP activity. In vivo study shows no significant difference among test groups at 4 weeks, but Col/nβ-TCP + L-PRF demonstrates more NBF compared to others (P \u3c 0.05) after 8 weeks. Conclusion The bilayer-collagen/nβ-TCP thick membrane shows promising physiochemical in vitro results and significant NBF, as ¾ of the defect is filled with lamellar bone when combined with L-PRF membrane