A Standardized Method of Applying Toluidine Blue Metachromatic Staining for Assessment of Chondrogenesis.

OBJECTIVES: Staining with toluidine blue is a well-established procedure for the histological assessment of cartilaginous- and chondrogenic-differentiated tissues. Being a cationic dye, toluidine blue staining visualizes proteoglycans in a tissue because of its high affinity for the sulfate groups in proteoglycans. It is generally accepted that metachromatic staining with toluidine blue represents cartilaginous matrix and that the degree of positive staining corresponds with the amount of proteoglycans. DESIGN: Articular cartilage and pellets of chondrocytes or bone marrow stromal cells were analyzed with a standardized staining procedure for toluidine blue. RESULTS: In the present study, we illustrate why such an assumption is invalid unless a detailed description of the procedure and/or reference to a detailed published method are provided. This is because the staining specificity and intensity depend, as we have shown, on the pH of the staining solution, the use of dehydration, and on staining time. CONCLUSIONS: We can, therefore, suggest a well-controlled standardized protocol for toluidine blue staining, which provides an easy and simple selective staining technique for the assessment of cartilage tissue and proteoglycan development in chondrogenic differentiation. If this procedure is not used, then investigators must provide sufficient technical information concerning the staining protocol to allow an assessment of the validity of the staining results.

Surface chemistry, substrate, and topography guide the behavior of human articular chondrocytes cultured in vitro.

Understanding the behavior of chondrocytes in contact with artificial culture surfaces is becoming increasingly important in attaining appropriate ex vivo culture conditions of chondrocytes in cartilage regeneration. Chondrocyte transplantation-based cartilage repair requires efficiently expanded chondrocytes, and the culture surface plays an important role in guiding the behavior of the cell. Micro- and nano-engineered surfaces make it possible to modulate cell behavior. We hypothesized that the combined influence of topography, substrate, and surface chemistry may affect the chondrocyte culturing in terms of proliferation and phenotypic means. Human chondrocytes were cultured on polystyrene fabricated microstructures, flat polydimethylsiloxane (PDMS), or polystyrene treated with fibronectin or oxygen plasma and cultured for 1, 4, 7, and 10 days. The behavior of chondrocytes was evaluated by proliferation, viability, chondrogenic gene expression, and cell morphology. Contrary to our hypothesis, microstructures in polystyrene did not significantly influence the behavior of chondrocytes neither under normoxic- nor hypoxic conditions. However, changes in the substrate stiffness and surface chemistry were found to influence cell viability, gene expression, and morphology of human chondrocytes. Oxygen plasma treatment was the most important parameter followed by the softer substrate type PDMS. The findings indicate the culture of human chondrocytes on softer substratum and surface activation by oxygen plasma may prevent dedifferentiation and may improve chondrocyte transplantation-based cartilage repair. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 2805-2816, 2018.

Cyanoacrylate medical glue application in intervertebral disc annulus defect repair: Mechanical and biocompatible evaluation.

In an attempt to find an ideal closure method during annulus defect repair, we evaluate the use of medical glue by mechanical and biocompatible test. Cyanoacrylate medical glue was applied together with a multilayer microfiber/nanofiber polycaprolactone scaffold and suture in annulus repair. Continuous axial loading and fatigue mechanical test was performed. Furthermore, the in vitro response of mesenchymal stem cell (MSC) to the glue was evaluated by cell viability assay. The in vivo response of annulus tissue to the glue and scaffold was also studied in porcine lumbar spine; histological sections were evaluated after 3 months. Cyanoacrylate glue significantly improved the closure effect in the experimental group with failure load 2825.7 ± 941.6 N, compared to 774.1 ± 281.3 N in the control group without glue application (p < 0.01). The experimental group also withstood the fatigue test. No toxic effect was observed by in vitro cell culture and in vivo implantation. On the basis of this initial evaluation, the use of cyanoacrylate medical glue improves closure effect with no toxicity in annulus defect repair. This method of annulus repair merits further effectiveness study in vivo. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 14-20, 2017.

Precipitant induced porosity augmentation of polystyrene preserves the chondrogenicity of human chondrocytes.

Cells constantly sense and receive chemical and physical signals from neighboring cells, interstitial fluid, and extracellular matrix, which they integrate and translate into intracellular responses. Thus, the nature of the surface on which cells are cultured in vitro plays an important role for cell adhesion, proliferation, and differentiation. Autologs chondrocyte implantation is considered the treatment of choice for larger cartilage defects in the knee. To obtain a sufficient number of chondrocytes for implantation multiple passaging is often needed, which raises concerns about the changes in the chondrogenic phenotype. In the present study, we analyzed the effect at cellular and molecular level of precipitant induced porosity augmentation (PIPA) of polystyrene surfaces on proliferation and differentiation of human chondrocytes. Human chondrocytes were isolated from healthy patients undergoing anterior cruciate ligament reconstruction and cultured on PIPA modified polystyrene surfaces. Microscopical analysis revealed topographically arranged porosity with micron pores and nanometer pits. Chondrocytes cultured on PIPA surfaces revealed no difference in cell viability and proliferation, but gene- and protein expressions of collagen type II were pronounced in the first passage of chondrocytes when compared to chondrocytes cultured on control surfaces. Additionally, an analysis of 40 kinases revealed that chondrocytes expanded on PIPA caused upregulated PI3K/mTOR pathway activation and inhibition of mTORC1 resulted in reduced sGAG synthesis. These findings indicate that PIPA modified polystyrene preserved the chondrogenicity of expanded human chondrocytes at gene and protein levels, which clinically may be attractive for the next generation of cell-culture surfaces for ex vivo cell growth. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 3073-3081, 2016.

Dental pulp-derived stromal cells exhibit a higher osteogenic potency than bone marrow-derived stromal cells in vitro and in a porcine critical-size bone defect model.

INTRODUCTION: The osteogenic differentiation of bone marrow-derived mesenchymal stromal cells (BMSCs) was compared with that of dental pulp-derived stromal cells (DPSCs) in vitro and in a pig calvaria critical-size bone defect model. METHODS: BMSCs and DPSCs were extracted from the tibia bone marrow and the molar teeth of each pig, respectively. BMSCs and DPSCs were cultured in monolayer and on a three-dimensional (3D) polycaprolactone (PCL) - hyaluronic acid - tricalcium phosphate (HT-PCL) scaffold. Population doubling (PD), alkaline phosphatase (ALP) activity, and calcium deposition were measured in monolayer. In the 3D culture ALP activity, DNA content, and calcium deposition were evaluated. Six non-penetrating critical-size defects were made in each calvarium of 14 pigs. Three paired sub-studies were conducted: (1) empty defects vs. HT-PCL scaffolds; (2) PCL scaffolds vs. HT-PCL scaffolds; and (3) autologous BMSCs on HT-PCL scaffolds vs. autologous DPSCs on HT-PCL scaffolds. The observation time was five weeks. Bone volume fractions (BV/TV) were assessed with micro-computed tomography (µCT) and histomorphometry. RESULTS AND DISCUSSION: The results from the in vitro study revealed a higher ALP activity and calcium deposition of the DPSC cultures compared with BMSC cultures. Significantly more bone was present in the HT-PCL group than in both the pure PCL scaffold group and the empty defect group in vivo. DPSCs generated more bone than BMSCs when seeded on HT-PCL. In conclusion, DPSCs exhibited a higher osteogenic potential compared with BMSCs both in vitro and in vivo, making it a potential cell source for future bone tissue engineering.

Improvement of Distribution and Osteogenic Differentiation of Human Mesenchymal Stem Cells by Hyaluronic Acid and ß-Tricalcium Phosphate-Coated Polymeric Scaffold In Vitro.

Bone tissue engineering requires a well-designed scaffold that can be biodegradable, biocompatible, and support the stem cells to osteogenic differentiation. Porous polycaprolactone (PCL) scaffold prepared by fused deposition modeling is an attractive biomaterial that has been used in clinic. However, PCL scaffolds lack biological function and osteoinductivity. In this study, we functionalized the PCL scaffolds by embedding them with a matrix of hyaluronic acid/ß-tricalcium phosphate (HA/TCP). Human mesenchymal stem cells (MSCs) were cultured on scaffolds with and without coating to investigate proliferation and osteogenic differentiation. The DNA amount was significantly higher in the HA/TCP-coated scaffold on day 21. At the gene expression level, HA/TCP coating significantly increased the expression of ALP and COLI on day 4. These data correlated with the ALP activity peaking on day 7 in the HA/TCP-coated scaffold. Scanning electron microscope and histological analysis revealed that the cell matrix and calcium deposition were distributed more uniformly in the coated scaffolds compared to scaffolds without coating. In conclusion, the HA/TCP coating improved cellular proliferation, osteogenic differentiation, and uniform distribution of the cellular matrix in vitro. The HA/TCP-PCL scaffold holds great promise to accommodate human bone marrow-derived MSCs for bone reconstruction purposes, which warrants future in vivo studies.

Topography-Guided Proliferation: Distinct Surface Microtopography Increases Proliferation of Chondrocytes In Vitro.

Chondrocyte-based cartilage repair techniques require control of articular chondrocyte expansion ex vivo. Articular chondrocytes have limited availability, and prolonged culturing to obtain a cell number sufficient for clinical use often results in phenotypic alterations and increased costs. In this study, we applied a screening library consisting of micrometer-sized topographical features, termed biosurface structure array (BSSA), to identify specific topographical microstructures affecting the proliferation of human chondrocytes in passage 1 (P1) or 2 (P2). The BSSA library comprised 10 patterns and 16 combinations of pillar size (X) and interpillar gap size (Y). Specific microstructures significantly increased the chondrocytes' proliferative responsiveness in term of patterns, X and Y for P2 compared with P1. The P1 and P2 chondrocytes responded independently to similar patterns after 4 days of culturing, whereas only chondrocytes at P2 responded to specific microstructures with Y = 1 µm and X = 2, 4 µm by a 2.3- and 4.4-fold increased proliferation, respectively. In conclusion, these findings indicate that specific surface topographies promote chondrocyte proliferation and may, indeed, be a tool to control the behavior of chondrocytes in vitro.

Gradient Fractionated Separation of Chondrogenically Committed Cells Derived from Human Embryonic Stem Cells.

Cartilage regeneration is a fast growing field that combines biotechnology and molecular techniques in creating new tissue mimicking the native microenvironment. Human embryonic stem cells (hESCs) are a highly potent cell source for cartilage regeneration owing to their infinite proliferation capacity and pluripotency. Thus, lineage-specific differentiation of hESCs often results in populations with cellular heterogeneity. Chondrogenesis was induced through high-density micromass culture of hESCs and by addition of chondrogenic medium; 1:100 ITS(+), 100 nM dexamethasone, 40 µg/ml l-proline, 50 µg/mL ascorbic acid-2-phosphate, 1:100 Knockout serum, and 10 ng/mL TGFß3. At day 14 micromasses were dissociated and chondrogenically committed cell separated in a fraction-based discontinuous density gradient. After fractionation the chondrogenically committed cells were analyzed with regard to embryonic- and chondrogenic gene expression and fraction F3 and F4 with histology. In general, we found that the chondrogenic condition compared with the control condition had a significant effect on the following gene expression levels: NANOG, OCT4, SOX5, SOX9, ACAN, and COL2A1 in all fractions. Furthermore, we found in the chondrogenic condition that NANOG, OCT4, and SOX9 were significantly higher in F4 compared with F3, whereas COL2A1 and the ratio COL2A1:COL1A1 were significantly lower. Additionally, toluidine blue pH 4 stains of pellet cultures of F3 and F4 revealed that cells from F3 were more homogenous in morphology than F4. In conclusion, we propose a simple strategy to obtain more homogenous population of chondrogenically committed cells from hESCs using micromass culture and discontinuous density gradient separation.

Functionalization of polycaprolactone scaffolds with hyaluronic acid and ß-TCP facilitates migration and osteogenic differentiation of human dental pulp stem cells in vitro.

In this study, we sought to assess the osteogenic potential of human dental pulp stem cells (DPSCs) on three different polycaprolactone (PCL) scaffolds. The backbone structure of the scaffolds was manufactured by fused deposition modeling (PCL scaffold). The composition and morphology was functionalized in two of the scaffolds. The first underwent thermal induced phase separation of PCL infused into the pores of the PCL scaffold. This procedure resulted in a highly variable micro- and nanostructured porous (NSP), interconnected, and isotropic tubular morphology (NSP-PCL scaffold). The second scaffold type was functionalized by dip-coating the PCL scaffold with a mixture of hyaluronic acid and ß-TCP (HT-PCL scaffold). The scaffolds were cylindrical and measured 5 mm in height and 10 mm in diameter. They were seeded with 1×10(6) human DPSCs, a cell type known to express bone-related markers, differentiate into osteoblasts-like cells, and to produce a mineralized bone-like extracellular matrix. DPSCs were phenotypically characterized by flow cytometry for CD90(+), CD73(+), CD105(+), and CD14(-). DNA, ALP, and Ca(2+) assays and real-time quantitative polymerase chain reaction for genes involved in osteogenic differentiation were analyzed on day 1, 7, 14, and 21. Cell viability and distribution were assessed on day 1, 7, 14, and 21 by fluorescent-, scanning electron-, and confocal microscopy. The results revealed that the DPSCs expressed relevant gene expression consistent with osteogenic differentiation. The NSP-PCL and HT-PCL scaffolds promoted osteogenic differentiation and Ca(2+) deposition after 21 days of cultivation. Different gene expressions associated with mature osteoblasts were upregulated in these two scaffold types, suggesting that the methods in which the scaffolds promote osteogenic differentiation, depends on functionalization approaches. However, only the HT-PCL scaffold was also able to support cell proliferation and cell migration resulting in even cell dispersion throughout the scaffold. In conclusion, DPSCs could be a possible alternate cell source for bone tissue engineering. The HT-PCL scaffold showed promising results in terms of promoting cell migration and osteogenic differentiation, which warrants future in vivo studies.

Preconditioning Human Mesenchymal Stem Cells with a Low Concentration of BMP2 Stimulates Proliferation and Osteogenic Differentiation In Vitro.

Clinical trials using bone morphogenetic protein-2 (BMP2) for bone reconstruction have shown promising results. However, the relatively high concentration needed to be effective raises concerns for efficacy and safety. The aim of this study was to investigate the osteogenic effect of an alternative treatment strategy in which human bone marrow-derived mesenchymal stem cells (hMSCs) are preconditioned with low concentrations of BMP2 for a short time in vitro. hMSCs in suspension were stimulated for 15 min with 10 and 20 ng/mL of BMP2. After the BMP2 was removed, the cells were seeded and cultured in osteogenic medium. The effects of preconditioning were analyzed with regard to proliferation and expression of osteogenic markers at both gene and protein level. The results were compared to those from cultures with continuous BMP2 stimulation. A significant increase in proliferation was seen with both precondition and continuous stimulation with BMP2, with no difference between the treatments. Preconditioning with BMP2 significantly increased gene expression of RUNX2, COLI, ALP, and OC, and protein levels of COLI and ALP. This was not found with continuous stimulation. The role of preconditioning with BMP2 in osteogenesis was validated by findings of increased gene expression of SMAD1 and an increase in dual phosphorylation of ser 463 and ser 465 in the SMAD 1/5/8 pathway. We concluded that preconditioning hMSCs with BMP2 stimulates osteogenesis: proliferation with matrix secretion and matrix maturation of hMSCs. This implies that preconditioning with BMP2 might be more effective at inducing proliferation and osteogenic differentiation of hMSCs than continuous stimulation. Preconditioning with BMP2 could benefit the clinical application of BMP2 since side effects from high-dose treatments could be avoided.