Paracrine effects of living human bone particles on the osteogenic differentiation of mesenchymal stem cells.

Bone autografting remains the clinical model of choice for resolving problematic fractures. The precise mechanisms through which the autograft promotes bone healing are unknown. The present study examined the hypothesis that cells within the autograft secrete osteogenic factors promoting the differentiation of mesenchymal stem cells (MSCs) into osteoblasts. Particles of human bone ("chips") were recovered at the time of joint replacement surgery and placed in culture. Then, conditioned media were added to cultures of human, adipose-derived MSCs under both basal and osteogenic conditions. Contrary to expectation, medium conditioned by bone chips reduced the expression of alkaline phosphatase and strongly inhibited mineral deposition by MSCs cultured in osteogenic medium. Real time PCR revealed the inhibition of collagen type I alpha 1 chain (Col1A1) and osteopontin (OPN) expression. These data indicated that the factors secreted by bone chips inhibited the osteogenic differentiation of MSCs. However, in late cultures, bone morphogenetic protein-2 (BMP-2) expression was stimulated, suggesting the possibility of a delayed, secondary osteogenic effect.

Chondrogenic potential of macroporous biodegradable cryogels based on synthetic poly(α-amino acids).

In this study, the potential of highly porous hydrogels based on biodegradable synthetic poly(α-amino acids) to support proliferation and chondrogenesis of human dental pulp stem cells (hDPSCs) was investigated. Covalently crosslinked gels with permanent pores were formed under cryogenic conditions by free-radical copolymerization of poly[N5-(2-hydroxyethyl)-l-glutamine-stat-N5-(2-methacryloyl-oxy-ethyl)-l-glutamine] (PHEG-MA) with 2-hydroxyethyl methacrylate (HEMA) and N-propargyl methacrylamide (PrMAAm) as minor co-monomers. PrMAAm provided alkyne groups for modifying the gels with cell-supporting moieties (RGDS peptides) by the azide-alkyne "click"-reaction. Two types of gels with different compressive moduli were prepared. Each type was modified with two different concentrations of RGDS peptide. X-ray computed nanotomography (nanoCT) was used to visualize and analyze the 3D-structure of the cryogels. It was shown that modifying the PHEG-MA cryogels within the range of RGDS concentrations examined here had a positive effect on the proliferation of hDPSCs. Immunofluorescence staining for collagen type 2 and aggrecan proved that there was differentiation of hDPSCs into chondrocytes.

The effect of accelerated mineral trioxide aggregate on odontoblastic differentiation in dental pulp stem cell niches.

AIM: To investigate the effect of accelerated-set mineral trioxide aggregate (MTA) on the proliferation and odontoblastic differentiation of human dental pulp cell niches (DPSC). METHODOLOGY: ProRoot White MTA (WMTA; Dentsply Tulsa Dental, Johnson City, TN, USA) was mixed with various additives, which included distilled water, 2.5% disodium hydrogen phosphate (Na2 HPO4 ; Merck, Darmstadt, Germany) and 5% calcium chloride (CaCl2 ; Merck). DPSC niches extracted from third molars were cultured directly on MTA in the culture medium. Cell viability was evaluated by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4- sulphophenyl)-2H-tetrazolium (MTS) assay. Cell growth and expression of odontoblastic differentiation markers (dentine sialophosphoprotein (DSPP) and collagen type 1 (COL1)) were determined using Real-Time Polymerase Chain Reaction analysis. Osteo-/odontogenic differentiation of DPSC niches was evaluated by measurement of alkaline phosphatase activity (ALP). Calcium deposition was assessed using von Kossa staining. The results were analysed statistically using Mann-Whitney tests and Kruskal-Wallis tests. RESULTS: MTA mixed with 5% CaCl2 and 2.5% Na2 HPO4 exhibited optimal cell viability (P < 0.05) compared to MTA mixed with distilled water. MTA mixed with 5% CaCl2 and 2.5% Na2 HPO4 significantly increased ALP activity (P < 0.05), significantly promoted mineralization nodule formation (P < 0.05) and significantly enhanced the mRNA expression level of the osteogenic/odontogenic markers (P < 0.05; DSPP and COL1) compared with MTA mixed with distilled water. CONCLUSIONS: MTA mixed with 5% CaCl2 and 2.5% Na2 HPO4 was biocompatible with dental pulp stem cell niches. Accelerated-set MTA promoted better differentiation in DPSC niches than conventional MTA. The accelerators could provide an alternative to MTA mixed with distilled water.

Isolation and characterization of stem cells derived from human third molar tooth germs of young adults: implications in neo-vascularization, osteo-, adipo- and neurogenesis.

A number of studies have reported in the last decade that human tooth germs contain multipotent cells that give rise to dental and peri-odontal structures. The dental pulp, third molars in particular, have been shown to be a significant stem cell source. In this study, we isolated and characterized human tooth germ stem cells (hTGSCs) from third molars and assessed the expression of developmentally important transcription factors, such as oct4, sox2, klf4, nanog and c-myc, to determine their pluri-potency. Flow-cytometry analysis revealed that hTGSCs were positive for CD73, CD90, CD105 and CD166, but negative for CD34, CD45 and CD133, suggesting that these cells are mesenchymal-like stem cells. Under specific culture conditions, hTGSCs differentiated into osteogenic, adipogenic and neurogenic cells, as well as formed tube-like structures in Matrigel assay. hTGSCs showed significant levels of expression of sox2 and c-myc messenger RNA (mRNA), and a very high level of expression of klf4 mRNA when compared with human embryonic stem cells. This study reports for the first time that hTGSCs express developmentally important transcription factors that could render hTGSCs an attractive candidate for future somatic cell re-programming studies to differentiate germs into various tissue types, such as neurons and vascular structures. In addition, these multipotential hTGSCs could be important stem cell sources for autologous transplantation.

Design of a 3D aligned myocardial tissue construct from biodegradable polyesters.

The heart does not regenerate new functional tissue when myocardium dies following coronary artery occlusion, or if it is defective. Ventricular restoration involves excising the infarct and replacing it with a cardiac patch to restore the heart to a more healthy condition. The goal of this study was to design and develop a clinically applicable myocardial patch to replace myocardial infarcts and improve long-term heart function. A basic design composed of 3D microfibrous mats that house mesenchymal stem cells (MSCs) was developed from human umbilical cord matrix (Wharton's Jelly) cells aligned in parallel to each other mimicking the native myocardium. Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), poly(L-D,L-lactic acid) (P(L-D,L)LA) and poly(glycerol sebacate) (PGS) were blended and electrospun into aligned fiber mats with fiber diameter ranging between 1.10 and 1.25 microm. The micron-sized parallel fibers of the polymer blend were effective in cell alignment and cells have penetrated deep within the mat through the fiber interstices, occupying the whole structure; 8-9 cell layers were obtained. Biodegradable macroporous tubings were introduced to serve as nutrient delivery route. It was possible to create a thick myocardial patch with structure similar to the native tissue and with a capability to grow.

Low-molecular-weight heparin-conjugated liposomes with improved stability and hemocompatibility.

Multilamellar vesicles (MLV) containing phosphatidyl choline (PC), cholesterol (CHOL), and stearylamine (SA) in the molar ratio of 7:2:0.2 were prepared by the thin film hydration method. Low-molecular-weight heparin (LMWH, MW: 3000) was conjugated with the MLV using carbodiimide (EDC). Infrared, Raman, and nuclear magnetic resonance spectra and DSC of each sample (MLV, LMWH, and MLV-LMWH) were obtained, enabling the authors to determine the chemical changes that occurred in the MLV structure at the end of the conjugation step. In addition, the changes in the chemical structures of the conjugated samples were revealed by the use of elemental analysis. Particle size analysis was used to determine the difference between the sizes of MLV and MLV-LMWH. In order to study the effect of LMWH on the behavior of MLV-LMWH in blood, osmotic fragility (in saline and plasma), hemolytic activity, and plasma recalcification time tests were carried out. These tests showed that it was possible to construct liposomes that would not induce reactions in the blood and would have potentially longer half-lives in the circulation.