Rat Bone Marrow Stem Cells Research Articles

Chondroinductive Hydrogel Pastes Composed of Naturally Derived Devitalized Cartilage.

Hydrogel precursors are liquid solutions that are prone to leaking from the defect site once implanted in vivo. Therefore, the objective of the current study was to create a hydrogel precursor that exhibited a yield stress. Additionally, devitalized cartilage extracellular matrix (DVC) was mixed with DVC that had been solubilized and methacrylated (MeSDVC) to create hydrogels that were chondroinductive. Precursors composed of 10% MeSDVC or 10% MeSDVC with 10% DVC were first evaluated rheologically, where non-Newtonian behavior was observed in all hydrogel precursors. Rat bone marrow stem cells (rBMSCs) were mixed in the precursor solutions, and the solutions were then crosslinked and cultured in vitro for 6weeks with and without exposure to human transforming growth factor β3 (TGF-β3). The compressive modulus, gene expression, biochemical content, swelling, and histology of the gels were analyzed. The DVC-containing gels consistently outperformed the MeSDVC-only group in chondrogenic gene expression, especially at 6weeks, where the relative collagen II expression of the DVC-containing groups with and without TGF-β3 exposure was 40- and 78-fold higher, respectively, than that of MeSDVC alone. Future work will test for chondrogenesis in vivo and overall, these two cartilage-derived components are promising materials for cartilage tissue engineering applications.

Influence of titanium dioxide nanorods with different surface chemistry on the differentiation of rat bone marrow mesenchymal stem cells.

In this study, four kinds of TiO2 nanorods (TiO2 NRs), with similar aspect ratios but different surface functional groups, i.e. amines (-NH2), carboxyl groups (-COOH) and poly(ethylene glycol) (-PEG), were used to study their interaction with rat bone marrow stem cells (MSCs). The aspect ratios of the TiO2 NRs were measured (50 to 65 nm in length and 8 nm in width) under transmission electron microscopy (TEM). The cellular uptake of the TiO2 NRs was qualitatively studied by TEM and then quantified by inductively coupled plasma mass spectrometry (ICP-MS). The results showed that the MSCs ingested larger amounts of TiO2-core NRs and TiO2-NH2 NRs than those of TiO2-COOH NRs and TiO2-PEG NRs, with similar intracellular distribution patterns. TiO2-core NRs induced the highest cytotoxicity, as a result of the highest intracellular level of reactive oxygen species (ROS), which was lowered upon surface functionalization. The genotoxicity of the TiO2 NRs was neglectable at tested concentrations, studied by the comet assay. The adipogenic and osteogenic differentiation potentials of the MSCs were firstly evaluated in terms of lipid droplet formation and calcium deposition respectively in the presence of the TiO2 NRs. All of the TiO2 NRs did not show an obvious influence on the adipogenic differentiation potential of the MSCs. But TiO2-COOH NRs showed a significant impairment on the osteogenic differentiation behaviors. The influence of TiO2 NRs on the osteogenic differentiation of the MSCs was further quantitatively studied by the expression of osteogenic markers (collagen type I and osteocalcein), at both gene and protein levels. The results confirmed the strongest hindrance of the osteogenic differentiation of the MSCs by TiO2-COOH NRs, due to the up-regulation of transforming growth factor beta 1 (TGF-β1) and fibroblast growth factor (FGF-2). The results provide new information that the differentiation potential of the MSCs can be influenced by the presence of TiO2 NRs with different surface functionalities, suggesting a careful analysis of the biological impact of nanomaterials.

Promoting chondroinduction with naturally derived hydrogel pastes

Event Abstract Back to Event Promoting chondroinduction with naturally derived hydrogel pastes Emily Beck1, Marilyn Barragan2, Madeleine H. Tadros3, Stevin H. Gehrke1, 4, Sarah L. Kieweg1, 5, Cory J. Berkland1, 4, 6 and Michael S. Detamore1, 4 1 University of Kansas, Bioengineering Program, United States 2 University of Kansas, Department of Molecular Biosciences, United States 3 Rice University, Department of Chemical and Biomolecular Engineering, United States 4 University of Kansas, Department of Chemical and Petroleum Engineering, United States 5 University of Kansas, Department of Mechanical Engineering, United States 6 University of Kansas, Department of Pharmaceutical Chemistry, United States Introduction: ECM-based materials are gaining wide attention in the tissue engineering field due to their ability to support cellular attachment, growth, and differentiation[1]. Although ECM materials have been incorporated into hydrogels and show great promise, hydrogels also have one major drawback because their precursors are liquid solutions that are prone to leaking after surgical placement[2]. Therefore, in this work, we mixed porcine devitalized cartilage (DVC) particles into methacrylated, solubilized, devitalized, porcine cartilage (MeSDVC) to achieve a hydrogel precursor that was not only paste-like to overcome the drawbacks of traditional hydrogels, but additionally to achieve a hydrogel that would induce chondrogensis and could be further crosslinked and analyzed. Methods: Porcine cartilage that was devitalized and cryoground into a fine powder was mixed with devitalized cartilage that was solubilized and then methacrylated, and the precursors were analyzed rheologically for yield stress. Formulations tested rheologically were 5% DVC, 10% DVC, 10% MeSDVC, 10% MeSDVC 10% DVC, and 10% MeSDVC 10% DVC + TGF-β3. The formulations containing MeSDVC were then mixed with rat bone marrow stem cells and UV crosslinked to further characterize them as solids. The gels were cultured in vitro for 6 weeks, where chondrogenic gene expression, the compressive modulus, swelling, and histology were also analyzed. Results and Discussion: All formulations tested exhibited a yield stress, where the yield stress of 10% MeSDVC and 10% DVC were 725 ± 55 and 57.9 ± 0.3, respectively. When 10% MeSDVC and 10% DVC were mixed, the yield stress increased by a factor of 2.6 compared to 10% MeSDVC alone (p<0.05). Because the yield stress is not additive, it suggests that there is some physiological or chemical interaction between MeSDVC and DVC that is creating the synergistic yield stress effect. After crosslinking, the only gels that were noted to not have contraction throughout culture were the gels containing DVC particles. At the 2 week and 3 week time points, Sox-9 and aggrecan expression were significantly higher in the DVC groups (p<0.05) and by 6 weeks, the relative collagen II expressions of the 10% MeSDVC 10% DVC and the 10% MeSDVC 10% DVC + TGF-β3 groups were 78 and 40 fold higher than 10% MeSDVC alone, respectively (p<0.05). The compressive moduli of all but one group ranged between 70 and 160 kPa, although the 20% MeSDVC acellular group had a modulus of 680 ± 130 kPa, which is much closer to that of native cartilage. Finally, aggrecan immunohistological staining showed increased staining around the cells at 6 weeks in the growth factor supplemented and the 10% MeSDVC group. Conclusion: Given that the mixtures of MeSDVC and DVC combined yielded hydrogel precursors that exhibited a yield stress and crosslinked to form hydrogels that did not contract over time, stimulated chondroinductive gene expression, as well as had a compressive moduli on the same order of magnitude as native cartilage, these MeSDVC and DVC mixtures may be promising materials for translational cartilage tissue engineering applications. NIH S10 RR024664; NSF Graduate Research Fellowship (E.B.); NSF Major Research Instrumentation Grant (0320648); Kansas Bioscience Authority Rising Star Award (M.D.); NIH R01 DE022472 (C.B.)

Proliferation and differentiation of rat bone marrow stem cells by 400 μT electromagnetic field

The interaction between environment electromagnetic field (EMF) and cells can effect on various physiological processes. EMF as an external inducing factor, could effect on proliferation or differentiation of cells. The purpose of this study was to evaluate the influence of the electromagnetic field on the viability, proliferation and differentiation rate of bone marrow stem cells (BMSCs) to neuron. BMSCs were obtained from 42 adult male rats. The cells incubated and cultured in 96-wells and 6-wells plates and exposed to electromagnetic field (40 or 400μT) with a selected waveform: AC (alternative current), rectified half wave (RHW) and rectified full wave (RFW), for a week. To assess the viability and proliferation rate of treated cells, MTT assay was done, and then immunocytochemistry staining Neu N was used to evaluate cell differentiation to neuron. Results showed that EMF decreases the viability and proliferation in treated groups. But in AC group’s reduction was significant. Minimum viability and proliferation rate was observed in RHW 400μT group compared with sham. Immunocytochemistry showed that EMF can induce BMSC differentiation into neuron in AC 400μT and RFW 400μT. Evidences of this research support the hypothesis that EMF can induce differentiation of BMSCs to neuron.

Role of Magnesium Transporter Subtype 1 (MagT1) in the Osteogenic Differentiation of Rat Bone Marrow Stem Cells.

In the present study, we investigated the role of magnesium transporter subtype 1 (MagT1), a selective Mg transporter protein, in the osteogenic differentiation of rat bone marrow stem cells (rBMSCs). Osteogenic differentiation was monitored by the expressions of alkaline phosphatase (ALP), osteocalcin (OCN), collagen-1 (COL-1) and runt-related transcription factor 2 (RUNX2), and extracellular matrix mineralization of rBMSCs. The expression of MagT1 increased with osteogenic differentiation of rBMSCs, suggesting the importance of intracellular Mg homeostasis to cell differentiation. Alteration of intracellular Mg homeostasis by culture condition with low extracellular Mg significantly reduced the osteogenic differentiation markers ALP, OCN, COL-1, and RUNX2 gene expressions. MagT1 knockdown during the differentiation period also reduced osteogenic differentiation and the extent of matrix mineralization of rBMSCs. In conclusion, our results indicate that Mg and MagT1 play an important role in osteogenic differentiation of rBMSCs and may be involved in the bone regeneration.

Bone Marrow Stem Cells Response to Collagen/Single-Wall Carbon Nanotubes-COOHs Nanocomposite Films with Transforming Growth Factor Beta 1.

Single-wall carbon nanotubes (SWNTs) have attractive biochemical properties such as strong cell adhesion and protein absorption, which are very useful for a cell cultivation scaffold. In this study, collagen/SWNT-COOHs nanocomposite films composed of regenerated fish collagen and SWNT-COOHs (0, 0.5, 1.0 and 2.0 weight percent) were prepared by mixing solubilized pepsin-soluble collagen with solutions of SWNT-COOHs. Morphological observation by SEM indicated the homogenous dispersion of SWNT-COOHs in the collagen matrix. The application of FTIR confirmed that the process we applied to prepare the composites did not destroy the native structures of collagen and composites were crosslinked by D-ribose. The biocompatibility was evaluated in vitro using SD rat bone marrow stem cells (BMSCs). Compared with films without transforming growth factor beta 1 (TGF-β1), films with TGF-β1 had superior performance on promotion of cell growth. Compared with pure collagen film with TGF-β1, SWNT-containing films might promote cellular functions by adsorbing more growth factors. In conclusion, the study suggested that the collagen/SWNT-COOHs nanocomposite films with TGF-β1 were expected to be useful scaffolds in cartilage tissue engineering.

Experimental research on the effects of calcitonin gene related peptide on biological characteristics of bone marrow stem cells

Objective To study the effect of the recombinant Lentivirus containing calcitonin gene related peptide (CGRP) gene on cells biological activity and differentiation of rat bone marrow stem cells(MSCs). Methods Rat MSCs were isolated and cultured by granulocytes adherent. MSCs were transfected with Lenti-EGFP-CGRP(MSCsCGRP+ /+ group), While MSCs were transfected with Lenti-EGFP as control group. Cell transfection rate was detected by flow cytometry, protein secretion in the above-mentioned MSCsCGRP+ /+ supernatant was detected using ELISA method. Cells surface markers weare detected by flow cytometry and immunohistochemistry. Trypan blue was used to examin the survive rate, β-galactosidase staining was used to examin aging of MSCs transfection, and MTT was used to examine cell vitality. Results At first day after transfecting with Lenti-EGFP-CGRP, fluorescence was not observed by fluorescence microscope, but a small amount of CGRP protein was detected by ELISA in MSCsCGRP+ /+ group, at 3 days and 4 days after transfecting with MSCs, strong fluorescence was observed by fluorescence microscope (the cell transfection rates were 77.87% and 79.58%). The CGRP expression was significantly higher in MSCsCGRP+ /+ group than in control group 〔(19.53±0.50) pg/ml vs. (3.12±0.00) pg/ml, t=48.964, P 0.05). At 14 days after transfection with Lenti-EGFP-CGRP, aging of cell were decreased in MSCsCGRP+ /+ group as compared with control group (t=2.446, P<0.05). Conclusions After MSCs are transfected with Lenti-EGFP-CGRP, biological characteristics of MSCs has no significant effects, there is still proliferation and differentiation activity. Cell secretion of CGRP can promote the endothelial cell differentiation, and inhibit the differentiation to smooth muscle cells. The CGRP modification of MSCs may play a role in the regulation of angiogenesis. Key words: Mesenchymal stem cells; Calcitonin gene-related peptide; Transfection; Cytological techniques

Therapeutic Effects of Bone Marrow Stem Cells in Diabetic Rats

Background: Currently, diabetes mellitus, specifically, Type 2 diabetes is a multifactorial metabolic disorder that affects more than 348 million people worldwide. It is considered to be one of the main causes of mortality. The pathway of type 2 diabetes is characterized both by insulin resistance in muscle, fat, and liver and a relative failure of the pancreatic β cell. Despite extensive study, yet no unifying hypothesis exists to explain these defects and the proper treatment. The key goal of diabetes treatment is to prevent complications because over time, diabetes can damage the heart, blood vessels, eyes, kidneys, and nerves. Therefore there is a great need to develop new and effective therapies for treating diabetic complications early before it cause irreparable tissue damage. Recently, advances experimental evidence empowers the idea that diabetic patients may greatly benefit from cell-based therapies, which include the use of adult stem and/or progenitor cells in disease therapy. In particular, therapeutic effect of bone marrow stem cell in treating the type 2 diabetic patients. Motivation: Mesenchymal stem cells (MSCs) are adherent and pluripotent and non-hematopoietic progenitor cells. Human bone marrow MSCs have been shown to inhibit antigen-dependent CD4+ and CD8+ T cell proliferation in an allogeneic setting in vitro. They have been found to reside in most organs and tissues investigated to date, including bone marrow, adipose, dermis, muscular tissue, hair follicles, the periodontal ligament and the placenta. In addition, recent studies have shown that adult bone marrow stem cells can differentiate into several types such as blood, liver, lung, skin, muscle, neuron and insulin producing cells. This has motivated us to explore potentials of their therapeutic applications in treating diabetes mellitus or type-2 diabetes. This article proposes a novel mechanism to isolate adult rat bone marrow stem cells and test their ability to treat diabetic rats. The main focus of this research is to investigate the therapeutic effect of mesenchymal stem cells on the diabetic rats. Experimental methods, data and results: The experimental studies were carried out based on twelveweek old healthy Sprague Dawley (S.D) rats and it was used for isolation and transplantation of stem cells. We carried a total number of 40 Sprague Dawley (S.D) male rats; 12-14 weeks old age and weighting 180-250 gm were used in the experimental study. Rats were obtained from Animal House of Nile Center for Experimental Researches, Mansoura, Egypt. Animals were housed in separate metal cages, fresh and clean drinking water was supplied adlibtium through specific nipple. The animals were anesthetized by halothane, and then the skin was sterilized with 70% ethyl alcohol before cutting the skin. The femurs and tibia were carefully dissected from adherent soft tissues. Then they were placed into sterilized beaker containing 70% ethyl alcohol for 1-2 min. The bones were put in Petri dish contain Phosphate buffer saline 1X (PBS) (Hyclone, USA) for wash. The bones were taken to laminar air flow (unilab biological safety cabinet class II, china) to extract the BM. The two ends of the bones were removed using sterile scissors. Conclusion and future work: Currently, the obtained results revealed that diabetes caused bad effects on the blood picture, pancreas and kidney functions, as well as the immune system represented by TNF. Treatment the diabetic rats by MSCs engrafting improved the tested parameters towards the stats of normal case. Nevertheless, the wide application of the stem cells engrafting still needs more investigations to be assured.

Effect of pulsed electromagnetic field therapy on the osteogenic and adipogenic differentiation of bone marrow mesenchymal stem cells.

We investigated the effects of pulsed electromagnetic fields (PEMFs) of 20 Hz/2 mT on the osteogenic and adipogenic differentiation of bone marrow stem cells (BMSCs). Sprague Dawley rat BMSCs were isolated and cultured in vitro. The BMSCs of the third passage were obtained and stimulated by PEMFs of 20 Hz/2 mT. The alkaline phosphatase (ALP) activity was measured according to the ALP assay kit manufacturer instructions, the BMSC osteogenic and adipogenic indicators were detected by semi-quantitative reverse transcription polymerase chain reaction (RT-PCR), and oil red O staining was used to observe the adipose-induced adipogenic differentiation of BMSCs. PEMFs of 20 Hz/2 mT significantly promoted the activity of ALP in the BMSCs (P < 0.01) and mRNA expression of osteogenic proteins (osteocalcin and osteopontin). The PEMFs inhibited the expression of adipogenic transcription factors such as adipokines and adipocyte-binding protein-2, and the adipogenic differentiation of BMSCs. PEMFs of 20 Hz/2 mT can promote osteogenic differentiation and inhibit adipogenic differentiation in BMSCs.

An in-vitro study on the proliferative potential of rat bone marrow stem cells on electrospun fibrous polycaprolactone scaffolds containing micro-hydroxyapatite particles

Hydroxyapatite has a chemical composition that closely resembles the mineral phase of natural hard tissues, making it a good material for use as a bone scaffold. Previous studies have demonstrated that the use of electrospun nanofibres that contain synthetic hydroxyapatite aids in bone regeneration. In this study, composite scaffolds of polycaprolactone and bovine-derived hydroxyapatite were produced by mixing bovine-derived hydroxyapatite powder into polycaprolactone solution, followed by electrospinning. Field emission scanning electron microscopy images show that the scaffolds contained an interconnected spheroidal porous network. Four groups of scaffolds containing different weight percentages of bovine-derived hydroxyapatite (0, 10, 30 and 50%) were prepared. The scaffolds were characterised by infrared spectroscopy and field emission scanning electron microscopy which was used to observe cell attachment to the scaffolds as well.

Influence of chondroitin sulfate and hyaluronic acid presence in nanofibers and its alignment on the bone marrow stromal cells: cartilage regeneration.

Cartilage degeneration is the major cause of disability and poses several challenges to repair and regenerate. Conventional surgical treatments often induce fibrous tissues and compromise its function. Alternative tissue engineering strategies utilized scaffolds, factors and cells alone or in combination with some degree of success. This study reports the use of nanostructured biomimetic scaffold system in regulating the rat bone marrow stem cells (rBMSCs) differentiation into chondrogenic lineage in vitro. The biometric scaffold is essentially a micro-porous polycaprolactone (PCL) spiral structure decorated with sparsely spaced bioactive PCL nanofibers. The bioactivity stems from the use of two major components of hyaline cartilage extracellular matrix (ECM) namely chondroitin sulfate (CS) and hyaluronic acid (HYA). The PCL spiral structure was surface functionalized with PCL nanofibers encapsulated with CS (20% (w/w)) and HYA (0.2% (w/w)). In order to retain and sustain the release of CS and HYA nanofibers were cross-linked using carbodiimide chemistry. This study also evaluated the effect of nanofiber alignment on rBMSCs differentiation and evaluated the production of characteristic hyaline cartilage proteins namely collagen type II and aggrecan in vitro up to 28 days. Rat bone marrow derived stem cells cultured on the aligned nanofibers expressed significantly elevated levels of collagen type II and aggrecan secretions (western blots) as compared to scaffolds decorated with random fibers and tissue culture polystyrene (TCPS). This fiber alignment dependent expression of collagen type II and aggrecan secretion were further confirmed through immunofluorescence staining. This biomimetic and bioactive scaffold may serve as a serve as an efficient scaffold system for cartilage regeneration.

Porous silk scaffolds for delivery of growth factors and stem cells to enhance bone regeneration.

Stem cell-based tissue engineering shows promise for bone regeneration and requires artificial microenvironments to enhance the survival, proliferation and differentiation of the seeded cells. Silk fibroin, as a natural protein polymer, has unique properties for tissue regeneration. The present study aimed to evaluate the influence of porous silk scaffolds on rat bone marrow stem cells (BMSCs) by lenti-GFP tracking both in vitro and in vivo in cranial bone defects. The number of cells seeded within silk scaffolds in rat cranial bone defects increased from 2 days to 2 weeks after implantation, followed by a decrease at eight weeks. Importantly, the implanted cells survived for 8 weeks in vivo and some of the cells might differentiate into endothelial cells and osteoblasts induced by the presence of VEGF and BMP-2 in the scaffolds to promote angiogenesis and osteogenesis. The results demonstrate that porous silk scaffolds provide a suitable niche to maintain long survival and function of the implanted cells for bone regeneration.

An in vitro study of the impact of 4mT static magnetic field to modify the differentiation rate of rat bone marrow stem cells into primordial germ cells

This investigation was performed to evaluate the differentiation capacity and alteration in genes expression patterns during in vitro differentiation of bone marrow stem cells into primordial germ cells using static magnetic field (4mT) and BMP-4 (25ng/ml). The rate of differentiation was investigated using the Real Time-PCR method for tracing expression of differentiation markers (Oct-4, Nanog, C-Myc, Fragilis, Mvh and Stella). Then, immunocytochemical reaction was carried out for detection of marker proteins (Oct4 and Mvh). Increasing of the exposure time of the 4mT SMF (24 and 48h) and treatment time with 25ng/ml BMP4 (48 and 96h) indicated a marked decrease in expression of pluripotency genes (Oct-4, Nanog and C-Myc) and Oct4 protein and increase in primordial germ cell-specific genes (Fragilis, Mvh and Stella) and Mvh protein compared with the control group. Final results showed that in a synergistic manner, the combination of SMF with BMP4 exaggerates the differentiation potential of BMSCs to PGCs by activating the MAPK pathway and altering the Ca2+ concentration.

In vitro and in vivo biocompatibility of multi-walled carbon nanotube/biodegradable polymer nanocomposite for bone defects repair

Biomaterials are extensively used in bone defect recovery caused by bone diseases. Multi-walled carbon nanotubes have been reported to reinforce synthetic polymeric materials. The aim of the study is to test poly(3-hydroxybutyrate- co-3-hydroxyvalerate) loaded with different amounts of multi-walled carbon nanotubes to fabricate nanocomposites. Mechanical, mineralization, and degradation properties were studied in vitro. The proliferation and differentiation of rat bone marrow stem cells were studied to determine biocompatibility in vivo. The incorporation of multi-walled carbon nanotubes greatly increased the mechanical properties of poly(3-hydroxybutyrate- co-3-hydroxyvalerate) and the strongest composite obtained was at 2% multi-walled carbon nanotubes. The 2% nanocomposite also had higher rat bone marrow stem cell adhesion, proliferation, and differentiation characteristics compared to the pure poly(3-hydroxybutyrate- co-3-hydroxyvalerate). The apoptosis in the later stage of rat bone marrow stem cells decreased in the 2% nanocomposites group at different time points. Based on histology and micro-computed tomography tests 6 weeks after in vivo implantation, the 2% multi-walled carbon nanotubes/poly(3-hydroxybutyrate- co-3-hydroxyvalerate) treated animals had a higher volume of bone formation compared to the pure poly(3-hydroxybutyrate- co-3-hydroxyvalerate) group. Thus, the presence of multi-walled carbon nanotubes has an apparent positive effect on poly(3-hydroxybutyrate- co-3-hydroxyvalerate) in assisting osteogenesis.

Nanostructured Titanate with Different Metal Ions on the Surface of Metallic Titanium: A Facile Approach for Regulation of rBMSCs Fate on Titanium Implants

Titanium (Ti) is widely used for load-bearing bio-implants, however, it is bio-inert and exhibits poor osteo-inductive properties. Calcium and magnesium ions are considered to be involved in bone metabolism and play a physiological role in the angiogenesis, growth, and mineralization of bone tissue. In this study, a facile synthesis approach to the in situ construction of a nanostructure enriched with Ca(2+) and Mg(2+) on the surface of titanium foil is proposed by inserting Ca(2+) and Mg(2+) into the interlayers of sodium titanate nanostructures through an ion-substitution process. The characteriz 0.67, and 0.73 nm ation results validate that cations can be inserted into the interlayer regions of the layered nanostructure without any obvious change of morphology. The cation content is positively correlated to the concentration of the solutions employed. The biological assessments indicate that the type and the amount of cations in the titanate nanostructure can alter the bioactivity of titanium implants. Compared with a Na(+) filled titanate nanostructure, the incorporation of divalent ions (Mg(2+) , Ca(2+) ) can effectively enhance protein adsorption, and thus also enhance the adhesion and differentiation ability of rat bone-marrow stem cells (rBMSCs). The Mg(2+) /Ca(2+) -titanate nanostructure is a promising implantable material that will be widely applicable in artificial bones, joints, and dental implants.

Delayed Administration of Bone Marrow Mesenchymal Stem Cell Conditioned Medium Significantly Improves Outcome After Retinal Ischemia in Rats

Delayed treatment after ischemia is often unsatisfactory. We hypothesized that injection of bone marrow stem cell (BMSC) conditioned medium after ischemia could rescue ischemic retina, and in this study we characterized the functional and histological outcomes and mechanisms of this neuroprotection. Retinal ischemia was produced in adult Wistar rats by increasing intraocular pressure for 55 minutes. Conditioned medium (CM) from rat BMSCs or unconditioned medium (uCM) was injected into the vitreous 24 hours after the end of ischemia. Recovery was assessed 7 days after ischemia using electroretinography, at which time we euthanized the animals and then prepared 4-μm-thick paraffin-embedded retinal sections. TUNEL and Western blot were used to identify apoptotic cells and apoptosis-related gene expression 24 hours after injections; that is, 48 hours after ischemia. Protein content in CM versus uCM was studied using tandem mass spectrometry, and bioinformatics methods were used to model protein interactions. Intravitreal injection of CM 24 hours after ischemia significantly improved retinal function and attenuated cell loss in the retinal ganglion cell layer. CM attenuated postischemic apoptosis and apoptosis-related gene expression. By spectral counting, 19 proteins that met stringent identification criteria were increased in the CM compared to uCM; the majority were extracellular matrix proteins that mapped into an interactional network together with other proteins involved in cell growth and adhesion. By restoring retinal function, attenuating apoptosis, and preventing retinal cell loss after ischemia, CM is a robust means of delayed postischemic intervention. We identified some potential candidate proteins for this effect.

Poly(ester-urethane) scaffolds: effect of structure on properties and osteogenic activity of stem cells

The present study aimed to investigate the effect of structure (design and porosity) on the matrix stiffness and osteogenic activity of stem cells cultured on poly(ester-urethane) (PEU) scaffolds. Different three-dimensional (3D) forms of scaffold were prepared from lysine-based PEU using traditional salt-leaching and advanced bioplotting techniques. The resulting scaffolds were characterized by differential scanning calorimetry (DSC), thermogravimetric analysis (TGA), scanning electron microscopy (SEM), mercury porosimetry and mechanical testing. The scaffolds had various pore sizes with different designs, and all were thermally stable up to 300 °C. In vitro tests, carried out using rat bone marrow stem cells (BMSCs) for bone tissue engineering, demonstrated better viability and higher cell proliferation on bioplotted scaffolds compared to salt-leached ones, most probably due to their larger and interconnected pores and stiffer nature, as shown by higher compressive moduli, which were measured by compression testing. Similarly, SEM, von Kossa staining and EDX analyses indicated higher amounts of calcium deposition on bioplotted scaffolds during cell culture. It was concluded that the design with larger interconnected porosity and stiffness has an effect on the osteogenic activity of the stem cells.

Effects of platelet rich plasma and platelet lysate on proliferation of rat bone marrow mesenchymal stem cells: a comparative study

Objective To compare the effects of platelet rich plasma(PRP)and platelet lysate(PL)on proliferation and cell cycle of cultured rat bone marrow stem cells(BMSCs).Methods BMSCs were obtained from twenty 4-week-old SD rats using the whole bone marrow isolation and cultivation method and were identified with flow cytometry.Blood samples were taken from the hearts of thirty 12-week-old SD rats and gradient centrifugation was used to prepare PRP,and PL was obtained after three times of centrifugation and repeated freezing and thawing.The third generation of BMSCs with good growth state were divided into seven groups according to different culture media:ordinary complete medium(A group),1% PRPconditioned medium(B group),1% PL-conditioned medium(C group),5% PRP-conditioned medium(D group),5% PLconditioned medium(E group),10% PRP-conditioned medium(F group),and 10% PL-conditioned medium(G group). The proliferation of BMSCs was assessed by MTT assay.The PCNA protein expression was assessed by immunofluorescence method.Cell cycle of BMSCs was tested by flow cytometry.Western blotting analysis was used to analyze CyclinD1and p27 Kip1protein expression of BMSCs.Results The proliferation of BMSCs was significantly promoted by 1%,5%,10% PRPand 1%,5%,10% PL-conditioned media after cultured for 24h,48h,and 72hin a time-and dose-dependent manner(P 0.05),with the effect of PRP and PL being similar at the same concentration(P0.05).Immunofluorescence assay showed that PRP and PL both significantly promoted PCNA protein expression in a dose-dependent manner(P0.01).Flow cytometry showed that different concentrations of PRP- and PL-conditioned media,compared with the ordinary complete medium,resulted in gradually reduced cells in G0/G1 phase,gradually increased cells in S,G2 /M phase,and significantly increased PI(P0.05),with the changes in a dose-dependent manner.However,the effects of the same concentration of PRP and PL on cell cycle were not significantly different(P0.05).Western blotting analysis showed that 5% PRP and 5% PL significantly up-regulated CyclinD1and down-regulated the expression of p27Kip1.Conclusion Different concentrations of PL and PRP can accelerate cell cycle progression of cultured rat BMSCs by up-regulation CyclinD1and inhibiting p27Kip1, promoting the proliferation of BMSCs in a time-and dose-dependent manner.PL and PRP at the same concentration have the same proliferation-promoting effect,indicating that PL may be used as an alternative of PRP to promote the repair of bone defects.

Bone marrow stromal cells inhibit caspase-12 expression in rats with spinal cord injury

The mechanisms underlying the potentially beneficial effect of bone marrow stem cells (BMSCs) on spinal cord injury (SCI) are unknown. Therefore, the aim of the present study was to explore the protective effect of BMSCs in rats with SCI. A total of 45 adult male Sprague-Dawley rats were randomly divided into three groups; the SCI group (n=15), the BMSC group (n=15) and the sham-operation group (n=15). In the SCI and BMSC treatment groups, a modified Allen’s weight-drop technique was used to induce SCI. The BMSC treatment group received an injection of BMSCs using a microneedle into the epicenter of the spinal cord 24 h after injury. Rats in the sham-operation group were not subjected to SCI; however, the corresponding vertebral laminae were removed. Seven days after transplantation, a rapid recovery was observed in the Basso, Beattie and Bresnahan (BBB) scores of the BMSC treatment group, whereas the BBB scores in the SCI group remained low (P<0.05). Caspase-12 expression in the SCI group was increased compared with that in the sham-operation group, whereas caspase-12 expression was attenuated 24 h after transplantation in the BMSC treatment group (P<0.05). In conclusion, the transplantation of BMSCs may improve locomotor function and attenuate caspase-12 expression following SCI. Therefore, it is likely to be an effective strategy for preventing severe injury of the spinal cord.

Adult Rat Bone Marrow-Derived Stem Cells Promote Late Fetal Type II Cell Differentiation in a Co-Culture Model

Bronchopulmonary dysplasia develops in preterm infants due to a combination of lung immaturity and lung injury. Cultured pluripotent bone marrow stem cells (BMSC) are known to reduce injury and induce repair in adult and in immature lungs, possibly through paracrine secretion of soluble factors. The paracrine relationship between BMSC and primary fetal lung epithelial type II cells is unknown. We determined the effects of BMSC on type II cell and fibroblast behavior using an in vitro co-culture model. Rat BMSC were isolated and co-cultured with primary fetal E21 rat type II cells or lung fibroblasts in a Transwell® system without direct cell contact. Effects of BMSC conditioned media (CM) on type II cell and fibroblast proliferation and on type II cell surfactant phospholipid (DSPC) synthesis and mRNA expression of surfactant proteins B and C (sftpb and sftpc) were studied. We also determined the effect of fibroblast and type II cell CM on BMSC proliferation and surface marker expression. Co-culture with BMSC significantly decreased type II cell and fibroblast proliferation to 72.5% and 83.7% of controls, respectively. Type II cell DSPC synthesis was significantly increased by 21% and sftpb and sftpc mRNA expressions were significantly induced (2.1 fold and 2.4 fold, respectively). BMSC proliferation was significantly reduced during the co-culture. Flow cytometry confirmed that BMSC retained the expression of undifferentiated stem cell markers despite their exposure to fetal lung cell CM. We conclude that BMSC induce fetal type II cell differentiation through paracrine release of soluble factors. These studies provide clues for how BMSC may act in promoting alveolar repair following injury.