Induction of Chondrogenic Differentiation in Human Mesenchymal Stem Cells Cultured on Human Demineralized Bone Matrix Scaffold under Hydrostatic Pressure

BACKGROUND: Articular cartilage damage is still a troublesome problem. Hence, several researches have been performed for cartilage repair. The aim of this study was to evaluate the chondrogenicity of demineralized bone matrix (DBM) scaffolds under cyclic hydrostatic pressure (CHP) in vitro. METHODS: In this study, CHP was applied to human bone marrow mesenchymal stem cells (hBMSCs) seeded on DBM scaffolds at a pressure of 5 MPa with a frequency of 0.5 Hz and 4 h per day for 1 week. Changes in chondrogenic and osteogenic gene expressions were analyzed by quantifying mRNA signal level of Sox9, collagen type I, collagen type II, aggrecan (ACAN), Osteocalcin, and Runx2. Histological analysis was carried out by hematoxylin and eosin, and Alcian blue staining. Moreover, DMMB and immunofluorescence staining were used for glycosaminoglycan (GAG) and collagen type II detection, respectively. RESULTS: Real-time PCR demonstrated that applying CHP to hBMSCs in DBM scaffolds increased mRNA levels by 1.3-fold, 1.2-fold, and 1.7-fold (p < 0.005) for Sox9, Col2, and ACAN, respectively by day 21, whereas it decreased mRNA levels by 0.7-fold and 0.8-fold (p < 0.05) for Runx2 and osteocalcin, respectively. Additionally, in the presence of TGF-β1 growth factor (10 ng/ml), CHP further increased mRNA levels for the mentioned genes (Sox9, Col2, and ACAN) by 1.4-fold, 1.3-fold and 2.5-fold (p < 0.005), respectively. Furthermore, in histological assessment, it was observed that the extracellular matrix contained GAG and type II collagen in scaffolds under CHP and CHP with TGF-β1, respectively. CONCLUSION: The osteo-inductive DBM scaffolds showed chondrogenic characteristics under hydrostatic pressure. Our study can be a fundamental study for the use of DBM in articular cartilage defects in vivo and lead to production of novel scaffolds with two different characteristics to regenerate both bone and cartilage simultaneously.

Effects of electromagnetic stimulation on gene expression of mesenchymal stem cells and repair of bone lesions

Objective: Most people experience bone damage and bone disorders during their lifetimes. The use of autografts is a suitable way for injury recovery and healing. Mesenchymal stem cells [MSCs] are key players in tissue engineering and regenerative medicine. Their proliferation potential and multipotent differentiation ability enable MSCs to be considered as appropriate cells for therapy and clinical applications. Differentiation of stem cells depends on their microenvironment and biophysical stimulations. The aim of this study is to analyze the effects of an electromagnetic field on osteogenic differentiation of stem cells

Materials and Methods: In this experimental animal study, we assessed the effects of the essential parameters of a pulsatile electromagnetic field on osteogenic differentiation. The main purpose was to identify an optimum electromagnetic field for osteogenesis induction. After isolating MSCs from male Wistar rats, passage-3 [P3] cells were exposed to an electromagnetic field that had an intensity of 0.2 millitesla [mT] and frequency of 15 Hz for 10 days. Flow cytometry analysis confirmed the mesenchymal identity of the isolated cells. Pulsatile electromagnetic field-stimulated cells were examined by immunocytochemistry and real-time polymerase chain reaction [PCR]

Results: Electromagnetic field stimulation alone motivated the expression of osteogenic genes. This stimulation was more effective when combined with osteogenic differentiation medium 6 hours per day for 10 days. For the in vivo study, an incision was made in the cranium of each animal, after which we implanted a collagen scaffold seeded with stimulated cells into the animals. Histological analysis revealed bone formation after 10 weeks of implantation

Conclusion: We have shown that the combined use of chemical factors and an electromagnetic field was more effective for inducing osteogenesis. These elements have synergistic effects and are beneficial for bone tissue engineering applications

[Effect of mechanical stimulations on the fate of stem cells - a review]

The provision of an adequate quantity of cells with proper function and purity is one of the main challenges of tissue engineering studies. Stem cells, with their self-renewal and differentiation capacity, are considered one of the main cell sources in the field of tissue engineering. Previously, the use of chemical factors seemed to be the only possible way for stem cell differentiation. However, scientists have recently realized that physiological processes of the human body are composed of chemical, mechanical and electrical signals. Mechanical stimulation is one of the current methods that produce cells with proper morphology and alignment in the scaffold. Specific differentiation, a higher rate of cell growth, proliferation and differentiation, and lower experiment costs can be achieved using mechanical stimulation. Different parameters such as the chemical environment, physical environment that surrounds the cell [including geometry, stiffness and topology of scaffold surface], amplitude, frequency, and duration of mechanical stimulation can affect the stem cell fate. In this study we have investigated the impact of all types of mechanical stimulations under different loading regimes on the fate of stem cells with respect to the target tissue. The result has been reflected in the design of a proper bioreactor

Healing potential of mesenchymal stem cells cultured on a collagen-based scaffold for skin regeneration

Wound healing of burned skin remains a major goal in public health. Previous reports showed that the bone marrow stem cells were potent in keratinization and vascularization of full thickness skin wounds. In this study, mesenchymal stem cells were derived from rat adipose tissues and characterized by flowcytometry. Staining methods were used to evaluate their differentiation ability. A collagen-chitosan scaffold was prepared by freeze-drying method and crosslinked by carbodiimide-based crosslinker. The results of immunecytochemistry and PCR experiments confirmed the adipose-derived stem cells [ASC] in differentiation to the keratinocytes under the treatment of keratinocyte growth factor. The isolated ASC were seeded on the scaffolds and implanted at the prepared wounds. The scaffolds without cells were considered as a control and implanted on the other side of the rat. Histopathological analyses confirmed the formation of new tissue on the scaffold-cell side after 14 days with the formation of dermis and epidermis. These results indicated the capacity of ASC in differentiation to keratinocytes and also wound healing in vivo

Relationship between cell compatibility and elastic modulus of silicone rubber/organoclay nanobiocomposites

Substrates in medical science are hydrophilic polymers undergoing volume expansion when exposed to culture medium that influenced on cell attachment. Although crosslinking by chemical agents could reduce water uptake and promote mechanical properties, these networks would release crosslinking agents. In order to overcome this weakness, silicone rubber is used and reinforced by nanoclay. Attempts have been made to prepare nanocomposites based on medical grade HTV silicone rubber [SR] and organo-modified montmorillonite [OMMT] nanoclay with varying amounts of clay compositions. Incorporation of nanocilica platelets into SR matrix was carried out via melt mixing process taking advantage of a Brabender internal mixer. The tensile elastic modulus of nanocomposites was measured by performing tensile tests on the samples. Produced polydimetylsiloxane [PDMS] composites with different flexibilities and crosslink densities were employed as substrates to investigate biocompatibility, cell compaction, and differential behaviors. The results presented here revealed successful nanocomposite formation with SRand OMMT, resulting in strong PDMS-based materials. The results showed that viability, proliferation, and spreading of cells are governed by elastic modulus and stiffness of samples. Furthermore, adipose derived stem cells [ADSCs] cultured on PDMS and corresponding nanocomposites could retain differentiation potential of osteocytes in response to soluble factors, indicating that inclusion of OMMT would not prevent osteogenic differentiation. Moreover, better spread out and proliferation of cells was observed in nanocomposite samples. Considering cell behavior and mechanical properties of nanobiocomposites it could be concluded that silicone rubber substrate filled by nanoclay are a good choice for further experiments in tissue engineering and medical regeneration due to its cell compatibility and differentiation capacity

[Effects of combined cyclic-static stretch on orientation and proliferation of human mesenchymal stem cells]

Cell vital function has correlation with mechanical loadings that cell experiences. Here, effects of in-vitro combined cyclic-static stretch on proliferation of human mesenchymal stem cell [HMSC] were evaluated. HMSCs were cultured on gelatin coated elastic membranes, and exposed to stretch loading. Four different regimes of cyclic, static, combined cyclic-static, and cyclic with a period of unloading were exerted on the elastic membrane. Duration of cyclic loading and static loading was 5 and 12 hours respectively. The results illustrate that 10% cyclic stretch causes cell alignment but there were no significant proliferation differences between control and test group. Combined cyclic-static stretch reduced proliferation significantly while cyclic stretch with an unloading period increased cell proliferation significantly. At last, static stretch did not affect cell proliferation significantly. Cell stretching regimes and post-loading duration are effective factors on cell proliferation

[Evaluation of effects of cyclic loading on structural properties of cultured endothelial cells]

Endothelium is a selective and permeable membrane for transferring nutrients and vital components to arterial wall. Endothelial damage might lead to altered biological function of endothelium and clinical consequences such as atherosclerosis. Blood pressure pulse always exerts circumferential tension to the arterial wall. Hence, such tension together with other loads, play important role in functional properties of endothelial cells. Previous studies verify effects of cyclic loading on adaptation and remodeling of endothelium. This study investigates structural properties of cultured endothelial cells subjected to uni-axial cyclic loading. Human umbilical vein endothelial cells, prepared from national cell bank of Iran [NCBI-C554], were cultured on silicon membrane, and then subjected to cyclic tension with 10% amplitude and 1 Hz frequency, and 2, 4, 6, 8 hour durations utilizing a custom made tensile device. Viscoelastic properties of endothelial cells were examined by micropipette aspiration technique. Results show increase in elastic modulus [E] of cells due to tensile cyclic loading which results in stiffening of cell body. Also results show primary increase then subsequent decrease in viscose modulus. Previous studies verify generation of stress fibers due to accumulation and increase in actin fibers in endothelial cells after tensile cyclic loading. Since mechanical and structural properties of endothelial cells depend on actin fibers, results of this study show tensile cyclic loading causes increase in stiffness of endothelial cells through generation of stress fibers