17-Beta estradiol enhances osteogenic and adipogenic differentiation of human adipose-derived stromal cells.

Adipose-derived stromal cells (ASCs) possess multiple differentiation potentials and may serve as a cell source, if effectively modulated, for regenerative medicine and tissue engineering. Due to estrogen's function in tissue and organ development through regulating cell proliferation and differentiation, we hypothesized that an estrogen supplement may effectively enhance the multiple differentiation potentials of human ASCs. 17-Beta estradiol (E2) was investigated for modulating in vitro osteogenic and adipogenic differentiation in human ASCs isolated from a healthy female donor. After ASCs' exposure to osteogenic and adipogenic differentiation medium supplemented with different concentrations of E2, osteogenic markers (alkaline phosphatase activity, extracellular matrix, calcium deposition, and osteocalcin expression) and adipogenic parameters (lipid accumulation and differentiated cell population) significantly improved. Estrogen's enhancement is dose dependent and linked to differing alpha and beta estrogen receptors. Our data preliminarily demonstrate that estrogen can modulate the differentiation, and potentially improve the efficiency of ASCs in stem cell-based tissue engineering and regeneration. However, further study is needed to verify the regulatory functions of estrogen on ASC differentiations of donors with different ages and genders.

Modulations of 17-beta estradiol on osteogenic and adipogenic differentiations of human mesenchymal stem cells.

Bone marrow mesenchymal stem cells (MSCs) are a promising cell source for tissue engineering and regenerative medicine applications. However, effective regulation to improve differentiation potentials of MSCs plays a critical role in promoting successful tissue formation. Because estrogen has been demonstrated to modulate tissue and organ development and differentiation, we hypothesized that adding estrogen could effectively improve the multiple differentiation potentials of human bone marrow MSCs in vitro. In the present study, 17-beta estradiol (E2) was investigated for in vitro osteogenic and adipogenic differentiations of MSCs isolated from a healthy male human donor. After MSCs were exposed to osteogenic differentiation medium supplemented with E2 at different concentrations, osteocalcin expression is upregulated and calcium deposition (21.0%) is significantly improved ( p < 0.01; n = 4). Under adipogenic stimulation, E2 increased 35.4% lipid accumulations more than that of the group without the E2 supplement ( p < 0.01; n = 4). Estrogen's effect on osteogenesis occurs via estrogen receptors (ER)-alpha and -beta, whereas the effect on adipogenesis is through ER-alpha. Estrogen's regulation of differentiations of MSCs is dose dependent. The present study indicated that estrogen could potentially improve the role of MSCs in tissue engineering and regeneration by serving as a modulator of differentiation.

MR assessment of osteogenic differentiation in tissue-engineered constructs.

Bone marrow stromal cells (MSC) are a promising source of osteoprogenitor cells for bone tissue engineering. However, the population of the osteoprogenitor cells and their differentiation potentials change with the gender, age, and health of the donor. Development of a noninvasive method to assess osteogenic progression is critical for successful bone tissue regeneration. High-resolution magnetic resonance imaging (MRI) (at 11.7 T, with spatial resolution of 62.5 x 62.5 microm in 500 microm slices) is used in the present study to monitor osteogenic differentiation of tissue-engineered constructs prepared by seeding human bone MSCs on gelatin sponge scaffolds. Quantitative measurements of the MR relaxation times (T1, T2) and the apparent diffusion coefficient (ADC) were performed for four successive weeks on control tissue constructs and constructs exposed to osteogenic differentiation medium. The T1 and T2 relaxation times and ADC were found to decrease as osteogenic progression proceeded in samples exposed to osteogenic differentiation medium. At week 4, the T1, T2, and ADC of TE constructs were 1.81 +/- 0.11 s, 19.5 +/- 11.02 ms, and 1.01 +/- 0.47 x 10(3) mm(2)/s, respectively, for osteogenic differentiated constructs, significantly different from control constructs 2.22 +/- 0.08 s, 50.39 +/- 5.57 ms, and 1.86 +/- 0.18 x 107(3) mm(2)/s (p < 0.05). The MR parameters were also highly correlated with the cell seeding densities and alkaline phosphatase (ALP) activities of the osteogenic constructs. In conclusion, periodic measurements of MR parameters (T1, T2, and ADC) provide a promising method for noninvasive monitoring of the status of tissue-engineered bone growth and differentiation.

Nondestructive evaluation of osteogenic differentiation in tissue-engineered constructs.

Conventional measurements of osteogenesis in tissue-engineered constructs are destructive to living cells and incapable to provide three-dimensional information. In the present study, noninvasive magnetic resonance (MR) microscopy was used to evaluate osteogenic differentiation in vitro in human mesenchymal stem cell-based tissue-engineered constructs. The constructs were prepared by seeding the cells (10(6)cells/ml) on 4 x 4 x 4 mm gelatin sponge cubes and subsequently exposing them to osteogenic differentiation or basic medium. During the 4-week experiment, alkaline phosphatase (ALP) activity and calcium content of differentiated constructs were significantly increased compared to the basic medium controls. The T1, T2, and apparent diffusion coefficient (ADC) of differentiated constructs were significantly lower than those of the control group at each time point (p < 0.05). The MR parameters of constructs were significantly correlated to their ALP activities (r to T1, T2, and ADC = -0.57, -0.78, and -0.81, respectively) and calcium content (r to T1, T2, and ADC = 0.48, 0.90, and 0.92, respectively) measured by biochemical techniques. MR microscopy can be a promising tool for noninvasive assessment of osteogenic differentiation and to provide three-dimensional information about tissue-engineered constructs.

Magnetic resonance microscopy for monitoring osteogenesis in tissue-engineered construct in vitro.

Magnetic resonance microscopy (MRM) is used to monitor osteogenesis in tissue-engineered constructs. Measurements of the developing tissue's MR relaxation times (T(1) and T(2)), apparent diffusion coefficient (ADC) and elastic shear modulus were conducted over a 4-week growth period using an 11.74 T Bruker spectrometer with an imaging probe adapted for MR elastography (MRE). Both the relaxation times and the ADC show a statistically significant decrease after only one week of tissue development while the tissue stiffness increases progressively during the first two weeks of in vitro growth. The measured MR parameters are correlated with histologically monitored osteogenic tissue development. This study shows that MRM can provide quantitative data with which to characterize the growth and development of tissue-engineered bone.

Multiple differentiation potentials of neonatal dura mater-derived cells.

OBJECTIVE: The involvement of the dura mater in calvarial development and bone healing lead to a hypothesis that progenitor cells with multiple differentiation potentials exist within this tissue. The present study investigated the differentiation potentials of dura mater-derived cells by driving them into several cell-restricted lineages. METHODS: Dissected dura mater tissue of neonatal rats was washed, finely minced, and enzymatically digested. The harvested cells were exposed to different differentiation (osteogenic, adipogenic, and chondrogenic) and basic media. RESULTS: At defined time points, dura mater-derived cells were observed to differentiate into osteoblastic, adipoblastic, and chondroblastic cells, evidenced by specific biochemical staining. In addition, gene expressions of osteogenesis (alkaline phosphatase, osteocalcin, and osteopontin), chondrogenesis (collagen Type II and aggrecan core protein) and adipogenesis (peroxisome proliferator activated receptor gamma-2) were up-regulated in the differentiated dura mater-derived cells, confirmed by polymerase chain reaction. CONCLUSION: Preliminarily, it was concluded that a subpopulation of multiple potential mesenchymal cells exists in neonatal dura mater, which explains the function of the dura mater on neurocranium development and calvarial bone healing.

Adipose tissue engineering by human adipose-derived stromal cells.

Tissue engineering has emerged as a promising alternative approach to current clinical treatments for restoration of soft tissue defects. The purpose of this study was to investigate adipose tissue formation in vitro and in vivo by using human adipose-derived stromal cells (ADSCs) utilizing a gelatin sponge (Gelform) as a scaffold. Adipogenic potentials of human ADSCs were demonstrated by Oil-O-red staining and cellular morphology. After seeding human ADSCs in a density of 3 x 10(6) cells/ml on three-dimensional gelatin sponges, tissue-engineered constructs were exposed to adipogenic differentiation medium for in vitro studies and implanted in the backs of severe combined immunodeficient (SCID) mice for in vivo adipose regeneration. Adipogenesis of ADSC-seeded gelatin sponges was confirmed by Oil-O-red staining after 4 weeks of in vitro incubation. The optical density of the elution from Oil-O-red staining of adipogenic constructs is significantly higher than that of the control group (p < 0.05, n = 4). With short-term in vitro differentiation, adipogenic constructs turned into fat tissue 4 weeks after in vivo implantation, confirmed by biochemical and immunohistochemical examination. No adipogenic-morphological change or fat formation was observed in in vitro or in vivo studies when ADSCs were exposed to a control medium without adipogenic stimulation. These results indicate that engineered adipose tissue can be achieved using human ADSCs and biocompatible and degradable gelatin sponges.

Comparison of osteogenic potentials of visceral and subcutaneous adipose-derived cells of rabbits.

BACKGROUND: The demand for a large amount of osteogenic cells required in bone tissue engineering warranted exploration of a new source of osteoprogenitor cells. Recent studies have demonstrated that adipose-derived stromal cells possess multiple differentiation capacities, including osteogenic potential, as bone marrow mesenchymal stem cells. In the present study, the authors compared the osteogenic potentials of adipose-derived stromal cells from different anatomical sites of rabbits. METHODS: Different adipose-derived stromal cells were isolated from rabbit visceral and subcutaneous adipose tissues by enzymatic digestion and in vitro differentiation into osteogenic lineage. Osteogenic markers representing differentiation potentials of adipose-derived stromal cells from different anatomical sites were compared by biochemical and immunohistochemical assessment (n = 3). RESULTS: Fibroblast-like cells were digested from both visceral and subcutaneous adipose tissues. After exposure to osteogenic differentiation medium, visceral adipose-derived cells were found to possess greater osteogenic potentials than cells isolated from subcutaneous adipose tissues, evidenced by significantly different amounts of osteogenic markers including alkaline phosphatase, osteocalcin, and mineral deposition. CONCLUSION: This study indicates that osteogenic potentials of adipose-derived cells vary by their anatomical sites, with visceral adipose-derived cells exhibiting higher osteogenic potential than those isolated from subcutis. However, the mechanism is still unidentified.