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Enhanced osteogenic potential of mesenchymal stem cells from cortical bone: a comparative analysis
Joseph S. Fernandez-Moure1,2,
Bruna Corradetti2,3,
Paige Chan2,
Jeffrey L. Van Eps1,2,
Trevor Janecek2,
Pranela Rameshwar5,
Bradley K. Weiner4 &
Ennio Tasciotti2
Mesenchymal stem cells (MSCs) hold great promise for regenerative therapies in the musculoskeletal system. Although MSCs from bone marrow (BM-MSCs) and adipose tissue (AD-MSCs) have been extensively characterized, there is still debate as to the ideal source of MSCs for tissue-engineering applications in bone repair.
MSCs were isolated from cortical bone fragments (CBF-MSCs) obtained from patients undergoing laminectomy, selected by fluorescence-activated cell sorting analysis, and tested for their potential to undergo mesodermic differentiation. CBF-MSCs were then compared with BM-MSCs and AD-MSCs for their colony-forming unit capability and osteogenic potential in both normoxia and hypoxia. After 2 and 4 weeks in inducing media, differentiation was assessed qualitatively and quantitatively by the evaluation of alkaline phosphatase (ALP) expression and mineral deposition (Von Kossa staining). Transcriptional activity of osteoblastogenesis-associated genes (Alp, RUNX2, Spp1, and Bglap) was also analyzed.
The cortical fraction of the bone contains a subset of cells positive for MSC-associated markers and capable of tri-lineage differentiation. The hypoxic conditions were generally more effective in inducing osteogenesis for the three cell lines. However, at 2 and 4 weeks, greater calcium deposition and ALP expression were observed in both hypoxic and normoxic conditions in CBF-MSCs compared with AD- and BM-MSCs. These functional observations were further corroborated by gene expression analysis, which showed a significant upregulation of Bglap, Alp, and Spp1, with a 22.50 (±4.55)-, 46.56 (±7.4)-, 71.46 (±4.16)-fold increase compared with their uninduced counterparts.
This novel population of MSCs retains a greater biosynthetic activity in vitro, which was found increased in hypoxic conditions. The present study demonstrates that quantitative differences between MSCs retrieved from bone marrow, adipose, and the cortical portion of the bone with respect to their osteogenic potential exist and suggests the cortical bone as suitable candidate to use for orthopedic tissue engineering and regenerative medicine.
Mesenchymal stem cells (also known as multipotent stromal cells, or MSCs) are a group of cells defined for their capacity to self-renew and differentiate toward the mesodermal lineage, becoming osteoblasts, adipocytes, and chondrocytes [1–3]. MSCs from the bone marrow (BM-MSCs) were originally described by Friedenstein et al. as non-phagocytic and non-hematopoietic cells and adherent to plastic [4]. Over the last 15 years, there has been an explosion in the reports of MSCs isolated from a variety of other adult sources, including skin [5], adipose tissue [6], umbilical cord blood [7, 8] and matrix, peripheral blood [9], tendons [10, 11], amnion [12–14], and bone [15–17]. Their plasticity, immunosuppressive potential, immuno-modulatory properties [18, 19], and trophic activity [20, 21] make MSCs critical players in tissue homeostasis. For these reasons, MSCs are considered a suitable tool for regenerative medicine and have been already introduced in a number of clinical trials for tissue repair [18, 22–25]. Although they share similar epitope profiles, MSCs derived from different tissues show significant differences in the differentiation, proliferation, and migration potential, which depend on the tissue they originate from as they receive inputs that directly affect their specification [2, 26, 27].
For regenerative medicine purposes, BM-MSCs represent the gold standard [4, 22–25] and their use in investigational and clinical orthopedic tissue engineering has been well characterized [18–20]. Following much debate, however, MSCs with true "stemness" have been shown to constitute only a very small proportion of cells in the bone marrow (0.01–0.001 % of nucleated cells) and their proliferative and differentiative potential inversely correlate with age and the passages in vitro [21, 28]. Furthermore, bone marrow aspiration is painful and can be associated with multiple complications [29]. Adipose tissue has arisen as a reliable source for MSCs [30, 31]. They can be obtained by the less invasive method of lipoaspiration and yield a greater quantity of tissue and thus a greater number of cells [26, 27, 32, 33]. AD-MSCs have been shown to have a greater potential for proliferation, higher rates of colony formation, and greatest tolerance to serum deprivation-induced apoptosis than their bone marrow counterparts [28, 34–37].
Although BM-MSCs and AD-MSCs have been extensively characterized, there is still debate regarding the ideal source of MSCs for orthopedic tissue-engineering applications [18, 35]. Orthopedic reconstructive procedures remain some of the most common procedures performed worldwide. Historically, surgical reconstruction has been predominated by the use of synthetic implants and bone grafts. Over the past 15 years, there has been an explosion in the use of cell-based therapies in orthopedics [38–40]. Although BM-MSCs are the most commonly used for this purpose, groups have described different compartments of the bone, mainly the trabecular [16, 41–43] and cortical [17, 44] portions, as reservoirs of multipotent cells with a greater osteogenic commitment [15, 44, 45]. Moreover, their characterization has been limited to animal models and no comparison in humans has yet been reported. The aims of the present study were to isolate and characterize native populations of MSCs from the human cortical bone fraction (CBF-MSCs) and to compare them with commercially available MSCs obtained from the two most studied sources: the adipose tissue and the bone marrow. Comparison was performed in terms of morphology, clonogenic capability (colony-forming units, or CFU), and multidifferentiative potential, with particular emphasis to the osteogenic commitment, which has been assessed through the evaluation of calcium deposition, alkaline phosphatase (ALP), and osteoblastogenesis-associated genes expression.
Isolation of human CBF-MSCs
CBF-MSC populations were extracted by processing bones of three patients undergoing laminectomy following a protocol previously described [17] with some modifications for humans. All three patients were age-matched and all underwent the same procedure, spinal laminectomy, for benign pathology. This extraction protocol was approved by the Institutional Review Board of the Houston Methodist Hospital (Houston, TX, USA). Samples were processed after written informed consent was obtained. Bone fragments were obtained following the routine dissection and extraction of bone as part of the standard operation. No modification to the original surgery was made for the removal of bone fragments. The fragments used would have otherwise been discarded and no excess tissue that would have not been removed as standard of care for the planned procedure was extracted.
Bones were cleaned of any fat, connective tissue, or muscle by sharp dissection. Any blood that remained on the specimen was flushed from the bony fragments with phosphate-buffered saline (PBS) (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 1 % antibiotic/antimycotic (Gibco, Grand Island, NY, USA) until they appeared clean of all debris. The cancellous portion of the bone was sharply transected from the cortical fraction and discarded. Cortical bone fragments were crushed into chips approximately 3–4 mm3 and transferred to 50-ml polypropylene tubes (BD Falcon, Bedford, MA, USA). Bone chips were suspended in alpha-modified Eagle's medium (α-MEM) containing 2 % (vol/vol) defined fetal bovine serum (FBS) (Gibco) in presence of 3 mg/ml collagenase type-I (Worthington Biochemical Corporation, Lakewood, NJ, USA) and 4 mg/ml dispase II (Roche, Indianapolis, IN, USA) and placed on a shaking platform at 37 °C for 3 h. Following digestion, bone chips were plated into new flasks and grown undisturbed for 3 days to allow cells to migrate out of the fragments. A mixture of cells obtained from digestion and migration from the bone chips was obtained. These cells were considered passage 0 (P0).
Histological staining of bone fragments
To assess the complete removal of cells from the bone matrix, histologic staining was performed. Bone fragments underwent enzymatic processing as mentioned above. Before enzymatic digestion and after migration of cells from the bone fragments, representative fragments were fixed in 10 % buffered formalin and then underwent decalcification in an 8 % hydrochloric acid/formic acid solution. Tissue samples were then embedded in paraffin and cut into 4-μm sections using a microtome. Three non-consecutive sections were routinely stained with hematoxylin and eosin (H&E) and inspected for presence of cells following digestion.
CBF-MSC expansion
Adherent cells were cultured in α-MEM (Sigma-Aldrich, St. Louis, MO, USA) containing 20 % (vol/vol) FBS supplemented with 1 % antibiotic/antimycotic (Gibco) and incubated at 37 °C in hypoxic (5 % oxygen) or normoxic (21 % oxygen) conditions and 90 % humidity. Media was changed every 48 h until the cells were at 60–80 % confluency at which point they were passaged at a split ratio of 1:3. Cultures were established in Dulbecco's modified Eagle's medium (DMEM) containing 10 % (vol/vol) FBS supplemented with 1 % antibiotic/antimycotic (Gibco). AD- and BM-MSC cultures were established in accordance with the instructions of the manufacturer (Lonza, Basel, Switzerland). Adherent P2 cells were serially passaged by using TripLE™ Express (Invitrogen, part of Thermo Fisher Scientific) upon reaching near confluence (80 %) and reseeded for culture maintenance.
To confirm the MSC-associated phenotype of cells obtained from the cortical portion of the bone and work with a homogeneous cell population, freshly isolated CBF-MSCs were selected by fluorescence-activated cell sorting (FACS) based on a panel of lineage-committed cell surface markers.
Fluorescence-activated cell sorting
Approximately 1 × 106 cells were collected and stained in accordance with guidelines of the manufacturer. Cells used in FACS were at P0. Tested markers included the 5′-nucleotidase, CD73 (BioLegend, San Diego, CA, USA), the receptor-linked protein tyrosine phosphatase, CD45 (BioLegend), B-lymphocyte antigen CD19 (eBioscience, San Diego, CA, USA), the major histocompatibility complex (MHC) class II cell surface receptor, HLA-DR (BioLegend), the surface glycoprotein and cell-cell adhesion factor, CD34 (Invitrogen, Burlington, ON, Canada), the glycoprotein CD44 (Invitrogen), and membrane glycoprotein, CD105 (Invitrogen). Cells (5 × 105) were incubated with directly conjugated antibodies in 0.1 % bovine serum albumin/PBS for 30 min at room temperature in the dark. Cells were first gated on the basis of light-scatter properties to screen out debris. MSC cell surface phenotypes were verified through a multiparameter panel permitting the selection of CD73+CD44+CD105+HLA-DR−CD19−CD34− cells on a BD FACSAria™ II cell sorter (Becton Dickinson, Franklin Lakes, NJ, USA) to obtain a near-pure subpopulation of CBF-MSCs. Sorted cells were collected and cultured to perform proliferation assays, colony-forming unit-fibroblastic-like (CFU-F) analysis, and multilineage differentiation.
Proliferation assays (doubling time) in normoxic and hypoxic conditions
Proliferation rate was determined in triplicate on AD-, BM-, and CBF-MSCs as previously reported [46, 47]. Doubling time was assessed from P1 to P10 in normoxic and hypoxic conditions to determine the effect of low-tension oxygen on cell proliferation. Data obtained from each cell line are reported as mean of the values. For CBF-MSCs, the doubling time at each passage has also been shown.
Colony-forming unit (CFU-F) assay
Colony-forming unit assay was performed on freshly isolated CBF-MSCs, AD-MSCs, and BM-MSCs as previously reported [12, 17, 48]. Briefly, cells at P2 were plated in six-well plates at different densities (100, 250, 500, and 1000 cells/cm2) and cultured over a 15-day period. Colonies were fixed with 4 % formalin, stained with 1 % methylene blue (Serva, Heidelberg, Germany) in 10 mM borate buffer pH 8.8 (Fluka BioChemika, Buchs, Switzerland) at room temperature, and washed twice. Colonies formed by 16–20 nucleated cells were counted under a BX71 microscope (Nikon Corporation, Tokyo, Japan).
Adipogenic, chondrogenic, and osteogenic differentiation
Multipotent differentiation capability of CBF-MSCs, AD-MSCs, and BM-MSCs was assessed in vitro at P3. AD-MSCs and BM-MSCs were purchased from (Lonza) and delivered at P0. They were expanded to P3 in DMEM containing 10 % (vol/vol) FBS and used for subsequent differentiation assays. Non-induced AD-MSCs, BM-MSCs, and CFB-MSCs were used as control and cultured for the same time in growth medium.
To assess chondrogenic differentiation, micromass cultures were generated by seeding 5-μl droplets of a 1.6 × 107cells/ml solution into the center of a multi-well culture vessel. The micromass was allowed to settle for 2 h and then complete StemPro replaced with complete StemPro Media (Gibco) was gently added so as to not perturb the micromass. Cells were allowed to undergo differentiation for 21 days with media change every 2 days. After induction, micromass cultures were stained by Alcian blue stain for glycosaminoglycans and mucopolysaccharides.
Cells undergoing adipogenic differentiation were seeded into culture vessels at the density of 1 × 104 cells/cm2 and incubated for 24 h in standard medium. Then the medium was replaced with complete StemPro Adipogenic Differentiation medium (Gibco) and incubated for 14 days. Media were changed every 3 days. After induction, cells were stained by Oil red O to highlight the presence of intracellular lipid vacuoles. Osteogenic induction was performed by seeding cells at the density of 5000 cells/cm2 in 12-well plates and culturing them until they reached approximately 80–90 % confluence.
The osteogenic potential of cells cultured in hypoxic or normoxic conditions was assessed in vitro. In both cases, induction was performed over 2 and 4 weeks by using StemPro Osteogenesis Differentiation Madium (Gibco). To confirm mineral deposition, conventional von Kossa and ALP stainings were performed by using a Vector Blue Alkaline Phosphatase Substrate Kit (Vector Labs, Burlingame, CA, USA). Equal numbers of cells were plated in eight-well plates and grown for 2 weeks in both hypoxia and normoxic conditions. Staining was then performed in accordance with the instructions of the manufacturer. Fluorescence intensity per field of view was quantified by using ImageJ software, and total fluorescence was normalized to cell number. Five fields of view per well were quantified. Similarly, cells were grown in hypoxic and normoxic condition for 2 and 4 weeks and then stained by Von Kossa. Five fields of view were chosen for analysis, and the area of positively staining material was quantified by using ImageJ software.
Molecular characterization
Quantitative reverse transcription-polymerase chain reaction analysis was used to evaluate the expression of the precursor-associated gene CD271 and specific osteogenesis-associated markers following induction. Total RNA was isolated from CBF-MSCs, AD-MSCs, and BM-MSCs by using Trizol reagent (Invitrogen). DNAse (Sigma-Aldrich) treatment followed the reaction. RNA concentration and purity were measured by using a NanoDrop ND1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA). The cDNA was synthesized from 1 μg total RNA by using an iScript retrotranscription kit (Bio-Rad Laboratories, Hercules, CA, USA), and quantitative polymerase chain reaction was run in an ABI 7500 Fast Sequence Detection System (Applied Biosystems, Foster City, CA, USA) using commercially available master mix. The following target probes (Applied Biosystems) were used to evaluate the expression of Runt-related transcription factor 2 (RUNX2: Hs00231692_m1) osteocalcin (Bglap; Hs01587814_g1), osteopontin (Spp1; Hs00959010_m1), and ALP (Alp; Hs01029144_m1) expression for osteogenesis. The expression of each gene was normalized to the level of glyceraldehyde 3-phosphate dehydrogenase (Gapdh; Hs02758991_g1) and presented in comparison with the values obtained from the control (uninduced cells). For the expression of CD271 (Hs00609977_m1), CBF-MSCs were compared with MSCs obtained from their bone-marrow counterparts.
All experiments were repeated a minimum of three times, and all data are presented as mean ± standard deviation. All statistical calculations were completed using GraphPad Prism software (GraphPad Software, Inc., La Jolla, CA, USA). A paired t test was used to analyze the statistical significance of hypoxic against normoxic conditions per cell line and to determine the statistical significance of one hypoxic cell line to another. Anything with a P value of less than 0.05 was considered statistically significant.
CBF-MSC collection and morphology
As shown by H&E stainings, after digestion with dispase and collagenase I, the bone fragments had little to no cells or cellular debris visible (Fig. 1a). Spindle-shaped cells appeared following isolation (Fig. 1b), and a confluent layer of cells was seen within 21 days following primary cell culture (Fig. 1c).
a Hematoxylin-and-eosin images showing the cortical portion of the bone before (left side) and after (right side) digestion. Black arrows indicate canaliculi. Fibroblast-like cells at 72 h (b) and at 5 days (c) after the initial culture. Magnification: 20×. Scale bar: 40 μm
Fluorescence-activated cell sorting analysis
In Fig. 2, the plot of our protocol allowed for the isolation of a heterogeneous cell population with a preponderance of cells displaying phenotypically similar surface markers from the cortical portion of the bone. Within this population, FACS analysis demonstrated that more than 72, 82, and 95 % of cells derived from CBF-MSCs stained positively for the typical MSC marker antigens CD73, CD44, and CD105, respectively (Fig. 2a). Sorting was done by combination staining and multicolor analysis to allow us to see whether cells were co-expressing surface markers that were chosen on the basis of previously published reports [14]. The cells from the three different donors were found to be phenotypically similar and thus a representative display of the expression plots is shown in Fig. 2. As for AD-MSCs and BM-MSCs, CBF-MSCs showed no expression of the hematopoietic markers CD34 and CD45, MHC class II maker HLA-DR, or B-lymphocyte antigen CD19. No differences in the expression of the MSC-precursor associated marker CD271 were found in sorted CBF-MSCs in comparison with BM-MSCs (Fig. 2b).
Immunophenotyping characterization. (a) Flow cytometric analysis showing morphological plots for CBF-MSCs soon after isolation (passage 0). Cells obtained from the cortical portion of the bone represent a homogeneous population that stains positively for mesenchymal stem cell-associated markers (CD44, CD73, and CD105) and negative for hematopoietic (CD45 and CD34)- and the major histocompatibility complex-class II (HLA-DR)- or B-lymphocyte antigen (CD19)-associated markers. Stained cells are represented in green, whereas unstained cells seen in red were used as controls. (b) Quantitative polymerase chain reaction analysis of RNA expression of the common progenitor-associated marker CD271 in BM- and CBF-MSCs. Data are represented as fold-change compared with BM-MSCs. Values are mean ± standard deviation (n = 3). BM-MSC bone marrow mesenchymal stem cell, CBF-MSC cortical bone fragment mesenchymal stem cells
Proliferation rate in normoxic and hypoxic conditions
In the doubling time values obtained in CBF-MSCs at each passage (from P1 to P10), the same trend was observed in normoxic and hypoxic conditions, showing a progressive decreasing value. The doubling time for the first passages was higher than the later passages, and mean values dropped from 4.58 ± 1 and 2.5 ± 0.15 days in normoxia and from 3.87 ± 0.25 to 1.69 ± 0.33 days in hypoxia. As expected, the proliferative capacity of CBF-MSCs in vitro was generally slower than that observed in BM-MSCs and AD-MSCs in normoxic conditions, where the average values have been addressed around 3.24 ± 0.33, 2.5 ± 0.4, and 2.34 ± 0.45 for CBF-MSCs, BM-MSCs, and AD-MSCs, respectively (Fig. 3b).
Cell proliferation in normoxic and hypoxic conditions. a Graph represents the doubling time in normoxic and hypoxic conditions during cell culture for AD-, BM-, and CBF-MSCs expressed as mean value between data obtained from P1 to P10. b Doubling time evaluated on CBF-MSCs at each passage in normoxic and hypoxic conditions. Asterisks represent statistically different doubling time means between normoxia and hypoxia, n = 3: *P < 0.05. AD-MSC adipose tissue mesenchymal stem cell, BM-MSC bone marrow mesenchymal stem cell, CBF-MSC cortical bone fragment mesenchymal stem cells, P passage
When MSC samples were examined and compared for clonogenicity, a significant increase in CFU-Fs was found at higher cell-seeding densities in each of the three cell lines tested (Fig. 4a). However, among AD-, BM-, and CBF-MSCs, statistically significant differences in the clonogenic potential have been found, with CBF-MSCs showing the lowest clonogenicity. CBF-MSCs demonstrated the fewest yet largest CFU-Fs per number of cells plated. In contrast, BM-MSCs and AD-MSCs had three to four times more CFU-Fs detected after plating when applying the same initial plating density. CBF-MSCs were noted to spontaneously differentiate toward the osteogenic lineage and stain positively for ALP (Fig. 4b).
Colony-forming unit (CFU-F) assay. a Graph showing the number of CFU-Fs (expressed as a measure of cell commitment) counted in AD-, BM-, and CBF-MSCs when cells were seeded at different densities. A marked reduction (**P < 0.01) in the number of colonies in CBF-MSCs compared with AD- and BM-MSCs. b Representative images showing colonies formed by CBF-MSCs that stained positively for alkaline phosphatase. Magnification: 20×. Scale bar: 20 μm. AD-MSC adipose tissue mesenchymal stem cell, BM-MSC bone marrow mesenchymal stem cell, CBF-MSC cortical bone fragment mesenchymal stem cells
Chondrogenic and adipogenic differentiation
Chondrogenic differentiation was demonstrated by micromass culture of cells for 21 days in chondrogenic media. After 21 days, all samples, irrespective of their origin, stained positively for Alcian blue, which is known to be a marker for glycosaminoglycans and mucopolysaccharides (Fig. 5a). Adipogenic potential was demonstrated by AD-MSCs, BM-MSCs, or CBF-MSCs in either adipogenic medium over a 14-day period. The three cell populations responded to adipocyte induction by accumulation of positively staining lipid vacuoles in the cytoplasm of the cell (Fig. 5b, yellow arrows).
Differentiation assay. a Chondrogenic (Alcian blue, 20× magnification) and (b) adipogenic (Oil red O, 20× magnification) differentiation of AD-, BM-, and CBF-MSCs. Yellow arrows show intracellular lipidic vacuoles that are representative of adipogenic induction. Magnification: 20×. Scale bar: 40 μm. AD-MSC adipose tissue mesenchymal stem cell, BM-MSC bone marrow mesenchymal stem cell, CBF-MSC cortical bone fragment
Alkaline phosphatase activity
To evaluate the osteogenic phenotype of the cell lines, ALP activity was assessed. Because ALP activity is recognized as an early marker of osteoblastic differentiation, it was measured at 2 weeks [49]. ALP activity was greatest in induced CBF-MSCs, and no significant differences were observed between the AD- and BM-MSCs (Fig. 6). Under hypoxic conditions, all cell types demonstrated increased ALP activity (Fig. 6b). Compared with AD- and BM-MSCs, CBF-MSCs showed nearly fourfold greater ALP activity in hypoxic conditions (P < 0.05). CBF-MSC ALP activity increased compared with the normoxic counterpart (P = 0.0004, Fig. 6a).
Effects of hypoxia on alkaline phosphatase activity in of AD-, BM-, and CBF-MSCs after 2 weeks in induction media. Fluorescent staining of alkaline phosphatase of induced and non-induced cells (control) of all three types in normoxic (a) and hypoxic (b) conditions and respective quantification. Values are mean ± standard deviation (n = 3). Asterisks represent significant differences between induced AD-, BM, and CBF-MSCs: **P < 0.05, ***P < 0.001, ****P = 0.0004. Magnification: 10×. Scale bar: 40 μm. AD-MSC adipose tissue mesenchymal stem cell, BM-MSC bone marrow mesenchymal stem cell, CBF-MSC cortical bone fragment mesenchymal stem cells, CTRL control
Von Kossa staining and quantification
A greater capability of CBF-MSCs to undergo osteoblastogenesis compared with AD-MSCs and BM-MSCs was demonstrated qualitatively and quantitatively by the formation of mineralized nodules as detected by von Kossa staining in normoxic and hypoxic conditions (Figs. 7 and 8). CBF-MSCs had a greater average amount of calcium deposition at both 2 (Fig. 7a) and 4 (Fig. 8a) weeks. The amount of mineralized nodules seen covering the wells in CBF-MSCs at 4 weeks was significantly greater than the average amount of calcium deposited by AD- and BM-MSCs following exposure to both normoxic and hypoxic conditions (Fig. 8). No significant differences were seen among the AD- and BM-MSCs. CBF-MSCs in either normoxic or hypoxic conditions. CBF-MSCs had greater amounts of mineralization compared with AD- and BM-MSCs (Fig. 8b). The increased calcium deposition exhibited by CBF-MSCs after 4 weeks suggests a continued and greatly enhanced potential for mineralization. Bone marrow-derived MSCs decreased their calcium deposition after 4 weeks in comparison with 2-week calcium deposition.
Osteogenic potential assessed on AD-, BM-, and CBF-MSCs after 2 weeks in induction media. Cells were cultured in normoxic (a) and hypoxic (b) conditions. Histological images showing mineral deposits produced by the three cell types. Cells grown in standard media for the same period are also shown (second and fourth rows). Magnification: 10×. Scale bar: 40 μm. (c) Quantification of mineral deposition following osteogenic induction in normoxia and hypoxia conditions represented as a percentage of total well area. Values are mean ± standard deviation (n = 3). Asterisks depict highly significant differences (P < 0.0005) between CBF-MSCs and AD-MSCs and BM-MSCs. AD-MSC adipose tissue mesenchymal stem cell, BM-MSC bone marrow mesenchymal stem cell, CBF-MSC cortical bone fragment
Osteogenic potential assessed on AD-, BM-, and CBF-MSCs after 4 weeks in induction media. Cells were cultured in normoxic (a) and hypoxic (b) conditions. von Kossa stainings showing mineral deposits produced by the three cell types. Cell grown in standard media for the same period are also showed (second and fourth rows). Magnification: 10×. Scale bar: 40 μm. c Quantification of mineral deposition following osteogenic induction in normoxia and hypoxia represented as a percentage of total well area. Values are mean ± standard deviation (n = 3). Asterisks depict highly significant differences (P < 0.0005) between CBF-MSCs and AD-MSCs and BM-MSCs. AD-MSC adipose tissue mesenchymal stem cell, BM-MSC bone marrow mesenchymal stem cell, CBF-MSC cortical bone fragment mesenchymal stem cells
Osteoblastogenesis-associated gene expression analysis
The expression of osteogenesis-associated genes confirmed the induction (Fig. 9). When induced, the three cell lines showed a marked upregulation of the tested genes. When CBF-MSCs were cultured in normoxic conditions, the expression levels increased to 13.21 (±1.01)-fold for Bglap, 14.66 (±2.99)-fold for Alp, 46.12 (±4.55)-fold for Spp1, and 14.92 (±1.22)-fold for Runx2 compared with the uninduced counterparts. Significant differences in Runx2 and Spp1 gene expression were found in BM-MSCs and AD-MSCs exposed to normoxic conditions when expression levels were compared with those obtained from CBF-MSCs (Fig. 9a). The hypoxic conditions were generally more effective in inducing osteogenesis for the three cell lines (Fig. 9b). In particular, CBF-MSCs were found to be more prone to differentiate toward the osteogenic lineage in hypoxic conditions. An upregulation of the genes Bglap, Alp, and Spp1 was observed, showing 22.50 (±4.55)-, 46.56 (±7.4)-, and 71.46 (±4.16)-fold increases compared with their uninduced counterparts. The hypoxic conditions seemed not to affect the expression of transcription factor Runx2.
Quantitative polymerase chain reaction analysis for the expression of the osteogenic markers (Bglap, Ssp1, Alp, and Runx2) for AD-, BM-, and CBF-MSCs at 4 weeks of induction in normoxic (a) and hypoxic (b) conditions. Data are represented as fold-change compared with the expression levels found in the uninduced cells, respectively. Values are mean ± standard deviation (n = 3). Asterisks depict significant (P < 0.05) and highly significant (P < 0.01) differences between CBF-MSCs and AD- or BM-MSCs. AD-MSC adipose tissue mesenchymal stem cell, BM-MSC bone marrow mesenchymal stem cell, CBF-MSC cortical bone fragment
Many tissues as sources of MSCs have been described, and for clinical applications, bone marrow is the most frequently used [50, 51]. As translation into practice becomes a reality, the ideal source for the intended application must be established.
Currently, the use of MSCs to promote bone regeneration in the clinical setting is limited to the use of bone marrow or iliac crest bone graft as sources of cells [51]. This has led researchers to identify alternative sources for acquisition of cells. Adipose has emerged as a reliable source because tissue can be easily obtained in larger quantities through lipoaspiration compared with bone marrow aspiration [30]. Based on this, several groups have compared the osteogenic potential of MSCs derived from the adipose tissue with that of MSCs obtained from bone marrow [31, 52–56]. Although results suggesting one source as favorable to another have been mixed [30–32], bone marrow overall was found to be superior as far as ALP activity, mineral deposition, and the expression of genes associated with osteogenesis are concerned [55]. Within the bone, besides the bone marrow [57], other tissues have already been proposed as sources of osteoprogenitor cells, including the cortical [17, 44, 58] and trabecular regions [16, 41–43]. The current literature, however, lacks of a direct comparison between human bone-derived presumptive MSCs in terms of osteogenic potential. Furthermore, a characterization under prolonged hypoxic conditions of CBF-MSCs or comparison of sources including compact bone under prolonged hypoxic conditions has not been reported. According to previously reported evidence for mouse [44] and rat [17] species, cells obtained represented a population which stained positively for the MSC-associated markers tested (CD44, CD105, and CD73) and negatively for the hematopoietic (CD45 and CD34) and the major histocompatibility-class II (HLA-DR) associated markers. In addition, once sorted, CBF-MSCs were analyzed for their expression of a recently described marker CD271 useful to define an MSC precursor subpopulation, in comparison with the gold standard BM-MSCs, thus suggesting, also in humans, the cortical portion of the bone as a rich source of progenitor cells [59, 60]. Despite sharing a similar phenotype with MSCs derived from the bone marrow and the adipose tissue, however, when the osteogenic potential was assessed in vitro our findings suggested the cortical fraction of the bone as a superior source of osteoprogenitor cells to use in clinic for bone healing and bone repair. Compared with AD- and BM-MSCs, CBF-MSCs demonstrated higher activity of ALP following osteogenic induction after 14 days and a greater calcium deposition at both 2 and 4 weeks than their bone marrow counterparts. Results obtained from the evaluation of the ALP activity might suggest the compact bone as a source for MSCs may prime cells for an osteoblastic lineage [17], thus holding significant impact in the field of osteoregenerative tissue engineering. At a molecular level, our data support these observations showing a marked upregulation in Alp, Runx2, and Spp1 expression. Specifically, Alp and Runx2 are considered to play a critical role in the regulation of osteoblastogenesis, whereas Bglap and Spp1 are associated with a more mature differentiation [61–63]. In our study, although the expression of Bglap was similar between BM-MSCs and CBF-MSCs, CBF-MSCs were found to express higher levels of SSP1, confirming the more mature osteoblastogenesis even compared with BM-MSCs [17]. Concomitantly with their marked osteogenic potential compared with the other sources, their reduced ability to form CFU-F corroborates the more committed nature of CBF-MSCs, according to previous evidence reported by us for rat [17]. Doubling time mean values further supported this statement, showing a generally slower proliferation rate for CBF-MSCs compared with the other cell lines in both normoxic and hypoxic conditions. As previously hypothesized for rat CBF-MSCs, the increased proliferation rate observed in human CBF-MSCs overtime might reflect the resting nature of osteoprogenitor cells lying on the bone surface, further confirming their superior osteogenic commitment. [17].
From the standpoint of translation, these findings have profound implications. One great downfall of transplanted stem cell therapeutics in tissue engineering is their inability to survive the harsh hypoxic environment of the recipient tissue [64–66]. Their ability to survive in an environment that is deprived of both oxygen and nutrient supply is necessary for successful tissue repair and angiogenesis [67]. Similarly, tissue engineering of cell-seeded constructs above a critical-size defect is often problematic because availability of oxygen and nutrient is limited to imbibition in the avascular recipient environment. Because of their increased and persistent biosynthetic activity in prolonged hypoxic conditions, the CBF-MSCs may be better suited for orthopedic tissue engineering of critical-size defects and other traumatic injuries compared with other sources of MSCs. One potential mechanism for the differences in osteogenesis seen here could be differential activation of hypoxia response pathways. Hypoxia-inducible factor (HIF) is the major regulator of cellular hypoxic responses and is known to be upregulated in MSCs [68]. Furthermore, it has been reported that HIF is essential for peripheral blood MSC mobilization seen in hypoxia [69]. A deeper understanding of the activation of hypoxia response pathways in the CBF-MSCs may elucidate the mechanisms leading to the enhanced biosynthetic activity and is the subject of our ongoing investigations in this cell population.
Our approach may not be suited for all orthopedic reconstruction as many times bone is not reamed or removed and thus a second procedure would be necessary. We believe, though, that in operative scenarios where, as part of the standard procedure, bone is exposed, resected, and discarded, fragments can be processed to isolate cells. Procedures such as spinal fusions, laminectomies, and traumatic orthopedic reconstruction would be ideal candidates for harvesting exposed bone tissue for cell isolation and potential re-implantation. Thus, the patient would be spared from additional procedures. These pieces previously thought of as waste house a population of cells with the potential for dramatic augmentation of bony regeneration without the need for an additional invasive procedure for the acquisition of tissue for processing and isolation of cells. More work needs to be done on the molecular characterization of these cells and how the effect of the tissue they originate from affects their commitment. As Shafiee et al. demonstrated with the preferred differentiation of AD-MSCs into adipocytes, this study represents a further example of how, regardless of phenotypic characterization prior to induction, the source the cells are derived from plays a critical role in their ultimate performance in translational applications [56]. Limitations to this and many other studies of its type are the conditions of cell culture used in this investigation. Generally speaking, the environment of intended use is three-dimensional (3D) with mechanical forces and cellular interactions [70, 71]. We acknowledge that to draw more relevant conclusions in vitro, culture conditions using bio-mimetic 3D scaffolds for growth should be sought.
In conclusion, our study demonstrates that quantitative differences between MSCs acquired from bone marrow, adipose, and the cortical portion of the bone exist with respect to their osteogenic potential. These differences are further augmented under conditions of prolonged low-oxygen tension. Our findings suggest the compact bone as a suitable candidate to replace bone marrow as the preferred cell source in selected situations of orthopedic tissue engineering and regenerative medicine. This confirms the presence of lineage predisposition in different stromal cell compartments that influences their ultimate differentiation and potential for use in clinical scenarios [72, 73]. Thus, the choice of cell source must be based on the intended surgical application and accessibility of patient tissue. In this setting, the identification of tissue-specific MSC epigenetic signature may help in the development of markers that are predictive of the in vivo biologic activity of MSCs and could potentially be used to screen MSCs prior to their use.
Although MSCs from various sources have been identified, the optimal source for orthopedic regeneration has yet to be identified. The cortical fraction of the bone has been shown to house a homogeneous population of cells with distinct immunologic and phenotypic characteristics. Compared with BM-MSCs and AD-MSCs, CBF-MSCs retain superior osteogenic potential in both normoxia and hypoxia. Altogether these properties may contribute to enhance regenerative potential in the harsh microenvironments of tissue engineered constructs and healing critical-size defects. More research needs to be done in vivo to characterize this cell source as the optimal source for orthopedic regeneration.
α-MEM:
Alpha-modified Eagle's medium
AD-MSC:
Adipose tissue mesenchymal stem cell
ALP:
Alkaline phosphatase
Bglap:
Osteocalcin
BM-MSC:
Bone marrow mesenchymal stem cell
CBF-MSC:
Cortical bone fragment- mesenchymal stem cells
CFU-F:
Colony-forming unit-fibroblastic-like
Dulbecco's modified Eagle's medium
FACS:
Hemotoxylin and eosin
HIF:
Hypoxia-inducible factor
MSC:
Runx2:
Runt-related transcription factor 2
Spp1:
Dominici M, Le Blanc K, Mueller I, Slaper-Cortenbach I, Marini F, Krause D, et al. Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement. Cytotherapy. 2006;8(4):315-7. doi:10.1080/14653240600855905.
Abbott S, Mackay G, Durdy M, Solomon S, Zylberberg C. Twenty years of the International Society for Cellular Therapies: the past, present and future of cellular therapy clinical development. Cytotherapy. 2014;16(4 Suppl):S112-9. doi:10.1016/j.jcyt.2014.01.001.
Simmons PJ, Leavesley DI, Levesque JP, Swart BW, Haylock DN, To LB, et al. The mobilization of primitive hemopoietic progenitors into the peripheral blood. Stem Cells. 1994;12 Suppl 1:187-201; discussion -2.
Friedenstein AJ, Petrakova KV, Kurolesova AI, Frolova GP. Heterotopic of bone marrow. Analysis of precursor cells for osteogenic and hematopoietic tissues. Transplantation. 1968;6(2):230-47.
Riekstina U, Muceniece R, Cakstina I, Muiznieks I, Ancans J. Characterization of human skin-derived mesenchymal stem cell proliferation rate in different growth conditions. Cytotechnology. 2008;58(3):153-62. doi:10.1007/s10616-009-9183-2.
Wang Y, Zhao L, Hantash BM. Support of human adipose-derived mesenchymal stem cell multipotency by a poloxamer-octapeptide hybrid hydrogel. Biomaterials. 2010;31(19):5122-30. doi:10.1016/j.biomaterials.2010.03.007.
Jin HJ, Bae YK, Kim M, Kwon SJ, Jeon HB, Choi SJ, et al. Comparative analysis of human mesenchymal stem cells from bone marrow, adipose tissue, and umbilical cord blood as sources of cell therapy. Int J Mol Sci. 2013;14(9):17986-8001. doi:10.3390/ijms140917986.
Bieback K, Kern S, Kluter H, Eichler H. Critical parameters for the isolation of mesenchymal stem cells from umbilical cord blood. Stem Cells. 2004;22(4):625-34. doi:10.1634/stemcells. 22-4-625.
Gutierrez-Aguirre CH, Gomez-De-Leon A, Alatorre-Ricardo J, Cantu-Rodriguez OG, Gonzalez-Llano O, Jaime-Perez JC, et al. Allogeneic peripheral blood stem cell transplantation using reduced-intensity conditioning in an outpatient setting in ABO-incompatible patients: are survival and graft-versus-host disease different? Transfusion. 2014;54(5):1269-77. doi:10.1111/trf.12466.
Lovati AB, Corradetti B, Cremonesi F, Bizzaro D, Consiglio AL. Tenogenic differentiation of equine mesenchymal progenitor cells under indirect co-culture. The International journal of artificial organs. 2012;35(11):996-1005. doi:10.5301/ijao.5000129.
Lovati AB, Corradetti B, Lange Consiglio A, Recordati C, Bonacina E, Bizzaro, D et al. Characterization and differentiation of equine tendon-derived progenitor cells. Journal of biological regulators and homeostatic agents. 2011;25(2 Suppl):S75-84.
Corradetti B, Meucci A, Bizzaro D, Cremonesi F, Lange Consiglio A. Mesenchymal stem cells from amnion and amniotic fluid in the bovine. Reproduction. 2013;145(4):391-400. doi:10.1530/REP-12-0437.
Lange-Consiglio A, Corradetti B, Bizzaro D, Magatti M, Ressel L, Tassan S, et al. Characterization and potential applications of progenitor-like cells isolated from horse amniotic membrane. Journal of tissue engineering and regenerative medicine. 2012;6(8):622-35. doi:10.1002/term.465.
Rutigliano L, Corradetti B, Valentini L, Bizzaro D, Meucci A, Cremonesi F, et al. Molecular characterization and in vitro differentiation of feline progenitor-like amniotic epithelial cells. Stem cell research & therapy. 2013;4(5):133. doi:10.1186/scrt344.
Qiao S, Ren H, Shi Y, Liu W. Allogeneic compact bone-derived mesenchymal stem cell transplantation increases survival of mice exposed to lethal total body irradiation: a potential immunological mechanism. Chin Med J (Engl). 2014;127(3):475-82.
Sottile V, Halleux C, Bassilana F, Keller H, Seuwen K. Stem cell characteristics of human trabecular bone-derived cells. Bone. 2002;30(5):699-704.
Corradetti B, Taraballi F, Powell S, Sung D, Minardi S, Ferrari M et al. Osteoprogenitor cells from bone marrow and cortical bone: understanding how the environment affects their fate. Stem cells and development. 2014;1;24(9):1112-23. doi:10.1089/scd.2014.0351.
Gao X, Usas A, Tang Y, Lu A, Tan J, Schneppendahl J, et al. A comparison of bone regeneration with human mesenchymal stem cells and muscle-derived stem cells and the critical role of BMP. Biomaterials. 2014;35(25):6859-70. doi:10.1016/j.biomaterials.2014.04.113.
Ahmed TA, Hincke MT. Mesenchymal stem cell - based tissue engineering strategies for repair of articular cartilage. Histol Histopathol. 2014;29(6):669-89.
Chen W, Liu J, Manuchehrabadi N, Weir MD, Zhu Z, Xu HH. Umbilical cord and bone marrow mesenchymal stem cell seeding on macroporous calcium phosphate for bone regeneration in rat cranial defects. Biomaterials. 2013;34(38):9917-25. doi:10.1016/j.biomaterials.2013.09.002.
Peng L, Jia Z, Yin X, Zhang X, Liu Y, Chen P, et al. Comparative analysis of mesenchymal stem cells from bone marrow, cartilage, and adipose tissue. Stem cells and development. 2008;17(4):761-73. doi:10.1089/scd.2007.0217.
Rappold I, Ziegler BL, Kohler I, Marchetto S, Rosnet O, Birnbaum D, et al. Functional and phenotypic characterization of cord blood and bone marrow subsets expressing FLT3 (CD135) receptor tyrosine kinase. Blood. 1997;90(1):111-25.
Simmons PJ, Przepiorka D, Thomas ED, Torok-Storb B. Host origin of marrow stromal cells following allogeneic bone marrow transplantation. Nature. 1987;328(6129):429-32. doi:10.1038/328429a0.
Gupta PK, Chullikana A, Parakh R, Desai S, Das A, Gottipamula S, et al. A double blind randomized placebo controlled phase I/II study assessing the safety and efficacy of allogeneic bone marrow derived mesenchymal stem cell in critical limb ischemia. J Transl Med. 2013;11:143. doi:10.1186/1479-5876-11-143.
Bianco P, Robey PG, Simmons PJ. Mesenchymal stem cells: revisiting history, concepts, and assays. Cell Stem Cell. 2008;2(4):313-9. doi:10.1016/j.stem.2008.03.002.
Katz AJ, Tholpady A, Tholpady SS, Shang H, Ogle RC. Cell surface and transcriptional characterization of human adipose-derived adherent stromal (hADAS) cells. Stem Cells. 2005;23(3):412-23. doi:10.1634/stemcells.2004-0021.
Nakagami H, Morishita R, Maeda K, Kikuchi Y, Ogihara T, Kaneda Y. Adipose tissue-derived stromal cells as a novel option for regenerative cell therapy. J Atheroscler Thromb. 2006;13(2):77-81.
Kern S, Eichler H, Stoeve J, Kluter H, Bieback K. Comparative analysis of mesenchymal stem cells from bone marrow, umbilical cord blood, or adipose tissue. Stem Cells. 2006;24(5):1294-301. doi:10.1634/stemcells.2005-0342.
Hjortholm N, Jaddini E, Halaburda K, Snarski E. Strategies of pain reduction during the bone marrow biopsy. Annals of hematology. 2013;92(2):145-9. doi:10.1007/s00277-012-1641-9.
Zuk PA, Zhu M, Ashjian P, De Ugarte DA, Huang JI, Mizuno H, et al. Human adipose tissue is a source of multipotent stem cells. Mol Biol Cell. 2002;13(12):4279-95. doi:10.1091/mbc.E02-02-0105.
De Ugarte DA, Morizono K, Elbarbary A, Alfonso Z, Zuk PA, Zhu M, et al. Comparison of multi-lineage cells from human adipose tissue and bone marrow. Cells Tissues Organs. 2003;174(3):101-9.
Schaffler A, Buchler C. Concise review: adipose tissue-derived stromal cells--basic and clinical implications for novel cell-based therapies. Stem Cells. 2007;25(4):818-27. doi:10.1634/stemcells.2006-0589.
Amos PJ, Kapur SK, Stapor PC, Shang H, Bekiranov S, Khurgel M, et al. Human adipose-derived stromal cells accelerate diabetic wound healing: impact of cell formulation and delivery. Tissue Eng Part A. 2010;16(5):1595-606. doi:10.1089/ten.TEA.2009.0616.
Park JY, Jeon HJ, Kim TY, Lee KY, Park K, Lee ES, et al. Comparative analysis of mesenchymal stem cell surface marker expression for human dental mesenchymal stem cells. Regen Med. 2013;8(4):453-66. doi:10.2217/rme.13.23.
Strioga M, Viswanathan S, Darinskas A, Slaby O, Michalek J. Same or not the same? Comparison of adipose tissue-derived versus bone marrow-derived mesenchymal stem and stromal cells. Stem cells and development. 2012;21(14):2724-52. doi:10.1089/scd.2011.0722.
Casteilla L, Planat-Benard V, Laharrague P, Cousin B. Adipose-derived stromal cells: Their identity and uses in clinical trials, an update. World J Stem Cells. 2011;3(4):25-33. doi:10.4252/wjsc.v3.i4.25.
Valenzuela CD, Allori AC, Reformat DD, Sailon AM, Allen RJ, Jr., Davidson EH, et al. Characterization of adipose-derived mesenchymal stem cell combinations for vascularized bone engineering. Tissue Eng Part A. 2013;19(11-12):1373-85. doi:10.1089/ten.TEA.2012.0323.
Diederichs S, Shine KM, Tuan RS. The promise and challenges of stem cell-based therapies for skeletal diseases: stem cell applications in skeletal medicine: potential, cell sources and characteristics, and challenges of clinical translation. Bioessays. 2013;35(3):220-30. doi:10.1002/bies.201200068.
Rothrauff BB, Tuan RS. Cellular therapy in bone-tendon interface regeneration. Organogenesis. 2014;10(1):13-28. doi:10.4161/org.27404.
Tuan RS. Regenerative medicine in 2012: the coming of age of musculoskeletal tissue engineering. Nat Rev Rheumatol. 2013;9(2):74-6. doi:10.1038/nrrheum.2012.235.
Sakaguchi Y, Sekiya I, Yagishita K, Ichinose S, Shinomiya K, Muneta T. Suspended cells from trabecular bone by collagenase digestion become virtually identical to mesenchymal stem cells obtained from marrow aspirates. Blood. 2004;104(9):2728-35. doi:10.1182/blood-2003-12-4452.
Tuli R, Seghatoleslami MR, Tuli S, Wang ML, Hozack WJ, Manner PA, et al. A simple, high-yield method for obtaining multipotential mesenchymal progenitor cells from trabecular bone. Mol Biotechnol. 2003;23(1):37-49. doi:10.1385/MB:23:1:37.
Tuli R, Tuli S, Nandi S, Wang ML, Alexander PG, Haleem-Smith H et al. Characterization of multipotential mesenchymal progenitor cells derived from human trabecular bone. Stem Cells. 2003;21(6):681-93. doi:10.1634/stemcells.21-6-681.
Zhu H, Guo ZK, Jiang XX, Li H, Wang XY, Yao HY, et al. A protocol for isolation and culture of mesenchymal stem cells from mouse compact bone. Nat Protoc. 2010;5(3):550-60. doi:10.1038/nprot.2009.238.
Short BJ, Brouard N, Simmons PJ. Prospective isolation of mesenchymal stem cells from mouse compact bone. Methods Mol Biol. 2009;482:259-68. doi:10.1007/978-1-59745-060-7_16.
Corradetti B, Correani A, Romaldini A, Marini MG, Bizzaro D, Perrini C, et al. Amniotic membrane-derived mesenchymal cells and their conditioned media: potential candidates for uterine regenerative therapy in the horse. PloS one. 2014;9(10):e111324. doi:10.1371/journal.pone.0111324.
Lange-Consiglio A, Tassan S, Corradetti B, Meucci A, Perego R, Bizzaro D, et al. Investigating the efficacy of amnion-derived compared with bone marrow-derived mesenchymal stromal cells in equine tendon and ligament injuries. Cytotherapy. 2013;15(8):1011-20. doi:10.1016/j.jcyt.2013.03.002.
Corradetti B, Lange-Consiglio A, Barucca M, Cremonesi F, Bizzaro D. Size-sieved subpopulations of mesenchymal stem cells from intervascular and perivascular equine umbilical cord matrix. Cell proliferation. 2011;44(4):330-42. doi:10.1111/j.1365-2184.2011.00759.x.
Golub EE, Boesze-Battaglia K. The role of alkaline phosphatase in mineralization. Current Opinion in Orthopaedics. 2007;18(5):444-8.
Veronesi F, Giavaresi G, Tschon M, Borsari V, Nicoli Aldini N, Fini M. Clinical use of bone marrow, bone marrow concentrate, and expanded bone marrow mesenchymal stem cells in cartilage disease. Stem cells and development. 2013;22(2):181-92. doi:10.1089/scd.2012.0373.
Myeroff C, Archdeacon M. Autogenous bone graft: donor sites and techniques. The Journal of bone and joint surgery American volume. 2011;93(23):2227-36. doi:10.2106/JBJS.J.01513.
De Ugarte DA, Alfonso Z, Zuk PA, Elbarbary A, Zhu M, Ashjian P, et al. Differential expression of stem cell mobilization-associated molecules on multi-lineage cells from adipose tissue and bone marrow. Immunology letters. 2003;89(2-3):267-70.
Park SH, Sim WY, Min BH, Yang SS, Khademhosseini A, Kaplan DL. Chip-based comparison of the osteogenesis of human bone marrow- and adipose tissue-derived mesenchymal stem cells under mechanical stimulation. PloS one. 2012;7(9):e46689. doi:10.1371/journal.pone.0046689.
Kang BJ, Ryu HH, Park SS, Koyama Y, Kikuchi M, Woo HM, et al. Comparing the osteogenic potential of canine mesenchymal stem cells derived from adipose tissues, bone marrow, umbilical cord blood, and Wharton's jelly for treating bone defects. Journal of veterinary science. 2012;13(3):299-310.
Liu TM, Martina M, Hutmacher DW, Hui JH, Lee EH, Lim B. Identification of common pathways mediating differentiation of bone marrow- and adipose tissue-derived human mesenchymal stem cells into three mesenchymal lineages. Stem Cells. 2007;25(3):750-60. doi:10.1634/stemcells.2006-0394.
Shafiee A, Seyedjafari E, Soleimani M, Ahmadbeigi N, Dinarvand P, Ghaemi N. A comparison between osteogenic differentiation of human unrestricted somatic stem cells and mesenchymal stem cells from bone marrow and adipose tissue. Biotechnology letters. 2011;33(6):1257-64. doi:10.1007/s10529-011-0541-8.
Honczarenko M, Le Y, Swierkowski M, Ghiran I, Glodek AM, Silberstein LE. Human bone marrow stromal cells express a distinct set of biologically functional chemokine receptors. Stem Cells. 2006;24(4):1030-41. doi:10.1634/stemcells.2005-0319.
Tydings JD, Martino LJ, Kircher M, Alfred R, Lozman J. The osteoinductive potential of intramedullary canal bone reamings. Current surgery. 1986;43(2):121-4.
Kuci S, Kuci Z, Kreyenberg H, Deak E, Putsch K, Huenecke S, et al. CD271 antigen defines a subset of multipotent stromal cells with immunosuppressive and lymphohematopoietic engraftment-promoting properties. Haematologica. 2010;95(4):651-9. doi:10.3324/haematol.2009.015065.
Jones EA, English A, Kinsey SE, Straszynski L, Emery P, Ponchel F, et al. Optimization of a flow cytometry-based protocol for detection and phenotypic characterization of multipotent mesenchymal stromal cells from human bone marrow. Cytometry Part B, Clinical cytometry. 2006;70(6):391-9. doi:10.1002/cyto.b.20118.
Komori T. Regulation of bone development and extracellular matrix protein genes by RUNX2. Cell and tissue research. 2010;339(1):189-95. doi:10.1007/s00441-009-0832-8.
McKee MD, Pedraza CE, Kaartinen MT. Osteopontin and wound healing in bone. Cells Tissues Organs. 2011;194(2-4):313-9. doi:10.1159/000324244.
Inman CK, Shore P. The osteoblast transcription factor Runx2 is expressed in mammary epithelial cells and mediates osteopontin expression. The Journal of biological chemistry. 2003;278(49):48684-9. doi:10.1074/jbc.M308001200.
Reinecke H, Murry CE. Cell grafting for cardiac repair. Methods Mol Biol. 2003;219:97-112.
Toma C, Pittenger MF, Cahill KS, Byrne BJ, Kessler PD. Human mesenchymal stem cells differentiate to a cardiomyocyte phenotype in the adult murine heart. Circulation. 2002;105(1):93-8.
Liu Y, Ma T. Metabolic regulation of mesenchymal stem cell in expansion and therapeutic application. Biotechnology progress. 2015;31(2):468-81. doi:10.1002/btpr.2034.
Zhang M, Methot D, Poppa V, Fujio Y, Walsh K, Murry CE. Cardiomyocyte grafting for cardiac repair: graft cell death and anti-death strategies. Journal of molecular and cellular cardiology. 2001;33(5):907-21. doi:10.1006/jmcc.2001.1367.
Palomaki S, Pietila M, Laitinen S, Pesala J, Sormunen R, Lehenkari P, et al. HIF-1alpha is upregulated in human mesenchymal stem cells. Stem Cells. 2013;31(9):1902-9. doi:10.1002/stem.1435.
Liu L, Yu Q, Lin J, Lai X, Cao W, Du K, et al. Hypoxia-inducible factor-1alpha is essential for hypoxia-induced mesenchymal stem cell mobilization into the peripheral blood. Stem Cells Dev. 2011;20(11):1961-71. doi:10.1089/scd.2010.0453.
Rocabado M. The importance of soft tissue mechanics in stability and instability of the cervical spine: a functional diagnosis for treatment planning. Cranio : the journal of craniomandibular practice. 1987;5(2):130-8.
Rundell SA, Guerin HL, Auerbach JD, Kurtz SM. Effect of nucleus replacement device properties on lumbar spine mechanics. Spine. 2009;34(19):2022-32. doi:10.1097/BRS.0b013e3181af1d5a.
Corradetti B, Taraballi F, Powell S, Sung D, Minardi S, Ferrari M, et al. Osteoprogenitor cells from bone marrow and cortical bone: understanding how the environment affects their fate. Stem Cells Dev. 2015;24(9):1112-23. doi:10.1089/scd.2014.0351.
Al-Nbaheen M, Vishnubalaji R, Ali D, Bouslimi A, Al-Jassir F, Megges M, et al. Human stromal (mesenchymal) stem cells from bone marrow, adipose tissue and skin exhibit differences in molecular phenotype and differentiation potential. Stem cell reviews. 2013;9(1):32-43. doi:10.1007/s12015-012-9365-8.
This work was supported by the Cullen Trust for Health Care Foundation for stem cell plasticity studies (Project ID: 18130014) and the Brown Foundation (Project ID: 18130011) (to ET). We acknowledge David Haviland and the Flow Cytometry Core of Houston Methodist Research Institute for their excellent support and technical consultation.
Houston Methodist Hospital Department of Surgery, Houston, USA
Joseph S. Fernandez-Moure
& Jeffrey L. Van Eps
Department of Nanomedicine, Houston Methodist Research Institute, 6670 Bertner Avenue, Houston, TX, 77030, USA
, Bruna Corradetti
, Paige Chan
, Jeffrey L. Van Eps
, Trevor Janecek
& Ennio Tasciotti
Department of Life and Environmental Sciences, Università Politecnica delle Marche, via Brecce Bianche, 60131, Ancona, Italy
Bruna Corradetti
Houston Methodist Hospital Department of Orthopedic Surgery, 6565 Fannin Street, Houston, TX, 77030, USA
Bradley K. Weiner
Department of Medicine, Rutgers New Jersey Medical School, 185 South Orange Avenue, Newark, NJ, 07103, USA
Pranela Rameshwar
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Correspondence to Ennio Tasciotti.
JSFM carried out cell isolation and differentiation, flow cytometry, histological studies, data analysis and interpretation, drafting of manuscript, and final approval. BC carried out cell expansion and differentiation, doubling assays, molecular characterization, histological interpretation, manuscript writing, and final approval. PC participated in quantitative polymerase chain reaction data collection, assembly of data (molecular studies and histological studies). JLV participated cell expansion and differentiation, histological staining, and manuscript drafting. TJ participated in maintenance of cells. PR participated in the overall design of the study and interpretation of data and helped edit the manuscript for intellectual and scientific content. BKW is the surgeon who helped provide tissue samples, conceive the project and draft the institutional review board and helped in design and coordination of the overall project. ET conceived the overall project and scope, coordinated participants in the work, and aided in the interpretation of data and drafting of the manuscript. All authors read, made edits as necessary, and approved the final draft.
Joseph S. Fernandez-Moure and Bruna Corradetti contributed equally to this work.
Fernandez-Moure, J.S., Corradetti, B., Chan, P. et al. Enhanced osteogenic potential of mesenchymal stem cells from cortical bone: a comparative analysis. Stem Cell Res Ther 6, 203 (2015) doi:10.1186/s13287-015-0193-z
Cortical bone
Multipotent differentiation ability
Osteogenic potential
Normoxia


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M.S. & Ph.D, 1996, Nuclear Engineering, University of Michigan | Ann Arbor, MI.
B.S., 1992, Nuclear Engineering, University of Michigan | Ann Arbor, MI.
Acting Division Cheif, (2017-Present), Air Force Office of Scientific Research.
Technical Advisor, (2016-Present), Air Force Office of Scientific Research.
Program Element Monitor, (2013-2014), Air Force Office of Scientific Research.
Program Manager, (2009-Present), Air Force Office of Scientific Research.
Senior Scientist, (2001-2003), Science Applications International Corp.
Senior Nuclear Engineer, (1998-2001), Air Force Research Laboratory.
Resident Post-Doctoral Researcher, (1996-1998), National Academy of Sciences.
Fellow, Oct 2016, AFRL, Fellow of the Air Force Research Laboratory - 0.2% of the lab.


Our art is made up of the current "we".
To feel aversion to easy consumption.
To feel the daily life happy.
To feel jealous of someone who is doing well.
To feel impatience to my clumsiness.
To feel convinced when it was possible to make a work peacefully.
What makes it over a long time has meaning.
The work sewn is ourselves.
Emotion at that time, feelings at that time.
At that time we have dialogue with cloth.
It does not come out except what is in us.
We hope they feel something.
© 248NISHIYA. All Rights Reserved.


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Stronger Earnings Are Likely. So What?
Many analysts would be embarrassed of the predictions they regularly make, and the indicators they regularly cite, if they would take ten minutes to analyze the historical data. But then, portfolio managers usually cut their teeth by analyzing companies and the economy based on relationships they are taught are important, rather than questioning whether those relationships actually exist in the numbers. That's not really their fault - they were taught by professors and analysts who don't look at the data either. If you poll 100 academic economists, I'd put money on it that fewer than half could tell you the current GDP of the U.S. within $3 trillion of its actual value*.
This is why analysts still believe that the Federal Funds rate has anything to do with the quantity of bank lending except during liquidity crises (the link between bank reserves and lending was eliminated in the early 1990's when reserve requirements were dropped on everything but demand deposits).
It's why analysts believe that government deficits predictably raise the level of interest rates, and that money growth is closely correlated with inflation (it's growth in unproductive forms of spending, regardless of how that spending is financed, that forces structural shifts in the level of interest rates and inflation on account of government policy).
It's also why analysts believe that short-term S&P 500 earnings growth is actually correlated with returns on the S&P 500 (the correlation between year-over-year earnings growth and annual total returns on the S&P is literally 0.0). That's not to deny that stock prices and earnings are related over the long-term (otherwise P/E ratios would be useless). Rather, it's to emphasize that annual fluctuations in S&P 500 earnings growth are entirely meaningless in driving the S&P.
If you're looking for considerations that have a large impact on investment returns, here's the dynamite: changes in market P/E ratios (particularly from extreme levels to historical norms or opposite extremes) can strongly affect the returns that investors earn over periods as long as 10-30 years. Fluctuations in earnings growth? Not so much. Whether one looks at S&P 500 earnings over 10, 20, 50 or 100 years, earnings have fluctuated within a very well-defined 6% long-term growth channel. Stocks are not a claim on a single year of earnings, and short-term earnings fluctuations around their long-term trend (e.g. peak-to-trough or trough-to-peak movements within that growth channel) have very little relationship with changes in the S&P 500 over the same period.
It's true that stock price changes have some ability to predict earnings. Over the past 50 years, there's been a slight positive correlation (0.12) between the annual return on the S&P 500 and the growth in S&P 500 earnings over the following year. But other indicators are much better for anticipating earnings. For example, the ISM Purchasing Managers Index has a meaningfully positive but not reliably tight 0.56 correlation with S&P 500 earnings growth over the following year. Interestingly, the PMI has a negative correlation with subsequent S&P 500 returns (-0.36). The fact that these correlations go in opposite directions underscores that short-term earnings strength and stock returns don't reliably go hand in hand.
[Geeks note - one might infer that the opposing correlations with the PMI imply that strong earnings accompany weak stock returns. In other words, if A implies both B and C, then B must accompany C. That inference would only be true if both correlations with A were 1.0 or -1.0].
As for valuation metrics, the price/peak-earnings ratio outperforms other commonly used ratios (price/reported earnings, price/operating earnings - though you have to estimate these if you want to go back beyond a couple of decades, price/dividend, price/book, and price/revenue), displaying a -0.34 correlation with S&P 500 returns over the following year. Even this is a fairly weak relationship and is not strongly predictive. Valuations are generally important only for their implications about long-term market returns.
In my view, the quality of market action (an indicator of investors' willingness to take risk) matters profoundly to whether an overvalued market declines, or simply becomes more overvalued. This is why I don't make forecasts. Even at high valuations, there is always the potential for investor risk preferences to shift. While I believe we can adequately measure those shifts, we can't forecast them. So we align our investment positions with the prevailing condition of valuations and market action at each point in time.
Prospects for earnings growth
Last week, the Federal Reserve opted for the expected quarter-point hike in the Federal Funds rate. Looking at history, S&P 500 earnings grew by an average of 19.6% in the 12-months following an initial rate hike, with negligible returns in the S&P 500 over the following 3 and 6 months, and below average returns over the following year. If the Market Climate was unfavorable at the time of the initial hike, there were no net gains on average for the S&P 500 over 3, 6 or 12 month horizons (though a shift to a favorable Climate occasionally made market risk worth taking over some intervening period). As usual, none of that should be taken as a forecast, but it does underscore the fact that earnings growth does not directly translate into investment returns.
Given the historical record, the still relatively strong ISM figures, and other economic factors, it's probable that S&P 500 earnings have not peaked. Though reported S&P 500 earnings (don't get me started on operating earnings) are at a new high of about $52, the current peak of that 6% long-term growth channel is closer to $62, which would require robust profit margin expansion from here, but is not unattainable. Again, that's about a 19% difference, so one doesn't need extraordinary assumptions to accept the prospect of further earnings growth here.
Though the current price/peak earnings multiple is over 21 here, it's tempting to ask, "Well then, if attainable earnings are $62 on the S&P, isn't the real price/peak earnings multiple just 18?" The issue here is that if you base multiples on "attainable" or "projected" earnings, you'd better compare the resulting multiples with historical norms calculated the same way. While the average price/peak earnings multiple is about 14 historically, the average price/"top of channel earnings" multiple is just 12.
So you can compare 21 with 14, or 18 with 12. Either way, valuations are well above average, and earnings multiples are by far the most charitable of all valuation multiples because profit margins and return on equity are currently quite high (making P/Es less extreme than price/revenue and price/book ratios) while dividend payout ratios are quite low (making P/Es less extreme than price/dividend ratios).
June employment probably won't derail the Fed
Last week's employment report came in decidedly weak. As a result, the notion immediately surfaced that the Fed might discontinue its program of rate hikes altogether, or at least postpone it meaningfully.
Don't count on it. If you look at the year-over-year CPI inflation rate, it's already above 3% (it's above 4.5% on the PPI). Even with fairly benign numbers in the upcoming July and August reports, that inflation rate will probably rise to 3.25% and 3.4%, respectively. In other words, the simple change in the inflation rate is already set to outstrip the pace of Fed tightening, even without velocity effects which will probably accelerate short-term inflation pressures. The hawks on the FOMC won't like that, and they were already pushing for more aggressive action months ago.
In short, be careful about what you believe, and when in doubt, stare at the data. At present, the risk of contraction in valuation multiples (particularly given still unfavorable market action) outweighs the very probable growth in earnings here. For those inclined to make predictions, the same strength in the PMI that argues for earnings strength also argues for subpar stock returns, as do the high level of valuations, unfavorable Market Climate, heavy insider selling, advisory sentiment, and other factors.
I don't like making predictions either way (and they're not required anyway). Very simply, if we observe a favorable shift in market action, that potential pressure on valuations will become less important, and despite high valuations, we would be willing to accept at least some speculative exposure to market risk. At present, we remain fully hedged in stocks.
As of last week, the Market Climate in stocks remained characterized by unusually unfavorable valuations and modestly unfavorable market action. Despite the fact that I view the fundamentals of both stocks and the economy with suspicion, those views don't compel other investors to agree (if they did, there would never have been a dot com bubble). If investors adopt a robust preference to accept risk, there is very little in the way of evidence and intellectual debate that will dissuade them.
As always, it's the quality of market action, not the extent or duration of a particular market fluctuation, that conveys information about risk preferences. We've seen very dull market action for a while here, but if it resolves into a broad pattern of accumulation across a wide range of industries, particularly on expanding volume, we would be inclined to lift a portion of our hedges. That's certainly no sort of prediction, but it is important for shareholders to understand that we are not attached to any particular market outlook, and that there is always evidence that could emerge to shift the Market Climate we identify.
For now, the Strategic Growth Fund remains fully invested in a broadly diversified portfolio of stocks, with an offsetting hedge to remove the impact of market fluctuations from the portfolio. What remains is "active risk" - the risk (and potential) for our stocks to behave differently than the broad market. When we are in a fully hedged position, this is always our primary source of risk, as well as our primary source of expected returns.
In bonds, the Market Climate is characterized by modestly unfavorable valuations and unfavorable market action. I continue to view Treasury Inflation Protected Securities as a relatively attractive alternative to straight bonds. In my view, inflation risks and pressures remain much higher than widely assumed, and while I certainly believe that we are likely to observe higher defaults and eventual economic softness that could make Treasury securities a safe haven, I don't believe that we are at that point yet, and we don't observe enough evidence on Market Climate considerations to take a significant exposure to straight Treasuries.
The fact is that the economic environment is bifurcated depending on whether you look at income statements or balance sheets. From an "income statement" perspective, we're likely to observe near term growth in earnings and GDP, but also inflationary pressures as monetary velocity presses higher. From a "balance sheet" perspective, the massive U.S. current account will continue to weigh on longer-term growth prospects for gross domestic investment, and heavy levels of consumer and corporate debt create real risks for a fresh "deleveraging cycle" in the economy, with its attendant defaults. The issue is one of timing, and at this point, the evidence continues to lean toward near term economic strength and inflation pressures, with balance sheet problems exerting themselves on a longer horizon. So while I do believe we'll find ourselves in a substantially more aggressive bond market position perhaps several quarters from now, we still have insufficient evidence to take that position here.
The Strategic Total Return Fund continues to carry a duration of about 3.25 years, primarily in TIPS. Our investment position in precious metals shares is now about 14% due to a combination of appreciation and modest purchases. As such, that position will probably continue to account for much of the day-to-day fluctuation in the Fund. Our strongest source of potential return here would be downward pressure on the U.S. dollar. Our primary risk would be a substantial increase long-term real interest rates, which might help to support the dollar despite large current account imbalances and revaluation pressures on the Chinese and Japanese currencies. If robust, sustained, and long-term GDP growth were likely in the U.S., the risk of higher real interest rates would be more pointed than it appears at present.
In short, though I don't believe that straight Treasury securities are priced to deliver substantial returns in the near term, our ability to hold various bond market alternatives - primarily TIPS and precious metals shares here - substantially expands our flexibility to navigate even an environment featuring inflation pressures and Fed tightening.

redpajama_set_name: RedPajamaCommonCrawl


Pexa has announced it will be exhibiting at Aero India 2019, held on 20-24 February in Bengaluru on stand F2.25. The company supplies a wide range of aerospace approved paints, solid film lubricants, surface treatments, sealants, adhesives and associated application equipment and consumables.
After three years of supplying high-tech surface coatings to India's fast-growing aerospace industry from its UK base, Pexa says it has now established its own company and premises in India. Pexa Aerospace Materials Pvt Ltd is located in South Bangalore close to India's main centres of aerospace production. This new facility will be a full player in the local market, receiving goods in bulk and splitting them down for local supply, stocking products to support customer programmes and moving further into value adding activities such as product mixing. In addition, it will act as the sales agent for Pexa for sales of products into India for export programmes.
Pexa will be exhibiting samples at the event from three of its manufacturers, Socomore, Nycote and Global Mask. Socomore manufactures critical surface treatment products, NDT material, adhesive shims and surface coatings under the Aeroglaze brand. Pexa are available to discuss Socomore's products together with their wide range of aerospace products and are happy to show product samples.
Global mask offers an extensive range of masking products to offer customers a complete solution for masking prior to painting. Their product range includes a full assortment of masking products for all wet paints and powder applications. The range includes, masking tapes, masking discs, masking die cuts, masking plugs and masking caps.


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'Lord of The Rings': Amazon Series Officially Adds 20 New Cast Members Including Benjamin Walker and Peter Mullan
Posted byChristopher Marc December 3, 2020 December 3, 2020 Posted inTelevision NewsTags:Amazon Studios, Benjamin Walker, Lord of The Rings, Middle-Earth, New Zealand, Peter Mullan, Prime Video
Amazon Studios have announced today 20 brand new casting additions to their massive Lord of The Rings series which is currently shooting in Auckland, New Zealand. After a pause due to COVID-19 had resumed filming back in September.
Their massive announcement sees the additions of actors such as Lenny Henry, Peter Mullan (Trainspotting, Children of Men), Lloyd Owen, Augustus Prew, Peter Tait, Benjamin Walker (Heart of The Sea, Abraham Lincoln: Vampire Hunter), Cynthia Addai-Robinson (Arrow, Power), Simon Merrells, Maxim Baldry (finally confirmed by Amazon), Ian Blackburn, Kip Chapman, Anthony Crum, Maxine Cunliffe, Trystan Gravelle, Thusitha Jayasundera, Fabian McCallum, Geoff Morrell, Alex Tarrant, Leon Wadham, and Sara Zwangobani.
They join a cast that already consists of Robert Aramayo, Owain Arthur, Nazanin Boniadi, Tom Budge, Morfydd Clark, Ismael Cruz Córdova, Ema Horvath, Markella Kavenagh, Joseph Mawle, Tyroe Muhafidin, Sophia Nomvete, Megan Richards, Dylan Smith, Charlie Vickers, and Daniel Weyman.
Amazon has yet to give an official synopsis of the series other than it will be taking place during the Second Age and covering the ancient kingdom of Numenor that did not appear in the previous two trilogies.
However, we are hearing that the Amazon series will attempt to deviate from the Peter Jackson series by potentially ignoring the established look of Middle-Earth along with redesigning both familiar creatures and the population. There is also a much more diverse cast this time around which hints this will be different enough from the past two trilogies.
It's looking like ILM and not Weta Digital will be handling the visual effects on the show. There sadly hasn't been even confirmation that Weta Workshop is involved either given that they're shooting in proximity of Weta makes this even more upsetting.
Also, the series set during the Second Age is said to cover Sauron's perversion of the advanced kingdom of Numenor (essentially J.R.R. Tolkien's Atlantis) leading to Morgoth (Middle-Earth's version of Lucifer and the first Dark Lord) cults and a splintering of the forces of man in the island kingdom by fueling their hatred towards the elves because of their immortality. Sauron (a Maia just like the wizards) in a much more pleasing form allows himself to be captured and taken to Numenor corrupting the population from within stoking resentment against the Elves rather than being an unseen villainous force hiding in the shadows.
We're expecting to see the creation of the rings of power and potentially the origins of the nine Nazgul/Ringwraiths that were once men tricked by Sauron to take rings of power turning them into his unnatural servants via dark magic. Three are supposedly Numenorean Lords including the Witch King of Angmar, who is their leader and had a significant role in The Return of The King. It's also expected we'll see the ultimate destruction of Numenor forcing surviving inhabitants to the mainland along with the eventual Last Alliance leading to the defeat of Sauron. The latter I wouldn't expect to happen for a very long time.
I'll be keen to see if they'll also attempt to purse the eastern part of Middle-Earth as that could be an equally interesting part to explore as the two Blue Wizards (Alatar/Morinehtar aka Darkness Slayer and Pallando/Rómestámo aka East Helper) operated there during the Second and Third ages, but have only been briefly referenced in the past. One of the Nazgul, Khamûl The Easterling aka Shadow of The East, is said to be a former Easterling King. We only got a glimpse of the Easterling forces in The Lord of The Rings as they were mostly made up of mercenaries or those loyal to Sauron, who had a strong presence in East along with Morgoth in the First Age.
There are also rumblings of Amazon taking a much more mature tone which wouldn't be shocking given that director John Boorman (Excalibur, Zardoz) had once attempted a live-action film based on Lord of The Rings in the 1970s that would have included sex and hard violence for the era likely would have landed an R-rating. The Second Age is described as extremely dark in comparison to the Third Age with men's lust for wealth/power/immortality combined with what is essentially devil worshiping cults and human sacrifices.
The streaming service has yet to announce when episodes will begin dropping on Prime Video.
SOURCE: AMAZON STUDIOS
'The Mandalorian': Rosario Dawson Would Love The Opportunity To Reprise Ahsoka Tano
'Dune', 'Godzilla vs Kong', 'The Suicide Squad', 'Mortal Kombat', 'Matrix 4', and Other WB Films Heading To HBO Max


CHICAGO -- A 17-year-old girl was shot and critically wounded early Saturday in the West Garfield Park neighborhood.
About 3:20 a.m., she was in the back seat of a vehicle with three other people traveling in the 4300 block of West Kinzie when a brown car started following them, according to Chicago Police.
Someone in the brown car then opened fire, shooting the girl in the back of the head, police said.
The driver took her to Mount Sinai Hospital, where she was listed in critical condition.
Police initially said the girl was shot on a party bus but later corrected that information.


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Home>All Posts>Tag: Danny McBride
'Ant-Man' Sequel, 'Arizona,' 'Anna Karenina' Series and More Available on Home Entertainment
Blu-Ray / DVD ReviewsNews By Angela Dawson|October 15, 2018	|0
By ANGELA DAWSON Front Row Features Movies Ant-Man and The Wasp Marvel Studios' "Ant-Man and The Wasp" lands on Blu-ray and Blu-ray 4K Ultra HD Tuesday Oct. 16. The follow-up to 2015's hit "Ant-Man," the 20th consecutive Marvel Cinematic Universe (MCU) film debuted at No. 1 during its opening wee [...]
News Bites: 'Ivan' Starts, Imogen Poots Signs, 'Arizona' Acquired, Elephant Doc Gathers
Film Features By Angela Dawson|May 2, 2018	|0
Compiled by a Front Row Features Staff Reporter Production has commenced at Pinewood Studios outside London on Disney's "The One and Only Ivan," a live-action/CG hybrid adaptation of the children's book by Katherine Applegate. Directed by Thea Sharrock ("Me before You"), the screenplay is by two- [...]


Design water features make wonderful additions to your outdoor or even your indoor landscape. These decorative items come in a seemingly endless array of styles and sizes. Following are some quick examples of lovely indoor and outdoor water features that you may want to consider for your space.
The first thing that comes to mind when someone mentions design water features is the outdoor landscape. Many of us adore the manmade ponds that are popping up on properties all around the country. This outdoor water feature seems like a practically insurmountable task but these design water features are actually quite easy to install and even easier to maintain.
The ease of installation is connected to the particular product you choose to purchase. I loved that my husband chose a design water feature that was ready to be installed. All we needed to do was create a nice spot for the unit and everything fell into place. In little time at all, our outdoor design water feature was up and running.
Maintenance has been a breeze and I'll give you one quick trick that will make your outdoor pond simple to maintain. Buy fish. Yes, the fish not only add beauty and interest to your outdoor design water feature, they also serve to keep the area clean and healthy as well.
Live in a place that suffers cold winters? Don't fret. Many of the outdoor fish that are used in these design water features hibernate during the cold months. They spring back to their old active selves in the springtime. Not only are the fish useful in maintaining your pond, they are also quite easy to care for themselves.
While the outdoor water feature is quite popular, many homeowners are bringing this natural element into the home. There are some great reasons for doing this. The design water features are not only beautiful they are calming as well. I love our small fountain that we added to the reading room. This element is very relaxing and I adore every moment I have in this space.
You have your choice of styles and designs and I can't even begin to scratch the surface when it comes to finding all of the different options available. My first recommendation is to start browsing online for various products to see what's available. You may find that the perfect design water feature is just a click away. Some other guy they buy some good designs from other Company like EGI Consulting, they had the best services like designing and they are best in Seo web design company in Dallas.


Lounge Pug® Velvet, Mega mammoth bean bags, are the ultimate giant sized comfort pieces for your relaxation space. This beanbag is a substantial round beast of a sofa chair which won't fail to impress. Its shape has been designed to provide excellent comfort whilst lounging out and, due to its size, it's more than possible to snuggle up with your children, pet or your other half.


Tasty Bites for April laziness – with the Herbs!
Spring is sprung and many people are out in the garden 'doing stuff'! If, like me, you prefer being outside to being in the kitchen, then this is the month to bridge that gap between slow cooking of hot, warming, food and the ease of a quick BBQ! In Spring, I always go for tasty bites. Warm. But quick to make.
I am a great believer in not using supermarkets if you can, and instead supporting the amazing small food businesses that we are lucky enough to have in and around Northamptonshire. And this month I would like to introduce you to Waterloo Cottage Farm (http://waterloocottagefarm.co.uk). You will know that I am a huge believer in only eating meat that has been 'kindly lived and kindly killed' and this amazing place meets those requirements for me. And, being an ex environmental lawyer, organic is the way to go for me. So off I popped for a visit and returned with enough goodies to make my first Tasty Bites for Spring!
What do you for each person?
1 large cabbage leaf, some butter/oil/lard, a slice of pork, a few Garlic Chives. And a pan!
Would you like something refreshing with that, to quench your thirst after all that outside work? Try making your own 'lemonade' using Lemon Balm (or Lemon Verbena or both!). There are a number of different ways of making it depending on which herbs you have, how intense you like the taste, and whether you would like to add sweetness.
Simplest way is to make some herbal tea with your Lemon Balm, leave it to cool. Drink it as a cold tea. Add Lemon Verbena or Lemon zest (if you have a lemon but the point about this drink is that you can substitute Lemon flavoured herbs for lemons) if you wish as part of the tea making brew. Add sugar, (or Sweet Cicely or Korean Mint which are great sugar substitutes) if you have a sweet tooth.
Or, for a little more of an intense flavour – take a pint of water, add about 10 long stems of Lemon Balm/Lemon Verbena to the water, bring it to the boil and then gently simmer it for about 10 minutes to get a more intense flavour than with the tea. Add sugar if you wish or the sugar substitute herbs already mentioned (I don't as I prefer a tarter lemon flavour than a sweeter one). Make sure you don't let it burn dry! Then let it cool. Drain it to remove the leaves. Add some fizzy water (for those of us of a retro era, the good old Soda Stream comes in handy here..) to taste.
I always make too much, and let it go cold, and then put it in freezer cubes. Ready to add to fizzy water on another occasion.
Herbs are great for lazy cooks! Like me… I lose the will sometimes to peel and chop garlic and onions (a combination I love) so I always grow Garlic Chives which combine both flavours. And sugar substitutes such as Sweet Cicely and Liquorice/Korean Mint are great for that sweetness of taste if you don't have sugar to hand.
Where can you find the herbs this month?
And look out for the new cookery courses coming up in Sudborough soon!
Dermasonic is a treatment that combines ultrasonic sound waves and LED light technology to improve the skin appearance. Ultrasonic will push products into the skin 400 times better than manual massage. It will also promote cell renewal and repair, firm and tighten skin, promote better skin circulation, and the LED light can treat skin conditions such as Aging, acne & pigmentation. Suitable for all skin types and ages.
I had my session after a Dermaplaning session with Dermatechnix – based in Changes Hair Salon in Thrapston.
After my Dermaplaning, Liz applied a collagen cream.
People that live, work and play in East Northants are being invited to join in with the district's largest game this spring.
Thousands of residents will compete to see if their school, community group or workplace can walk, run, scoot, or cycle the furthest by entering the free 'Beat the Street' challenge that will take place from 27 March to 8 May.
More than 50 special sensors called 'Beat Boxes' will appear from Rushden to Thrapston. Players tap the Beat Boxes with cards and fobs to track their journey and earn points for themselves and their team – the more Beat Boxes people swipe during a journey, the more points they earn.
Schools and community groups will be competing against each other to see if they can travel the furthest, climb the leaderboards and win prizes including sports, crafts and book vouchers.
Families are encouraged to play for their local school while the wider community can create their own teams by emailing team.eastnorthants@beatthestreet.me.
In addition to the leaderboard prizes, registered players can also win weekly 'Lucky Tap' prizes for playing along – including vouchers and experience days.
Beat the Street is being delivered by Intelligent Health and is funded by The National Lottery on behalf of Sport England, and East Northamptonshire Council in partnership with Freedom Leisure, which manage leisure facilities across the district on behalf of the council.
"We are excited to be bringing Beat the Street to East Northamptonshire and we look forward to seeing how far everyone is able to walk, run, cycle and scoot during the challenge.
Want to find out more about Beat the Street? Visit www.beatthestreet.me/eastnorthants for more information.
Mar. 13 Information Pages, Local News, Thrapston & Oundle Comments Off on Do you Struggle with Sleep?
Next comes facial acupressure with a serum suited to your skin type.
Box breathing exercises will be practiced during each treatment and mind clearing.
You will be sent home ready for bed with a sleep balm and pillow spray to use. All this with the addition of Reiki throughout the treatment means total relaxation and a good night's sleep.
The Sweet Dreams package is £80. This includes two x 45 minute long treatments ( to be taken a week apart) and your sleep related items to take home.


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Q: How to batch SQL statements in Postgres using Golang What is the fastest way to send batch requests to Postgres database using Golang? Each request contains 500-200000 rows.
Methods I know about are-
 1. Using database/sql package's transaction Begin, Prepare, Commit.
 2. Sending all data in one statement.
 3. Sending a list of statements using sql.Exec() method.
Is there some other way to send batch requests without making a connection at every statement? If not which is the best way of these?
This question is similar to question at- Golang how do I batch sql statements with package database.sql

A: There is a bit old depesz blog post on that. His programs are Perl scripts, but if you concentrate on SQL... Anyway - from DB perspective, you can use COPY, or INSERT with many rows in VALUES. It looks that around 20 is good choice, but it is worth to test that in your case. If performance is key factor, I would put around 2000-5000 rows per transaction. Also, from DB perspective transaction, and session are two separate things. So you can open session, and to many transactions in it.
For PostgreSQL starting new session per operation is really bad idea - DB spawns new process for each session. One of answers for the question you referred contains this. So you open connection, and then transaction, as it should be done.

redpajama_set_name: RedPajamaStackExchange


Posted on November 27, 2018, in Uncategorized and tagged #Mutt, DNA, Goldendoodle, Great Pyrenees, Rescue dog, Stevie Nicks, Terrier, Wisdom Panel. Bookmark the permalink.	1 Comment.
She is a dog of all dogs. You couldn't ask for more.


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EXCLUSIVE: Family says caretaker stole thousands from elderly veteran
by: Amanda Mueller
Posted: Nov 15, 2019 / 10:15 PM CST / Updated: Nov 19, 2019 / 09:18 AM CST
A Henderson family says a home caretaker victimized their elderly father, an 89-year-old veteran, after stealing thousands from his credit cards and forging checks.
The family says that caretaker is 44-year-old Misty Bennett, who is charged with fraudulent use of a credit card and forgery.
They said they hired Bennett to care for their elderly parents.
But just months after their mother passed away from lung cancer, they knew something was very wrong.
"We thought she was a godsend to our family."
The victim's daughter, Dana Viars says at first, Bennett was not just a godsend, but part of the family.
"We had her come to Christmas dinners, Thanksgiving dinners."
They were all united over the love of their 89-year-old veteran father. And everything seemed picture perfect, until it wasn't.
"All of sudden we started noticing that my father was acting funny."
According to a warrant obtained by Eyewitness News, the bank noticed it too. They called the police.
"They asked him if he had been paying her bills, and he said no."
According to that warrant, Bennett told police at the bank that she had a signed note allowing her to use his account, which the victim denied. But the problems kept piling up.
"We found where she had used my mother's credit card… somehow loaded it on her phone or remembered the number."
Credit card records show over $2,000 worth of purchases on that card, including numerous Ipsy and Birchbox make-up boxes.
On October 30th, police arrested Bennett, but she bonded out. The Viars say they're still fighting.
"What we are told of at this point is that she has another person's finances that she's taking care of…"
They filed an abuse claim with Adult Protective Services, which the Cabinet substantiated.
But they say the damage has already been done to their father.
"He doesn't like to see the bad in people… and he had grown very attached to her. […] He doesn't trust anybody anymore."
Bennett, who is also listed as Misty Walters, will be arraigned in Henderson County Circuit Court on November 26th.
"We just want people to realize that just because you hire somebody to come into your home, it doesn't always mean that they're there for the right reasons. Because we really thought she was there for us. We thought she was part of our family."


Singer-songwriter and frontman for Old '97s, Rhett Miller is rolling through World Cafe Live tonight. The alt-rock/country musician is no stranger to the scene and is sure to put on a masterful show. Grab your tickets and some more information at the WXPN Concert Calendar. You can watch the video for "Most in the Summertime" off his latest record The Traveller, below.
Tonight at the Hard Rock Cafe, WXPN presents our Volunteer Spotlight Concert held in support of the nonprofit Musicians on Call. The event itself is free of admission, but proceeds from purchases of any Hard Rock Heals merchandise will benefit this organization that brings live and recorded music to the bedsides of hospital patients all around our region. You can find more information on the event by clicking here, and also watch a video about MOC below.