Cyclic Tensile Strain Enhances Osteogenesis and Angiogenesis in Mesenchymal Stem Cells from Osteoporotic Donors

We have shown that the uniaxial cyclic tensile strain of magnitude 10% promotes and enhances osteogenesis of human mesenchymal stem cells (hMSC) and human adipose-derived stem cells (hASC) from normal, nonosteoporotic donors. In the present study, MSC from osteoporotic donors were analyzed for changes in mRNA expression in response to 10% uniaxial tensile strain to identify potential mechanisms underlying the use of this mechanical loading paradigm for prevention and treatment of osteoporosis. Human MSC isolated from three female, postmenopausal osteoporotic donors were analyzed for their responses to mechanical loading using microarray analysis of over 47,000 gene probes. Human MSC were seeded in three-dimensional collagen type I constructs to mimic the organic extracellular matrix of bone and 10% uniaxial cyclic tensile strain was applied to promote osteogenesis. Seventy-nine genes were shown to be regulated within hMSC from osteoporotic donors in response to 10% cyclic tensile strain. Upregulation of six genes were further confirmed with real-time RT-PCR: jun D proto-oncogene (JUND) and plasminogen activator, urokinase receptor (PLAUR), two genes identified as potential key molecules from network analysis; phosphoinositide-3-kinase, catalytic, delta polypeptide (PIK3CD) and wingless-type MMTV integration site family, member 5B (WNT5B), two genes with known importance in bone biology; and, PDZ and LIM domain 4 (PDLIM4) and vascular endothelial growth factor A (VEGFA), two genes that we have previously shown are significantly regulated in hASC in response to this mechanical stimulus. Function analysis indicated that 10% cyclic tensile strain induced expression of genes associated with cell movement, cell proliferation, and tissue development, including development in musculoskeletal and cardiovascular systems. Our results demonstrate that hMSC from aged, osteoporotic donors are capable of enhanced osteogenic differentiation in response to 10% cyclic tensile strain with significant increases in the expression of genes associated with enhanced cell proliferation, musculoskeletal development, and angiogenesis. Surprisingly, cyclic tensile strain of magnitude 10% not only enhanced osteogenesis in hMSC from osteoporotic donors, but also enhanced expression of angiogenic factors. Better understanding and methodologies to promote osteogenesis in hMSC from elderly, osteoporotic donors may greatly facilitate achieving long-term success in bone regeneration and functional bone tissue engineering for this ever-growing patient population

Enhanced cellular infiltration of human adipose-derived stem cells in allograft menisci using a needle-punch method

Abstract Background The meniscus plays a crucial role in knee joint stability, load transmission, and stress distribution. Meniscal tears are the most common reported knee injuries, and the current standard treatment for meniscal deficiency is meniscal allograft transplantation. A major limitation of this approach is that meniscal allografts do not have the capacity to remodel and maintain tissue homeostasis due to a lack of cellular infiltration. The purpose of this study was to provide a new method for enhanced cellular infiltration in meniscal allografts. Methods Twenty medial menisci were collected from cadaveric human sources and split into five experimental groups: (1) control native menisci, (2) decellularized menisci, (3) decellularized menisci seeded with human adipose-derived stem cells (hASC), (4) decellularized needle-punched menisci, and (5) decellularized needle-punched menisci seeded with hASC. All experimental allografts were decellularized using a combined method with trypsin EDTA and peracetic acid. Needle punching (1-mm spacing, 28 G microneedle) was utilized to improve porosity of the allograft. Samples were recellularized with hASC at a density of 250 k/g of tissue. After 28 days of in vitro culture, menisci were analyzed for mechanical, biochemical, and histological characteristics. Results Menisci maintained structural integrity and material properties (compressive equilibrium and dynamic moduli) throughout preparations. Increased DNA content was observed in the needle-punched menisci but not in the samples without needle punching. Histology confirmed these results, showing enhanced cellular infiltration in needle-punched samples. Conclusions The enhanced infiltration achieved in this study could help meniscal allografts better remodel post-surgery. The integration of autologous adipose-derived stem cells could improve long-term efficacy of meniscal transplantation procedures by helping to maintain the meniscus in vivo

Betulinic acid decreases lipid accumulation in adipogenesis-induced human mesenchymal stem cells with upregulation of PGC-1α and UCP-1 and post-transcriptional downregulation of adiponectin and leptin secretion

Background Controlling cellular functions, including stem cell growth and differentiation, can be the key for the treatment of metabolic disorders, such as type II diabetes mellitus (T2DM). Previously identified as peroxisome proliferator-activated receptor gamma (PPARγ) antagonist, betulinic acid (BA) may have the capability to control stem cell homeostasis, benefiting T2DM treatment. In this study, the effects of BA on osteogenesis and adipogenesis mechanisms of human mesenchymal stem cells (hMSCs) were investigated. Results We observed that BA increased hMSC osteogenesis by enhancing the alkaline phosphatase activity, calcium deposition, and mRNA expressions of osteogenic markers, namely, runt-related transcription factor 2, osteocalcin, and osteopontin. In addition, BA decreased hMSC adipogenesis with the decrease in glycerol-3-phosphate dehydrogenase activity, reduced intracellular lipid accumulations, down-regulated CCAAT-enhancer-binding protein alpha, and suppressed post-transcriptional adiponectin and leptin secretion. BA increased the brown adipocyte characteristics with the increase in the ratio of small lipid droplets and glucose uptake. Furthermore, the mRNA expressions of brown adipocyte markers, namely, PPARγ coactivator one alpha, uncoupling protein 1, and interleukin-6 increased. Conclusions Our results uncovered the mechanisms of how BA improved glucose and lipid metabolisms by decreasing white adipogenesis and increasing brown adipogenesis. Altogether, BA may be used for balancing glucose metabolisms without the potential side effects on bone loss or weight gain

Distinctions in bone matrix nanostructure, composition, and formation between osteoblast-like cells, MG-63, and human mesenchymal stem cells, UE7T-13

Osteoblast-like cells and human mesenchymal stem cells (hMSCs) are frequently employed as osteoprogenitor cell models for evaluating novel biomaterials in bone healing and tissue engineering. In this study, the characterization of UE7T-13 hMSCs and MG-63 human osteoblast-like cells was examined. Both cells can undergo osteogenesis and produce calcium extracellular matrix; however, calcium nodules produced by MG-63 lacked a central mass and appeared flatter than UE7T-13. The absence of growing calcium nodules in MG-63 was discovered by SEM-EDX to be associated with the formation of alternating layers of cells and calcium extracellular matrix. The nanostructure and composition analysis showed that UE7T-13 had a finer nanostructure of calcium nodules with a higher calcium/phosphate ratio than MG-63. Both cells expressed high intrinsic levels of collagen type I alpha 1 chain, while only UE7T-13 expressed high levels of alkaline phosphatase, biomineralization associated (ALPL). High ALP activity in UE7T-13 was not further enhanced by osteogenic induction, but in MG-63, low intrinsic ALP activity was greatly induced by osteogenic induction. These findings highlight the differences between the two immortal osteoprogenitor cell lines, along with some technical notes that should be considered while selecting and interpreting the pertinent in vitro model

Dual-Analyte Fluorescent Sensor Based on [5]Helicene Derivative with Super Large Stokes Shift for the Selective Determinations of Cu²⁺ or Zn²⁺ in Buffer Solutions and Its Application in a Living Cell

A new fluorescent sensor, <b>M201-DPA</b>, based on [5]­helicene derivative was utilized as dual-analyte sensor for determination of Cu²⁺ or Zn²⁺ in different media and different emission wavelengths. The sensor could provide selective and bifunctional determination of Cu²⁺ in HEPES buffer containing Triton-X100 and Zn²⁺ in Tris buffer/methanol without interference from each other and other ions. In HEPES buffer, <b>M201-DPA</b> demonstrated the selective ON–OFF fluorescence quenching at 524 nm toward Cu²⁺. On the other hand, in Tris buffer/methanol, <b>M201-DPA</b> showed the selective OFF–ON fluorescence enhancement upon the addition of Zn²⁺, which was specified by the hypsochromic shift at 448 nm. Additionally, <b>M201-DPA</b> showed extremely large Stokes shifts up to ∼150 nm. By controlling the concentration of Zn²⁺ and Cu²⁺ in a living cell, the imaging of a HepG2 cellular system was performed, in which the fluorescence of <b>M201-DPA</b> in the blue channel was decreased upon addition of Cu²⁺ and was enhanced in UV channel upon addition of Zn²⁺. The detection limits of <b>M201-DPA</b> for Cu²⁺ and Zn²⁺ in buffer solutions were 5.6 and 3.8 ppb, respectively. Importantly, the Cu²⁺ and Zn²⁺ detection limits of the developed sensors were significantly lower than permitted Cu²⁺ and Zn²⁺ concentrations in drinking water as established by the U.S. EPA and WHO

Cyclic Tensile Strain Enhances Osteogenesis and Angiogenesis in Mesenchymal Stem Cells from Osteoporotic Donors

We have shown that the uniaxial cyclic tensile strain of magnitude 10% promotes and enhances osteogenesis of human mesenchymal stem cells (hMSC) and human adipose-derived stem cells (hASC) from normal, nonosteoporotic donors. In the present study, MSC from osteoporotic donors were analyzed for changes in mRNA expression in response to 10% uniaxial tensile strain to identify potential mechanisms underlying the use of this mechanical loading paradigm for prevention and treatment of osteoporosis. Human MSC isolated from three female, postmenopausal osteoporotic donors were analyzed for their responses to mechanical loading using microarray analysis of over 47,000 gene probes. Human MSC were seeded in three-dimensional collagen type I constructs to mimic the organic extracellular matrix of bone and 10% uniaxial cyclic tensile strain was applied to promote osteogenesis. Seventy-nine genes were shown to be regulated within hMSC from osteoporotic donors in response to 10% cyclic tensile strain. Upregulation of six genes were further confirmed with real-time RT-PCR: jun D proto-oncogene (JUND) and plasminogen activator, urokinase receptor (PLAUR), two genes identified as potential key molecules from network analysis; phosphoinositide-3-kinase, catalytic, delta polypeptide (PIK3CD) and wingless-type MMTV integration site family, member 5B (WNT5B), two genes with known importance in bone biology; and, PDZ and LIM domain 4 (PDLIM4) and vascular endothelial growth factor A (VEGFA), two genes that we have previously shown are significantly regulated in hASC in response to this mechanical stimulus. Function analysis indicated that 10% cyclic tensile strain induced expression of genes associated with cell movement, cell proliferation, and tissue development, including development in musculoskeletal and cardiovascular systems. Our results demonstrate that hMSC from aged, osteoporotic donors are capable of enhanced osteogenic differentiation in response to 10% cyclic tensile strain with significant increases in the expression of genes associated with enhanced cell proliferation, musculoskeletal development, and angiogenesis. Surprisingly, cyclic tensile strain of magnitude 10% not only enhanced osteogenesis in hMSC from osteoporotic donors, but also enhanced expression of angiogenic factors. Better understanding and methodologies to promote osteogenesis in hMSC from elderly, osteoporotic donors may greatly facilitate achieving long-term success in bone regeneration and functional bone tissue engineering for this ever-growing patient population