Mesenchymal stem cells (MSCs) within their native environment of the stem cell niche in bone receive biochemical stimuli from surrounding cells. These stimuli likely influence how MSCs differentiate ...to become bone precursors. The ability of MSCs to undergo osteogenic differentiation is well established in vitro;however, the role of the natural cues from bone's regulatory cells, osteocytes and osteoblasts in regulating the osteogenic differentiation of MSCs in vivo are unclear. In this study we delineate the role of biochemical signalling from osteocytes and osteoblasts, using conditioned media and co-culture experiments, to understand how they direct osteogenic differentiation of MSCs. Furthermore, the synergistic relationship between osteocytes and osteoblasts is examined by transwell co-culturing of MSCs with both simultaneously. Osteogenic differentiation of MSCs was quantified by monitoring alkaline phosphatase (ALP) activity, calcium deposition and cell number. Intracellular ALP was found to peak earlier and there was greater calcium deposition when MSCs were co-cultured with osteocytes rather than osteoblasts, suggesting that osteocytes are more influential than osteoblasts in stimulating osteogenesis in MSCs. Osteoblasts initially stimulated an increase in the number of MSCs, but ultimately regulated MSC differentiation down the same pathway. Our novel co-culture system confirmed a synergistic relationship between osteocytes and osteoblasts in producing biochemical signals to stimulate the osteogenic differentiation of MSCs. This study provides important insights into the mechanisms at work within the native stem cell niche to stimulate osteogenic differentiation and outlines a possible role for the use of co-culture or conditioned media methodologies for tissue engineering applications.
Osteoporotic bone loss and fracture have long been regarded to arise upon depletion of circulating estrogen, which increases osteoclastogenesis and bone resorption. Osteoblasts from human ...osteoporotic patients also display deficient osteogenic responses to mechanical loading. However, while osteoblasts play an important role in regulating osteoclast differentiation, how this relationship is affected by estrogen deficiency is unknown. This study seeks to determine how mechanically stimulated osteoblasts regulate osteoclast differentiation and matrix degradation under estrogen deficiency. Here, we report that osteoblast-induced osteoclast differentiation (indicated by tartrate-resistant acid phosphatase, cathepsin K, and nuclear factor of activated T cells, cytoplasmic 1) and matrix degradation were inhibited by estrogen treatment and mechanical loading. However, estrogen-deficient osteoblasts exacerbated osteoclast formation and matrix degradation in conditioned medium and coculture experiments. This was accompanied by higher expression of cyclooxygenase-2 and macrophage colony-stimulating factor, but not osteoprotegerin, by osteoblasts under estrogen deficiency. Interestingly, this response was exacerbated under conditions that block the Rho-Rho-associated protein kinase signaling pathway. This study provides an important, but previously unrecognized, insight into bone loss in postmenopausal osteoporosis, whereby estrogen-deficient osteoblasts fail to produce inhibitory osteoprotegerin after mechanical stimulation but upregulate macrophage colony-stimulating factor and cyclooxygenase-2 expression and, thus, leave osteoclast activity unconstrained.
Mechanobiology has underpinned many scientific advances in understanding how biophysical and biomechanical cues regulate cell behavior by identifying mechanosensitive proteins and specific signaling ...pathways within the cell that govern the production of proteins necessary for cell-based tissue regeneration. It is now evident that biophysical and biomechanical stimuli are as crucial for regulating stem cell behavior as biochemical stimuli. Despite this, the influence of the biophysical and biomechanical environment presented by biomaterials is less widely accounted for in stem cell-based tissue regeneration studies. This Review focuses on key studies in the field of stem cell mechanobiology, which have uncovered how matrix properties of biomaterial substrates and 3D scaffolds regulate stem cell migration, self-renewal, proliferation and differentiation, and activation of specific biological responses. First, we provide a primer of stem cell biology and mechanobiology in isolation. This is followed by a critical review of key experimental and computational studies, which have unveiled critical information regarding the importance of the biophysical and biomechanical cues for stem cell biology. This review aims to provide an informed understanding of the intrinsic role that physical and mechanical stimulation play in regulating stem cell behavior so that researchers may design strategies that recapitulate the critical cues and develop effective regenerative medicine approaches.
Summary
This study delineates the time sequence of changes in bone tissue mineralisation in ovariectomised rats. We report that changes in bone mineral distribution arise secondary to the initial ...rapid bone loss but coincide with trabecular thickening. We propose that these changes compensate for elevated stresses in remaining trabeculae after bone resorption.
Introduction
Recent studies have shown that osteoporosis is not simply a disease of bone loss and microarchitectural degradation but that important changes in tissue composition also occur. Such changes may be a secondary response to early bone loss, but the time sequence of changes in bone mineral distribution is not fully understood. The objective of this study was to quantify the temporal effects of estrogen deficiency on trabecular mineral distribution in the tibia of ovariectomised (OVX) rats.
Methods
Weekly in vivo micro-CT scans and morphometric and bone mineral density distribution analyses of the proximal tibia were conducted for the first 4 weeks of estrogen deficiency and then at 8, 14 and 34 weeks.
Results
Here we report that although trabecular bone volume and architecture are significantly deteriorated within the first 4 weeks of estrogen deficiency, there is no change in the distribution of bone mineral within trabeculae during this initial period. The rate of bone loss in OVX animals dramatically reduced between week 4 and week 14, which coincided with the initiation of increases in trabecular thickness and mineralisation in the OVX group.
Conclusions
Together this study reveals for the first time that alterations in bone mineralisation and trabecular thickening arise secondary to the initial rapid bone loss. We propose that these secondary mineralisation changes act to reinforce the trabecular network in an attempt to compensate for the increased loading that ensues after severe bone loss. This study provides an insight into temporal changes in bone mineral distribution in estrogen deficiency.
Current drug treatments for post-menopausal osteoporosis cannot eliminate bone fractures, possibly because the mechanisms responsible for bone loss are not fully understood. Although research within ...various disciplines has significantly advanced the state of knowledge, fundamental findings are not widely understood between different disciplines. For that reason, this paper presents noteworthy experimental findings from discrete disciplines focusing on post-menopausal osteoporosis. These studies have established that, in addition to bone loss, significant changes in bone micro-architecture, tissue composition and micro-damage occur. Cellular processes and molecular signalling pathways governing pathological bone resorption have been identified to a certain extent. Ongoing studies endeavour to determine how such changes are initiated at the onset of oestrogen deficiency. It emerges that, because of the discrete nature of previous research studies, the sequence of events that lead to bone fracture is not fully understood. In this paper, two sequences of multi-scale changes are proposed and the experimental challenges that need to be overcome to fully define this sequence are outlined. Future studies must comprehensively characterize the time sequence of molecular-, cellular- and tissue-level changes to attain a coherent understanding of the events that ultimately lead to bone fracture and inform the future development of treatments for post-menopausal osteoporosis.
Extracellular matrix (ECM) stiffness and cell density can regulate osteoblast differentiation in two dimensional environments. However, it is not yet known how osteoblast-osteocyte differentiation is ...regulated within a 3D ECM environment, akin to that existing in vivo. In this study we test the hypothesis that osteocyte differentiation is regulated by a 3D cell environment, ECM stiffness and cell density. We encapsulated MC3T3-E1 pre-osteoblastic cells at varied cell densities (0.25, 1 and 2 × 106 cells/mL) within microbial transglutaminase (mtgase) gelatin hydrogels of low (0.58 kPa) and high (1.47 kPa) matrix stiffnesses. Cellular morphology was characterised from phalloidin-FITC and 4',6-diamidino-2-phenylindole (DAPI) dilactate staining. In particular, the expression of cell dendrites, which are phenotypic of osteocyte differentiation, were identified. Immunofluorescent staining for the osteocytes specific protein DMP-1 was conducted. Biochemical analyses were performed to determine cell number, alkaline phosphatase activity and mineralisation at 2.5 hours, 3, 21 and 56 days. We found that osteocyte differentiation and the formation of an interconnected network between dendritic cells was significantly increased within low stiffness 3D matrices, compared to cells within high stiffness matrices, at high cell densities. Moreover we saw that this network was interconnected, expressed DMP-1 and also connected with osteoblast-like cells at the matrix surface. This study shows for the first time the role of the 3D physical nature of the ECM and cell density for regulating osteocyte differentiation and the formation of the osteocyte network in vitro. Future studies could apply this method to develop 3D tissue engineered constructs with an osteocyte network in place.
The aim of this study was to evaluate midpalatal suture maturation in adults, as observed in cone beam computed tomography (CBCT) images. CBCT scans from 78 subjects (64 female and 14 male, age range ...from 18 to 66 years) were evaluated. Midpalatal suture maturation was verified on the central cross-sectional axial slice in the superior–inferior dimension of the palate, using methods validated previously. Intra-examiner agreement was analyzed by weighted kappa test. Multinomial logistic regression was used to test whether sex and chronological age (adults <30 years or ≥30 years) could be used as a predictor for the maturational stages of the midpalatal suture. The majority of the adults presented a fused midpalatal suture in the palatine (stage D) and/or maxillary bones (stage E). However, the midpalatal suture was not fused in 12% of the subjects. Sex and chronological age were not significant predictors of the maturational stages of the midpalatal suture. The individual assessment of midpalatal suture maturation by way of CBCT images may provide reliable information critical to making the clinical decision between rapid maxillary expansion and surgically assisted rapid maxillary expansion for the treatment of maxillary atresia in adults.
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