Journal of regenerative medicine & tissue engineering最新文献

Background: It has been known for more than 70 years that citrate is a major component of bone; comprising 1-2% weight of bone, and a concentration that is ~5-25-fold greater than the citrate concentration of most other tissues. This relationship exists in humans and in all vertebrates; which reveals that it is an indispensible and essential structural/functional component of bone. However, its implications relating to the structure and properties of bone, to the process of bone formation and regeneration, to bone disorders, and other issues have remained largely unknown and unaddressed. Recent studies have identified citrate as a structural component of the apatite nanocrystal/collagen complex, which is essential for imparting the bone properties of stability, strength, and resistance to fracture. This raises the issues of the status of citrate, and its source in normal bone formation.

Methods: The present report investigated the association of citrate with the hydroxyapatite (mineral) component and with the collagen component of human cortical bone preparations. The bone preparations were subjected to demineralization procedures to extract the mineral component; followed by extraction of the collagen component in the residual demineralized bone. The extracts were assayed for citrate, calcium, and collagen.

Results: The results reveal, for the first time, the existence of two major pools of citrate in bone. One pool comprising ~65-80% of the total citrate is associated with the hydroxyapatite component; and another pool comprising ~20-35% of the total citrate is tightly bound to the collagen component of the apatite nanocrystal/collagen complex.

Conclusions: Citrate is an indispensible chemical and structural component of the apatite nanocrystal/collagen complex; and is required for manifestation of the biomechanical properties of bone. These results lead to a new concept of bone formation in which citrate incorporation ("citration") in concert with mineralization must be included in the process of bone formation. Along with this relationship, osteoblast citrate production has recently been identified as the likely source of citrate. It is now evident that the role of citrate in normal bone formation and its implications in bone disorders and defects, and in bone repair and regeneration, now requires renewed attention and support for much needed research.

Background: Survival and colony formation by transplanted tissue derived connective tissue progenitor cells (CTPs) are thought to be important factors in the success of clinical tissue engineering strategies for bone regeneration. Transplantation of cells into defects larger than a few millimeters expose cells to a profoundly hypoxic environment. This study tested the hypothesis that delaying the onset of hypoxia will improve the survival and performance of CTPs in vitro.

Methods: To mimic declines seen in an avascular in vivo bone defect, colony forming efficiency by marrow derived nucleated cells was assessed under osteogenic conditions. Variation in the rate of oxygen decline from an oxygen tension of 21% to 0.1% oxygen was explored using an incubator with programmable active control of gas concentrations. The effect of doping cultures with defined concentrations of RBCs was also used to evaluate the potential for RBCs to serve as a natural buffer in the setting of declining oxygen levels.

Results: A delay in onset of hypoxia over 96 hours resulted in a 3-fold increase in the relative colony forming efficiency (rCFE) of CTPs as compared to an immediate onset of hypoxia. The presence of RBCs in vitro inhibited the rCFE of CTPs. Given the negative effects of RBCs, methods of RBC removal were evaluated and compared for their effectiveness of RBC removal and retention of colony forming efficiency.

Conclusions: These data suggest that conditions of hypoxia compromise colony forming efficiency in marrow derived CTPs. However, slowing the rate of decline of oxygen preserved colony forming efficiency at levels achieved in a stable normoxic (3% O₂) environment. These data also suggest that RBCs are detrimental to the rCFE of CTPs and that buffy coat is an effective and preferred method for removing RBCs from marrow aspirates while preserving CTPs. These findings may inform clinical strategies for CTP transplantation.

Stem cells are highly proliferating cells that have the potential for differentiation leading to the development of specialized functional cell types. The process of stem cell differentiation requires an increase in the recruitment and population of the undifferentiated stem cells, which are then differentiated to specific functional cell types. Genetic/metabolic transformations in the cellular intermediary energy metabolism are required to provide the bioenergetic, synthetic, and catabolic requirements of the stem cells during this process. However, the identification of the intermediary energy metabolism pathways and their alterations during the proliferation and differentiation of stem cells remain largely unknown; mainly due to the lack of attention and/or required research that focuses on this relationship. In the absence of such information, a full understanding of the factors and conditions required to promote stem cell differentiation leading to development of normal functional metabolic specialized cells cannot be achieved. The purpose of this review is to provide the background and bring attention to the essential relationship of altered cellular intermediary metabolism in the context of the process of stem cell proliferation and differentiation. Citrate metabolism is central to the genetic and metabolic transformation leading to the development of the specialized functional cells. This review identifies the involvement of altered citrate metabolism and the associated genetic alterations of key pathways, enzymes, and transporters; as well as the bioenergetic implications. The importance is emphasized for identification and employment of required conditions to insure that the process of experimental stem cell differentiation results in the development of specialized cells that represent the functional metabolic characteristics and capabilities of their native specialized cells. This is an essential requirement for the successful application of stem cell therapy and regenerative medicine for many pathological conditions.

Background: Pluripotent human embryonic stem cells (hESCs) proffer cures for a wide range of neurological disorders by supplying the diversity of human neuronal cell types in the developing CNS for repair. However, realizing the therapeutic potential of hESC derivatives has been hindered by generating neuronal cells from pluripotent cells through uncontrollable and inefficient multi-lineage differentiation. Previously, we used a defined platform to identify retinoic acid as sufficient to induce the specification of neuroectoderm direct from the pluripotent state of hESCs and trigger uniform neuronal lineage-specific progression to human neuronal progenitors (hESC-I hNuPs) and neurons (hESC-I hNus) in the developing CNS with high efficiency.

Methods: Having achieved uniformly conversion of pluripotent hESCs to a neuronal lineage, in this study, the expression and intracellular distribution patterns of a set of chromatin modifiers in hESC-I hNuPs were examined and compared to the two prototypical neuroepithelial-like human neural stem cells (hNSCs) either derived from hESCs or isolated directly from the human fetal neuroectoderm in vivo.

Results: These hESC-I hNuPs expressed high levels of active chromatin modifiers, including acetylated histone H3 and H4, HDAC1, Brg-1, and hSNF2H, retaining an embryonic acetylated globally active chromatin state. Consistent with this observation, several repressive chromatin remodeling factors regulating histone H3K9 methylation, including SIRT1, SUV39H1, and Brm, were inactive in hESC-I hNuPs. These Nurr1-positive hESC-I hNuPs, which did not express the canonical hNSC markers, yielded neurons efficiently and exclusively, as they did not differentiate into glial cells. Following engraftment in the brain, hESC-I hNuPs yielded well-dispersed and well-integrated human neurons at a high prevalence.

Conclusions: These observations suggest that, unlike the prototypical neuroepithelial-like nestin-positive hNSCs, these in vitro neuroectoderm-derived Nurr1-positive hESC-I hNuPs are a more neuronal lineage-specific and plastic human stem cell derivative, providing an engraftable human embryonic neuronal progenitor in high purity and large supply with adequate neurogenic potential for scale-up CNS regeneration as stem cell therapy to be translated to patients in clinical trials.