Citrate-based materials fuel human stem cells by metabonegenic regulation.
ABSTRACT: A comprehensive understanding of the key microenvironmental signals regulating bone regeneration is pivotal for the effective design of bioinspired orthopedic materials. Here, we identified citrate as an osteopromotive factor and revealed its metabonegenic role in mediating citrate metabolism and its downstream effects on the osteogenic differentiation of human mesenchymal stem cells (hMSCs). Our studies show that extracellular citrate uptake through solute carrier family 13, member 5 (SLC13a5) supports osteogenic differentiation via regulation of energy-producing metabolic pathways, leading to elevated cell energy status that fuels the high metabolic demands of hMSC osteodifferentiation. We next identified citrate and phosphoserine (PSer) as a synergistic pair in polymeric design, exhibiting concerted action not only in metabonegenic potential for orthopedic regeneration but also in facile reactivity in a fluorescent system for materials tracking and imaging. We designed a citrate/phosphoserine-based photoluminescent biodegradable polymer (BPLP-PSer), which was fabricated into BPLP-PSer/hydroxyapatite composite microparticulate scaffolds that demonstrated significant improvements in bone regeneration and tissue response in rat femoral-condyle and cranial-defect models. We believe that the present study may inspire the development of new generations of biomimetic biomaterials that better recapitulate the metabolic microenvironments of stem cells to meet the dynamic needs of cellular growth, differentiation, and maturation for use in tissue engineering.
Project description:Biomaterial approaches for engineering orthopedic interfaces such as the tendon-bone junction (TBJ) are limited by a lack of understanding of how insoluble (microstructure, composition) and soluble regulators of stem cell fate work in concert to promote bioactivity and differentiation. One strategy for regenerating the interface is to design biomaterials containing spatially graded structural properties sufficient to induce divergent mesenchymal stem cell (MSC) differentiation into multiple interface-specific phenotypes. This work explores the hypothesis that selective structural modification to a 3D collagen-glycosaminoglycan (CG) scaffold combined with biochemical supplementation can drive human bone-marrow-derived MSC differentiation down tenogenic, osteogenic, and chondrogenic lineages. Tenogenic differentiation is enhanced in geometrically anisotropic scaffolds versus a standard isotropic control. Notably, blebbistatin treatment abrogates this microstructurally driven effect. Further, enhanced osteogenic differentiation and new mineral synthesis are achieved by incorporation of a calcium phosphate mineral phase within the CG scaffold along with the use of osteogenic induction media. Finally, chondrogenic differentiation is optimally driven by combining chondrogenic induction media with a reduced density scaffold that promotes increased cellular condensation, significantly higher expression of chondrogenic genes, and increased GAG deposition. Together these data provide critical insight regarding design rules for elements of an integrated biomaterial platform for orthopedic interface regeneration.
Project description:Citrate is essential to biomineralization of the bone especially as an integral part of apatite nanocomposite. Citrate precipitate of apatite is hypothesized to be derived from mesenchymal stem/stromal cells (MSCs) upon differentiation into mature osteoblasts. Based on 13C-labeled signals identified by solid-state multinuclear magnetic resonance analysis, boosted mitochondrial activity and carbon-source replenishment of tricarboxylic acid cycle intermediates coordinate to feed forward mitochondrial anabolism and deposition of citrate. Moreover, zinc (Zn2+) is identified playing dual functions: (i) Zn2+ influx is influenced by ZIP1 which is regulated by Runx2 and Osterix to form a zinc-Runx2/Osterix-ZIP1 regulation axis promoting osteogenic differentiation; (ii) Zn2+ enhances citrate accumulation and deposition in bone apatite. Furthermore, age-related bone loss is associated with Zn2+ and citrate homeostasis; whereas, restoration of Zn2+ uptake alleviates age-associated declining osteogenic capacity and amount of citrate deposition. Together, these results indicate that citrate is not only a key metabolic intermediate meeting the emerging energy demand of differentiating MSCs but also participates in extracellular matrix mineralization, providing mechanistic insight into Zn2+ homeostasis and bone formation.
Project description:Hydrogels derived from naturally occurring polymers are attractive matrix for tissue engineering. Here, we report a biofunctional hydrogel for specific use in bone regeneration by introducing Arg-Gly-Asp (RGD)-containing cell adhesive motifs and phosphorylated serine residues, which are prevalent in native bone extracellular matrix and known to promote osteogenesis by enhancing cell-matrix interactions and hydroxyapatite nucleation, into photopolymerizable methacrylated glycol chitosan (MeGC). Incorporation of phosphoserine into MeGC hydrogels increased the ability of the hydrogels to nucleate mineral on their surfaces. RGD incorporation enhanced cell-matrix interactions by supporting attachment, spreading, and proliferation of bone marrow stromal cells (BMSCs) encapsulated in the hydrogels. Moreover, co-modification of MeGC hydrogels with RGD and phosphoserine synergistically increased osteogenic differentiation of encapsulated BMSCs in vitro. The bone healing capacity of the modified hydrogels was further confirmed in a mouse calvarial defect model. These findings suggest a promising hydrogel platform with a specific microenvironment tailored to promote osteogenesis for clinical bone repair.
Project description:Stem cells are a centerpiece of regenerative medicine research, and the recent development of adult stem cell-based therapy systems has vigorously expanded the scope and depth of this scientific field. The regeneration of damaged and/or degraded bone tissue in orthopedic, dental, or maxillofacial surgery is one of the main areas where stem cells and their regenerative potential could be used successfully, requiring tissue engineering solutions incorporating an ideal stem cell type paired with the correct mechanical support. Our contribution to this ongoing research provides a new model of in vitro osteogenic differentiation using blood-derived stem cells (BDSCs) and rapamycin, visibly expressing typical osteogenic markers within ten days of treatment. In depth imaging studies allowed us to observe the adhesion, proliferation, and differentiation of BDSCs to both titanium and bone scaffolds. We demonstrate that BDSCs can differentiate towards the osteogenic lineage rapidly, while readily adhering to the scaffolds we exposed them to. Our results show that our model can be a valid tool to study the molecular mechanisms of osteogenesis while tailoring tissue engineering solutions to these new insights.
Project description:Phosphoserine aminotransferase (PSAT) is a pyridoxal 5'-phosphate (PLP)-dependent enzyme that catalyzes the conversion of 3-phosphohydroxypyruvate (3-PHP) to 3-phosphoserine (PSer) in an L-glutamate (Glu)-linked reversible transamination reaction. This process proceeds through a bimolecular ping-pong mechanism and in plants takes place in plastids. It is a part of the phosphorylated pathway of serine biosynthesis, one of three routes recognized in plant organisms that yield serine. In this three-step biotransformation, 3-phosphoglycerate (3-PGA) delivered from plastidial glycolysis and Calvin cycle is oxidized by 3-PGA dehydrogenase. Then, 3-PHP is subjected to transamination with Glu to yield PSer and ?-ketoglutarate (AKG). In the last step of the pathway, serine is produced by the action of phosphoserine phosphatase. Here we present the structural characterization of PSAT isoform 1 from Arabidopsis thaliana (AtPSAT1), a dimeric S-shaped protein that truncated of its 71-residue-long chloroplast-targeting signal peptide. Three crystal structures of AtPSAT1 captured at different stages of the reaction: (i) internal aldimine state with PLP covalently bound to the catalytic K265, (ii) holoenzyme in complex with pyridoxamine-5'-phosphate (PMP) after transfer of the amino group from glutamate and (iii) the geminal diamine intermediate state wherein the cofactor is covalently bound to both, K265 and PSer. These snapshots over the course of the reaction present detailed architecture of AtPSAT1 and allow for the comparison of this plant enzyme with other PSATs. Conformational changes of the protein during the catalytic event concern (i) the neighborhood of K265 when the amino group is transferred to the cofactor to form PMP and (ii) movement of the gate-keeping loop (residues 391-401) upon binding of 3-PHP and PSer. The latter conformational change of the loop may likely be one of key elements that regulate catalytic activity of PSATs.
Project description:BACKGROUND:Restoration of massive bone defects remains a huge challenge for orthopedic surgeons. Insufficient vascularization and slow bone regeneration limited the application of tissue engineering in bone defect. The effect of electromagnetic field (EMF) on bone defect has been reported for many years. However, sinusoidal EMF (SEMF) combined with tissue engineering in bone regeneration remains poorly investigated. METHODS:In the present study, we investigated the effect of SEMF and vascular endothelial growth factor (VEGF) on osteogenic and vasculogenic differentiation of rat bone marrow-derived mesenchymal stem cells (rBMSCs). Furthermore, pretreated rBMSC- laden polycaprolactone-hydroxyapatite (PCL/HA) scaffold was constructed and implanted into the subcritical cranial defect of rats. The bone formation and vascularization were evaluated 4 and 12?weeks after implantation. RESULTS:It was shown that SEMF and VEGF could enhance the protein and mRNA expression levels of osteoblast- and endothelial cell-related markers, respectively. The combinatory effect of SEMF and VEGF slightly promoted the angiogenic differentiation of rBMSCs. The proteins of Wnt1, low-density lipoprotein receptor-related protein 6 (LRP-6), and ?-catenin increased in all inducted groups, especially in SEMF + VEGF group. The results indicated that Wnt/?-catenin pathway might participate in the osteogenic and angiogenic differentiation of rBMSCs. Histological evaluation and reconstructed 3D graphs revealed that tissue-engineered constructs significantly promoted the new bone formation and angiogenesis compared to other groups. CONCLUSION:The combinatory effect of SEMF and VEGF raised an efficient approach to enhance the osteogenesis and vascularization of tissue-engineered constructs, which provided a useful guide for regeneration of bone defects.
Project description:Dehydroalanine (Dha) is a nonproteinogenic electrophilic amino acid that is a synthetic intermediate or product in the biosynthesis of several bioactive cyclic peptides such as lantibiotics, thiopeptides, and microcystins. Dha also enables labeling of proteins and synthesis of post-translationally modified proteins and their analogues. However, current chemical approaches to introducing Dha into peptides have substantial limitations. Using in vitro selection, here we show that DNA can catalyze Zn(2+) or Zn(2+)/Mn(2+)-dependent formation of Dha from phosphoserine (pSer), i.e., exhibit pSer lyase activity, a fundamentally new DNA-catalyzed reaction. Two new pSer lyase deoxyribozymes, named Dha-forming deoxyribozymes 1 and 2 (DhaDz1 and DhaDz2), each function with multiple turnover on the model hexapeptide substrate that was used during selection. Using DhaDz1, we generated Dha from pSer within an unrelated linear 13-mer peptide. Subsequent base-promoted intramolecular cyclization of homocysteine into Dha formed a stable cystathionine (thioether) analogue of the complement inhibitor compstatin. These findings establish the fundamental catalytic ability of DNA to eliminate phosphate from pSer to form Dha and suggest that with further development, pSer lyase deoxyribozymes will have broad practical utility for site-specific enzymatic synthesis of Dha from pSer in peptide substrates.
Project description:Hyperbaric oxygen (HBO) therapy has been reported to be beneficial for treating many conditions of inflammation-associated bone loss. The aim of this work was to in vitro investigate the effect of HBO in the course of osteogenesis of human Mesenchymal Stem Cells (MSCs) grown in a simulated pro-inflammatory environment. Cells were cultured with osteogenic differentiation factors in the presence or not of the pro-inflammatory cytokine Tumor Necrosis Factor-? (TNF-?), and simultaneously exposed daily for 60 min, and up to 21 days, at 2,4 atmosphere absolute (ATA) and 100% O2. To elucidate osteogenic differentiation-dependent effects, cells were additionally pre-committed prior to treatments. Cell metabolic activity was evaluated by means of the MTT assay and DNA content quantification, whereas osteogenic and vasculogenic differentiation was assessed by quantification of extracellular calcium deposition and gene expression analysis. Metabolic activity and osteogenic properties of cells did not differ between HBO, high pressure (HB) alone, or high oxygen (HO) alone and control if cells were pre-differentiated to the osteogenic lineage. In contrast, when treatments started contextually to the osteogenic differentiation of the cells, a significant reduction in cell metabolic activity first, and in mineral deposition at later time points, were observed in the HBO-treated group. Interestingly, TNF-? supplementation determined a significant improvement in the osteogenic capacity of cells subjected to HBO, which was not observed in TNF-?-treated cells exposed to HB or HO alone. This study suggests that exposure of osteogenic-differentiating MSCs to HBO under in vitro simulated inflammatory conditions enhances differentiation towards the osteogenic phenotype, providing evidence of the potential application of HBO in all those processes requiring bone regeneration.
Project description:Phosphorylation is an abundant post-translational modification involved in a myriad of cell signaling pathways. Herein, we have engineered the class II lantipeptide synthetase ProcM to generate a variety of peptides containing O-phosphoserine (pSer) and O-phosphothreonine (pThr) residues, either in vitro or in vivo.
Project description:Bone marrow-derived mesenchymal stem cells are an important source of osteoblasts critical for both bone homeostasis and repair. The ability to isolate, or specifically target, mesenchymal stem cells committed to the osteogenic lineage is necessary for orthopedic translational therapy efforts; however the precise molecular signature of these cells remains elusive. Previously, we identified a population of osteoprogenitor cells expressing the Wnt signaling agonist Lgr6, which contributes to the development and regeneration of the mouse digit tip bone. In our present study we build upon this data and investigate the expression of Lgr6 more broadly in the skeleton. We find that Lgr6, and closely related Lgr4, are expressed in mouse primary calvarial cells, bone marrow cells, and bone marrow-derived mesenchymal stem cells. In addition, our data demonstrates that Lgr4 expression is modestly increased throughout the differentiation and mineralization of mesenchymal stem cells. In contrast, we find Lgr6 expression to be strikingly increased upon osteogenic induction and subsequently decreased upon differentiation and mineralization. These findings provide evidence for Lgr6 as a novel marker of osteoprogenitor cells in bone marrow, which could prove useful for isolation of this population toward future research and clinical applications.