ABSTRACT
Human mesenchymal stromal cells (MSCs) are promising candidates for cell therapy due to their ease of isolation and expansion and their ability to secrete antiapoptotic, pro-angiogenic, and immunomodulatory factors. Three-dimensional (3D) aggregation "self-activates" MSCs to augment their pro-angiogenic and immunomodulatory potential, but the microenvironmental features and culture parameters that promote optimal MSC immunomodulatory function in 3D aggregates are poorly understood. Here, we generated MSC aggregates via three distinct methods and compared them with regard to their (a) aggregate structure and (b) immunomodulatory phenotype under resting conditions and in response to inflammatory stimulus. Methods associated with fast aggregation kinetics formed aggregates with higher cell packing density and reduced extracellular matrix (ECM) synthesis compared to those with slow aggregation kinetics. While all three methods of 3D aggregation enhanced MSC expression of immunomodulatory factors compared to two-dimensional culture, different aggregation methods modulated cells' temporal expression of these factors. A Design of Experiments approach, in which aggregate size and aggregation kinetics were systematically covaried, identified a significant effect of both parameters on MSCs' ability to regulate immune cells. Compared to small aggregates formed with fast kinetics, large aggregates with slow assembly kinetics were more effective at T-cell suppression and macrophage polarization toward anti-inflammatory phenotypes. Thus, culture parameters including aggregation method, kinetics, and aggregate size influence both the structural properties of aggregates and their paracrine immunomodulatory function. These findings underscore the utility of engineering strategies to control properties of 3D MSC aggregates, which may identify new avenues for optimizing the immunomodulatory function of MSC-based cell therapies.
Subject(s)
Mesenchymal Stem Cells , Cell Aggregation , Cell Proliferation , Cells, Cultured , Extracellular Matrix , Immunomodulation , T-LymphocytesABSTRACT
Stem cell-derived organoids and other 3D microtissues offer enormous potential as models for drug screening, disease modeling, and regenerative medicine. Formation of stem/progenitor cell aggregates is common in biomanufacturing processes and critical to many organoid approaches. However, reproducibility of current protocols is limited by reliance on poorly controlled processes (e.g., spontaneous aggregation). Little is known about the effects of aggregation parameters on cell behavior, which may have implications for the production of cell aggregates and organoids. Here we introduce a bioengineered platform of labile substrate arrays that enable simple, scalable generation of cell aggregates via a controllable 2D-to-3D "self-assembly". As a proof-of-concept, we show that labile substrates generate size- and shape-controlled embryoid bodies (EBs) and can be easily modified to control EB self-assembly kinetics. We show that aggregation method instructs EB lineage bias, with faster aggregation promoting pluripotency loss and ectoderm, and slower aggregation favoring mesoderm and endoderm. We also find that aggregation kinetics of EBs markedly influence EB structure, with slower kinetics resulting in increased EB porosity and growth factor signaling. Our findings suggest that controlling internal structure of cell aggregates by modifying aggregation kinetics is a potential strategy for improving 3D microtissue models for research and translational applications.
Subject(s)
Cell Differentiation , Cell Lineage , Embryoid Bodies/cytology , Human Embryonic Stem Cells/cytology , Organoids/cytology , Pluripotent Stem Cells/cytology , Cell Culture Techniques , Cells, Cultured , Humans , Signal TransductionABSTRACT
Despite significant advances in the fabrication of bioengineered scaffolds for tissue engineering, delivery of nutrients in complex engineered human tissues remains a challenge. By taking advantage of the similarities in the vascular structure of plant and animal tissues, we developed decellularized plant tissue as a prevascularized scaffold for tissue engineering applications. Perfusion-based decellularization was modified for different plant species, providing different geometries of scaffolding. After decellularization, plant scaffolds remained patent and able to transport microparticles. Plant scaffolds were recellularized with human endothelial cells that colonized the inner surfaces of plant vasculature. Human mesenchymal stem cells and human pluripotent stem cell derived cardiomyocytes adhered to the outer surfaces of plant scaffolds. Cardiomyocytes demonstrated contractile function and calcium handling capabilities over the course of 21 days. These data demonstrate the potential of decellularized plants as scaffolds for tissue engineering, which could ultimately provide a cost-efficient, "green" technology for regenerating large volume vascularized tissue mass.
Subject(s)
Perfusion/methods , Plant Leaves/chemistry , Plant Vascular Bundle/chemistry , Stem Cells/cytology , Stem Cells/physiology , Tissue Engineering/instrumentation , Tissue Scaffolds , Batch Cell Culture Techniques/instrumentation , Cell-Free System/chemistry , Cells, Cultured , Equipment Design , Extracellular Matrix/chemistry , Humans , Petroselinum/chemistry , Spinacia oleracea/chemistry , Tissue Engineering/methodsABSTRACT
To date, there is no periadventitial drug delivery method available in the clinic to prevent restenotic failure of open vascular reconstructions. Resveratrol is a promising anti-restenotic natural drug but subject to low bioavailability when systemically administered. In order to reconcile these two prominent issues, we tested effects of periadventitial delivery of resveratrol on all three major pro-restenotic pathologies including intimal hyperplasia (IH), endothelium impairment, and vessel shrinkage. In a rat carotid injury model, periadventitial delivery of resveratrol either via Pluronic gel (2-week), or polymer sheath (3-month), effectively reduced IH without causing endothelium impairment and vessel shrinkage. In an in vitro model, primary smooth muscle cells (SMCs) were stimulated with elevated transforming growth factor (TGFß) and its signaling protein Smad3, known contributors to IH. TGFß/Smad3 up-regulated Kruppel-like factor (KLF5) protein, and SMC de-differentiation which was reversed by KLF5 siRNA. Furthermore, TGFß/Smad3-stimulated KLF5 production and SMC de-differentiation were blocked by resveratrol via its inhibition of the Akt-mTOR pathway. Concordantly, resveratrol attenuated Akt phosphorylation in injured arteries. Taken together, periadventitial delivery of resveratrol produces durable inhibition of all three pro-restenotic pathologies - a rare feat among existing anti-restenotic methods. Our study suggests a potential anti-restenotic modality of resveratrol application suitable for open surgery.
Subject(s)
Cell Differentiation/drug effects , Coronary Restenosis/prevention & control , Gene Expression Regulation/drug effects , Muscle, Smooth, Vascular/cytology , Smad3 Protein/metabolism , Stilbenes/pharmacology , Transforming Growth Factor beta/metabolism , Animals , Antioxidants/pharmacology , Cell Proliferation/drug effects , Cells, Cultured , Coronary Restenosis/metabolism , Coronary Restenosis/pathology , Male , Muscle, Smooth, Vascular/drug effects , Muscle, Smooth, Vascular/metabolism , Phosphorylation/drug effects , Rats , Rats, Sprague-Dawley , Resveratrol , Signal Transduction/drug effectsABSTRACT
UNLABELLED: Mesenchymal stem cell (MSC)-based therapies are under broad investigation for applications in tissue repair but suffer from poor cell persistence and engraftment upon transplantation. MSC spheroids exhibit improved survival, anti-inflammatory, and angiogenic potential in vitro, while also promoting vascularization when implanted in vivo. However, these benefits are lost once cells engage the tissue extracellular matrix and migrate from the aggregate. The efficacy of cell therapy is consistently improved when using engineered materials, motivating the need to investigate the role of biomaterials to instruct spheroid function. In order to assess the contribution of adhesivity on spheroid activity in engineered materials and promote the bone-forming potential of MSCs, we compared the function of MSC spheroids when entrapped in Arg-Gly-Asp (RGD)-modified alginate hydrogels to nonfouling unmodified alginate. Regardless of material, MSC spheroids exhibited reduced caspase activity and greater vascular endothelial growth factor (VEGF) secretion compared with equal numbers of dissociated cells. MSC spheroids in RGD-modified hydrogels demonstrated significantly greater cell survival than spheroids in unmodified alginate. After 5 days in culture, spheroids in RGD-modified gels had similar levels of apoptosis, but more than a twofold increase in VEGF secretion compared with spheroids in unmodified gels. All gels contained mineralized tissue 8 weeks after subcutaneous implantation, and cells entrapped in RGD-modified alginate exhibited greater mineralization versus cells in unmodified gels. Immunohistochemistry confirmed more diffuse osteocalcin staining in gels containing spheroids compared with dissociated controls. This study demonstrates the promise of cell-instructive biomaterials to direct survival and function of MSC spheroids for bone tissue engineering applications. SIGNIFICANCE: Mesenchymal stem cell (MSC) spheroids exhibit improved therapeutic potential in vitro compared with dissociated MSCs, yet spheroids are directly injected into tissues, ceding control of cell function to the extracellular matrix and potentially limiting the duration of improvement. Cell delivery using adhesive biomaterials promotes cell retention and function. These studies explored the role of adhesion to the surrounding matrix on spheroid function. When entrapped in an adhesive biomaterial, MSC spheroids exhibited improved survival and proangiogenic growth factor secretion in vitro and bone formation in vivo compared with cells in nonadhesive hydrogels. These findings demonstrate the value of deploying MSC spheroids in instructive biomaterials to improve cell function.
Subject(s)
Mesenchymal Stem Cell Transplantation , Mesenchymal Stem Cells/cytology , Spheroids, Cellular/cytology , Tissue Engineering , Alginates/administration & dosage , Alginates/chemistry , Biocompatible Materials/administration & dosage , Biocompatible Materials/chemistry , Cell Survival/drug effects , Extracellular Matrix , Glucuronic Acid/administration & dosage , Glucuronic Acid/chemistry , Hexuronic Acids/administration & dosage , Hexuronic Acids/chemistry , Humans , Hydrogels/administration & dosage , Hydrogels/chemistry , Mesenchymal Stem Cells/drug effects , Osteogenesis/drug effects , Spheroids, Cellular/drug effectsABSTRACT
Vascular endothelial growth factor (VEGF) spatial and temporal activity must be tightly controlled during angiogenesis to form perfusable vasculature in a healing wound. The native extracellular matrix (ECM) regulates growth factor activity locally via sequestering, and researchers have used ECM-mimicking approaches to regulate the activity of VEGF in cell culture and in vivo. However, the impact of dynamic, affinity-mediated growth factor sequestering has not been explored in detail with biomaterials. Here, we sought to modulate VEGF activity dynamically over time using poly(ethylene glycol) microspheres containing VEGF-binding peptides (VBPs) and exhibiting varying degradation rates. The degradation rate of VBP microspheres conferred a differential ability to up- or down-regulate VEGF activity in culture with primary human endothelial cells. VBP microspheres with fast-degrading crosslinks reduced VEGF activity and signaling, while VBP microspheres with no inherent degradability sequestered and promoted VEGF activity in culture with endothelial cells. VBP microspheres with degradable crosslinks significantly reduced neovascularization in vivo, but neither non-degradable VBP microspheres nor bolus delivery of soluble VBP reduced neovascularization. The covalent incorporation of VBP to degradable microspheres was required to reduce neovascularization in a mouse model of choroidal neovascularization in vivo, which demonstrates a potential clinical application of degradable VBP microspheres to reduce pathological angiogenesis. The results herein highlight the ability to modulate the activity of a sequestered growth factor by changing the crosslinker identity within PEG hydrogel microspheres. The insights gained here may instruct the design and translation of affinity-based growth factor sequestering biomaterials for regenerative medicine applications.
Subject(s)
Microspheres , Neovascularization, Physiologic , Vascular Endothelial Growth Factor A/metabolism , Animals , Choroidal Neovascularization/pathology , Cross-Linking Reagents/chemistry , Disease Models, Animal , Human Umbilical Vein Endothelial Cells/metabolism , Humans , Hydrogel, Polyethylene Glycol Dimethacrylate/pharmacology , Induced Pluripotent Stem Cells/cytology , Mice, Inbred C57BL , Neovascularization, Physiologic/drug effects , Peptides/metabolism , Polyethylene Glycols/chemistry , Signal TransductionABSTRACT
UNLABELLED: Current protocols for inducing osteogenic differentiation in mesenchymal stem/stromal cells (MSCs) in culture for tissue engineering applications depend on the use of biochemical supplements. However, standard in vitro culture conditions expose cells to ambient oxygen concentrations and high levels of serum (21% O2, 10% FBS) that do not accurately recapitulate the physiological milieu. While we and others have examined MSC behavior under hypoxia, the synergistic effect of low serum levels, such as those present in ischemic injury sites, on osteogenic differentiation has not been clearly examined. We hypothesized that a concomitant reduction of serum and O2 would enhance in vitro osteogenic differentiation of MSCs by more accurately mimicking the fracture microenvironment. We show that serum deprivation, in conjunction with hypoxia, potentiates osteogenic differentiation in MSCs. These data demonstrate the role of serum levels in regulating osteogenesis and its importance in optimizing MSC differentiation strategies. HIGHLIGHTS: Serum levels, in addition to hypoxia, have a significant effect on MSC osteogenic differentiation. Both naïve and osteogenically induced MSCs exhibit higher osteogenic markers in reduced serum. MSCs deposit the most calcium under 5% O2 in osteogenic media supplemented with 5% FBS. Standard culture conditions (21% O2, 10% FBS) may not be optimal for MSC osteogenic differentiation.
Subject(s)
Cell Culture Techniques , Cell Differentiation/genetics , Mesenchymal Stem Cells/cytology , Osteogenesis/genetics , Apoptosis/drug effects , Cell Differentiation/drug effects , Cell Hypoxia/drug effects , Cell Hypoxia/genetics , Cell Proliferation , Culture Media, Serum-Free/pharmacology , Humans , Mesenchymal Stem Cells/drug effectsABSTRACT
The presentation and controlled release of bioactive signals to direct cellular growth and differentiation represents a widely used strategy in tissue engineering. Historically, work in this field has primarily focused on the delivery of large cytokines and growth factors, which can be costly to manufacture and difficult to deliver in a sustained manner. There has been a marked increase over the past decade in the pursuit of lipid mediators due to their wide range of effects over multiple cell types, low cost, and ease of scale-up. Lysophosphatidic acid (LPA) and sphingosine-1-phosphate (S1P) are two bioactive lysophospholipids (LPLs) that have gained attention for use as pharmacological agents in tissue engineering applications. While these lipids can have similar effects on cellular response, they possess distinct chemical backbones, mechanisms of synthesis and degradation, and signaling pathways using a discrete set of G-protein-coupled receptors (GPCRs). LPA and S1P predominantly act extracellularly on their GPCRs and can directly regulate cell survival, differentiation, cytokine secretion, proliferation, and migration--each of the important functions that must be considered in regenerative medicine. In addition to these potent physiological functions, these LPLs play pivotal roles in a number of pathophysiological processes. To capitalize on the promise of these molecules in tissue engineering, these lipids have been incorporated into biomaterials for in vivo delivery. Here, we survey the effects of LPA and S1P on both cellular- and tissue-level phenotypes, with an eye toward regulating stem/progenitor cell growth and differentiation. In particular, we examine work that has translational applications for cell-based tissue engineering strategies in promoting cell survival, bone and cartilage engineering, and therapeutic angiogenesis.
Subject(s)
Cell Differentiation/drug effects , Lysophospholipids/pharmacology , Receptors, G-Protein-Coupled/metabolism , Sphingosine/analogs & derivatives , Stem Cells/metabolism , Tissue Engineering/methods , Animals , Cell Movement/drug effects , Humans , Lysophospholipids/metabolism , Sphingosine/metabolism , Sphingosine/pharmacologyABSTRACT
Mesenchymal stem/stromal cells (MSCs) are under examination for use in cell therapies to repair bone defects resulting from trauma or disease. MSCs secrete proangiogenic cues and can be induced to differentiate into bone-forming osteoblasts, yet there is limited evidence that these events can be achieved in parallel. Manipulation of the cell delivery vehicle properties represents a candidate approach for directing MSC function in bone healing. We hypothesized that the biophysical properties of a fibrin gel could simultaneously regulate the proangiogenic and osteogenic potential of entrapped MSCs. Fibrin gels were formed by supplementation with NaCl (1.2, 2.3, and 3.9% w/v) to modulate gel biophysical properties without altering protein concentrations. MSCs entrapped in 1.2% w/v NaCl gels were the most proangiogenic in vitro, yet cells in 3.9% w/v gels exhibited the greatest osteogenic response. Compared to the other groups, MSCs entrapped in 2.3% w/v gels provided the best balance between proangiogenic potential, osteogenic potential, and gel contractility. The contribution of MSCs to bone repair was then examined when deployed in 2.3% w/v NaCl gels and implanted into an irradiated orthotopic bone defect. Compared to acellular gels after 3 weeks of implantation, defects treated with MSC-loaded fibrin gels exhibited significant increases in vessel density, early osteogenesis, superior morphology, and increased cellularity of repair tissue. Defects treated with MSC-loaded gels exhibited increased bone formation after 12 weeks compared to blank gels. These results confirm that fibrin gel properties can be modulated to simultaneously promote both the proangiogenic and osteogenic potential of MSCs, and fibrin gels modified by supplementation with NaCl are promising carriers for MSCs to stimulate bone repair in vivo.
Subject(s)
Fibrin/chemistry , Hydrogels/chemistry , Mesenchymal Stem Cells/metabolism , Neovascularization, Physiologic , Osteogenesis , Humans , Mesenchymal Stem Cells/cytologyABSTRACT
The survival of transplanted cells and their resulting efficacy in cell-based therapies is markedly impaired due to serum deprivation and hypoxia (SD/H) resulting from poor vascularization within tissue defects. Lysophosphatidic acid (LPA) is a platelet-derived growth factor with pleiotropic effects on many cell types. Mesenchymal stromal cells (MSC) exhibit unique secretory and stimulatory characteristics depending on their differentiation state. In light of the potential of MSC in cell-based therapies, we examined the ability of LPA to abrogate SD/H-induced apoptosis in human MSC at increasing stages of osteogenic differentiation in vitro and assessed MSC survival in vivo. Undifferentiated MSC were rescued from SD/H-induced apoptosis by treatment with both 25 and 100 µM LPA. However, MSC conditioned with osteogenic supplements responded to 25 µM LPA, and cells conditioned with dexamethasone-containing osteogenic media required 100 µM LPA. This rescue was mediated through LPA1 in all cases. The addition of 25 µM LPA enhanced vascular endothelial growth factor (VEGF) secretion by MSC in all conditions, but VEGF availability was not responsible for protection against apoptosis. We also showed that codelivery of 25 µM LPA with MSC in alginate hydrogels significantly improved the persistence of undifferentiated MSC in vivo over 4 weeks as measured by bioluminescence imaging. Osteogenic differentiation alone was protective of SD/H-induced apoptosis in vitro, and the synergistic delivery of LPA did not enhance persistence of osteogenically induced MSC in vivo. These data demonstrate that the capacity of LPA to inhibit SD/H-induced apoptosis in MSC is dependent on both the differentiation state and dosage. This information will be valuable for optimizing osteogenic conditioning regimens for MSC before in vivo implementation.
Subject(s)
Apoptosis/drug effects , Cell Differentiation/drug effects , Cytoprotection/drug effects , Lysophospholipids/pharmacology , Mesenchymal Stem Cells/cytology , Animals , Cell Hypoxia/drug effects , Cell Proliferation/drug effects , Cell Survival/drug effects , Culture Media, Serum-Free/chemistry , Humans , Mesenchymal Stem Cells/drug effects , Mesenchymal Stem Cells/metabolism , Mice , Osteogenesis/drug effects , Receptors, Lysophosphatidic Acid/metabolism , Vascular Endothelial Growth Factor A/biosynthesisABSTRACT
Recombinant proteins and cytokines are under broad preclinical and clinical investigation to promote angiogenesis, but their success is limited by ineffective delivery, lack of long-term stability and excessive cost. Mesenchymal stem/stromal cells (MSC) secrete bioactive trophic factors, and thus, may provide an effective alternative to address these challenges. Glycine-Histidine-Lysine (GHK) is a peptide fragment of osteonectin, a matricellular protein with reported proangiogenic potential. We examined the capacity of GHK to up-regulate secretion of proangiogenic factors from human MSC in culture and when covalently coupled to alginate hydrogels. GHK had no apparent cytotoxic effects on MSC in culture over a wide range of concentrations. We detected a dose-dependent increase in vascular endothelial growth factor (VEGF) concentration in media conditioned by GHK-treated MSC, which increased endothelial cell proliferation, migration and tubule formation. We covalently coupled GHK to alginate using carbodiimide chemistry, and human MSC were entrapped in alginate hydrogels to assess VEGF secretion. Similar to monolayer culture, MSC responded to GHK-modified gels by secreting increased concentrations of VEGF and basic fibroblast growth factor compared to unmodified gels. The pre-treatment of MSC with antibodies to α6 and ß1 integrins prior to entrapment in GHK-modified gels abrogated VEGF secretion, suggesting that the proangiogenic response of MSC was integrin-mediated. These data demonstrate that the proangiogenic potential of MSC can be significantly increased by the presentation of GHK with a biodegradable carrier, therefore increasing their clinical potential when used for tissue repair.
Subject(s)
Alginates/pharmacology , Hydrogels/pharmacology , Mesenchymal Stem Cells/metabolism , Oligopeptides/pharmacology , Vascular Endothelial Growth Factor A/metabolism , Cell Adhesion/drug effects , Cell Death/drug effects , Cell Movement/drug effects , Cell Survival/drug effects , Colony-Forming Units Assay , Culture Media, Conditioned/pharmacology , Endothelial Cells/cytology , Endothelial Cells/drug effects , Endothelial Cells/metabolism , Glucuronic Acid/pharmacology , Hexuronic Acids/pharmacology , Humans , Integrins/metabolism , Mesenchymal Stem Cells/drug effects , Mitogens/pharmacologyABSTRACT
Ischemic diseases such as peripheral vascular disease (PVD) affect more than 15% of the general population and in severe cases result in ulcers, necrosis, and limb loss. While the therapeutic delivery of growth factors to promote angiogenesis has been widely investigated, large-scale implementation is limited by strategies to effectively deliver costly recombinant proteins. Multipotent adipose-derived stromal cells (ASC) and progenitor cells from other tissue compartments secrete bioactive concentrations of angiogenic molecules, making cell-based strategies for in situ delivery of angiogenic cytokines an exciting alternative to the use of recombinant proteins. Here, we show that the phospholipid lysophosphatidic acid (LPA) synergistically improves the proangiogenic effects of ASC in ischemia. We found that LPA upregulates angiogenic growth factor production by ASC under two- and three-dimensional in vitro models of serum deprivation and hypoxia (SD/H), and that these factors significantly enhance endothelial cell migration. The concurrent delivery of LPA and ASC in fibrin gels significantly improves vascularization in a murine critical hindlimb ischemia model compared to LPA or ASC alone, thus exhibiting the translational potential of this method. Furthermore, these results are achieved using an inexpensive lipid molecule, which is orders-of-magnitude less costly than recombinant growth factors that are under investigation for similar use. Our results demonstrate a novel strategy for enhancing cell-based strategies for therapeutic angiogenesis, with significant applications for treating ischemic diseases.
Subject(s)
Cell- and Tissue-Based Therapy/methods , Lysophospholipids/pharmacology , Neovascularization, Physiologic/drug effects , Adipose Tissue/cytology , Adult , Cell Movement/drug effects , Endothelial Cells/cytology , Endothelial Cells/drug effects , Fibroblast Growth Factor 2/genetics , Gene Expression Regulation/drug effects , Humans , Ischemia/pathology , Ischemia/physiopathology , Ischemia/therapy , Male , Middle Aged , Receptors, Lysophosphatidic Acid/metabolism , Stem Cells/cytology , Stromal Cells/cytology , Stromal Cells/drug effects , Vascular Endothelial Growth Factor A/geneticsABSTRACT
Fibrin gels are a promising material for use in promoting bone repair and regeneration due to their ease of implant formation, tailorability, biocompatibility, and degradation by natural processes. However, these materials lack necessary osteoconductivity to nucleate calcium, integrate with surrounding bone, and promote bone formation. Polymeric substrata formed from poly(lactide-co-glycolide) (PLG) are widely used in bone tissue engineering. A carbonated apatite layer of bone-like mineral can be successfully grown on the surface of PLG microspheres after a multiday incubation process in modified simulated body fluid. Such coatings improve the osteoconductivity of the polymer, provide nucleation sites for cell-secreted calcium, and enhance the potential osseointegration with host tissue. We examined the capacity of mineralized polymeric microspheres suspended within fibrin hydrogels to enhance the osteoconductivity of fibrin gels and increase the osteogenic potential of these materials. The inclusion of microparticles, both nonmineralized and mineralized, reduced the capacity of mesenchymal stem cells (MSCs) to contract the gel. When cultured in osteogenic media, we detected a near linear increase in both calcium and phosphate incorporation in gels containing mineralized microspheres and entrapped MSCs. The osteoconductivity of acellular fibrin gels with mineralized and nonmineralized microspheres was assessed in a rodent calvarial bone defect over 12 weeks. Compared to untreated rodent calvarial bone defects, we detected significant increases in early vascularization when treated with fibrin gels, with greater vascularization, on average, occurring with gels containing microspheres. We detected a trend for increased bone mineral density in gels containing mineralized microspheres after 12 weeks. These findings demonstrate that the osteoconductivity of fibrin gels can be increased by inclusion of mineralized microspheres, but additional signals may be required to rapidly accelerate bone repair.
Subject(s)
Apatites/chemistry , Fibrin/chemistry , Microspheres , Polymers/chemistry , Apoptosis/physiology , Cell Movement/physiology , Fibrinogen/chemistry , Humans , Mesenchymal Stem Cells/cytology , Osteogenesis/physiology , Thrombin/chemistryABSTRACT
In nature, plants generate protective secondary metabolites in response to environmental stresses. Such metabolites include terpenoid indole alkaloids (TIAs), which absorb UV-B light and serve putatively to protect the plant from harmful radiation. Catharanthus roseus plants, multiple shoot cultures, and cell suspension cultures exposed to UV-B light show significant increases in the production of TIAs, including precursors to vinblastine and vincristine, which have proven effective in the treatment of leukemia and lymphoma. Here, the effect of UV-B light on C. roseus hairy roots was examined. Analysis of alkaloid concentrations up to 168 h after UV-B exposure shows significant increases in the concentrations of lochnericine and significant decreases in the concentration of hörhammericine over time (ANOVA, P < 0.05). Our results also indicate that increasing UV-B exposure time up to 20 min caused significant increases in lochnericine, serpentine, and ajmalicine and a decrease in hörhammericine (t-test, p < 0.05).