Hydrogel biophysical properties instruct coculture‐mediated osteogenic potential

Murphy, Kaitlin C.; Stilhano, Roberta Sessa; Mitra, Debika; Zhou, Daohong; Batarni, Samir; Silva, Eduardo A.; Leach, J. Kent

doi:10.1096/fj.15-279984

Cited by 18 publications

(20 citation statements)

References 48 publications

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“…The supplementation of the pre-gel solution with NaCl provides a means to enhance hydrogel material properties, is rapidly eluted over 24 hours and does not alter the osmolality of the cell microenvironment, and does not impair cell viability [20]. We previously demonstrated that changing the NaCl content in the pre-gel solution alters gel stiffness, pore size, fiber diameter, and permeability [20–22]. While the mechanism of how NaCl affects material properties is not fully characterized, thrombin possesses Na + binding sites, which may regulate activation kinetics and network organization.…”

Section: Discussionmentioning

confidence: 99%

“…Diabetic HMVECs were expanded in standard culture conditions in EGM-2 MV media with Lonza’s SingleQuot supplements (hydrocortisone, gentamycin, VEGF, bFGF, EGF, insulin-like growth factor [IGF], and heparin) and further supplemented with 5% FBS and 1% P/S. Growth-factor deficient media (GF-Def EGM-2 MV) was prepared with serum-containing EGM-2 but lacking VEGF, FGF, and IGF [10, 22]. Raw264.7 murine macrophages (ATCC, Manassas, VA) were used without further characterization and expanded as adherent cultures in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% FBS and 1% P/S.…”

Section: Methodsmentioning

confidence: 99%

“…The contraction of fibrin gels due to activity of entrapped MSCs was measured by visually following morphologic changes in gel area. MSC spheroid-containing gels were imaged over 7 days in culture using a Canon PowerShot A470 camera, and the area of each construct were measured using ImageJ [21, 22]. To visualize the gross morphology of acellular fibrin hydrogels scanning electron microscopy (Hitachi 3500-N, Hitachi Science Systems Ltd, Tokyo, Japan) was used at 3 kV.…”

Section: Methodsmentioning

confidence: 99%

“…To test the bioactivity of conditioned media (CM) from spheroids entrapped in fibrin gels, we measured the mitogenic response of diabetic HMVECs as we previously described [22]. Briefly, diabetic HMVECs were seeded at 7,500 cells/cm 2 in EGM-2 MV in 12-well culture plates and allowed to attach overnight.…”

Section: Methodsmentioning

confidence: 99%

“…Fibrin is found naturally in the body as a scaffold for leukocytes and endothelial cells during tissue regeneration [17, 18]. Additionally, the bulk stiffness, degradability, and porosity of fibrin gels can be easily tailored to direct the lineage-specific differentiation and secretome of entrapped MSCs [19–22]. Compared to hydrogels formed from collagen that is found in mature tissues, fibrin gels direct associated cells to secrete reparative growth factors and extracellular matrix components to stimulate tissue repair [23].…”

Section: Introductionmentioning

confidence: 99%

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Engineering fibrin hydrogels to promote the wound healing potential of mesenchymal stem cell spheroids

et al. 2017

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Mesenchymal stem cells (MSCs) secrete endogenous factors such as vascular endothelial growth factor (VEGF) and prostaglandin E2 (PGE2) that promote angiogenesis, modulate the inflammatory microenvironment, and stimulate wound repair, and MSC spheroids secrete more trophic factors than dissociated, individual MSCs. Compared to injection of cells alone, transplantation of MSCs in a biomaterial can enhance their wound healing potential by localizing cells at the defect site and upregulating trophic factor secretion. To capitalize on the therapeutic potential of spheroids, we engineered a fibrin gel delivery vehicle to simultaneously enhance the proangiogenic and anti-inflammatory potential of entrapped human MSC spheroids. We used multifactorial statistical analysis to determine the interaction between four input variables derived from fibrin gel synthesis on four output variables (gel stiffness, degradation rate, and secretion of VEGF and PGE2). Manipulation of the four input variables tuned fibrin gel biophysical properties to promote the simultaneous secretion of VEGF and PGE2 by entrapped MSC spheroids while maintaining overall gel integrity. MSC spheroids in stiffer gels secreted the most VEGF, while PGE2 secretion was highest in more compliant gels. Simultaneous VEGF and PGE2 secretion was greatest using hydrogels with intermediate mechanical properties, as small increases in stiffness increased VEGF secretion while maintaining PGE2 secretion by entrapped spheroids. The fibrin gel formulation predicted to simultaneously increase VEGF and PGE2 secretion stimulated endothelial cell proliferation, enhanced macrophage polarization, and promoted angiogenesis when used to treat a wounded three-dimensional human skin equivalent. These data demonstrate that a statistical approach is an effective strategy to formulate fibrin gel formulations that enhance the wound healing potential of human MSCs.

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Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Engineering fibrin hydrogels to promote the wound healing potential of mesenchymal stem cell spheroids

et al. 2017

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Exogenous Lysyl Oxidase-Like 2 and Perfusion Culture Induce Collagen Crosslink Formation in Osteogenic Grafts

et al. 2018

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Lysyl oxidase (LOX)-mediated collagen crosslinking can regulate osteoblastic phenotype and enhance mechanical properties of tissues, both areas of interest in bone tissue engineering. The objective of this study is to investigate the effect of lysyl oxidase-like 2 (LOXL2) on osteogenic differentiation of mesenchymal stem cells (MSCs) cultured in perfusion bioreactors, enzymatic collagen crosslink formation in the extracellular matrix (ECM), and mechanical properties of engineered bone grafts. Exogenous LOXL2 to MSCs seeded in composite scaffolds under perfusion culture for up to 28 days is administered. Constructs treated with LOXL2 appear brown in color and possess greater DNA content and osteogenic potential measured by a twofold increase in bone sialoprotein gene expression. Collagen expression of LOXL2-treated scaffolds is lower than untreated controls. Functional outputs such as calcium deposition, osteocalcin expression, and compressive modulus are unaffected by LOXL2 supplementation. Excitingly, LOXL2-treated constructs contain 1.8- and 1.4-times more pyridinoline (PYD) crosslinks per mole of collagen and per wet weight, respectively, than untreated constructs. Despite these increases, compressive moduli of LOXL2-treated constructs are similar to untreated constructs over the 28-day culture duration. This is the first report of LOXL2 application to engineered, three-dimensional bony constructs. The results suggest a potentially new strategy for engineering osteogenic grafts with a mature ECM by modulating crosslink formation.

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Efficiency of coculture with angiogenic cells or physiological BMP‐2 administration on improving osteogenic differentiation and bone formation of MSCs

Zhang

Yang

Devit

et al. 2018

J Biomedical Materials Res

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Cell‐based bone regeneration with mesenchymal stem cells (MSCs) represents the current challenge toward repair of bone defects and fractures. The supposed hurdles for satisfactory performance of cell‐based constructs include inadequate vascularization and osteogenic signals. Considering the reported beneficial role of angiogenic cells in promoting vascularization and osteogenic differentiation and the osteogenic potential of bone morphogenetic protein 2 (BMP‐2), we here evaluated the efficiency of coculture with angiogenic cells or a physiological dose of BMP‐2 on improving osteogenic differentiation of MSCs and bone formation in vivo. In three dimensional (3D) collagen hydrogels in vitro, cocultured human umbilical vein endothelial cells (HUVECs) in a 1:1 ratio or with a physiological dose of BMP‐2 (2 ng/μL) promoted the osteogenic potential of MSCs evidenced by enhanced alkaline phosphatase activity and gene expression of osteogenic markers. Notably, HUVECs evoked similar osteogenic stimulation as BMP‐2, albeit in a delayed manner. When their bone formation capacity was further evaluated in a mouse subcutaneous implantation model, MSCs with BMP‐2 demonstrated the highest efficiency with reproducible bone formation. In contrast, MSCs cocultured with HUVECs constructs displayed substantial blood vessel‐like structures with fibrous tissue rather than ectopic bone as MSC monoculture controls. Our findings confirm the priority of generating cell‐based bone constructs with physiological BMP‐2 administration and indicate the potential of using angiogenic cells to develop vascularized constructs. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 107A: 643–653, 2019.

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Hydrogel biophysical properties instruct coculture‐mediated osteogenic potential

Cited by 18 publications

References 48 publications

Engineering fibrin hydrogels to promote the wound healing potential of mesenchymal stem cell spheroids

Engineering fibrin hydrogels to promote the wound healing potential of mesenchymal stem cell spheroids

Exogenous Lysyl Oxidase-Like 2 and Perfusion Culture Induce Collagen Crosslink Formation in Osteogenic Grafts

Efficiency of coculture with angiogenic cells or physiological BMP‐2 administration on improving osteogenic differentiation and bone formation of MSCs

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