Multifactorial Experimental Design to Optimize the Anti-Inflammatory and Proangiogenic Potential of Mesenchymal Stem Cell Spheroids.

Mesenchymal stem cell therapies promote wound healing by manipulating the local environment to enhance the function of host cells. Aggregation of mesenchymal stem cells (MSCs) into three-dimensional spheroids increases cell survival and augments their anti-inflammatory and proangiogenic potential, yet there is no consensus on the preferred conditions for maximizing spheroid function in this application. The objective of this study was to optimize conditions for forming MSC spheroids that simultaneously enhance their anti-inflammatory and proangiogenic nature. We applied a design of experiments (DOE) approach to determine the interaction between three input variables (number of cells per spheroid, oxygen tension, and inflammatory stimulus) on MSC spheroids by quantifying secretion of prostaglandin E2 (PGE2 ) and vascular endothelial growth factor (VEGF), two potent molecules in the MSC secretome. DOE results revealed that MSC spheroids formed with 40,000 cells per spheroid in 1% oxygen with an inflammatory stimulus (Spheroid 1) would exhibit enhanced PGE2 and VEGF production versus those formed with 10,000 cells per spheroid in 21% oxygen with no inflammatory stimulus (Spheroid 2). Compared to Spheroid 2, Spheroid 1 produced fivefold more PGE2 and fourfold more VEGF, providing the opportunity to simultaneously upregulate the secretion of these factors from the same spheroid. The spheroids induced macrophage polarization, sprout formation with endothelial cells, and keratinocyte migration in a human skin equivalent model-demonstrating efficacy on three key cell types that are dysfunctional in chronic non-healing wounds. We conclude that DOE-based analysis effectively identifies optimal culture conditions to enhance the anti-inflammatory and proangiogenic potential of MSC spheroids. Stem Cells 2017;35:1493-1504.

Hydrogel biophysical properties instruct coculture-mediated osteogenic potential.

Cell-based approaches for bone formation require instructional cues from the surrounding environment. As an alternative to pharmacological strategies or transplanting single cell populations, one approach is to coimplant populations that can establish a new vasculature and differentiate to bone-forming osteoblasts. Mesenchymal stem/stromal cells (MSCs) possess osteogenic potential and produce numerous angiogenic growth factors. Endothelial colony-forming cells (ECFCs) are a subpopulation of endothelial progenitor cells capable of vasculogenesis in vivo and may provide endogenous cues to support MSC function. We investigated the contribution of the carrier biophysical properties to instruct entrapped human MSCs and ECFCs to simultaneously promote their osteogenic and proangiogenic potential. Compared with gels containing MSCs alone, fibrin gels engineered with increased compressive stiffness simultaneously increased the osteogenic and proangiogenic potential of entrapped cocultured cells. ECFCs produced bone morphogenetic protein-2 (BMP-2), a potent osteoinductive molecule, and increases in BMP-2 secretion correlated with gel stiffness. Coculture of MSCs with ECFCs transduced to knockdown BMP-2 production abrogated the osteogenic response to levels observed with MSCs alone. These results demonstrate that physical properties of engineered hydrogels modulate the function of cocultured cells in the absence of inductive cues, thus increasing the translational potential of coimplantation to speed bone formation and repair.

Simultaneous arthroscopic cystectomy and unicompartmental knee arthroplasty for the management of partial knee osteoarthritis with a popliteal cyst: A case report.

Introduction: Popliteal cysts are secondary to degenerative changes in the knee joint. After total knee arthroplasty (TKA), 56.7% of patients with popliteal cysts at 4.9 years follow-up remained symptomatic in the popliteal area. However, the result of simultaneous arthroscopic cystectomy and unicompartmental knee arthroplasty (UKA) was uncertain. Case presentation: A 57-year-old man was admitted to our hospital with severe pain and swelling in his left knee and the popliteal area. He was diagnosed with severe medial unicompartmental knee osteoarthritis (KOA) with a symptomatic popliteal cyst. Subsequently, arthroscopic cystectomy and unicompartmental knee arthroplasty (UKA) were performed simultaneously. A month after the operation, he returned to his normal life. There was no progression in the lateral compartment of the left knee and no recurrence of the popliteal cyst at the 1-year follow-up. Conclusion: For KOA patients with a popliteal cyst seeking UKA, simultaneous arthroscopic cystectomy and UKA are feasible with great success if managed appropriately.

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

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, gel contraction, 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. STATEMENT OF SIGNIFICANCE: Mesenchymal stem cells (MSCs) are under investigation for wound healing applications due to their secretion of bioactive factors that enhance granulation tissue formation, blood vessel ingrowth, and reduce inflammation. However, the effectiveness of cell-based therapies is reduced due to poor engraftment and high rates of cell death when transplanted into harsh environments characteristic of large wounds. Compared to dissociated cells, MSCs exhibit increased overall function when aggregated into three-dimensional spheroids, and transplantation of cells using biomaterials is one strategy for guiding cell function in the defect site. The present study demonstrates that the biophysical properties of fibrin hydrogels, designed for use as a cell carrier, can be engineered to dictate the secretion of bioactive factors by entrapped MSC spheroids. This strategy enables MSCs to contribute to wound healing by synergistically promoting neovascularization and modulating the inflammatory milieu.

Measurement of oxygen tension within mesenchymal stem cell spheroids.

Spheroids formed of mesenchymal stem cells (MSCs) exhibit increased cell survival and trophic factor secretion compared with dissociated MSCs, making them therapeutically advantageous for cell therapy. Presently, there is no consensus for the mechanism of action. Many hypothesize that spheroid formation potentiates cell function by generating a hypoxic core within spheroids of sufficiently large diameters. The purpose of this study was to experimentally determine whether a hypoxic core is generated in MSC spheroids by measuring oxygen tension in aggregates of increasing diameter and correlating oxygen tension values with cell function. MSC spheroids were formed with 15 000, 30 000 or 60 000 cells per spheroid, resulting in radii of 176 ± 8 µm, 251 ± 12 µm and 353 ± 18 µm, respectively. Oxygen tension values coupled with mathematical modelling revealed a gradient that varied less than 10% from the outer diameter within the largest spheroids. Despite the modest radial variance in oxygen tension, cellular metabolism from spheroids significantly decreased as the number of cells and resultant spheroid size increased. This may be due to adaptive reductions in matrix deposition and packing density with increases in spheroid diameter, enabling spheroids to avoid the formation of a hypoxic core. Overall, these data provide evidence that the enhanced function of MSC spheroids is not oxygen mediated.

Mesenchymal Stem Cell Spheroids Retain Osteogenic Phenotype Through α2ß1 Signaling.

UNLABELLED: : The induction of mesenchymal stem cells (MSCs) toward the osteoblastic lineage using osteogenic supplements prior to implantation is one approach under examination to enhance their bone-forming potential. MSCs rapidly lose their induced phenotype upon removal of the soluble stimuli; however, their bone-forming potential can be sustained when provided with continued instruction via extracellular matrix (ECM) cues. In comparison with dissociated cells, MSC spheroids exhibit improved survival and secretion of trophic factors while maintaining their osteogenic potential. We hypothesized that entrapment of MSC spheroids formed from osteogenically induced cells would exhibit better preservation of their bone-forming potential than would dissociated cells from monolayer culture. Spheroids exhibited comparable osteogenic potential and increased proangiogenic potential with or without osteogenic preconditioning versus monolayer-cultured MSCs. Spheroids were then entrapped in collagen hydrogels, and the osteogenic stimulus was removed. In comparison with entrapped dissociated MSCs, spheroids exhibited significantly increased markers of osteogenic differentiation. The capacity of MSC spheroids to retain their osteogenic phenotype upon withdrawal of inductive cues was mediated by α2ß1 integrin binding to cell-secreted ECM. These results demonstrate the capacity of spheroidal culture to sustain the mineral-producing phenotype of MSCs, thus enhancing their contribution toward bone formation and repair. SIGNIFICANCE: Despite the promise of mesenchymal stem cells (MSCs) for cell-based therapies for tissue repair and regeneration, there is little evidence that transplanted MSCs directly contribute to new bone formation, suggesting that induced cells rapidly lose their osteogenic phenotype or undergo apoptosis. In comparison with dissociated cells, MSC spheroids exhibit increased trophic factor secretion and improved cell survival. The loss of phenotype represents a significant clinical challenge for cell therapies, yet there is no evidence for whether MSC spheroids retain their osteogenic phenotype upon entrapment in a clinically relevant biomaterial. These findings demonstrate that MSC spheroids retain their osteogenic phenotype better than do dissociated MSCs, and this is due to integrin engagement with the cell-secreted extracellular matrix. These data provide evidence for a novel approach for potentiating the use of MSCs in bone repair.