Bone marrow aspirates provide a rich source of cells for use in tissue engineering of bone and other clinical indications. However, progenitor cells such as mesenchymal stem cells (MSCs) account for a small fraction of nucleated cells in bone marrow aspirate (BMA), requiring extensive culture expansion. Accessory cell populations such as endothelial or hematopoietic cells can potentiate the bone-forming potential of MSCs, and cell-secreted extracellular matrix (ECM) can increase cell seeding efficiency and osteogenic differentiation of heterogeneous cell populations. In this study, we hypothesized that cell-secreted ECM could be used to sequester MSCs and accessory cells from BMA for bone regeneration. To generate 3D implantable constructs, BMA was resuspended in media with or without type I collagen or ECM and injected into a perfusion bioreactor system. The addition of protein coatings increased cell seeding efficiency compared to uncoated scaffolds. Compared to fresh BMA, the culture of BMA on all scaffolds reduced the proportion of CD45+ myeloid cells and increased CD31+CD45- endothelial cells. Compared to uncoated scaffolds, we observed a 143- and 30-fold increase in MSCs when fresh BMA was cultured on ECM- or collagen-coated scaffolds, respectively. Upon subcutaneous implantation, ECM-coated scaffolds promoted cell survival and early vascularization. However, bone formation was comparable across all implant groups, suggesting additional osteogenic cues are necessary to increase the bone forming potential of fresh BMA. These results motivate further investigation into strategies which elicit more robust bone regeneration by tissue aspirates.

Bone marrow aspirates provide a rich source of cells for use in tissue engineering of bone and other clinical indications. However, progenitor cells such as mesenchymal stem cells (MSCs) account for a small fraction of nucleated cells in bone marrow aspirate (BMA), requiring extensive culture expansion. Accessory cell populations such as endothelial or hematopoietic cells can potentiate the bone-forming potential of MSCs, and cell-secreted extracellular matrix (ECM) can increase cell seeding efficiency and osteogenic differentiation of heterogeneous cell populations. In this study, we hypothesized that cell-secreted ECM could be used to sequester MSCs and accessory cells from BMA for bone regeneration. To generate 3D implantable constructs, BMA was resuspended in media with or without type I collagen or ECM and injected into a perfusion bioreactor system. The addition of protein coatings increased cell seeding efficiency compared to uncoated scaffolds. Compared to fresh BMA, the culture of BMA on all scaffolds reduced the proportion of CD45+ myeloid cells and increased CD31+CD45- endothelial cells. Compared to uncoated scaffolds, we observed a 143- and 30-fold increase in MSCs when fresh BMA was cultured on ECM- or collagen-coated scaffolds, respectively. Upon subcutaneous implantation, ECM-coated scaffolds promoted cell survival and early vascularization. However, bone formation was comparable across all implant groups, suggesting additional osteogenic cues are necessary to increase the bone forming potential of fresh BMA. These results motivate further investigation into strategies which elicit more robust bone regeneration by tissue aspirates.

The therapeutic potential of mesenchymal stem/stromal cells (MSCs) is limited by acquired senescence following prolonged culture expansion and high-passage numbers. However, the degree of cell senescence is dynamic, and cell-cell communication is critical to promote cell survival. MSC spheroids exhibit improved viability compared with monodispersed cells, and actin-rich tunneling nanotubes (TNTs) may mediate cell survival and other functions through the exchange of cytoplasmic components. Building upon our previous demonstration of TNTs bridging MSCs within these cell aggregates, we hypothesized that TNTs would influence the expression of senescence markers in MSC spheroids. We confirmed the existence of functional TNTs in MSC spheroids formed from low-passage, high-passage, and mixtures of low- and high-passage cells using scanning electron microscopy, confocal microscopy, and flow cytometry. The contribution of TNTs toward the expression of senescence markers was investigated by blocking TNT formation with cytochalasin D (CytoD), an inhibitor of actin polymerization. CytoD-treated spheroids exhibited decreases in cytosol transfer. Compared with spheroids formed solely of high-passage MSCs, the addition of low-passage MSCs reduced p16 expression, a known genetic marker of senescence. We observed a significant increase in p16 expression in high-passage cells when TNT formation was inhibited, establishing the importance of TNTs in MSC spheroids. These data confirm the restorative role of TNTs within MSC spheroids formed with low- and high-passage cells and represent an exciting approach to use higher-passage cells in cell-based therapies.

Lipoaspirates contain a readily accessible heterogeneous cell source for use in bone regeneration collectively referred to as the stromal vascular fraction (SVF). However, the osteogenic potential of SVF is inferior to other progenitor cell populations, thereby requiring alternative strategies to potentiate its effective use in cell-based therapies of bone repair. Cell-secreted extracellular matrix (ECM) is a promising substrate to guide cell phenotype or for use in biomaterial design, yet the instructional capacity of ECMs produced by various cell types is unknown. To determine whether the bioactivity of cell-secreted ECM was dependent on cell source, we assessed the osteogenic response of human SVF on ECMs secreted by bone marrow-derived mesenchymal stem cells (MSCs), adipose stromal cells (ASCs), and human dermal fibroblasts (HDFs). Tissue culture plastic (TCP), type I collagen, and ECM induced expression of integrin subunits α₂, α₅, and β₁ in SVF, yet seeding efficiency was only improved on MSC-derived ECM. Regardless of ECM source, SVF deposited over 8- and 1.3-fold more calcium compared to TCP and collagen-coated controls, respectively. Flow cytometry confirmed that SVF cultured on ECM retained CD31 and CD34 positive cell populations better than TCP. After depleting accessory cells, ASCs deposited significantly less calcium compared to donor-matched SVF. This function was partially restored in the presence of MSC-derived ECM when donor-matched endothelial cells (ECs) were added in an ASC/EC co-culture, confirming a role for ECs in osteogenic differentiation. These findings support the use of cell-derived ECM as a means to promote cell retention and osteogenic differentiation of SVF.

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 α2β1 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.

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.

We investigate the use of a fiber-based, multispectral fluorescence lifetime imaging (FLIm) system to nondestructively monitor changes in mechanical properties of collagen hydrogels caused by controlled application of widely used cross-linking agents, glutaraldehyde (GTA) and ribose. Postcross-linking, fluorescence lifetime images are acquired prior to the hydrogels being processed by rheological or tensile testing to directly probe gel mechanical properties. To preserve the sterility of the ribose-treated gels, FLIm is performed inside a biosafety cabinet (BSC). A pairwise correlation analysis is used to quantify the relationship between mean hydrogel fluorescence lifetimes and the storage or Young's moduli of the gels. In the GTA study, we observe strong and specific correlations between fluorescence lifetime and the storage and Young's moduli. Similar correlations are not observed in the ribose study and we postulate a reason for this. Finally, we demonstrate the ability of FLIm to longitudinally monitor dynamic cross-link formation. The strength of the GTA correlations and deployment of our fiber-based FLIm system inside the aseptic environment of a BSC suggests that this technique may be a valuable tool for the tissue engineering community where longitudinal assessment of tissue construct maturation in vitro is highly desirable.

There is a critical need to develop noninvasive, nondestructive methods for assessing the quality of engineered constructs prior to implantation. Currently, the composition and maturity of engineered tissues are assessed using destructive, costly, and time-consuming biochemical and mechanical analyses. The goal of this study was to use noninvasive, multimodal imaging to monitor osteogenic differentiation and matrix deposition by human mesenchymal stem/stromal cells (MSCs) during in vitro culture. MSCs were encapsulated in alginate hydrogels and cultured in osteogenic conditions for 4 weeks. Samples were evaluated using fluorescence lifetime imaging (FLIm) and ultrasound backscatter microscopy (UBM) prior to traditional biochemical and mechanical testing. Using linear regression analysis, we identified strong correlations between imaging parameters (e.g., fluorescence lifetime and acoustic attenuation coefficient) and destructive mechanical and biochemical tests to assess the maturation of osteogenically induced constructs. These data demonstrate the promise of nondestructive label-free imaging techniques to noninvasively ascertain the progression and maturity of tissue engineered bone grafts.

The maintenance and direction of stem cell lineage after implantation remains challenging for clinical translation. Aggregation and encapsulation into instructive biomaterials after preconditioning can bolster retention of differentiated phenotypes. Since these procedures do not depend on cell type or lineage, we hypothesized we could use a common, tunable platform to engineer formulations that retain and enhance multiple lineages from different cell populations. To test this, we varied alginate stiffness and adhesive ligand content, then encapsulated spheroids of varying cellularity. We used Design-of-Experiments to determine the effect of these parameters and their interactions on phenotype retention. The combination of parameters leading to maximal differentiation varied with lineage and cell type, inducing a 2-4-fold increase over non-optimized levels. Phenotype was also retained for 4 weeks in a murine subcutaneous model. This widely applicable approach can facilitate translation of cell-based therapies by instructing phenotype in situ without prolonged induction or costly growth factors.

The maintenance and direction of stem cell lineage after implantation remains challenging for clinical translation. Aggregation and encapsulation into instructive biomaterials after preconditioning can bolster retention of differentiated phenotypes. Since these procedures do not depend on cell type or lineage, we hypothesized we could use a common, tunable platform to engineer formulations that retain and enhance multiple lineages from different cell populations. To test this, we varied alginate stiffness and adhesive ligand content, then encapsulated spheroids of varying cellularity. We used Design-of-Experiments to determine the effect of these parameters and their interactions on phenotype retention. The combination of parameters leading to maximal differentiation varied with lineage and cell type, inducing a 2-4-fold increase over non-optimized levels. Phenotype was also retained for 4 weeks in a murine subcutaneous model. This widely applicable approach can facilitate translation of cell-based therapies by instructing phenotype in situ without prolonged induction or costly growth factors.