Mesenchymal stromal cell spheroids in sulfated alginate enhance muscle regeneration.

The therapeutic efficacy of mesenchymal stromal cells (MSCs) for tissue regeneration is critically linked to the potency of the complex mixture of growth factors, cytokines, exosomes, and other biological cues that they secrete. The duration of cell-based approaches is limited by rapid loss of cells upon implantation, motivating the need to prolong cell viability and extend the therapeutic influence of the secretome. We and others demonstrated that the secretome is upregulated when MSCs are formed into spheroids. Although the efficacy of the MSC secretome has been characterized in the literature, no studies have reported the therapeutic benefit of in situ sequestration of the secretome within a wound site using engineered biomaterials. We previously demonstrated the capacity of sulfated alginate hydrogels to sequester components of the MSC secretome for prolonged presentation in vitro, yet the efficacy of this platform has not been evaluated in vivo. In this study, we used sulfated alginate hydrogels loaded with MSC spheroids to aid in the regeneration of a rat muscle crush injury. We hypothesized that the use of sulfated alginate to bind therapeutically relevant growth factors from the MSC spheroid secretome would enhance muscle regeneration by recruiting host cells into the tissue site. The combination of sulfated alginate and MSC spheroids resulted in decreased collagen deposition, improved myogenic marker expression, and increased neuromuscular junctions 2 weeks after injury. These data indicate that MSC spheroids delivered in sulfated alginate represent a promising approach for decreased fibrosis and increased functional regeneration of muscle. STATEMENT OF SIGNIFICANCE: The therapeutic efficacy of mesenchymal stromal cells (MSCs) for tissue regeneration is attributed to the complex diversity of the secretome. Cell-based approaches are limited by rapid cell death, motivating the need to extend the availability of the secretome. We previously demonstrated that sulfated alginate hydrogels sequester components of the MSC secretome for prolonged presentation in vitro, yet no studies have reported the in situ sequestration of the secretome. Herein, we transplanted MSC spheroids in sulfated alginate hydrogels to promote muscle regeneration. MSC spheroids in sulfated alginate decreased collagen deposition, improved myogenic marker expression, and increased neuromuscular junctions. These data indicate that MSC spheroids delivered in sulfated alginate represent a promising approach for decreasing fibrosis and increasing functional muscle regeneration.

Skeletal muscle progenitors are sensitive to collagen architectural features of fibril size and cross linking.

Muscle stem cells (MuSCs) are essential for the robust regenerative capacity of skeletal muscle. However, in fibrotic environments marked by abundant collagen and altered collagen organization, the regenerative capability of MuSCs is diminished. MuSCs are sensitive to their extracellular matrix environment but their response to collagen architecture is largely unknown. The present study aimed to systematically test the effect of underlying collagen structures on MuSC functions. Collagen hydrogels were engineered with varied architectures: collagen concentration, cross linking, fibril size, and fibril alignment, and the changes were validated with second harmonic generation imaging and rheology. Proliferation and differentiation responses of primary mouse MuSCs and immortal myoblasts (C2C12s) were assessed using EdU assays and immunolabeling skeletal muscle myosin expression, respectively. Changing collagen concentration and the corresponding hydrogel stiffness did not have a significant influence on MuSC proliferation or differentiation. However, MuSC differentiation on atelocollagen gels, which do not form mature pyridinoline cross links, was increased compared with the cross-linked control. In addition, MuSCs and C2C12 myoblasts showed greater differentiation on gels with smaller collagen fibrils. Proliferation rates of C2C12 myoblasts were also higher on gels with smaller collagen fibrils, whereas MuSCs did not show a significant difference. Surprisingly, collagen alignment did not have significant effects on muscle progenitor function. This study demonstrates that MuSCs are capable of sensing their underlying extracellular matrix (ECM) structures and enhancing differentiation on substrates with less collagen cross linking or smaller collagen fibrils. Thus, in fibrotic muscle, targeting cross linking and fibril size rather than collagen expression may more effectively support MuSC-based regeneration.

Conditioning of myoblast secretome using mesenchymal stem/stromal cell spheroids improves bone repair.

Local muscle loss associated with open fractures remains an obstacle to functional recovery and bone healing. Muscle cells secrete bioactive myokines that elicit autocrine and paracrine effects and initiate signaling pathways for regenerating damaged muscle and bone. Mesenchymal stem/stromal cells (MSCs) are under investigation for the regeneration of both muscle and bone through their potent secretome. Compared to monodisperse cells, MSC spheroids exhibit a more complex secretome with heightened therapeutic potential. We hypothesized that the osteogenic potential of myokines would be enhanced when myoblasts were exposed to the MSC spheroid secretome. Conditioned media from MSC spheroids increased osteogenic response of MC3T3 pre-osteoblasts compared to myokines from L6 myoblasts alone. This effect was synergistically enhanced when conditioned media of MSC spheroids was serially delivered to myoblasts and then osteoprogenitor cells in vitro. We then delivered myoblast-stimulated conditioned media in the presence or absence of syngeneic rat bone marrow stromal cells (rBMSCs) from alginate hydrogels to a rat critical-sized segmental defect. We observed increased bone formation in defects treated with conditioned media compared to rBMSCs alone, while bone formation was greatest in defects treated with both conditioned media and rBMSCs over 12 weeks. This foundational study demonstrates a novel approach for capitalizing on the paracrine signaling of muscle cells to promote bone repair and provides additional evidence of the synergistic interaction between muscle and bone.

Multimodal Label-Free Imaging for Detecting Maturation of Engineered Osteogenic Grafts.

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.

Engineering principles for guiding spheroid function in the regeneration of bone, cartilage, and skin.

There is a critical need for strategies that effectively enhance cell viability and post-implantation performance in order to advance cell-based therapies. Spheroids, which are dense cellular aggregates, overcome many current limitations with transplanting individual cells. Compared to individual cells, the aggregation of cells into spheroids results in increased cell viability, together with enhanced proangiogenic, anti-inflammatory, and tissue-forming potential. Furthermore, the transplantation of cells using engineered materials enables localized delivery to the target site while providing an opportunity to guide cell fate in situ, resulting in improved therapeutic outcomes compared to systemic or localized injection. Despite promising early results achieved by freely injecting spheroids into damaged tissues, growing evidence demonstrates the advantages of entrapping spheroids within a biomaterial prior to implantation. This review will highlight the basic characteristics and qualities of spheroids, describe the underlying principles for how biomaterials influence spheroid behavior, with an emphasis on hydrogels, and provide examples of synergistic approaches using spheroids and biomaterials for tissue engineering applications.

Extraction of microRNAs from biological matrices with titanium dioxide nanofibers.

MicroRNAs (miRNAs) are small RNAs that bind to mRNA targets and regulate their translation. A functional study of miRNAs and exploration of their utility as disease markers require miRNA extraction from biological samples, which contain large amounts of interfering compounds for downstream RNA identification and quantification. The most common extraction methods employ silica columns or the TRIzol reagent but give out low recovery for small RNAs probably due to their short strand lengths. Herein, we fabricated the titanium dioxide nanofibers using electrospinning to facilitate miRNA extraction and developed the optimal buffer conditions to improve miRNA recovery from biological matrices of cell lysate and serum. We found that our TiO2 fibers could obtain a recovery of 18.0 ± 3.6% for miRNA fibers while carrying out the extraction in the more complex medium of cell lysate, much higher than the 0.02 ± 0.0001% recovery from the commercial kit. The much improved extraction of miRNAs from our fibers could be originated from the strong coordination between TiO2 and RNA's phosphate backbone. In addition, the binding, washing, and elution buffers judiciously developed in the present study can achieve selective extraction of small RNA shorter than 500 nucleotides in length. Our results demonstrate that TiO2 nanofibers can work as a valuable tool for extraction of miRNAs from biological samples with high recovery. Graphical abstract Schematic for extraction of small RNAs using TiO2 nanofibers.