Stem cell spheroids incorporating fibers coated with adenosine and polydopamine as a modular building blocks for bone tissue engineering.

Although stem cell spheroids offer great potential as functional building blocks for bottom-up bone tissue engineering, delivery of bioactive signals remain challenging. Here, we engineered adenosine-ligand-modified fiber fragments to create a 3D cell-instructive microenvironment for bone. Briefly, the Poly(ι-lactic acid) (PLLA) nanofiber sheet was partially degraded into fragmented fibers (FFs) through aminolysis and adenosine was stably incorporated via one-step polydopamine coating. The SEM and XPS analysis demonstrated that polydopamine assisted adenosine coating efficiency was significantly increased, which led to high coating efficiency of adenosine and its significant retention. The engineered fibers were then assembled into stable spheroids with human-adipose-derived stem cells (hADSCs). The adenosine in the spheroids effectively stimulated A2bR (1.768 ± 0.08) signaling, which further significantly induced the expression of osteogenic markers such as Runx2 (3.216 ± 0.25), OPN (4.136 ± 0.14), OCN (10.16 ± 0.34), and OSX (2.27 ± 0.11) with improved mineral deposition (1.375 ± 0.05 µg per spheroid). In contrast, the adipogenic differentiation of hADSCs was significantly suppressed within the engineered spheroids. Transplantation of engineered spheroids strongly induced osteogenic differentiation of hADSCs in ectopic subcutaneous tissue. Finally, the bone regeneration was significantly enhanced by implanting AP-FF group (59.97 ± 18.33%) as compared to P-FF (27.96 ± 11.14) and defect only (7.97 ± 3.76%). We propose that stem cell spheroids impregnated with engineered fibers enabling adenosine delivery could be promising building blocks for a bottom-up approach to create large tissues for regeneration of damaged bone.

Hybrid-spheroids incorporating ECM like engineered fragmented fibers potentiate stem cell function by improved cell/cell and cell/ECM interactions.

Extracellular matrix (ECM) microenvironment is critical for the viability, stemness, and differentiation of stem cells. In this study, we developed hybrid-spheroids of human turbinate mesenchymal stem cells (hTMSCs) by using extracellular matrix (ECM) mimicking fragmented fibers (FFs) for improvement of the viability and functions of hTMSCs. We prepared FFs with average size of 68.26 µm by partial aminolysis of poly L-lactide (PLLA) fibrous sheet (FS), which was coated with polydopamine for improved cell adhesion. The proliferation of hTMSCs within the hybrid-spheroids mixed with fragmented fibers was significantly increased as compared to that from the cell-only group. Cells and fragmented fibers were homogenously distributed with the presence of pore like empty spaces in the structure. LOX-1 staining revealed that the hybrid-spheroids improved the cell viability, which was potentially due to enhanced transport of oxygen through void space generated by engineered ECM. Transmission electron microscopy (TEM) analysis confirmed that cells within the hybrid-spheroid formed strong cell junctions and contacts with fragmented fibers. The expression of cell junction proteins including connexin 43 and E-cadherin was significantly upregulated in hybrid-spheroids by 16.53 ±â€¯0.04 and 28.26 ±â€¯0.11-fold greater than that from cell-only group. Similarly, expression of integrin α2, α5, and ß1 was significantly enhanced at the same group by 25.72 ±â€¯0.13, 27.48 ±â€¯0.49, and 592.78 ±â€¯0.06-fold, respectively. In addition, stemness markers including Oct-4, Nanog, and Sox2 were significantly upregulated in hybrid-spheroids by 96.56 ±â€¯0.06, 158.95 ±â€¯0.06, and 115.46 ±â€¯0.47-fold, respectively, relative to the cell-only group. Additionally, hTMSCs within the hybrid-spheroids showed significantly greater osteogenic differentiation under osteogenic media conditions. Taken together, our hybrid-spheroids can be an ideal approach for stem cell expansion and serve as a potential carrier for bone regeneration. STATEMENT OF SIGNIFICANCE: Cells are spatially arranged within extracellular matrix (ECM) and cell/ECM interactions are crucial for cellular functions. Here, we developed a hybrid-spheroid system incorporating engineered ECM prepared from fragmented electrospun fibers to tune stem cell functions. Conventionally prepared cell spheroids with large diameters (>200 µm) is often prone to hypoxia. In contrast, the hybrid-spheroids significantly enhanced viability and proliferation of human turbinate mesenchymal stem cells (hTMSCs) as compared to spheroid prepared from cell only. Under these conditions, the presence of fragmented fibers also improved maintenance of stemness of hTMSCs for longer time cultured in growth media and demonstrated significantly greater osteogenic differentiation under osteogenic media conditions. Thus, the hybrid-spheroids can be used as a delivery carrier for stem cell based therapy or a 3D culture model for in vitro assay.