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Stem cell spheroids incorporating fibers coated with adenosine and polydopamine

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, the delivery of bioactive signals remains challenging. Here, they 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 the AP-FF group (59.97 ± 18.33%) as compared to P-FF (27.96 ± 11.14) and defect only (7.97 ± 3.76%). They 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 the regeneration of damaged bone.


Bone tissue engineering has emerged as a strategy to address the limitations associated with allografts and autografts, including insufficient availability, morbidity, disease transfer, and contamination. Classic bone tissue engineering commonly involves a top-down approach, in which cells are seeded onto biomaterial scaffolds with signaling molecules to provide a structural framework for cell growth and large-sized tissue formation. However, top-down processes are limited by inhomogeneous cell distribution, insufficient nutrients diffusion, and, most importantly, an inability to recapitulate the complex architecture of bone. In fact, compact bone is composed of repeating microscale units called osteons that are hierarchically organized into a large bone structure. By contrast, a bottom-up approach aims to recapitulate microstructural features of tissues by engineering microscale building blocks that can be assembled into large and functional tissue constructs. Previously, cell sheets, spheroids, and cell-laden microgels have been developed as building blocks for engineering large-sized tissue by cell-sheet stacking, spheroid fusion, random packing, or direct assembly. However, cell-based building blocks are hampered by a lack of extracellular matrix (ECM) deposition on initial stages and limited control over viability and differentiation of cells.


Because 3D spheroid cell cultures can mimic key aspects of native cellular microenvironments, they have attracted a great deal of attention from regenerative medicine researchers. Their uniform circular size and potential for spontaneous fusion render them ideal for bottom-up tissue engineering. Stem cell spheroids are also known for their ability to differentiate into many cell types. For example, mesenchymal stem cells (MSCs) in spheroids have shown significant promise in maintaining pluripotential characteristics in general growth conditions and multi-lineage differentiation in induction media. In particular, MSCs isolated from adipose tissue are promising sources for in vivo cell transplantation due to their ease of isolation, fast proliferation, and multipotent differentiation. However, multipotent differentiation of stem cells may hinder therapeutic outcomes due to non-specific and uncontrolled differentiation upon transplantation. Transplanted MSCs reportedly fail to regenerate bone and induce fibrous tissue formation under low oxygen conditions or induce adipocyte formation under inflammation. Stem cell transplantation for bone regeneration therefore often requires co-delivery of growth factors such as bone morphogenetic protein 2 (BMP-2). However, soluble delivery of growth factors with stem cells has not been effective due to rapid degradation and diffusion from the target site. Moreover, exogenous delivery of growth factors to spheroids or other cell-based building blocks can impede penetration of the core of the construct due to compact cellular assembly.





a) Schematic representation of fabrication of spheroids of human-derived adipose stem cells (hADSCs) with AP-FF. b) Phase contrast microscope images, H &E staining, and Live and dead staining of spheroids at days 1 and 14. c) The area of spheroids at days 1, 4, 7, and 14. d) The proliferation of hADSCs within the spheroid. e) SEM images of spheroids. The white dashed line indicates the area of interest for magnified images.T. Ahmad, et al.Biomaterials 230 (2020) 1196526


The incorporation of biomaterials with cell-instructive cues into spheroids can modulate cellular functions. Biomaterials modified with growth factors (BMP-2, PDGF, and bFGF), small peptide sequences from growth factors (osteogenic peptide from BMP-2 and osteogenic growth peptide from BMP-7), and small molecules (lovastatin, simvastatin, and SVAK-12) have exhibited significant potential in bone regeneration in various top-down tissue-engineering applications. Similarly, attempts have been made to improve stem cell functions by delivering growth factors into spheroids that incorporate microparticles or nanomaterials. For example, incorporation of BMP-2–loaded mineralized microparticles in spheroids significantly stimulated osteogenic differentiation of hMSCs. However, these approaches have difficulty mimicking the extracellular matrix (ECM), show an uneven distribution of microparticles, hinder cell-cell contact, offer no information about the fate of cells after in vivo transplantation, and present unwanted outcomes of growth factors such as BMP-2–mediated uncontrolled bone formation and cancer development. Recently, adenosine receptor ligation, involving the adenosine 2b receptor (A2bR) in particular, has been proposed as an alternative potent signal for regulation of inflammation and induction of bone healing, including enhanced osteoblastic differentiation and inhibition of adipogenic differentiation of stem cells. However, adenosine delivery for therapeutic applications remains challenging due to its extremely short life. Local delivery of adenosine via protective carriers may be critical for prolonged activation of adenosine receptors for therapeutic applications that prevent off-target activation of adenosine signaling.




a) Schematic representation of transplantation of spheroids into subcutaneous tissue. b and d) H and E staining of specimens retrieved from transplanted tissue after 8 weeks. c and e) Alizarin Red S staining of specimen retrieved from transplanted tissue after 8 weeks. f) The high-magnification images of white dashed boxes indicate H and E and Alizarin staining from images b, d, and e. g) Immunohistochemistry of the tissue specimen for OPN. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)


Given that, they designed a proof-of-concept approach to incorporate cell-instructive materials within stem cell spheroids to tune their functions for bottom-up tissue-engineering applications. They hypothesized that a one-step coating with polydopamine could be exploited for the protective storage of adenosine on a biomaterial surface. To that end, they stably introduced an adenosine-ligand on ECM-inspired engineered fibers using polydopamine chemistry. They then developed human-adipose-derived stem cell (hADSCs) spheroids as modular building blocks for bone regeneration. They evaluated the effects of adenosine-ligand-engineered fibers on stimulation of A2bR signaling, osteogenic differentiation, and inhibition of adipogenic differentiation of hADSCs within a 3D microenvironment.



a) Interactions and adhesion of AP-FF with stem cells assisted by polydopamine and adenosine binding with A2bR. b) A2bR immunofluorescence staining of hADSCs in spheroids cultured for 14 days. Quantitative analysis of mRNA expression of c) A2bR, d) A1R, e) A2aR, and f) A3R in hADSCs within spheroids (*, P < 0.05 compared with FF). GM and OM represent growth and osteogenic media, respectively.


Finally, they transplanted the engineered spheroids into mice and examined ectopic osteogenic differentiation of hADSCs in the subcutaneous tissue and bone regeneration in calvarial defects1.


  1. Ahmad, T., Byun, H., Lee, J., Madhurakat Perikamana, S. K., Shin, Y. M., Kim, E. M., & Shin, H. (2020). Stem cell spheroids incorporating fibers coated with adenosine and polydopamine as a modular building blocks for bone tissue engineering. Biomaterials, 230, 119652. https://doi.org/10.1016/j.biomaterials.2019.119652


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