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1. Notch signaling and fluid shear stress in regulating osteogenic differentiation

   https://doi.org/10.3389/fbioe.2022.1007430  

   Zhao, Yuwen ; Richardson, Kiarra ; Yang, Rui ; Bousraou, Zoe ; Lee, Yoo Kyoung ; Fasciano, Samantha ; Wang, Shue ( October 2022 , Frontiers in Bioengineering and Biotechnology)

   Osteoporosis is a common bone and metabolic disease that is characterized by bone density loss and microstructural degeneration. Human bone marrow-derived mesenchymal stem cells (hMSCs) are multipotent progenitor cells with the potential to differentiate into various cell types, including osteoblasts, chondrocytes, and adipocytes, which have been utilized extensively in the field of bone tissue engineering and cell-based therapy. Although fluid shear stress plays an important role in bone osteogenic differentiation, the cellular and molecular mechanisms underlying this effect remain poorly understood. Here, a locked nucleic acid (LNA)/DNA nanobiosensor was exploited to monitor mRNA gene expression of hMSCs that were exposed to physiologically relevant fluid shear stress to examine the regulatory role of Notch signaling during osteogenic differentiation. First, the effects of fluid shear stress on cell viability, proliferation, morphology, and osteogenic differentiation were investigated and compared. Our results showed shear stress modulates hMSCs morphology and osteogenic differentiation depending on the applied shear and duration. By incorporating this LNA/DNA nanobiosensor and alkaline phosphatase (ALP) staining, we further investigated the role of Notch signaling in regulating osteogenic differentiation. Pharmacological treatment is applied to disrupt Notch signaling to investigate the mechanisms that govern shear stress induced osteogenic differentiation. Our experimental results provide convincingmore »evidence supporting that physiologically relevant shear stress regulates osteogenic differentiation through Notch signaling. Inhibition of Notch signaling mediates the effects of shear stress on osteogenic differentiation, with reduced ALP enzyme activity and decreased Dll4 mRNA expression. In conclusion, our results will add new information concerning osteogenic differentiation of hMSCs under shear stress and the regulatory role of Notch signaling. Further studies may elucidate the mechanisms underlying the mechanosensitive role of Notch signaling in stem cell differentiation.« less
2. Tuning Thermal Dosage to Facilitate Mesenchymal Stem Cell Osteogenesis in Pro-Inflammatory Environment

   https://doi.org/10.1115/1.4047660  

   Sunderic, Kristifor ; Li, Chenghai ; Ahmed, A.H. Rezwanuddin ; Dawkins, Dionne ; Azar, Tala ; Cardoso, Luis ; Wang, Sihong ( June 2020 , Journal of Biomechanical Engineering)

   Abstract Mesenchymal stem cells (MSCs) are multipotent cells that can replicate and differentiate to different lineages of mesenchymal tissues, potentiating their use in regenerative medicine. Our previous work and other studies have indicated that mild heat shock enhances osteogenesis. However, the influence of pro-inflammatory cytokines on osteogenic differentiation during mildly elevated temperature conditions remains to be fully explored. In this study, human MSCs (hMSCs) were cultured with Tumor Necrosis Factor-alpha (TNF-a), an important mediator of the acute phase response, and Interleukin-6 (IL-6) which plays a role in damaging chronic inflammation, then heat shocked at 39ºC in varying frequencies - 1 hour per week (low), 1 hour every other day (mild), and 1 hour intervals three times per day every other day (high). DNA data showed that periodic mild heating inhibited suppression of cell growth caused by cytokines and induced maximal proliferation of hMSCs while high heating had the opposite effect. Quantitative osteogenesis assays show significantly higher levels of alkaline phosphatase activity and calcium precipitation in osteogenic cultures following mild heating compared to low heating or non-heated controls. These results demonstrate that periodic mild hyperthermia may be used to facilitate bone regeneration using hMSCs, and therefore may influence the design ofmore »heat-based therapies in vivo.« less
3. Cell-Laden Composite Hydrogel Bioinks with Human Bone Allograft Particles to Enhance Stem Cell Osteogenesis

   https://doi.org/10.3390/polym14183788  

   Gharacheh, Hadis ; Guvendiren, Murat ( September 2022 , Polymers)

   There is a growing demand for bone graft substitutes that mimic the extracellular matrix properties of the native bone tissue to enhance stem cell osteogenesis. Composite hydrogels containing human bone allograft particles are particularly interesting due to inherent bioactivity of the allograft tissue. Here, we report a novel photocurable composite hydrogel bioink for bone tissue engineering. Our composite bioink is formulated by incorporating human allograft bone particles in a methacrylated alginate formulation to enhance adult human mesenchymal stem cell (hMSC) osteogenesis. Detailed rheology and printability studies confirm suitability of our composite bioinks for extrusion-based 3D bioprinting technology. In vitro studies reveal high cell viability (~90%) for hMSCs up to 28 days of culture within 3D bioprinted composite scaffolds. When cultured within bioprinted composite scaffolds, hMSCs show significantly enhanced osteogenic differentiation as compared to neat scaffolds based on alkaline phosphatase activity, calcium deposition, and osteocalcin expression.
4. Spatial organization of biochemical cues in 3D-printed scaffolds to guide osteochondral tissue engineering

   https://doi.org/10.1039/D1BM00859E  

   Camacho, Paula ; Behre, Anne ; Fainor, Matthew ; Seims, Kelly B. ; Chow, Lesley W. ( October 2021 , Biomaterials Science)

   Functional repair of osteochondral (OC) tissue remains challenging because the transition from bone to cartilage presents gradients in biochemical and physical properties necessary for joint function. Osteochondral regeneration requires strategies that restore the spatial composition and organization found in the native tissue. Several biomaterial approaches have been developed to guide chondrogenic and osteogenic differentiation of human mesenchymal stem cells (hMSCs). These strategies can be combined with 3D printing, which has emerged as a useful tool to produce tunable, continuous scaffolds functionalized with bioactive cues. However, functionalization often includes one or more post-fabrication processing steps, which can lead to unwanted side effects and often produce biomaterials with homogeneously distributed chemistries. To address these challenges, surface functionalization can be achieved in a single step by solvent-cast 3D printing peptide-functionalized polymers. Peptide-poly(caprolactone) (PCL) conjugates were synthesized bearing hyaluronic acid (HA)-binding (HAbind–PCL) or mineralizing (E3–PCL) peptides, which have been shown to promote hMSC chondrogenesis or osteogenesis, respectively. This 3D printing strategy enables unprecedented control of surface peptide presentation and spatial organization within a continuous construct. Scaffolds presenting both cartilage-promoting and bone-promoting peptides had a synergistic effect that enhanced hMSC chondrogenic and osteogenic differentiation in the absence of differentiation factors compared to scaffolds without peptidesmore »or only one peptide. Furthermore, multi-peptide organization significantly influenced hMSC response. Scaffolds presenting HAbind and E3 peptides in discrete opposing zones promoted hMSC osteogenic behavior. In contrast, presenting both peptides homogeneously throughout the scaffolds drove hMSC differentiation towards a mixed population of articular and hypertrophic chondrocytes. These significant results indicated that hMSC behavior was driven by dual-peptide presentation and organization. The downstream potential of this platform is the ability to fabricate biomaterials with spatially controlled biochemical cues to guide functional tissue regeneration without the need for differentiation factors.« less
5. Primary Cilia Exhibit Mechanosensitivity to Cyclic Tensile Strain and Lineage-Dependent Expression in Adipose-Derived Stem Cells

   https://doi.org/10.1038/s41598-019-43351-y  

   Bodle, Josephine ; Hamouda, Mehdi S. ; Cai, Shaobo ; Williams, Ramey B. ; Bernacki, Susan H. ; Loboa, Elizabeth G. ( May 2019 , Scientific Reports)

   Non-motile primary cilia are dynamic cellular sensory structures and are expressed in adipose-derived stem cells (ASCs). We have previously shown that primary cilia are involved in chemically-induced osteogenic differentiation of human ASC (hASCs)in vitro. Further, we have reported that 10% cyclic tensile strain (1 Hz, 4 hours/day) enhances hASC osteogenesis. We hypothesize that primary cilia respond to cyclic tensile strain in a lineage dependent manner and that their mechanosensitivity may regulate the dynamics of signaling pathways localized to the cilium. We found that hASC morphology, cilia length and cilia conformation varied in response to culture in complete growth, osteogenic differentiation, or adipogenic differentiation medium, with the longest cilia expressed in adipogenically differentiating cells. Further, we show that cyclic tensile strain both enhances osteogenic differentiation of hASCs while it suppresses adipogenic differentiation as evidenced by upregulation ofRUNX2gene expression and downregulation ofPPARGandIGF-1, respectively. This study demonstrates that hASC primary cilia exhibit mechanosensitivity to cyclic tensile strain and lineage-dependent expression, which may in part regulate signaling pathways localized to the primary cilium during the differentiation process. We highlight the importance of the primary cilium structure in mechanosensing and lineage specification and surmise that this structure may be a novel target in manipulating hASC formore »in tissue engineering applications.

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