Fig 2: ZC4H2 overexpression enhanced BMP-induced osteogenesis of C2C12 cells. (a) Real-time PCR assay showed that ZC4H2 mRNA level in different plasmids transfected C2C12 cells during BMP-induced osteogenesis. C2C12 cells were firstly transfected with the indicated plasmids and then treated with 50 ng ml−1 rhBMP2 for 3 day to induce osteogenesis. Then total RNAs were isolated and then real-time PCR assays were carried out. (b–e) ZC4H2 overexpression enhanced the expression levels of BMP-induced osteogenic markers ALP (b), Osteocalcin (c), Osteorix (d) and Type I Collegan (e). The experiments were carried out as described above for panel (a). (f,g) ZC4H2 overexpression increased rhBMP induced ALP activity in C2C12 cells. C2C12 cells were treated for osteogenesis as described in panel (a). After induction, ALP activity was analysed by a ALP fluorometric kit (f) or NBT/BCIP staining (g). n.s., not significant; **p < 0.01.

Fig 3: Osteogenic differentiation of encapsulated ADSCs after cryopreservation. (A) Qualitative images of ARS-stained calcium nodules, taken as gross images of the well, and microscopic images. ADSCs within the hydrogel were either incubated in basal culture media or induced toward osteogenic differentiation for 14 days after cryopreservation with either 10% DMSO, 0.5 M trehalose, or 0.75 M trehalose. Cryopreserved ADSCs, purportedly released from the hydrogels, were observed to have attached onto the TCPS surface of the cell culture plates. Alizarin red staining was done to identify calcium deposits (dotted white circles) secreted by successfully differentiated cells (scale bar = 20 μm). (B) Quantification of ARS-stained calcium nodules by measuring the blank-corrected absorbance of their CPC extract at 570 nm. (C) Quantification of intracellular ALP after osteogenic differentiation for 5 days by measuring the blank-corrected fluorescence of 4-MU. Data are presented as mean ± SD, n = 2, and nsp > 0.05.

Fig 4: Graphical summary of results. Upon agonist-induced platelet activation, ATP and ADP are released. These are converted to AMP by platelet CD39 activity. AMP is converted to adenosine by MSC-expressed CD73 and to a low extent by alkaline phosphatase. Adenosine signals vial A2AR and other P1 receptors to raise cAMP levels and to induce VASP phosphorylation. This reduces further platelet activation. Used inhibitors indicated in red. ADA adenosine deaminase, ADP adenosine diphosphate, ALP alkaline phosphatase, AMP adenosine monophosphate, ATP adenosine triphosphate, TRAP thrombin receptor activator for peptide, VASP vasodilator-stimulated phosphoprotein

Fig 5: Alkaline phosphatase and adenosine deaminase activity and function blocking. a Alkaline phosphatase (ALP) and b adenosine deaminase (ADA) activity in different cell types. Individual biological replicates depicted as dots. c Effects induced by adding ALP inhibitor levamisole or ADA in platelet–cell cocultures. Data normalized against control activated by TRAP-6 without cells (dotted line at value 1). w/o n = 4–11, BM-MSCs n = 9, LA-MSCs n = 7–12, CB-MSCs n = 5–9, HUVECs n = 6 biological replicates; HeLa cells n = 3, adenosine n = 4. **p < 0.01. BM bone marrow, CB cord blood, HUVEC human umbilical vein endothelial cell, LA lipoaspirate, MFI mean fluorescence intensity, w/o without