Anti-BGLAP (aa 1-22) polyclonal antibody

Hydrogels represent an attractive material platform for realization of three-dimensional (3D) tissue-engineered constructs, as they have tunable mechanical properties, are compatible with different types of cells, and resemble elements found in natural extracellular matrices. So far, numerous hydrogel-cartilage/bone tissue engineering (TE)-related studies were performed by utilizing a single cell encapsulation approach. Although multicellular spheroid cultures exhibit advantageous properties for cartilage or bone TE, the chondrogenic or osteogenic differentiation potential of stem cell microspheroids within hydrogels has not been investigated much. This study explores, for the first time, how stiffness of gelatin-based hydrogels (having a storage modulus of 538, 3584, or 7263 Pa) affects proliferation and differentiation of microspheroids formed from telomerase-immortalized human adipose-derived stem cells (hASC/hTERT). Confocal microscopy indicates that all tested hydrogels supported cell viability during their 3- to 5-week culture period in the control, chondrogenic, or osteogenic medium. Although in the softer hydrogels cells from neighboring microspheroids started outgrowing and interconnecting within a few days, their protrusion was slower or limited in stiffer hydrogels or those cultured in chondrogenic medium, respectively. High expressions of chondrogenic markers (SOX9, ACAN, COL2A1), detected in all tested hydrogels, proved that the chondrogenic differentiation of hASC/hTERT microspheroids was very successful, especially in the two softer hydrogels, where superior cartilage-specific properties were confirmed by Alcian blue staining. These chondrogenically induced samples also expressed COL10A1, a marker of chondrocyte hypertrophy. Interestingly, the hydrogel itself (with no differentiation medium) showed a slight chondrogenic induction. Regardless of the hydrogel stiffness, in the samples stimulated with osteogenic medium, the expression of selected markers RUNX2, BGLAP, ALPL, and COL1A1 was not conclusive. Nevertheless, the von Kossa staining confirmed the presence of calcium deposits in osteogenically stimulated samples in the two softer hydrogels, suggesting that these also favor osteogenesis. This observation was also confirmed by Alizarin red quantification assay, with which higher amounts of calcium were detected in the osteogenically induced hydrogels than in their controls. The presented data indicate that the encapsulation of adipose-derived stem cell microspheroids in gelatin-based hydrogels show promising potential for future applications in cartilage or bone TE. Impact Statement Osteochondral defects represent one of the leading causes of disability in the world. Although numerous tissue engineering (TE) approaches have shown success in cartilage and bone tissue regeneration, achieving native-like characteristics of these tissues remains challenging. This study demonstrates that in the presence of a corresponding differentiation medium, gelatin-based hydrogels support moderate osteogenic and excellent chondrogenic differentiation of photo-encapsulated human adipose-derived stem cell microspheroids, the extent of which depends on hydrogel stiffness. Because photosensitive hydrogels are a convenient material platform for creating stiffness gradients in three dimensions, the presented microspheroid-hydrogel encapsulation strategy holds promise for future strategies of cartilage or bone TE.

SLIT2, a member of neuronal guidance cues, has been reported to regulate inflammation and cancer progression. Periodontitis is an oral inflammatory disease that degenerates periodontal tissue, alveolar bone and tooth. This study aims to explore the expression pattern of SLIT2 in periodontitis and its role in disease progression and bone loss. Gingival tissue of 20 periodontitis patients and 20 healthy-controls was obtained. Ligature-induced periodontitis (LIP) mice-model was developed inSlit2-Tgand wild-type mice. The effect of SLIT2 on inflammation, immune cell infiltration, M1 macrophage polarization, and alveolar bone loss in periodontitis was analyzed extensively. In periodontitis-affected gingival-tissue, SLIT2 expression was 4.4-fold higher compared to healthy-volunteers. LIP enhanced SLIT2 expression in mice periodontitis-affected periodontal tissue (PAPT) and blood circulation of wild-type mice by 4. 6-, and 5.0-fold, respectively. In Slit2-Tg-mice PAPT, SLIT2 expression was 1.8-fold higher compared to wild-type mice. Micro-CT and histomorphometric analysis revealed a 1.3-fold higher cement-enamel-junction to the alveolar-bone-crest (CEJ-ABC) distance and alveolar bone loss in LIP Slit2-Tg-mice compare to LIP wild-type mice. Results from RNA-sequencing, RT-qPCR, and ELISA showed a higher expression of Cxcr2, Il-18, TNF alpha, IL-6, and IL-1 beta in Slit2-Tg-mice PAPT compared to wild-type-mice. Slit2-Tg-mice PAPT showed a higher number of osteoclasts, M1 macrophages, and the upregulation of Robo1 expression. Slit2-Tg-mice PAPT showed upregulation of M1 macrophage marker CD16/32 and osteoclastogenic markersAcp5,Ctsk, andNfatc1, but osteogenic markers (Alp, Bglap) remained unchanged. Immunohistochemistry unveiled the higher vasculature and infiltration of leucocytes and macrophages in Slit2-Tg-mice PAPT. RNA-sequencing, GO-pathway enrichment analysis, and western blot analysis revealed the activation of the MAPK signaling pathway inSlit2-Tg mice PAPT. In conclusion, SLIT2 overexpression in periodontitis intensifies inflammation, immune cells infiltration, M1 macrophage polarization, osteoclastogenesis, and alveolar bone loss, possibly via activation of MAPK signaling, suggesting the role of SLIT2 on exacerbation of periodontitis and alveolar bone loss.