Sulfated glycosaminoglycans (GAG) are components of the bone marrow stem cell niche and to a minor extent of mature bone tissue with important functions in regulating stem cell lineage commitment and differentiation. We anticipated that artificial extracellular matrices (aECM) composed of collagen I and synthetically oversulfated GAG derivatives affect preferentially the differentiation of osteoblast-precursor cells andearlyosteoblasts. A set of gradually sulfated chondroitin sulfate and hyaluronan derivatives was used for the preparation of aECM. All these matrices were analysed with human bone marrow stromal cells to identify the most potent aECM and to determine the influence of the degree and position of sulfate groups and the kind of disaccharide units on the osteogenic differentiation. Oversulfated GAG derivatives with a sulfate group at the C-6 position of the N-acetylglycosamine revealed the most pronounced proosteogenic effect as determined by tissue nonspecific alkaline phosphatase activity and calcium deposition. A subset of the aECM was further analysed with different primary osteoblasts and cell lines reflecting different maturation stages to test whether the effect of sulfated GAG derivatives depends on the maturation status of the cells. It was shown that the proosteogenic effect of aECM was most prominent inearlyosteoblasts.

The extracellular matrix (ECM) is a highly dynamic network constantly remodeled by a fine-tuned protein formation and degradation balance. Matrix metalloproteinases (MMPs) constitute key orchestrators of ECM degradation. Their activity is controlled by tissue inhibitors of metalloproteinases (TIMPs) and glycosaminoglycans (GAG). Here, we investigated the molecular interplay of MMP2 with different GAG (chondroitin sulfate, hyaluronan (HA), sulfated hyaluronan (SH) and heparin (HE)) and the impact of GAG on MMP2/TIMP3 complex formation using in vitro-experiments with human bone marrow stromal cells, in silico docking and molecular dynamics simulations. SH and HE influenced MMP2 and TIMP3 protein levels and MMP2 activity. Only SH supported the alignment of both proteins in fibrillar-like structures, which, based on our molecular models, would be due to a stabilization of the interactions between MMP2-hemopexin domain and TIMP3-C-terminal tail. Dependent on the temporal sequential order in which the final ternary complex was formed, our models indicated that SH and HA can affect TIMP3-induced MMP2 inhibition through precluding or supporting their interactions, respectively. Our combined experimental and theoretical approach provides valuable new insights on how GAG interfere with MMP2 activity and MMP2/TIMP3 complex formation. The results obtained evidence GAG as promising molecules for fine-balanced intervention of ECM remodeling.

Bone transplantation is regarded as the preferred therapy to treat a variety of bone defects. Autologous bone tissue is often lacking at the source, and the mesenchymal stem cells (MSCs) responsible for bone repair mechanisms are extracted by invasive procedures. This study explores the potential of autologous mesenchymal stem cells derived from the hair follicle outer root sheath (MSCORS). We demonstrated that MSCORS have a remarkable capacity to differentiate in vitro towards the osteogenic lineage. Indeed, when combined with a novel gelatin-based hydrogel called Osteogel, they provided additional osteoinductive cues in vitro that may pave the way for future application in bone regeneration. MSCORS were also compared to MSCs from adipose tissue (ADMSC) and bone marrow (BMMSC) in a 3D Osteogel model. We analyzed gel plasticity, cell phenotype, cell viability, and differentiation capacity towards the osteogenic lineage by measuring alkaline phosphatase (ALP) activity, calcium deposition, and specific gene expression. The novel injectable hydrogel filled an irregularly shaped lesion in a porcine wound model displaying high plasticity. MSCORS in Osteogel showed a higher osteo-commitment in terms of calcium deposition and expression dynamics of OCN, BMP2, and PPARG when compared to ADMSC and BMMSC, whilst displaying comparable cell viability and ALP activity. In conclusion, autologous MSCORS combined with our novel gelatin-based hydrogel displayed a high capacity for differentiation towards the osteogenic lineage and are acquired by non-invasive procedures, therefore qualifying as a suitable and expandable novel approach in the field of bone regeneration therapy.

Transglutaminases (TGs) catalyze the covalent crosslinking of proteins via isopeptide bonds. The most prominent isoform, TG2, is associated with physiological processes such as extracellular matrix (ECM) stabilization and plays a crucial role in the pathogenesis of e.g. fibrotic diseases, cancer and celiac disease. Therefore, TG2 represents a pharmacological target of increasing relevance. The glycosaminoglycans (GAG) heparin (HE) and heparan sulfate (HS) constitute high-affinity interaction partners of TG2 in the ECM. Chemically modified GAG are promising molecules for pharmacological applications as their composition and chemical functionalization may be used to tackle the function of ECM molecular systems, which has been recently described for hyaluronan (HA) and chondroitin sulfate (CS). Herein, we investigate the recognition of GAG derivatives by TG2 using an enzyme-crosslinking activity assay in combination with in silico molecular modeling and docking techniques. The study reveals that GAG represent potent inhibitors of TG2 crosslinking activity and offers atom-detailed mechanistic insights.