**6. Conclusions**

Such studies have convinced that the mechanical cues can influence the fate of the stem cells; however, the mechanistic insights as to how these cues direct the differentiation of stem cells is just beginning to be unravelled. Stem cells can sense the stiff microenvironment and transduce the signals through the Rho kinase [86, 87], TGF-β [88, 89], Src family kinases [90] and phosphor-tyrosine signalling pathways [91, 92]. Other studies by Dupont et al. and Swift et al. have shown that yes-associated protein and transcriptional coactivator with PDZ-binding motif also have a significant role in regulating the stem cell differentiation mechanism in response to mechanical parameters. While more and more mechanistic data begins to emerge, it is only clear that mechanical cues are potent physical parameters in the regulation of stem cell differentiation [93–96]. The release and beneficial effects of trophic factors from the stem cells will also depend on the microenvironment it is residing in. Changes in the microenvir‐ onment with respect to the biological and mechanical cues can affect the release of trophic factors which can have profound implications in their functionality. More studies are now beginning to emerge to decipherthese concepts using various artificial platforms. For example, Abdeen et al. studied the combined role of stiffness and matrix protein on the secretory profile of MSCs and their effects on human microvascular endothelial cells. In this work, the condi‐ tioned media from MSCs adherent to polyacrylamide hydrogel with controlled matrix rigidity and protein composition was collected and applied to a model angiogenesis assay using HMVECs within Matrigel. The result from this study showed that secretion of the trophic factors was related to a combined effect of stiffness and adhesion protein for directing proangiogenic signalling [97]. Jose et al. pretreated the MSCs with glycine-histidine-lysine (GHK), a peptide fragment of osteonectin and a matrix cellular protein with reported proangiogenic potential. The study revealed a dose-dependent increase in VEGF concentra‐ tion in media conditioned by GHK-treated MSC, which increased endothelial cell prolifera‐ tion, migration and tubule formation. This study suggested that microenvironment of the stem cells can have significant influence on the trophic factors and their functionality [98]. Furthermore, Silva et al. showed that the secretome of the bone marrow MSCs were affected when the cells were cultured on fibronectin peptide-modified hydrogels as compared to the unmodified gels and this change in the secretome-induced higher metabolic viabilities and neuronal cell densities [99]. Hoch et al. also showed that cell-secreted decellularized extracel‐ lular matrices can preserve the bone-forming phenotype of the differentiated MSC. In this study, osteogenically induced MSCs were cultured on the decellularized matrices and the osteogenic and angiogenic potential was measured after the withdrawal of the induction media. It was found that culturing osteogenically induced MSCs on decellularized matrix can enhance calcium deposition and secretion of proangiogenic factor such as VEGF [100].

366 Advanced Techniques in Bone Regeneration

It has also been noted that the changes in the microenvironment of the stem cells due to biomolecules can also affect the trophic factors secretion by the stem cells and this can in turn affect the functionality of the cells by our group and others. We have shown that short-term exposure of human osteoblasts to tumour necrosis factor (TNF-α) can promote osteogenic differentiation and also stimulate human osteoblasts to secrete soluble factors that can foster a microenvironment favouring osteogenic differentiation of ADSC [101]. A similar study was performed by Czekanska et al., whereby MSCs were stimulated with interleukin-1β (IL1β), granulocyte-colony stimulating factor (GCSF), stromal cell-derived factor 1 (SDF1) and stem

Stem cells are in a way considered to be the "building blocks" of regenerative medicine and are believed to possess solutions to many types of injuries and diseases. Enormous amount of research is focussed on deciphering and understanding the functionalities of these cells through the cell-based therapy, through tissue engineering or purely through their para‐ crine activities.

The idea of "stem cell free regenerative" medicine has undoubtedly captured a great deal of attention in the recent few years. Most studies suggest that the use of stem cells secretory molecules such as trophic factors, microvesicles or exosomes can be advantageous and valuable in the treatment of injuries or diseases compared to the cell-based therapies. There are more to be explored in terms of their mechanisms at a molecular level, the effect of microenvironment on their release, and the long-term effects of these kinds of treatments in an in vivo scenario. Answers to these questions can help in validating cell-free regenerative technology as a potential therapeutic tool.
