Mild heat treatment of human mesenchymal stem cells for tissue engineering applications

Abstract

Mesenchymal stem cells (MSCs) have garnered significant attention for various biomedical applications, including cell-based therapies. However, several practical issues, including limited in vitro expansion due to quality control and insufficient therapeutic activity, still remain to be addressed. Therefore, research focused on preserving or enhancing MSC quality during in vitro expansion, as well as maximizing their therapeutic efficacy for in vivo applications, is essential. Mild heat treatment (MHT) has been shown to enhance various functions of MSCs, including survival and differentiation, likely by upregulating the expression of protective proteins, such as heat shock proteins (HSPs). This thesis aimed to i) investigate the short- and long-term responses of MSCs to MHT, ii) synthesize effective magnetic nanoparticles for remote heating, and iii) develop an MSC-encapsulated magnetic hydrogel for bone tissue engineering applications. First, the effects of MHT conditions (temperature, duration, and repetition) on the characteristics of adipose tissue-derived MSCs were systematically explored in vitro using multiple assays. Comprehensive studies with various MHT conditions revealed that thermal stimulation of MSCs at 41ºC or 44ºC for 1 hour upregulated the expression of HSPs and stemness markers and enhanced differentiation potential. Moreover, periodic MHT prolonged the growth rate and maintained the stemness of MSCs for up to an additional 10 passages, significantly delaying their spontaneous aging during in vitro culture. RNA sequencing indicated that MHT downregulated the genes associated with aging and apoptosis of MSCs. To leverage the advantages of MHT for tissue engineering applications, magnetic nanoparticles (MNPs) were synthesized for enhanced magnetic hyperthermia and biocompatibility. As conventional iron oxide magnetic Ph.D./MS 20194009 nanoparticles (IONPs) have several issues in heating efficiency, biocompatibility, and colloidal stability, highly optimized magnetic nanocomposites (MNCs) were synthesized by embedding IONPs in a poly(L-lactic acid) (PLA) matrix via nanoemulsion. MNCs containing 13% Fe (w/w) were found to show the highest heating efficiency, with a two-fold increase compared to non-embedded IONPs. These MNCs demonstrated excellent colloidal stability, heating efficiency under physiological conditions, and biocompatibility in vitro and in vivo. Lastly, a magnetic hydrogel embedding MSCs and MNCs was developed for remote MHT and bone tissue regeneration. Specifically, magnetic collagen hydrogels (MCHs) encapsulating MSCs were developed to remotely induce osteogenic differentiation and enhance bone regeneration. In vitro experiments demonstrated that AMF- induced MHT (15 min, 2X HT), which resulted in 41°C, significantly upregulated HSP70 expression and promoted MSC osteogenesis. Implantation of MCHs into a rat femur defect model and subsequent AMF-induced heat treatment facilitated bone regeneration compared to non-treated groups. X-ray imaging confirmed accelerated bone regeneration with MHT by evaluating bone density and structural integrity. In conclusion, this thesis successfully explored the effects of MHT on MSC characteristics and developed a remote system using MNCs for MSC-based bone tissue engineering applications.