As populations age across the world, osteoporosis and osteoporosis-related fractures are becoming the most prevalent degenerative bone diseases. More than 75 million patients suffer from osteoporosis in the USA, the EU and Japan. Furthermore, it is anticipated that the number of patients affected by osteoporosis will increase by a third by 2050. Although conventional therapies including bisphosphonates, calcitonin and oestrogen-like drugs can be used to treat degenerative diseases of the bone, they are often associated with serious side effects including the development of oesophageal cancer, ocular inflammation, severe musculoskeletal pain and osteonecrosis of the jaw.The use of autologous mesenchymal stromal cells/mesenchymal stem cells (MSCs) is a possible alternative therapeutic approach to tackle osteoporosis while overcoming the limitations of traditional treatment options. However, osteoporosis can cause a decrease in the numbers of MSCs, induce their senescence and lower their osteogenic differentiation potential.Three-dimensional (3D) cell culture is an emerging technology that allows a more physiological expansion and differentiation of stem cells compared to cultivation on conventional flat systems.This review will discuss current understanding of the effects of different 3D cell culture systems on proliferation, viability and osteogenic differentiation, as well as on the immunomodulatory and anti-inflammatory potential of MSCs.

The anti-inflammatory and immunomodulatory properties of human mesenchymal stromal cells (MSCs) are a focus within regenerative medicine. However, 2D cultivation of MSCs for extended periods results in abnormal cell polarity, chromosomal changes, reduction in viability, and altered differentiation potential. As an alternative, various 3D hydrogels have been developed which mimic the endogenous niche of MSCs. Nevertheless, imaging cells embedded within 3D hydrogels often suffers from low signal-to-noise ratios which can be at least partly attributed to the high light absorbance and light scattering of the hydrogels in the visible light spectrum. In this study, human adipose tissue-derived MSCs (ADSCs) are cultivated within an anionic nanofibrillar cellulose (aNFC) hydrogel. It is demonstrated that aNFC forms nanofibres arranged as a porous network with low light absorbance in the visible spectrum. Moreover, it is shown that aNFC is cytocompatible, allowing for MSC proliferation, maintaining cell viability and multilineage differentiation potential. Finally, aNFC is compatible with scanning electron microscopy (SEM) and light microscopy including the application of conventional dyes, fluorescent probes, indirect immunocytochemistry, and calcium imaging. Overall, the results indicate that aNFC represents a promising 3D material for the expansion of MSCs whilst allowing detailed examination of cell morphology and cellular behaviour.

Due to the ageing population, there is a steadily increasing incidence of osteoporosis and osteoporotic fractures. As conventional pharmacological therapy options for osteoporosis are often associated with severe side effects, bone grafts are still considered the clinical gold standard. However, the availability of viable, autologous bone grafts is limited making alternative cell-based strategies a promising therapeutic alternative. Adipose-derived stem cells (ASCs) are a readily available population of mesenchymal stem/stromal cells (MSCs) that can be isolated within minimally invasive surgery. This ease of availability and their ability to undergo osteogenic differentiation makes ASCs promising candidates for cell-based therapies for bone fractures. Recent studies have suggested that both exposure to electrical fields and cultivation in 3D can positively affect osteogenic potential of MSCs. To elucidate the osteoinductive potential of a combination of these biophysical cues on ASCs, cells were embedded within anionic nanofibrillar cellulose (aNFC) hydrogels and exposed to electrical stimulation (ES) for up to 21 days. ES was applied to ASCs in 2D and 3D at a voltage of 0.1 V/cm with a duration of 0.04 ms, and a frequency of 10 Hz for 30 min per day. Exposure of ASCs to ES in 3D resulted in high alkaline phosphatase (ALP) activity and in an increased mineralisation evidenced by Alizarin Red S staining. Moreover, ES in 3D aNFC led to an increased expression of the osteogenic markers osteopontin and osteocalcin and a rearrangement and alignment of the actin cytoskeleton. Taken together, our data suggest that a combination of ES with 3D cell culture can increase the osteogenic potential of ASCs. Thus, exposure of ASCs to these biophysical cues might improve the clinical outcomes of regenerative therapies in treatment of osteoporotic fractures.

Adipose-derived mesenchymal stromal cells (ASCs) are multipotent stem cells which can differentiate into various cell types, including osteocytes and adipocytes. Due to their ease of harvesting, multipotency, and low tumorigenicity, they are a prime candidate for the development of novel interventional approaches in regenerative medicine. ASCs exhibit slow, spontaneous Ca2+ oscillations and the manipulation of Ca2+ signalling via electrical stimulation was proposed as a potential route for promoting their differentiation in vivo. However, the effects of differentiation-inducing treatments on spontaneous Ca2+ oscillations in ASCs are not yet fully characterised. In this study, we used 2-photon live Ca2+ imaging to assess the fraction of cells showing spontaneous oscillations and the frequency of the oscillation (measured as interpeak interval—IPI) in ASCs undergoing osteogenic or adipogenic differentiation, using undifferentiated ASCs as controls. The measurements were carried out at 7, 14, and 21 days in vitro (DIV) to assess the effect of time in culture on Ca2+ dynamics. We observed that both time and differentiation treatment are important factors associated with a reduced fraction of cells showing Ca2+ oscillations, paralleled by increased IPI times, in comparison with untreated ASCs. Both adipogenic and osteogenic differentiation resulted in a reduction in Ca2+ dynamics, such as the fraction of cells showing intracellular Ca2+ oscillations and their frequency. Adipogenic differentiation was associated with a more pronounced reduction of Ca2+ dynamics compared to cells differentiating towards the osteogenic fate. Changes in Ca2+ associated oscillations with a specific treatment had already occurred at 7 DIV. Finally, we observed a reduction in Ca2+ dynamics over time in untreated ASCs. These data suggest that adipogenic and osteogenic differentiation cell fates are associated with specific changes in spontaneous Ca2+ dynamics over time. While this observation is interesting and provides useful information to understand the functional correlates of stem cell differentiation, further studies are required to clarify the molecular and mechanistic correlates of these changes. This will allow us to better understand the causal relationship between Ca2+ dynamics and differentiation, potentially leading to the development of novel, more effective interventions for both bone regeneration and control of adipose growth.