Physiological cyclic hydrostatic pressure induces osteogenic lineage commitment of human bone marrow stem cells: a systematic study

Abstract

BackgroundPhysical loading is necessary to maintain bone tissue integrity. Loading-induced fluid shear is recognised as one of the most potent bone micromechanical cues and has been shown to direct stem cell osteogenesis. However, the effect of pressure transients, which drive fluid flow, on human bone marrow stem cell (hBMSC) osteogenesis is undetermined. Therefore, the objective of the study is to employ a systematic analysis of cyclic hydrostatic pressure (CHP) parameters predicted to occur in vivo on early hBMSC osteogenic responses and late-stage osteogenic lineage commitment.MethodshBMSC were exposed to CHP of 10 kPa, 100 kPa and 300 kPa magnitudes at frequencies of 0.5 Hz, 1 Hz and 2 Hz for 1 h, 2 h and 4 h of stimulation, and the effect on early osteogenic gene expression of COX2, RUNX2 and OPN was determined. Moreover, to decipher whether CHP can induce stem cell lineage commitment, hBMSCs were stimulated for 4 days for 2 h/day using 10 kPa, 100 kPa and 300 kPa pressures at 2 Hz frequency and cultured statically for an additional 1–2 weeks. Pressure-induced osteogenesis was quantified based on ATP release, collagen synthesis and mineral deposition.ResultsCHP elicited a positive, but variable, early osteogenic response in hBMSCs in a magnitude- and frequency-dependent manner, that is gene specific. COX2 expression elicited magnitude-dependent effects which were not present for RUNX2 or OPN mRNA expression. However, the most robust pro-osteogenic response was found at the highest magnitude (300 kPa) and frequency regimes (2 Hz). Interestingly, long-term mechanical stimulation utilising 2 Hz frequency elicited a magnitude-dependent release of ATP; however, all magnitudes promoted similar levels of collagen synthesis and significant mineral deposition, demonstrating that lineage commitment is magnitude independent. This therefore demonstrates that physiological levels of pressures, as low as 10 kPa, within the bone can drive hBMSC osteogenic lineage commitment.ConclusionOverall, these findings demonstrate an important role for cyclic hydrostatic pressure in hBMSCs and bone mechanobiology, which should be considered when studying pressure-driven fluid shear effects in hBMSCs mechanobiology. Moreover, these findings may have clinical implication in terms of bioreactor-based bone tissue engineering strategies.Electronic supplementary materialThe online version of this article (10.1186/s13287-018-1025-8) contains supplementary material, which is available to authorized users.

Pressure‐induced mesenchymal stem cell osteogenesis is dependent on intermediate filament remodeling

“…We contribute this response to a possible physiologic mechanism, as pressure transients fluctuate within the bone in response to mechanical loading (7,47). However, we recognize that differences in the bioreactor setup and stimulation protocols may also contribute to divergent observations (15). Nonetheless, our observations that physiologic low-magnitude cyclic pressure transients, which represent the precursor to fluid shear in vivo, are sufficient to directly drive MSC osteogenesis independent of fluid flow.…”

“…A number of recent studies have demonstrated that pressures up to 300 kPa can drive MSC osteogenesis as depicted by up-regulation of the osteogenic-associated genes cyclooxygenase 2 (Cox2), runt related transcription factor 2 (Runx2), distal-less homeobox 5, osterix, osteopontin (Opn), and alkaline phosphatase (8,9). Moreover, low bone physiologic pressures (kilopascal range) were sufficient to promote mineralization of human (h)MSCs, further supporting the importance of pressure in regulating osteogenic lineage commitment (8)(9)(10)(11)(12)(13)(14)(15). Despite this clear osteogenic response to pressure stimulation, little is known about how MSCs sense and transduce pressure mechanical cues, with only one study to date highlighting the involvement of mechanosensitive ion channel Piezo 1 (9).…”

“…A previously described custom pressure bioreactor was utilized to administer HP and cyclic HP (CHP) (15). The bioreactor consists of the following: a rectangular chamber that houses a glass slide onto which the cells are cultured, and an inlet port that supports the infusion of finite volumes of fluid via a syringe pump to achieve transient or static pressurization of the fluid interfaced with the cells.…”

Human bone marrow stem/stromal cell osteogenesis is regulated via mechanically activated osteocyte-derived extracellular vesicles

“…These concentrations represent minimal levels for the support of osteogenesis. 32,37 The role Figure 3C, and listed in Table 1. Within these proteins, significant enrichment (enrichment factor > 1.7, P < 10 -4 ) of several "extracellular" GOCC terms was shown in comparison to the total 393 identified proteins using Fisher's exact test, with enrichment of UniProt keywords "secreted" and "signal" (enrichment factor > 1.6, P < 10 -5 ) also occurred ( Figure 3D).…”

Effects of mechanobiological signaling in bone marrow on skeletal health

“…Dissecting the individual contributions of bone‐resident versus marrow‐resident cells on bone adaptation is challenging. Individual bone marrow–resident cells have been studied in in vitro culture and subjected to mechanical loads, demonstrating that the relevant cells are mechanosensitive. Coculture has further demonstrated that both mesenchymal and hematopoietic lineage cells in the marrow interact with osteocytes and osteoblast precursors through various signaling pathways .…”