Mechanobiological Approaches for Stimulating Chondrogenesis of Stem Cells

Abstract

Chondrogenesis is the process of differentiation of stem cells into mature chondrocytes. Such a process consists of chemical, functional, and structural changes which are initiated and mediated by the host environment of the cells. To date, the mechanobiology of chondrogenesis has not been fully elucidated. Hence, experimental activity is focused on recreating specific environmental conditions for stimulating chondrogenesis and to look for a mechanistic interpretation of the mechanobiological response of cells in the cartilaginous tissues. There are a large number of studies on the topic that vary considerably in their experimental protocols used for providing environmental cues to cells for differentiation, making generalizable conclusions difficult to ascertain. The main objective of this contribution is to review the mechanobiological stimulation of stem cell chondrogenesis and methodological approaches utilized to date to promote chondrogenesis of stem cells in vitro. In vivo models will also be explored, but this area is currently limited. An overview of the experimental approaches used by different research groups may help the development of unified testing methods that could be used to overcome existing knowledge gaps, leading to an accelerated translation of experimental findings to clinical practice.

Introduction

Chondrogenesis includes the differentiating process by which stem cells undergo chemical, functional, and structural changes to become mature chondrocytes. It begins with the aggregation of the chondroprogenitors into condensations, which is signaled by the presence of transforming growth factor beta (TGF-β) [1]. Chondrocyte differentiation and proliferation is marked by increases in chondrogenic gene expression, such as SOX9, aggrecan, and collagen II [1]. Levels of other biomarkers can indicate poor chondrogenesis, or hypertrophy, such as increased expression of collagen I and X [1].

It is well known that chondrocytes are sensitive to their mechanical environment. As in vivo chondrocytes function within an environment constantly subjected to a range of mechanical loading stimulations, these conditions are thought to accelerate chondrogenesis of progenitor cells [2 –4]. Thus, these conditions are mimicked in in vitro experimentation. A range of studies have shown how mechanical stimulation can be used to modulate cell metabolism. In particular, the type and magnitude of mechanical load may affect proliferation and apoptosis rates, as well as gene expression in chondrocytes and differentiation of stem cells.

To date, the mechanobiology of chondrogenesis has not been fully elucidated, despite the numerous studies published. Specifically aimed at providing mechanistic interpretation of the mechanobiological response of cells in the cartilaginous tissues, in vitro studies on cells or on tissue explants have been carried out. However, these studies vary considerably in their experimental protocols used for load application (eg, static vs. dynamic) and type of load [eg, mechanical vs. chemical using hydrostatic pressure (HP)] making generalizable conclusions difficult to ascertain [2 –4]. Moreover, the development of organ-on-a-chip technology provides a closer look at the interactions of cells with their environment [5 –8]. However, even in this case, experimental protocols for mechanical loading greatly vary from one study to another.

Research has also been conducted on animal models and human subjects to further test mechanobiological driven research hypotheses and potential treatments for cartilaginous degenerative diseases [2 –4]. Similar to in vitro studies, the methods adopted to modulate the mechanical environment of cells varied between passive (through robotic systems and/or external manipulation) and active loading mechanisms (eg, specific rehabilitative protocol) [2 –4].

The objective herein is to review the mechanobiological stimulation of stem cell chondrogenesis and methodological approaches utilized to date to promote chondrogenesis of stem cells in vitro. The limited number of current in vivo studies will also be explored, mainly in terms of future directions for the field of cartilage regeneration. In turn, the adoption of standard testing methods could be used to overcome existing knowledge gaps leading to an accelerated translation of experimental findings to clinical care. Current research on the optimal culture conditions for chondrogenic differentiation seeks to elaborate on the principles of environmental cues and cell cultures. The studies reviewed henceforth represent recent work and novel discoveries in regard to the effects of these factors on the chondrogenesis of stem cells. The different techniques for simulating chondrogenic mechanobiology, as well as their success in inducing chondrogenesis, will be explored.

Cell Environment

Earlier studies of various types of stem cells have shown that their differentiation varies according to mechanical cues from their culture environment [9]. In embryonic development, both intrinsic and extrinsic forces influence cell fate. Intrauterine breathing creates tensile forces in the bronchial epithelium, which supports smooth muscle development in the lungs [10]. The development of cardiac tissues and fetal hematopoiesis are influenced by shear forces from maternal blood flow [11,12], which in turn signals kidney morphogenesis through the primary cilia [13,14]. Finally, stress, strain, HP, and fluid flow have all been shown to contribute to proper ossification and bone collar formation in limb development [15].

Studies of the effects of chemical immobilization of chick embryos have proven that mechanical stimulation is vital for chondrogenesis and joint development [13,14]. Compared to embryos that were allowed to develop normally, immobilized embryos showed impaired joint cavity formation, abnormal limb shape, and failure to form synovial joint elements when paralysis was induced during early development [16]. When immobilized at later points of development, the chick embryos displayed a decrease in cartilage matrix production, cartilage volume, and hyaluronan content of articular surfaces, as well as decreased mechanical properties [17].

The differentiation of progenitor cells is heavily influenced by the mechanical properties of their culture environment, with the most notable property being matrix stiffness. In a study by Zeng et al., the addition of stiffening components to fibrous scaffolds seeded with porcine chondrocytes led to increased new cartilage formation in vivo after 16 weeks, compared to fibrous scaffolds that did not contain stiffening agents [18]. When substrate stiffness is similar to that of brain, muscle, or bone tissue, mesenchymal stem cells (MSCs) have been shown to differentiate into neurons, myoblasts, and osteoblasts, respectively, demonstrating that the mechanical properties of the culture material also influence MSC fate [14]. In general, osteogenic differentiation is more likely to occur in elastic materials with a storage modulus (G′) between 25 and 40 kPa, and differentiation of brain cells occurs in the softest matrices, with a G′ of less than 1 kPa. Chondrogenic differentiation has been shown to occur at intermediate substrate stiffness, and ongoing research has aimed to pinpoint the ideal matrix stiffness for chondrogenesis [9].

Recent studies have observed enhanced chondrogenesis across a wide range of matrix storage moduli. In static cultures, MSCs cultured in hydrogels of varying stiffnesses show the highest expression of chondrogenic markers in the stiffest hydrogels, with G′ values of 21 and 25 kPa [19,20]. When combining different matrix stiffnesses with cyclic HP, chondrogenic markers were also increased in stiffer matrices (17.5 kPa), but better morphological outcomes were observed in softer matrices (5.2 kPa) [21]. These results are supported by an in vivo study of bone-marrow derived stromal cell (BMSC) laden hydrogels implanted in mice after a 2-week preculture, with enhanced chondrogenesis observed in the stiffer constructs that had a G′ of 10 kPa compared to those with a G′ of 1 kPa [22].

Although the previous studies point toward an ideal G’ for chondrogenesis in the kPa range, a study by Feng et al. examining the effect of crosslinking (CL) density of microgels prepared three gelatin-hyaluronic acid (Gel-HA) systems with average G′ values of 238 Pa (low CL density), 510 Pa (medium CL density), and 772 Pa (high CL density). After a 4-week culture, all three gels produced high cell viability, with BMSCs showing trends of differentiating into hyaline cartilage in gels with low crosslinking density and fibrocartilage in gels with medium and high crosslinking densities [23]. This indicates that further research should be conducted to determine the ideal G′ ranges for differentiation of different types of cartilage.

Another important matrix property is the 3D network microstructure. In a study by Yang et al., fibrous and porous collagen hydrogels with identical chemical composition and similar mechanical strengths demonstrated varied effects on BMSC culture. Both in vitro and in vivo experiments suggested that the fibrous network hydrogel facilitates better chondrogenic differentiation and may slow down extracellular matrix (ECM) calcification [24]. Further research should seek to confirm these results and identify the best combination of network structure and mechanical strength for successful chondrogenesis.

Environmental Cues to Chondrogenesis

This section provides a comprehensive review of chondrogenic responses of stem cells to various physical stimuli. A summary of previous findings is provided in Table 1.

Table 1.

Chondrogenic Responses of Stem Cells to Environmental Cues

Type of stimulation	Loading conditions	Cell source	Scaffold information	Key findings	Reference
Compressive loading and strain	Sinusoidal dynamic compression at 10 kPa with frequency of 0.25 Hz, 1 h/day; either early (day 1) or late (day 28) start	Human synovium derived MSCs	Agarose hydrogel	Dynamic compression at early-point downregulated chondrogenesis and hypertrophy, while late-start compression enhanced chondrogenesis and suppressed hypertrophy	[45]
	10% strain, 2 h per day, 5 days per week at 1 Hz after a 0–6 week priming period	Human bone marrow stromal cells	Fibrin hydrogel	GAG and collagen II deposition significantly increased as priming time increased Increased expression of chondrogenic genes with loading for all priming times. Loading suppressed expression of osteogenic genes in cultures with less than 6 weeks of priming time	[55]
	Vibrations at magnitude 0.49 g, frequency 40 Hz, for 30 min every 24 h for a period of 1, 3, 5, 7, 14, and 21 days	Rat BMSCs	Plate culture with chondrogenic media	Low magnitude high frequency vibration promotes BMSC chondrogenic differentiation	[63]
	Dynamic stretching simulation: 5% strain at 0.5 Hz frequency for 6 h continuous or intermittent with 20 min/h for 6 h	Rat ADSCs	PDMS with immobilized collagen or collagen and TGF -β1	Surface chemical characteristics and external mechanical stimulation could synergistically affect efficacy of chondrogenic differentiation	[155]
	Perfusion (fluid flow), volumetric flow rate of 0.2 mL/min, 21 days	Human BMSCs	3D porous poly(ɛ-caprolactone) (PCL)	Perfused constructs show similar cell metabolic activity and significantly higher sGAG production in comparison to their nonperfused counterparts	[156]
	Uniaxial cyclic compressive force (0.6 kPa, 0.05 Hz) on every culturing day starting on day 1 for 60 min	Cells from early-stage chicken embryos	Pellets	Cyclic uniaxial mechanical load applied for 1 hr for a period of 6 days entrains the molecular clockwork in chondroprogenitor cells during chondrogenesis in limb-bud derived micromass cultures	[47]
	Sinusoidal loading with a magnitude of 15% strain at a frequency of 1 Hz for 4 h/day	Rabbit BMSCs	Agarose culture	TGF-β signal transduction and activities of activating protein-1 (AP-1) and SOX9 are involved in the early stages of BMSC chondrogenesis promoted by dynamic compressive loading	[48]
	Air pressure driven bioreactor induced strain of 10% of hydrogel constructs at 1 Hz frequency for 1, 2, 2.5, or 4 h/day for 1, 2, and 3 weeks	BMSCs and embryonic stem cell derived cells	Hydrogel	Mechanical stimulation quantitatively increased gene expression of cartilage-related markers independently from presence of TGF-β	[49]
	In the bioreactor, gels subjected to 5% static strain for 5 min followed by sinusoidal 10% strain for 4 h, followed for 3 days	Human MSCs	Agarose culture	Variations in gel component concentration and stimulus frequency can be modified such that a significant chondrogenic response can be achieved by human MSCs in fibrin constructs after 8 hr of compression spread out over 2 days	[50]
	Loading protocol of 10% dynamic strain superimposed on static 2% tare strain for 5 days/week for 3 weeks	Calf BMSCs	Suspension	Dynamic compressive loading initiated after a sufficient period of chondroinduction and with sustained TGF-β exposure enhances matrix distribution and the mechanical properties of MSC-seeded constructs	[51]
	Preload of 0.01 N, then dynamic compression of 10% strain amplitude at a frequency of 1 Hz for 1 h/day, 5 days/week; either continuous (6 weeks) or delayed (only week 4–6)	Porcine BMSCs	Cylindrical agarose construct	Continuous exposure to low oxygen tension is a more potent pro-chondrogenic stimulus than 1 h/day of dynamic compression for porcine MSCs embedded in agarose culture	[52]
	Dynamic compression protocol consisted of 10% strain amplitude superimposed on 0.01 N/construct preload at a frequency of 1 Hz employed for 1 h/day, 5 days/week; begin at day 0 or 21	Porcine femur MSCs	Agarose hydrogel	Application of dynamic compression from day 0 inhibited chondrogenesis, which was not observed if MSCs waited for 21 days before	[53]
	Hydrogels subjected to 4 h daily intermittent dynamic compressive loading (0.3 Hz, 15% amplitude strain) for up to 14 days	Human BMSCs	Cylindrical hydrogel constructs	Loading regimen had inhibitory effect on chondrogenesis and osteogenesis	[54]
	10% compressive sinusoidal strain at 1 Hz frequency, superimposed on 5% compressive tare strain for 4 h/day and 5 days/week	Human MSCs	Methacrylated hyaluronic acid (MeHA) hydrogel discs	Dynamic compressive loading increased GAG and collagen, and reduced hypertrophy, in seeding density dependent manner (higher seeding density had more positive results)	[55]
	Prestrain of 10% followed by sinusoidal compressive load of 10% amplitude at a frequency of 1 Hz	Epiphyseal chondro-progenitor cells	Different hydroxyethyl methacrylate (HEMA)-based scaffolds	Scaffolds with energy dissipation level close to the one of cartilage favor chondrogenic expression when dynamic loading is present	[56]
	Cyclic compression at 0.03, 0.15, or 0.33 Hz for 2 h and load durations of 12, 54, or 120 min evaluated while holding frequency constant at 0.33 Hz	Chick limb bud MSCs	Agarose gel	Significant increase in chondrocyte differentiation for loading at 0.15 Hz and 0.33 Hz; also for 54 and 120 min tests	[57]
	Dynamic compression at strain amplitude of 15% and frequency of 1 Hz in presence/absence of TGF-β	Human BMSCs	Alginate or pellet culture	Upregulation in chondrogenic markers	[58]
	Intermittent dynamic compressive strain applied daily for 1 h at 5% peak-to-peak strain and 1 Hz in a sinusoidal waveform followed by 23 h of rest	Adult human MSCs	Hydrogel	Loading inhibited hypertrophy while maintaining chondrogenesis	[59]
	Preload with 5% static strain for 300 s, then sinusoidal compressive loading with 10% magnitude at frequency of 1 Hz for 1 h for 3, 7, and 14 days	Rabbit BMSCs	2% agarose gel	Cyclic compressive loading can promote the chondrogenesis of BSCs by inducing the synthesis of TGF-β	[60]
	Dynamic compressive strains applied at 5% peak to peak strain	Human skin fibroblasts (iPSCs)	Suspension	Supported chondrogenesis, but delayed transition to hypertrophy	[61]
	Cyclic displacement of 10% strain at 1 Hz	Human epiphyseal chondro-progenitor cells	Poly(2-hydroxyethylmethacrylate) (pHEMA) porous hydrogels	Coexistence of thermomechanical cues had superior effect on chondrogenic gene expression	[62]
	Ceramic hip ball (32 mm diameter) pressed onto scaffold and oscillates at ±25° at 1 Hz in addition to dynamic compression at 1 Hz with 10% sinusoidal strain, superimposed on 10% static offset strain	Human BMSCs	Composite scaffold	Mechanical load promotes chondrogenesis of human BMSCs	[64 –69]
	Cyclic pressure applied at 0.5 Hz frequency	Human MSCs	Alginate hydrogel beads	More ECM and upregulation of chondrogenic markers	[70]
Hydrostatic pressure (HP)	Cyclic HP applied for 4 h/day; 1 Hz sinusoidal waveform with minimum pressure of 0.55 MPa and a peak pressure of 5.03 MPa; 2 groups loaded 4 h on single day, 1 group loaded 4 h/day for days 1–7	Human mesenchymal progenitor cells from bone marrow	Aggregates in tubes with flexible rubber seals on caps	Single day loading did not demonstrate significant changes in proteoglycan and collagen contents; multiload day aggregates showed statistically significant increases in proteoglycan and collagen contents	[71]
	0.1, 1, and 10 MPa of intermittent HP at a frequency of 1 Hz for 4 h/day for 3, 7, and 14 days	Adult human BMSCs	Pellets	Different levels of HP differently modulate human BMSC chondrogenesis in presence of TGF-β	[72]
	1 MPa intermittent HP at frequency of 1 Hz for 4 h/day for 10 days	Human bone marrow stromal cells	Porous type I collagen sponges	HP enhanced differentiation of MSCs in presence of multipotent differentiation factors in vitro	[73]
	Sealed bags in pressure vessel; HP applied at amplitude of 1 MPa and frequency of 1 Hz for 4 h/day, beginning on day 2, until day 8	Mice embryonic limb bud cells	Pellets in suspension	HP had stabilizing effect on chondrogenesis, reducing expression of hypertrophic marker genes	[74]
	Sealed bags placed in pressure vessel and HP at amplitude of 2 MPa and frequency of 1 Hz for duration of 4 h/day, for 7 days	MSCs/marrow stromal cells	Collagen-alginate interpenetrating network hydrogel	Stiffness and timing of HP exposure impacted chondrogenesis of MSCs	[21]
	Tubes with flexible caps; cyclic HP at 650 PSI at 1 Hz for 4 h on day 1 or day 3 or days 1–7	Human bone marrow derived mesenchymal progenitor cells	Aggregate of pelleted cells	HP loading increased proteoglycan production; multiday loading enhanced proteoglycan and collagen production	[75]
	Steady (7.5 MPa) or ramped (1 MPa-7.5 MPa over 14 days) cyclic HP for 4 h/day at f = 1 Hz for 14 days	Human BMSCs	Agarose constructs in bags	HP may induce chondrogenesis in BMSC-seeded agarose constructs without TGF-β	[76]
	Intermittent HP applied to hMSCs in pellet culture at level of 10 MPa and frequency of 1 Hz for 4 h/day for periods of 3, 7, and 14 days	Adult human MSCs	Pellets	Adjunctive effects of HP on TGF-β induced chondrogenesis and mechanical loading can facilitate articular cartilage tissue engineering	[77]
	hASCs in 2% agarose hydrogels exposed to 7.5 MPa of cyclic HP for 4 h/day at a frequency of 1 Hz for up to 21 days	Human adipose derived stem cells	3D agarose hydrogels	HP initiates hASC chondrogenic differentiation, even in absence of soluble chondrogenic inductive factors	[78]
	Pellets subjected to 10 MPa of cyclic HP (4 h/day) and different concentrations of TGF-β (0, 1, and 10 ng/mL) for 14 days	Porcine chondrocytes, synovial membrane derived stem cells, infrapatellar fat pad derived stem cells	Pellets	At low concentrations of TGF-β, HP enhanced chondrogenesis; HP appears to play a key role in maintenance of chondrogenic phenotype	[79]
	Samples loaded for 4 h/day with 0.5–3 MPa cyclic HP at 1 Hz	Bovine bone marrow mesenchymal stromal cells	Pellets	Pressure had no influence on GAG/DNA content	[80]
	Dynamic sinusoidal HP at 1 Hz for 4 h, 0.55–5.03 MPa from days 1 to 7	Human meniscal cells	Suspension	Loading increased size and had enhanced chondrogenesis	[81]
	Continuous compressive load with sinusoidal waveform of 0.33 Hz and peak stress of 7994 Pa over period of 4 h/day for 7 days	Human bone marrow derived mesenchymal progenitor cells	Hyaluronan-gelatin composite scaffolds	7 days of loading upregulated chondrogenic markers; 21 days of loading significantly increased proteoglycan and collagen levels	[82]
	Scaffolds stimulated with HP of 10 MPa and 1 Hz for 2 h/day	Porcine MSCs	PCL, PCL-HA, or PCL-bioglass scaffolds	Scaffold composition modulated response to HP: positive effect on PCL and BG, but not for HA	[83]
	Agarose or fibrin and exposed to 10 MPa of cyclic HP at 1 Hz for 4 h/day, 5 days/week for 3 week	Porcine bone marrow derived MSCs	Agarose or fibrin hydrogels	Agarose hydrogels better supported chondrogenesis of MSCs	[84]
	10 MPa of cyclic HP at 1 Hz for 1 h/day, 5 days/week; either begin at day 0 or day 21	Porcine bone marrow derived MSCs	Cylindrical agarose constructs	Response to HP is donor dependent	[85]
	Cyclic load of 0.05 Hz, 600 Pa for 30 min applied on days 2 and 3	Chondroprogenitor cells isolated from limb buds of chicken embryos	Suspension	Proper mechanical stimuli augment in vitro cartilage formation by promoting both differentiation and matrix production of chondrogenic cells	[86]
	ASCs and BSCs exposed to 3 MPa cyclic HP	Human adipose derived MSCs and bone marrow MSCs	Seeding on silicone rubber substrates	Enhanced expression of chondrogenic markers	[87]
	Intermittent HP of 0.20, 0.10, 0.05, and 0.02 MPa for 2 h/day for 7 days starting on day 2	Rabbit MSCs and chondrocytes	Alginate beads	Stimulation with higher magnitudes of HP was more effective on proliferation and differentiation of cocultured MSCs	[88]
	Dynamic pressure with cell beads at 13–36 kPa and a frequency of 0.25 Hz for 1, 3, 5, and 7 days	Rat bone marrow derived MSCs	Pellets	Gene expression of chondrocyte-specific markers was upregulated by dynamic compressive stress introduced at the 8th day of chondrogenic differentiation in vitro	[89]
	Chondrocytes treated with static pressure of 100 kPa for 0, 1, 2, 3, and 4 h in a chamber	Rabbit mandibular condylar chondrocytes	Suspension	Static pressure for 3–4 h resulted in significant increase in COL2A1, SOX9, ALP, and Runx2	[90]
	Chondrocytes pressed with static pressures of 50 kPa, 100 kPa, and 200 kPa for 3 h	Rabbit mandibular condylar chondrocytes	Suspension	Chondrocyte proliferation increased at 100 kPa and decreased at 200 kPa	[91]
	Bioreactor offers steady oscillating laminar flow (max shear stress of 250 dyne/cm²) and hydrodynamic pressure (increased from 0 to 15 PSI)	Rabbit periosteal explants	PCL scaffold	Cartilage yield was 75%–85%, which is better than the 65%–75% shear stress only bioreactor and 17% static culture	[92]
	5–37 kPa at 0.5 Hz for 1 h/day for 3 days	Mouse prechondrogenic cell line	Suspension	HP promoted chondrogenesis in as little as 3 days	[93]
Static magnetic field	280 mT SMF from permanent magnet for 3, 7, or 14 days	Mandibular bone marrow MSCs	Indirect coculture with mandibular condylar chondrocytes	Moderate-intensity SMF improved the chondrogenesis and proliferation of MBMSCs in the coculture system, and it might be a promising approach to repair condylar cartilage defects in clinical settings	[94]
Static magnetic field	0.1, 0.2, 0.4, and 0.6 T SMF on BMSCs cultured as pellets for 3 weeks; then 0.4 T only for 1, 2, or 3 weeks	Adult human BMSCs	Pellets	Moderate-strength magnetic fields can induce chondrogenesis in BMSCs through a TGF-β dependent pathway	[99]
Pulsed magnetic field	PEMF: flux density of 2 mT, frequency of 15 Hz, magnetic field of 20 G, pulsed period of 67.1 msSPEMF: single pulse repeated in adjustable times and magnetic fields	Human ADSCs	3D pellet culture system	Both PEMF and SPEMF enhanced chondrogenic gene expression of ADSCs cultured in 2D- hyaluronan and 3D-pellet	[101]
Pulsed magnetic field	20 min at magnetic strength of 100 mT and a frequency of 40–70 Hz	Rat BMSCs	Magnetic hydrogel	PEMF boosted chondrogenesis of MSCs grown on the magnetic hydrogel	[102]
	Barrage of magnetic pulses of 6 ms duration at a repetition rate of 15 Hz and at flux densities between 1 and 4 mT	Human BMSCs	Pellets	Optimized PEMF regimens (10 min, 2 mT, 6 ms burst, 15 Hz, one time at onset of chondrogenic induction) have clear clinical implications for future regenerative strategies for cartilage	[103]
	0–3 mT for 10 min in X, Y, or Z-direction	Human BMSCs	Electrospun fibrous scaffolds	MSCs on randomly aligned (RND) scaffolds underwent greatest chondrogenic differentiation in response to 10 min at 1 mT in Z-direction (perpendicular)	[104]
Electromagnetic powered bioreactors	Soft, magnetic hydrogel that was vertically moved by magnetic field (0.8 T) induced by external electromagnet; 30 min cyles every 1.5 h for 8 h/day during 3 weeks	Human MSCs	Magnetic hydrogel	Pressure-less, soft mechanical stimulation precipitated by the cyclic deformation of soft, magnetic hydrogel scaffolds with an external magnetic field, can induce chondrogenesis in MSCs without any additional chondrogenesis transcription factors	[95]
Electromagnetic powered bioreactors	Input (1 A and 5 V) was applied to actuator coil so that electromagnet-derived force could be generated, it attracted metal caps and produced compressive stress on the samples; 1 Hz frequency for 10 min twice a day for 7 days	Rabbit BMSCs	3D alginate layer culture	Micromechanically exerted cyclic compressive loading by the cell exciter was very effective in enhancing the chondrogenic differentiation of MSCs, even without using exogenous TGF-β	[96]
Magnetic particles or conductive scaffold stimulated by electromagnetism	Magnetic nanoparticles 100 nm in diameter and cross-linked with dextran iron oxide composite particles with an iron oxide core; stimulated with permanent, external magnetic field	Human ADSCs	Magnetic nanoparticles (MNPs)	MNPs incorporated in human ADSCs under influence of external magnetic field have potential to induce differentiation toward osteogenic and chondrogenic lineages	[97]
	Magnetic particles incorporated into cell sheet and exposed to variable rotations around permanent magnets (1.45 T)	Human BMSCs	Magnetic sheets	Magnetic field-induced stimulation by magnetic particles did not affect cartilage formation	[98]
	TPT with 2.5% or 5% TA doped with camphorsulfonic acid (CSA), 28 days	Ovine chondroprogenitor cells	3D printed CSA-doped tetraaniline-b-polycaprolactone-b-tetraaniline (TPT) mixed with high molecular weight PCL	Greater sGAG and type II collagen production in 5% scaffolds	[100]
	21-day chondrogenic induction period, followed by 7 days of alternating magnetic field exposure for 10 min every 2 h	Human ADSCs and Wharton Jelly MSCs	Pellet culture in chondrogenic media	Magnetic field exposure significantly increased GAG deposition in human ADSCs, but significantly decreased GAG production in Wharton Jelly MSCs	[106]
	10 mV/cm, 60 kHz for 30 min, 4 times/day, 21 days	Porcine BMSCs	Gelatin-HA hydrogel	Increased proliferation, stimulated production of SOX-9 and aggrecan	[107]
Continuous ultrasound	Ultrasound (US) stimulation at frequency of 0.8 MHz and intensity of 200 mW/cm² for 10 min/day up to 4 weeks	BMSCs	PGA mesh	Total collagen and GAG increased more significantly in the US-stimulated group than in the control; US has some stimulatory effects in vivo on chondrogenesis of MSCs	[108]
	Wave with intensity of 200 mW/cm² and frequency of 1 MHz for 1–4 weeks	Rabbit MSCs	Fibrin-HA hydrogel	Chondrogenesis of MSCs in fibrin-HA was shown to be further enhanced by treatment with LIUS; combination of fibrin-HA and LIUS is useful tool in constructing high-quality cartilage tissues from MSCs	[112]
	LIUS applied 20 m/d until 1–2 weeks at frequency of 1 MHz and intensity of 200 mW/cm²	Human BMSCs	3D alginate cultures	LIUS treatment could be valuable tool in cartilage tissue engineering using MSCs as it enhances cell viability and directs the chondrogenic differentiation process	[114]
	3 levels of US were applied to the plates: 1.5, 5, and 8.5 MHz; duration of application was 161 s for 1.5 MHz, 51 s for 5 MHz, and 24 s for 8.5 MHz; signal applied twice over 24 h	Human cartilage chondrocytes	Porous 3D scaffold	Chondrocytes when stimulated with continuous US for predetermined time intervals possessed higher cellular viability (1.2–1.4 times) and higher levels of type II collagen and aggrecan mRNA expression compared to controls	[115]
	Stimulation applied for 20 min/day for a week at a frequency of 0.8 MHz and intensity of 200 mW/cm²	Rabbit MSCs	PGA nonwoven mesh	LIUS preconditioning in vitro could be an effective cue to upregulate chondrogenic differentiation of MSCs in vivo	[116]
	Wave at 1 MHz and intensity of 200 mW/cm² were applied for 10 min/day	Human ADSCs	Pellets	LIUS resulted in early chondrogenesis, so it may be an applicable, safe, efficient, and cheap tool for chondrogenic differentiation of ADSCs in cartilage tissue engineering	[117]
	14 kPa (5 MHz, 2.5 Vpp), 5 m/application and 6 applications/day at a regular interval	Human BMSCs	Pellets	US could be used to induce chondrogenic differentiation of MSCs for cartilage tissue engineering	[118, 119]
	cLIUS applied at 5 MHz, 2 MHz, or 8 MHz at a constant pressure amplitude of 14 kPa one time for 5 min	Human MSCs	2D suspension	Preconditioning of MSCs by cLIUS resulted in nuclear localization of SOX-9, phosphorylation of ERK 1 and 2, and disruption of actin filaments, and the expression of SOX-9 was dependent on the phosphorylation of ERK 1 and 2 under cLIUS	[120]
	14 kPa (5 MHz, 2.5 Vpp), 10 min/application, and 4 applications/day	Human BMSCs	Alginate:collagen hydrogels	Chondroinductive potential of cLIUS was preserved as noted by increased expression of SOX-9 and deposition of collagen II	[121]
Pulsed ultrasound	4 intensities (20, 30, 40, or 50 mW/cm²) of low intensity pulsed ultrasound (LIPUS) (on-off ratio of 20%, frequency of 3 MHz) waves; stimulation 20 min/day for 10 days	Rat BMSCs	Suspension	LIPUS promoted MSC migration; in vivo results demonstrate that it significantly enhances cartilage repair effects of MSCs on OA	[109]
	Performed experiments varying intensity (10–300 mW/cm²), duty cycle (0.02%–80%), frequency (1.5–3.5 MHz) and excitation duration (1–5 min) – 30 mW/cm², 1.5 MHz, and 20% was best	Human MSCs	3D printed poly(ethylene glycol) (PEG) scaffold	Integrating LIPUS and microbubbles is a promising strategy for enhanced human MSC growth and chondrogenic differentiation	[110]
	US signal with spatial and temporal average intensities of 15, 30, 60, and 120 mW/cm² with a frequency of 1 MHz and a 200 μs tone burst repeating at 1 kHz; 30 min/day	Human MSCs	Pellets	LIUS enhances TGF-β mediated chondrocyte differentiation of MSCs in pellet culture, and the application of US may facilitate larger preparations of chondrocytes and the formation of mature cartilage tissue	[113]
	Spatial- and temporal-average intensities of 30 mW/cm², with a frequency of 1.5 MHz with 200 μs-burst sine waves repeated at 1 kHz	Rat chondrocytes	Aggregate chondrocyte culture system	LIPUS promotes the proliferation and retains the differentiation state of chondrocytes in the aggregate culture and that TGF-β plays an important role in mediating the LIPUS effects in chondrocytes	[122]
	30 mW/cm² for 0, 20, and 40 min/day, for the first 7 consecutive days of culture; carrier frequency of 1.5 MHz and 200 μs tone burst repeating at 1 kHz	Human BMSCs	Hyaluronan–gelatin scaffolds	LIPUS can be used to enhance chondrogenesis of MSCs in cell aggregates and cell–scaffold constructs	[123]
	1 MHz, pulsed 1:4 (2 ms “on” and 8 ms “off”) and intensity of 200 mW/cm² with a repetition rate of 100 Hz	Human BMSCs	Suspension seeded on plates	LIPUS alone increases osteogenic differentiation of human BMSCs, and LIPUS enhances TD-mediated chondrogenic differentiation of human BMSCs	[124]
	3.5 MHz, 27.5 mW/cm², and 5 min/application	Chondrogenic progenitor cells from bovine	Suspension	LIPUS might be utilized to promote cartilage healing by inducing migration of Chondrogenic progenitor cells to injured sites, which could delay or prevent the onset of post-traumatic osteoarthritis	[125]
	Frequency of 3 MHz and average intensities of 20, 30, 40, and 50 mW/cm² for 20 min/day for 10 days	Rat BMSCs	Pellets	LIPUS promoted TGF-β induced chondrogenesis of BMSCs, represented by increased expression of COL2, aggrecan, and SOX-9 genes and decreased expression of COL1	[126]
	Average intensity of 30 mW/cm², pulse repetition rate of 1 kHz, and duty cycle of 20% for 20 min/day for 4 weeks	Rat BMSCs	Exosomes	LIPUS enhances the promoting effects of BMSC-derived exosomes on osteoarthritic cartilage regeneration, mainly by strengthening the inhibition of inflammation and further enhancing chondrocyte proliferation and cartilage matrix synthesis	[127]
Piezoelectric stimulation by ultrasound	Range of 1–20 mW/cm² delivered to MSCs on a quartz coverslip that localizes electric charges by vibrating	MSCs	AT-cut quartz plate	Drove clustering of MSCs and consequently facilitated chondrogenesis of MSCs without the use of differentiation media	[111]

ADSC, adipose-derived stem cell; BMSC, bone-marrow derived stromal cell; MSC, mesenchymal stem cell; iPSC, induced pluripotent stem cell; HP, hydrostatic pressure; RCCS, rotary cell culture system; TGF-β, transforming growth factor beta; PGA, polyglycolic acid.

Dynamic/intermittent versus static cultures

During daily activities, articular cartilage experiences a combination of extrinsic and intrinsic compressive, tensile, and shear forces [25,26]. In healthy individuals, major load-bearing joints such as the knee and hip are subjected to thousands of compressive loading cycles per day [27]. For example, compressive forces can be static (up to 1 MPa while standing) or dynamic (up to 20 MPa when jumping) [28]. The presence of static pressure is also necessary within joints to control the growth and differentiation of chondroprogenitor cells into chondrocytes. If proliferation and ECM deposition continue to increase without the introduction of sufficient surrounding pressure, such as provided by the perichondrium in developing tissues, an accelerated hypertrophy may occur, leading to ectopic cartilage formation [25,29,30]. It then follows that dynamic stem cell cultures may be more suitable than static cultures to mimic the in vivo conditions that both promote and regulate chondrogenesis.

Dynamic loading on human chondrocyte cultures has been shown to increase cartilage matrix deposition and improve the mechanical qualities of engineered cartilage tissues [31]. In uniaxial loading studies, chondrocytes have been known to respond positively to moderate dynamic compressive loading in vitro, in the range of 2%–10% strain [32,33], 0.5–1.0 MPa [34,35] applied at 0.1–1.0 Hz [25,26]. In contrast, HP and shear loading have produced mixed effects on chondrocyte macromolecule synthesis and metabolism, respectively [36 –40]. More recent studies simulating multiaxial loading by combining compression and shear loading have observed superior chondrogenesis and increased glycosaminoglycan (GAG) and collagen II deposition compared to static culture [41], and even the suppression of factors that contribute to joint destruction in osteoarthritis (OA) [42]. In dynamic in vitro cultures, loading methods combined with chondrogenic induction media mimic the conditions that differentiating chondrocytes are subjected to in vivo. The benefits of dynamic culture over static culturing have previously been observed for inducing chondrogenesis in stem cells [43].

In a review by Fahy et al., studies of human, rabbit, and bovine MSCs all demonstrated increases in chondrogenic markers when the cells were exposed to cyclic compression, HP, or a combination of cyclic compression and surface shear [13]. Mechanical agitation of 3D cultures has corroborated these results, with the added benefits of increased proliferation and chondrogenic differentiation of BMSCs compared to 2D cultures [44]. Loading methods, when combined with chondrogenic induction media, mimic the conditions that differentiating chondrocytes are subjected to in vivo. Mechanical agitation of 3D cultures has corroborated these results, with the added benefits of increased proliferation and chondrogenic differentiation of BMSCs compared to 2D cultures [44].

Several groups have sought to evaluate whether dynamic culture conditions would enhance chondrogenesis, rather than how preculturing in static conditions would affect the outcome. As such, some recent studies have shifted toward determining if allowing for a static differentiation period before dynamic culture is beneficial, and whether there is an ideal amount of priming time which will best encourage chondrogenesis and cartilage maturation. Subjecting cultures to 7 days of cyclic HP or dynamic compression after a 7- or 21-day preculture, respectively, showed enhanced chondrogenesis and suppressed hypertrophy compared to loading without a preculture [21,45]. McDermott et al. varied the priming times before dynamic compression even further and found that shorter priming times may preserve chondrocyte homeostasis, while longer priming times may support cartilage maturation [46]. In all cases, applying loading at the beginning of a culture period tended to drive cells toward osteogenesis, while allowing for a priming period encouraged chondrogenesis, suggesting that priming time does affect the effectiveness of physical loading.

Compressive loading and strain

Some groups evaluated the effect of unconfined compressive strain on chondrogenesis. This loading may impact chondrogenesis through the entraining of the molecular clock within cells [47]. For these compression experiments, samples were exposed to a strain with a magnitude of 2%–20% (or less than 10 kPa) at a frequency of 1 Hz or lower [45,47 –59]. The strain cycle was (1) cyclic through sinusoidal loading [45,47 –49,52,54,58,59], (2) dynamic strain superimposed on static loading [51,53,55], or (3) a brief, initial static preload followed by a longer period of sinusoidal loading [50,56]. Custom-made bioreactors within incubators were used to generate the compressive forces, several of which were driven by air pressure [45,47 –57,60 –62]. Groups tested the ideal length of time that cell cultures should receive loading, which was around 1–4 h per day [49,57].

In addition, it was determined that cells should not be placed in a loading environment until after they have had adequate time to undergo chondrogenic differentiation [45,53]. Delayed time point dynamic compression (beginning around day 20) enhanced chondrogenesis and suppressed hypertrophy, while early time point compression suppressed chondrogenesis [45,51,53]. Temperature increases from compression are an unavoidable by-product of loading. Nasrollahzadeh et al. examined this widely neglected effect and found that the coexistence of thermomechanical cues had a superior effect on chondrogenic gene expression compared to either signal alone [62].

In addition, it seems that the need to add TGF-β to cultures is dependent on the type of stem cell used [49]. For example, MSCs and induced pluripotent stem cells (iPSCs) were found to show increased expression of cartilage-related markers in response to loading, independent from the presence of TGF-β [48,49,58,61]. Yet, it is believed that addition of TGF-β would only synergistically improve the chondrogenic response, to an extent [51,53,58,61]. However, embryonic stem cells required the addition of TGF-β to the existing loading to achieve these positive results [49].

Both the type of hydrogel and the concentration of cells within it are also important components of load testing. Scaffolds with higher dissipation levels lead to greater upregulation of chondrogenic markers [56]. As shown by Pelaez et al., fibrin gel is suitable for supporting cyclic compression-induced chondrogenesis of MSCs, and the gel works optimally at a concentration of 40 mg/mL [50]. Bian et al. concluded that MSCs in hyaluronic acid (HA) hydrogels with higher seeding densities (60 million cells/mL) led to increased mechanical properties and chondrogenesis [55].

Studies of shear strain induced by mechanical agitation of 3D cultures [44] or through low magnitude, high frequency vibration [63] supplemented with chondrogenic induction media have resulted in enhanced chondrogenesis within their dynamic culture groups. Surface shear strain superimposed on axial compression also has the potential to enhance chondrogenesis.

A 32 mm diameter ceramic hip ball is part of a bioreactor that is used to apply this complex dual loading to cells [64 –69]. The ball is pressed onto a cell-seeded scaffold and oscillates at ±25° at 1 Hz about an axis perpendicular to the scaffold axis, producing interface shear motion [64 –69]. This oscillation is in addition to the normal 1 Hz 10%–20% sinusoidal strain and/or 10% static strain [64 –69]. A representation of this type of loading apparatus is depicted in Fig. 1. Results showed that this type of loading promotes chondrogenesis of MSCs through the TGF-β pathway by upregulating TGF-β gene expression and protein synthesis [64,66,69]. The combination of compression and shear leads to significant increases in chondrogenesis, while either stimulation alone was insufficient [67]. In addition, asymmetrically seeded scaffolds led to improved tissue development [68].

FIG. 1.

A compressive loading and strain bioreactor: A 32 mm diameter ceramic hip ball bioreactor is held by a mobile metallic holder. The hip ball is pressed against an MSC-seeded scaffold and compresses the cell seeded scaffold dynamically at a strain amplitude that is a percentage of its height. It also oscillates at ±25° perpendicular to the scaffold to produce mechanical shear stress. The resulting complex dual loading upregulates TGF-β expression to increase chondrogenesis significantly. MSC, mesenchymal stem cell; TGF-β, transforming growth factor beta.

A slight alteration to the ceramic hip ball bioreactor is the metal roller [70]. This device can also apply both shear and cyclic compressive forces to the cell culture [70]. Results showed that this bioreactor upregulates positive chondrogenic markers only when both shear and compressive forces are administered [70].

Hydrostatic pressure

Repetitive HP is one component of the mechanical stresses present within joints, and the physiologic pressure range for humans is 3–10 MPa [71,72]. HP does appear to induce chondrogenesis in vivo, where chemical signals for multiple differentiation pathways are present [73]. Thus, in vitro HP can mimic the natural joint loading environment [74]. To create this dynamic mechanical stimulation, groups followed a similar experimental setup. Cell aggregates were placed in tubes with flexible caps that were then placed in the stainless steel HP vessel within a water-filled temperature-controlled incubator bath (set at 37°C) or aggregate pellets or tubes were placed in sterile plastic bags before being placed into the chamber (Fig. 2) [21,71 –85].

FIG. 2.

Examples of HP bioreactors: (A) Cell aggregates or scaffolds are placed within a chondrogenic medium-filled tube with a flexible rubber seal in the center of the cap. These tubes are then placed within a temperature-controlled HP chamber, where cyclic pressure is applied. (B) Cell-laden hydrogels are placed within sterile plastic bags that are filled with growth medium. These bags are then placed within the chamber where cyclic HP is applied. HP, hydrostatic pressure.

Some systems were powered by electromagnets [82,86]. Cyclic HP was consistently applied for 4 h per day at a frequency of 1 Hz [21,71 –82,85]. Although early studies examined a range of pressures from 0.1 to 10 MPa [71,75,77], by 2006 most studies chose to use (1) under 5 MPa [21,73,74,80 –82,87,88], (2) 7.5 MPa [76,78], or (3) 10 MPa [72,79,83 –85,89]. As shown by Finger et al. in 2007, this pressurization could either be instantly achieved or progressively ramped up to the final intended value in daily increments, with the steady application producing better effects [76]. In addition, HP could either be applied immediately on day 0 of culture or delayed to a later day further along in the differentiation process of the cells. Interestingly, delayed application of HP resulted in improved chondrogenesis [21,89].

Finally, the stiffness of the scaffold in which cells are encapsulated in also likely plays a role in the results, with softer hydrogels mimicking the in vivo cartilage matrix [21,83,84]. Even the type of stem cell and the donor have been shown to influence how loading impacts chondrogenesis [85,87]. Only two studies were identified in which static pressure was generated within a pressure chamber. In these reports, done by the same group, cells were loaded into wells and transferred into the machine producing 50–200 kPa of static pressure for 0–4 h [90,91].

Many groups sought to develop an in vitro loading system that could examine the effect of cyclic HP on a particular line of stem cells undergoing chondrogenesis with adequate growth medium [21,71,73 –75,77,80 –89,92,93]. Results showed that multiday loading could enhance proteoglycan and collagen production of the differentiating stem cells compared to unloaded controls [71,73,75,81,82,88]. Chondrogenic markers such as SOX9, type II collagen, and aggrecan levels were upregulated to different degrees by different loading pressures [73,74,77,82,86]. Size changes were noted in HP samples, with these constructs being more uniform and compact [71,73,75,77]. Similar results were obtained with static pressure, as chondrogenic marker expression increased for pressures of around 100–150 kPa applied for 3 h [90,91]. However, all of these studies only looked at HP indirectly since conditions that promoted chondrogenic differentiation were used. Thus, other groups looked at whether cyclic HP is a functional stimulus for chondrogenesis, either alone or in the presence of TGF-β [72,76,78,79].

Even without TGF-β, groups found that stem cells exposed to cyclic HP showed early increased SOX9, type II collagen, and aggrecan levels [72,78,87,88]. Effects were additive if TGF-β was added, with greater concentrations producing better effects to an upper limit [72,79]. Finger et al. found that HP without TGF-β initially increased collagen I expression, then later increased SOX9, suggesting that HP alone may be sufficient to begin the induction of chondrogenesis in stem cells [76]. Puetzer et al. came to a similar conclusion, as chondrogenic expression levels were only upregulated with HP for the first 7 days of loading [78]. In contrast, Vinardell et al. concluded that HP alone cannot induce robust chondrogenesis of stem cells, but can act synergistically with TGF-β to increase chondrogenic differentiation and inhibit collagen type X [79]. Still, HP, with or without TGF-β, was also shown to create cell pellets that were more compact and uniform in structure [72,79].

In addition, HP superimposed on another mechanical stimulation, such as fluid shear stress, upregulates chondrogenesis more than either loading alone [86,87,92]. This is likely due to complex loading patterns that better mimic the native in vivo cartilage environment.

Electromagnetic stimulation

Magnetic forces do have excellent prospects for application as a noninvasive physical stimulation for chondrogenic differentiation [94]. These forces can be used to power mechanical stimulation bioreactors [95,96]. They can also be incorporated into nanoparticles that are internally incorporated into cell cultures and stimulated by external magnetic fields [97,98]. The external magnetic forces that are directly applied to stem cell constructs, with or without magnetic nanoparticles, can be generated by either (1) static magnetic fields (SMFs) or (2) pulsed magnetic fields (PMFs). These two stimulation techniques are discussed below.

Biophysical forces such as SMF have attracted interest for their effect on cartilage regeneration [99]. SMF changes the structure and function of cells by exposing them to permanent magnets that are stable in direction and intensity [94]. Although some reports at the time suggested that SMF may be useful in stimulating chondrogenic differentiation of precursor cells, a study by Amin et al. in 2014 provided a detailed look into the chondrogenesis-promoting in vitro technique with adult human BMSCs [99]. The group cultured BMSCs in a SMF of 0.1, 0.2, 0.4, and 0.6 T of SMF for 3 weeks, and they also exposed samples to 0.4 T SMF for 1, 2, or 3 weeks since this field strength was shown to be optimal in previous reports [99]. Results showed that SMFs stimulated sulfated-GAG (sGAG) production (a key indicator of terminal chondrogenic differentiation) and collagen II deposition, and maximal effects were seen in the 14–21 days 0.4 T groups [99].

In addition, SOX9, aggrecan gene (ACAN), and collagen type I gene (COL2A1) expression levels were elevated in SMF conditions compared to controls, and the TGF-β pathway was even stimulated by SMF [99]. Thus, a moderate-strength SMF promoted chondrogenic differentiation of BMSCs [99]. Zhang et al. found similar results in 2021 when the group investigated the effects of another moderate-strength SMF on the chondrogenesis of a mandibular bone marrow mesenchymal stem cell (MBMSC)/mandibular condylar chondrocyte (MCC) coculture [94]. The group used a 0.280 T SMF and found that this regimen promoted proliferation of cells, as well as chondrogenesis [94]. Within a week, the cells displayed Alcian blue staining and elevated SOX9, COL2A1, and ACAN expression [94].

In addition, bioelectricity plays a key role in a multitude of physiological processes, with recent evidence suggesting that cells respond to bioelectric signals during embryonic development and regenerative repair [100]. Current research on the electromagnetic mechanobiology of stem cells has centered around finding the ideal combination of electrical and magnetic field stimulation for enhanced chondrogenesis in vitro.

In 2013, Chen et al. examined a pulsed electromagnetic field (PEMF) and a single-pulsed electromagnetic field (SPEMF) to compare chondrogenic induction of adipose-derived stem cells (ADSCs) cultured in both 2D and 3D [101]. For the PEMF condition, the following characteristics were implemented: magnetic flux density of 2 mT, frequency of 15 Hz, magnetic field of 20 G, and pulsed period of 67.1 ms, all for 8 h per day [101]. For the SPEMF condition, the following characteristics were implemented: period of 5 ms, magnetic field magnitude of 0.6–1 T, magnetic flux density of 1 T, all for 3 min per day [101]. In both the 2D and 3D conditions, both PEMF and SPEMF stimulation increased chondrogenic marker genes and increased sGAG synthesis [101]. The group concluded SPEMF to be a superior treatment since it had reduced administration times and did not induce osteogenesis [101].

In 2019, Huang et al. expanded the knowledge of PEMF by examining its combined benefit with hydrogel embedded with magnetic nanoparticles [102]. The group found that the hydrogel together with the PEMF treatment promoted chondrogenesis, as evidenced by strong Toluidine blue staining and increased collagen II, SOX9, COL2, and ACAN expression [102]. It was hypothesized that these positive results were due to PEMF enhancing the cellular interactions and nutrient perfusion [102].

Also adding to the knowledge of the effects of PEMF on stem cells, Parate et al. varied the field amplitude, exposure duration, and dosage to determine the briefest and lowest amplitude exposure to produce a significant outcome [103]. MSCs were subject to 0–4 mT for 10 min, with 2 mT producing maximal effects in terms of SOX9, aggrecan, and COL2 expression, leading this strength to then be tested for 5–60 min [103]. The 10 min treatment proved to be best, and it was also found that the PEMF produced the best effects (increased collagen II levels) when administered once per day [103].

Celik et al. expanded on the work of Parate et al. by examining the influence of directionally applied PEMFs on chondrogenic differentiation of MSCs onto topographically-variant scaffolds [104]. MSCs were exposed to 1 mT of PEMF for 10 min per day in the X, Y, and Z directions (Fig. 3) [104]. A testing protocol of 1 mT and 10 min per day was chosen because these parameters optimized the upregulation of SOX9, COL2, and ACAN [104]. Results showed that PEMF in the Z-direction elevated cartilage markers, while the X-direction had little effect and the Y-direction inhibited chondrogenesis [104]. Most recently, as reviewed by Varani et al., rabbit, bovine, and human MSCs have shown enhanced chondrogenic differentiation, paracrine activity, migration, and proliferation in response to PEMFs at various magnitudes and frequencies.

FIG. 3.

An electromagnetic stimulation bioreactor: The directionality of the magnetic field influences chondrogenesis for MSCs in a fibrous scaffold of different alignments. For an aligned scaffold, Y-directed fields (parallel to cells) demonstrated strong chondrogenic inhibition. X-directed fields (perpendicular to cells) had no effect on chondrogenesis when cells were aligned and did increase hypertrophy. Z-directed fields significantly increased chondrogenic expression, but only for MSCs randomly aligned on a scaffold.

Yet, the impact of SMF, PMF, SPEMF, and PEMF is not wholly proven. Electromagnetic stimulation has been found to both enhance and inhibit chondrogenesis [104 –106]. In a study with rat BM-MSCs, PEMF exposure resulted in the inhibition of maintaining a cartilaginous phenotype [105]. While both exposure to electrical fields and the use of conductive polymer scaffolds have led to increased chondrogenesis separately [100,107], these results are yet to be expanded to in vivo conditions, and the effects of external electrical stimulation in conjunction with conductive scaffolds are yet to be investigated.

In studies focusing on magnetic field stimulation, the effectiveness of SMF or PMF exposure at encouraging chondrogenesis has been mixed. One study found that even after allowing for a chondrogenic induction period, SMF exposure only increased chondrogenesis in human ADSCs, while it decreased chondrogenesis in Wharton Jelly MSCs, demonstrating that not all types of stem cells react equally to MF exposure [106]. Another study focusing on the directionalities of magnetic fields found that Z-directed fields had the best effect on chondrogenesis, while X-directed fields inhibited chondrogenesis in randomly oriented scaffolds. Interestingly, the same study observed that PEMFs did not promote chondrogenic differentiation on either polystyrene tissue culture plastic or aligned scaffolds, irrespective of field directionality, and that multiple PMF exposures could also inhibit chondrogenesis in all types of scaffolds tested [104].

Dikina et al. found in 2017 that neither static nor variable magnetic fields generated by 1.44–1.45 T permanent magnets affected cartilage formation in a scaffold-free MSC sheet [98]. Even those samples that used magnetic nanoparticles did not make an impact on chondrogenesis [98]. These results suggest that the success of chondrogenesis in response to MF exposure depends on many different factors. Overall, further research should be conducted to determine ideal stimulation parameters, categorize the effects of MF exposure on the chondrogenesis multiple types of stem cells, and to confirm the efficacy of the conditions presented above for enhancing chondrogenesis.

Ultrasound signaling

Ultrasound is a mechanically vibratory form of acoustic wave energy that travels through a given medium [108,109]. Over the past few decades, medical use of ultrasound has been extended beyond imaging and diagnosis toward therapeutic applications [110]. Ultrasound can be used to induce piezoelectric stimulation, which has been shown to facilitate MSC chondrogenesis [111]. Yet, most notably, low intensity ultrasound (LIUS) has been used to induce localized chondrogenic differentiation and enhance cell viability and cartilage matrix synthesis [112,113]. This type of ultrasound uses a frequency of 0.75–1.15 MHz and an intensity lower than 200 mW/cm² [110]. These waves are characterized as either (1) continuous (cLIUS) or (2) pulsed (pLIUS). These two stimulation wave forms are discussed below.

All groups that used a cLIUS wave found that this biomechanical stimulation treatment can accelerate chondrocyte differentiation and improve cellular viability in vitro or in vivo [108,112 –121]. An example of this type of apparatus is provided in Fig. 4. In 2004, Ebisawa et al. tested a continuous signal with intensities of 15, 30, 60, and 120 mW/cm², finding the 30 mW/cm² treatment to produce the best effects [113]. Subsequent groups chose an intensity of 200 mW/cm² [108,112,114,116,117] or less than 30 mW/cm² [115,120]. Signal frequency and treatment time ranged from 0.8 MHz to 8.5 MHz and 1 min/day to 80 min/day, respectively [108,112 –121].

FIG. 4.

An ultrasound signaling bioreactor: A transducer emits the low-intensity ultrasound waves through the culture medium to stimulate the cell-laden hydrogel scaffold or cell pellet culture. The transducer surface is aligned perpendicularly to face the cells. Treatment is either pulsed or continuous and varies in terms of intensity, frequency, and time per day.

Results for cLIUS treatments showed that chondrogenesis was significantly upregulated, as evident through increased GAG content [108,112,116], ECM synthesis [108,116], aggrecan expression [108,114 –116], SOX9 expression [114,116,118 –121], collagen type II content [112,114 –117,119 –121], and cellular viability [114,119]. Osteogenic differentiation, as measured by markers for type X collagen, was also downregulated [116,117]. Yet, there are conflicting findings on whether cLIUS can act in the absence of TGF-β. Ebisawa et al. reported in 2004 that application of cLIUS alone showed little chondrogenic enhancement [113]. They found that cLIUS could only enhance the effects of TGF-β mediated chondrocyte differentiation [113]. Yet, experiments conducted by others showed that cLIUS treatment could positively impact chondrogenesis in the absence of exogenously added growth factors [114,118,119]. cLIUS was shown to perhaps even be better than TGF-β treatment, which results in unwanted production of the osteogenic collagen type X [117,121].

pLIUS uses low energy and pulsates it with very short intervals to produce pulsed pressure waves [122]. Similar to cLIUS, pLIUS is also shown to enhance chondrogenic differentiation and repair [109,110,122 –127]. Most groups chose an intensity of around 20–50 mW/cm² [109,110,122,123,125 –127] or 200 mW/cm² [124]. A frequency of 1.5 MHz was commonly used, with 200 μs-burst sine waves repeated at 1 KHz [110,122,123,127]. Yet, some groups did use 1–5 MHz [109,124,125] with a 100 Hz repetition rate [124]. pLIUS treatments were administered once per day, for 20–40 min/day [109,110,122 –127]. Results showed that pLIUS promotes the proliferation and stimulates differentiation of chondrocytes [110,122,123]. Expression of collagen type II [122,123,127], aggrecan [122], ECM [123],7 SOX9 [127], and proteoglycans [110,123] was increased, while collagen type X levels decreased [122]. However, most groups determined that TGF-β was required for pLIUS to produce these positive effects [109,122,124,126].

Alternative Cell Culture Approaches

This section provides a comprehensive review of alternative cell culture approaches. A summary of previous findings is provided in Table 2.

Table 2.

Alternative Cell Culture Approaches

Type of stimulation	Loading conditions	Cell source	Scaffold information	Key findings	Reference
Organ on a chip	Perfusion flow rate of 30 μL/min, average bottom wall shear stresses in chambers 1–4 in uniform regions were 1.089, 0.231, 0.055, and 0.009 dyne/cm²	Rat MSCs and primary chondrocytes	Suspension	Increase in stimulation magnitude increased osteogenesis and dedifferentiation for chondrocytes	[128]
	Dynamic sinusoidal unidirectional compression at 1 Hz for 2 h/day, 6 days/week for 3 and 7 days; air pressure of 21 kPa, corresponding to strain magnitudes of 20%–0% in reach row; flow rate of 250 μL/h for the 20 wells	Rabbit ADSCs	Alginate hydrogel	Dynamic mechanical compression positively influenced cellular viability and upregulated collagen II, SOX-9, and aggrecan in absence of exogenous growth factors in ADSCs; 10% found to be optimal	[129]
	Membrane deformation at 0.3 Hz for 2 days and compressed air at 5–6 kPa applied below wells	Rabbit periosteal cells	Suspension	Chondrocytes could be differentiation only under large and nearly single dimensional tensile strain	[130]
Shaking	See-saw shaker, 0, 0.3, or 0.5 Hz for 28 days after a 3 day preculture	Mouse iPSCs	Suspension	Shaking significantly enhanced chondrogenesis, with no significant differences between 0.3 and 0.5 Hz	[131]
Sliding contact	20% axial strain at 2.5 mm/s for 21 day, 3 h/day, 5 days/week	Calf MSCs	Agarose strips	Increased expression of aggrecan and type II collagen	[132]
Microgravity	RCCS container initially at speed of 10–12 rpm, then adjusted to 12–14 rpm after 24 h	Rabbit bone marrow stromal cells and MSCs	Suspension	RCCS provided a dynamic culture microenvironment conductive for cell proliferation, aggregation, and differentiation	[133 –135]
	Rotating Wall Vessel (RWV) bioreactor set to speed of 11 rpm for 14 days	Human ADSCs	Pellets	Increased cell proliferation, but not as enhanced chondrogenesis as the cyclic HP samples	[136]
	Rotation speed of the RCCS varied between 11–25 rpm and increased steadily with time for 21 days	Human adipose derived MSCs	Pellets	Microgravity upregulated chondrocyte-specific gene expression and matrix production (with TGF-β 1)	[137]
	Rotation speed of the RCCS vessel varied between 4.2 and 7.2 rpm and increased to maintain cells in free floating position; applied in last 7 days of 21 day differentiation period	Adult human MSCs	Pellets	Simulated Microgravity (SMG) reduced hypertrophy of MSCs during chondrogenic differentiation, but collagen II expression was reduced	[138]
	Rotation speed of the RCCS increased from 20 to 30 rpm over the course of 21 days	Human meniscus cells and adipose derived MSCs	Coculture	SMG significantly enhanced cartilaginous matrix formation in cocultures of meniscus cells and MSCs	[139]
	Microgravity rotating culture with 20 rpm for 2 weeks	BMSCs	Suspension	Compared with routine 3D static culture, the microgravity rotating culture system was more effective for the construction of tissue-engineered cartilage in vitro	[140]
	Rotational speed of 6.5 rpm	Rabbit BMSCs	Pellets	Independent, spontaneous differentiation of MSCs toward a nucleus pulposus-like phenotype in SMG occurred without addition of any external bioactive stimulators	[141]
	Rotation speed adjusted to the velocity at which the pellet was maintained in free-fall condition at ∼7 rpm between days 15 and 21	Adult human BMSCs	Pellets	SMG reduced collagen II expression	[142]
	Rotating bioreactor with 4 sponges	Rat chondrocytes	poly(lactic-co-glycolic acid) (PLGA) sponge	Higher yield of cell attachment and spatially uniform cell distribution was achieved when dynamic seeding was used; promoted growth and infiltration throughout sponge structure and showed formation of cartilage tissue	[143]

On a chip

Microfluidic-based microdevices accelerate the execution of experimental testing by decreasing the sample size and platform and better controlling the surrounding microenvironment compared to conventional methods. These devices have been used to examine the effects on extrinsic factors, such as mechanical stimulation, on cell fate. Recently, several groups have used this technique to examine the effect of (1) shear stresses or (2) strains on the chondrogenesis of stem cells.

Shear stresses within the microfluidic device can potentially play a role in chondrocyte differentiation, as demonstrated by Zhong et al. in 2013. These stresses mirror fluid flow stimuli within the cartilage matrix in vivo that regulates cell behavior and functions [128]. The microdevice used by Zhong et al. consisted of four cell culture chambers connected to different resistance channels, and fluid flow was driven by a pump (Fig. 5A) [128]. Both articular cartilage chondrocytes and primary MSCs were isolated from rats and injected into a cell chamber [128]. Average stresses administered to the cell cultures ranged from 1.089 to 0.009 dyne-cm² [128]. Results showed that the proliferation rates of both chondrocytes and MSCs increased with increasing magnitude of fluid shear stress [128]. However, SOX9 levels in the differentiating MSCs were not altered for the different shear stresses [128]. With increasing levels of fluid flow, chondrocytes began to synthesize collagen I, while collagen II expression diminished [128].

FIG. 5.

Examples of organ on a chip bioreactors: (A) An integrated microfluidic perfusion device generated multiple-parameter fluid shear stresses on MSCs. The four cell culture chambers experienced different stresses due to different resistance channels. While increasing fluid shear stresses did increase cellular proliferation, these higher levels did reduce chondrogenesis to favor osteogenesis. (B) A multilayer microdevice is used to apply unidirectional compressive stimulation to adipose-derived stem cells in a 3D alginate hydrogel cell culture. Uniform axial strain is applied to the cell-laden alginate hydrogel by moving the middle layer upwards as the pressure in the air chamber increases. Strain levels of 0%–20% were examined. This dynamic compression increased chondrogenesis, even without TGF-β supplementation. (C) A membrane-type microsystem provides cell stretching stimulation in two directions to periosteal cells. Circular (1:1) and oval cell (1:2 and 1:3) culture wells generate uniform and nonuniform tensile strain at the actuated pressures of 5 and 6 kPa for 2 days. Chondrogenic differentiation only increased when a large and two dimensional strain was applied to the cell culture.

In 2018, Kowsari et al. published the first known report on the effect of different uniaxial strains on chondrogenic differentiation [129]. The group used a novel semiconfined design that applied a wide range of static and dynamic mechanical compressive strains to cell-laden cylindrical hydrogels [129]. The microbioreactor is a 5 × 4 well array of 20 actuation cavities that enable simultaneous various compressive strains by a single pressurized air source [129]. When pressure in the air chamber of the microdevice increases above atmospheric pressure, an elastic polydimethylsiloxane membrane moves upwards and creates uniform cyclic axial strain in the hydrogel [129]. A diagram of this organ on a chip model is given in Fig. 5B.

Numerical methods and equations were used to ensure that the strain distribution was uniform and the microenvironment was suitable for the vitality of the cell culture [129]. For testing, the group used ADSCs from rabbits that were expanded and mixed with an alginate-sodium solution before being poured into the microwells of the microdevice [129]. Loading occurred for 2 h a day, 6 days a week, at 1 Hz with strain magnitudes of 20%, 15%, 10%, 5%, and 0% in each row of the array [129]. Results showed that loading tended to increase cell viability compared to controls [129]. In addition, collagen II was maximally upregulated in the 10% strain condition, while aggrecan and SOX9 were more highly expressed in all of the loaded groups [129].

This publication was followed by a 2020 report by Chiu et al. that looked at chondrogenesis under uniform and nonuniform 2-axial strain in another membrane-type microdevice that was also driven by a pneumatic system [130]. The microdevice used 1:1 circular, 1:2 oval, and 1:3 oval culture wells to generate uniform and nonuniform strains on cultured rabbit periosteal cells within the wells (Fig. 5C) [130]. Results showed that cell viability was not reduced by loading and that SOX9 expression was upregulated when cells were stretched under 6 kPa pressure in the 1:3 oval well [130]. Thus, chondrocytes were differentiated from stem cells under a large and nearly single dimensional strain [130].

Shaking

To date, there is only one study that uses shaking culture methods for chondrogenic induction of stem cells. The 2020 study by Limraksasin et al. used 3D mouse iPSC cultures that were treated with hydrodynamic stress at specific frequencies in a simple see-saw shaker (Fig. 6) [131]. The single cell suspensions were added to low-cell attachment 96-well U bottom plates, where they were maintained for 24 h before chondrogenic induction medium was added [131]. After 3 days, the cells were transferred to low-cell attachment plates and subjected to suspension shaking culture at a frequency of 0 (static), 0.3, or 0.5 Hz [131].

FIG. 6.

A shaking bioreactor: The see-saw shaker bioreactor administers noncontact hydrodynamic stimulation to iPSCs in vitro. Shaking cultures at 0.3 and 0.5 Hz increased expression of chondrogenic-related genes (compared to static cultures) by increasing TGF-β expression and Wnt signaling. iPSC, induced pluripotent stem cell.

The frequencies of 0.3 and 0.5 Hz were chosen as the shaking frequencies because 0.1–1 Hz has been well established by previous studies on mechanical loading and the present apparatus did not allow for shaking above frequencies of 0.5 Hz due to medium overflow [131]. Results showed that the shaking cultures produced chondrogenically induced iPSC constructs that were larger in size, surrounded by more extensive ECM lacunae, and more differentiated and mature as determined by gene expression compared to static cultures [131].

Sliding contact

Physiological joint loading often includes sliding contact between two contacting cartilage layers, such as in the knee and hip [132]. This type of loading is characterized by moving contact and fluid pressurization [132]. Thus, in 2012, Huang et al. constructed a displacement controlled bioreactor that applies sliding contrast in both the X and Y directions to MSC-seeded hydrogel strips [132]. The cell-hydrogel strips were subjected to 5%–20% axial strain applied at a velocity of 2.5 mm/s [132]. Results showed that short term sliding contact improved chondrogenic gene expression [132]. However, this success was dependent on the axial strain applied and supplementation with TGF-β [132].

Microgravity

Both a 3D culture environment and appropriate mechanical stimulation are important for chondrogenic differentiation of stem cells to mimic in vivo conditions [133]. A rotary cell culture system (RCCS) is a 3D microgravity culture system that allows culture materials to establish a suspension track similar to a homogenous fluid on a horizontal axis (Fig. 7) [134,135]. It is thought to potentially induce chondrogenic differentiation, and the device was first used by NASA for microgravity testing [133,136,137]. In this machine's container, gravity, buoyancy, and shear force achieve a balance in a suspension orbit, which constitutes an environment conducive to cell aggregation [133,134]. A free fall status is maintained, similar to if the culture was in space, and cells attach to microcarriers cultured in a 3D scaffold material that can freely rotate with the rotation of the base [134,135,138]. Specific biomechanical stimuli, such as shear stress, joint compression force, and HP, that support tissue development are also provided by the RCCS system [135,139,140].

FIG. 7.

A microgravity bioreactor: The RCCS is used to rotate cell-microcarrier cultures or cell pellets and create a microgravity environment. Rotation speeds are increased over time to ensure that the cells do not come in contact with the bioreactor's walls and instead are in free-fall. SHH and IHH likely play a role in regulating chondrogenesis under microgravity conditions. RCCS, rotary cell culture system; SHH, Sonic Hedgehog; IHH, Indian Hedgehog.

All groups increased the rotation speed of the RCCS container from the initial speed over time to create a true microgravity environment. Several groups did this in a stepwise manner. These groups initially set the rotation speed of the container to 10–12 rpm to ensure full contact between the cells and their microcarriers [133 –135]. Then, after 24 h, the rotation speed was increased to 12–14 rpm [133 –135]. This was done so that the culture material in the container no longer came into contact with the container and remained in free-fall during rotation [133 –135]. Other groups increased rotation speed gradually as time progressed. Luo et al. initiated studies at a speed of 6.5 rpm, which was increased as aggregates expanded [141]. Mayer-Wagner et al. varied rotational speed between 4.2 and 7.2 rpm during experimentation to maintain cells in a free floating position [138,142]. Yu et al. varied rotation speed between 11 and 25 rpm and increased it steadily over time for 21 days [137]. Still, some groups began with a speed of 20–30 rpm, which was further increased over the course of the study [139,140].

Most reports on the use of an RCCS to generated microgravity deem that it is an effective technique for chondrogenesis induction in stem cells. The dynamic 3D culture seems to increase the growth rate and distribution of seeded cells [141,143]. In addition, the rotating action continuously transfers culture medium, gasses, and nutrients to the differentiating stem cells, producing higher metabolic activity and accelerated tissue growth [139,143]. Perhaps this explains why cells in microgravity conditions tend to be larger in size and express upregulation of type II collagen, some proteoglycans, and some chondrogenic signaling proteins [137,139 –141].

Some reports, such as that by Mayer-Wagner, still conclude that microgravity produces reduced expression of collagen II and aggrecan [138]. Although it is shown by some to suppress hypertrophic differentiation by reducing collagen X and its gene (COL10A1) [138], other reports show that RCCS does not produce these intended effects, as COL10A1 and matrix metalloproteinase 13 (MMP-13) expression was enhanced [139]. Thus, the chondrogenic and hypertrophic effects of microgravity are still quite disputed.

Several groups examined the effects of RCCS on Indian Hedgehog (IHH) and/or Sonic Hedgehog (SHH) induced chondrogenic differentiation to elucidate the role of microgravity in this process [134]. Chen et al. found that transfection of BMSCs with IHH and SHH induced a significant chondrogenic response over time in the RCCS group, compared to nonmicrogravity controls, as levels of collagen II and ACAN were elevated [134]. Similarly, the same group found that BMSCs transfected with SHH and cultured in RCCS resulted in significantly upregulated mRNA levels of SOX9, ACAN, and collagen II [133]. Liu confirmed that IHH transfected BMSCs in RCCS produced increased mRNA expression of collagen II and aggrecan compared to conventionally cultured cells [135].

The benefit of microgravity appears to be that chondrogenic hypertrophy and aging are alleviated during differentiation, which could be due to continued mechanical stress from medium flow and low shear stresses [134]. The free-fall environment is thought to facilitate cellular aggregation and enhance cell–cell and cell–matrix communication [135]. In the RCCS, cells and nutrients are homogenously distributed, improving the transport of nutrients and removal of metabolic wastes in the microenvironment and thus improving the culture [133 –135].

Yet, the chondrogenic effects of simulated microgravity are still conflicting, especially compared to more proven mechanical stimuli techniques such as electromagnetic forces (EMFs) and HP. Mayer-Wagner et al. sought to determine the single and combined effects of low-frequency EMF and microgravity on chondrogenesis of hMSCs [142]. Results showed that microgravity alone reduced collagen II, COL2A1, and aggrecan levels significantly, compared to EMF and combined groups [142]. In addition, Mellor et al. compared cyclic HP with simulated microgravity to better understand the effect of these stimuli on chondrogenesis of human adipose stem cells (hASCs) [136]. Again, results showed that the microgravity group had reduced aggrecan and sGAG compared to the HP controls [136]. Wnt signaling was increased in the microgravity condition, which has been suggested to inhibit chondrogenesis and stimulate chondrocyte hypertrophy [136].

Conclusion and Future Directions

Chondrocytes and chondroprogenitors transduce and respond to a wide array of in vivo environmental mechanical stimuli during development and throughout adulthood [144]. Joint loading produces direct stresses and strains, as well as indirect factors such as HP and fluid flow [144]. As described in this review, the various cell culture techniques that model these stimuli have shown much promise in inducing chondrogenesis in stem cells. Cell ECM metabolism on the gene level, including remodeling and organization, is likely very influential in cartilage tissue engineering [144]. Mechanical stimuli induce ion channel changes, such as with calcium or potassium, which could be an underlying mechanical signaling pathway [144]. The primary cilium is also suggested to contribute to mechanical signal transduction [144]. Nuclear or cytoskeleton deformation also has been shown to play a role in the cellular response to biophysical stimuli [144]. These underlying cellular changes induced by individual or combined physical stimulants could be accelerating chondrogenesis.

Models that use compression, sliding, fluidics, and HP clearly directly mimic forces found in the natural cartilage environment. However, factors such as electromagnetism, ultrasound, shaking, sliding, and microgravity are less traditional in how they constitute mechanical stimulation. Electromagnetism stimulation works by promoting cell differentiation and proliferation through the modulation of extracellular calcium entry to the cells [99,103]. Ultrasound is a mechanically vibratory form of energy that creates displacement waves within a medium, likely generating a deforming compressive environment [108]. Shaking cultures create noncontract hydrodynamic forces that impact cellular properties [131]. Microgravity conditions are created with an RCCS container in which shear force, gravity, and buoyancy are balanced to create an environment conducive to cell aggregation [133].

Mechanical stimuli for cartilage formation during embryonic and fetal development

Previous studies using models of cartilage growth and skeletal development have demonstrated that development of the joints is modulated by muscle-generated forces during the embryonic and early fetal stages. Cells residing in cartilaginous biphasic ECM can experience HP, shear, compression, and tension under loading. Those mechanical stimuli orchestrate the control of chondrogenesis of stem cells during the embryonic and early fetal stages. For example, when morphology of the joint is changing due to mechanical stimulation [145,146], articular cartilage may regenerate in some locations and calcify to form bone or degenerate in others. A better understanding of micromechanical environments during joint development at the embryonic and early fetal stages could enable development of new mechanobiological approaches to promote cartilage regeneration of stem cells. To do so, advanced computational methods, such as multiphasic models, could be the best approach that allows us to predict in situ biomechanical environment favoring cartilage formation to aid cartilage tissue engineering strategies.

Mechanotransduction of stem cells—a combined approach

Signaling pathways have extensively studied for cellular mechanotransduction under mechanical stimulation (eg, HP, shear, compression, and tension) [147]. As described previously, cells may experience a variety of mechanical stimuli simultaneously in tissues. Different mechanical stimuli may affect stem cell synergistically. For instance, HP can further enhance ECM synthesis of chondrocytes promoted by shear stress [148] while inhibiting nitric oxide release from chondrocytes induced by shear stress [149]. A combination of shear and dynamic compressive forces enhanced chondrogenic differentiation in MSCs [67,70]. Also in MSCs, shear stress combined with cyclic HP showed increased collagen II and SOX-9 [87].

Chondrocytes subjected to an electromagnetic field combined with mechanical stimulation from a ceramic hip ball had increased gene expression for the collagen II/I ratio and a more chondrogenic phenotype [150]. Therefore, combining the mechanical systems described earlier can allow us to study interplay between signaling pathways in stem cells under complex mechanical environments and optimize mechanobiological strategies for chondrogenesis of stem cells.

There is some understanding on the mechanisms underlying the effects of mechanical cues on stem cells. Mechanical forces activate signaling pathways within the stem cells, thus transducing mechanical messages into actions within the cells [151]. These actions can include responses such as but not limited to the production of growth factors or the synthesis of ECM proteins [151].

There are a number of mechanotransduction signaling pathways, and each relies on concentration changes of intracellular ions and molecules in response to a particular mechanical stimulus [151]. Tension, compression, shear stress, HP, stiffness, elasticity, and viscoelasticity are all stimuli with specific proteins that serve as transmitters in mediating the cellular response to them [147]. Downstream signaling events that are activated by these mechanical forces include YAP/TAZ, Rho/ROCK, FAK, mitogen activated protein kinase, and G protein related calcium signaling [151]. These proteins mount the cellular response to the particular mechanical stimulus [151]. A comprehensive summary of the protein response for each different mechanical stimulus covered in this review is provided in Table 3.

Table 3.

Signaling Pathways Triggered by Different Mechanical Stimuli

Mechanical stimuli	Proteins	Reference
Compressive loading and strain	Collagen, vimentin, F-actin ROCK, myosin regulatory light chain, Wnt/β-catenin	[147,157]
Hydrostatic pressure	Shc1, integrins, collagen, TGF-β, F-actin	[147,158]
Electromagnetic stimulation	Voltage-gated calcium channels, calmodulin, nitric oxide	[159,160]
Ultrasound signaling	Progranulin, TGF-β, focal adhesion kinase (FAK), nuclear factor kappa B (NF-κβ), heat shock protein 70, integrin-mTOR	[113,125 –127,161,162]
Shear stress	PECAM1, VEGFR, ERK1/2, PGTS2, IER3, ERG1, IGF1, IGFBP1, Integrin, TGF-β, β-catenin, MAPK, laminin-5, F-actin, PI3K	[147,158, 163]
Shaking	TGF-β, Wnt	[131]
Microgravity	SHH, IHH, p38 MAPK	[133 –135,137]

Translation of in vitro findings into clinical applications

Articular cartilage plays a crucial mechanical role in diarthrodial joints providing a low friction surface to minimize wear. The tissue further acts to absorb and distribute force within the joint. In vitro studies have demonstrated that chondrogenesis of stem cells can be promoted by mechanical stimulation. After an analysis of the different cell types used, as specified in Tables 1 and 2, it appears that the most suitable stem cell type for chondrogenesis studies are MSCs derived from adipose or bone marrow. These cells are collected from humans or animals such as rabbits or rats. However, present methods for differentiating MSCs into stable articular cartilage chondrocytes that contribute to joint regeneration have not been effective to date, despite extensive investigation.

Cell-based tissue engineering methods are being explored as a solution for the treatment of chondral defects, often an early precursor to OA. Fundamental to any successful technique will be an understanding of the loading environment that is required for the maintenance of a healthy articular cartilage phenotype. Herein may be the major obstacle from extrapolating from in vitro studies to the human condition.

Some in vivo studies have shown promise in using mechanical stimuli to better induce chondrogenesis. For example, Cui et al. used US to noninvasively condition rabbit bone-marrow derived MSCs in polyglycolic acid scaffolds that have been implanted into mice. In the US treatment group, collagen and GAG content increased significantly, and mechanical tests showed that these cells were stronger and the tissue was more cartilage like. This study suggests that US could be used to stimulate chondrogenesis in vivo [108].

Regenerative rehabilitation is an emerging field that integrates regenerative medicine with rehabilitation approaches and principles. Recent preclinical studies have demonstrated that exercise and continuous passive motion enhance cartilage repair after introducing stem cells into osteochondral defects [152,153]. However, current rehabilitative protocols for joint injury involve complex mechanical stimulation that may not provide consistent therapeutic effects as described in previous in vitro studies.

Our recent study uses an internally designed and fabricated porcine knee joint loading device which can apply controlled dynamic compression to articular cartilage by mimicking in vitro loading conditions to demonstrate similar therapeutic effects on articular cartilage as described previously [154]. Developing new rehabilitative protocols or devices to closely translate therapeutic in vitro loading conditions to in vivo settings may (1) enable us to precisely translate in vitro findings into clinical applications and (2) provide controlled mechanical stimulation for us to quantitatively optimize therapeutic effectiveness clinically. A prototype human device is under development at this time that is based on anatomic scaling of the porcine to human anatomy. Finally, it is evident that the biological and mechanical environments both play a role and interact and should be considered when devising novel strategies to possibly stimulate chondrogenesis in the human.

Footnotes

Author Disclosure Statement

No competing financial interests exist.

Funding Information

Funding for this work was provided by The Soffer Family Foundation and the Upwelling Foundation.

References

Goldring

, Tsuchimochi

and Ijiri

. (2006). The control of chondrogenesis. J Cell Biochem, 97:33–44.

Schumann

, Kujat

, Nerlich

and Angele

. (2006). Mechanobiological conditioning of stem cells for cartilage tissue engineering. Biomed Mater Eng, 16:S37–S52.

Huang

, Farrell

. and Mauck

. (2010). Mechanics and mechanobiology of mesenchymal stem cell-based engineered cartilage. J Biomech, 43:128–136.

Kelly

and Jacobs

. (2010). The role of mechanical signals in regulating chondrogenesis and osteogenesis of mesenchymal stem cells. Birth Defects Res C Embryo Today, 90:75–85.

Bhatia

and Ingber

. (2014). Microfluidic organs-on-chips. Nat Biotechnol, 32:760–772.

Gottardi

(2019). Load-induced osteoarthritis on a chip. Nat Biomed Eng, 3:502–503.

Lopa

, Mondadori

, Mainardi

, Talò

, Costantini

, Candrian

, W Święszkowski and Moretti

. (2018). Translational application of microfluidics and bioprinting for stem cell-based cartilage repair. Stem Cells Int 2018: Article ID. 6594841.

Thompson

, Fu

, Heywood

, Knight

and Thorpe

. (2020). Mechanical stimulation: a crucial element of organ-on-chip models. Front Bioeng Biotechnol, 8:602646.

Vining

and Mooney

. (2017). Mechanical forces direct stem cell behaviour in development and regeneration. Nat Rev Mol Cell Biol, 18:728–742.

10.

Yang

, Beqaj

, Kemp

, Ariel

and Schuger

. (2000). Stretch-induced alternative splicing of serum response factor promotes bronchial myogenesis and is defective in lung hypoplasia. J Clin Invest, 106:1321–1330.

11.

North

, Goessling

, Peeters

, Li

, Ceol

, Lord

, Weber

, Harris

, Cutting

, et al. (2009). Hematopoietic stem cell development is dependent on blood flow. Cell, 137:736–748.

12.

Hove

, Köster

R. W

, Forouhar

, Acevedo-Bolton

, Fraser

and Gharib

. (2003). Intracardiac fluid forces are an essential epigenetic factor for embryonic cardiogenesis. Nature, 421:172–177.

13.

Fahy

, Alini

and Stoddart

. (2018). Mechanical stimulation of mesenchymal stem cells: implications for cartilage tissue engineering. J Orthop Res, 36:52–63.

14.

Responte

, Lee

, Hu

and Athanasiou

. (2012). Biomechanics-driven chondrogenesis: from embryo to adult. FASEB J, 26:3614–3624.

15.

Nowlan

, Murphy

and Prendergast

. (2008). A dynamic pattern of mechanical stimulation promotes ossification in avian embryonic long bones. J Biomech, 41:249–258.

16.

Roddy

, Prendergast

and Murphy

. (2011). Mechanical influences on morphogenesis of the knee joint revealed through morphological, molecular and computational analysis of immobilised embryos. PLoS One, 6:e17526.

17.

Mikic

, Isenstein

and Chhabra

. (2004). Mechanical modulation of cartilage structure and function during embryogenesis in the chick. Ann Biomed Eng, 32:18–25.

18.

Zeng

, Li

, Liu

, Yang

, Zhou

, Fan

and Jiang

. (2021). PLLA porous microsphere-reinforced silk-based scaffolds for auricular cartilage regeneration. ACS Omega, 6:3372–3383.

19.

Mohammed

, Lai

and Lin

. (2021). Substrate stiffness and sequence dependent bioactive peptide hydrogels influence the chondrogenic differentiation of human mesenchymal stem cells. J Mater Chem B, 9:1676–1685.

20.

Zhan

(2020). Effect of matrix stiffness and adhesion ligand density on chondrogenic differentiation of mesenchymal stem cells. J Biomed Mater Res A, 108:675–683.

21.

Aprile

and Kelly

. (2020). Hydrostatic pressure regulates the volume, aggregation and chondrogenic differentiation of bone marrow derived stromal cells. Front Bioeng Biotechnol, 8:619914.

22.

, Wu

, Hu

, Zhu

, Pan

, Jiao

, Li

, Luo

, Zhou

and Lu

. (2020). Stress-relaxing double-network hydrogel for chondrogenic differentiation of stem cells. Mater Sci Eng C Mater Biol Appl, 107:110333.

23.

Feng

, Gao

, Wen

, Huang

, Li

, Liang

, Liu

, Dong

and Cao

. (2020). Engineering the cellular mechanical microenvironment to regulate stem cell chondrogenesis: insights from a microgel model. Acta Biomater, 113:393–406.

24.

Yang

, Li

, Liu

, Li

, Zhang

, Wang

, Xiao

and Zhang

. (2019). Influence of hydrogel network microstructures on mesenchymal stem cell chondrogenesis in vitro and in vivo. Acta Biomater, 91:159–172.

25.

Chen

, Yan

and Lu

. (2016). The function of mechanical loading on chondrogenesis. Front Biosci (Landmark Ed), 21:1222–1232.

26.

Salinas

, Hu

and Athanasiou

. (2018). A guide for using mechanical stimulation to enhance tissue-engineered articular cartilage properties. Tissue Eng Part B Rev, 24:345–358.

27.

Darling

and Athanasiou

. (2003). Articular cartilage bioreactors and bioprocesses. Tissue Eng, 9:9–26.

28.

Urban JP. (1994). The chondrocyte: a cell under pressure. Br J Rheumatol, 33:901–908.

29.

Long

and Linsenmayer

. (1998). Regulation of growth region cartilage proliferation and differentiation by perichondrium. Development (Cambridge, England), 125:1067–1073.

30.

Rooney

and Archer

. (1992). The development of the perichondrium in the avian ulna. J Anat 181, (Pt 3)::393–401.

31.

Xie

, Fritch

, He

, Fu

, Hong

and Lin

. (2021). Dynamic loading enhances chondrogenesis of human chondrocytes within a biodegradable resilient hydrogel. Biomater Sci, 9:5011–5024.

32.

Wong

, Siegrist

and Cao

. (1999). Cyclic compression of articular cartilage explants is associated with progressive consolidation and altered expression pattern of extracellular matrix proteins. Matrix Biol, 18:391–399.

33.

Sah

, Kim

, Doong

, Grodzinsky

, Plaas

and Sandy

. (1989). Biosynthetic response of cartilage explants to dynamic compression. J Orthop Res, 7:619–636.

34.

Steinmeyer

, Ackermann

and Raiss

. (1997). Intermittent cyclic loading of cartilage explants modulates fibronectin metabolism. Osteoarthritis Cartilage, 5:331–341.

35.

Parkkinen

, Ikonen

, Lammi

, Laakkonen

, Tammi

and Helminen

. (1993). Effects of cyclic hydrostatic pressure on proteoglycan synthesis in cultured chondrocytes and articular cartilage explants. Arch Biochem Biophys, 300:458–465.

36.

Islam

, Haqqi

, Jepsen

, Kraay

, Welter

, Goldberg

and Malemud

. (2002). Hydrostatic pressure induces apoptosis in human chondrocytes from osteoarthritic cartilage through up-regulation of tumor necrosis factor-alpha, inducible nitric oxide synthase, p53, c-myc, and bax-alpha, and suppression of bcl-2. J Cell Biochem, 87:266–278.

37.

Trindade

, Shida

, Ikenoue

, Lee

, Lin

, Yaszay

, Yerby

, Goodman

, Schurman

and Smith

. (2004). Intermittent hydrostatic pressure inhibits matrix metalloproteinase and pro-inflammatory mediator release from human osteoarthritic chondrocytes in vitro. Osteoarthritis Cartilage, 12:729–735.

38.

Hall

, Urban

and Gehl

. (1991). The effects of hydrostatic pressure on matrix synthesis in articular cartilage. J Orthop Res, 9:1–10.

39.

Toyoda

, Seedhom

, Yao

, Kirkham

, Brookes

and Bonass

. (2003). Hydrostatic pressure modulates proteoglycan metabolism in chondrocytes seeded in agarose. Arthritis Rheum, 48:2865–2872.

40.

Toyoda

, Seedhom

, Kirkham

and Bonass

. (2003). Upregulation of aggrecan and type II collagen mRNA expression in bovine chondrocytes by the application of hydrostatic pressure. Biorheology, 40:79–85.

41.

Pötter

, Westbrock

, Grad

, Alini

, Stoddart

, Schmal

, Kubosch

, Salzmann

and Kubosch

. (2021). Evaluation of the influence of platelet-rich plasma (PRP), platelet lysate (PL) and mechanical loading on chondrogenesis in vitro. Sci Rep, 11:20188.

42.

Antunes

, Vainieri

, Alini

, Monsonego-Ornan

, Grad

and Yayon

. (2020). Enhanced chondrogenic phenotype of primary bovine articular chondrocytes in Fibrin-Hyaluronan hydrogel by multi-axial mechanical loading and FGF18. Acta Biomater, 105:170–179.

43.

Foster

, Henstock

, Reinwald

and El Haj

. (2015). Dynamic 3D culture: models of chondrogenesis and endochondral ossification. Birth Defects Res C Embryo Today, 105:19–33.

44.

Sulaiman

, Chowdhury

, Fauzi

, Rani

, Yahaya

NHM

, Tabata

, Hiraoka

, Binti Haji Idrus

and Min Hwei

. (2020). 3D Culture of MSCs on a gelatin microsphere in a dynamic culture system enhances chondrogenesis. Int J Mol Sci, 21:2688.

45.

, Li

, Wang

, Li

, Teng

and Jiang

. (2021). Effects of mechanical compression on chondrogenesis of human synovium-derived mesenchymal stem cells in agarose hydrogel. Front Bioeng Biotechnol, 9:697281.

46.

McDermott

, Eastburn

, Kelly

and Boerckel

. (2021). Effects of chondrogenic priming duration on mechanoregulation of engineered cartilage. J Biomech, 125:110580.

47.

Vágó

, ÉKatona, Takács

, Zákány

, van der Veen

and Matta.

(2021). Cyclic uniaxial mechanical load enhances chondrogenesis through entraining the molecular circadian clock. DOI: 10.1101/2021.10.26.465847.

48.

Huang

, Reuben

and Cheung

. (2005). Temporal expression patterns and corresponding protein inductions of early responsive genes in rabbit bone marrow-derived mesenchymal stem cells under cyclic compressive loading. Stem Cells, 23:1113–1121.

49.

Terraciano

, Hwang

, Moroni

, Park

, Zhang

, Mizrahi

, Seliktar

and Elisseeff

. (2007). Differential response of adult and embryonic mesenchymal progenitor cells to mechanical compression in hydrogels. Stem Cells, 25:2730–2738.

50.

Pelaez

, Huang

and Cheung

. (2009). Cyclic compression maintains viability and induces chondrogenesis of human mesenchymal stem cells in fibrin gel scaffolds. Stem Cells Dev, 18:93–102.

51.

Huang

, Farrell

, Kim

and Mauck

. (2010). Long-term dynamic loading improves the mechanical properties of chondrogenic mesenchymal stem cell-laden hydrogel. Eur Cell Mater, 19:72–85.

52.

Meyer

, Buckley

, Thorpe

and Kelly

. (2010). Low oxygen tension is a more potent promoter of chondrogenic differentiation than dynamic compression. J Biomech, 43:2516–2523.

53.

Thorpe

, Buckley

, Vinardell

, O'Brien

, Campbell

and Kelly

. (2010). The response of bone marrow-derived mesenchymal stem cells to dynamic compression following TGF-beta3 induced chondrogenic differentiation. Ann Biomed Eng, 38:2896–2909.

54.

Steinmetz

and Bryant

. (2011). The effects of intermittent dynamic loading on chondrogenic and osteogenic differentiation of human marrow stromal cells encapsulated in RGD-modified poly(ethylene glycol) hydrogels. Acta Biomater, 7:3829–3840.

55.

Bian

, Zhai

, Zhang

, Mauck

and Burdick

. (2012). Dynamic compressive loading enhances cartilage matrix synthesis and distribution and suppresses hypertrophy in hMSC-laden hyaluronic acid hydrogels. Tissue Eng Part A, 18:715–724.

56.

Abdel-Sayed

, Darwiche

, Kettenberger

and Pioletti

. (2014). The role of energy dissipation of polymeric scaffolds in the mechanobiological modulation of chondrogenic expression. Biomaterials, 35:1890–1897.

57.

Elder

, Goldstein

, Kimura

, Soslowsky

and Spengler

. (2001). Chondrocyte differentiation is modulated by frequency and duration of cyclic compressive loading. Ann Biomed Eng, 29:476–482.

58.

Campbell

, Lee

and Bader

. (2006). Dynamic compressive strain influences chondrogenic gene expression in human mesenchymal stem cells. Biorheology, 43:455–470.

59.

Aisenbrey

and Bryant

. (2019). The role of chondroitin sulfate in regulating hypertrophy during MSC chondrogenesis in a cartilage mimetic hydrogel under dynamic loading. Biomaterials, 190–191:51–62.

60.

Huang

, Hagar

, Frost

, Sun

and Cheung

. (2004). Effect of cyclic compressive loading on chondrogenesis of rabbit bone-marrow derived mesenchymal stem cells. Stem Cells, 22:313–323.

61.

Aisenbrey

, Bilousova

, Payne

and Bryant

. (2019). Dynamic mechanical loading and growth factors influence chondrogenesis of induced pluripotent mesenchymal progenitor cells in a cartilage-mimetic hydrogel. Biomater Sci, 7:5388–5403.

62.

Nasrollahzadeh

, Karami

, Wang

, Bagheri

, Guo

, Abdel-Sayed

, Applegate

and Pioletti

. (2022). Temperature evolution following joint loading promotes chondrogenesis by synergistic cues via calcium signaling. eLife, 11:e72068.

63.

Hou

, Zhang

, Feng

and Zhou

. (2020). Low magnitude high frequency vibration promotes chondrogenic differentiation of bone marrow stem cells with involvement of β-catenin signaling pathway. Arch Oral Biol, 118:104860.

64.

, Kupcsik

, Yao

, Alini

and Stoddart

. (2010). Mechanical load modulates chondrogenesis of human mesenchymal stem cells through the TGF-beta pathway. J Cell Mol Med, 14,(6A)::1338–1346.

65.

Kupcsik

, Stoddart

, Li

, Benneker

and Alini

. (2010). Improving chondrogenesis: potential and limitations of SOX9 gene transfer and mechanical stimulation for cartilage tissue engineering. Tissue Eng, 6:1845–1855.

66.

, Yao

S-J

, Alini

and Stoddart

. (2010). Chondrogenesis of human bone marrow mesenchymal stem cells in fibrin-polyurethane composites is modulated by frequency and amplitude of dynamic compression and shear stress Tissue Eng, 16:575–584.

67.

Schatti

, Goldhahn

, Salzmann

, Li

, Alini

and Stoddart

. (2011). A combination of shear and dynamic compression leads to mechanically induced chondrogenesis of human mesenchymal stem cells. Eur Cells Mater, 22:214–225.

68.

Gardner

OFW

, Musumeci

, Neumann

, Eglin

, Archer

, Alini

and Stoddart

. (2017). Asymmetrical seeding of MSCs into fibrin-poly(ester-urethane) scaffolds and its effect on mechanically induced chondrogenesis. J Tissue Eng Regen Med, 11:2912–2921.

69.

Cochis

, Grad

, Stoddart

, Fare

, Altomare

, Azzimonti

, Alini

and Rimondini

. (2017). Bioreactor mechanically guided 3D mesenchymal stem cell chondrogenesis using a biocompatible novel thermo-reversible methylcellulose-based hydrogel. Sci Rep, 7:45018.

70.

Guo

, Yu

, Lim

, Goodley

, Xiao

, Placone

, Ferlin

, Nguyen

, Hsieh

and Fisher

. (2016). Effect of dynamic culture and periodic compression on human mesenchymal stem cell proliferation and chondrogenesis. Ann Biomed Eng, 44:2103–2113.

71.

Angele

, Yoo

, Smith

, Mansour

, Jepsen

, Nerlich

and Johnstone

. (2003). Cyclic hydrostatic pressure enhances the chondrogenic phenotype of human mesenchymal progenitor cells differentiated in vitro. J Orthop Res, 21:451–457.

72.

Miyanishi

, Trindade

, Lindsey

, Beaupre

, Carter

, Goodman

, Schurman

and Smith

. (2006). Dose- and time-dependent effects of cyclic hydrostatic pressure on transforming growth factor-beta-3-induced chondrogenesis by adult human mesenchymal stem cells in vitro. Tissue Eng, 12:2253–2261.

73.

Wagner

, Lindsey

, Li

, Tummala

, Chandran

, Smith

, Longaker

, Carter

and Beaupre

. (2008). Hydrostatic pressure enhances chondrogenic differentiation of human bone marrow stromal cells in osteochondrogenic medium. Ann Biomed Eng, 36:813–820.

74.

Saha

, Rolfe

, Carroll

, Kelly

and Murphy

. (2017). Chondrogenesis of embryonic limb bud cells in micromass culture progresses rapidly to hypertrophy and is modulated by hydrostatic pressure. Cell Tissue Res, 368:47–59.

75.

Angele

, Smith

, Mansour

, Jepsen

, Johnstone

and Yoo

. (2000). Effects of cyclic hydrostatic pressure on the chondrogenic differentiation of mesenchymal progenitor cells. Orthop Res Soc,, 25:645.

76.

Finger

, Sargent

, Dulaney

, Bernacki

and Loboa

. (2007). Differential effects on messenger ribonucleic acid expression by bone marrow-derived human mesenchymal stem cells seeded in agarose constructs due to ramped and steady applications of cyclic hydrostatic pressure. Tissue Eng, 13:1151–1158.

77.

Miyanishi

, Trindade

, Lindsey

, Beaupre

, Carter

, Goodman

, Schurman

and Smith

. (2006). Effects of hydrostatic pressure and transforming growth factor-beta 3 on adult human mesenchymal stem cell chondrogenesis in vitro. Tissue Eng, 12:1419–1427.

78.

Puetzer

, Williams

, Gillies

, Bernacki

and Loboa

. (2013). The effects of cyclic hydrostatic pressure on chondrogenesis and viability of human adipose- and bone marrow-derived mesenchymal stem cells in three-dimensional agarose constructs. Tissue Eng Part A, 19:299–306.

79.

Vinardell

, Rolfe

, Buckley

, Meyer

, Ahearne

, Murphy

and Kelly

. (2012). Hydrostatic pressure acts to stabilize a chondrogenic phenotype in porcine joint tissue derived stem cells. Eur Cells Mater, 23:121–134.

80.

Zeiter

, Lezuo

and Ito

. (2009). Effect of TGF beta1, BMP-2 and hydraulic pressure on chondrogenic differentiation of bovine bone marrow mesenchymal stromal cells. Biorheology, 46:45–55.

81.

Zellner

, Mueller

, Xin

, Krutsch

, Brandl

, Kujat

, Nerlich

and Angele

. (2015). Dynamic hydrostatic pressure enhances differentially the chondrogenesis of meniscal cells from the inner and outer zone. J Biomech, 48:1479–1484.

82.

Angele

, Schumann

, Angele

, Kinner

, Englert

, Hente

, Fuchtmeier

, Nerlich

, Nuemann

and Kujat

. (2004). Cyclic, mechanical compression enhances chondrogenesis of mesenchymal progenitor cells in tissue engineering scaffolds. Biorheology, 41:335–346.

83.

Rodenas-Rochina

, Kelly

, Gómez Ribelles

and Lebourg

. (2016). Compositional changes to synthetic biodegradable scaffolds modulate the influence of hydrostatic pressure on chondrogenesis of mesenchymal stem cells. Biomed Phys Eng Express, 2:035005.

84.

Steward

, Thorpe

, Vinardell

, Buckley

, Wagner

and Kelly

. (2012). Cell-matrix interactions regulate mesenchymal stem cell response to hydrostatic pressure. Acta Biomater, 8:2153–2159.

85.

Meyer

, Buckley

, Steward

and Kelly

. (2011). The effect of cyclic hydrostatic pressure on the functional development of cartilaginous tissues engineered using bone marrow derived mesenchymal stem cells. J Mech Behav Biomed Mater, 4:1257–1265.

86.

Juhasz

, Matta

, Somogyi

, Katona

, Takacs

, Soha

, Szabo

, Cserhati

, Szody

, et al. (2014). Mechanical loading stimulates chondrogenesis via the PKA/CREB-Sox9 and PP2A pathways in chicken micromass cultures. Cell Signal, 26:468–482.

87.

Hosseini

, Tafazzoli-Shadpour

, Haghighipour

, Aghdami

and Goodarzi

. (2015). The synergistic effects of shear stress and cyclic hydrostatic pressure modulate chondrogenic induction of human mesenchymal stem cells. Int J Artif Organs, 38:557–564.

88.

Jeong

, Park

, Shin

, Kang

, Han

and Shin

. (2012). Effects of intermittent hydrostatic pressure magnitude on the chondrogenesis of MSCs without biochemical agents under 3D co-culture. J Mater Sci Mater Med, 23:2773–2781.

89.

, Zhao

, Yang

, Liu

, Wang

, Li

and Liu

. (2009). p38 MAPK mediated in compressive stress-induced chondrogenesis of rat bone marrow MSCs in 3D alginate scaffolds. J Cell Physiol, 221:609–617.

90.

Huang

, Cai

, Li

, Xie

, Zhang

and Yang

. (2015). The effects of static pressure on chondrogenic and osteogenic differentiation in condylar chondrocytes from temporomandibular joint. Arch Oral Biol, 60:622–630.

91.

, Huang

, Xie

, Cai

, Yang

, Wang

and Zhang

. (2017). Study on the effects of gradient mechanical pressures on the proliferation, apoptosis, chondrogenesis and hypertrophy of mandibular condylar chondrocytes in vitro. Arch Oral Biol, 73:186–192.

92.

Tarng

, Huang

and Su

. (2012). A novel recirculating flow-perfusion bioreactor for periosteal chondrogenesis. Int Orthop, 36:863–868.

93.

Kim

, Montagne

, Ushida

and Furukawa

. (2015). Enhanced chondrogenesis with upregulation of PKR using a novel hydrostatic pressure bioreactor. Biosci Biotechnol Biochem, 79:239–241.

94.

Zhang

, Li

, He

and Xu

. (2021). Static magnetic fields enhance the chondrogenesis of mandibular bone marrow mesenchymal stem cells in coculture systems. Biomed Res Int, 2021:9962861.

95.

Fuhrer

, Hofmann

, Hild

, Vetsch

, Herrmann

, Grass

and Stark

. (2013). Pressureless mechanical induction of stem cell differentiation is dose and frequency dependent. PLoS One, 8:e81362.

96.

Park

S-H

, Young

, Park

, Yang

, Choi

, Park

and Min

B-H

. (2006). An electromagnetic compressive force by cell exciter stimulates chondrogenic differentiation of bone marrow-derived mesenchymal stem cells. Tissue Eng, 12:3107–3117.

97.

Lima

, Gonçalves

, Rodrigues

, Reis

and Gomes

. (2015). The effect of magnetic stimulation on the osteogenic and chondrogenic differentiation of human stem cells derived from the adipose tissue (hASCs). J Magnetism Magn Mater, 393:526–536.

98.

Dikina

, Lai

, Cao

, Zborowski

and Alsberg

. (2017). Magnetic field application or mechanical stimulation via magnetic microparticles does not enhance chondrogenesis in mesenchymal stem cell sheets. Biomater Sci, 5:1241–1245.

99.

Amin

, Brady

, St-Pierre

, Stevens

, Overby

and Ethier

. (2014). Stimulation of chondrogenic differentiation of adult human bone marrow-derived stromal cells by a moderate-strength static magnetic field. Tissue Eng Part A, 20:1612–1620.

100.

Prasopthum

, Deng

, Khan

, Yin

, Guo

and Yang

. (2020). Three dimensional printed degradable and conductive polymer scaffolds promote chondrogenic differentiation of chondroprogenitor cells. Biomater Sci, 8:4287–4298.

101.

Chen

, Lin

, Fu

, Wang

, Wu

, Wang

, Eswaramoorthy

, Wang

, et al. (2013). Electromagnetic fields enhance chondrogenesis of human adipose-derived stem cells in a chondrogenic microenvironment in vitro. J Appl Physiol (1985), 114:647–655.

102.

Huang

, Liang

, Huang

, Zhao

, Liang

, Liu

, Duan

, Liu

, Zhu

, et al. (2019). Magnetic enhancement of chondrogenic differentiation of mesenchymal stem cells. ACS Biomater Sci Eng, 5:2200–2207.

103.

Parate

, Franco-Obregon

, Frohlich

, Beyer

, Abbas

, Kamarul

, HP Hui

and Yang

. (2017). Enhancement of mesenchymal stem cell chondrogenesis with short-term low intensity pulsed electromagnetic fields. Sci Rep, 7:9421.

104.

Celik

, Franco-Obregon

, Lee

, Hui

and Yang

. (2021). Directionalities of magnetic fields and topographic scaffolds synergise to enhance MSC chondrogenesis. Acta Biomater, 119:169–183.

105.

Varani

, Vincenzi

, Pasquini

, Blo

, Salati

, Cadossi

and De Mattei

. (2021). Pulsed electromagnetic field stimulation in osteogenesis and chondrogenesis: signaling pathways and therapeutic implications. Int J Mol Sci, 22:809.

106.

Labusca

, Herea

, Emanuela Minuti

, Stavila

, Danceanu

, Plamadeala

, Chiriac

and Lupu

. (2021). Magnetic nanoparticles and magnetic field exposure enhances chondrogenesis of human adipose derived mesenchymal stem cells but not of Wharton Jelly mesenchymal stem cells. Front Bioeng Biotechnol, 9:737132.

107.

Vaca-González

, Clara-Trujillo

, Guillot-Ferriols

, Ródenas-Rochina

, Sanchis

, LG Ribelles

, Garzón-Alvarado

and Ferrer

. (2020). Effect of electrical stimulation on chondrogenic differentiation of mesenchymal stem cells cultured in hyaluronic acid - Gelatin injectable hydrogels. Bioelectrochemistry, 134:107536.

108.

Cui

, Park

and Min

B-H

. (2006). Effects of low intensity ultrasound on chondrogenic differentiation of mesenchymal stem cells embedded in polyglycolic acid: an in vivo study. Tissue Eng, 12:75–82.

109.

Wang

, Lin

, Zhang

, Wang

, Cheng

, Gao

, Xia

and Li

. (2019). Low-intensity pulsed ultrasound promotes chondrogenesis of mesenchymal stem cells via regulation of autophagy. Stem Cell Res Ther, 10:41.

110.

Aliabouzar

, Zhang

and Sarkar

. (2016). Lipid Coated Microbubbles and Low Intensity Pulsed Ultrasound Enhance Chondrogenesis of Human Mesenchymal Stem Cells in 3D Printed Scaffolds. Sci Rep, 6:37728.

111.

Chu

, Lim

, Hwang

, Lin

and Wang

. (2020). Piezoelectric stimulation by ultrasound facilitates chondrogenesis of mesenchymal stem cells. J Acoust Soc Am, 148:EL58.

112.

Choi

, Choi

, Park

, Pai

, Li

, Min

and Park

. (2013). Mechanical stimulation by ultrasound enhances chondrogenic differentiation of mesenchymal stem cells in a fibrin-hyaluronic acid hydrogel. Artif Organs, 37:648–655.

113.

Ebisawa

, Hata

K-I

, Okada

, Kimata

, Ueda

, Torii

and Waanabe

. (2004). Ultrasound enhanes transforming growth factor beta-mediated chondrocyte differentiation of human mesenchymal stem cells. Tissue Eng, 10:921–929.

114.

Lee

, Choi

, Min

and Park

. (2007). Low-intensity ultrasound inhibits apoptosis and enhances viability of human mesenchymal stem cells in three-dimensional alginate culture during chondrogenic differentiation. Tissue Eng, 13:1049–1057.

115.

Noriega

, Mamedov

, Turner

and Subramanian

. (2007). Intermittent applications of continuous ultrasound on the viability, proliferation, morphology, and matrix production of chondrocytes in 3D matrices. Tissue Eng, 13:611–618.

116.

Cui

, Park

, Choi

and Min

. (2007). Preconditioning of mesenchymal stem cells with low-intensity ultrasound for cartilage formation in vivo. Tissue Eng, 13:351–360.

117.

Shafaei

, Esfandiari

, Esmaeili

, Razavi

, Hashemibeni

, Nasr Esfahani

, Shiran

, Isfahani

and Mardani

. (2013). Optimizing a novel method for low intensity ultrasound in chondrogenesis induction. Adv Biomed Res, 2:79.

118.

Guha Thakurta

, Budhiraja

and Subramanian

. (2015). Growth factor and ultrasound-assisted bioreactor synergism for human mesenchymal stem cell chondrogenesis. J Tissue Eng, 6:2041731414566529.

119.

Thakurta

, Sahu

, Miller

, Budhiraja

, Akert

, Viljoen

and Subramanian

. (2016). Long-term culture of human mesenchymal stem cell-seeded constructs under ultrasound stimulation: evaluation of chondrogenesis. Biomed Phys Eng Express, 2:055016.

120.

Sahu

, Budhiraja

and Subramanian

. (2020). Preconditioning of mesenchymal stromal cells with low-intensity ultrasound: influence on chondrogenesis and directed SOX9 signaling pathways. Stem Cell Res Ther, 11:6.

121.

Bhogoju

, Khan

, Wang

and Subramanian

. (2021). Continuous low-intensity ultrasound preserves mitochondrial potential, inhibits NFkB activation and rescues chondrogenesis of mesenchymal stromal cells. Res Square DOI: 10.21203/rs.3.rs-342462/v1

122.

Mukai

, Ito

, Nakagawa

, Akiyama

, Miyamoto

and Nakamura

. (2005). Transforming growth factor-beta1 mediates the effects of low-intensity pulsed ultrasound in chondrocytes. Ultrasound Med Biol, 31:1713–1721.

123.

Schumann

, Kujat

, Zellner

, Angele

, Nerlich

, Mayr

and Angele

. (2006). Treatment of human mesenchymal stem cells with pulsed low intensity ultrasound. Biorheology, 43:431–443.

124.

Lai

, Chen

, Chiu

, Yang

, Tsai

, Zuo

, Chang

and Lai

. (2010). Effects of low-intensity pulsed ultrasound, dexamethasone/TGF-beta1 and/or BMP-2 on the transcriptional expression of genes in human mesenchymal stem cells: chondrogenic vs. osteogenic differentiation. Ultrasound Med Biol, 36:1022–1033.

125.

Jang

, Ding

, Seol

, Lim

, Buckwalter

and Martin

. (2014). Low-intensity pulsed ultrasound promotes chondrogenic progenitor cell migration via focal adhesion kinase pathway. Ultrasound Med Biol, 40:1177–1186.

126.

Xia

, Wang

, Qu

, Lin

, Cheng

, Gao

, Ren

, Zhang

and Li

. (2017). TGF-beta1-induced chondrogenesis of bone marrow mesenchymal stem cells is promoted by low-intensity pulsed ultrasound through the integrin-mTOR signaling pathway. Stem Cell Res Ther, 8:281.

127.

Liao

, Li

, Xiao

, Zeng

, Liu

, Yuan

and Liu

. (2021). Low-intensity pulsed ultrasound promotes osteoarthritic cartilage regeneration by BMSC-derived exosomes via modulating the NF-kappaB signaling pathway. Int Immunopharmacol, 97:107824.

128.

Zhong

, Tian

, Zheng

, Li

, Zhang

, Wang

and Qin

. (2013). Mesenchymal stem cell and chondrocyte fates in a multishear microdevice are regulated by Yes-associated protein. Stem Cells Dev, 22:2083–2093.

129.

Kowsari-Esfahan

, Jahanbakhsh

, Saidi

and Bonakdar

. (2018). A microfabricated platform for the study of chondrogenesis under different compressive loads. J Mech Behav Biomed Mater, 78:404–413.

130.

Chiu

C-H

, Tong

Y-W

, Yu

J-F

, Lei

and Chen

AC-Y

. (2020). Osteogenesis and chondrogenesis of primary rabbit periosteal cells under Non-uniform 2-axial tensile strain. BioChip J, 14:438–446.

131.

Limraksasin

, Kosaka

, Zhang

, Horie

, Kondo

, Okawa

, Yamada

and Egusa

. (2020). Shaking culture enhances chondrogenic differentiation of mouse induced pluripotent stem cell constructs. Sci Rep, 10:14996.

132.

Huang

, Baker

, Ateshian

and Mauck

. (2012). Sliding contact loading enhances the tensile properties of mesenchymal stem cell-seeded hydrogels. Eur Cell Mater, 24:29–45.

133.

Chen

, Liu

, Li

and Wu

. (2019). Sonic hedgehog promotes chondrogenesis of rabbit bone marrow stem cells in a rotary cell culture system. BMC Dev Biol, 19:18.

134.

Chen

, Liu

, Li

and Wu

. (2019). Chondrogenic differentiation of bone marrow-derived mesenchymal stem cells following transfection with Indian hedgehog and sonic hedgehog using a rotary cell culture system. Cell Mol Biol Lett, 24:16.

135.

Liu

P-CL

, Liu

J-F

, Xia

, Chen

L-Y

and Wu

. (2016). Transfection of the IHH gene into rabbit BMSCs in a simulated microgravity environment promotes chondrogenic differentiation and inhibits cartilage aging. Oncotarget, 7:62873–62885.

136.

Mellor

, Steward

, Nordberg

, Taylor

and Loboa

. (2017). Comparison of simulated microgravity and hydrostatic pressure for chondrogenesis of hASC. Aerosp Med Hum Perform, 88:377–384.

137.

, Yu

, Cao

, Zhao

, Long

, Liu

, Tang

and Zhu

. (2011). Simulated microgravity using a rotary cell culture system promotes chondrogenesis of human adipose-derived mesenchymal stem cells via the p38 MAPK pathway. Biochem Biophys Res Commun, 414:412–418.

138.

Mayer-Wagner

, Hammerschmid

, Redeker

, Schmitt

, Holzapfel

, Jansson

, Betz

and Muller

. (2014). Simulated microgravity affects chondrogenesis and hypertrophy of human mesenchymal stem cells. Int Orthop, 38:2615–2621.

139.

Weiss

, Mulet-Sierra

, Kunze

, Jomha

and Adesida

. (2017). Coculture of meniscus cells and mesenchymal stem cells in simulated microgravity. NPJ Microgravity, 3:28.

140.

, Li

, Lou

and Chen

. (2013). The effect of the microgravity rotating culture system on the chondrogenic differentiation of bone marrow mesenchymal stem cells. Mol Biotechnol, 54:331–336.

141.

Luo

, Xiong

, Qiu

, Lv

, Li

and Li

. (2011). Differentiation of mesenchymal stem cells towards a nucleus pulposus-like phenotype utilizing simulated microgravity In vitro. J Huazhong Univ Sci Technolog Med Sci, 31:199.

142.

Mayer-Wagner

, Hammerschmid

, Blum

, Krebs

, Redeker

, Holzapfel

, Jansson

and Muller

. (2018). Effects of single and combined low frequency electromagnetic fields and simulated microgravity on gene expression of human mesenchymal stem cells during chondrogenesis. Arch Med Sci, 14:608–616.

143.

Emin

, Koc

, Durkut

, Elcin

and Elcin

. (2008). Engineering of rat articular cartilage on porous sponges: effects of tgf-beta 1 and microgravity bioreactor culture. Artif Cells Blood Substit Immobil Biotechnol, 36:123–137.

144.

O'Conor

, Case

and Guilak

. (2013). Mechanical regulation of chondrogenesis. Stem Cell Res Ther, 4:61.

145.

Giorgi

, Carriero

, Shefelbine

and Nowlan

. (2014). Mechanobiological simulations of prenatal joint morphogenesis. J Biomech, 47:989–995.

146.

Heegaard

, Beaupre

and Carter

. (1999). Mechanically modulated cartilage growth may regulate joint surface morphogenesis. J Orthop Res, 17:509–517.

147.

Argentati

, Morena

, Tortorella

, Bazzucchi

, Porcellati

, Emiliani

and Martino

. (2019). Insight into mechanobiology: how stem cells feel mechanical forces and orchestrate biological functions. Int J Mol Sci, 20:5337.

148.

Nazempour

, Quisenberry

, Abu-Lail

and Van Wie

. (2017). Combined effects of oscillating hydrostatic pressure, perfusion and encapsulation in a novel bioreactor for enhancing extracellular matrix synthesis by bovine chondrocytes. Cell Tissue Res, 370:179–193.

149.

Lee

, Trindade

, Ikenoue

, Schurman

, Goodman

and Smith

. (2003). Intermittent hydrostatic pressure inhibits shear stress-induced nitric oxide release in human osteoarthritic chondrocytes in vitro. J Rheumatol, 30:326–328.

150.

Hilz

, Ahrens

, Grad

, Stoddart

, Dahmani

, Wilken

, Sauerschnig

, Niemeyer

, Zwingmann

, et al. (2014). Influence of extremely low frequency, low energy electromagnetic fields and combined mechanical stimulation on chondrocytes in 3-D constructs for cartilage tissue engineering. Bioelectromagnetics, 35:116–128.

151.

Naqvi

, and McNamara

. (2020). Stem cell mechanobiology and the role of biomaterials in governing mechanotransduction and matrix production for tissue regeneration. Front Bioeng Biotechnol, 8:597661.

152.

Yamaguchi

, Aoyama

, Ito

, Nagai

, Iijima

, Tajino

, Zhang

, Kiyan

and Kuroki

. (2016). The effect of exercise on the early stages of mesenchymal stromal cell-induced cartilage repair in a rat osteochondral defect model. PLoS One, 11:e0151580.

153.

Wang

, Lin

, Chang

, Hsu

and Yeh

. (2019). Continuous passive motion promotes and maintains chondrogenesis in autologous endothelial progenitor cell-loaded porous PLGA scaffolds during osteochondral defect repair in a rabbit model. Int J Mol Sci, 20:259.

154.

Hazbun

, Martinez

, Best

, Kaplan

and Huang

C-Y

. (2021). Anti-inflammatory effects of tibial axial loading on knee articular cartilage post traumatic injury. J Biomech, 128:110736.

155.

Tseng

, Wu

, Cheng

and Lin

. (2020). Studies of surface grafted collagen and transforming growth factor β1 combined with cyclic stretching as a dual chemical and physical stimuli approach for rat adipose-derived stem cells (rADSCs) chondrogenesis differentiation. J Mech Behav Biomed Mater, 112:104062.

156.

Silva

, Moura

, Borrecho

, de Matos

APA

, da Silva

, Cabral

JMS

, Bártolo

, Linhardt

and Ferreira

. (2020). Extruded Bioreactor Perfusion Culture Supports the Chondrogenic Differentiation of Human Mesenchymal Stem/Stromal Cells in 3D Porous Poly(ɛ-Caprolactone) Scaffolds. Biotechnol J, 15:e1900078.

157.

Takemoto

, Ishihara

, Mizutani

, Kawabata

and Haga

. (2015). Compressive stress induces dephosphorylation of the myosin regulatory light chain via RhoA phosphorylation by the adenylyl cyclase/protein kinase A signaling pathway. PLoS One, 10:e0117937.

158.

Kunnen

, Malas

, Semeins

, Bakker

and Peters

DJM

. (2018). Comprehensive transcriptome analysis of fluid shear stress altered gene expression in renal epithelial cells. J Cell Physiol, 233:3615–3628.

159.

Pall ML. (2013). Electromagnetic fields act via activation of voltage-gated calcium channels to produce beneficial or adverse effects. J Cell Mol Med, 17:958–965.

160.

Petecchia

, Sbrana

, Utzeri

, Vercellino

, Usai

, Visai

, Vassalli

and Gavazzo

. (2015). Electro-magnetic field promotes osteogenic differentiation of BM-hMSCs through a selective action on Ca2+-related mechanisms. Sci Rep, 5:13856.

161.

Uddin

, Richbourgh

, Yi

and Liu

. (2015). Synergistic effects of progranulin and low intensity pulsed ultrasound on chondrocyte differentiation, migration and metabolism. Osteoarthritis Cartilage, 23:A144–A145.

162.

Nam

, Seo

and Kim

. (2014). Pulsed and continuous ultrasound increase chondrogenesis through the increase of heat shock protein 70 expression in rat articular cartilage. J Phys Ther Sci, 26:647–650.

163.

Tzima

, Irani-Tehrani

, Kiosses

, Dejana

, Schultz

, Engelhardt

, Cao

, DeLisser

and Schwartz

. (2005). A mechanosensory complex that mediates the endothelial cell response to fluid shear stress. Nature, 437:426–431.