Table 2.
Summary of relevant studies and results for the use of LIPUS to improve 3D bone engineering techniques.
| Study | Cell and Scaffold Type | Ultrasound Parameters | Findings |
|---|---|---|---|
| Veronick et al. (2016) [18] | Cell Type: MC3T3 mouse osteoblast cells Scaffold Material: type 1 collagen hydrogels |
Frequency: 1 MHz wave with 1 kHz repetition frequency Pulse mode: 20, 50, or 100% duty cycle Intensity: 30 mW/cm2 |
LIPUS produced a measurable force and hydrogel deformation. LIPUS increased alkaline phosphatase and osteocalcin gene expression. The effect on gene expression was indirectly proportional to hydrogel stiffness and directly proportional to duty cycle. |
| Zhou et al. (2016) [20] | Cell Type: human mesenchymal cells (hMSCs) Scaffold Material: polyethylene glycol diacrylate bio inks containing RGDS or nHA |
Intensity: 150 mW/cm2 Frequency: 1.5 MHz Duty cycle: 20% |
LIPUS increased MSC proliferation, alkaline phosphatase activity, mineralization, and total protein content in a 3D printed RGDS nHA scaffold. |
| Feng et al. (2019) [21] | Cell type: MC3T3-E1 mouse pre-osteoblast cells Scaffold Material: Ti6Al4V |
Intensity: 40 mW/cm2 Pulse Length: 1 ms Frequency: 1 MHz and 3.2 MHz Exposure: 20 min daily for either 3 weeks or 6 weeks. |
LIPUS had no significant impact on cell proliferation, increased alkaline phosphatase activity and osteocalcin expression, and increased volume and amount of new bone formation No significant difference was found between 1 MHz and 3.2 MHz frequencies. The 1 MHz frequency was slightly better for ALP activity, OCN content, scaffold pore occupancy, bone area percentage, and calcium deposition, but the difference was not statistically significant. |
| Kuang et al. (2019) [22] | Cell Type: dental follicle cells (DFCs) Scaffold Material: OsteoBoneTM ceramic |
Intensity: 90 mW/cm2 Frequency: 1.5 MHz Pulse Repetition: 1 kHz Pulse Duration: 200 μs Exposure: 20 min daily for 3, 5, 7, 9, or 21 days |
In vitro, LIPUS increased ALP, Runx2, OSX, and COL-I gene expression and the formation of mineralized nodules. In vivo, LIPUS treatment improved fibrous tissue and blood vessel growth. |
| Wu et al. (2015) [23] | Cell Type: MC3T3-E1 mouse pre-osteoblast cells Scaffold Material: silicon carbide (SiC) |
Intensity: 30 mW/cm2 Frequency: 1 MHz Pulse length: 1 ms Pulse repetition: 100 Hz Exposure: 20 min for 4 or 7 days |
LIPUS improved cell density, cell ingrowth, dsDNA content, and alkaline phosphatase activity |
| Carina et al. (2017) [37] | Cell Type: human mesenchymal stem cells (hMSCs) Scaffold Material: magnesium dopped hydroxyapatite and type 1 collagen composite (MgHA/Coll) |
Intensity: 20 mW/cm2 Frequency: 1.5 MHz Pulse repetition: 1 kHz Burst length: 200 μs Exposure: 20 min per day for 5 d/wk for 1 or 2 weeks |
LIPUS improved hMSC viability and upregulated several osteogenic genes (ALPL, BGLAP, MAPK1, MAPK6, and VEGF). |
| Zhu et al. (2020) [44] | Cell Type: MC3T3-E1 mouse pre-osteoblast cells (for in vitro Alizarin red staining experiments) Scaffold Material: poly-L-lactic acid/polylactic-co-glycolic acid/poly-ε-caprolactone (PLLA/PLGA/PCL) |
Intensity: 30 mW/cm2 Exposure: 20 min daily for 12 weeks |
LIPUS improved load carrying capacity, accelerated bone formation, angiogenesis, and differentiation. LIPUS was used to alleviate the effects of osteonecrosis. |
| Iwai et al. (2007) [72] | Cell Type: MC3T3-E1 mouse pre-osteoblast cells Scaffold Material: hydroxyapatite |
Intensity: 30 mW/cm2 Frequency: 1.5 MHz Burst width: 200 μs Wave Repetition: 1 kHz Exposure: not specified |
LIPUS did not affect biomechanics/compressive strength of hydroxyapatite ceramic LIPUS improved osteoblast number and bone area in the center of implanted, porous scaffold. LIPUS improved volume of mineralized tissue and MC3T3-E1 migration. |
| Wang, J et al. (2014) [73] | Cell Type: bone marrow stromal cells (BMSCs) Scaffold Material: β-tricalcium phosphate composite |
Frequency: 1.5 MHz Burst width: 200 μs Wave Repetition: 1 kHz Intensity: 30 mW/cm2 Exposure: 20 min daily for 5, 10, 25, or 50 days |
LIPUS increased ALP activity and OCN content. Additionally, LIPUS improved the degree of soft tissue repair, increased blood flow, and resulted in more extensive bone repair. LIPUS did not impact the compressive strength of the β-TCP scaffold. |
| Hui et al. (2011) [74] | Cell Type: mesenchymal stem cell derived osteogenic cells Scaffold Material: tricalcium phosphate |
Frequency: 1.5 MHz Burst width: 200 μs Wave Repetition: 1 kHz Intensity: 30 mW/cm2 Exposure: 20 min daily; 5 d/wk, 7 weeks |
LIPUS increased spinal fusion at L5 and L6 in New Zealand white rabbits. |
| Cao et al. (2017) [75] | Cell Type: MC3T3-E1 pre-osteoblast cells Scaffold Material: Ti6Al4V |
Frequency: 1 MHz Pulse length: 1 ms Pulse repetition: 100 Hz Intensity: 30 mW/cm2 Exposure: 20 min daily for: 1, 4, or 7 days (in vitro) 3 or 6 weeks (in vivo) |
An intensity of 30 mW/cm2 was found to be most effective at promoting osteogenic differentiation In vitro: LIPUS had no effect on cell proliferation but increased ALP activity, OCN content, and cell ingrowth into the scaffold. In vivo: LIPUS increased/improved amount and volume of new bone formed and the bone maturity. |
| Liu et al. (2020) [76] | Cell Type: bone marrow stromal cells Scaffold Material: Ti6Al4V coated with BaTiO3 |
Frequency: 1.5 MHz sine wave repeating at 1 kHz Pulse duration: 200 μs Intensity: 30 mW/cm2 Exposure: 10 min daily for 7 or 14 days |
When combined with BaTiO3 LIPUS increased ALP activity and expression of Runx-2, Col-1, and OPN on a titanium scaffold. LIPUS improved the amount of new bone formed (greater volume and filled the scaffold pores to a greater degree). |
| Fan et al. (2020) [77] | Cell Type: bone marrow mesenchymal stem cells Scaffold Material: Ti6Al4V with BaTiO3 coating |
Intensity: 30 mW/cm2 Frequency: 1.5 MHz Pulse Repetition: 1 kHz Pulse duration: 200 μs Exposure: 10 min daily for 4, 7, or 14 days |
In vitro: LIPUS improved cell adhesion, proliferation, and gene expression on a titanium scaffold especially when paired with BaTiO3 coating to induce the piezoelectric effect. In vivo: LIPUS improved new bone formation, osteointegration, mineral apposition rate (MAR), and bonding strength of bone and scaffold. |
| Veronick et al. (2018) [78] | Cell Type: MC3T3-E1 mouse pre-osteoblast cells Scaffold Material: type 1 collagen hydrogels |
Frequency: 1 MHz wave with 1 kHz repetition frequency Pulse mode: 20, 50, or 100% duty cycle Intensity: 30 mW/cm2 |
Hydrogel deformation was a function of hydrogel stiffness and duty cycle. LIPUS upregulated COX-2 and PGE2 expression. Effects of LIPUS and hydrogel encapsulation were additive. |
| Wang, Y et al. (2014) [79] | Cell Type: human bone marrow derived mesenchymal stem cells (hMSCs) Scaffold Material: RGD grafted oxidized sodium alginate/N-succinyl chitosan hydrogel (RGD-OSA/NSC) |
Duty Cycle: 20% Frequency: 1 MHz Intensity: 200 mW/cm2 Exposure: 10 min daily for 1, 3, 7,10, 14, 0r 21 days |
LIPUS improved cell proliferation, ALP activity, and mineralization. |
| Hsu et al. (2011) [80] | Cell Type: MG63 osteoblast-like cells Scaffold Material: commercial purity titanium (CP-Ti) |
Intensity: 0, 50, 150, and 300 mW/cm2 Frequency: 1 MHz Pulse Repetition: 100 Hz Exposure: 3 min daily for 5 days (in vitro); 10 min daily for 20 or 30 days (in vivo) |
LIPUS improved cell viability and ALP activity in vitro. LIPUS improved blood flow and the maturation of collagen fibers. Pulsed ultrasound was better than continuous ultrasound for |
| Nagasaki et al. (2015) [81] | Cell Type: adipose derived stem cells (ADSCs) Scaffold Material: nanohydroxyapatite (nHA) |
Intensity: 60 mW/cm2 Frequency: 3.0 MHz sine waves repeated at 100 Hz Exposure: 10 min daily for 7, 14, or 21 days |
LIPUS increased calcium and phosphate deposition and bone thickness for adipose derived stem cells in a nHA scaffold. |