MC3T3 infiltration and proliferation in bovine trabecular scaffold regulated by dynamic flow bioreactor and augmented by low-intensity pulsed ultrasound

Abstract

Background: Low-intensity pulsed ultrasound (LIPUS) has been used in both basic research and clinical settings for its therapeutic potential in promoting tissue healing. Clinical data has shown that LIPUS can accelerate fresh fracture healing. However, the treatment for aging osteoporosis and non-union is still unclear. In addition, the mechanism of ultrasound promoted bone healing has remained unknown.

Objective: It is proposed that noninvasive ultrasound treatment can enhance local fluid flow within the tissue to initiate remodeling and regeneration. The goal of this study was to evaluate the effects of dynamic ultrasound in promoting cellular mechanotransduction within bioengineered organic scaffolds to trigger osteogenesis and mineralization.

Methods: The experiment was designed in two-fold: to evaluate the role of LIPUS on osteoblastic-like (MC3T3) cell proliferation and mineralization in response to acoustic waves, using biomechanical rate-dependent signals in a bioreactor; and, to evaluate the new scaffold experimentation techniques, in order to generate a potential implantable biomaterial for orthopedic tissue regeneration and repair.

Results: LIPUS treatment on MC3T3 cells yielded enhanced cellular mineralization (**p < 0.001) in 3-D scaffolding, but reduced the total cell numbers (*p < 0.05), using Alizarin Red staining and cell counting analyses, respectively, in comparison to the control.

Conclusion: This study suggests that LIPUS, if applied at proper frequency and duty cycle, can promote cell mineralization within the 3-D organic scaffold under in vitro setting. The translational component of this experiment seeks to draw a parallel to the potential pre-treatment of scaffolds for implantation before orthopedic surgery, which could prove to greatly benefit the patient in accelerating fracture healing and tissue regeneration.

The translational potential of this article: LIPUS stimulation was critical in contributing to the mechanical signaling transductions that activated bone enhancement parameters in MC3T3 cells regulated by bioreactor, and thus has potential to change how we pretreat scaffolds for orthopedic surgery and noninvasively accelerate healing in the future, e.g., in an extreme condition such as long-term space mission.

Keywords: Bioreactor; Bone tissue engineering and regeneration; Low-intensity pulsed ultrasound; Scaffold.

Figure 1

Bioreactor setup in incubator and probes. The control panel was installed into the hood for technical reasons. The Masterflex rotor could then be operated using the control panel to circulate media at the specified flow rate of 0.1 mL/min to the OsteoGen chambers. This setup has the LIPUS console outside of the incubator. This allowed control LIPUS stimulation without opening the incubator, thus minimizing environmental perturbations. Probes were coupled to the chambers through a polyurethane mold and gel to remove airspace and seal coupling. To ensure the consistence of the treatment, we have set the ultrasound exposure in the far field, which operated by an acoustic lens (∼7 mm), acoustic coupling gel (∼1–2 mm) and the thickness of the bioreactor chamber (∼3 mm). Within the ultrasound far field zone, the energy is consistent and is controlled at approximately 30 mW/cm². LIPUS = low-intensity pulsed ultrasound.

Figure 2

LIPUS decreases the cell count after 5 days of stimulation. The average number of cells counted after control (5.70 × 10⁴) and LIPUS treatment (3.52 × 10⁴) on the scaffolds in conditions of bioreactor (*p < 0.05) demonstrates results in favour of the null hypothesis regarding the effect of LIPUS treatment on cell proliferation.

Figure 3

Alizarin Red Stain on fixed MC3T3 cells. Images A and C depict the control under 4× and 10× magnification, respectively. Images B and D depict the LIPUS-treated cells under 4× and 10× magnification, respectively. It is difficult to qualify the difference in calcific deposition by the images alone. Scale bar 4× = 5000 μm and 10× = 2000 μm. LIPUS = low-intensity pulsed ultrasound.

Figure 4

LIPUS increases matrix mineralization after 5 days of stimulation. Alizarin Red quantification demonstrated a 62% increase in extracellular matrix calcific depositions measured by a significant increase in absorbance - 2.18 control vs. 2.69 ultrasound (**p < 0.001). LIPUS = low-intensity pulsed ultrasound.