Developing and Investigating a Nanovibration Intervention for the Prevention/Reversal of Bone Loss Following Spinal Cord Injury

Osteoporosis disrupts the fine-tuned balance between bone formation and resorption, leading to reductions in bone quantity and quality and ultimately increasing fracture risk. Prevention and treatment of osteoporotic fractures is essential for reductions in mortality, morbidity, and the economic burden, particularly considering the aging global population. Extreme bone loss that mimics time-accelerated osteoporosis develops in the paralyzed limbs following complete spinal cord injury (SCI). In vitro nanoscale vibration (1 kHz, 30 or 90 nm amplitude) has been shown to drive differentiation of mesenchymal stem cells toward osteoblast-like phenotypes, enhancing osteogenesis and inhibiting osteoclastogenesis simultaneously. Here, we develop and characterize a wearable device designed to deliver and monitor continuous nanoamplitude vibration to the hindlimb long bones of rats with complete SCI. We investigate whether a clinically feasible dose of nanovibration (two 2 h/day, 5 days/week for 6 weeks) is effective at reversing the established SCI-induced osteoporosis. Laser interferometry and finite element analysis confirmed transmission of nanovibration into the bone, and microcomputed tomography and serum bone formation and resorption markers assessed effectiveness. The intervention did not reverse SCI-induced osteoporosis. However, serum analysis indicated an elevated concentration of the bone formation marker procollagen type 1 N-terminal propeptide (P1NP) in rats receiving 40 nm amplitude nanovibration, suggesting increased synthesis of type 1 collagen, the major organic component of bone. Therefore, enhanced doses of nanovibrational stimulus may yet prove beneficial in attenuating/reversing osteoporosis, particularly in less severe forms of osteoporosis.


Systems
The electronics for the animal device consist of two main elements; the wave generator circuit and the accelerometer amplification circuit.The wave generator circuit is based on that used to generate a sine wave voltage signal in the Nanokick bioreactor (Campsie et al., 2019).An AD9833 low power, programmable waveform generator (Analog Devices, Massachusetts, USA) produces the required sine wave with the output frequency and phase programmed using an ATMega328 microcontroller (Atmel, California, USA).Filtering is required at the output of the AD9833 to significantly reduce higher frequency components, created during the digital synthesis of the primary signal, using a seventh order LC elliptical reconstruction filter.A noninverting amplifier circuit, using an OPA37 ultra-low noise OP-AMP (Texas Instruments, Texas, USA), is utilised to boost the amplitude of the filtered sine wave before it reaches the final amplification stage.A 10 kΩ rotary potentiometer was used for the N40 rats in the non-inverting amplifier circuit allows the gain, and therefore the amplitude of the sine wave to be adjusted by the user.This was switched to a 20 kΩ potentiometer for the N100 rats to allow higher gain, and therefore higher displacement amplitudes from the transducer.
Equation 1 shows the gain for a non-inverting op-amp.Increasing the maximum value of the potentiometer (  ), which is acting as the feedback resistor in our circuit, increases the gain of the amplifier.
Finally, the sine wave signal is amplified with a MAX98306 3.7W power amplifier (Maxim Integrated, California, USA) to provide the bone conduction transducer with the voltage and current needed to function.The power amplification stage was purchased as a standalone printed circuit board (PCB) (Adafruit Industries, New York, USA).A block diagram of circuitry described above can be seen in Figure 1 and the wave generator and power amplifier PCBs can be seen in Figure 2.   measurement range of 1 -200 nm the gradient of the calibration curve can be weighted by data points at the higher end of the scale, giving conversions at the lower end of the scale a larger error.
For wide range measurements, the amplitude of the sine wave is incrementally increased from 2.5 nm to 200 nm and the ACH-01 voltage measured with an oscilloscope (V rms and V peak-to-peak ), see Figure 4B, and for shorter range measurements data is taken from 1 -10 nm in incremental steps of 1 nm, see Figure 4A.For the displacements detected during animal experiments it was determined that the calibration curve for the lower range measurements would give the most accurate results.

Figure 1 :
Figure 1: Block diagram of main components of the wave generator PCB used in the rat study.

Figure 2 :
Figure 2: (A) Photograph of the wave generator PCB with main components highlighted (B)

Figure 3 :
Figure 3: Block diagram of main components of the accelerometer circuitry and data acquisition

Figure 4 :
Figure 4: (A) Plot of the calibration data for ACH-01 device 4 over a short range of displacement

Figure 6B .
Figure 6B.Representative µCT-based images of the nanovibrated (right) and contralateral control

Figure 7B .
Figure 7B.Mean morphometric outcome measures of the nanovibrated (right) and contralateral

Figure 7C .
Figure 7C.Mean morphometric outcome measures for the proximal tibial epiphyseal secondary

Figure 7E .
Figure 7E.Mean morphometric outcome measures of the nanovibrated (right) and contralateral

Figure 7F .
Figure 7F.Mean morphometric outcome measures for the distal femur metaphyseal secondary

Figure 7H .
Figure 7H.Mean morphometric outcome measures of the nanovibrated (right) and contralateral

Figure 7I .
Figure 7I.Mean morphometric outcome measures for the distal femur epiphyseal trabecular VOI

Figure 8B .
Figure 8B.Representative µCT-based images of the nanovibrated (right) and contralateral control

Figure 8C .
Figure 8C.Representative µCT-based images of the tibial mid-diaphyseal cortical bone VOI